Patent application title: STEADY STATE ANAEROBIC DENITRIFYING CONSORTIUM FOR APPLICATION IN IN-SITU BIOREMEDIATION OF HYDROCARBON-CONTAMINATED SITES AND ENHANCED OIL RECOVERY
Inventors:
Edwin R. Hendrickson (Hockessin, DE, US)
Edwin R. Hendrickson (Hockessin, DE, US)
Abigail K. Luckring (West Chester, PA, US)
Sharon Jo Keeler (Bear, DE, US)
Michael P. Perry (Downingtown, PA, US)
Michael P. Perry (Downingtown, PA, US)
Eric R. Choban (Williamstown, NJ, US)
Assignees:
E. I. DU PONT DE NEMOURS AND COMPANY
IPC8 Class: AC12N120FI
USPC Class:
4352524
Class name: Micro-organism, per se (e.g., protozoa, etc.); compositions thereof; proces of propagating, maintaining or preserving micro-organisms or compositions thereof; process of preparing or isolating a composition containing a micro-organism; culture media therefor bacteria or actinomycetales; media therefor mixed culture
Publication date: 2014-07-10
Patent application number: 20140193884
Abstract:
Enriched steady state microbial consortiums for microbial enhanced oil
recovery and in situ bioremediation of hydrocarbon-contaminated sites,
under anaerobic denitrifying conditions, are disclosed.Claims:
1. An isolated consortium of microbial species wherein said consortium
comprises; a) at least one first species of the genus Thauera having a
16S rDNA nucleic acid molecule having the nucleic acid sequence that has
at least 95% identity to SEQ ID NO: 15; b) at least one second species
having 16S rDNA nucleic acid molecule having the nucleic acid sequence
that has at least 95% identity to a sequence selected from the group
consisting of SEQ ID NOs:16, 19, 21, 23, 24-28, 30-41, 67 and 68; and c)
at least one third species having a 16S rDNA nucleic acid molecule having
the nucleic acid sequence that has at least 95% identity to a sequence
selected from the group consisting of SEQ ID NOs: 17, 18, 20, 22, 29, 54,
69 and 86 and combinations thereof.
2. The isolated consortium of microbial species of claim 1 further comprising species having 16S rDNA nucleic acid molecule having the nucleic acid sequence that has at least 95% identity to a sequences selected from the group consisting of SEQ ID NOs: 55, 63, 75, 76 and 81 and having the nucleic acid sequence that has at least 95% identity to a sequence selected from the group consisting of SEQ ID NOs: 42, 45, 50-52, 64-66 and 79 and having the nucleic acid sequence that has at least 95% identity to a sequence selected from the group consisting of SEQ ID NOs: 48, 49 and 82.
3. The isolated consortium of microbial species of claim 1 further comprising species having 16S rDNA nucleic acid molecule having the nucleic acid sequence that has at least 95% identity to a sequence selected from the group consisting of SEQ ID NOs: 53, 58 and 87 and having the nucleic acid sequence that has at least 95% identity to a sequence selected from the group consisting of SEQ ID NOs: 56 and 77.
4. The isolated consortium of microbial species of claim 1 further comprising species having 16S rDNA nucleic acid molecule having the nucleic acid sequence that has at least 95% identity to a sequence selected from the group consisting of SEQ ID NO: 43.
5. The isolated consortium of microbial species of claim 1 further comprising species having 16S rDNA nucleic acid molecule having the nucleic acid sequence that has at least 95% identity to a sequence selected from the group consisting of SEQ ID NOs: 60-62, 80 and 83 and having the nucleic acid sequence that has at least 95% identity to a sequence selected from the group consisting of SEQ ID NOs: 20, 44, 46, 57, 70-74, 84 and 85.
6. The isolated consortium of microbial species of claim 1 further comprising species having 16S rDNA nucleic acid molecule having the nucleic acid sequence that has at least 95% identity to a sequence selected from the group consisting of SEQ ID NOs: 47 and 59.
7. The isolated consortium of microbial species of claim 1 further comprising species having 16S rDNA nucleic acid molecule having the nucleic acid sequence that has at least 95% identity to a sequence selected from the group consisting of SEQ ID NO: 78.
Description:
[0001] This application claims the benefit of U.S. Provisional Application
61/154,542, filed Feb. 23, 2009 and the U.S. National application Ser.
No. 12/704,609 filed Feb. 12, 2010.
FIELD OF INVENTION
[0002] This disclosure relates to the field of environmental microbiology. More specifically, a steady state consortium of anaerobic denitrifying microorganisms is developed with functionality in environmental microbiology and its population is defined at molecular levels. This consortium is used for enhanced oil recovery and in situ bioremediation of hydrocarbon-contaminated sites.
BACKGROUND OF THE INVENTION
[0003] The challenge to meet the ever increasing demand for oil has resulted in increasing activities in crude oil recovery from oil reservoirs for refinery processes and various other applications. These activities have resulted in contaminating various environments such as soil, groundwater, sand, drinking water, etc, with hydrocarbons. There are now two worldwide challenges that need to be met: 1) recovering the petroleum deposits for oil reservoirs; and 2) remediating the hydrocarbon-contaminated environmental sites.
[0004] Heavy crude oil in the form of petroleum deposits and oil reservoirs are distributed worldwide and because of its relatively high viscosity, it is essentially immobile and cannot be easily recovered by conventional primary and secondary means. Expanding efforts to develop alternative cost efficient oil recovery processes have been documented (Kianipey, S. A. and Donaldson, E. C. 61st Annual Technical Conference and Exhibition, New Orleans, La., USA, Oct. 5-8, 1986).
[0005] Microbial Enhanced Oil Recovery (MEOR) is a methodology for increasing oil recovery by the action of microorganisms (Brown, L. R., et al., SPE 59306, SPE/DOE Improved Oil Recovery Symposium, Oklahoma, Apr. 3-5, 2000). MEOR research and development is an ongoing effort directed at discovering techniques to use microorganisms to modify crude oil properties to benefit oil recovery (Sunde. E., et al., SPE 24204, SPE/DOE 8th Symposium on enhanced Oil Recovery, Tulsa, Okla., USA, Apr. 22-24, 1992). In MEOR processes, useful microbes are typically hydrocarbon-utilizing, non-pathogenic microorganisms, which use hydrocarbons as their source of energy to grow or excrete natural bio-products such as alcohols, gases, acids, surfactants and polymers. These bio-products change the physio/chemical properties of the crude oil and stimulate changes in the oil-water-rock interactions to improve oil recovery.
[0006] Remediation of hydrocarbon-contaminated sites is difficult due to the structural properties of the crude oil. Crude oil is characterized by apolar C--C and C--H bonds and lacks functional chemical groups that contribute to the crude oil's recalcitrant nature. Crude oil consists of alkanes, alkenes, alkynes, aromatic polycyclic hydrocarbons, asphaltenes and resins. Conventional methods used to remediate hydrocarbons include solvent treatment and polymeric particles having covalently bound to a polymeric component as described in U.S. Pat. No. 7,449,429B2, U.S. Pat. No. 6,852,234B2, U.S. Pat. No. 7,465,395, U.S. Pat. No. 7,201,804B2, U.S. Pat. No. 7,473,672B2, U.S. Pat. No. 7,442,313B2; site excavation as practiced by Ground Remediation Systems, LTD, UK; and pump and treat, which involves pumping out contaminated groundwater with the use of a submersible or vacuum pump The extracted groundwater is then purified by slowly proceeding through a series of vessels that contain materials designed to adsorb the contaminants from the groundwater and vacuum extraction (U.S. Pat. No. 7,172,688B2). These processes are costly, time consuming and leave undesirable environmental footprints.
[0007] Alternatively, microorganisms may be used for in situ bioremediation of hydrocarbon-contaminated sites. For example, biodegradation of contaminants by indigenous microbial populations is common in many aerobic environments (Gibson, D. T., Microbial Degradation of Organic Compounds, 1984, Marcel Dekker, NY). Addition of oxygen and nutrients to stimulate the growth of indigenous microorganisms can be an effective bioremediation tool in the cleanup of crude oil spill. An alternative approach, reported for soils contaminated with crude oil or petroleum hydrocarbons, is the introduction into the soils of microbes capable of degrading these chemicals. These processes rely on oxidative degradation under aerobic conditions, and the microbes use the hydrocarbon contaminant as the carbon and energy source (U.S. Pat. No. 6,652,752B2). However, in many cases aerobic bioremediation is impractical because of the anoxic nature of the natural environments of the hydrocarbon-contaminated sites, such as soil, groundwater aquifers, fresh water and marine sediments and oil reservoirs.
[0008] Since application of microorganisms for MEOR and in-situ bioremediation is a promising alternative to traditional oil recovery or in situ remediation means, developing methods for identifying microorganisms useful in these processes, which would allow cost-effective processes for MEOR and bioremediation, is important. Previously described methods for such applications, for example, include obtaining the sample under specific conditions with a defined nutrient medium in the presence of anaerobic gas mixtures (U.S. Patent Application No. 200710092930A1). A process for stimulating the in situ activity of a microbial consortium to produce methane for oil was described (U.S. Pat. No. 6,543,535B2). However, such processes are time consuming, labor-intensive and therefore costly.
[0009] Thus, there is a need for developing methods to: 1) obtain a steady state population of consortium of microorganisms that can grow in or on oil under anaerobic denitrifying conditions; 2) identify the members of the steady state consortium for properties that might be useful in oil modification and/or degradation and 3) use said steady state consortium of microorganisms, in a cost-effective way, for enhanced oil recovery from wells or reservoirs or in situ bioremediation of hydrocarbon-contaminated sites.
SUMMARY ON THE INVENTION
[0010] Enriched steady state microbial consortiums for microbial enhanced oil recovery and in situ bioremediation of hydrocarbon-contaminated sites, under anaerobic denitrifying conditions, are disclosed. The consortium is identified by obtaining environmental samples comprising indigenous microbial populations exposed to crude oil and enriching said populations per an enrichment protocol. The enrichment protocol employs a chemostat bioreactor to provide a steady state population. The steady state population may be characterized by using phylogenetic DNA sequence analysis techniques, which include 16S rDNA profiling and/or DGGE fingerprint profiling as described herein. The steady state population is further characterized as an enriched consortium comprising microbial constituents having relevant functionalities for improving oil recovery or in situ bioremediation of hydrocarbon-contaminated environmental sites. The steady state enriched consortium may grow in situ, under reservoir conditions, using one or more electron acceptors and the reservoir's crude oil as the carbon source for microbial enhancement of oil recovery or in situ bioremediation of hydrocarbon-contaminated environmental sites. The steady state consortium may be used with other microorganisms to enhance oil recovery in reservoirs or wells or in situ bioremediation of hydrocarbon-contaminated environmental sites with analogous reservoir conditions of the selected/targeted wells.
[0011] In one aspect, a method for in situ bioremediation of hydrocarbon-contaminated environmental sites or enhancing oil recovery from an oil reservoir using an enriched steady state microbial consortium is provided, said method comprising:
[0012] a. at least one first species of the genus Thauera having a 16S rDNA nucleic acid molecule having the nucleic acid sequence that has at least 95% identity to SEQ ID NO: 15;
[0013] b. at least one second species having 16S rDNA nucleic acid molecule having the nucleic acid sequence that has at least 95% identity to a sequence selected from the group consisting of SEQ ID NOs:16, 19, 21, 23, 24-28, 30-41, 67 and 68; and
[0014] c. at least one third species having 16S rDNA nucleic acid molecule having the nucleic acid sequence that has at least 95% identity to a sequence selected from the group consisting of SEQ ID NOs: 17, 18, 20, 22, 29, 54, 69 and 86 and combinations thereof is provided.
[0015] In another aspect, a composition for enhancing oil recovery or for in situ bioremediation comprising: an isolated consortium of microbial species, comprising at least one Thauera strain and at least two other strains selected from the group consisting of Azoarcus species, Pseudomonas species, Azotobacter species, Bacteroides species, Clostridium species, Anaerovorax species, Finegoldia species, Spirochetes species, Deferribacter species, Flexistipes species, Chloroflexi species and Ochrobactrum species is provided.
BRIEF DESCRIPTION OF FIGURES OF THE INVENTION
[0016] FIG. 1: Distribution of microorganisms in the parent POG1 consortium after three months in second-generation parent populations as determined by 16S rDNA identities.
[0017] FIGS. 2A and 2B: Distribution of microorganisms in the parent POG1 consortium after 190 days in second- and third-generation parent populations determined by 16S rDNA identities. FIG. 2A: Population distribution of third-generation parent at 190 days while 6400 ppm Nitrate had been reduced. FIG. 2B: Population distribution of second-generation parent at 240 days while 6400 ppm Nitrate had been reduced
[0018] FIG. 3: Diagram of the anaerobic chemostat bioreactor for denitrifying growth studies with the steady state POG1 consortium: A) Reverse flow bubbler; B) Nitrogen manifold; C) Feed sampling syringe and relief valve (5 psi); D) Feed syringe pump; E) Feed reservoir head space nitrogen gas port; F) Feed input port on chemostat bioreactor; G) Feed medium reservoir (minimal and nitrate); H)Chemostat Bioreactor; I) Minimal salt medium and consortium culture; J) Magnetic stirrer; K) Crude oil supplement; L) Effluent reservoir; M) Effluent exit port on chemostat bioreactor; N) Effluent reservoir head space nitrogen gas port; O) Effluent syringe port; P) Effluent sampling syringe and relief valve (5 psi); Q) Inoculation and sampling port on chemostat bioreactor; R) Extra port and plug; S) Chemostat bioreactor head space nitrogen gas port.
[0019] FIG. 4: Distribution of microorganisms in the steady state POG1 as determined by 16S rDNA identities. Consortium constituents at 0, 28 and 52 day, were compared to the parent populations.
[0020] FIG. 5: Denaturing gradient gel electrophoresis fingerprint profile of the bacterial 16S rRNA gene fragments derived from community DNA extracted from the steady state POG1 chemostat bioreactor using primers SEQ ID NO: 12 and SEQ ID NO: 14 for region V4-5. (A) Thauera strain AL9:8 is a prominent species of a consortium as described herein. (B) Pseudomonas stutzeri LH4:15 is also a represented species of the consortium. (C) Ochrobactrum oryzae AL1:7 is the minor species. Minor bacterial species (D through L) are present in all samples. Bacterial species (C & M through 0) are less important members of population and are selected against.
[0021] FIG. 6: Microsand column oil release--Using oil on North Slope sand, the 3rd generation parent POG1 consortium culture EH40:1 (2400 ppm Nitrate).
[0022] The following sequences conform to 37 C.F.R. §1.821-1.825 ("Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures--the Sequence Rules") and are consistent with the World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5 (a-bis), and Section 208 and Annex C of the Administrative Instructions. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
TABLE-US-00001 TABLE 1 PRIMER SEQUENCES USED IN THIS INVENTION SEQ ID NO: Description Nucleic acid 8F 1 Bacterial 16S rDNA forward universal primer 1492 R 2 Bacterial 16S rDNA reverse universal primer 1407 R 3 Bacterial 16rDNA reverse universal primer U518R, 4 16S rDNA universal reverse primer UB 357F 5 Bacterial 16S rDNA forward universal primer dG•UB 357F 6 DGGE Bacterial 16S rDNA universal forward primer with 5' 40-bp GC-rich clamp UA 341F1 7 Archaeal 16S rDNA universal forward primer dG•UA 341F1 8 DGGE Archaeal 16S rDNA universal forward primer with 5' 40-bp GC-rich clamp UA 341F2 9 Archaeal 16S rDNA universal forward primer dG•UA 341F2 10 DGGE Archaeal rDNA universal forward 16S primer with 5' 40-bp GC-rich clamp U 519F 11 Universal 16S rDNA forward primer dG•U 519F 12 DGGE Universal 16S rDNA forward primer with 5' 40-bp GC-rich clamp UA958R, 13 Archaeal universal 16S rDNA reverse primer UB 939R, Bacterial 16S rRNA universal 14 reverse primer
[0023] The following DNA sequences were consensus sequences of unique cloned PCR sequences, which were generated using universal 16S primers with DNA isolated from whole POG1 community:
SEQ ID NO: 15 is the consensus DNA sequence, clones ID: 1A: Thauera sp AL9:8 SEQ ID NO: 16 is the consensus DNA sequence, clones ID: 1B: Thauera sp R26885 SEQ ID NO: 17 is the consensus DNA sequence, clones ID: 1C: Azoarcus sp mXyN1 SEQ ID NO: 18 is the consensus DNA sequence, clones IDI: Azoarcus sp mXyN1 SEQ ID NO: 19 is the consensus DNA sequence, clones ID: 1E: Thauera sp R26885 SEQ ID NO: 20 is the consensus DNA sequence, clones ID: 1F: Azotobacter beijerinckii SEQ ID NO: 21 is the consensus DNA sequence, clones ID: 1G: Thauera sp R26885 SEQ ID NO: 22 is the consensus DNA sequence, clones ID: 1H: Azoarcus sp mXyN1 SEQ ID NO: 23 is the consensus DNA sequence, clones ID: 1I: Thauera aromatica SEQ ID NO: 24 is the consensus DNA sequence, clones ID: 1J: Thauera aromatica SEQ ID NO: 25 is the consensus DNA sequence, clones ID: 1: Thauera aromatica SEQ ID NO: 26 is the consensus DNA sequence, clones ID: 1L: Thauera aromatica SEQ ID NO: 27 is the consensus DNA sequence, clones ID: 1M: Thauera aromatica SEQ ID NO: 28 is the consensus DNA sequence, clones ID: 1N: Thauera aromatica SEQ ID NO: 29 is the consensus DNA sequence, clones ID: 1O: Azoarcus sp. EH1O SEQ ID NO: 30 is the consensus DNA sequence, clones ID: 1P: Thauera sp R26885 SEQ ID NO: 31 is the consensus DNA sequence, clones ID: 1Q: Thauera aromatica SEQ ID NO: 32 is the consensus DNA sequence, clones ID: 1R: Thauera aromatica SEQ ID NO: 33 is the consensus DNA sequence, clones ID: 1S: Thauera aromatica SEQ ID NO: 34 is the consensus DNA sequence, clones ID: 1T: Thauera aromatica SEQ ID NO: 35 is the consensus DNA sequence, clones ID: 1U: Thauera aromatica SEQ ID NO: 36 is the consensus DNA sequence, clones ID: 1V: Thauera aromatica SEQ ID NO: 37 is the consensus DNA sequence, clones ID: 1W: Thauera aromatica SEQ ID NO: 38 is the consensus DNA sequence, clones ID: 1X: Thauera aromatica SEQ ID NO: 39 is the consensus DNA sequence, clones ID: 1Y: Thauera aromatica SEQ ID NO: 40 is the consensus DNA sequence, clones ID: 1Z: Thauera aromatica SEQ ID NO: 41 is the consensus DNA sequence, clones ID: 1AZ: Thauera aromatica SEQ ID NO: 42 is the consensus DNA sequence, clones ID: 2: Finegoldia magna SEQ ID NO: 43 is the consensus DNA sequence, clones ID: 3 Spirochaeta sp MET-E SEQ ID NO: 44 is the consensus DNA sequence, clones ID: 4: Azotobacter beijerinckii SEQ ID NO: 45 is the consensus DNA sequence, clones ID: Finegoldia magna SEQ ID NO: 46 is the consensus DNA sequence, clones ID: 6: Azotobacter beijerinckii SEQ ID NO: 47 is the consensus DNA sequence, clones ID: 7: Ochrobactrum sp mp-5 SEQ ID NO: 48 is the consensus DNA sequence, clones ID: 8A: Anaerovorax sp. EH8A SEQ ID NO: 49 is the consensus DNA sequence, clones ID: 8B: Anaerovorax sp. EH8B SEQ ID NO: 50 is the consensus DNA sequence, clones ID: 9A: Finegoldia magna SEQ ID NO: 51 is the consensus DNA sequence, clones ID: 9B: Finegoldia magna SEQ ID NO: 52 is the consensus DNA sequence, clones ID: 9C: Finegoldia magna SEQ ID NO: 53 is the consensus DNA sequence, clones ID: 10: Flexistipes sp vp180 SEQ ID NO: 54 is the consensus DNA sequence, clones ID: 11: Azoarcus sp._EH11 SEQ ID NO: 55 is the consensus DNA sequence, clones ID: 12: Clostridium chartatabidium SEQ ID NO: 56 is the consensus DNA sequence, clones ID: 13: Deferribacter desulfuricans SEQ ID NO: 57 is the consensus DNA sequence, clones ID: 14A: Azotobacter beijerinckii SEQ ID NO: 58 is the consensus DNA sequence, clones ID: 14B: Flexistipes sp vp180 SEQ ID NO: 59 is the consensus DNA sequence, clones ID: 15: Ochrobactrum lupini SEQ ID NO: 60 is the consensus DNA sequence, clones ID: 16A: Pseudomonas pseudoalcligenes SEQ ID NO: 61 is the consensus DNA sequence, clones ID: 16B: Pseudomonas putida SEQ ID NO: 62 is the consensus DNA sequence, clones ID: 17A: Pseudomonas pseudoalcligenes SEQ ID NO: 63 is the consensus DNA sequence, clones ID: 17B: Clostridium chartatabidium SEQ ID NO: 64 is the consensus DNA sequence, clones ID: 18A: Finegoldia magna SEQ ID NO: 65 is the consensus DNA sequence, clones ID: 18B: Finegoldia magna SEQ ID NO: 66 is the consensus DNA sequence, clones ID: 18C: Finegoldia magna SEQ ID NO: 67 is the consensus DNA sequence, clones ID: 19: Thauera aromatica SEQ ID NO: 68 is the consensus DNA sequence, clones ID: 20: Thauera aromatica SEQ ID NO: 69 is the consensus DNA sequence, clones ID: 21: Azoarcus sp. EH21 SEQ ID NO: 70 is the consensus DNA sequence, clones ID: 22: Azotobacter beijerinckii SEQ ID NO: 71 is the consensus DNA sequence, clones ID: 23: Azotobacter beijerinckii SEQ ID NO: 72 is the consensus DNA sequence, clones ID: 24: Azotobacter beijerinckii SEQ ID NO: 73 is the consensus DNA sequence, clones ID: 25: Azotobacter beijerinckii SEQ ID NO: 74 is the consensus DNA sequence, clones ID: 26: Azotobacter beijerinckii SEQ ID NO: 75 is the consensus DNA sequence, clones ID: 27: Clostridium chartatabidium SEQ ID NO: 76 is the consensus DNA sequence, clones ID: 28: Clostridium aceticum SEQ ID NO: 77 is the consensus DNA sequence, clones ID: 29: Deferribacter desulfuricans SEQ ID NO: 78 is the consensus DNA sequence, clones ID: 30: Bacteroides sp. EH30 SEQ ID NO: 79 is the consensus DNA sequence, clones ID: 31: Finegoldia magna SEQ ID NO: 80 is the consensus DNA sequence, clones ID: 32: Pseudomonas putida SEQ ID NO: 81 is the consensus DNA sequence, clones ID: 33: Clostridium aceticum SEQ ID NO: 82 is the consensus DNA sequence, clones ID: 34: Anaerovorax sp. EH34 SEQ ID NO: 83 is the consensus DNA sequence, clones ID: 35: Pseudomonas putida SEQ ID NO: 84 is the consensus DNA sequence, clones ID: 36: Azotobacter beijerinckii SEQ ID NO: 85 is the consensus DNA sequence, clones ID: 37: Azotobacter beijerinckii SEQ ID NO: 86 is the consensus DNA sequence, clones ID: 38: Azoarcus sp. EH36 SEQ ID NO: 87 is the consensus DNA sequence, clones ID: 39: Flexistipes sp vp180
DETAILED DESCRIPTION OF THE INVENTION
[0024] Applicants specifically incorporate the entire content of all cited references in this disclosure. Unless stated otherwise, all percentages, parts, ratios, etc., are by weight. Trademarks are shown in upper case. Further, when an amount, concentration, or other value or parameter is given as either a range, preferred range or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the invention be limited to the specific values recited when defining a range.
[0025] The components of, means, methods and procedures for providing an enriched steady state consortium having one or more relevant functionality to enhance the release and recovery of oil from a petroleum reservoir or in situ bioremediation of hydrocarbon-contaminated sites are disclosed.
[0026] The following definitions are provided for the terms and abbreviations used in this application:
[0027] The term "environmental sample" means any substance exposed to hydrocarbons of the contaminated site, including a mixture of water, soil and oil comprising microorganisms. As used herein, environmental samples include water, soil and oil samples that comprise indigenous microorganisms and/or populations of microorganisms of varying genus and species that may be characterized by 16S rDNA profiling or DNA fingerprinting techniques as described in detail below. The environmental samples may comprise a microbial consortium unique to a geographic region or the target hydrocarbon-contaminated site, or, alternatively the microbial consortium may be adaptable to other environmental sites, geographies and reservoirs.
[0028] The term "enriching for one or more steady state consortium" as used herein means that an environmental sample may be enriched in accordance with the invention by culturing the sample in a chemostat bioreactor under desired conditions such as anaerobic denitrifying conditions using a basic minimal medium, such as SL-10 as described in Table 2, and a sample of the target oil or its components or a soil or water sample from the hydrocarbon-contaminated site as a carbon source.
[0029] The term "core flood assay" refers to water-flooding the core of an oil reservoir after application of an oil recovery technique, i.e., a MEOR technology, to the reservoir. An increase in oil release represents the ability of applied microbes to aid in the release of oil from the core matrix.
[0030] The term "hydrocarbon-contaminated site" as used herein means an environmental site that has received heavy spills of either crude oil or other mixtures of various aliphatic, aromatic and asphaltene hydrocarbons.
[0031] The term "bioremediation of hydrocarbon-contaminated site" as used herein means degradation of the hydrocarbons that have contaminated the site through action of the microbial constituents of the steady state consortium.
[0032] The term "components of the POG1 consortium" refers to members or microbial constituents (both major and minor) of the POG1 consortium. These may be indigenous to the consortium or may be added strains. Additional components such as electron acceptors and combination of electron acceptors could be present too.
[0033] The terms "steady state consortium" and "enriched steady state microbial consortium" refers to a mixed culture of microorganisms and/or microbial populations grown in a chemostat bioreactor and in a medium under specific growth conditions to enrich for growth of particular populations of microorganisms, and once enriched, to reach a stable condition such that the consortium does significantly change over time under a given set of conditions. The steady state is controlled by a limiting nutrient. In an embodiment the steady state consortium is provided by enriching the microorganisms in a defined minimal, denitrifying medium, under anaerobic denitrifying conditions, using crude oil or a hydrocarbon-contaminated environmental sample as the carbon source, until the population has reached its steady state. In the present case the electron acceptor, nitrate, is limiting and is fed at a constant flow. The consortium may comprise microbial populations from environmental samples or from pure or mixed non-indigenous cultures.
[0034] The term "POG1 consortium" as used herein refers to a consortium derived from a hydrocarbon-contaminated environmental enrichment that was obtained from a soil sample contaminated with polycyclic aromatic hydrocarbons.
[0035] The term "crude oil" refers to a naturally occurring, flammable liquid found in rock formations and comprises a complex mixture of hydrocarbons of various molecular weights, plus other organic compounds. The crude oil may contain, for example, a mixture of paraffins, aromatics, asphaltenes, aliphatic, aromatic, cyclic, polycyclic and polyaromatic hydrocarbons. The crude oil may be generic or may be from a reservoir targeted for enhanced oil recovery, or from a hydrocarbon-contaminated environmental site targeted for in situ bioremediation.
[0036] The term "electron acceptor" refers to a molecule or compound that receives or accepts an electron during cellular respiration.
[0037] The terms "denitrifying" and "denitrification" mean reducing nitrate for use as an electron acceptor in respiratory energy generation.
[0038] The term "nitrates" and "nitrites" refers to any salt of nitrate (NO3) or nitrite (NO2).
[0039] The term "relevant functionalities" means that the consortium has the ability to function in ways that promotes oil recovery or in situ bioremediation. Certain such functionalities include:
[0040] (a) alteration of the permeability of the subterranean formation for improved water sweep efficiency;
[0041] (b) modification of the hydrocarbon components of the contaminated site
[0042] (c) production of biosurfactants to decrease surface and interfacial tensions;
[0043] (d) change in wettability;
[0044] (e) production of polymers other than surfactants that facilitate mobility of petroleum or availability of hydrocarbons;
[0045] (f) production of low molecular weight acids which cause rock dissolution;
[0046] (g) generation of gases to increase formation pressure;
[0047] (h) reduction in oil viscosity; and
[0048] (i) degradation of oil hydrocarbons or hydrocarbon components.
[0049] The ability to demonstrate such functionalities in the present invention is dependent upon the consortium's ability to (1) grow under anaerobic conditions while reducing nitrates or nitrites; (2) use at least one component available in the oil well or hydrocarbon-contaminated site as a carbon source; (3) use at least one component in the injected or produced water; (4) grow in the presence of oil; (5) grow optimally in the oil well or in the hydrocarbon-contaminated environment; and (6) achieve combinations of the above.
[0050] The term "modifying the environment of oil reservoir" includes the ability of the enriched steady state microbial consortium to affect an oil bearing formation in the following ways (per the relevant functionalities) 1) alter the permeability of the subterranean formation (sweep efficiency), (2) produce biosurfactants which decrease surface and interfacial tensions, (3) mediate changes in wettability, (4) produce polymers, which facilitate mobility of petroleum or availability of hydrocarbons; and (5) generate gases (predominantly CO2) that increase formation pressure; and (6) reduce oil viscosity.
[0051] The terms "well" and "reservoir" may be used herein interchangeably and refer to a subterranean or seabed formation from which oil may be recovered. The terms well and reservoir include the physical/chemical composition of the soil-rock-sediment structure of the reservoir below the surface.
[0052] The terms "target oil reservoir" and "target reservoir" may be used herein interchangeably and refer to a subterranean or seabed formation from which enhanced oil recovery is desired and to which the enriched steady state microbial consortium may be applied.
[0053] The term "growing on oil" means the microbial species capable of metabolizing aliphatic, aromatic and polycyclic aromatic hydrocarbons or any other organic components of the crude petroleum as a nutrient to support growth. The ability to grow on oil according to an embodiment of the invention eliminates the need for supplying certain nutrients, such as additional carbon sources, for using the microbial consortium for improved oil recovery or for in situ bioremediation of the hydrocarbon-contaminated site.
[0054] The term "chemostat bioreactor" refers to a bioreactor used for a continuous flow culture to maintain microbial populations or a consortium of microorganism in a steady state growth phase. This is accomplished by regulating a continuous supply of medium to the microbes, which maintains the electron donor or electron receptor in limited quantities in order to control the growth rate of the culture.
[0055] The term "fingerprint profile" refers to the process of generating a specific pattern of DNA bands on a denaturing gradient electrophoresis gel that are defined by their length and sequence and is used to identify and describe the predominant microbial population of a culture assessing microbial diversity and population stability at any particular metabolic state.
[0056] The term "promotes in situ bioremediation" as used herein means growing the microbial consortium in the contaminated site under anaerobic conditions to provide for modification of the oil in the site as defined above by a relevant functionality which may result in a change in the oil content of the hydrocarbon-contaminated site. Such changes support release of oil or its components from sand or soil to enhance bioremediation of the hydrocarbon-contaminated site.
[0057] The term "rDNA typing" or "rDNA profiling" means the process of comparing the 16S rDNA gene sequences found in the experimental samples to rDNA sequences maintained in several international databases to identify, by sequence homology, the "closest relative" of microbial species.
[0058] The term "signature sequences" herein will refer to unique sequences of nucleotides in the 16S rRNA gene sequence that can be used specifically to phylogenetically define an organism or group of organisms. These sequences are used to distinguish the origin of the sequence from an organism at the kingdom, domain, phylum, class, order, genus, family, species and even an isolate at the phylogenic level of classification.
[0059] The term "structural domain" herein refers to specific sequence regions in the 16S rRNA gene sequence that when aligned reveal a pattern in which relatively conserved stretches of primary sequence and a secondary sequence alternate with variable regions that differ remarkably in sequence length, base composition and potential secondary structure. These structural domains of 16S rRNA gene sequence are divided into three categories: the universally conserved or "U" regions, semi conserved or "S" regions and the variable or "V" regions. All of the structural domains contain signature sequence regions that phylogenetically define a microorganism. (Neefs, J-M et al., Nucleic acids Res., 18: 2237, 1990, Botter, E. C., ASM News 1996).
[0060] The term "phylogenetics" refers to the study of evolutionary relatedness among various groups of organisms (e.g., bacterial or archaeal species or populations).
[0061] The term "phylogenetic typing", "phylogenetic mapping" or "phylogenetic classification" may be used interchangeably herein and refer to a form of classification in which microorganisms are grouped according to their ancestral lineage. The methods herein are specifically directed to phylogenetic typing on environmental samples based on 16S ribosomal DNA (rDNA) sequencing. In this context, approximately 1400 base pair (bp) length of the 16S rDNA gene sequence is generated using 16S rDNA universal primers identified herein and compared by sequence homology to a database of microbial rDNA sequences. This comparison is then used to help taxonomically classify pure cultures for use in enhanced oil recovery.
[0062] The abbreviation "DNA" refers to deoxyribonucleic acid.
[0063] "Gene" is a specific unit on a DNA molecule that is composed of a nucleotide sequence that encodes a distinct genetic message for regulatory regions, transcribed structural regions or functional regions.
[0064] The abbreviation "rDNA" refers to ribosomal operon or gene sequences encoding ribosomal RNA on the genomic DNA sequence.
[0065] The abbreviation "NTPs" refers to ribonucleotide triphosphates, which are the chemical building blocks or "genetic letters" for RNA.
[0066] The abbreviation "dNTPs" refers to deoxyribonucleotide triphosphates, which are the chemical building blocks or "genetic letters" for DNA.
[0067] The term "rRNA" refers to ribosomal structural RNA, which includes the 5S, 16 S and 23S rRNA molecules. The term "rRNA operon" refers to an operon that produces structural RNA, which includes the 5S, 16 S and 23S ribosomal structural RNA molecules.
[0068] The term "mRNA" refers to an RNA molecule that has been transcribed from a gene coded on a DNA template and carries the genetic information for a protein to the ribosomes to be translated and synthesized into the protein.
[0069] The term "hybridize" is used to describe the formation base pairs between complementary regions of two strands of DNA that were not originally paired.
[0070] The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another.
[0071] For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine.
[0072] The abbreviation "cDNA" refers to DNA that is complementary to and is derived from either messenger RNA or rRNA.
[0073] The abbreviation "NCBI" refers to the National Center for Biotechnology Information.
[0074] The term "GenBank" refers to the National Institute of Health's genetic sequence database.
[0075] The term "nutrient supplementation" refers to the addition of nutrients that benefit the growth of microorganisms that are capable of using crude oil as their main carbon source but grow optimally with other non-hydrocarbon nutrients, i.e., yeast extract, peptone, succinate, lactate, formate, acetate, propionate, glutamate, glycine, lysine, citrate, glucose, and vitamin solutions.
[0076] The abbreviation "NIC" refers to non-inoculum, negative controls in microbial culture experiments.
[0077] The abbreviation "ACO" (autoclaved crude oil) refers to crude oil that has been steam sterilized using an autoclave, and is assumed to be devoid of living microbes.
[0078] The term "bacterial" means belonging to the bacteria--Bacteria are an evolutionary domain or kingdom of microbial species separate from other prokaryotes based on their physiology, morphology and 16S rDNA sequence homologies.
[0079] The term "microbial species" means distinct microorganisms identified based on their physiology, morphology and phylogenetic characteristics using 16S rDNA sequences.
[0080] The term "archaeal" means belongings to the Archaea. Archaea are an evolutionary domain or kingdom of microbial species separate from other prokaryotes based on their physiology, morphology and 16S rDNA sequence homologies.
[0081] The term "sweep efficiency" means the ability of injected water employed in water flooding oil recovery techniques to `push` oil through a geological formation toward a producer well.
[0082] The term "biofilm" means a film made up of a matrix of a compact mass of microorganisms consisting of structural heterogeneity, genetic diversity, complex community interactions, and an extracellular matrix of polymeric substances.
[0083] The term "irreducible water saturation" is the minimal water saturation that can be achieved in a porous core plug when flooding with oil to saturation. It represents the interstitial water content of the matrix where the water is never completely displaced by the oil because a minimal amount of water must be retained to satisfy capillary forces.
[0084] The term "ribotyping" or "riboprint" refers to fingerprinting of genomic DNA restriction fragments that contain all or part of the rRNA operon encoding for the 5S, 16S and 23S rRNA genes. Ribotyping, as described herein, is where restriction fragments, produced from microbial chromosomal DNA, are separated by electrophoresis, transferred to a filter membrane and probed with labeled rDNA operon probes. Restriction fragments that hybridize to the label probe produce a distinct labeled pattern or fingerprint/barcode that is unique to a specific microbial strain. The ribotyping procedure can be entirely performed on the Riboprinter® instrument (DuPont Qualicon, Wilmington, Del.).
[0085] The term "percent identity", as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by sequence comparisons. In the art, "identity" also means the degree of sequence relatedness or homology between polynucleotide sequences, as determined by the match between strings of such sequences and their degree of invariance. The term "similarity" refers to how related one nucleotide or protein sequence is to another. The extent of similarity between two sequences is based on the percent of sequence identity and/or conservation. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in "Computational Molecular Biology, Lesk, A. M., ed. Oxford University Press, NY, 1988"; and "Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NY, 1993"; and "Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, NJ, 1994"; and "Sequence Analysis in Molecular Biology, von Heinje, G., ed., Academic Press, 1987"; and "Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, NY, 1991". Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs.
[0086] The term "sequence analysis software" refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. "Sequence analysis software" may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul, S. F. et al., J. Mol. Biol. 215: 403-410, 1990), DNASTAR (DNASTAR, Inc., Madison, Wis.), and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, W. R., Comput. Methods Genome Res., Proc. Int. Symp, Meeting Date 1992, 111-120. eds.; Suhai, Sandor. Publisher: Plenum, New York, N.Y., 1994). Within the context of this application, it will be understood that where sequence analysis software is used for analysis, the results of the analysis will be based on the "default values" of the program referenced, unless otherwise specified. As used herein "default values" will mean any set of values or parameters that load with the software when first initialized.
[0087] The term "denaturing gradient gel electrophoresis" or "DGGE" refers to a molecular fingerprinting method that separates polymerase chain reaction-generated (PCR-generated) DNA products based on their length and sequence. The separation of the PCR product fragment of the same size, but with a different sequence reflects differential denaturing characteristics of the DNA due to their sequence variation. During DGGE, PCR products encounter increasingly higher concentrations of chemical denaturant as they migrate through a polyacrylamide gel. The rDNA PCR products are generated from the mixed microbial population being characterized. The weaker melting domains of certain double-stranded PCR sequences will begin to denature, slowing the electrophoretic migration dramatically. The different sequences of DNA (that are generated from different bacteria) will denature at different denaturant concentrations resulting in a pattern of bands that can be collectively referred to as the "community fingerprint profile". In theory, each band in a given DGGE fingerprint profile represents an individual bacterial species present in the community. Once generated, the data represents a fingerprint profile of the population at a given point in time and under certain growth conditions. The DGGE fingerprint profile can be uploaded into database to compare profiles of the consortium under prescribed growth conditions. Thus DGGE is used to generate the finger prints of a microbial community and to resolve the genetic diversity of complex microbial populations.
[0088] The present method provides for enhanced microbial oil recovery from oil reservoirs and enhanced in situ bioremediation of hydrocarbon-contaminated sites using an enriched steady state microbial consortium comprising the following steps: 1) obtaining an environmental samples comprising indigenous microbial populations; 2) developing an enriched steady state microbial consortium wherein said consortium is enriched under anaerobic denitrifying conditions, using crude oil from the target oil reservoir or hydrocarbon component samples from the specific contaminated site as the carbon source, until the population has reached its steady state; 3) developing fingerprint profiles of samples of the steady state consortium using 16S rDNA profiling methods of said samples; 4) selecting samples of the consortium comprising various microbial genera, for example, one or more Thauera species and other additional species selected from the group consisting of Rhodocyclaceae, Pseudomonadales., Bacteroidaceae., Clostridiaceae, Incertae Sedis., Spirochete, Spirochaetaceaes., Deferribacterales, Brucellaceae and Chloroflexaceae; 5) identifying at least one relevant functionality of the selected enriched steady state consortium for use in MEOR of oil reservoir or in situ bioremediation of the hydrocarbon-contaminated site; 6) growing the selected enriched steady state consortium having at least one relevant functionality to a concentration sufficient for oil well or hydrocarbon-contaminated site inoculation; 7) inoculating a subsurface matrix of an oil reservoir or hydrocarbon-contaminated site with said sufficient concentration of the steady state consortium and injection water or further additives comprising one or more electron acceptors wherein the consortium grows in the reservoir or environmental matrix (soil, groundwater, sandstone, rock or any combinations of all within the matrix) and wherein it promotes enhanced oil recovery or in situ bioremediation.
Environmental Samples for Development of a Microbial Consortium
[0089] The sample source used for enrichment cultures and development of a consortium for use in MEOR or in situ bioremediation may be: 1) the oil well itself in the form of: a water sample (injection, power or production water), soil from a reservoir core or from a sample of the targeted oil; 2) an environmental sample that has been exposed to crude oil or any one or combination of its components, such as paraffins, aromatics, asphaltenes, etc.; or (3) a preexisting consortium that meet the criteria for growth in the presence of the targeted oil. The sample must be in contact with or near the oil formation since sample constituents are specific to an area. Sampling near an intended location is preferred. The sample volume and the number of microbial cells per milliliter may vary from 1 mL to 5 L and from 105 to 1010 cells/mL, depending upon the specific requirements of the intended application. For the purposes of this invention, the cell density in the sample may be 107 cells per milliliter. To these samples, a basic mineral salt medium, which is required for microbial growth, vitamins and electron acceptors, may be added in addition to the sample of the crude oil from the desired contaminated location and the mixture may be incubated at a suitable temperature to allow development of the desired consortium with specific functionalities.
[0090] In another embodiment an environmental sample may be provided from an oil well or reservoir environment or a hydrocarbon-contaminated site located in the oil fields or contaminated sites, which include, but not limited to Texas, Alaska the industrial North Eastern and Midwestern United States, Oklahoma, California, the Gulf of Mexico, West Africa, the Middle East, India, China, North and Eastern South America, North Sea and the Old Soviet Union.
Microbial Chemostat Bioreactor
[0091] The environmental samples comprising microbial populations may be grown in a chemostat bioreactor using enrichment techniques. The enrichment conditions may include growing an environmental sample under anaerobic denitrifying conditions in bottles while limiting the concentration of electron acceptor provided during anaerobic respiration since the rate of manual feed is often too slow to keep up with reduction of nitrate. In addition, if too high a concentration of nitrate (e.g., >2500 ppm) were to be applied, it may either inhibit growth of some microbes or be toxic and kill some other species. Conversely, denitrifying bacteria stop growing when nitrate is completely reduced, hence allowing other microbial populations to dominate the composition of the consortium through reducing other trace metals, minerals and unsaturated hydrocarbons or organic molecules. Fluctuations in nitrate levels may affect changes in the microbial composition of the consortium and unduly influence the definition of the composition of the population in it. The non-limiting examples provided herein describe how to manipulate these conditions to enrich for and identify desired constituents of a steady state microbial consortium.
[0092] Chemostat bioreactors are systems for the cultivation of microbial communities or single microbial species and provide for maintaining conditions for microbial growth and populations at a steady state by controlling the volumetric feed rate of a growth dependant factor. The chemostat setup consists of a sterile fresh nutrient reservoir connected to a growth reactor. Fresh medium containing nutrients essential for cell growth is continuously pumped to the chamber from the medium reservoir. The medium contains a specific concentration of one or more growth-limiting nutrient that allows for growth of the consortium in a controlled physiological steady state. Varying the concentration of the growth-limiting nutrients will, in turn, change the steady state concentration of cells. The effluent, consisting of unused nutrients, metabolic wastes and cells, is continuously removed from the vessel, pumped from the chemostat bioreactor to the effluent reservoir and monitored for complete reduction of nitrate. To maintain constant volume, the flow of nutrients and the removal of effluent are maintained at the same rate and are controlled by synchronized syringe pumps.
Enrichment Conditions
[0093] As stated above an environmental sample may be enriched in accordance with the invention herein by culturing the sample in a chemostat bioreactor under desired conditions such as anaerobic denitrifying conditions. Additional enrichment conditions include use of a basic minimal medium, such as SL-10 as described in Table 2.
[0094] The chemostat bioreactor may be held at a room temperature that may fluctuate from about 15° C. to about 35° C.
[0095] The steady state consortium may be enriched under anaerobic, denitrifying conditions using a nitrate salt as the electron acceptor. The enrichment culture thus may include nitrate concentrations from 25 ppm to 10,000 ppm. More specifically, the nitrate concentration may be from 25 ppm to 5000 ppm. Most specifically, the nitrate concentration may be from 100 ppm to 2000 ppm.
[0096] In one embodiment an enriched steady state microbial consortium designated POG1 was developed under denitrifying conditions with a nitrate salt as the anoxic electron acceptor. Other suitable anaerobic reducing conditions would use selective electron acceptors that include, but are not limited to: iron (III), manganese (IV), sulfate, carbon dioxide, nitrite, ferric ion, sulfur, sulfate, selenate, arsenate, carbon dioxide and organic electron acceptors that include, but not limited the chloroethenes, fumarate, malate, pyruvate, acetylaldehyde, oxaloacetate and similar unsaturated hydrocarbon compounds may also be used.
[0097] The enrichment of the consortium may include a minimal growth medium supplemented with additional required nutritional supplements, e.g., vitamins and trace metals, and crude oil as the carbon source as described in details below.
[0098] This consortium may be grown at a pH from 5.0 to 10. More specifically the pH could be from 6.0 to about 9.0. Most specifically the pH could be from 6.5 to 8.5. In addition, the steady state consortium should have an OD550 from about 0.8 to about 1.2 and should actively reduce the electron acceptor.
Characterization of Microbial Populations in the Enriched Steady State Microbial Consortium
[0099] Constituents or the microbial populations of the enriched steady state consortium may be identified by molecular phylogenetic typing techniques. Identification of microbial populations in a consortium provides for selection of a consortium with certain microbial genera and species described to have relevant functionalities for enhancing oil recovery or in situ bioremediation of the hydrocarbon-contaminated sites.
[0100] In an embodiment of the invention, an enriched steady state consortium (referred to as "POG1") was developed, as described above, from a parent mixed culture, enriched from an environmental sample, using crude oil from the targeted hydrocarbon-contaminated site as the energy source. Various constituents of the consortium were characterized using fingerprint profiles of their 16S rDNA as described below, using signature regions within the variable sequence regions found in the 16S rRNA gene of microorganisms (see Muyzer, G., et al., supra). DNA sequences of the V3 region of 16S rRNA genes in a mix population were targeted and PCR amplified as described in detail below. Using this method a consortium comprising members from Thauera, Rhodocyclaceae, Pseudomonadales, Bacteroidaceae, Clostridiaceae, Incertae Sedis, Spirochete, Spirochaetaceaes, Deferribacterales, Brucellaceae and Chloroflexaceae were characterized (FIG. 1). The Thauera strain AL9:8 was the predominant microorganism in the consortium. It represented between 35 to 70% of the constituents during sampling processes. There were 73 unique sequences (SEQ ID NOs: 15-87), which were grouped into eight phylum of bacteria, which included alpha-Proteobacteria, beta-Proteobacteria, gamma-Proteobacteria, Deferribacteraceae, Spirochaetes, Bacteroidetes, Chloroflexi (Green sulfur bacteria) and Firmicutes/Clostridiales.
[0101] The phylum beta-Proteobacteria, which constitutes Gram negative and chemoautotrophic bacteria. They were represented by a large diverse group of the members of Thauera/Azoarcus group. There were 31 unique 16S rDNA sequences whose sequence differences occurred in the primary signature sequences of the variable regions. Thauera strain AL9:8 of this group was the predominant microorganism in the consortium and represented between 35 to 70% of the constituents during sampling processes and were represented in the consortium samples by (SEQ ID NOs: 15, 16, 19, 21, 23, 24-28, 30-41, 67 and 68). The Azoarcus species of this phylum in the steady state consortium were represented by (SEQ ID NOs: 17, 18, 20, 22, 29, 54, 69 and 86).
[0102] The phylum Firmicutes, order Clostridia, which consist of spore-forming, Gram-positive, obligate anaerobes that are mostly obligate fermenters was represented by Clostridium species, Anaerovorax species and Finegoldia species. In the consortium, Firmicutes/Clostridiales group was diverse with 16 unique sequences that include constituents from the Clostridia, Anaerovorax and Finegoldia genera. Further analyses using fingerprint profiling may allow assigning the DNA bands in the DGGE DNA fingerprint to some of these sequences. The Clostridia species in the consortium were represented by (SEQ ID NOs: 55, 63, 75, 76 and 81). The Anaerovorax species were characterized by (SEQ ID NO: 48, 49 and 82). The Finegoldia species were characterized by (SEQ ID NOs: 42, 45, 50-52, 64-66 and 79).
[0103] The phylum Deferribacteraceae are obligate, fermentative anaerobes and use nitrate and a wide variety of metal ion as electron acceptors. This phylum was represented by Deferribacter and Flexistipes species, which were represented by (SEQ ID NO: 56 and 77) and (SEQ ID NO: 56 and 77) respectively in the steady state consortium.
[0104] The phylum Spirochaetes are obligate, fermentative anaerobes that have a unique morphology. Spirochaetaceae are a tightly coiled slender and flexuous in shape and flagella are attached each pole and fold back from each pole and into the protoplasmic cylinder and remain located in the periplasm of the cell and are called endoflagella. The Spirochaeta species were represented by (SEQ ID NO: 43).
[0105] The phylum gamma-Proteobacteria and the Pseudomonadales order, which consists of Gram negative bacteria that are spiral or spherical or rod-shaped, usually motile by polar flagella and are facultative anaerobes that have the ability to degrade organic compounds under denitrifying conditions was represented by various Pseudomonas and Azotobacter species. The Pseudomonas species were represented by (SEQ ID NOs: 60-62, 80 and 83) and the Azotobacter species were represented by (SEQ ID NOs: 20, 44, 54, 70-74, 84 and 85) in the steady state consortium.
[0106] The phylum alpha-Proteobacteria, order Rhizobiales, family Brucellaceae was represented by Ochrobactrum species. They are Gram negative, rod-shaped, motile, chemoorganotrophic, facultative anaerobes. The Ochrobactrum species were represented by (SEA ID NOs: 47 and 59) in the steady state consortium.
[0107] The phylum Chloroflexi are filamentous anoxygenic phototrophs (formerly known as green non-sulfur) bacteria that produce energy through photosynthesis. During various stages of the enrichment of the POG1 consortium, Chloroflexi species were present. However, upon further enrichment of other species, they become undetectable in the steady state consortium.
[0108] The phylum Bacteroidetes, which are Gram negative rod shape, non-endospore-forming, anaerobes, and may be either motile or non-motile bacteria. The Bacteroides species were represented by (SEQ ID NO: 78) in the steady state consortium.
[0109] Based on these characterizations of samples of an enriched steady state microbial consortium, an embodiment of the invention includes an enriched steady state consortium comprising: Thauera, alpha-Proteobacteria, gamma-Proteobacteria, Deferribacteraceae, Bacteroides/Chloroflexi and Firmicutes/Clostridiales species.
[0110] In addition, the co-pending U.S. application Ser. No. 12/194,749, describes specifically, the one or more microbial cultures may be selected from the group consisting of Marinobacterium georgiense (ATCC#33635), Thauera aromatica T1 (ATCC#700265), Thauera chlorobenzoica (ATCC#700723), Petrotoga miotherma (ATCC#51224), Shewanella putrefaciens (ATCC#51753), Thauera aromatica S100 (ATCC#700265), Comamonas terrigena (ATCC#14635), Microbulbifer hydrolyticus (ATCC#700072), and mixtures thereof, having relevant functionalities for enhanced oil recovery or in situ bioremediation.
[0111] Comparing the components of an enriched steady state consortium to the phylogeny of known microorganisms having the ability to enhance oil recovery or bioremediate hydrocarbon-contaminated sites provides a mechanism for selecting a consortium useful for these processes. Further, such known microorganisms may be added to a steady state consortium to further enhance oil recovery or in situ bioremediation.
Phylogenetic Typing
[0112] The following description provides mechanisms for characterizing the constituents of the enriched steady state microbial consortium.
[0113] Methods for generating oligonucleotide probes and microarrays for performing phylogenetic analysis are known to those of ordinary skill in the art (Loy, A., et al., Appl. Environ. Microbiol. 70: 6998-700, 2004) and (Loy A., et al., Appl. Environ. Microbiol. 68: 5064-5081, 2002) and (Liebich, J., et al., Appl. Environ. Microbiol. 72: 1688-1691, 2006). These methods are applied herein for the purpose of identifying microorganisms present in an environmental sample.
Specifically, conserved sequences of the 16S ribosomal RNA coding region of the genomic DNA were used herein. However there are other useful methodologies for phylogenetic typing noted in the literature. These include: 23S rDNA or gyrase A genes or any other highly conserved gene sequences. 16S rDNA is commonly used because it is the largest database of comparative known phylogenetic genotypes and has proven to provide a robust description of major evolutionary linkages (Ludwig, W., et al., Antonie Van Leewenhoek, 64: 285, 1993 and Brown, J. R. et al., Nature Genet., 28: 631, 2001).
[0114] The primers described herein were chosen as relevant to environmental samples from an oil reservoir (Grabowski, A., et al., FEMS Micro. Eco. 544: 427-443, 2005) and by comparisons to other primer sets used for other environmental studies. A review of primers available for use herein can be found in Baker et al (G. C. Baker, G. C. et al., Review and re-analysis of domain-specific primers, J. Microbiol. Meth. 55: 541-555, 2003). Any primers which generate a part or whole of the 16S rDNA sequence would be suitable for the claimed method.
[0115] DNA extraction by phenol/chloroform technique is known in the art and utilized herein as appropriate for extracting DNA from oil contaminated environmental samples. However, there are other methodologies for DNA extraction in the literature that may be used in accordance with the present invention.
[0116] DNA sequencing methodologies that generate >700 bases of high quality sequence may be used for the type of plasmid based sequencing in accordance with the present invention in conjunction with other sequence quality analysis programs. The comparisons by homology using the BLAST algorithms to any comprehensive database of 16S rDNAs would achieve an acceptable result for identifying the genera of microorganisms present in the environmental sample. The most widely used databases are ARB (Ludwig, W., et al., ARB: a software environment for sequence data. Nucleic Acid Res., 32: 1363-1371, 2004) and NCBI.
Fingerprint Profiling
[0117] Fingerprint profiling is a process of generating a specific pattern of DNA bands on an electrophoresis gel that are defined by their length and sequence. This profile is used to identify and describe the predominant microbial population of a culture assessing microbial diversity and population stability at particular metabolic state. For example, each band and its intensity in a given DGGE fingerprint profile represent an individual bacterial species present in the community and its relative representation in the population. Once generated, the data represents a fingerprint profile of the population at a given point in time and under certain growth conditions. The DGGE fingerprint profile can be compared to profiles of the consortium under prescribed growth conditions.
Denaturing Gradient Gel Electrophoresis
[0118] This technique has been adopted to analyze PCR amplification products by targeting variable sequence regions in conserved genes such as one of the nine variable regions found in the 16S rRNA gene of microorganisms (Gerard Muyzer et al., supra and Neefs, J-M et al. supra, and Botter, E. C., ASM News 1996). DGGE provides a genetic fingerprint profile for any given population.
[0119] Denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) are electrophoresis-gel separation methods that detect differences in the denaturing behavior of small DNA fragments (50-600 bp), separating DNA fragments of the same size based on their denaturing or "melting" profiles related to differences in their base sequence. This is in contrast to non-denaturing gel electrophoresis where DNA fragments are separated only by size.
[0120] The DNA fragments are electrophoresed through a parallel DGGE gel, so called because the linear gradient of denaturant ˜30-60% (urea/formamide) is parallel to the gel's electric field. Using DGGE, two strands of a DNA molecule separate or melt, when a chemical denaturant gradient is applied at constant temperature between 55°-65° C. The denaturation of a DNA duplex is influenced by two factors: 1) the hydrogen bonds formed between complimentary base pairs (since GC rich regions melt at higher denaturing conditions than regions that are AT rich); and 2) the attraction between neighboring bases of the same strand, or "stacking". Consequently, a DNA molecule may have several melting domains, depending upon the denaturing conditions, which are characteristic of and determined by their nucleotide sequence. DGGE exploits the fact that virtually identical DNA molecules that have the same length and similar DNA sequence, which may differ by only one nucleotide within a specific denaturing domain, will denature at different conditions. Thus, when the double-stranded (ds) DNA fragment moves (by electrophoresis) through a gradient of increasing chemical denaturant, urea, formamide or both, it begins to denature and undergoes both conformational and mobility changes. At some point the two strands of the DNA to will come completely apart (also called "melting"). However, at some intermediate denaturant concentrations, as the denaturing environment increases, the two strands will become partially separated, with some segments of the molecules still being double-stranded and others being single-stranded, specifically at the particular low denaturing domains; thus, forming variable and intermediate denatured structures, which begin to retard the movement of the fragments through the gel denaturant gradients. The dsDNA fragment will travel faster than a denatured single-stranded (ss) DNA fragment. The more denatured fragment will travel slower through the gel matrix. The DGGE gel electrophoresis method offers a "sequence dependent, size independent method" for separating DNA molecules.
[0121] In practice, the DGGE electrophoresis is conducted at a constant temperature (60° C.) and chemical denaturants are used at concentrations that will result in 100% of the DNA molecules being denatured (i.e., 40% formamide and 7M urea). This variable denaturing gradient is created using a gradient maker, such that the composition of denaturants in the gel gradually decreases from the bottom of the gel to the top, where the fragments are loaded, e.g., 60% to 30%.
[0122] The principle used in DGGE profiling can also be applied to a second method, Temperature Gradient Gel Electrophoresis (TGGE), which uses a temperature gradient instead of a chemical denaturant gradient. This method makes use of a temperature gradient to induce the conformational change of dsDNA to ssDNA to separate fragments of equal size with different sequences. As in DGGE, DNA fragments will become immobile at different positions in the gel depending upon their different nucleotide sequences.
[0123] For characterizing microbial communities, DGGE fingerprint profiling has been applied to identify and characterize the genetic diversity of complex microbial populations much as, riboprinting has been applied to identify new environmental isolates by their rRNA fingerprint profile as being the same or different from previously described strains.
[0124] In practicing DGGE profiling, the variable sequence regions found in the 16S rRNA gene of microorganisms are targeted in PCR amplification of whole DNA isolated from a mix population (Gerard Muyzer, et al., supra). The variable or "V" regional segment not only differs in nucleotide sequence, but in length and secondary structure in the sequence. It is only recognizable as similar sequence in only closely related microorganisms. There are nine variable regions in the bacterial/archaeal 16S gene. These variable regions are designated by the letter V plus the number 1 through 9. Two V regions are most useful in using DGGE profile analysis, the V3 region and the V4/V5 region. Both V regions are flanked by universally conserved U regions.
[0125] The V3 region is flanked by two U sequences. The first at base coordinates 341 to 357 where bacteria and archaeal signature sequences exist. Bacterial universal primer, UB357F (SEQ NO: 5) and archaeal universal primers 341F1 and 341F2, (SEQ NO: 7 and SEQ NO: 9 respectively) are designed from this region. The other U region, which is universally conserved in all phylogenetic domains, is found at base coordinates, 518 to 534. The domain universal reverse primer, UB518R (SEQ NO: 5) is designed from this region.
[0126] The V4/V5 region is also flanked by two universal conserved sequences. The first as above is the domain universal region at base coordinates, 518 to 534. The domain universal forward, U519F (SEQ NO: 11) was designed from this region. The other region at base coordinates 918 to 960, where additional universal bacterial and archaeal signature sequences exist. The bacterial universal reverse primer, UB939R (SEQ NO: 14) and Archaeal universal primer UA958R (SEQ NO: 13) in this application were designed from this region.
[0127] A 40-bp GC-rich clamp in the 5' end of one of the PCR primers makes the method robust for genetic fingerprint profiling analysis of microbial populations. For profile analysis of region V3, the GC-clamp was designed into the bacterial universal primer, designated dG•UB357F (SEQ NO: 6) and archaeal universal primers designated dG•341F1 and dG•341F2, (SEQ NOs: 8 and 10 respectively) and for the V4/V5 region, the domain universal forward, designated dG•U 519F (SEQ NO: 12) was designed from this region. Using this method, PCR amplification of the total DNA from a diverse microbial population produces amplified fragments consisting of heterogeneous sequences of approximately 193 bp in length. These 16S rDNA fragments, when analyzed by DGGE analysis, demonstrate the presence of multiple distinguishable bands in the separation pattern, which are derived from the many different species constituting the population. Each band thereby, represents a distinct member of the population. Intensity of each band is most likely representative of the relative abundance of a particular species in the population, after the intensity is corrected for rRNA gene copies in one microbe versus the copies in others. The banding pattern also represents a DGGE profile or fingerprint of the populations. Using this method, it is possible to identify constituents, which represent only 1% of the total population. Changes in the DGGE fingerprint profile of the population can signal changes in the parameters, e.g., the electron donors and electron acceptors that determine the growth and metabolism of the community as a whole.
Relevant Functionalities of Characterized, Enriched Steady State Microbial Consortium
[0128] Once an enriched steady state microbial consortium has been characterized, or in certain embodiments prior to genetic characterization of the constituents, the consortium may be assayed for one or more relevant functionality related to enhancing oil recovery or bioremediation of a hydrocarbon-contaminated site, including ability to degrade crude oil under the conditions of interest. Assays for the relevant functionalities include microsand column release assay and the LOOS (Liberation of Oil Off Sand) test (see Example 8) and the "sand packed slim tube or core flood test".
Inoculation of an Oil Well for Enhanced Oil Recovery
[0129] The following steps are taken to inoculate an oil well/reservoir:
[0130] a) Inoculating the microbial consortium in a bioreactor containing an anaerobic minimal salts medium, the target crude oil and an appropriate electron acceptor (e.g., nitrate herein).
[0131] b) Incubating the microbial consortium of step (a) at a temperature similar to the target well to obtain a seed population of the microbial consortium (e.g., 30° C., or in the range of room temperature, +/-5° C. in this disclosure).
[0132] c) Inoculating the seed microbial consortium of step (b) under anaerobic condition into anaerobic reservoir injection water.
[0133] d) Injecting the biological mixture of step (c) in to the reservoir, followed by injection water with dissolved electron acceptor to push the consortium mixture into the reservoir subterranean matrix, allowing the microbial consortium to grow and propagate resulting in dissociation and release of the crude oil from the reservoir matrix.
Inoculation of a Hydrocarbon-Contaminated Environmental Site for In Situ Bioremediation
[0134] The following steps are taken to inoculate a hydrocarbon-contaminated environmental site:
[0135] a) Inoculating the microbial consortium in a bioreactor containing an anaerobic minimal salts medium, the target crude oil and an appropriate electron acceptor (e.g., nitrate in this disclosure).
[0136] b) Incubating the microbial consortium of step (a) at a temperature similar to the target site to obtain a seed population of the microbial consortium (e.g., 30° C., or in the range of room temperature, +/-5° C. in this disclosure).
[0137] c) Inoculating the seed microbial consortium of step (b) under anaerobic condition into contaminated site's subsurface.
[0138] d) Injecting the biological mixture of step (c) in to the subsurface, followed by injection water with dissolved electron acceptor to push the consortium mixture into the subterranean matrix, allowing the microbial consortium to grow and propagate resulting in degradation of the hydrocarbon contaminants.
Benefits of Enhancing Oil Recovery or In Situ Bioremediation Using Enriched Steady State Microbial Consortium
[0139] In this application, methods are disclosed to provide an enriched steady state consortium of microbial population, under denitrifying conditions, using a chemostat bioreactor. The enriched steady state consortium population anaerobically degrades crude oil components under reservoir conditions or environmental conditions to modify the physiochemical properties of the crude oil and/or the reservoir environmental matrix, resulting in enhanced recovery of the crude oil. Furthermore, modifying the hydrocarbons of a hydrocarbon-contaminated environmental site by this consortium, results in its in situ bioremediation. The ideal consortium would be developed and enriched from an indigenous microbial population.
[0140] An additional benefit of the application of the present microbial consortium may be in the prevention of the damage to the oil pipeline and oil recovery hardware. Corrosion of the oil pipeline and other oil recovery hardware may be defined as the destructive attack on metals by some microbial, chemical or electrochemical mechanisms. Microbially induced corrosion in oil pipelines is known (EP3543361 B and U.S. Pat. No. 4,879,240A) and is caused by a variety of microorganisms including, but not limited to, aerobic bacteria, anaerobic bacteria, acid forming bacteria, slime formers, and sulfate reducing bacteria (SRB). In an anaerobic environment, corrosion is most commonly attributed to the growth of dissimilatory SRB. This group of bacteria is responsible for possibly 50% of all instances of corrosion. The control of microbial corrosion in oil recovery operations generally incorporates both physical or mechanical and chemical treatments.
[0141] The use of nitrate as a means of controlling the activity of SRB and removing hydrogen sulfide from oil pipeline and other oil recovery hardware is well documented (The stimulation of nitrate-reducing bacteria (nrb) in oilfield systems to control sulfate-reducing bacteria (srb), microbiologically influenced corrosion (mic) and reservoir souring an introductory review, published by the Energy Institute, London, 2003). Because nitrate is a better electron acceptor than sulfide, nrb have a competitive advantage over srb. Nitrate produces a higher growth yield than sulfide reduction does. Application of denitrifying microorganisms for enhancing oil recovery, therefore, may provide a cost effective, efficient and environmentally acceptable means of controlling SRB and remediating hydrogen sulfide contaminated systems, avoiding the use of expensive and environmentally unacceptable organic biocides. The use of the POG1 consortium therefore, may not only be beneficial to oil recovery, it may also prevent costly damage to the oil pipeline and other oil recovery hardware.
[0142] While aerobic in situ bioremediation of crude oil or its hydrocarbon components is in many cases it is impractical because of the anoxic nature of the natural environments contaminated with hydrocarbons, they may be bioremediated using by anaerobic microorganisms. Theoretically, the differences in energy release from the organic carbon oxidation by the different electron acceptors will be the controlling factor for the different anaerobic redox environments developing around the carbon source. Anaerobic oxidation of hydrocarbon compounds occurs under specific redox conditions for each electron acceptor, which include nitrate, iron (III), manganese (IV), sulfate, carbon dioxide, nitrite, ferric ion, sulfur, sulfate, selenate, arsenate, carbon dioxide and organic electron acceptors that include the chloroethenes, fumarate, malate, pyruvate, acetylaldehyde oxaloacetate and similar unsaturated hydrocarbon compounds. The rate of degradation in these redox zones is relevant to the abundance of the relevant microbes, the availability of the hydrocarbon via diffusion, the kinetics and energetics of the initial hydrocarbon-activating reaction which is dependant on the redox potential of the contaminated area.
[0143] Denitrifying bacteria provide an excellent choice for in situ bioremediation, because they grow rapidly under anaerobic conditions and yield substantial cell mass. In addition, denitrifying microorganisms from the genera Thauera, Azoarcus and Dechloromonas have been shown to breakdown hydrocarbons such as benzene, toluene, ethylbenzene, and xylenes (BTEX), which are constituents of crude oil (see above for references). In situ bioremediation remains potentially the most cost-effective cleanup technology for removing these compounds from contaminated sites. Application of the POG1 consortium may provide a custom bacterial culture that may be used to remediate crude oil, BTEX and other related hydrocarbon contaminated sites. Bioremediation may take place when the steady state consortium cells are exposed to hydrocarbons and convert them into products such as carbon dioxide, water, and oxygen or growth of the steady state consortium cells may allow for the release of high molecular weight hydrocarbons to the surface for subsequent removal by physical clean up methods. In some embodiments, the steady state consortium may be incubated in the environment to be bioremediated without any added co-substrate, or other carbon or energy source. The bioremediation process may be monitored by periodically taking samples of the contaminated environment, extracting the hydrocarbons, and analyzing the extract using methods known to one skilled in the art. Contaminated substrates that may be treated with the steady state consortium include, but are not limited to, beach sand, harbor dredge spoils, sediments, wastewater, sea water, soil, sand, sludge, air, and refinery wastes.
[0144] In another embodiment, the contaminated target may be an oil pipeline or refinery equipment. Hydrocarbon incrustation and sludge build-up are significant causes of decreased pipeline performance and can eventually lead to failure of the pipeline. Because of the ability of the steady state consortium to release hydrocarbons, its application to an oil pipeline containing incrusted hydrocarbons or hydrocarbon-containing sludge may be useful in the removal of the unwanted hydrocarbons from the pipeline.
General Methods
Growth of Microorganisms
[0145] Techniques for growth and maintenance of anaerobic cultures are described in "Isolation of Biotechnological Organisms from Nature", (Labeda, D. P. ed. p 117-140, McGraw-Hill Publishers, 1990). Anaerobic growth was measured by nitrate depletion from the growth medium over time. Nitrate was utilized as the primary electron acceptor under the growth conditions used in this invention. The reduction of nitrate to nitrogen has been previously described (Moreno-Vivian, C., et al., J. Bacteriol. 181: 6573-6584, 1999). In some cases, nitrate reduction processes lead to nitrite accumulation, which is subsequently, further reduced to nitrogen. Accumulation of nitrite is therefore also considered evidence for active growth and metabolism by these microorganisms.
Description of the Chemostat Bioreactor Used in this Disclosure
[0146] In this disclosure, a chemostat bioreactor was used as a bioreactor to maintain the consortium population in a steady state, using crude oil in excess as the sole energy source and a limiting nitrate supply, as the electron acceptor. FIG. 3 shows a diagram of the chemostat bioreactor used in this disclosure. The chemostat bioreactor was designed and used as a continuous-cultivation system, using a constant feed of medium and nitrate to develop a steady state population designated "POG1 consortium". The chemostat bioreactor was operated under anaerobic conditions, at room temperature, pH 7.4 and one atmosphere pressure, using the targeted crude oil (Milne Pont reservoir, North Slop of Alaska) as the carbon source (primary source of electron donors), and supplying a minimal salts medium (Table 2) containing minimal essential minerals, salts, vitamins and nitrate, as the primary electron acceptor, for growth.
TABLE-US-00002 TABLE 2 Composition of the SL10 minimal salts medium - The pH of the medium was adjusted to between 7.4-7.8 Growth component Final Concentration Chemical Source Nitrogen 18.7 μM NH4Cl Phosphorus 3.7 μM KH2PO4 Magnesium 984 μM MgCl2•6H2O Calcium 680 μM CaCL2•2H2O Sodium chloride 172 mM NaCl Trace metals 670 μM nitrilotriacetic acid 15.1 μM FeCl2•4H2O 1.2 μM CuCl2•2H2O 5.1 μM MnCL2•4H2O 12.6 μM CoCl2•6H2O 7.3 μM ZnCl2 1.6 μM , H3BO3 0.4 μM Na2MoO4•2H2O 7.6 μM NiCl2•6H2O Selenium-tungstate 22.8 nM Na2SeO3•5H2O 24.3 nM Na2WO4•2H2O PH buffer/Bicarbonate 23.8 nM NaHCO3 vitamins 100 μg/L vitamin B12 80 μg/L p-amino-benzoic acid 20 μg/L nicotinic acid 100 μg/L calcium pantothenate 300 μg/L pyridoxine hydrochloride 200 μg/L thiamine-HCL•2H2O 50 μg/L alpha-lipoic acid Electron acceptor 0.4 g/L NaNO3
[0147] The chemostat bioreactor was set up in a chemical hood at room temperature (20 to 25° C.). All headspaces were anaerobic, using a blanket of nitrogen and an open-ended nitrogen flow (<1 psi) system, with a reverse double bubbler system, containing 5 mL mineral oil closing off the system from the atmosphere. Both the initial SL10 medium in the bioreactor and in the medium feed reservoir were degassed with an anaerobic mix of carbon dioxide and nitrogen (20/80 on a % basis) for 10 min, the pH checked and then titrated with either CO2/N2 mix or just N2 until it was pH7.4. The SL10 minimal salts medium (114 in the bioreactor, was initially supplemented with 800 ppm nitrate and 400 mL of the targeted crude oil. The bioreactor was inoculated with 50 mL of the 3rd generation (3rd gen) parent POG1 from enrichment culture (designated EH50:1) grown on the target crude oil and 1600 ppm nitrate for 1 week and incubated at room temperature while shaking at 100 rpm. A magnetic stirrer at the bottom of the reactor was stirring the culture at 40 to 50 rpm.
[0148] The SL10 medium, supplemented with 3800 ppm nitrate, was pumped from the medium reservoir (FIG. 3: G) into the chemostat bioreactor by means of the feed syringe pump (KDS230 Syringe Pump, KD Scientific, Holliston, Mass.) (FIG. 3: D). A sampling port was attached to and inline with the feed syringe pump. A 5 mL Becton-Dickinson (BD) sterile plastic polypropylene syringe (FIG. 3: C) (Becton-Dickinson, Franklin Lakes, N.J.) was attached to the sampling port and had a double function: 1) as a sampling syringe for the input feed and 2) as a 5 psi pressure release valve for the feed syringe pump. The effluent from the chemostat bioreactor was pumped into an effluent reservoir (FIG. 3: L) by means of the effluent syringe pump (supra) (FIG. 3: O). A second sampling port was attached to and inline with the effluent syringe pump. The effluent sampling port also had a 5 mL BD sterile plastic polypropylene syringe (supra) attached (FIG. 3: P). Again, it functioned both as a sampling syringe for effluent and as a 5 psi pressure release valve for the effluent syringe pump.
Obtaining the Environmental Sample
[0149] In this disclosure, soil or water samples obtained from anaerobic and microaerophilic (aerobic microorganisms that requires lower levels of oxygen to survive) locations on a hydrocarbon-contaminated site, which had been exposed to tar, creosol and polycyclic aromatic hydrocarbons (PAHs) were used for developing the microbial consortium. Soil samples were taken from locations where PAHs had been shown to be at elevated levels. Soil samples were placed in 500 mL brown bottles, filled to the top, sealed with no air space and, then shipped back to the lab on ice in a cooler. Once in the lab, the samples were placed in a Coy Type B anaerobic chamber (Coy Laboratories, Grass Lake, Mich.), filled with a specific anaerobic gas mixture (oxygen free anaerobic mix of hydrogen, carbon dioxide and nitrogen, 5%, 10% and 85%, respectively) for further processing.
Ion Chromatography
[0150] An ICS2000 chromatography unit (Dionex, Banockburn, Ill.) was used to quantitate nitrate and nitrite ions in the growth medium. Ion exchange was accomplished on an AS15 anion exchange column using a gradient of 2 to 50 mM potassium hydroxide. Standard curves were generated and used for calibrating nitrate and nitrite concentrations.
Genomic DNA Extractions from Bacterial Cultures
[0151] To extract genomic DNA from liquid bacterial cultures, cells were harvested and concentrated by filtration onto a 0.2 micron Supor® Filter (Pall Corp, Ann Arbor, Mich.) or by centrifugation. An aliquot (2-5 mL) of a bacterial culture was passed through a 0.2 micron, 25 mm filter disk in a removable cartridge holder using either vacuum or syringe pressure. The filters were removed and placed in the following lysis buffer (100 mM Tris-HCL, 50 mM NaCl, 50 mM EDTA, pH8.0) followed by agitation using a Vortex mixer. The following reagents were then added to a final concentration of 2.0 mg/mL lysozyme, 10 mg/mL SDS, and 10 mg/mL Sarkosyl to lyse the cells. After further mixing with a Vortex mixer, 0.1 mg/mL RNase and 0.1 mg/mL Proteinase K were added to remove the RNA and protein contaminants and the mixture was incubated at 37° C. for 1.0-2.0 hr. Post incubation, the filters were removed and samples were extracted twice with an equal volume of a phenol: chloroform: isoamyl alcohol (25:24:1, v/v/v) and once with chloroform: isoamyl alcohol (24:1, v/v). One-tenth volume of 5.0M NaCl and two volumes of 100% ethanol were added to the aqueous layer and mixed. The tubes were frozen at -20° C. overnight and then centrifuged at 15,000×g for 30 min at room temperature to pellet chromosomal DNA. The pellets were washed once with 70% ethanol, centrifuged at 15,000×g for 10 min, dried, resuspended in 100 μL of de-ionized water and stored at -20° C. An aliquot of the extracted DNA was analyzed on an agarose gel to ascertain the quantity and quality of the extracted DNA.
Population Analysis of the Microorganisms of the Steady State Consortium and Parent Enrichment Cultures Using Cloned 16S rDNA Libraries
[0152] Primer sets were chosen from Grabowski et al. (FEMS Microbiol. Ecol., 54: 427-443, 2005) to generate 16S rDNA of microbial species in DNA samples prepared from the consortium. The combination of forward primer (SEQ ID NO: 1) and reverse primers (SEQ ID NOs: 2 or 3) were chosen to specifically amplify the bacterial 16S rDNA sequences.
[0153] The PCR amplification mix included: 1.0× GoTaq PCR buffer (Promega), 0.25 mM dNTPs, 25 pmol of each primer, in a 50 μL reaction volume. 0.5 μL of GoTaq polymerase (Promega) and 1.0 μL (20 ng) of sample DNA were added. The PCR reaction thermal cycling protocol used was 5.0 min at 95° C. followed by 30 cycles of: 1.5 min at 95° C., 1.5 min at 53° C., 2.5 min at 72° C. and final extension for 8 min at 72° C. in a Perkin Elmer 9600 thermal-cycler (Waltham, Mass.). This protocol was also used with cells from either purified colonies or mixed species from enrichment cultures.
[0154] The 1400 base pair amplification products for a given DNA pool were visualized on 0.8% agarose gels. The PCR reaction mix was used directly for cloning into pPCR-TOPO4 vector using the TOPO TA cloning system (Invitrogen) as recommended by the manufacturer. DNA was transformed into TOP10 chemically competent cells selecting for ampicillin resistance. Individual colonies (˜48-96 colonies) were selected and grown in microtiter plates for sequence analysis.
Plasmid Template Preparation
[0155] Large-scale automated template purification systems used Solid
Phase Reversible Immobilization (SPRI, Agencourt, Beverly, Mass.) (DeAngelis, M. M., et al., Nucleic Acid Res., 23: 4742-4743, 1995). The SPRI® technology uses carboxylate-coated, iron-core, paramagnetic particles to capture DNA of a desired fragment length based on tuned buffering conditions. Once the desired DNA is captured on the particles, they can be magnetically concentrated and separated so that contaminants can be washed away.
[0156] The plasmid templates were purified using a streamlined SprintPrep® SPRI protocol (Agencourt). This procedure harvests plasmid DNA directly from lysed bacterial cultures by trapping both plasmid and genomic DNA to the functionalized bead particles and selectively eluting only the plasmid DNA. Briefly, the purification procedure involves addition of alkaline lysis buffer (containing RNase A) to the bacterial culture, addition of alcohol based precipitation reagent including paramagnetic particles, separation of the magnetic particles using custom ring based magnetic separator plates, 5× washing of beads with 70% ETOH and elution of the plasmid DNA with water.
rDNA Sequencing, Clone Assembly and Phylogenetic DNA Analysis
[0157] DNA templates were sequenced in a 384-well format using BigDye® Version 3.1 reactions on ABI3730 instruments (Applied Biosystems, Foster City, Calif.). Thermal cycling was performed using a 384-well thermal-cycler. Sequencing reactions were purified using Agencourt's CleanSeq® dye-terminator removal kit as recommended by the manufacturer. The reactions were analyzed with a model ABI3730XL capillary sequencer using an extended run module developed at Agencourt. All sequence analyses and calls were processed using Phred base calling software (Ewing et al., Genome Res., 8: 175-185, 1998) and constantly monitored against quality metrics.
Assembly of rDNA Clones
[0158] A file for each rDNA clone was generated. The assembly of the sequence data generated for the rDNA clones was performed by the PHRAP assembly program (Ewing, et al., supra). Proprietary scripts generate consensus sequence and consensus quality files for greater than one overlapping sequence read.
Analysis of rDNA Sequences
[0159] Each assembled sequence was compared to the NCBI (rDNA database; ˜260,000 rDNA sequences) using the BLAST algorithm program (Altschul, supra). The BLAST hits were used to group the sequences into homology clusters with ≧90% identity to the same NCBI rDNA fragment. The homology clusters were used to calculate proportions of particular species in any sample. Because amplification and cloning protocols were identical for analysis of each sample, the proportions could be compared from sample to sample. This allowed comparisons of population differences in samples taken for different enrichment selections and or at different sampling times for the same enrichment consortium culture.
Using Fingerprint Profiles to Characterize the Genetic Diversity of Complex Microbial Populations
[0160] For characterizing microbial communities, DGGE fingerprint profiling (as described above) has been applied to identify and characterize the genetic diversity of complex microbial communities. Targeting the variable sequence regions found in the 16S rRNA gene of microorganisms, Muyzer, G., et al (supra) PCR amplified DNA sequence of the V3 region of 16S rRNA genes in a mixed population. As stated above, the region is flanked by two universal conserved primer regions one at 341 to 357 and the other at 518 to 534. A 40-bp GC-rich clamp in the 5' end of one of the forward PCR primers, which included: universal bacterial primer 357, universal archaeal primers, 341F1, 341F2, (SEQ ID NOs: 5, 7 and 9) were designed as dG•UB 357, dG•UA 341F1 and dG•UA 341F2, respectively (SEQ ID NOs: 6, 8 and 10). As described above, the rDNA PCR products were electrophoresed on a linear gradient of denaturant ˜30-60% (urea/formamide) which is parallel to the gel's electric field. DGGE gels were cast and electrophoresed using a D Gene®: Denaturing Electrophoresis System from BIORAD (Hercules, Calif.) following manufacturer's suggested protocols. rDNA samples were electrophoresed at a constant temperature of 60° C. for 8-24 hr at an appropriate voltage depending upon the 16S rDNA fragment population being analyzed. The electrophoresis buffer (1×TAE) was preheated to the target temperature in the D GENE chamber prior to electrophoresis. DGGE gels were stained with SYBR® GOLD nucleic acid stain (Invitrogen, Carlsbad, Calif.) for visualization and imaged on a Kodak imaging station 440. Multiple distinguishable bands, which were visualized in the separation pattern, were derived from the different species which constituted the POG1 population. Each band thereby, represented a distinct member of the population. Intensity of each band was most likely representative of the relative abundance of a particular species in the population, after the intensity was corrected for rRNA gene copies in one microbe versus the copies in others. The banding pattern also represented a DGGE profile or fingerprint of the populations. It is possible to identify constituents, which represent only 1% of the total population. Changes in the DGGE fingerprint profile of the population can signal changes in the parameters, e.g., the electron donors and electron acceptors that determine the growth and metabolism of the community as a whole. Thus the method described above provided a unique and powerful tool for conclusive identification of various microbial species within a mixed population.
Microsand Column Oil Release Test
[0161] Isolated bacterial strains were examined for their ability to release oil from sand using a microsand column assay to visualize oil release. The microsand column consisted of an inverted glass Pasteur pipette containing the sand (10 to 100 microns) from the Alaskan North Slope oil reservoirs, which had been coated with crude oil and allowed to age for at least one week. Specifically, oil and sand were autoclaved separately to sterilize. Autoclaved sand samples are then transferred to a vacuum oven and dried at 180° C. for a minimum of one week. Sterilized dried sand and oil were then combined ˜1:1 v/v in an anaerobic environment. The mixtures were stirred and allowed to age for a minimum of seven days in an anaerobic environment. The barrels of glass Pasteur pipette (53/4 inches) were cut to approximately half height (3 inches) and autoclaved. The cut end of the pipette was plunged into the sand/oil mix and the core filled to about 0.5 inches in height from the bottom of the pipette barrel. Next, the cut-end of the pipette, which contained the oil/sand mixture, was then placed (with the tapered end of the pipette pointing upward) into the 13 mm glass test tube. A test inoculum in four milliliters of minimal salts medium was added to the 13 mm glass tube. The apparatus was sealed inside 23×95 mm glass vials in an anaerobic environment. Oil released from the sand collects in the narrow neck of the Pasteur pipettes or as droplets on the surface of the sand layer. Cultures that enhanced release of oil over background (sterile medium) were presumed to have altered the interaction of the oil with the sand surface, demonstrating the potential to contribute to enhancing oil recovery in a petroleum reservoir.
Gas Chromatography
[0162] A flame ionization detector gas chromatography (GC FID) method was developed to analyze the wet sand from the sacrificed slim tubes for residual oil. An empirical relationship was determined based on North Slope sand and the intrinsic pore volume of packed sand, e.g., for 240 g of packed sand there was a pore volume of 64 mL. Weights of the individual sand samples were obtained and the oil on the sand was extracted with a known amount of toluene. A sample of this toluene with extracted oil was then analyzed by GC. The samples were analyzed using an Agilent Model 5890 Gas Chromatograph (Agilent, Wilmington, Del.) fitted with equipped with a flame photoionization detector, a split/splitless injector and capillary column, DB5 column (length 30 m×thickness 0.32 mm, film thickness 0.25 μm). An aliquot of 2 μL was injected with an analysis of 42 min. The injector temperature was at 300° C. and the detector temperature kept at 300° C. The carrier gas was helium, flowing at 2 mL/min. The FID detector gases were air and hydrogen flowing at 300 mL/min and 30 mL/min, respectively. A calibration curve was generated and used to determine the amount of oil in toluene on a weight percent basis. The calibration curve used 0.01, 0.1, 1, 5, and 10 wt % dissolved crude oil in toluene.
EXAMPLES
[0163] The present disclosure is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the disclosure, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications to the disclosure to adapt it to various usages and conditions.
[0164] In the present disclosure, it was intended to develop a steady state consortium of microorganisms, under anaerobic denitrifying conditions, using crude oil as the carbon source would maintain the relative abundance of various microbial species of the consortium hence allowing the consortium's optimal operation under specific environmental conditions for enhanced oil recovery or in situ bioremediation of hydrocarbon-contaminated sites as compared to the ability of a single major species on the consortium as shown below.
[0165] Additional abbreviations used in the Examples below are as follows: "hr" means hour(s), "min" means minute(s), "L" means liter(s), "mL" means milliliters, "4" means microliters, "g" means gram, "mg/mL" means milligram per milliliter, "M" means molar, "mM" means millimolar, "mmoles" means millimoles, "μmoles" means micromoles, pmoles means picomole(s), "° C." means degrees Centigrade, "bp" means base pair(s), "rpm" refers to revolutions per minute, "ppm" means part per million, "v/v" means volume for volume, "v/v/v" means volume for volume for volume, "w/v" means weight for volume, "mL/hr" means milliliter per hour, "mL/min" means milliliter per minute, "%" means percent, "g" means gravitational force, "nm" means nano meter, "psi" means per square inch, "sec" means second, "LB" means Luria Broth culture medium, "R2A" means Reasoner's 2A culture medium, "PCR" means polymerase chain reaction and "SDS" means sodium dodecyl sulfate.
Example 1
Enrichment of a Microbial Consortium on Targeted Oil, as the Carbon Source, Under Denitrifying Anaerobic Conditions
Development of the Parent POG1 Consortium
[0166] For the present Example, parent enrichment cultures and a screening protocol were developed to identify microbes capable of growth under anoxic conditions on either crude oil or its components or samples from a hydrocarbon-contaminated site as the sole source of carbon. Nitrate was used as the primary electron acceptor as described herein. Soil samples were diluted at a 1 to 10 w/v ratio (10 g in 100 mL medium) and incubated in the SL10 medium and 250 ppm sodium nitrate as the electron acceptor for 72 hr as described below. These soil suspensions were used as an inoculum into 60 mL serum vials that contained 2:1 v/v of the minimal salts medium (20 mL) and the autoclaved crude oil (10 mL). Inoculations for the enrichment cultures were performed in the Coy anaerobic glove bag as described above. All crude oil used in the present Examples was from Milne Point, Prudhoe Bay on the Alaskan North Slop. The enrichment cultures were maintained anaerobically in the gas tight, septa sealed vials. These cultures were grown with moderate shaking (100 rpm) at ambient temperatures for weeks to months and sampled regularly for nitrate depletion and nitrite accumulation, visible turbidity and visible altered oil viscosity or oil adherence to glass. Cultures were occasionally sampled for analysis of their structure of microbial populations by rDNA sequence typing.
[0167] After 10 to 15 days, a biomass had developed in the original enrichment cultures that used crude oil for as the carbon source. Using these enrichments as an inoculum, a new series of enrichment parent subcultures were prepared. These second set of enrichment subcultures were designated "1st generation parent cultures" (1st gen) and were inoculated, capped and sealed in the anaerobic chamber. The 60 mL subculture serum vials contained 30 mL of the SL10 minimal salts medium (Table 2) with 250 ppm sodium nitrate and 15 mL autoclaved crude oil. The 1st gen subcultures were grown with moderate shaking (100 rpm) at ambient temperatures for several weeks to three months and sampled regularly for nitrate depletion and nitrite accumulation, or in some cases, nitrite depletion. Changes observed included: visible turbidity, biofilms observed on the glass bottles or on the oil aqueous interface, oil-water emulsion, and visible altered oil viscosity or oil adherence to glass. Cultures were intermittently sampled for 16S rDNA phylogenetic typing.
[0168] When all available nitrates and produced-nitrites were reduced, the cultures were anaerobically subcultured into fresh medium supplemented with additional 250 ppm of sodium nitrate. Culture sampling was performed as before. After three months of growth and one to three subcultures, the resulting subculture populations were characterized using 16S rDNA typing (see above). The enrichment populations consisted of both facultative and strict anaerobes. These included various species of beta-Proteobacteria, primarily Thauera species and other species from: beta-Proteobacteria (Rhodocyclaceae), alpha-Proteobacteria, gamma-Proteobacteria, Deferribacteraceae, Bacteroidetes, Chloroflexi and Firmicutes/Clostridiales phyla (FIG. 1).
[0169] Since the individual enrichment populations were similar to each other, they were anaerobically pooled and inoculated into one liter of SL10 medium with 250 ppm sodium nitrate. The inoculated medium was then divided into 250 mL portions and each aliquot was inoculated into one of four 500 mL-serum bottles containing 125 mL of sterile crude oil. All bottles were anaerobically sealed. The cultures were referred to as "second-generation parent cultures" (2nd gen). Enrichments samples (designated EH36:1A, EH36:1B, EH36:1C, EH36:1D) (see Table 5) of the 2nd gen cultures, were grown with moderate shaking (100 rpm) at ambient temperatures for several weeks and sampled regularly for nitrate and nitrite depletion. Nitrate was replenished to 250 ppm on four separate occasions. After the fourth depletion of nitrate, a 10 mL aliquot from one of the cultures was anaerobically inoculated and sealed into a 500 mL serum bottle containing 200 mL of SL10 medium with 2400 ppm sodium nitrate and 100 mL sterile crude oil, and designated as "third-generation parent" (3rd gen) (designated EH40:1 and EH44:1). The 2nd gen cultures were continued on 250 ppm sodium nitrate, by removing 150 mL of culture and adding back 150 mL of sterile SL 10 minimal salts medium plus nitrate. All consortium cultures were incubated as described above for several weeks and regularly sampled for nitrate and nitrite depletion. After the 3rd gen parent cultures had depleted the 2400 ppm sodium nitrate and all of the produced nitrite, all enrichment cultures were replenished with 2400 ppm sodium nitrate. After 190 days, all 2nd and 3rd gen enrichments had reduced 6600 ppm nitrate. Cultures were then sampled for 16S rDNA phylogenetic typing to characterize their populations (FIG. 2). The members of population profiles of the enrichments were similar to what had been detected in previous enrichments.
Example 2
Monitoring Denitrification and Growth of a Steady State Consortium in a Chemostat Bioreactor
[0170] Growth of the steady state POG1 consortium in the chemostat was monitored by optical density (OD550) and nitrate reduction through taking daily samples for six weeks and then every second to third day for the next nine weeks. The nitrate and nitrite concentrations were determined by ion chromatography as described above. For the first two weeks, nitrate was fed at 14 ppm/day and thereafter at 69 ppm/day. Table 3 shows that equilibrium for nitrate reduction was reached after 9 days, where all of the nitrate, as well as the produced nitrite, were completely reduced. The culture completely reduced its nitrate supply for the next 97 days. Cell density equilibrium was reached after 32 days, two weeks after the nitrate feed had been increased by approximately five fold. The optical densities remained relatively constant for the next 74 days. At 35 to 43 days, the cells started to aggregate together and form biofilms at the oil-aqueous interface and oil water emulsions were observed. These culture characteristics made it difficult to obtain homogenous samples for growth measurements. Between 30 and 32 days into the experiment, the magnetic stirrer had stopped mixing and nitrate reduction was interrupted due to incomplete mixing of the culture in the bioreactor. Once the stirrer was restarted, nitrate was completely reduced within two days and the chemostat returned to equilibrium.
[0171] The steady state POG1 consortium consumed 6662 mg or 107.5 mol of nitrate in 106 days before nitrate reduction began to decrease as indicated by the presence of 27 ppm nitrite in the effluent after 106 days. The decreased rate of nitrate reduction seemed to indicate that the target component of the oil was becoming limiting. The denitrification of nitrate and its reduced nitrite to nitrogen is equivalent to 537.3 mmol of electrons consumed in crude oil oxidation (Rabus, R., et al., Arch Microbiol., 163: 96-103, 1995). It follows that the equivalent of 1.23 g of decane (8.6 mmol) was degraded to carbon dioxide. Therefore since 400 g of crude oil had been added to the chemostat bioreactor, theoretically approximately 0.31% of the oil had been dissimilated.
TABLE-US-00003 TABLE 3 Monitoring the optical density, nitrate feed and denitrification of the POG1 consortium in the chemostat bioreactor Time (days) 0 4 9 11 18 32 42 57 71 85 91 106 OD550 nm .04 0.553 0.584 0.586 0.717 1.151 1.469 0.870 0.994 0.814 0.989 0.906 Total 583.0 631.4 699.5 763.4 1045 2002 2654 3448 4337 5226 5636 6662 Nitrate fed Nitrate in 356.1 5.7 0 0 0 150 0 0 0 0 0 0 Effluent ppm Nitrite in 0 4.7 1.4 0 1 26.6 0 0 0 0 0 27.1 Effluent ppm
[0172] After 106 days of incubation, biofilm was seen on the glass of the bioreactor at or near the oil/aqueous fraction. The oil and aqueous fractions showed signs of emulsification. To observe emulsification, samples were examined using dark field and bright field phase microscopy at 400× magnification (Zeiss Axioskop 40, Carl Zeiss Micro Imaging, Inc, Thornwood, N.Y.). Microbes adhered to both the glass slide and the cover slip, demonstrating a positive hydrophobic response. This assay is a modified version of a procedure which indirectly measures hydrophobicity through the attachment of microbes to polystyrene plates (Pruthi, V. and Cameotra, S., Biotechnol. Tech., 11: 671-674, 1997). In addition, tiny, emulsified oil droplets (around 3 to 40 micron in diameter) were seen in the aqueous phase. Bacteria were also seen in a biofilm-like attachments to some of these emulsified oil droplets.
[0173] An aliquot (14) of the steady state POG1 consortium with an emulsified oil drop was placed on a microscope slide and covered with a 20 mm-square No. 1 coverslip and examined using a phase imaging microscopy under an oil emersion lens at 1000× magnification. Microbes were also found in the oil phase in irregular "pockets" formed around aggregated bacteria.
[0174] Normally water droplets that are trapped in oil will take on a near circular shaped form. The aqueous-oil interface was moving toward the bottom of the slide, the bacteria were being captured at the interface within these aggregated hydrophobic forms, which were eventually "pinched-off" and left in the oil phase.
[0175] Microbes were also seen aggregated at the aqueous-oil interface. Bacteria are usually attracted to the interface but not in mass; they often stream quickly along the interface in one direction, one bacterium at a time. In this Example, the microbes were attracted to the interface as a non-motile aggregate of 30 to 50 microns wide. These observations demonstrate formation of a hydrophobic aggregate mass that may contribute to the formation of the biofilm at the aqueous-oil interface or with an oil/aqueous emulsion. This structure allows microbes to interact with oil and use some of its components as their carbon source.
[0176] The members of population profiles of the steady state were similar to what had been detected in previous enrichments and are shown in Table 4 below. There were 73 unique sequences (SEQ ID NOs: 15-87), which were grouped into seven classes of bacteria, which included alpha-Proteobacteria, beta-Proteobacteria, gamma-Proteobacteria, Deferribacteraceae, Spirochaetes, Bacteroidetes and Firmicutes/Clostridiales and Incertae Sedis. The primary Genera continued to be the beta-Proteobacteria, Thauera. Thauera strain AL9:8 was the dominant constituent. The diversity among the members of Thauera/Azoarcus group (Rhodocyclaceae) is significant since there are 31 unique 16S rDNA sequences in this group whose sequence differences occur in the primary signature regions of the variable regions. Also the Firmicutes/Clostridiales group are diverse with 16 unique sequences that include constituents from the Clostridia, Anaerovorax and Finegoldia genera.
TABLE-US-00004 TABLE 4 Unique strains in consortium population based on 16S rDNA sequences GenBank Accession SEQ ID Class Genus Highest Identity species No. NO. Beta-Proteobacteria Thauera Thauera strain AL9:8 AJ315680 15 Thauera Thauera aromatica U95176 23, 24, 25, 26, 27, 28, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 67, 68 Thauera sp. R26885 AM084104 16, 19, 21, 30 Azoarcus Azoarcus sp mXyN2 X83533 17, 18, 22 Azoarcus sp AY570623 29, 54, 69, 86 Gamma- Azotobacter Azotobacter beijerinckii AJ30831 20, 44, Proteobacteria 46, 57, 70, 71, 72, 73, 74, 84, 85 Pseudomonas Pseudomonas putida EU930815 61, 80, 83 Pseudomonas AB109012 60, 62 pseudoalcligenes Deferribacteraceae Deferribacter Deferribacter AB086060 56, 77 desulfuricans Flexistipes Flexistipes sp vp180 AF220344 53, 58, 87 Alpha- Ochrobactrum Ochrobactrum sp mp- AY331579 47 Proteobacteria 57 Ochrobactrum lupini AY457038 59 Spirochaetes Spirochaeta Spirochaeta sp MET-_E AY800103 43 Bacteroidetes/ Bacteroides Uncultured DQ238269 78 Chloroflexi group Bacteroides/Cytophaga Firmicutes Clostridia Clostridium aceticum Y181183 76, 81 Clostridiales Clostridium X71850 55, 63, chartatabidium 75 Anaerovorax Anaerovorax sp EU498382 48, 49, 82, Finegoldia Finegoldia magna NC010376 42, 45, 50, 51, 52, 64, 65, 66, 79
Example 3
Population Analysis of the Steady State POG1 Consortium and Parent POG1 Cultures Using Cloned 16S rDNA Libraries
[0177] DNA was extracted as described above from the 3rd gen POG1 parent enrichment cultures and from the steady state POG1 chemostat culture samples and used to make cloned 16S rDNA libraries. Briefly, the 1400 base pair 16S rDNA amplification products for a given DNA pool were visualized on 0.8% agarose gels. The PCR reaction mix was used directly for cloning into pPCR-TOPO4 vector using the TOPO TA cloning system (Invitrogen) following the manufacturer's recommended protocol. DNA was transformed into TOP10 chemically competent cells selecting for ampicillin resistance. Individual colonies (-48-96 colonies) were selected, grown in microtiter plates, prepared and submitted for sequence analysis as described above.
Results of 16S rDNA Sequence Analysis
[0178] An overall 16S profile was compiled for 1st gen, 2nd gen and 3rd gen parent POG1 cultures described herein. 16S rDNA profiles were also prepared from samples taken at several different time points from the ongoing steady state POG1 chemostat culture. A minimum of 48 16S rDNA clones for each enrichment and/or steady state time sample were sent to Agencourt for sequencing. The 16S rDNA sequence obtained was subsequently blasted (BLASTn) against the NCBI database. Sequences were grouped into homology clusters with at 90% identity to the same NCBI rDNA fragment. The homology clusters obtained for all parent POG1 cultures and steady state culture were used to calculate the proportions of particular bacteria in any sample. The populations' results obtained from selected parent enrichment cultures verses steady state is shown FIG. 4.
[0179] Analysis indicated that 50-90% of the total 16S rDNAs sequenced belonged to the taxonomic class of beta-Proteobacteria, family Rhodocyclaceae. Members of the beta-Proteobacteria phylum subclass, Thauera in particular, were the most abundant microorganism in the steady state POG1 consortium at any given time. Strains of Thauera have been shown to grow on oil and or oil constituents under anaerobic conditions without the need for additional nutrient supplementation (Anders et. al. Int. J. Syst. Evol. Microbiol. 45: 327-333, 1995).
[0180] Sequences belonging to the phyla Bacteroides, Firmicutes/Clostridiales (low G+C gram-positive bacteria), Deferribacteres and Spirochaetes represented between 4-23% of the microbial population and were consistently represented in the POG1 consortium steady state samples and its parent enrichments. The sample size of cloned 16S rDNAs (n=47) for steady state POG1 samples most likely under report the incidences of these organisms in the microbial population. Sequences affiliated with members of the gamma-Proteobacteria, Pseudomonadales, were also represented at a consistently low level in steady state POG1 time samples. This is in contrast to 16S rDNA profiles obtained for several of the initial parent enrichments of this consortium, which did not contain Pseudomonadales 16S rDNA sequences indicating that members of this phylotype may not be critical to steady state POG1 function in MEOR or in-situ bioremediation.
[0181] Lastly, a low level of sequences (≦3%) associated with phylotypes representing the Chloroflexi, Synergistes, delta-Proteobacteria, and alpha-Proteobacteria were frequently detected in the POG1 parent enrichment cultures.
[0182] In summary, the distribution of 16S rDNA sequences described for the steady state POG1 culture as well as the POG1 parent enrichment cultures describes the composition of organisms that define the steady state POG1 consortium. This selected composition of microorganisms may enhance the oil recovery and may be effective in in-situ bioremediation of the hydrocarbon-contaminated sites.
Example 4 (Partially Prophetic)
Analysis of Microbial Community by DGGE
[0183] The distribution of individual microbial populations in the steady state POG1 consortium's community was analyzed using the 16S rDNA variable region analysis by DGGE. DNA for DGGE community fingerprinting was isolated from samples taken from the steady state POG1 consortium crude oil chemostat over the course of two months. PCR amplified fragments were generated using primers dG.UB357 and U518R for bacteria (SEQ ID NOs: 6 and 4) and dG. UA341F1 and F2 with U518R for Archaea (SEQ ID NOs: 8, 10 and 4). This produced an approximately 200 bp sequence from the V3 region of the bacterial and archaeal 16S rDNA which were then analyzed by DGGE. In addition, PCR amplified fragments for the V4/V5 region of the bacterial and archaeal 16S rDNA sequences were also generated producing fragments of approximately 400 bp generated using primers dG.U519F and UB 936R for bacteria (SEQ ID NOs: 12 and 14) and dG.U519F and UA 9958R for Archaea (SEQ ID NOs: 12 and 15). These PCR fragments were separated by length and nucleotide sequence using DGGE.
[0184] Denaturing gradient gel electrophoresis for fingerprint profiling was performed using a Bio-Rad DGGE DCode System (Bio-Rad Laboratories, Hercules, Calif.). Fingerprint profiles of the amplified rRNA gene fragments were resolved by electrophoresis at 60° C. at 35 V for 16 hr on 8% (w/v) denaturing polyacrylamide gels containing from 30% to 60% denaturant concentration gradient (w/v, 7M urea and 40% formamide in 1×TAE (50×TAE: 2M Tris-Acetate, 50 mM EDTA, pH 8.0)). FIG. 5 is an example of a community DGGE profile of the V4N5 region from time zero to 52 days. The profiles of the steady state POG1 consortium test samples (days, 0, 4, 28, 44, 52) on the left side appear to have stabilized after 28 days. The controls, on the right half of the gel, include the parent POG1 startup inoculum EH50:1 and a Thauera strain AL9:8. Also included as controls were two strains isolated from the Alaskan North Slop production oil, strain LH4:15 (Pseudomonas stutzeri) and strain AL1:7 (Ochrobactrum sp., from the Brucellaceae family), respectively. The last two strains were chosen as controls to see if the steady state POG1 population included microorganisms that have been seen as major constituents of an oil field population. The major band in all consortium profiles (A) correlated with the band observed for Thauera strain AL9:8.
[0185] The second band, (B), which correlates with strain LH4:15, appears to decrease as a major constituent of the population in profiles from day 4 through day 52. The third band (C), which correlates with strain AL1:7 is less dense and is a constituent of the population in profiles for zero through 28 days. However, this band disappears in the later stages of denitrification. Bands D through L are also detectable as minor constituent bands of the population in all samples.
[0186] The Following Steps are Prophetic:
[0187] To identify these steady state POG1 profile bands, previously identified 16S rDNA clones representing constituents from the steady state POG1 consortium, may be applied to DGGE analysis to identify individual DGGE bands as was done to identify to bands A through C in FIG. 5. The V4/V5 region from cloned constituent 16S rDNAs may be used to analyze and identify the remaining bands D through L of the steady state POG1 DGGE profile. The results should closely correlate with the profile bands with major constituents of the consortium identified in the earlier 16S rDNA profile in FIG. 5. Table 4 in Example 2 lists the isolated 16S rDNA clones, obtained from POG1 16S rDNA population profile studies. The clones used to obtain these sequences may be used to generate PCR produces using the DGGE PCR products to identify and correlate the individual bands (A-L) of the DGGE 16S V4/V5 rDNA. Table 4 also includes the associated NCBI rDNA database Accession number ID obtained for these reference clones. These clones represent the major groups of bacteria comprising the POG1 consortium, which include beta-Proteobacteria, primarily Thauera aromatica species (Rhodocyclaceae), and from Pseudomonadales, Bacteroidaceae, Clostridiaceae, Incertae Sedis., Spirochete, Spirochaetaceaes., Deferribacterales Brucellaceae and Chloroflexaceae. PCR amplified fragments for the V4/V5 region of the microbial 16S rDNA may then be generated from both the cloned rDNA (plasmid DNA) that were identified as POG1 constituents and genomic DNA from correlated POG1 samplings as well as POG1 cultures started form frozen culture stocks. Miniprep DNA from POG1 16S rDNA clones may be prepared using a Qiagen Miniprep Kit (Valencia, Calif.) following the manufacturer's protocol. PCR amplified fragments from the V4/V5 region of approximately 400 bp may be generated using primers dG.U519F and UB 936R for bacteria (SEQ ID NOs: 12 and 14). Amplified fragments may be separated by length and nucleotide sequence using DGGE as described above.
Example 5 (Partially Prophetic)
Long-Term Storage and Recovery of the Consortium for Field Inoculations
[0188] An important criterion for the application of any consortium is its viability and function following its long term storage. An aliquot (20 mL) of the steady state POG1 consortium was taken during the steady state growth in the chemostat. The 16S rDNA community sequence and a DGGE fingerprint profiles were performed to define the composition of the community at the sampling time point. The anaerobic sample was placed in a 15-20% glycerol mix (e.g., 150 μL of sterile degassed glycerol into 650 μL of the sample) in the Coy anaerobic chamber, dispensed into sterile 2.0 mL cryogenic polypropylene tubes and treated as described above. The tubes were quickly frozen on dry ice and stored in a -70° C. freezer until needed.
[0189] To test the viability of the steady state POG1 freezer culture or to use it as an inoculum, a cryogenic tube was removed from a -70° C. freezer and thawed on wet ice in an anaerobic chamber. An aliquot (50 μL) of the sample was used to start a seed culture for a larger inoculum for the chemostat bioreactor. The seed culture was inoculated into 20 mL of SL10 minimal medium supplemented with 300 ppm nitrate and 10 mL of the autoclaved-targeted crude oil in a 60 mL sterile serum bottle. The anaerobic bottle was sealed with a septum, incubated outside the anaerobic chamber at room temperature (20° C. to 25° C.) while shaking at 100 rpm on an orbital shaker. Culture turbidity, which is indicative of growth of the constituents of the consortium, was visually observed.
[0190] The Following Steps are Prophetic:
[0191] In addition, with a revived consortium, reduction of nitrate to nitrite is expected to occur after three days. When nitrate concentration reaches about 50 ppm or less, a sample may be taken for isolating the microbial community's DNA for 16S rDNA typing and DGGE fingerprint profiling. It would be expected that the DGGE profile and the 16S rDNA typing of the freezer seed culture would be similar to the profiles obtained for the steady state POG1 consortium. If the freezer culture were stable as expected, a seed culture may be prepared as an anaerobic inoculum for the chemostat bioreactor for nitrate assimilation analysis. The revived frozen consortium may also be used in an oil release sandpack or core flood assay. Furthermore, the revived frozen consortium may be used a reservoir growth-injection tank, which is a vessel next to the oil well for holding the culture prior to injection or it can be used for growth of the culture prior to injecting the culture it the oil well. In addition, it could be used as a seed culture for inoculating the initial culture that might be used for in situ bioremediation of the hydrocarbon-contaminated sites.
Example 6
Oil Release Sandpack or Core Flood Assay
[0192] The application of the steady state POG1 consortium to a sandpack saturated with oil to evaluate its use in MEOR and as a denitrifying consortium, growing in pipelines as possible method to impede the effects of SRB strains producing corrosion in pipelines or refinery pipes. This was accomplished using the sandpack technique in an in-house developed Teflon® shrink-wrapped sandpack apparatus that simulates packed sand of sandstone.
[0193] The process described herein was used for making two column sets, a "control" set and a "test" set, which was inoculated with the steady state POG1 consortium to test its efficacy to release oil from the sand column. Using a 1.1 inches thick, and 7 inches long Teflon heat shrink tube, an aluminum inlet fitting with Viton® O-ring was attached to one end of the tube using a heat gun. North Slope sand was added to the column which was vibrated with an engraver to pack down the sand and release trapped air. A second aluminum inlet fitting with Viton® O-ring was attached to the other end of the tube and sealed with heat a gun. The sandpack was then put in an oven at 275° C. for 7 min to evenly heat and shrink the wrap. The sandpack was removed and allowed to cool to room temperature. A second Teflon® heat shrink tube was installed over the original pack and heated in the oven as described above. After the column had cooled, a hose clamp was attached on the pack on the outer wrap over the O-ring and then tightened.
[0194] Both column sets (two columns in each set) were then flooded horizontally (at 60 mL/hr) with four pore volumes of "Brine" (sterile, anaerobic SL 10 medium, supplemented with 250 ppm nitrate and 3 mM phosphate buffer, pH 7.4) by means of a syringe pump and a 60 mL sterile plastic polypropylene syringe. Both sets of sandpacks were then flooded with anaerobic autoclaved crude oil to irreducible water saturation, which was predetermined to be two pore volumes. The oil was flooded, at a rate of 0.4 mL/hr, using a 10 mL sterile syringe and a syringe pump. The crude oil was aged on the sand by shutting-in the columns for seven days. One column set was anaerobically inoculated with one half of a pore volume at 0.4 mL/hr with a sample of the consortium removed anaerobically from the chemostat. Simultaneously a control inoculation using anaerobic "Brine" was also loaded on the control column set using the same procedure. The inocula were shut-in for incubation with the oil for seven days and the columns were then flooded with four pore volumes of anaerobic sterile "Brine" at 0.4 mL/hr.
[0195] At the conclusion of the production flood, the 7 inches long slim tubes were sacrificed into 5× one-inch sections labeled A-E. One inch was skipped at the beginning and at the exit of the slim tube to avoid edge effects during analysis. Section "A" came from the front end of the column. Sections A, C, and E were analyzed for residual oil saturation on the sand. The amount of oil on the wet sand from the sacrificed slim tubes for residual oil was measured by GC as described above. This value was multiplied by the total amount of toluene used to extract the oil resulting in the total amount of oil on the sand. The value obtained was then divided by the total sample weight to yield the percent of oil with respect to the total sample weight. The weight percent of oil of the sample was then multiplied by the ratio of the empirically derived characteristic of packed North Slope sand (total weight of sample after being flooded with brine divided by total sand weight, 1.27). This relationship is equal to the amount of oil on dry sand. This value was then multiplied by the ratio of the weight of the North Slope sand to the weight of the fluid trapped in the pore space of the sand, 3.75. The resulting value reflected the residual oil left on the sand in units of g of oil/g of total fluid in the pore space. As shown in Table 5, residual oil left on the column, in fractions A and C of the test column, were less than the controls confirming that the columns inoculated with the POG1 consortium released more oil than those that were not inoculated.
TABLE-US-00005 TABLE 5 Residual oil left on sand along the tube length after flooding with anaerobic sterile "Brine" Average Percent Residual Oil on Sand Column A C E Fraction Assay Column Test columns 23.2% 22.2% 18.5% Control 27.3% 22.3% 18.2% columns
Example 7
Ability of the Parent POG1 Consortium to Enhance Oil Release and Grow Using Oil as the Carbon Source
[0196] The parent POG1 consortium cultures were examined for their ability to release oil from sand in a visual oil release assay using the microsand column described above. This Example was used evaluate the consortium for enhanced oil recovery and also as a denitrifying culture in pipelines as possible method to impede the effects of SRB strains producing corrosion in pipelines or refinery pipes, using oil as the carbon source. Inocula from early parallel enrichment cultures of the 2nd gen parent POG1 consortium e.g., EH36:1A, EH36:1B, EH36:1C, EH36:1D each with ˜250 ppm nitrate and one 3rd gen culture (EH40:1) with high nitrate concentration (˜1600 ppm) were tested in this assay. All enrichment cultures were grown anaerobically in the SL10 minimal salts medium (Table 2) using ACO oil as the carbon source and nitrate as the electron acceptor until turbidity was observed. All operations for preparation of the microsand columns, inoculation and growth were done in an anaerobic chamber using sterile techniques. A 4.0 mL aliquot of each inoculum was added to the 13 mm glass tubes either directly or diluted 1:2 with the minimal salts medium. The microsand columns (filled with oil-saturated sand as described above) were placed in each glass tube, immersed in the medium/cell inoculum with the tapered neck of the Pasteur pipettes pointing up. The outer vials were sealed in the anaerobic Coy chamber and allowed to incubate at ambient temperatures for the next several weeks. Each column was periodically checked for oil release. Cultures that enhanced release of oil over background (sterile medium) were presumed to have altered the interaction of the oil with the sand surface.
[0197] Oil released from the sand was visualized by the released oil collecting in the tapered neck of the Pasteur pipettes or forming droplets on the surface of the sand layer (FIG. 6). Oil release was observed for some of the POG1 parent enrichment cultures as rapidly as only 3 hr after inoculation. Oil release was also observed with the pure Thauera strain AL9:8, isolated from the 1st gen POG1 parent enrichment cultures. Microsand columns were then observed over the course of several weeks. An increase in the initial amount of oil released was observed after 3 months of incubation. Uninoculated controls did not show visual release of oil over the course of the experiment. Triton® X-100 (Rohm & Haas Co), a nonionic surfactant was used as a positive assay for the release of oil from sand. Table 6 lists the enrichment cultures tested and the observations of oil release after 7 days and 3 months incubation at ambient temperatures. These results indicated that the parent POG1 consortium interacted with oil-wet sands at the water/oil/sand interface and induced oil release from the sand's surface. Results described in Example 6 and 7 clearly underline the ability of the POG1 steady state consortium in the release of oil from sand. In addition, it is anticipated that this consortium may be used in applications such as for cleaning oil or refinery pipelines.
TABLE-US-00006 TABLE 6 Release of oil from microsand columns by enrichment cultures the steady state POG1 consortium Inoculum Oil release Oil release ID dilution T = 7 days T = 3 months Controls 1.0% Triton no +++ ++++ 1.0% Triton 1/2 ++ +++ NIC (medium) no - - Parent Environmental Enrichment Cultures EH36:1A no - + EH36:1B no + ++ EH36:1C no - - EH36:1C 1/2 + + EH36:1D no + + EH40:1 no - +/- EH40:1 1/2 + + Thauera strain AL9:8 no + ++ 1. Microsand columns were scored for oil release on a scale of 1 to 5 (+) in order of increased oil release; (-) = no release of oil, 5 = complete release of oil from oil coated sand, as judged visually.
Example 8
The Ability of the Steady State Consortium to Release Oil from Sand Particles
[0198] In order to screen the enrichment cultures for the ability to release oil from the nonporous silica medium, a microtiter plate assay was developed to measure the ability of the microbes to release oil/sand from oil-saturated North Slope sand and evaluate its use in growing a denitrifying culture in pipelines as a possible method to impede the effects of SRB strains producing corrosion in pipelines or refinery pipes. The assay is referred to as the LOOS test (Liberation of Oil Off Sand).
[0199] A microtiter plate assay was developed to measure the ability of the enrichment cultures and the consortium to release oil/sand from the oil-saturated Alaskan North Slope sand. North Slope sand was autoclaved and then dried under vacuum at 160° C. for 48 hr and 20 g of this dried sand was then mixed with 5 mL of autoclaved, degassed crude oil obtained from Milne point, North Slope. The oil-coated sand was then allowed to adsorb to the sand and age anaerobically at room temperature for at least a week. Microtiter plate assays were set up in the Coy anaerobic chamber. An aliquot of the undiluted steady state POG1 consortium (20 mL) was added into the wells of a 12-well microtiter plate. The POG1 was grown anaerobically in SL10 minimal medium with 2000 ppm sodium nitrate and North Slope crude oil. The control wells contained 2 mL of the SL10/2000 ppm NaNO3 medium alone. Approximately 40 mg of oil-coated sand was then added to the center of each well. Samples were then monitored over time for the release and accumulation of "free" sand collecting in the bottom of the wells. Approximate diameters (in millimeters) of the accumulated total sand released were measured daily. A score of 3 mm and above indicated the microbes' potential to release oil from a nonporous silica medium such as sand.
[0200] Table 7 shows the relative sand release by the steady state POG1 consortium over a period of four weeks. After about 15 days, a 4 mm zone of released sand was observed in the bottom of the wells containing the steady state POG1 consortium. No release was observed for the medium alone. The results indicate that the steady state POG1 consortium may be used to release oil from nonporous silicate substrates. The consortium may be also used to grow this denitrifying culture in pipelines as a possible method to impede the effects of SRB strains producing corrosion in pipelines or refinery pipes.
TABLE-US-00007 TABLE 7 Relative sand release by the steady state POG1 consortium over a period of four weeks (Values 2 or greater represent significant oil release) Sample Day 1 Day 6 Day 16 Day 24 Steady state POG1 0 2 4 4 Consortium in SL10 medium SL10 medium alone 0 0 0 0 (control)
Example 9
Emulsification Of Crude Oil by the 3rd Generation Parent Consortium
[0201] Microorganisms isolated from the crude oil reservoir sample, refinery environmental samples or environmental samples, containing crude oil or its components, have been shown to form a stable emulsion when grown on crude oil or at least low molecular weight organic acids (LMWOA), e.g., succinate, propionate, lactate, acetate and formate, as a carbon source. The purpose of this Example was to demonstrate the ability of microorganisms, either as isolated species or as a consortium, to form a stable emulsion in the crude oil organic phase.
[0202] To test the ability of the 3rd gen POG1 consortium to develop an oil-water phase emulsion, a test system was developed using pure strains isolated from sample exposed to crude oil or its organic components. The 3rd gen POG1 consortium was anaerobically grown in 32 mL SL10 medium with 1600 ppm NaNO3 and 16 mL autoclaved crude oil (ACO). One sample contained only ACO as the carbon source. The other test samples contained 0.2% of one of the following LMWOAs e.g., succinate, propionate, lactate or acetate. Each emulsion test set contained one vial that had been inoculated with the parent consortium and the second vial that was the control. These were all sealed anaerobically and incubated for two weeks at room temperature. All inoculated samples had completely reduced the nitrate to nitrite after two weeks. An aliquot (2 mL) was removed from each vial and centrifuged at 14,000 rpm for 5 min in a Thermo 5519 microcentrifuge (Thermo Fisher Scientific Inc., Waltham, Mass.). The supernatant was added to a 4 mL Wheaton 225142 sample vial (Wheaton Science Products, Millville, N.J.) containing 1 mL of 2,2,4,4,6,8,8-heptamethyl-nonane (HMN) (Sigma-Aldrich, St Louis, Mo.) and a straight chain liquid organic solvent as the organic phase. The vials were securely fastened in a test tube-rack. The test tube-rack was placed on the lab bench, twelve inches away from the front of a Canon Powershot A530 digital camera, which was set to its macro picture function. A control picture was taken of the 10 vials to record their two liquid-phases in their initial state containing 2 mL of aqueous phase and 1 mL of organic phase. The vials and their contents were shaken by rapidly turning the rack head-over-tail 12 times. They were then placed down on the lab bench, at the same position where the control picture had been taken. A picture was taken immediately to record the initial emulsion state of each vial at time zero. To record the dissipation or stability over time of the emulsion formed by mixing the solutions, a picture was taken at 15 sec intervals until 300 sec had elapsed. The digital frames were studied to measure the dissipation of the emulsion. An emulsion was formed in the organic phase in all vials, including those that had not been inoculated with the consortium. The results are scored on a scale of 1 to 5 and shown in Table 8. The emulsion was scored on a scale of zero to five to indicate the thickness of the emulsion phase at the organic-water interface, where five was the finest and thickest emulsion. The emulsion became more coarse and thinner at the interface as the number decreased to one. A completely dissipated emulsion was scored zero. The non-inoculated controls dissipated either completely or almost completely within the first 15 seconds. An exception was observed with the control sample containing 0.2% acetate which remained somewhat stable for 75 sec before it completely dissipated. Cultures that had only ACO, crude oil plus acetate and ACO plus lactate were stable beyond 5 min and were actually stable for one hour. The inoculated sample containing lactate formed the most stable emulsion in thickness and fineness in comparison with all other samples. Succinate fed cultures did not form a stable emulsion, and propionate fed cultures formed a stable emulsion that was short lived, less than three minutes. These results indicate that several microorganisms within the consortium could emulsify crude oil and that this ability could be enhanced using low molecular weight organic acids supplements such as lactate and acetate.
TABLE-US-00008 TABLE 8 Modification of the autoclaved crude oil by the 3rd gen microorganisms in the presence of various low molecular weight organic acids (Values 2 or greater represent significant oil release and reflects the stability of the emulsion formed as described (5 > 4 > 3 > 2 > 1)) Carbon Time (Min) source 0 15 30 45 60 75 90 105 120 150 180 210 240 300 ACO + 5 3 2 2 2 2 2 2 2 1 1 1 1 1 Inoculum ACO only 5 1 1 0 0 0 0 0 0 0 0 0 0 0 ACO + 5 5 3 3 3 3 3 3 3 1 1 1 1 1 Acetate + Inoculum ACO + 5 5 3 2 2 2 0 0 0 0 0 0 0 0 Acetate only ACO + 5 5 4 3 3 3 2 2 2 1 0 0 0 0 Propionate + Inoculum ACO + 5 1 1 1 1 1 1 1 1 1 0 0 0 0 Propionate only ACO + 5 4 4 4 4 3 3 3 3 3 3 3 3 3 Lactate + Inoculum ACO + 5 0 0 0 0 0 0 0 0 0 0 0 0 0 Lactate only ACO + 5 0 0 0 0 0 0 0 0 0 0 0 0 0 Succinate + Inoculum ACO + 5 0 0 0 0 0 0 0 0 0 0 0 0 0 Succinate only
Example 10
Comparison of Growth of the POG1 Consortium and the Pure Strain Thauera AL9:8 on Targeted Oil Under Anaerobic Denitrifying Conditions
[0203] Growth rates of the POG1 consortium and Thauera strain AL9:8 in oil enrichments under anaerobic denitrifying conditions were compared. Thauera strain AL9:8 represents the major microbial constituent of the POG1 consortium. Equivalent inocula of about 106 cells of the consortium and the purified strain were used to inoculate 60 mL serum vials containing a 1:2 ratio of minimal salts medium to autoclaved crude oil under anaerobic conditions. SL10 medium (20 mL) (Table 2) with added nitrate (final concentration of 1100 to 1200 ppm) and 10.0 mL of autoclaved crude oil was used. The medium and crude oil had been deoxygenated by sparging with a mixture of nitrogen and carbon dioxide followed by autoclaving. All manipulations of bacteria were done in an anaerobic chamber. Samples were inoculated in triplicates, were incubated at ambient temperatures for several days and monitored for nitrate and nitrite levels for visible turbidity and gross visible changes to the integrity of the oil phase. POG1 inoculated vials consistently reduced nitrate at a faster rate than did pure cultures of Thauera strain AL9:8. Table 9 summarizes the results of the average nitrate reduction for the triplicate cultures of POG1 consortium verses pure cultures of Thauera strain AL9:8.
TABLE-US-00009 TABLE 9 Anaerobic growth in oil enrichments Average1 % of Microbial Average1 ppm Average1 ppm Nitrate reduced after inoculum Nitrate Day 0 Nitrate Day 5 6 days POG1 consortium 971 117 95% Strain AL9:8 1323 789 43% 1Nitrate values are the average of three replicates per microbial test inoculum
[0204] The POG1 consortium consistently developed biofilms under anaerobic denitrifying conditions in oil enrichments, a phenomenon not observed consistently in oil enrichments of Thauera strain AL9:8. Table 10 summarizes the results obtained for a set of oil enrichments cultured anaerobically as above in the SL10 medium and autoclaved crude oil (2:1) ratio. These cultures were initially incubated with ˜300 ppm nitrate and then further supplemented with nitrate to a final concentration of 1100-1200 ppm for 6 days. Formation of a stable biofilm was observed on the surface of the glass vial [after 3-5 days]. These results underline the synergistic effect of various components of the POG1 consortium, whose major constituent is Thauera strain AL 9:8, on forming a biofilm compared to that formed by Thauera strain AL9:8 alone.
[0205] The results demonstrate that the selected denitrifying consortium may have a more synergistic affect that contributes to a higher growth rate on nitrate than its primary constituent, Thauera strain AL9:8. This may imply that the consortium may have a competitive advantage in the presence of SRB under denitrifying conditions. Additionally, this may support its use as denitrifying culture in pipelines as possible method to impede the effects of SRB strains, which produce corrosion in pipelines or refinery pipes.
TABLE-US-00010 TABLE 10 Biofilm formation of microbes in oil enrichments Microbial Oil Enrichment Biofilm Formation POG1 consortium + POG1 consortium + POG1 consortium + POG1 consortium + POG1 consortium + Strain AL9:8 - Strain AL9:8 - Strain AL9:8 - Strain AL9:8 - Strain AL9:8 -
Sequence CWU
1
1
87120DNAartificial sequenceprimer - 8F 1agagtttgat ymtggctcag
20219DNAartificial sequenceprimer
1492R 2ggwtaccttg ttacgactt
19317DNAartificial sequenceprimer 1407R 3gacgggggtg wgtrcaa
17417DNAartificial sequenceprimer
U518R 4attaccgcgg ctgctgg
17517DNAartificial sequenceprimer UB357F 5cctacgggag gcagcag
17657DNAartificial
sequenceprimer dG UB 357F 6cgcccgccgc gccccgcgcc cgtcccgccg cccccgcccg
cctacgggag gcagcag 57716DNAartificial sequenceprimer UA 341F1
7tayggggcgc agcagg
16858DNAartificial sequenceprimer dG UA341F1 8cgcccgccgc gccccgcgcc
cgtcccgccg cccccgcccg cctayggggc gcagcagg 58918DNAartificial
sequenceprimer UA 341F2 9cctacggggc gcagaggg
181058DNAartificial sequenceprimer dG UA341F2
10cgcccgccgc gccccgcgcc cgtcccgccg cccccgcccg cctacggggc gcagaggg
581118DNAartificial sequenceprimer U519F 11cagcmgccgc ggtaatwc
181258DNAartificial sequenceprimer
U519F with 40 bp 12cgcccgccgc gccccgcgcc cgtcccgccg cccccgcccg cagcmgccgc
ggtaatwc 581319DNAartificial sequenceprimer UA958R 13yccggcgttg
amtccaatt
191423DNAartificial sequenceprimer UB 939R 14cttgtgcggg cccccgtcat ttc
23151510DNAunknownUnknown clone
from enriched environmental sample that by rDNA sequence analysis
has highest identity to the genus Thauera 15tggctcagat tgaacgctgg
cggcatgctt tacacatgca agtcgaacgg cagcgggggc 60ttcggcctgc cggcgagtgg
cgaacgggtg agtaatgcat cggaacgtgc ccatgtcgtg 120ggggataacg tatcgaaagg
tacgctaata ccgcatacgt cctgagggag aaagcggggg 180atcttcggac ctcgcgcgat
tggagcggcc gatgtcggat tagctagtag gtgaggtaaa 240ggctcaccta ggcgacgatc
cgtagcgggt ctgagaggat gatccgccac actgggactg 300agacacggcc cagactccta
cgggaggcag cagtggggaa ttttggacaa tgggcgcaag 360cctgatccag ccatgccgcg
tgagtgaaga aggccttcgg gttgtaaagc tctttcggcc 420gggaagaaat cgtggtctct
aacataggcc atggatgacg gtaccggact aagaagcacc 480ggctaactac gtgccagcag
ccgcggtaat acgtagggtg cgagcgttaa tcggaattac 540tgggcgtaaa gcgtgcgcag
gcggttttgt aagacagatg tgaaatcccc gggctcaacc 600tgggaactgc gtttgtgact
gcaaggctag agtacggcag aggggggtgg aattcctggt 660gtagcagtga aatgcgtaga
gatcaggagg aacaccgatg gcgaaggcag ccccctgggc 720ctgtactgac gctcatgcac
gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 780ccacgcccta aacgatgtcg
actagtcgtt cggagcagca atgcactgag tgacgcagct 840aacgcgtgaa gtcgaccgcc
tggggagtac ggccgcaagg ttaaaactca aaggaattga 900cggggacccg cacaagcggt
ggatgatgtg gattaattcg atgcaacgcg aaaaacctta 960cctacccttg acatgccagg
aaccttgccg agaggcgagg gtgccttcgg gagcctggac 1020acaggtgctg catggctgtc
gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1080gagcgcaacc cttgtcacta
gttgccatca tttggttggg cactctagtg agactgccgg 1140tgacaaaccg gaggaaggtg
gggatgacgt caagtcctca tggcccttat gggtagggct 1200tcacacgtca tacaatggtc
ggtacagagg gttgccaagc cgcgaggtgg agccaatccc 1260ttaaagccga tcgtagtccg
gatcgtagtc tgcaactcga ctacgtgaag tcggaatcgc 1320tagtaatcgc agatcagcat
gctgcggtga atacgttccc gggtcttgta cacaccgccc 1380gtcacaccat gggagtgggt
ttcaccagaa gtaggtagct taaccttcgg gagggcgctt 1440accacggtga gattcatgac
tggggtgaag tcgtaacaag gtaaccgaag ggcgaattcg 1500cggccgctaa
1510161489DNAunknownUnknown
clone from enriched environmental sample that by rDNA sequence
analysis has highest identity to tThauera sp.R26885 16tggctcagat
tgaacgctgg cggcatgctt tacacatgca agtcgaacgg cagcgggggc 60ttcggcctgc
cggcgagtgg cgaacgggtg agtaatgcat cggaacgtgc ccatgtcgtg 120ggggataacg
tatcgaaagg tacgctaata ccgcatacgc cctgaggggg aaagcggggg 180attcttcgga
acctcgcgcg attggagcgg ccgatgtcgg attagctagt aggtgaggta 240aaggctcacc
taggcgacga tccgtagcgg gtctgagagg atgatccgcc acactgggac 300tgagacacgg
cccagactcc tacgggaggc agcagtgggg aattttggac aatgggcgca 360agcctgatcc
agccatgccg cgtgagtgaa gaaggccttc gggttgtaaa gctctttcgg 420ccgggaagaa
atcgcattct ctaatatagg atgtggatga cggtaccgga ctaagaagca 480ccggctaact
acgtgccagc agccgcggta atacgtaggg tgcgagcgtt aatctgaatt 540actgggcgta
aagcgtgcgc aggcggtttt gtaagacaga tgtgaaatcc ccgggcttaa 600cctgggaact
gcgtttgtga ctgcaaggct agagtacggc agaggggggt ggaattcctg 660gtgtagcagt
gaaatgcgta gatatcggga ggatcaccta tggcgagggc agccccctgg 720gcttgtactg
acgctcatgc acgaaagcgt ggggagcaaa caggattaga taccctggta 780gtccacgccc
taaacgatgt cgactagtcg ttcggagcag caatgcactg agtgacgcag 840ctaacgcgtg
aagtcgaccg cctggggagt acggccgcaa ggttaaaact caaaggaatt 900gacggggacc
cgcacaagcg gtggatgatg tggattaatt cgatgcaacg cgaaaaacct 960tacctaccct
tgacatgtct ggaaccttgg tgagagccga gggtgccttc gggagccaga 1020acacaggtgc
tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca 1080acgagcgcaa
cccttgtcat tagttgccat catttagttg ggcactctaa tgagactgcc 1140ggtgacaaac
cggaggaagg tggggatgac gtcaagtcct catggccctt atgggtaggg 1200cttcacacgt
catacaatgg tcggtacaga gggttgccaa gccgcgaggt ggagccaatc 1260ccttaaagcc
gatcgtagtc cggatcgtag tctgcaactc gactacgtga agtcggaatc 1320gctagtaatc
gcagatcagc atgctgcggt gaatacgttc ccgggtcttg tacacaccgc 1380ccgtcacacc
atgggagtgg gtttcaccag aagtaggtag cttaaccttc gggagggcgc 1440ttaccacggt
gagattcatg actggggtga agtcgtaaca aggtaaccg
1489171489DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Azoarcus sp.
mXyN1 17tggctcagat taaacgctgg cggcatgctt tacacatgca agtcgaacgg cagcgggggc
60ttcggcctgc cggcgagtgg cgaacgggtg agtaatgcat cggaacgtgc ccatgtcgtg
120ggggataacg tatcgaaagg tacgctaata ccgcatacgc cctgaggggg aaagcggggg
180attcttcgga acctcgcgcg attggagcgg ccgatgtcgg attagctagt aggtgaggta
240aaggctcacc taggcgacga tccgtagcgg gtctgagagg atgatccgcc acactgggac
300tgagacacgg cccagactcc tacgggaggc agcagtgggg aattttggac aatgggcgca
360agcctgatcc agccatgccg cgtgagtgaa gaaggccttc gggttgtaaa gctctttcgg
420ccgggaagaa atcgcattct ctaatatagg atgtggatga cggtaccgga ctaagaagca
480ccggctaact acgtgccagc agccgcggta atacgtaggg tgcgagcgtt aatcggaatt
540actgggcgta aagcgtgcgc aggcggtttt gtaagacaga tgtgaaatcc ccgggcttaa
600cctgggaact gcgtttgtga ctgcaaggct agagtacggc agaggggggt ggaattcctg
660gtgtancant gaaatgcgta aatatcagga ggaacaccga tggcgaaggc agccccctgg
720gcctgtactg acgctcatgc acgaaagggt ggggagcaaa caggattaga taccctggta
780gtccacgccc taaacgatgt cgactagtcg ttcggagcag caatgcactg agtgacgcag
840ctaacgcgtg aagtcgaccg cctggggagt acggccgcaa ggttaaaact caaaggaatt
900gacggggacc cgcacaagcg gtggatgatg tggattaatt cgatgcaacg cgaaaaacct
960tacctaccct tgacatgcca ggaaccttgc cgagaggcga gggtgccttc gggagcctgg
1020acacaggtgc tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca
1080acgagcgcaa cccttgtcac tagttgccat catttggttg ggcactctag tgagactgcc
1140ggtgacaaac cggaggaagg tggggatgac gtcaagtcct catggccctt atgggtaggg
1200cttcacacgt catacaatgg tcggtacaga gggttgccaa gccgcgaggt ggagccaatc
1260ccttaaagcc gatcgtagtc cggatcgtag tctgcaactc gactacgtga agtcggaatc
1320gctagtaatc gcagatcagc atgctgcggt gaatacgttc ccgggtcttg tacacaccgc
1380ccgtcacacc atgggagtgg gtttcaccag aagtaggtag cttaaccttc gggagggcgc
1440ttaccacggt gagattcatg actggggtga agtcgtaaca aggtaaccg
1489181487DNAunknownunkown clone 18tggctcagat tgaacgctgg cggcatgctt
tacacatgca agtcgaacgg cagcgggggc 60ttcggcctgc cggcgagtgg cgaacgggtg
agtaatgcat cggaacgtgc ccatgtcgtg 120ggggataacg tatcgaaagg tacgctaata
ccgcatacgt cctgagggag aaagcggggg 180atcttcggac ctcgcgcgat tggagcggcc
gatgtcggat tagctagtag gtgaggtaaa 240ggctcaccta ggcgacgatc cgtagcgggt
ctgagaggat gatccgccac actgggactg 300agacacggcc cagactccta cgggaggcag
cagtggggaa ttttggacaa tgggcgcaag 360cctgatccag ccatgccgcg tgagtgaaga
aggccttcgg gttgtaaagc tctttcggcc 420gggaagaaat cgcattctct aatataggat
gtggatgacg gtaccggact aagaagcacc 480ggctaactac gtgccagcag ccgcggtaat
acgtagggtg cgagcgttaa tcggaattac 540tgggcgtaaa gcgtgcgcag gcggttttgt
aagacagatg tgaaatcccc gggcttaacc 600tgggaactgc gtttgtgact gcaaggctag
agtacggcag aggggggtgg aattcctggt 660gtaccagtga aatgcgtaaa gatcaagacg
aacaccgatg gcgaaggcag ccccctgggc 720ctgtactgac gctcatgcac aaaagcgtgg
ggagcaaaca ggattagata ccctggtagt 780ccacgcccta aacgatttcg actagtcgtt
tggagcagca atgcactgag tgacgcagct 840aacgcgtgaa gtcgaccgcc tggggagtac
ggccgcaagg ttaaaactca aaggaattga 900cggggacccg cacaagcggt ggatgatgtg
gattaattng atgcaacgcg aaaaacctta 960cctacccttg acatgccagg aaccttgccg
agaggcgagg gtgccttcgg gagcctggac 1020acaggtgctg catggctgtc gtcagctcgt
gtcgtgagat gttgggttaa gtcccgcaac 1080gagcgcaacc cttgtcatta gttgccatca
tttagttggg cactctaatg agactgccgg 1140tgacaaaccg gaggaaggtg gggatgacgt
caagtcatca tggcccttac ggcctgggct 1200tcacacgtca tacaatggtc ggtacagagg
gttgccaagc cgcgaggtgg agccaatccc 1260ttaaagccga tcgtagtccg gatcgtagtc
tgcaactcga ctacgtgaag tcggaatcgc 1320tagtaatcgc agatcagcat gctgcggtga
atacgttccc gggtcttgta cacaccgccc 1380gtcacaccat gggagtgggt ttcaccagaa
gtaggtagct taaccttcgg gagggcgctt 1440accacggtga gattcatgac tggggtgaag
tcgtaacaag gtaaccg 1487191489DNAunknownUnknown clone from
enriched environmental sample that by rDNA sequence analysis has
highest identity to Thauera sp. R26885 19tggctcagat tgaacgctgg
cggcatgctt tacacatgca agtcgaacgg cagcgggggc 60ttcggcctgc cggcgagtgg
cgaacgggtg agtaatgcat cggaacgtgc ccatgtcgtg 120ggggataacg tatcgaaagg
tacgctaata ccgcatacgt cctgagggag aaagcggggg 180attcttcgga acctcgcgcg
attggagcgg ccgatgtcgg attagctagt aggtgaggta 240aaggctcacc taggcgacga
tccgtagcgg gtctgagagg atgatccgcc acactgggac 300tgagacacgg cccagactcc
tacgggaggc agcagtgggg aattttggac aatgggggca 360accctgatcc agccatgccg
cgtgagtgaa gaaggccttc gggttgtaaa gctctttcgg 420ccgggaagaa atcgcgcact
ctaacatagt gtgtggatga cggtaccgga ctaagaagca 480ccggctaact acgtgccagc
agccgcggta atacgtaggg tgcgagcgtt aatcggaatt 540actgggcgta aagcgtgcgc
aggcggtttt gtaagacgga tgtgaaatcc ccgggctcaa 600cctgggaact gcgtttgtga
ctgcaaggct agagtacggc agaggggggt ggaattcctg 660gtgtagcagt gaaatgcgta
gatatcagga ggaacaccga tggcgaaggc agccccctgg 720gcctgtactg acgctcatgc
acgaaagcgt ggggagcaaa caggattaga taccctggta 780gtccacgccc taaacgatgt
cgactagtcg ttcggagcag caatgcactg agtgacgcag 840ctaacgcgtg aagtcgaccg
cctggggagt acggccgcaa ggttaaaact caaaggaatt 900gacggggacc cgcacaagcg
gtggatgatg tggattaatt cgatgcaacg cgaaaaacct 960tacctaccct tgacatgtct
ggaaccttgg tgagagccga gggtgccttc gggagccaga 1020acacaggtgc tgcatggctg
tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca 1080acgagcgcaa cccttgtcac
tagttgccat catttggttg ggcactctag tgagactgcc 1140ggtgacaaac cggaggaagg
tggggatgac gtcaagtcct catggccctt atgggtaggg 1200cttcacacgt catacaatgg
tcggtacaga gggttgccaa gccgcgaggt ggagccaatc 1260ccttaaagcc gatcgtagtc
cggatcgtag tctgcaactc gactacgtga agtcggaatc 1320gctagtaatc gcagatcagc
atgctgcggt gaatacgttc ccgggtcttg tacacaccgc 1380ccgtcacacc atgggagtgg
gtttcaccag aagtaggtag cttaaccttc gggagggcgc 1440ttaccacggt gagattcatg
actggggtga agtcgtaaca aggtaaccg 148920894DNAunknownUnknown
clone from enriched environmental sample that by rDNA sequence
analysis has highest identity to Azotobacter beijerinckii
20cttaacctgg gaactgcgtt tgtgactgca aggctagagt acggcagagg ggggtggaat
60tccacgtgta acagtgaaat gcgtagagat gtggaggaac accgatggcg aaggcagccc
120cctgggcctg tactgacgct catgcacgaa agcgtgggga gcaaacagga ttagataccc
180tggtagtcca cgccctaaac gatgtcgact agtcgttcgg agcagcaatg cactgagtga
240cgcagctaac gcgtgaagtc gaccgcctgg ggagtacggc cgcaaggtta aaactcaaag
300gaattgacgg ggacccgcac aagcggtgga tgatgtggat taattcgatg caacgcgaaa
360aaccttacct acccttgaca tgtctggaac cttggtgaga gccgagggtg ccttcgggag
420ccagaacaca ggtgctgcat ggctgtcgtc agctcgtgtc gtgagatgtt gggttaagtc
480ccgcaacgag cgcaaccctt gtcactagtt gccatcattt ggttgggcac tctagtgaga
540ctgccggtga caaaccggag gaaggtgggg atgacgtcaa gtcctcatgg cccttatggg
600tagggcttca cacgtcatac aatggtcggt acagagggtt gccaagccgc gaggtggagc
660caatccctta aagccgatcg tagtccggat cgtagtctgc aactcgacta cgtgaagtcg
720gaatcgctag taatcgcaga tcagcatgct gcggtgaata cgttcccggg tcttgtacac
780accgcccgtc acaccatggg agtgggtttc accagaagta ggtagcttaa ccttcgggag
840ggcgcttacc acggtgagat tcatgactgg ggtgaagtcg taacaaggta accg
894211486DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Thauera sp.
R26885 21tggctcagat tgaacgctgg cggcatgctt tacacatgca agtcgaacgg
cagcgggggc 60ttcggcctgc cggcgagtgg cgaacgggtg agtaatgcat cggaacgtgc
ccatgtcgtg 120ggggataacg tatcgaaagg tacgctaata ccgcatacgt cctgagggag
aaagcggggg 180atcttcggac ctcgcgcgat tggagcggcc gatgtcggat tagctagtag
gtgaggtaaa 240ggctcaccta ggcgacgatc cgtagcgggt ctgagaggat gatccgccac
actgggactg 300agacacggcc cagactccta cgggaggcag cagtggggaa ttttggacaa
tgggcgcaag 360cctgatccag ccatgccgcg tgagtgaaga aggccttcgg gttgtaaagc
tctttcggcc 420gggaagaaat cgtggtctct aacatgggcc atggatgacg gtaccggact
aagaagcacc 480ggctaactac gtgccagcag ccgcggtaat acgtagggtg cgagcgttaa
tcggaattac 540tgggcgtaaa gcgtgcgcag gcggttttgt aagacagatg tgaaatcccc
gggctcaacc 600tgggaactgc gtttgtgact gcaaggctag agtacggcag aggggggtgg
aattcctggt 660gtagcagtga aatgcgtaaa gatcaggagg aacaccgagg ggaaggcagc
cccctgggcc 720tgtatgaagg ctcaggcagg aaagcgtggg gagcaaacag gaatagatac
cctggtagtc 780cacgccctaa acgatgtcga ctagtcgttc ggagcagcaa tgcactgagt
gacgcagcta 840acgcgtgaag tcgaccgcct ggggagtacg gccgcaaggt taaaactcaa
aggaattgac 900ggggacccgc acaagcggtg gatgatgtgg attaattcga tgcaacgcga
aaaaccttac 960ctacccttga catgtctgga accttggtga gagccgaggg tgccttcggg
agccagaaca 1020caggtgctgc atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag
tcccgcaacg 1080agcgcaaccc ttgtcactag ttgccatcat ttggttgggc actctagtga
gactgccggt 1140gacaaaccgg aggaaggtgg ggatgacgtc aagtcctcat ggcccttatg
ggtagggctt 1200cacacgtcat acaatggtcg gtacagaggg ttgccaagcc gcgaggtgga
gccaatccca 1260aaaagccgat cgtagtccgg atcgtagtct gcaactcgac tacgtgaagt
cggaatcgct 1320agtaatcgca gatcagcatg ctgcggtgaa tacgttcccg ggtcttgtac
acaccgcccg 1380tcacaccatg ggagtgggtt tcaccagaag taggtagctt aaccttcggg
agggcgctta 1440ccacggtgag attcatgact ggggtgaagt cgtaacaagg taaccg
1486221489DNAunknownUnknown clone from enriched environmental
sample that by rDNA sequence analysis has highest identity to
Azoarcus sp. mXyN1 22tggctcagat tgaacgctgg cggcatgctt tacacatgca
agtcgaacgg cagcgggggc 60ttcggcctgc cggcgagtgg cgaacgggtg agtaatgcat
cggaacgtgc ccatgtcgtg 120ggggataacg tatcgaaagg tacgctaata ccgcatacgt
cctgagggag aaagcggggg 180attcttcgga acctcgcgcg attggagcgg ccgatgtcgg
attagctagt aggtgaggta 240aaggctcacc taggcgacga tccgtagcgg gtctgagagg
atgatccgcc acactgggac 300tgaggcacgg cccagactcc tacgggaggc agcagtgggg
aattttggac aatgggggca 360accctgatcc agccatgccg cgtgagtgaa gaaggccttc
gggttgtaaa gctctttcgg 420ccgggaagaa atcgcgcact ctaacatagt gtgtggatga
cggtaccgga ctaagaagca 480ccggctaact acgtgccagc agccgcggta atacgtaggg
tgcgagcgtt aatcggaatt 540actgggcgta aagcgtgcgc aggcggtttt gtaagacaga
tgtgaaatcc ccgggctcaa 600cctgggaact gcgtttgtga ctgcaaggct agagtacggc
agaggggggt ggaattcctg 660gtgtagcagt gaaatgcgta gatatcagga ggaacaccga
tggcgaaggc agccccctgg 720gcctgtactg acgctcatgc acgaaagcgt ggggagcaaa
caggattaga taccctggta 780gtccacgccc taaacgatgt cgactagtcg ttcggagcag
caatgcactg agtgacgcag 840ctaacgcgtg aagtcgaccg cctggggagt acggccgcaa
ggttaaaact caaaggaatt 900gacggggacc cgcacaagcg gtggatgatg tggattaatt
cgatgcaacg cgaaaaacct 960tacctaccct tgacatgcca ggaaccttgc cgagaggcga
gggtgccttc gggagcctgg 1020acacaggtgc tgcatggctg tcgtcagctc gtgtcgtgag
atgttgggtt aagtcccgca 1080acgagcgcaa cccttgtcac tagttgccat catttggttg
ggcactctag tgagactgcc 1140ggtgacaaac cggaggaagg tggggatgac gtcaagtcct
catggccttt atgggtaggg 1200cttcacacgt catacaatgg tcggtacaga gggttgccaa
gccgcgaggt ggagccaatc 1260ccttaaagcc gattgtagtc cggatcgtag tctgcaactc
gactacgtga agtcggaatc 1320gctagtaatc gcagatcagc atgctgcggt gaatacgttc
ccgggtcttg cacacaccgc 1380ccgtcacacc atgggagtgg gtttcaccag aagtaggtag
cttaaccttc gggagggcgc 1440ttaccacggt gagattcatg actggggtga agtcgtaaca
aggtaaccg 1489231487DNAunknownUnknown clone from enriched
environmental sample that by rDNA sequence analysis has highest
identity to Thauera aromatica 23tggctcagat tgaacgctgg cggcatgctt
tacacatgca agtcgaacgg cagcgggggc 60ttcggcctgc cggcgagtgg cgaacgggtg
agtaatgcat cggaacgtgc ccatgtcgtg 120ggggataacg tatcgaaagg tacgctaata
ccgcatacgt cctgagggag aaagcggggg 180atcttcggac ctcgcgcgat tggagcggcc
gatgtcggat tagctagtag gtgaggtaaa 240ggctcaccta ggcgacgatc cgtagcgggt
ctgagaggat gatccgccac actgggactg 300agacacggcc cagactccta cgggaggcag
cagtggggaa ttttggacaa tgggcgcaag 360cctgatccag ccatgccgcg tgagtgaaga
aggccttcgg gttataaagc tctttcggcc 420gggaagaaat cgtggtctct aacataggcc
atggatgacg gtaccggact aagaagcacc 480ggctaactac gtgccagcag ccgcggtaat
acgtagggtg cgagcgttaa tcggagttac 540tgggcgtaaa gcgtgcgcag gcggttttgt
aagacagatg tgaaatcccc gggctcaacc 600tgggaactgc gtttgtgact gcaaggctag
agtacggcag aggggggtgg aattcctggt 660gtaacagtga aatgcgtaga gatcaggagg
aacaccgatg gcgaaggcag ccccctgggc 720ctgtactgac gctcatgcac gaaagcgtgg
ggagcaaaaa ggattaaata ccctggtagt 780ccacgcccta aacgatgtcg actagtcgtt
cggagcagca atgcactgag tgacgcagct 840aacgcgtgaa gtcgaccgcc tggggagtac
ggccgcaagg ttaaaactca aaggaattga 900cggggacccg cacaagcggt ggatgatgtg
gattaattcg atgcaacgcg aaaaacctta 960cctacccttg acatgccagg aaccttgccg
agaggcgagg gtgccttcgg gagcctggac 1020acaggtgctg catggctgtc gtcagctcgt
gtcgtgagat gttgggttaa gtcccgcaac 1080gagcgcaacc cttgtcacta gttgccatca
tttggttggg cactctagtg agactgccgg 1140tgacaaaccg gaggaaggtg gggatgacgt
caagtcctca tggcccttat gggtagggct 1200tcacacgtca tacaatggtc ggtacagagg
gttgccaagc cgcgaggtgg agccaatccc 1260ttaaagccga tcgtagtccg gatcgtagtc
tgcaactcga ctacgtgaag tcggaatcgc 1320tagtaatcgc agatcagcat gctgcggtga
atacgttccc gggtcttgta cacaccgccc 1380gtcacaccat gggagtgggt ttcaccagaa
gtaggtagct taaccttcgg gagggcgctt 1440accacggtga gattcatgac tggggtgaag
tcgtaacaag gtaaccg 1487241487DNAunknownUnknown clone from
enriched environmental sample that by rDNA sequence analysis has
highest identity to Thauera aromatica 24tggctcagat tgaacgctgg
cggcatgctt tgcacatgca agtcgaacgg cagcgggggc 60ttcggcctgc cggcgagtgg
cgaacgggtg agtaatgcat cggaacgtgc ccatgtcgtg 120ggggataacg tatcgaaagg
tacgctaata ccgcatacgt cctgagggag aaagcggggg 180atcttcggac ctcgcgcgat
tggagcggcc gatgtcggat tagctagtag gtgaggtaaa 240ggctcaccta ggcgacgatc
cgtagcgggt ctgagaggat gatccgccac actgggactg 300agacacggcc cagactccta
cgggaggcag cagtggggaa ttttggacaa tgggcgcaag 360cctgatccag ccatgccgcg
tgagtgaaga aggccttcgg gttgtaaagc tctttcggcc 420gggaagaaat cgtggtctct
aacataggcc atggatgacg gtaccggact aagaagcacc 480ggctaactac gtgccagcag
ccgcggtaat acgtagggtg cgagcgttaa tcggaattac 540tgggcgtaaa gcgtgcgcag
gcggttttgt aagacagatg tgaaatcccc gggctcagcc 600tgggaactgc gtttgtgact
gcaaggctag agtacggcag aagggggtgg aattcctggt 660gtagcagtga aatgcgttga
gatcaggagg aacaccgatg gcgaaggcag ccccctgggc 720ctgtactgac gctcatgtac
aaaagcgtgg ggagcaaaca ggattagata ccctggtagt 780ccacgcccta aacgatgtcg
actagtcgtt cggagcagca atgcactgag tgacgcagct 840aacgcgtgaa gtcgaccgcc
tggggagtac ggccgcaagg ttaaaactca aaggaattga 900cggggacccg cacaagcggt
ggatgatgtg gattaattcg atgcaacgcg aaaaacctta 960cctacccttg acatgccagg
aaccttgccg agaggcgagg gtgccttcgg gagcctggac 1020acaggtgctg catggctgtc
gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1080gagcgcaacc cttgtcacta
gttgccatca tttggttggg cactctagtg agactgccgg 1140tgacaaaccg gaggaaggtg
gggatgacgt caagtcctca tggcccttat gggtagggct 1200tcacacgtca tacaatggtc
ggtacagagg gttgccaagc cgcgaggtgg agccaatccc 1260ttaaagccga tcgtagtccg
gatcgtagtc tgcaactcga ctacgtgaag tcggaatcgc 1320tagtaatcgc agatcagcat
gctgcggtga atacgttccc gggtcttgta cacaccgccc 1380gtcacaccat gggagtgggt
ttcaccagaa gtaggtagct taaccttcgg gagggcgctt 1440accacggtga gattcatgac
tggggtgaag tcgtaacaag gtaaccg 1487251487DNAunknownUnknown
clone from enriched environmental sample that by rDNA sequence
analysis has highest identity to Thauera aromatica 25tggctcagat
tgaacgctgg cggcatgctt tacacatgca agtcgaacgg cagcgggggc 60ttcggcctgc
cggcgagtgg cgaacgggtg agtaatgcat cggaacgtgc ccatgtcgtg 120ggggataacg
tatcgaaagg tacgctaata ccgcatacgt cctgagggag aaagcggggg 180atcttcggac
ctcgcgcgat tggagcggcc gatgtcggat tagctagtag gtgaggtaaa 240ggctcaccta
ggcgacgatc cgtagcgggt ctgagaggat gatccgccac actgggactg 300agacacggcc
cagactccta cgggaggcag cagtggggaa ttttggacaa tgggcgcaag 360cctgatccag
ccatgccgcg tgagtgaaga aggccttcgg gttgtaaagc tctttcggcc 420gggaagaaat
cgtggtctct aacataggcc atggatgacg gtaccggact aagaagcacc 480ggctaactac
gtgccagcag ccgcggtaat acgtagggtg cgagcgttaa tcggaattac 540tgggcgtaaa
gcgtgcgcag gcggttttgt aagacagatg tgaaatcccc gggctcaacc 600tgggaactgc
gtttgtgact gcaaggctag agtacggcag aggggggtgg aattcctggt 660ttagcagtga
aatgcgtaga gatcaagagg aacaccgatg gcgaaggcag ccccctgggc 720ctgtactgac
gctcatgcac gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 780ccacgcccta
aacgatgtcg actagtcgtt cggagcagca atgcactgag tgacgcagct 840aacgcgtgaa
gtcgaccgcc tggggagtac ggccgcaagg ttaaaactca aaggaattga 900cggggacccg
cacaagcggt ggatgatgtg gattaattcg atgcaacgcg aaaaacctta 960cctacccttg
acatgccagg aaccttgccg agaggcgagg gtgccttcgg gagcctggac 1020acaggtgctg
catggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1080gagcgcaacc
cttgtcacta gttgccatca tttggttggg cactctagtg agactgccgg 1140tgacaaaccg
gaggaaggtg gggatgacgt caagtcctca tggcccttat gggtagggct 1200tcacacgtca
tacaatggtc ggtacagagg gttgccaagc cgcgaggtgg agccaatccc 1260ttaaagccga
tcgtagtccg gatcgtagtc tgcaactcga ctacgtgaag tcggaatcgc 1320tagtaatcgc
agatcagcat gctgcggtga atacgttccc gggtcttgta cacaccgccc 1380gtcacaccat
gggagtgggt ttcaccagaa gtaggtagct taaccttcgg gagggcgctt 1440accacggtga
gattcatgac tggggtgaag tcgtaacaag gtaaccg
1487261487DNAunknownunknown clone 26tggctcagat tgaacgctgg cggcatgctt
tacacatgca agtcgaacgg cagcgggggc 60ttcggcctgc cggcgagtgg cgaacgggtg
agtaatgcat cggaacgtgc ccatgtcgtg 120ggggataacg tatcgaaagg tacgctaata
ccgcatacgt cctgagggag aaagcggggg 180atcttcggac ctcgcgcgat tggagcggcc
gatgtcggat tagctagtag gtgaggtaaa 240ggctcaccta ggcgacgatc cgtagcgggt
ctgagaggat gatccgccac actgggactg 300agacacggcc cagactccta cgggaggcag
cagtggggaa ttttggacaa tgggcgcaag 360cctgatccag ccatgccgcg tgagtgaaga
aggccttcgg gttgtaaagc tctttcggcc 420gggaagaaat cgtggtctct aacataggcc
atggatgacg gtaccggact aagaagcacc 480ggctaactac gtgccagcag ccgcggtaat
acgtagggtg cgagcgttaa tcggaattac 540tgggcgtaaa gcgtgcgcag gcggttttgt
aagacagatg tgaaatcccc gggctcaacc 600tgggaactgc gtttgtgact gcaaggctag
agtacggcag aggggggtgg aattcctggt 660gtagcagtga aatgcgtaga gatcaagagg
aacaccgatg gcgaaggcag ccccctgggc 720ctgtactgac gctcatgcac gaaagcgtgg
ggagcaaaca ggattagata ccctggtagt 780ccacgcccta aacgatgtcg actagtcgtt
cggagcagca atgcactgag tgacgcagct 840aacgcgtgaa gtcgaccgcc tggggagtac
ggccgcaagg ttaaaactca aaggaattga 900cggggacccg cacaagcggt ggatgatgtg
gattaattcg atgcaacgcg aaaaacctta 960cctacccttg acctgccagg aaccttgccg
agaggcgagg gtgccttcgg gagcctggac 1020acaggtgctg catggctgtc gtcagctcgt
gtcgtgagat gttgggttaa gtcccgcaac 1080gagcgcaacc cttatcacta gttgccatca
tttggttggg cactctagtg agactgccgg 1140tgacaaaccg gaggaaggtg gggatgacgt
caagtcctca tggcccttat gggtagggct 1200tcacacgtca tacaatggtc ggtacagagg
gttgccaagc cgcgaggtgg agccaatccc 1260ttaaagccga tcgtagtccg gatcgtagtc
tgcaactcga ctacgtgaag tcggaatcgc 1320tagtaatcgc agatcagcat gctgcggtga
atacgttccc gggtcttgta cacaccgccc 1380gtcacaccat gggagtgggt ttcaccagaa
gtaggtagct taaccttcgg gagggcgctt 1440accacggtga gattcatgac tggggtgaag
tcgtaacaag gtaaccg 1487271488DNAunknownUnknown clone from
enriched environmental sample that by rDNA sequence analysis has
highest identity to Thauera aromatica 27tggctcagat tgaacgctgg
cggcatgctt tacacatgca agtcgaacgg cagcgggggc 60ttcggcctgc cggcgagtgg
cgaacgggtg agtaatgcat cggaacgtgc ccatgtcgtg 120ggggataacg tatcgaaagg
tacgctaata ccgcatacgt cctgagggag aaagcggggg 180atcttcggac ctcgcgcgat
tggagcggcc gatgtcggat tagctagtag gtgaggtaaa 240ggctcaccta ggcgacgatc
cgtagcgggt ctgagaggat gatccgccac actgggactg 300agacacggcc cagactccta
cgggaggcag cagtggggaa ttttggacaa tgggcgcaag 360cctgatccag ccatgccgcg
tgagtgaaga aggccttcgg gttgtaaagc tctttcggcc 420gggaagaaat cgtggtctct
aacataggcc atggatgacg gtaccggact aagaagcacc 480ggctaactac gtgccagcag
ccgcggtaat acgtagggtg cgagcgttaa tcggaattac 540tgggcgtaaa gcgtgcgcag
gcggttttgt aagacagatg tgaaatcccc gggctcaacc 600tgggaactgc gtttgtgact
gcaaggctag agtacggcag aggggggtgg aattcctggt 660gtagcagtga aatgcgtaga
gatcaagagg aacaccgatg gcggaagcag cccccctggg 720cctgtactga cgttcatgca
cgaaagcgtg gggagcaaac aggattagat acctggtaag 780tccacgccct aaacgatgtc
gactagtcgt tcggagcagc aatgcactga gtgacgcagc 840taacgcgtga agtcgaccgc
ctggggagta cggccgcaag gttaaaactc aaaggaattg 900acggggaccc gcacaagcgg
tggatgatgt ggattaattc gatgcaacgc gaaaaacctt 960acctaccctt gacatgccag
gaaccttgcc gagaggcgag ggtgccttcg ggagcctgga 1020cacaggtgct gcatggctat
cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1080cgagcgcaac ccttgtcact
agttgccatc atttggttgg gcactctagt gagactgccg 1140gtgacaaacc ggaggaaggt
ggggatgacg tcaagtcctc atggccctta tgggtagggc 1200ttcacacgtc atacaatggt
cggtacagag ggttgccaag ccgcgaggtg gagccaatcc 1260cttaaagccg atcgtagtcc
ggatcgtagt ctgcaactcg actacgtgaa gtcggaatcg 1320ctagtaatcg cagatcagca
tgctgcggtg aatacgttcc cgggtcttgt acacaccgcc 1380cgtcacacca tgggagtggg
tttcaccaga agtaggtagc ttaaccttcg ggagggcgct 1440taccacggtg agattcatga
ctggggtgaa gtcgtaacaa ggtaaccg 1488281489DNAunknownUnknown
clone from enriched environmental sample that by rDNA sequence
analysis has highest identity to Thauera aromatica 28tggctcagat
tgaacgctgg cggcatgctt tacacatgca agtcgaacgg cagcgggggc 60ttcggcctgc
cggcgagtgg cgaacgggtg agtaatgcat cggaacgtgc ccatgtcgtg 120ggggataacg
tatcgaaagg tacgctaata ccgcatacgt cctgagggag aaagcggggg 180atcttcggac
ctcgcgcgat tggagcggcc gatgtcggat tagctagtag gtgaggtaaa 240ggctcaccta
ggcgacgatc cgtagcgggt ctgagaggat gatccgccac actgggactg 300agacacggcc
cagactccta cgggaggcag cagtggggaa ttttggacaa tgggcgcaag 360cctgatccag
ccatgccgcg tgagtgaaga aggccttcgg gttgtaaagc tctttcggcc 420gggaagaaat
cgtggtctct aacataggcc atggatgacg gtaccggact aagaagcacc 480ggctaactac
gtgccagcag ccgcggtaat acgtagggtg cgagcgttaa tcggaattac 540tgggcgtaaa
gcgtgcgcag gcggttttgt aagacagatg tgaaatcccc gggctcaacc 600tgggaactgc
gtttgtgact gcaaggctag agtacggcag aggggggtgg aatttctggt 660gtagcagtaa
aatgcgtaga gatcaagagg aacaccgtat ggcgaagcca gcccctgggg 720cttgtactga
cgttcatgca cgaaagggtg gggagcaaac aggattagat acccctggta 780gtccacgccc
taaacgatgt cgactagtcg ttcggagcag caatgcactg agtgacgcag 840ctaacgcgtg
aagtcgaccg cctggggagt acggccgcaa ggttaaaact caaaggaatt 900gacggggacc
cgcacaagcg gtggatgatg tggattaatt cgatgcaacg cgaaaaacct 960tacctaccct
tgacatgcca ggaaccttgc cgagaggcga gggtgccttc gggagcctgg 1020acacaggtgc
tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca 1080acgagcgcaa
cccttgtcac tagttgccat catttggttg ggcactctag tgagactgcc 1140ggtgacaaac
cggaggaagg tggggatgac gtcaagtcct catggccctt atgggtaggg 1200cttcacacgt
catacaatgg tcggtacaga gggttgccaa gccgcgaggt ggagccaatc 1260ccttaaagcc
gatcgtagtc cggatcgtag tctgcaactc gactacgtga agtcggaatc 1320gctagtaatc
gcagatcagc atgctgcggt gaatacgttc ccgggtcttg tacacaccgc 1380ccgtcacacc
atgggagtgg gtttcaccag aagtaggtag cttaaccttc gggagggcgc 1440ttaccacggt
gagattcatg actggggtga agtcgtaaca aggtaaccg
148929749DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Azoarcus ap.
EH1O 29tggctcagat cgaacgctgg cggcatgctt tacacatgca agtcgaacgg cagcgggggc
60ttcggcctgc cggcgagtgg cgaacgggtg agtaatgcat cggaacgtgc ccatgtcgtg
120ggggataacg tatcgaaagg tacgctaata ccgcatacgc cctgaggggg aaagcggggg
180attcttcgga acctcgcgcg attggagcgg ccgatgtcgg attagctagt aggtgaggta
240aaggctcacc taggcgacga tccgtagcgg gtctgagagg atgatccgcc acactgggac
300tgagacacgg cccagactcc tacgggaggc agcagtgggg aattttggac aatgggggca
360accctgatcc agccatgccg cgtgagtgaa gaaggccttc gggttgtaaa gctctttcgg
420ccgggaagaa atcgcgcact ctaacatagt gtgtggatga cggtaccgga ctaagaagca
480ccggctaact acgtgccagc agccgcggta atacgtaggg tgcgagcgtt aatcggaatt
540actgggcgta aagcgtgcgc aggcggtttt gtaagacaga tgtgaaatcc ccgggctcaa
600cctgggaact gcgtttgtga ctgcaaggct agagtacggc agaggggggt ggaattcctg
660gtgtagcagt gaaatgcgta aatatcagga ggaacaccga tggcgaaggc agccccctgg
720gcctgtactg acgctcatgc acgaaagcg
749301487DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Thauera sp.
R26885 30tggctcagat tgaacgctgg cggcatgctt tacacatgca agtcgaacgg
cagcgggggc 60ttcggcctgc cggcgagtgg cgaacgggtg agtaatgcat cggaacgtgc
ccatgtcgtg 120ggggataacg tatcgaaagg tacgctaata ccgcatacgt cctgagggag
aaagcggggg 180atcttcggac ctcgcgcgat tggagcggcc gatgtcggat tagctagtag
gtgaggtaaa 240ggctcaccta ggcgacgatc cgtagcgggt ctgagaggat gatccgccac
actgggactg 300agacacggcc cagactccta cgggaggcag cagtggggaa ttttggacaa
tgggcgcaag 360cctgatccag ccatgccgcg tgagtgaaga aggccttcgg gttgtaaagc
tctttcggcc 420gggaagaaat cgtggtctct aacataggcc atggatgacg gtaccggact
aagaagcacc 480ggctaactac gtgccagcag ccgcggtaat acgtagggtg cgagcgttaa
tcggaattac 540tgggcgtaaa gcgtgcgcag gtggttttgt aagacagatg tgaaatcccc
gggctcaacc 600tgggaactgc gtttgtgact gcaaggctag agtacggcag aggggggtgg
aattcctggt 660gtagcagtga aatgcgtaaa gatcaagagg aacaccgatg gcgaaggcag
ccccctgggc 720ctgtactgac gttcatgcac gaaagcgtgg ggagcaaaca ggattagata
ccctggtagt 780ccacgcccta aacgatgtcg actagtcgtt cggagcagca atgcactgag
tgacgcagct 840aacgcgtgaa gtcgaccgcc tggggagtac ggccgcaagg ttaaaactca
aaggaattga 900cggggacccg cacaagcggt ggatgatgtg gattaattcg atgcaacgcg
aaaaacctta 960cctacccttg acatgtctgg aaccttggtg agagccgagg gtgccttcgg
gagccagaac 1020acaggtgctg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa
gtcccgcaac 1080gagcgcaacc cttgtcatta gttgccatca tttagttggg cactctaatg
agactgccgg 1140tgacaaaccg gaggaaggtg gggatgacgt caagtcctca tggcccttat
gggtagggct 1200tcacacgtca tacaatggtc ggtacagagg gttgccaagc cgcgaggtgg
agccaatccc 1260ttaaagccga tcgtagtccg gatcgtagtc tgcaactcga ctacgtgaag
tcggaatcgc 1320tagtaatcgc agatcagcat gctgcggtga atacgttccc gggtcttgta
cacaccgccc 1380gtcacaccat gggagtgggt ttcaccagaa gtaggtagct taaccttcgg
gagggcgctt 1440accacggtga gattcatgac tggggtgaag tcgtaacaag gtaaccg
1487311487DNAunknownUnknown clone from enriched environmental
sample that by rDNA sequence analysis has highest identity to
Thauera aromatica 31tggctcagat tgaacgctgg cggcatgctt tacacatgca
agtcgaacgg cagcgggggc 60ttcggcctgc cggcgagtgg cgaacgggtg agtaatgcat
cggaacgtgc ccatgtcgtg 120ggggataacg tatcgaaagg tacgctaata ccgcatacgt
cctgagggag aaagcggggg 180atcttcggac ctcgcgcgat tggagcggcc gatgtcggat
taactagtag gtgaggtaaa 240ggctcaccta ggcgacgatc cgtagcgggt ctgagaggat
gatccgccac actgggactg 300agacacggcc cagactccta cgggaggcag cagtggggaa
ttttggacaa tgggcgcaag 360cctgatccag ccatgccgcg tgagtgaaga aggccttcgg
gttgtaaagc tctttcggcc 420gggaagaaat cgtggtctct aacataggcc atggatgacg
gtaccggact aagaagcacc 480ggctaactac gtgccagcag ccgcggtaat acgtagagtg
cgagcgttaa tcggaattac 540tgggcgtaaa gcgtgcgcag gcggttttgt aagacagatg
tgaaatcccc gggctcaacc 600tgggaactgc gtttgtgact gcaaggctag agtacggcag
aggggggtgg aattcctggt 660gtagcagtga aatgcgtaaa gatcaagagg aacaccgatg
gcgaatgcaa ccccctgggc 720ctgtactgac gctcatgcac gaaagcgtgg ggagcaaaca
ggattagata ccctggtagt 780ccacgcccta aacgatgtcg actagtcgtt cggagcagca
atgcactgag tgacgcagct 840aacgcgtgaa gtcgaccgcc tggggagtac ggccgcaagg
ttaaaactca aaggaattga 900cggggacccg cacaagcggt ggatgatgtg gattaattcg
atgcaacgcg aaaaacctta 960cctacccttg acatgccagg aaccttgccg agaggcgagg
gtgccttcgg gagcctggac 1020acaggtgctg catggctgtc gtcagctcgt gtcgtgagat
gttgggttaa gtcccgcaac 1080gagcgcaacc cttgtcacta gttgccatca tttggttggg
cactctagtg agactgccgg 1140tgacaaaccg gaggaaggtg gggatgacgt caagtcctca
tggcccttat gggtagggct 1200tcacacgtca tacaatggtc ggtacagagg gttgccaagc
cgcgaggtgg agccaatccc 1260ttaaagccga tcgtagtccg gatcgtagtc tgcaactcga
ctacgtgaag tcggaatcgc 1320tagtaatcgc agatcagcat gctgcggtga atacgttccc
gggtcttgta cacaccgccc 1380gtcacaccat gggagtgggt ttcaccagaa gtaggtagct
taaccttcgg gagggcgctt 1440accacggtga gattcatgac tggggtgaag tcgtaacaag
gtaaccg 1487321499DNAunknownUnknown clone from enriched
environmental sample that by rDNA sequence analysis has highest
identity to Thauera aromatica 32cggttacctt gttacgactt caccccagtc
atgaatctca ccgtggtaag cgccctcccg 60aaggttaagc tacctacttc tggtgaaacc
cacccccatg gtgtgacggg cggtgtgtac 120aagacccggg aacgtattca ccgcagcatg
ctgatctgcg attactagcg attccgactt 180cacgtagtcg agttgcagac tacgatccgg
actacgatcg gctttaaggg attggctcca 240cctcgcggct tggcaaccct ctgtaccgac
cattgtatga cgtgtgaagc cctacccata 300agggccatga ggacttgacg tcatccccac
cttcctccgg tttgtcaccg gcagtctcac 360tagagtgccc aaccaaatga tggcaactag
tgacaagggt tgcgctcgtt gcgggactta 420acccaacatc tcacgacacg agctgacgac
agccatgcag cacctgtgtc caggctcccg 480aaggcaccct cgcctctcgg caaggttcct
ggcatgtcaa gggtaggtaa ggtttttcgc 540gttgcatcga attaatccac atcatccacc
gcttgtgcgg gtccccgtca attcctttga 600gttttaacct tgcggccgta ctccccaggc
ggtcgacttc acgcgttagc tgcgtcactc 660agtgcattgc tgctccgaac gactagtcga
catcgtttag ggcgtggact accagggtat 720ctaatcctgt ttgctcccca cgctttcgtg
catgagcgtc agtacaggcc cagggggctg 780ccttcgccat cggtgttcct cctgatctct
gcgcatttca ctgctacacc aggaattcca 840cccccctctg ccgtactcta gccttgcagt
cacaaacgca gttcccaggt tgagcccggg 900gatttcacat ctgtcttaca aaaccgcctg
cgcacgcttt acgcccagta attccgatta 960acgctcgcac cctacgtatt accgcggctg
ctggcacgta gttagccggt gcttcttagt 1020ccggtaccgt catccatggc ctatgttaga
gaccacgatt tcttcccggc cgaaagagct 1080ttacaacccg aaggccttct tcactcacgc
ggcatggctg gatcaggctt gcgcccattg 1140tccaaaattc cccactgctg cctcccgtag
gagtctgggc cgtgtctcag tcccagtgtg 1200gcggatcatc ctctcagacc cgctacggat
cgtcgcctag gtgagccttt acctcaccta 1260ctagctaatc cgacatcggc cgctccaatc
gcgcgaggtc cgaagatccc ccgctttctc 1320cctcaggacg tatgcggtat tagcgtacct
ttcgatacgt tatcccccac gacatgggca 1380cgttccgatg cattactcac ccgttcgcca
ctcgccggca ggccgaagcc cccgctgccg 1440ttcgacttgc atgtgtaaag catgccgcca
gcgttcaatc tgagccatga tcaaactct 1499331429DNAunknownUnknown clone from
enriched environmental sample that by rDNA sequence analysis has
highest identity to Thauera aromatica 33tgaacgctgg cggcatgctt
tacacatgca agtcgaacgg cagcgggggc ttcggcctgc 60cggcgagtgg cgaacgggtg
agtaatgcat cggaacgtgc ccatgtcgtg ggggataacg 120tatcgaaagg tacgctaata
ccgcatacgt cctgagggag aaagcggggg attttcggac 180ctcgcgcgat tggagcggcc
gatgtcggat tagctagtag gtgaggtaaa ggctcaccta 240ggcgacgatc cgtagcgggt
ctgagaggat gatccgccac actgggactg agacacggcc 300cagactccta cgggaggcag
cagtggggaa ttttggacaa tgggcgcaag cctgatccag 360ccatgccgcg tgagtgaaga
aggccttcgg gttgtaaagc tctttcggcc gggaagaaat 420cgtggtctct aacataggcc
atggatgacg gtaccggact aagaagcacc ggctaactac 480gtgccagcag ccgcggtaat
atgtagggtg cgagcgttaa tcggaattac tgggcgtaaa 540gcgtgcgcag gcggttttgt
aagacagatg tgaaatcccc gggctcaacc tgggaactgc 600gtttgtgact gcaaggctag
agtacggcgg aggggggtgg aattcctggt gtagcagtga 660aatgcgtaga gatcaggagg
aacaccgatg gcgaaggcag ccccctgggc ctgtactgac 720gctcatgcac gaaagcgtgg
ggagcaaaca ggattagata ccctggtagt ccacgcccta 780aacgatgtcg actagtcgtt
cggagcagca atgcactgag tgacgcagct aacgcgtgaa 840gtcgaccgcc tggggagtac
ggccgcaagg ttaaaactca aaggaattga cggggacccg 900cacaagcggt ggatgatgtg
gattaattcg atgcaacgcg aaaaacctta cctacccttg 960acatgccagg aaccttgccg
agaggcgagg gtgccttcgg gagcctggac acaggtgctg 1020catggctgtc gtcagctcgt
gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1080cttgtcacta gttgccatca
tttggttggg cactctagtg agactgccgg tgacaaaccg 1140gaggaaggtg gggatgacgt
caagtcctca tggcccttat gggtagggct tcacacgtca 1200tacaatggtc ggtacagagg
gttgccaagc cgcgaggtgg agccaatccc ttaaagccga 1260tcgtagtccg gatcgtagtc
tgcaactcga ctacgtgaag tcggaatcgc tagtaatcgc 1320agatcagcat gctgcggtga
atacgttccc gggtcttgta cacaccgccc gtcacaccat 1380gggagtgggt ttcaccagaa
gtaggtagct taaccttcgg gagggcgct 1429341439DNAunknownUnknown
clone from enriched environmental sample that by rDNA sequence
analysis has highest identity to Thauera aromatica 34tggctcagat
tgaacgctgg cggcatgctt tacacatgca agtcgaacgg cagcgggggc 60ttcggcctgc
cggcgagtgg cgaacgggtg agtaatgcat cggaacgtgc ccatgtcgtg 120ggggataacg
tatcgaaagg tacgctaata ccgcatacgc cctgagggag aaagcggggg 180atcttcggac
ctcgcgcgat tggagcggcc gatgtcggat tagctagtag gtgaggtaaa 240ggctcaccta
ggcgacgatc cgtagcgggt ctgagaggat gatccgccac actgggactg 300agacacggcc
cagactccta cgggaggcag cagtggggaa ttttggacaa tgggcgcaag 360cctgatccag
ccatgccgcg tgagtgaaga aggccttcgg gttgtaaagc tctttcggcc 420gggaagaaat
cgtggtctct aacataggcc atggatgacg gtaccggact aagaagcacc 480ggctaactac
gtgccagcag ccgcggtaat acgtagggtg cgagcgttaa tcggaattac 540tgggcgtaaa
gcgtgcgcag gcggttttgt aagacagatg tgaaatcccc gggctcaacc 600tgggaactgc
gtttgtgact gcaaggctag agtacggcag aggggggtgg aattcctggt 660gtagcagtga
aatgcgtaaa gatcaggagg aacaccgatg gcgaaggcag ccccctgggc 720ctgtactgac
gctcatgcac gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 780ccacgcccta
aacgatgtcg actagtcgtt cggagcagca atgcactgag tgacgcagct 840aacgcgtgaa
gtcgaccgcc tggggagtac ggccgcaagg ttaaaactca aaggaattga 900cggggacccg
cacaagcggt ggatgatgtg gattaatttg atgcaacgcg aaaaacctta 960cctacccttg
acatgccagg aaccttgccg agaggcgagg gtgccttcgg gagcctggac 1020acaggtgctg
catggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1080gagcgcaacc
cttgtcacta gttgccatca tttggttggg cactctagtg agactgccgg 1140tgacaaaccg
gaggaaggtg gggatgacgt caagtcctca tggcccttat gggtagggct 1200tcacacgtca
tacaatggtc ggtacagagg gttgccaagc cgcgaggtgg agccaatccc 1260ttaaagccga
tcgtagtccg gatcgtagtc tgcaactcga ctacgtgaag tcggaatcgc 1320tagtaatcgc
agatcagcat gctgcggtga atacgttccc gggtcttgta cacaccgccc 1380gtcacaccat
gggagtgggt ttcaccagaa gtaggtagct taaccttcgg gagggcgct
1439351440DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Thauera
aromatica 35tggctcagat tgaacgctgg cggcatgctt tacacatgca agtcgaacgg
cagcgggggc 60ttcggcctgc cggcgagtgg cgaacgggtg agtaatgcat cggaacgtgc
ccatgtcgtg 120ggggataacg tatcgaaagg tacgctaata ccgcatacgt cctgagggag
aaagcggggg 180atcttcggac ctcgcgcgat tggagcggcc gatgtcggat tagctagtag
gtgaggtaaa 240ggctcaccta ggcgacgatc cgtagcgggt ctgagaggat gatccgccac
actgggactg 300agacacggcc cagactccta cgggaggcag cagtggggaa ttttggacaa
tgggcgcaag 360cctgatccag ccatgccgcg tgagtgaaga aggccttcgg gttgtaaagc
tctttcggcc 420gggaagaaat cgtggtctct aacataggcc atggatgacg gtaccggact
aagaagcacc 480ggctaactac gtgccagcag ccgcggtaat acgtagggtg cgagcgttaa
tcggaattac 540tgggcgtaaa gcgtgcgcag gcggttttgt aagacagatg tgaaatcccc
gggctcaacc 600tgggaactgc gtttgtgact gcaaggctag agtacggcag aggggggtgg
aattcctggt 660gtagcagtga aatgcgtaga gatcaggagg aacaccgatg gcgaaggcag
ccccctgggc 720ctgtactgac gctcatgcac gaaagcctgg gggagcaaca ggattagata
ccctggtaag 780tccacgccct aaacgatgtc gactagtcgt tcggagcagc aatgcactga
gtgacgcagc 840taacgcgtga agtcgaccgc ctggggagta cggccgcaag gttaaaactc
aaaggaattg 900acggggaccc gcacaagcgg tggatgatgt ggattaattc gatgcaacgc
gaaaaacctt 960acctaccctt gacatgccag gaaccttgcc gagaggcgag ggtgccttcg
ggagcctgga 1020cacaggtgct gcatggctgt cgtcagctcg tgtcgtgaaa tgttgggtta
agtcccgcaa 1080cgagcgcaac ccttgtcact agttgccatc atttggttgg gcactctagt
gagactgccg 1140gtgacaaacc ggaggaaggt ggggatgacg tcaagtcctc atggccctta
tgggtagggc 1200ttcacacgtc atacaatggt cggtacagag ggttgccaag ccgcgaggtg
gagccaatcc 1260cttaaagccg atcgtagtcc ggatcgtagt ctgcaactcg actacgtgaa
gtcggaatcg 1320ctagtaatcg cagatcagca tgctgcggtg aatacgttcc cgggtcttgt
acacaccgcc 1380cgtcacacca tgggagtggg tttcaccaga agtaggtagc ttaaccttcg
ggagggcgct 1440361440DNAunknownUnknown clone from enriched
environmental sample that by rDNA sequence analysis has highest
identity to Thauera aromatica 36tggctcagat tgaacgctgg cggcatgctt
tacacatgca agtcgaacgg cagcgggggc 60ttcggcctgc cggcgagtgg cgaacgggtg
agtaatgcat cggaacgtgc ccatgtcgtg 120ggggataacg tatcgaaagg tacgctaata
ccgcatacgt cctgagggag aaagcggggg 180atcttcggac ctcgcgcgat tggagcggcc
gatgtcggat tagctagtag gtgaggtaaa 240ggctcaccta ggcgacgatc cgtagcgggt
ctgagaggat gatccgccac actgggactg 300agacacggcc cagactccta cgggaggcag
cagtggggaa ttttggacaa tgggcgcaag 360cctgatccag ccatgccgcg tgagtgaaga
aggccttcgg gttgtaaagt tctttcggcc 420gggaagaaat cgtggtctct aacataggcc
atggatgacg gtaccggact aagaagcacc 480ggctaactac gtgccagcag ccgcggtaat
acgtagggtg cgagcgttaa tcggaattac 540tgggcgtaaa gcgtgcgcag gcggttttgt
aagacagatg tgaaatcccc gggctcaacc 600tgggaactgc gtttgtgact gcaaggctag
agtacggcag aagggggtgg aattcctggt 660gtagcagtga aatgcgtaga gatcaggagg
aacaccgatg gcgaaggcag cccccttggg 720cctgtactga cgctcatgca cgaaagcgtg
gggagcaaac aggattagat accctggtag 780tccacgccct aaacgatgtc gactagtcgt
tcggagcagc aatgcactga gtgacgcagc 840taacgcgtga agtcgaccgc ctggggagta
cggccgcaag gttaaaactc aaaggaattg 900acggggaccc gcacaagcgg tggatgatgt
ggattaattc gatgcaacgc gaaaaacctt 960acctaccctt gacatgccag gaaccttgcc
gagaggcgag ggtgccttcg ggagcctgga 1020cacaggtgct gcatggctgt cgtcagctcg
tgtcgtgaga tgttgggtta agtcccgcaa 1080cgagcgcaac ccttgtcact agttgccatc
atttggttgg gcactctagt gagactgccg 1140gtgacaaacc ggaggaaggt ggggatgacg
tcaagtcctc atggccctta tgggtagggc 1200ttcacacgtc atacaatggt cggtacagag
ggttgccaag ccgcgaggtg gagccaatcc 1260cttaaagccg atcgtagtcc ggatcgtagt
ctgcaactcg actacgtgaa gtcggaatcg 1320ctagtaatcg cagatcagca tgccgcggtg
aatacgttcc cgggtcttgt acacaccgcc 1380cgtcacacca tgggagtggg tttcaccaga
agtaggtagc ttaaccttcg ggagggcgct 1440371439DNAunknownUnknown clone from
enriched environmental sample that by rDNA sequence analysis has
highest identity to Thauera aromatica 37tggctcagat tgaacgctgg
cggcatgctt tacacatgca agtcgaacgg cagcgggggc 60ttcggcctgc cggcgagtgg
cgaacgggtg agtaatgcat cggaacgtgc ccatgtcgtg 120ggggataacg tatcgaaagg
tacgctaata ccgcatacgt cctgagggag aaagcggggg 180atcttcggac ctcgcgcgat
tggagcggcc gatgtcggat tagctagtag gtgaggtaaa 240ggctcaccta ggcgacgatc
cgtagcgggt ctgagaggat gatccgccac actgggactg 300agacacggcc cagactccta
cgggaggcag cagtggggaa ttttggacaa tgggcgcaag 360cctgatccag ccatgccgcg
tgagtgaaga aggccttcgg gttgtaaagc tctttcggcc 420gggaagaaat cgtggtctct
aacataggcc atggatgacg gtaccggact aagaagcacc 480ggctaactac gtgccagcag
ccgcggtaat acgtagggtg cgagcgttaa tcggaattac 540tgggcgtaaa gcgtgcgcag
gcggttttgt aagacagatg tgaaatcccc gggctcaacc 600tgggaactgc gtttgtgact
gcaaggctag agtacggcag aggggggtgg aattcctggt 660gtagcagtga aatgcgtaga
gatcaagagg aacaccgatg gcgaaggcag ccccctgggc 720ctgtactgac gttcatgcac
gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 780ccacgcccta aacgatgtcg
actagtcgtt cggagcagca atgcactgag tgacgcagct 840aacgcgtgaa gtcgaccgcc
tggggagtac ggccgcaagg ttaaaactca aaggaattga 900cggggacccg cacaagcggt
ggatgatgtg gattaattcg atgcaacgcg aaaaacctta 960cctacccttg acatgccagg
aaccttgccg agaggcgagg gtgccttcgg gagcctggac 1020acaggtgctg catggctgtc
gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1080gagcgcaacc cttgtcacta
gttgccatca tttggttggg cactctagtg agactgccgg 1140tgacaaaccg gaggaaggtg
gggatgacgt caagtcctca tggcccttat gggtagggct 1200tcacacgtca tacaatggtc
ggtacagagg gttgccaagc cgcgaggtgg agccaatccc 1260ttaaagccga tcgtagtccg
gatcgtagtc tgcaactcga ctacgtgaag tcggaatcgc 1320tagtaatcgc agatcagcat
gctgcggtga atacgttccc gggtcttgta cacaccgccc 1380gtcacaccat gggagtgggt
ttcaccagaa gtaggtagct taaccttcgg gagggcgct 1439381439DNAunknownUnknown
clone from enriched environmental sample that by rDNA sequence
analysis has highest identity to Thauera aromatica 38tggctcagat
tgaacgctgg cggcatgctt tacacatgca agtcgaacgg cagcgggggc 60ttcggcctgc
cggcgagtgg cgaacgggtg agtaatgcat cggaacgtgc ccatgtcgtg 120ggggataacg
tatcgaaagg tacgctaata ccgcatacgt cctgagggag agagcggggg 180atcttcggac
ctcgcgcgat tggagcggcc gatgtcggat tagctagtag gtgaggtaaa 240ggctcaccta
ggcgacgatc cgtagcgggt ctgagaggat gatccgccac actgggactg 300agacacggcc
cagactccta cgggaggcag cagtggggaa ttttggacaa tgggcgcaag 360cctgatccag
ccatgccgcg tgagtgaaga aggccttcgg gttgtaaagc tctttcggcc 420gggaagaaat
cgtggtctct aacataggcc atggatgacg gtaccggact aagaagcacc 480ggctaactac
gtgccagcag ccgcggtaat acgtagggtg cgagcgttaa tcggaattac 540tgggcgtaaa
gcgtgcgcag gcggttttgt aagacagatg tgaaatcccc gggctcaacc 600tgggaactgc
gtttgtgact gcaaggctag agtacggcag aggggggtgg aattcctggt 660gtagcagtga
aatgcgtaga gatcaggagg aacaccgatg gggaaggcag ccccctgggc 720ctgtactgac
gctcatgcac gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 780ccacgcccta
aacgatgtcg actagtcgtt cggagcagca atgcactgag tgacgcagct 840aacgcgtgaa
gtcgaccgcc tggggagtac ggccgcaagg ttaaaactca aaggaattga 900cggggacccg
cacaagcggt ggatgatgtg gattaattcg atgcaacgcg aaaaacctta 960cctacccttg
acatgccagg aaccttgccg agaggcgagg gtgccttcgg gagcctggac 1020acaggtgctg
catggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1080gagcgcaacc
cttgtcacta gttgccatca tttggttggg cactctagtg agactgccgg 1140tgacaaaccg
gaggaaggtg gggatgacgt caagtcctca tggcccttat gggtagggct 1200tcacacgtca
tacaatggtc ggtacagagg gttgccaagc cgcgaggtgg agccaatccc 1260ttaaagccga
tcgtagtccg gatcgtagtc tgcaactcga ctacgtgaag tcggaatcgc 1320tagtaatcgc
agatcagcat gctgcggtga atacgttccc gggtcttgta cacaccgccc 1380gtcacaccat
gggagtgggt ttcaccagaa gtaggtagct taaccttcgg gagggcgct
1439391439DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Thauera
aromatica 39tggctcagat tgaacgctgg cggcatgctt tacacatgca agtcgaacgg
cagcgggggc 60ttcggcctgc cggcgagtgg cgaacgggtg agtaatgcat cggaacgtgc
ccatgtcgtg 120ggggataacg tatcgaaagg tacgctaata ccgcatacgt cctgagggag
aaagcggggg 180atcttcggac ctcgcgcgat tggagcggcc gatgtcggat tagctagtag
gtgaggtaaa 240ggctcaccta ggcgacgatc cgtagcgggt ctgagaggat gatccgccac
actgggactg 300agacacggcc cagactccta cgggaggcag cagtggggaa ttttggacaa
tgggcgcaag 360cctgatccag ccatgccgcg tgagtgaaga aggccttcgg gttgtaaagc
tctttcggcc 420gggaagaaat cgtggtctct aacataggcc atggatgacg gtaccggact
aagaagcacc 480ggctaactac gtgccagcag ccgcggtaat acgtagggtg cgagcgttaa
tcggaattac 540tgggcgtaaa gcgtgcgcag gcggttttgt aagacagatg tgaaatcccc
gggctcaacc 600tgggaactgc gtttgtgact gcaaggctag agtacggcag aggggggtgg
aattcctggt 660gtatcagtga aatgcgtaaa gatcaagagg aacaccgatg gggaaggcag
ccccctgggc 720ctgtactgac gttcatgcac gaaagcgtgg ggagcaaaca ggattagata
ccctggtagt 780ccacgcccta aacgatgtcg actagtcgtt cggagcagca atgcactgag
tgacgcagct 840aacgcgtgaa gtcgaccgcc tggggagtac ggccgcaagg ttaaaactca
aaggaattga 900cggggacccg cacaagcggt ggatgatgtg gattaattcg atgcaacgcg
aaaaacctta 960cctacccttg acatgccagg aaccttgccg agaggcgagg gtgccttcgg
gagcctggac 1020acaggtgctg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa
gtcccgcaac 1080gagcgcaacc cttgtcacta gttgccatca tttggttggg cactctagtg
agactgccgg 1140tgacaaaccg gaggaaggtg gggatgacgt caagtcctca tggcccttat
gggtagggct 1200tcacacgtca tacaatggtc ggtacagagg gttgccaagc cgcgaggtgg
agccaatccc 1260ttaaagccga tcgtagtccg gatcgtagtc tgcaactcga ctacgtgaag
tcggaatcgc 1320tagtaatcgt agatcagcat gctgcggtga atacgttccc gggtcttgta
cacaccgccc 1380gtcacaccat gggagtgggt ttcaccagaa gtaggtagct taaccttcgg
gagggcgct 1439401439DNAunknownUnknown clone from enriched
environmental sample that by rDNA sequence analysis has highest
identity to Thauera aromatica 40tggctcagat tgaacgctgg cggcatgctt
tacacatgca agtcgaacgg cagcgggggc 60ttcggcctgc cggcgagtgg cgaacgggtg
agtaatgcat cggaacgtgc ccatgtcgtg 120ggggataacg tatcgaaagg tacgctaata
ccgcatacgt cctgagggag aaagcggggg 180atcttcggac ctcgcgcgat tggagcggcc
gatgtcggat tagctagtag gtgaggtaaa 240ggctcaccta ggcgacgatc cgtagcgggt
ctgagaggat gatccgccac actgggactg 300agacacggcc cagactccta cgggaggcag
cagtggggaa ttttggacaa tgggcgcaag 360cctgatccag ccatgccgcg tgagtgaaga
aggccttcgg gttgtaaagc tctttcggcc 420gggaagaaat cgtggtctct aacataggcc
atggatgacg gtaccggact aagaagcacc 480ggctaactac gtgccagcag ccgcggtaat
acgtagggtg cgagcgttaa tcggaattac 540tgggcgtaaa gcgtgcgcag gcggttttgt
aagacagatg tgaaatcccc gggctcaacc 600tgggaactgc gtttgtgact gcaaggctag
agtacggcag aggggggtgg aactcctggt 660gtagcagtga aatgcgtaga gatcaggagg
aacaccgatg gcgaaggcag ccccctgggc 720ttgtactgac gctcatgcac gaaagcgtgg
ggagcaaaca ggattagata ccctggtagt 780ccacgcccta aacgatgtcg actagtcgtt
cggagcagca atgcactgag tgacgcagct 840aacgcgtgaa gtcgaccgcc tggggagtac
ggccgcaagg ttaaaactca aaggaattga 900cggggacccg cacaagcggt ggatgatgtg
gattaattcg atgcaacgcg aaaaacctta 960cctacccttg acatgccagg aaccttgccg
agaggcgagg gtgccttcgg gagcctggac 1020acaggtgctg catggctgtc gtcagctcgt
gtcgtgagat gttgggttaa gtcccgcaac 1080gagcgcaacc cttgtcacta gttgccatca
tttggttggg cactctagtg agactgccgg 1140tgacaaaccg gaggaaggtg gggatgacgt
caagtcctca tggcccttat gggtagggct 1200tcacacgtca tacaatggtc ggtacagagg
gttgccaagc cgcgaggtgg agccaatccc 1260ttaaagccga tcgtagtccg gatcgtagtc
tgcaactcga ctacgtgaag tcggaatcgc 1320tagtaatcgc agatcagcat gctgcggtga
atacgttccc gggtcttgta cacaccgccc 1380gtcacaccat gggagtgggt ttcaccagaa
gtaggtagct taaccttcgg gagggcgct 1439411439DNAunknownUnknown clone from
enriched environmental sample that by rDNA sequence analysis has
highest identity to Thauera aromatica 41tggctcagat tgaacgctgg
cggcatgctt tacacatgca agtcgaacgg cagcgggggc 60ttcggcctgc cggcgagtgg
cgaacgggtg agtaatgcat cggaacgtgc ccatgtcgtg 120ggggataacg tatcgaaagg
tacgctaata ccgcatacgt cctgagggag aaagcggggg 180atcttcggac ctcgcgcgat
tggagcggcc gatgtcggat tagctagtag gtgaggtaaa 240ggctcaccta ggcgacgatc
cgtagcgggt ctgagaggat gatccgccac actgggactg 300agacacggcc cagactccta
cgggaggcag cagtggggaa ttttggacaa tgggcgcaag 360cctgatccag ccatgccgcg
tgagtgaaga aggccttcgg gttgtaaagc tctttcggcc 420gggaagaaat cgtggtctct
aacataggcc atggatgacg gtaccggact aagaagcacc 480ggctaactac gtgccagcag
ccgcggtaat acgtagggtg cgagcgttaa tcggaattac 540tgggcgtaaa gcgtgcgcag
gcggttttgt aagacagatg tgaaatcccc gggctcaacc 600tgggaactgc gtttgtgact
gcaaggctag agtacggcag aggggggtgg aattcctggt 660gtagcagtga aatgcgtaga
gatcaggagg aacgccgatg gcgaagacag ccccctgggc 720ctgtactgac gctcatgcac
gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 780ccacgcccta aacgatgtcg
actagtcgtt cggagcagca atgcactgag tgacgcagct 840aacgcgtgaa gtcgaccgcc
tggggagtac ggccgcaagg ttaaaactca aaggaattga 900cggggacccg cacaagcggt
ggatgatgtg gattaattcg atgcaacgcg aaaaacctta 960cctacccttg acatgccagg
aaccttgccg agaggcgagg gtgccttcgg gagcctggac 1020acaggtgctg catggctgtc
gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1080gagcgcaacc cttgtcacta
gttgccatca tttggttggg cactctagtg agactgccgg 1140tgacaaaccg gaggaaggtg
gggatgacgt caagtcctca tggcccttat gggtagggct 1200tcacacgtca tacaatggtc
ggtacagggg gttgccaagc cgcgaggtgg agccaatccc 1260ttaaagccga tcgtagtccg
gatcgtagtc tgcaactcga ctacgtgaag tcggaatcgc 1320tagtaatcgc agatcagcat
gctgcggtga atacgttccc gggtcttgta cacaccgccc 1380gtcacaccat gggagtgggt
ttcaccagaa gtaggtagct taaccttcgg gagggcgct 1439421475DNAunknownUnknown
clone from enriched environmental sample that by rDNA sequence
analysis has highest identity to Finegoldia magna 42tggctcagga
cgaacgctgg cggcgtgcct aacacatgca agtcgagcga agtgactctg 60agagaaattt
tcggatggat cgaagagtca tcttagcggc ggacgggtga gtaacgcgtg 120agaaacctgc
ctttcacaaa gggatagcct cgggaaactg ggattaatac cttatgaaac 180tgaattaccg
catggtagat cagtcaaagc gaataagcgg tgaaagatgg tctcgcgtcc 240tattagctag
ttggtgaggt aacggctcac caaggcttcg ataggtagcc ggcctgagag 300ggtgaacggc
cacactggaa ctgagacacg gtccagactc ctacgggagg cagcagtggg 360gaatattgca
caatggagga aactctgatg cagcgacgcc gcgtgaatga tgaaggcctt 420cgggttgtaa
agttctgtcc ttggggaaga taatgacggt acccaaggag gaagccccgg 480ctaactacgt
gccagcagcc gcggtaatac gtagggggcg agcgttgtcc ggaattattg 540ggcgtaaagg
gttcgcaggc ggtctgataa gtcagatgtg aaaggcgtag gctcaaccta 600cgtaagcatt
tgaaactgtc agacttgagt taaggagagg aaagtggaat tcctagtgta 660gcggtgaaat
gcgtagatat taggaggaat accagtggcg aagggsgact ttctggactt 720atactgacgc
tgaggaacga aagcgtgggg agsaaacagg attagatacc ctggtagttc 780cacgccgtaa
acgawgagtg ctaggtgktg ggggtcaaac ctcggtgccg caasctaacg 840cattaagcac
tccgcctggg gggtacgtac gcmagtatga aactcaaagg aattgacggg 900gacccgcaca
agcggtggat gatgtggatt aattcgatgc aacgcgaaaa accttaccta 960cccttgacat
gccaggaacc ttgccgagag gcgagggtgc cttcgggagc ctggacacag 1020gtgctgcatg
gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc 1080gcaacccttg
tcactagttg ccatcatttg gttgggcact ctagtgagac tgccggtgac 1140aaaccggagg
aaggtgggga tgacgtcaag tcctcatggc ccttatgggt agggcttcac 1200acgtcataca
atggtcggta cagagggttg ccaagccgcg aggtggagcc aatcccttaa 1260agccgatcgt
agtccggatc gtagtctgca actcgactac gtgaagtcgg aatcgctagt 1320aatcgcagat
cagcatgctg cggtgaatac gttcccgggt cttgtacaca ccgcccgtca 1380caccatggga
gtgggtttca ccagaagtag gtagcttaac cttcgggaac cacggtgaga 1440ttcatgactg
gggtgaagtc gtaacaaggt aaccg
1475431481DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Spirochaeta
sp. MET-E 43tggctcagaa cgaacgctgg cggcgcgttt taagcatgca agtcgagcgg
caagggcctt 60tgggccccta gagcggcgga cgggtgagta acacgtggac aatctgcccc
ccggccgggg 120atagcccagg gaaacctgga ttaataccgg atgagacggg acgcacgatg
gtgcggtccg 180ggaaaggcgc tgcggcgccg ccgggggatg agtccgcgac ccattagctg
gacggcgggg 240taaaggccca ccgtggcgac gatgggtagc cggcctgaga gggtggacgg
ccacattgga 300actgagacac ggtccagact cctacgggag gcagcagcta agaatcttcc
gcaatgggcg 360aaagcctgac ggagcgacgc cgcgtgaacg aagaaggccg tgaggttgta
aagttctttt 420cgggaggggg aattaccgtg gcagggaatg gccgcgggat gacgtgaatc
ccggaacaag 480ccccggctaa ctacgtgcca gcagccgcgg taacacgtag ggggcgagcg
ttgttcggaa 540tcattgggcg taaagggcgt gcaggcggca ctgcaagtcc ggcgtgaaag
accccggccc 600aaccgggggg gtgcgctgga aactgcggtg cttgagtaca ggaggggatg
ccggaattcc 660aggtgtaggg gtgaaatctg tagatatctg gaagaacacc gatggcgaag
gcaggcatct 720ggccatgtac tgacgctgag acgcgaaggt gcggggagca aacaggttta
gataccctgg 780tagtccgcac agtaaacgat gtgcaccagg gtggcggggg tagaaccccc
ggtaccgtag 840caaacgcatt aagtgcaccg cctggggagt atgctcgcaa gggtgaaact
caaaggaatt 900gacgggggcc cgcacaagcg gaggagcatg tggtttaatt cgatgatacg
cgaggaacct 960tacctgggct cgaacgtaag atgactgtag gtgaaagctt acatctcttc
ggagcatttt 1020acgaggtgct gcatggttgt cgtcagctcg tgccgtgagg tgtcgggtta
agtcccataa 1080cgagcgcaac ccctaccttt agttgccatc aggtaatgct ggggactcta
aaggaactgc 1140ctacgcaagt agtgaggaag gcggggatga cgtcaaatca gcacggccct
tacgtccagg 1200gctacacacg tgctacaatg gccgatacag agggcagcta cctggtgaca
ggatgcaaat 1260ctccaaagtc ggtctcagtt cggatcggag tctgcaaccc gactccgtga
agttggattc 1320gctagtaatc gcgcatcagc catggcgcgg tgaatacgtt cccgggcctt
gtacacaccg 1380cccgtcaagc catggaagct ggggggacct aaagtcgata accgcaagga
gtcgcctagg 1440gtaaaaccag tgactggggc taagtcgtaa caaggtaacc g
1481441488DNAunknownUnknown clone from enriched environmental
sample that by rDNA sequence analysis has highest identity to
Azotobacter beijerinckii 44tggctcagat tgaacgctgg cggcaggcct aacacatgca
agtcgagcgg atgagtggag 60cttgctccat gattcagcgg cggacgggtg agtaatgcct
aggaatctgc ctggtagtgg 120gggacaacgt ttcgaaagga acgctaatac cgcatacgtc
ctacgggaga aagtggggga 180tcttcggacc tcacgctatc agatgagcct aggtcggatt
agctagttgg cgaggtaaag 240gctcaccaag gcgacgatcc gtaactggtc tgagaggatg
atcagtcaca ctggaactga 300gacacggtcc agactcctac gggaggcagc agtggggaat
attggacaat gggcgaaagc 360ctgatccagc catgccgcgt gtgtgaagaa ggccttcggg
ttgtaaagct ctttcggccg 420ggaagaaatc gtggtctcta acataggcca tggatgacgg
taccggacta agaagcaccg 480gctaactacg tgccagcagc cgcggtaata cgtagggtgc
gagcgttaat cggaattact 540gggcgtaaag cgtgcgcagg cggttttgta agacagatgt
gaaatccccg ggctcaacct 600gggaactgcg tttgtgactg caaggctaga gtacggcaga
ggggggtgga attcctggtg 660tagcagtgaa atgcgtagag atcaggagga acaccgatgg
cgaaggcagc cccctgggcc 720tgtactgacg ctcatgcacg aaagcgtggg gagcaaacag
gattagatac cctggtagtc 780cacgccctaa acgatgtcga ctagtcgttc ggagcagcaa
tgcactgagt gacgcagcta 840acgcgtgaag tcgaccgcct ggggagtacg gccgcaaggt
taaaactcaa aggaattgac 900ggggacccgc acaagcggtg gatgatgtgg attaattcga
tgcaacgcga aaaaccttac 960ctacccttga catgccagga accttgccga gaggcgaggg
tgccttcggg agcctggaca 1020caggtgctgc atggctgtcg tcagctcgtg tcgtgagatg
ttgggttaag tcccgcaacg 1080agcgcaaccc ttgtcactag ttgccatcat ttggttgggc
actctagtga gactgccggt 1140gacaaaccgg aggaaggtgg ggatgacgtc aagtcctcat
ggcccttatg ggtagggctt 1200cacacgtcat acaatggtcg gtacagaggg ttgccaagcc
gcgaggtgga gccaatccct 1260taaagccgat cgtagtccgg atcgtagtct gcaactcgac
tacgtgaagt cggaatcgct 1320agtaatcgca gatcagcatg ctgcggtgaa tacgttcccg
ggccttgtac acaccgcccg 1380tcacaccatg agagttggca atacccgaag tccgtggggc
aaccgtttac ggagccagcg 1440gccgaaggta gggtcagcga ttggggtgaa gtcgtaacaa
ggtaaccg 1488451481DNAunknownUnknown clone from enriched
environmental sample that by rDNA sequence analysis has highest
identity to Finegoldia magna 45cggttacctt gttacgactt caccccagtc
attgccccta ccttcgacag ctgccccctt 60ttatggttag ctcactggct tcgggtattg
acaactccca tggtgtgacg ggcggtgtgt 120acaagacccg ggaacgcatt caccgcggca
ttctgatccg cgattactag caactccgac 180ttcatgcagg cgagttgcag cctgcaatcc
gaactgggat cggcttttag agatttgctt 240gccatcgctg acttgcttct cgttgtaccg
accattgtag cacgtgtgta gcccaggaca 300taaagggcat gatgatttga cgtcatcccc
accttcctcc gatttgtcat cggcagtctc 360tttagagtgc ccaacttaat gatggcaact
aaagacaagg gttgcgctcg ttgcgggact 420taacccaaca tctcacgaca cgagctgacg
acaaccatgc accacctgtg tccgctgtac 480cccgaaggat aaagatctat ctctaaaccg
gtcagcggca tgtcaagccc tggtaaggtt 540cttcgcgttg cttcgaatta aaccacatgc
tccgctgctt gtgcgggtcc ccgtcaattc 600ctttgagttt catacttgcg tacgtactcc
ccaggcggag tgcttaatgc gttagctgcg 660gcaccgaggt ttgaccccca acacctagca
ctcatcgttt acggcgtgga ctaccagggt 720atctaatcct gtttgctccc cacgctttcg
ttcctcagcg tcagtataag tccagaaagt 780cgccttcgcc actggtattc ctcctaatat
ctacgcattt caccgctaca ctaggaattc 840cactttcctc tccttaactc aagtctgaca
gtttcaaatg cttacgtagg ttgagcctac 900gcctttcaca tctgacttat cagaccgcct
gcgaaccctt tacgcccaat aattccggac 960aacgctcgcc ccctacgtat taccgcggct
gctggcacgt agttagccgg ggcttcctcc 1020ttgggtaccg tcattatctt ccccaaggac
agaactttac aacccgaagg ccttcatcat 1080tcacgcggcg tcgctgcatc agagtttcct
ccattgtgca atattcccca ctgctgcctc 1140ccgtaggagt ctggaccgtg tctcagttcc
agtgtggccg ttcaccctct caggccggct 1200acctatcgaa gccttggtga gccgttacct
caccaactag ctaataggac gcgagaccat 1260ctttcaccgc ttattcgctt tgactgatct
accatgcggt aattcagttt cataaggtat 1320taatcccagt ttcccgaggc tatccctttg
tgaaaggcag gtttctcacg cgttactcac 1380ccgtccgccg ctaagatgac tcttcgatcc
atccgaaaay ttctctcmga gtcacttcgc 1440ggcacgccgc cagcgttcgt cctgagccak
aatcaaactc t 1481461486DNAunknownUnknown clone from
enriched environmental sample that by rDNA sequence analysis has
highest identity to Azotobacter beijerinckii 46tggctcagat tgaacgctgg
cggcaggcct aacacatgca agtcgagcgg atgagtggag 60cttgctccat gattcagcgg
cggacgggtg agtaatgcct aggaatctgc ctggtagtgg 120gggacaacgt ttcgaaagga
acgctaatac cgcatacgtc ctacgggaga aagtggggga 180tcttcggacc tcacgctatc
agatgagcct aggtcggatt agctagttgg tgaggtaaag 240gctcaccaag gcgacgatcc
gtaactggtc tgagaggatg atcagtcaca ctggaactga 300gacacggtcc agactcctac
gggaggcagc agtggggaat attggacaat gggcgaaagc 360ctgatccagc catgccgcgt
gtgtgaagaa ggtcttcgga ttgtaaagca ctttaagttg 420ggaggaaggg cagtaagtta
ataccttgct gttttgacgt taccgacaga ataagcaccg 480gctaacttcg tgccagcagc
cgcggtaata cgaagggtgc aagcgttaat cggaattact 540gggcgtaaag cgcgcgtagg
tggttcgtta agttggatgt gaaagccccg ggctcaacct 600gggaactgca tccaaaactg
gcgagctaga gtatggcaga tggtggtgga atttcctgtg 660tagcggtgaa atgcgtacat
ataggaagga acaccagtgg cgaaggcgac cacctgggct 720aatactgaca ctgaggtgcg
aaagcgtggg gagcaaacag gattagatac cctggtagtc 780cacgccctaa acgatgtcga
ctagtcgttc ggagcagcaa tgcactgagt gacgcagcta 840acgcgtgaag tcgaccgcct
ggggagtacg gccgcaaggt taaaactcaa aggaattgac 900ggggacccgc acaagcggtg
gatgatgtgg attaattcga tgcaacgcga aaaaccttac 960ctacccttga catgccagga
accttgccga gaggcgaggg tgccttcggg agcctggaca 1020caggtgctgc atggctgtcg
tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1080agcgcaaccc ttgtcactag
ttgccatcat ttggttgggc actctagtga gactgccggt 1140gacaaaccgg aggaaggtgg
ggatgacgtc aagtcctcat ggcccttatg ggtagggctt 1200cacacgtcat acaatggtcg
gtacagaggg ttgccaagcc gcgaggtgga gccaatccct 1260taaagccgat cgtagtccgg
atcgtagtct gcaactcgac tacgtgaagt cggaatcgct 1320agtaatcgca gatcagcatg
ctgcggtgaa tacgttcccg ggtcttgtac acaccgcccg 1380tcacaccatg ggagtgggtt
tcaccagaag taggtagctt aaccttcggg agggcgctta 1440ccacggtgag attcatgact
ggggtgaagt cgtaacaagg taaccg 1486471442DNAunknownUnknown
clone from enriched environmental sample that by rDNA sequence
analysis has highest identity to Ochrobactrum sp. mp-5 47tggctcagaa
cgaacgctgg cggcaggctt aacacatgca agtcgagcgc cccgcaaggg 60gagcggcaga
cgggtgagta acgcgtggga acgtaccttt tgctacggaa taactcaggg 120aaacttgtgc
taataccgta tgtgcccttc gggggaaaga tttatcggca aaggatcggc 180ccgcgttgga
ttagctagtt ggtgaggtaa aggctcacca aggcgacgat ccatagctgg 240tctgagagga
tgatcagcca cactgggact gagacacggc ccagactcct acgggaggca 300gcagtgggga
atattggaca atgggcgcaa gcctgatcca gccatgccgc gtgagtgatg 360aaggccctag
ggttgtaaag ctctttcacc ggtgaagata atgacggtaa ccggagaaga 420agccccggct
aacttcgtgc cagcagccgc ggtratacga agggggctag cgttgttcgg 480atttactggg
cgtaaagcgc acgtaggcgg acttttaagt caggggtgaa atcccggggc 540tcaaycccgg
aactgccttt gatactggaa gtcttgagta tggtagaggt gagtggaatt 600ccgagtgtag
aggtgaaatt cgtagatatt cggaggaaca ccagtggcga aggcggctca 660ctggaccatt
actgacgctg aggtgcgaaa gcgtggggag caaacaggat tagataccct 720ggtagtccac
gccgtaaacg atgaatgtta gccgttgggg agtttactct tcggtggcgc 780agctaacgca
ttaaacattc cgcctgggga gtacggtcgc aagattaaaa ctcaaaggaa 840ttgacggggg
cccgcacaag cggtggagca tgtggtttaa ttygaagcaa cgcgcagaac 900cttaccagcc
cttgacatac cggtcgcgga cacagagatg tgtctttcag ttcggctgga 960ccggatacag
gtgctgcatg gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1020cgcaacgagc
gcaaccctcg cccttagttg ccagcattta gttgggcact ctaaggggac 1080tgccggtgat
aagccgagag gaaggtgggg atgacgtcaa gtcctcatgg cccttacggg 1140ctgggctaca
cacgtgctac aatggtggtg acagtgggca gcgagcacgc gagtgtgagc 1200taatctccaa
aagccatctc agttcggatt gcactctgca actcgagtgc atgaagttgg 1260aatcgctagt
aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca 1320ccgcccgtca
caccatggga gttggtttta cccgaaggcg ctgtgctaac cgcaaggagg 1380caggcgacca
cggtagggtc agcgactggg gtgaagtcgt aacaaggtaa ccgaagggcg 1440at
1442481478DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Anaeurovorax
sp/EH8A 48tggctcagga tgaacgctgg cggcgtgcct aacacatgca agtcgagcgg
tatatagtgg 60aatgaaactt cggtcgagtg aagctataga gagcggcgga cgggtgagta
acgcgtaggc 120aacctgcccc atacagaggg atagcctcgg gaaaccggga ttaaaacctc
ataacgcgag 180gagttcacat ggactgctcg ccaaagattc atcggtatgg gatgggcctg
cgtctgatta 240gctagttggt gaggtaacgg ctcaccaagg cgacgatcag tatccgacct
gagagggtaa 300tcggccacat tggaactgag acacagtcca aactcctaca ggaggcagca
gtggggaata 360ttgcacaatg ggcgaaagcc tgatgcaaca acgccgcgtg agcgatgaac
gcctttgggt 420cgtaaagctc tgtccttggg gaagaaacaa atgacggtac ccttggaaga
agccccggct 480aactacgtgc cagcagccgc ggtaatacgt agggggcgag cgttatccgg
aattattggg 540cgtaaagagt gcgtacgtgg ctatgtaagc gcgaggtgaa aggcaatagc
ttaactattg 600taagccttgc gaactgtgtg gcttgggtgc aggacaggaa agtggaattc
ctattgtagc 660ggtgaaatgc gtagatatta ggaggaacac cactggcgaa ggcgactttc
tggactgtaa 720ctgacactga ggcacgaaag cgtggggagc aaacaggatt agataccctg
gtagtccacg 780ccgtaaacga tgagcactag gtgtaggggt cgcaagactt cggtgccgca
gttaacgcat 840taagtgctcc gcctggggag tacgcacgca agtgtgaaac tcaaaggaat
tgacggggac 900ccgcacaagc agcggagcat gtggtttaat tcgaagcaac gcgaagaacc
ttatcagggc 960ttgacatccg tatgacagtc cgttaaccgg gacgttcttc ggacagagga
gacaggtggt 1020gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa
cgagcgcaac 1080ccttgtcttt agttgccatc atttggttgg gcactctagt gagactgccg
gtgacaaacc 1140ggaggaaggt ggggatgacg tcaagtcctc atggccctta tgggtagggc
ttcacacgtc 1200atacaatggt cggtacagag ggttgccaag ccgcgaggtg gagccaatcc
cttaaagccg 1260atcgtagtcc ggatcgtagt ctgcaactcg actacgtgaa gtcggaatcg
ctagtaatcg 1320cagatcagca tgctgcggtg aatacgttcc cgggtcttgt acacaccgcc
cgtcacacca 1380tgggagtggg tttcaccaga agtaggtagc ttaaccttcg ggagggcgct
taccacggtg 1440agattcatga ctggggtgaa gtcgtaacaa ggtaaccg
147849883DNAunknownUnknown clone from enriched environmental
sample that by rDNA sequence analysis has highest identity to
Anaerovorax sp. EH8A 49tggctcagga tgaacgctgg cggcgtgcct aacacatgca
agtcgagcgg tatatagtgg 60aacgaaactt cggtcgagtg aagccataga gagcggcgga
cgggtgagta acgcgtaggc 120aacctgcccc atacagaggg atagcctcgg gaaaccggga
ttaaaacctc ataacgcgag 180gagttcacat ggacttctcg ccaaagattc atcggtatgg
gatgggcctg cgtctgatta 240gctagttggt gaggtaacgg ctcaccaagg cgacgatcag
tagccgacct gagagggtaa 300tcggccacat tggaactgag acacggtcca aactcctacg
ggaggcagca gtggggaata 360ttgcacaatg ggcgaaagcc tgatgcagca acgccgcgtg
agcgatgaag gcctttgggt 420cgtaaagctc tgtccttggg gaagaaacaa atgacggtac
ccttggagga agccccggct 480aactacgtgc cagcagccgc ggtaatacgt agggggcgag
cgttatccgg aattattggg 540cgtaaagagt gcgtaggtgg ccatgtaagc gcggggtgaa
aggcaatagc ttaactattg 600taagccttgc gaactgtgtg gcttgagtgc aggagaggaa
agtggaattc ctagtgtagc 660ggtgaaatgc gtagatatta ggaggaacac cagtggcgaa
ggcgactttc tggactgtaa 720ctgacactga ggcacgaaag cgtggggagc aaacaggatt
agataccctg gtagtccacg 780ccgtaaacga tgagcactag gtgtcggggt cgcaagactt
cggtgccgca gttaacgcat 840taagtgctcc gcctggggag tacgcacgca agtgtgaaac
tca 88350521DNAunknownUnknown clone from enriched
environmental sample that by rDNA sequence analysis has highest
identity to Finegoldia magna 50acatgccact gaccgcatca gagatggtgc
tttaccttcg ggtacagtgg acacaggtgg 60tgcatggttg tcgtcagctc gtgtcgtgag
atgttgggtt aagtcccgca acgagcgcaa 120cccctgtttc tagttgccag cattaagttg
ggcactctag agagactgcc gatgacaaat 180cggaggaagg tggggatgac gtcaaatcat
catgcccttt atgccctggg ctacacacgt 240gctacaatgg tcggtacaac gaggagcaaa
ccagcgatgg caagcaaatc tctaaaagcc 300gatcccagtt cggattgcag gctgcaactc
gcctgcatga agtcggagtt gctagtaatc 360gcggatcaga atgtcgcggt gaatgcgttc
ccgggtcttg tacacaccgc ccgtcacacc 420atgggagttg tcaatacccg aagccagtga
gctaaccagt aatggaggca gctgtcgaag 480gtaggggcga tgactggggt gaagtcgtaa
caaggtaacc g 52151888DNAunknownUnknown clone from
enriched environmental sample that by rDNA sequence analysis has
highest identity to Finegoldia magna 51tacgtaagca tttgaaactg
tcagacttga gttaaggaga ggaaagtgga attcctagtg 60tagcggtgaa atgcgtagat
attaggagga ataccagtgg cgaaggcgac tttctggact 120tatactgacg ctgaggaacg
aaagcgtggg gagcaaacag gattagatac cctggtagtc 180cacgccgtaa acgatgagtg
ctaggtgttg ggggtcaaac ctcggtgccg cagctaacgc 240attaagcact ccgcctgggg
agtacgtacg caagtatgaa actcaaagga attgacgggg 300acccgcacaa gcagcggagc
atgtggttta attcgaagca acgcgaagaa ccttaccagg 360gcttgacatg ccgctgaccg
gtgcagagat gcatctttat ccttcggggt acagcggaca 420caggtggtgc atggttgtcg
tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 480agcgcaaccc ttgtctttag
ttgccatcat taagttgggc actctaaaga gactgccgat 540gacaaatcgg aggaaggtgg
ggatgacgtc aaatcatcat gccctttatg tcctgggcta 600cacacgtgct acaatggtcg
gtacaacgag aagcaagtca gcgatggcaa gcaaatctct 660aaaagccgat cccagttcgg
attgcaggct gcaactcgcc tgcatgaagt cggagttgct 720agtaatcgcg gatcagaatg
ccgcggtgaa tgcgttcccg ggtcttgtac acaccgcccg 780tcacaccatg ggagttgtca
atacccgaag ccagtgagct aaccataaaa ggaggcagct 840gtcgaaggta ggggcaatga
ctggggtgaa gtcgtaacaa ggtaaccg 88852888DNAunknownUnknown
clone from enriched environmental sample that by rDNA sequence
analysis has highest identity to Finegoldia magna 52tacgtaagca
tttgaaactg tcagacttga gttaaggaga ggaaagtgga attcctagtg 60tagcggtgaa
atgcgtagat attaggagga ataccagtgg cgaaggcgac tttctggact 120tatactgacg
cggaggaacg aaagcgtggg gagcaaacag gattagatac cctggtagtc 180cacgccgtaa
acgatgagtg ctaggtgttg ggggtcaaac ctcggtgccg cagctaacgc 240attaagcact
ccgcctgggg agtacgtacg caagtatgaa actcaaagga attgacgggg 300acccgcacaa
gcagcggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg 360gcttgacatg
ccgctgaccg gtttagagat agatctttac ccttcggggt acagcggaca 420caggtggtgc
atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 480agcgcaaccc
ttgtctttag ttgccatcat taagttgggc actctaaaga gactgccgat 540gacaaatcgg
aggaaggtgg ggatgacgtc aaatcatcat gccctttatg tcctgggcta 600cacacgtgct
acaatggtcg gtacaacgag aagcaagtca gcgatggcaa gcaaatctct 660aaaagccgat
cccagttcgg attgcaggct gcaactcgcc tgcatggagt cggagttgct 720agtaatcgcg
gatcagaatg ccgcggtgaa tgcgttcccg ggtcttgtac acaccgcccg 780tcacaccatg
ggagttgtca atacccgaag ccagtgagct aaccataaaa gggggcagct 840gtcgaaggta
ggggcaatga ctggggtgaa gtcgtaacaa ggtaaccg
888531502DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Flexistipes
sp. vp180 53tggctcagaa cgaacgctgg cggcgtgctt aacacatgca agtcaaggag
aaagtctctt 60cggaggcgag taaactggcg cacgggtgag taacgcgtga ggaacctgcc
catatgtctg 120ggataacctg ctgaaaagcg ggctaatact ggatatattg tttaccgcat
ggtgaacaag 180gaaagttggt gcaagctaac gcatatggat ggtctcgcgt ctgattagct
agttggtggg 240gtaaaggctc accaaggcga cgatcagtag ccggtctgag agggtggccg
gccacactgg 300gactgagaca cggcccagac tcctacggga ggcagcagtg gggaattttg
cacaatgggg 360gcaaccctga tgcagcgacg ccgcgtgaac gaggaaggcc ttcgggtcgt
aaagttcttt 420cgacggggaa gaaatgttat acgagtaact gcgtataatt tgacggtacc
tgtagaagca 480gccccggcta actccgtgcc agcagccgcg gtaatacgga gggggcgagc
gttgttcgga 540gttactgggc gtaaagcgca cgtaggcggt gcggtaagtc aggggttaaa
ggtcacagct 600caactgtgat aaggcctttg atactatcgt gctagagtgt cagagagggt
agcggaattc 660ccggtgtagc ggtgaaatgc gtatatatcg ggaggaacac cagtggcgaa
gggcggctac 720ctggctgata actgacgctg aggtgcgaga gcgtggggag caaacaggat
tagataccct 780ggtagtccac gctgtaaacg atggacgtta ggtgttgggg gaaccgaccc
cctcagtgcc 840gaagctaacg cgttaaacgt cccgcctggg gagtacggcc gcaaggttga
aactcaaagg 900aattgacggg ggcccgcaca agcggtggag cacgtggttt aattcgatgc
taaccgaaga 960accttacctg ggtttgacat ccctcgaatc ctgtagagat atgggagtgc
ctggcttgcc 1020aggagcgagg agacaggtgc tgcatggctg tcgtcagctc gtgccgtgag
gtgttgggtt 1080aagtcccgca acgagcgcaa cccctatttt tagttgccat cacgttaagg
tgggcactct 1140aaagagaccg ccggggataa cccggaggaa ggtggggatg acgtcaagtc
atcatggccc 1200ttatgtccag ggctacacac gtgctacaat ggtgcataca gagggcagcg
agacagcgat 1260gttaagcgaa tcccttaaag tgtacctcag ttcggattgc agtctgcaac
tcgactgtat 1320gaagccggaa tcgctagtaa tcgcaggtca gcaaaactgc ggtgaatacg
ttcccgggcc 1380ttgtacacac cgcccgtcac accacgggag tcggttgtac ctgaagccgg
tggcccaacc 1440gcaagggggg agccgtctat ggtatggctg gtaactgggg tgaagtcgta
acaaggtaac 1500cg
1502541499DNAunknownUnknown clone from enriched environmental
sample that by rDNA sequence analysis has highest identity to
Azoarcus sp. EH11 54agagtttgat tatggctcag attgaacgct ggcggcatgc
tttacacatg caagtcgaac 60ggcagcgggg gcttcggcct gccggcgagt ggcgaacggg
tgagtaatgc atcggaacgt 120gcccatgtcg tgggggataa cgtatcgaaa ggtacgctaa
taccgcatac gtcctgaggg 180agaaagcggg ggatcttcgg acctcgcgcg attggagcgg
ccgatgtcgg attagctagt 240aggtgaggta aaggctcacc taggcgacga tccgtagcgg
gtctgagagg atgatccgcc 300acactgggac tgagacacgg cccagactcc tacgggaggc
agcagtgggg aattttggac 360aatgggcgca agcctgatcc agccatgccg cgtgagtgaa
gaaggccttc gggttgtaaa 420gctctttcgg ccgggaagaa atcgtggtct ctaacatagg
ccatggatga cggtaccgga 480ctaagaagca ccggctaact acgtgccagc agccgcggta
atacgtaggg tgcgagcgtt 540aatcggaatt actgggcgta aagcgtgcgc aggcggtttt
gtaagacaga tgtgaaatcc 600ccgggctcaa cctgggaact gcgtttgtga ctgcaaggct
agagtacggc agaggggggt 660ggaattcctg gtgtagcagt gaaatgcgta gagatcagga
ggaacaccga tggcgaaggc 720agccccctgg gcctgtactg acgctcatgc acgaaagcgt
ggggagcaaa caggattaga 780taccctggta gtccacgccg taaacgatga gtgctaggtg
ttgggggtca aacctcggtg 840ccgcagctaa cgcattaagc actccgcctg gggagtacgt
acgcaagtat gaaactcaaa 900ggaattgacg gggacccgca caagcagcgg agcatgtggt
ttaattcgaa gcaacgcgaa 960gaaccttacc agggcttgac atgccgctga ccggtttaga
gatagacctt tatccttcgg 1020ggtacagcgg acacaggtgg tgcatggttg tcgtcagctc
gtgtcgtgag atgttgggtt 1080aagtcccgca acgagcgcaa cccttgtcac tagttgccag
catttagttg ggcactctgg 1140tgagactgcc ggtgacaaac cggaggaagg tggggatgac
gtcaagtcct catggccctt 1200atgggtaggg cttcacacgt catacaatgg tcggtacaga
gggttgccaa gccgcgaggt 1260ggagccaatc ccttaaagcc gaccgtagtc cggatcgtag
tctgcaactc gactacgtga 1320agtcggaatc gctagtaatc gcagatcagc atgctgcggt
gaatacgttc ccgggtcttg 1380tacacaccgc ccgtcacacc atgggagtgg gtttcaccag
aagtaggtag cttaaccttc 1440gggagggcgc ttaccacggt gagattcatg actggggtga
agtcgtaaca aggtaaccg 1499551475DNAunknownUnknown clone from enriched
environmental sample that by rDNA sequence analysis has highest
identity to Clostridium chartatabidium 55tggctcagga cgaacgctgg
cggcgtgcct aacacatgca agtcgagcgg agaatgcaga 60aatgtttaca tggaagtatt
cttagcggcg gacgggtgag taacacgtgg gtaacctgcc 120tcaaagtggg ggatagcctt
ccgaaaggaa gattaatacc gcataagcct acagtgccgc 180atggcacagc aggaaaagga
gcaatccgct ttgagatgga cccgcggcgc attagctagt 240tggtgaggta acggctcacc
aaggcgacga tgcgtagccg acctgagagg gtgatcggcc 300acattggaac tgagacacgg
tccagactcc tacgggaggc agcagtgggg aatattgcac 360aatgggcgaa agcctgatgc
agcaacgccg cgtgagtgat gaaggccttc gggtcgtaaa 420gctctttgat cagggatgat
aatgacagta cctgaaaaac aagccacggc taactacgtg 480ccagcagccg cggtaatacg
taggtggcga gcgttgtccg gaattactgg gcgtaaagga 540tgcgtaggtg gatacttaag
tgggatgtga aatccccggg ctcaacccgg gaactgcatt 600ccaaactggg tatctagagt
gcaggagagg aaagcggaat tcctagtgta gcggtgaaat 660gcgtagagat caggaggaac
accgatggcg aaggcagccc cctgggcctg tactgacgct 720catgcacgaa agcgtgggga
gcaaacaggg atagataccc tggtagtcca cgccctaaac 780gatgtcgaat aagtcgttcc
gaccagcaat gcactgagtg acgcagctaa cgcgtgaagt 840cgaccgcctg gggagtacgg
ccgcaaggtt aaaactcaaa ggaattgacg gggacccgca 900caagcggtgg atgatgtgga
ttaattcgat gcaacgcgaa aaaccttacc tacccttgac 960atgccaggaa ccttgccgag
aggcgagggt gccttcggga gcctggacac aggtgctgca 1020tggctgtcgt cagctcgtgt
cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1080tgtcactagt tgccatcatt
tggttgggca ctctagtgag actgccggtg acaaaccgga 1140ggaaggtggg gatgacgtca
agtcctcatg gcccttatgg gtagggcttc acacgtcata 1200caatggtcgg tacagagggt
tgccaagccg cgaggtggag ccaatccctt aaagccgatc 1260gtagtccgga tcgtagtctg
caactcgact acgtgaagtc ggaatcgcta gtaatcgcag 1320atcagcatgc tgcggtgaat
acgttcccgg gtcttgtaca caccgcccgt cacaccatgg 1380gagtgggctt caccagaagt
aggtagctta accttcggga gggcgcttac cacggtgaga 1440ttcatgactg gggtgaagtc
gtaacaaggt aaccg 1475561492DNAunknownUnknown
clone from enriched environmental sample that by rDNA sequence
analysis has highest identity to Deferribacter desulfuricans
56tggctcagaa cgaacgctgg cggcgtgctt aacacatgca agtcaaggag aaagtctctt
60cggaggcgag taaactggcg cacgggtgag taacgcgtga ggaacctgcc catatgtctg
120ggataacctg ctgaaaagcg ggctaatact ggatatattg tttaccgcat ggtgaacaag
180gaaagttggt gcaagctaac gcatatggat ggtctcgcgt ctgattagct agttggtggg
240gtaaaggctc accaaggcaa cgatcagtag ccggtctgag agggtggccg gccacactgg
300gactgagaca cggcccagac tcctacggga ggcagcagtg gggaattttg cacaatgggg
360gcaaccctga tgcagcgacg ccgcgtgaac gaggaaggcc ttcgggtcgt aaagttcttt
420cgacggggaa gaaatgttat acgagtaact gcgtataatt tgacggtacc cgtagaagca
480gccccggcta actccgtgcc agcagccgcg gtaatacgga gggggcgagc gttgttcgga
540gttactgggc gtaaagcgca cgtacgcggt gcggtaagtc aggggttaaa ggtcacagct
600caactgtgat aaggcctttg atactatcgt gctagagtgt cagagagggt agcggaattc
660ccggtgtagc ggtgaaatgc gtagatatcg ggaggaacac cagtagcgaa ggcggctacc
720tggctgataa ctgacgctga ggtgcgagag cgtggggagc aaacaggatt agataccctg
780gtagtccacg ccctaaacga tgtcgactag tcgttcggag cagcaatgca ctgagtgacg
840cagctaacgc gtgaagtcga ccgcctgggg agtacggccg caaggttaaa actcaaagga
900attgacgggg acccgcacaa gcggtggatg atgtggatta attcgatgca acgcgaaaaa
960ccttacctac ccttgacatg ccaggaacct tgccgagagg cgagggtgcc ttcgggagcc
1020tggacacagg tgctgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc
1080gcaacgagcg caacccttgt cactagttgc catcatttgg ttgggcactc tagtgagact
1140gccggtgaca aaccggagga aggtggggat gacgtcaagt cctcatggcc cttatgggta
1200gggcttcaca cgtcatacaa tggtcggtac agagggttgc caagccgcga ggtggagcca
1260atcccttaaa gccgatcgta gtccggatcg tagtctgcaa ctcgactacg tgaagtcgga
1320atcgctagta atcgcagatc agcatgctgc ggtgaatacg ttcccgggtc ttgtacacac
1380cgcccgtcac accatgggag tgggtttcac cagaagtagg tagcttaacc ttcgggaggg
1440cgcttaccac ggtgagattc atgactgggg tgaagtcgta acaaggtaac cg
1492571519DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Azotobacter
beijerinckii 57tggctcagaa cgaacgctgg cggcgtgctt aacacatgca agtcaagggg
aaagtctctt 60cggaggcgag taaactggcg cacgggtgag taacgcgtga ggaacctgcc
catatgtctg 120ggataacctg ctgaaaagcg ggctaatact ggatatattg tttaccgcat
ggtgaacaag 180gaaagttggt gcaagctaac gcatatggat ggtctcgcgt ctgattagct
agttggtggg 240gtaaaggctc accaaggcaa cgatccgtag cgggtctgag aggatggtcc
gccacactgg 300gactgagaca cggcccagac tcctacggga ggcagcagtg gggaattttg
gacaatgggc 360gcaagcctga tccagccatg ccgcgtgagt gaagaaggcc ttcgggttgt
aaagctcttt 420cggccgggaa gaaatcgtgg tctctaacat aggccatgga tgacggtacc
ggactaagaa 480gcaccggcta actacgtgcc agcagccgcg gtaatacgta gggtgcgagc
gttaatcgga 540attactgggc gtaaagcgtg cgcaggcggt tttgtaagac agatgtgaaa
tccccgggct 600caacctggga actgcgtttg tgactgcaag gctagagtac ggcagagggg
ggtggaattc 660ctggtgtagc agtgaaatgc gtagagatca ggaggaacac cgatggcgaa
ggcagccccc 720tgggcctgta ctgacgctca tgcacgaaag cgtggggagc aaacaggatt
agataccctg 780gtagtccacg ccctaaacga tgtcgactag tcgttcggag cagcaatgca
ctgagtgacg 840cagctaacgc gtgaagtcga ccgcctgggg agtacggccg caaggttaaa
actcaaagga 900attgacgggg acccgcacaa gcggtggatg atgtggatta attcgatgca
acgcgaaaaa 960ccttacctac ccttgacatg ccaggaacct tgccgagagg cgagggtgcc
ttcgggagcc 1020tggacacagg tgctgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg
gttaagtccc 1080gcaacgagcg caacccttgt cactagttgc catcatttgg ttgggcactc
tagtgagact 1140gccggtgaca aaccggagga aggtggggat gacgtcaagt cctcatggcc
cttatgggta 1200gggcttcaca cgtcatacaa tggtcggtac agagggttgc caagccgcga
ggtggagcca 1260atcccttaaa gccgatcgta gtccggatcg tagtctgcaa ctcgactacg
tgaagtcgga 1320atcgctagta atcgcagatc agcatgctgc ggtaaatacg ttcccgggtc
ttgtacacac 1380cgcccgtcac accatgggag tgggtttcac cagaagtagg tagcttaacc
ttcgggaggg 1440cgcttaccac ggtgagattc atgactgggg tgaagtcgta acaaggtaac
cgaagggcga 1500atcaatcgcc tatgactgg
151958373DNAunknownUnknown clone from enriched environmental
sample that by rDNA sequence analysis has highest identity to
Flexistipes sp. vp180 58tggctcagaa cgaacgctgg cggcgtgctt aacacatgca
agtcaaggag aaaatctctt 60cgggggcgag taaactggcg cacgggtgag taacgcgtga
ggaacctgcc catatgtctg 120ggataacctg ctgaaaagcg ggctaatact ggatatattg
tttaccgcat ggtggacaag 180gaaagttggt gtaagctaac gcatatggat ggtctcgcgt
ctgattagct agttggtggg 240gtaaaggctc accaaggcga cgatcagtag ccggtctgag
agggtggccg gccacactgg 300gactgagaca cggcccatac tcctacggga ggcagcagtg
gggaattttg cacaatgggg 360gcaaccctga tgc
373591431DNAunknownUnknown clone from enriched
environmental sample that by rDNA sequence analysis has highest
identity to Ochrobactrum lupini 59tggctcagaa cgaacgctgg cggcaggctt
aacacatgca agtcgagcgc cccgcaagga 60gagcggcaga cgggtgagta acgcgtggga
acgtaccttt tgctacggaa taactcaggg 120aaacttgtgc taataccgta tgtgcccttc
gggggaaaga tttatcggca aaggatcggc 180ccgcgttgga ttagctagtt ggtgaggtaa
aggctcacca aggcgacgat ccatagctgg 240tctgagagga tgatcagcca cactgggact
gagacacggc ccagactcct acgggaggca 300gcagtgggga atattggaca atgggcgcaa
gcctgatcca gccatgccgc gtgagtgatg 360aaggccctag ggttgtaaag ctctttcacc
ggtgaagata atgacggtaa ccggagaaga 420agccccggct aacttcgtgc cagcagccgc
ggtaatacga agggggctag cgttgttcgg 480atttactggg cgtaaagcgc acgtaggcgg
acttttaagt caggggtgaa atcccggggc 540tcaaccccgg aactgccttt gatactggaa
gtcttgagta tggtagaggt gagtggaatt 600ccgagtgtag aggtgaaatt cgtagatatt
cggaggaaca ccagtggcga aggcggctca 660ctggaccatt actgacgctg aggtgcgaaa
gcgtggggag caaacaggat tagataccct 720ggtagtccac gccgtaaacg atgaatgtta
gccgttgggg agtttactct tcggtggcgc 780agctaacgca ttaaacattc cgcctgggga
gtacggtcgc aagattaaaa ctcaaaggaa 840ttgacgggga cccgcacaag cggtggatga
tgtggattaa ttcgatgcaa cgcgaaaaac 900cttacctacc cttgacatgc caggaacctt
gccgagaggc gagggtgcct tcgggagcct 960ggacacaggt gctgcatggc tgtcgtcagc
tcgtgtcgtg agatgttggg ttaagtcccg 1020caacgagcgc aacccttgcc actagttgcc
atcatttggt tgggcactct agtgagactg 1080ccggtgacaa accggaggaa ggtggggatg
acgtcaagtc ctcatggccc ttatgggtag 1140ggcttcacac gtcatacaat ggtcggtaca
gagggttgcc aagccgcgag gtggagccaa 1200tcccttaaag ccgatcgtag tccggatcgt
agtctgcaac tcgactacgt gaagtcggaa 1260tcgctagtaa tcgcagatca gcatgctgcg
gtgaatrcgt tcccgggtct tgtacacacc 1320gcccgtcaca ccatgggagt gggtttcacc
agaagtaggt agcttaacct tcgggagggc 1380acttaccacg gtgagattca tgactggggt
gaagtcgtaa caaggtaacc g 1431601454DNAunknownUnknown clone from
enriched environmental sample that by rDNA sequence analysis has
highest identity to Pseudomonas pseudoalcligenes 60tggctcagat
tgaacgctgg cggcaggcct aacacatgca agtcgagcgg atgagtggag 60cttgctccat
gattcagcgg cggacgggtg agtaatgcct aggaatctgc ctggtagtgg 120gggacaacgt
ttcgaaagga acgctaatac cgcatacgtc ctacgggaga aagtggggga 180tcttcggacc
tcacgctatc agatgagcct aggtcggatt agctagttgg cgaggtaaag 240gctcaccaag
gcgacgatcc gtaactggtc tgagaggatg atcagtcaca ctggaactga 300gacacggtcc
agactcctac gggaggcagc agtggggaat attggacaat gggcgaaagc 360ctgatccagc
catgccgcgt gtgtgaagaa ggtcttcgga ttgtaaagca ctttaagttg 420ggaggaaggg
cagtaagtta ataccttgct gttttgacgt taccgacaga ataagcaccg 480gctaacttcg
tgccagcagc cgcggtaata cgaagggtgc aagcgttaat cggaattact 540gggcgtaaag
cgcgcgtagg tggttcgtta agttggatgt gaaagccccg ggctcaacct 600gggaactgca
tccaaaactg gcgagctaag ttatggcaga ggggggtgga aatttcctgt 660gtagcggtga
aatgggtaga tataggaagg aacaccagtg gcgaaggcga ccacctgggc 720taatactgac
actgaggtgc gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 780ccacgccgta
aacgatgtcg actagccgtt gggatccttg agatcttagt ggcgcagcta 840acgcattaag
tcgaccgcct ggggagtacg gccgcaaggt taaaactcaa atgaattgac 900gggggcccgc
acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac 960caggccttga
catgctgaga acctgccaga gatggcgggg tgccttcggg aactcagaca 1020caggtgctgc
atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgtaacg 1080agcgcaaccc
ttgtccttag ttaccagcac gttatggtgg gcactctaag gagactgccg 1140gtgacaaacc
ggaggaaggt ggggatgacg tcaagtcatc atggccctta cggcctgggc 1200tacacacgtg
ctacaatggt cggtacaaag ggttgccaag ccgcgaggtg gagctaatcc 1260cataaaaccg
atcgtagtcc ggatcgcagt ctgcaactcg actgcgtgaa gtcggaatcg 1320ctagtaatcg
tgaatcagaa tgtcacggtg aatacgttcc cgggccttgt acacaccgcc 1380cgtcacacca
tgggagtggg ttgctccaga agtagctagt ctaaccttcg gggggacggt 1440taccacggag
tgat
145461854DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Pseudomonas
purida 61tgggaactgc atccaaaact ggcgagctag agtatggcag agggtggtgg
aatttcctgt 60gtagcggtga aatgcgtaga tataggaagg aacaccagtg gcgaaggcga
ccacctgggc 120taatactgac actgaggtgc gaaagcgtgg agagcaaaca ggattagata
ccctggtagt 180ccacgccgta aacgatgtcg actagccgtt gggatccttg agatcttagt
ggcgcagcta 240acgcattaag tcgaccgcct ggggagtacg gccgcaaggt taaaactcaa
atgaattgac 300gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga
agaaccttac 360caggccttga catgcagaga actttccaga gatggattgg tgccttcggg
agctctgaca 420caggtgctgc atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag
tcccgtaacg 480agcgcaaccc ttgtccttag ttaccagcac gttaaggtgg gcactctaag
gagactgccg 540gtgacaaacc ggaggaaggt ggggatgacg tcaagtcatc atggccctta
cggcctgggc 600tacacacgtg ctacaatggt cggtacaaag ggttgccaag ccgcgaggtg
gagctaatcc 660cataaaaccg atcgtagtcc ggatcgcagt ctgcaactcg actgcgtgaa
gtcggaatcg 720ctagtaatcg tgaatcagaa tgtcacggtg aatacgttcc cgggccttgt
acacaccgcc 780cgtcacacca tgggagtggg ttgctccaga agtagctagt ctaaccttcg
gggggacggt 840taccacggag tgat
85462854DNAunknownUnknown clone from enriched environmental
sample that by rDNA sequence analysis has highest identity to
Pseudomonas pseudoalcligenes 62tgggaactgc atccaaaact ggcgagctag
agtatggcag agggtggtgg aatttcctgt 60gtagcggtga aatgcgtaga tataggaagg
aacaccagtg gcgaaggcga ccacctgggc 120taatactgac actgaggtgc gaaagcgtgg
agagcaaaca ggattagata ccctggtagt 180ccacgccgta aacgatgtcg actagccgtt
gggatccttg agatcttagt ggcgcagcta 240acgcattaag tcgaccgcct ggggagtacg
gccgcaaggt taaaactcaa atgaattgac 300gggggcccgc acaagcggtg gagcatgtgg
tttaattcga agcaacgcga agaaccttac 360caggccttga catgcagaga actttccaga
gatggattgg tgccttcggg agctctgaca 420caggtgctgc atggctgtcg tcagctcgtg
tcgtgagatg ttgggttaag tcccgtaacg 480agcgcaaccc ttgtccttag ttaccagcac
gttaaggtgg gcactctaag gagactgccg 540gtgacaaacc ggaggaaggt ggggatgacg
tcaagtcatc atggccctta cggcctgggc 600tacacacgtg ctacaatggt cggtacaaag
ggttgccaag ccgcgaggtg gagctaatcc 660cataaaaccg atcgtagtcc ggatcgcagt
ctgcaactcg actgcgtgaa gtcggaatcg 720ctagtaatcg tgaatcagaa tgtcacggtg
aatacgttcc cgggccttgt acacaccgcc 780cgtcacacca tgggagtggg ttgctccaga
agtagctagt ctaaccttcg gggggacggt 840taccacggag tgat
854631448DNAunknownUnknown clone from
enriched environmental sample that by rDNA sequence analysis has
highest identity to Clostridium chartatabidium 63tggctcagga
cgaacgctgg cggcgtgcct aacacatgca agtcgagcgg agaatgcaga 60aatgtttaca
tggaagcgtt cttagcggcg gacgggtgag taacacgtgg gtaacctgcc 120tcaaagtggg
ggatagcctt ccgaaaggaa gattaatacc gcataagcct acagtgccgc 180atggcacagc
aggaaaagga gcaatccgct ttgagatgga cccgcggcgc attagctagt 240tggtgaggta
acggctcacc aaggcgacga tgcgtagccg acctgagagg gtgatcggcc 300acattggaac
tgagacacgg tccagactcc tacgggaggc agcagtgggg aatattgcac 360aatgggcgaa
agcctgatgc agcaacgccg cgtgagtgat gaaggccttc gggtcgtaaa 420gctctttgat
cagggatgat aatgacagta cctgaaaaac aagccacggc taactacgtg 480ccagcagccg
cggtaatacg taggtggcga gcgttgtccg gaattactgg gcgtaaagga 540tgcgtaggtg
gatacttaag tgggatgtga aatccccggg ctcaacccgg gaactgcatt 600ccaaactggg
tatctagagt gcaggagagg aaagcggaat tcctagtgta gcggtgaaat 660gcgtagatat
taggaggaac accagtggcg aaggcggctt tctggactgt aactgacact 720gaggcatgaa
agcgtgggta gcaaacagga ttagataccc tggtagtcca cgccgtaaac 780gatgggtact
aggtgtagga ggtatcgacc ccttctgtgc cgtcgttaac acaataagta 840ccccgcctgg
ggagtacggt cgcaagacta aaactcaaag gaattgacgg gggcccgcac 900aagcagcgga
gcatgtggtt taattcgaag caacgcgaag aaccttacct agacttgaca 960tctcctgaat
tacccttaac cggggaagcc cttcggggca ggaagacagg tggtgcatgg 1020ttgtcgtcag
ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttat 1080ttttagttgc
taccatttgg ttgagcactc taaagagact gcccgggtta accgggagga 1140aggtggggat
gacgtcaaat catcatgccc cttatgtcta gggctacaca cgtgctacaa 1200tggtgagaac
aaagagacgc gagaccgcga ggtggagcaa atctcataaa actcatccca 1260gttcggattg
caggctgaaa ctcgcctgca tgaagccgga gttgctagta atcgcgaatc 1320agcatgtcgc
ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accatgagag 1380ttggcaatac
ccgaagtccg tggggcaacc agttaatgga gccagcggcc gaaggtaggg 1440tcagcgat
1448641486DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Finegoldia
magna 64tggctcagga cgaacgctgg cggcgtgcct aacacatgca agtcgagcga agtgactcgg
60agagaagttt tcgaatggat cgaagagtca tcttagcggc ggacgggtga gtaacgcgtg
120agaaacctgc ctttcacaaa gggatagcct cgggaaactg ggattaatac cttatgaaac
180tgaattaccg catggtagat cagtcaaagc gaataagcgg tgaaagatgg tctcgcgtcc
240tattagctag ttggtgaggt aacggctcac caaggcttcg ataggtagcc ggcctgagag
300ggtgaacggc cacactggaa ctgagacacg gtccagactc ctacgggagg cagcagtggg
360gaatattgca caatggagga aactctgatg cagcgacgcc gcgtgaatga tgaaggcctt
420cgggttgtaa agttctgtcc ttggggaaga taatgacggt acccaaggag gaagccccgg
480ctaactacgt gccagcagcc gcggtaatac gtagggggcg agcgttgtcc ggaattattg
540ggcgtaaagg gttcgcaggc ggtctgataa gtcagatgtg aaaggcgtag gctcaaccta
600cgtaagcatt tgaaactgtc agacttgagt taaggagagg aaagtggaat tcctagtgta
660gcggtgaaat gcgtagatat taagaggaat accagtggcg aaggcgactt tctggactta
720tactgacgct taggaacgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca
780cgccgtaaac gatgagtgct aggtgttggg ggtcaaacct cggtgccgca gctaacgcat
840taagcactcc gcctggggag tacgtacgca agtatgaaac tcaaaggaat tgacggggac
900ccgcacaagc agcggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccagggc
960ttgacatgcc gctgaccggt ttagagatag acctttatcc ttcggggtac agcggacaca
1020ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag
1080cgcaaccctt gtctttagtt gccatcatta agttgggcac tctaaagaga ctgccgatga
1140caaatcggag gaaggtgggg gtgacgtcaa atcatcatgc cctttatgtc ctgggctaca
1200cacgtgctac aatggtcggt acaacgagaa gcaagccagc gatggcaagc aaatctctaa
1260aagccgatcc cagttcggat tgcaggctgc aactcgcctg catgaagtcg gagttgctag
1320taatcgcgga tcagaatgcc gcggtgaatg cgttcccggg tcttgtacac accgcccgtc
1380acaccatggg agttgtcaat acccgaagcc agtgagctaa ccataaaagg gggcagctgt
1440cgaaggtagg ggcaatgact ggggtgaagt cgtaacaagg taaccg
148665881DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Finegoldia
magna 65tggctcagga cgaacgctgg cggcgtgcct aacacatgca agtcgagcga agtgactcgg
60agagaaattt tcggatggat cgaagagtca tcttagcggc ggacgggtga gtaacgcgtg
120agaaacctgc ctttcacaaa gggatagcct cgggaaactg ggattaatac cttatgaaac
180tgaattaccg catggtagat cagtcaaagc gaataagcgg tgaaagatgg tctcgcgtcc
240tattagctag ttggtgaggt aacggctcac caaggcttcg ataggtagcc ggcctgagag
300ggtgaacggc cacactggaa ctgagacacg gtccagactc ctacgggagg cagcagtggg
360gaatattgca caatggagga aactctgatg cagcgacgcc gcgtgaatga tgaaggcctt
420cgggttgtaa agttctgtcc ttggggaaga taatgacggt acccaaggag gaagccccgg
480ctaactacgt gccagcagcc gcggtaatac gtagggggcg agcgttgtcc ggaattattg
540ggcgtaaagg gttcgcaggc ggtctgataa gtcagatgtg aaaggcgtag gctcaaccta
600cgtaagcatt tgaaactgtc agacttgagt taaggagagg aaagtggaat tcctagtgta
660gcggtgaaat gcgtagatat taggaggaat accagtggcg aaggcgactt tctggactta
720tactgacgct gaggaacgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca
780cgccgtaaac gatgagtgct aggtgttggg ggtcaaacct cggtgccgca gctaacgcat
840taagcactcc gcctggggag tacgtacgca agtatgaaac t
881661393DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Finegoldia
magna 66tggctcagga cgaacgctgg cggcgtgcct aacacatgca agtcgagcga agtgactctg
60agagaaattt tcggatggat cgaagagtca tcttagcggc ggacgggtga gtaacgcgtg
120agaaacctgc ctttcacaaa gggatagcct cgggaaactg ggattaatac cttatgaaac
180tgaattaccg catggtagat cagtcaaagc gaataagcgg tgaaagatgg tctcgcgtcc
240tattagctag ttggtgaggt aacggctcac caaggcttcg ataggtagcc ggcctgagag
300ggtgaacggc cacactggaa ctgagacacg gtccagactc ctacgggagg cagcagtggg
360gaatattgca caatggagga aactctgatg cagcgacgcc gcgtgaatga tgaaggcctt
420cgggttgtaa agttctgtcc ttggggaaga taatgacggt acccaaggag gaagccccgg
480ctaactacgt gccagcagcc gcggtaatac gtagggggcg agcgttgtcc ggaattattg
540ggcgtaaagg gttcgcaggc ggtctgataa gtcagatgtg aaaggcgtag gctcaaccta
600cgtaagcatt tgaaactgtc agacttgagt taaggagagg aaagtggaat tcctagtgta
660gcggtgaaat gcgcagatat taggaggaat accagtggcg aaggcgactt tctggactta
720tactgacgct gaggaacgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca
780cgccgtaaac gatgagtgct aggtgttggg ggtcaaacct cggtgccgca gctaacgcat
840taagcactcc gcctggggag tacgtacgca agtatgaaac tcaaaggaat tgacggggac
900ccgcacaagc agcggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccagggc
960ttgacatgcc gctgaccggt ttagagatag atctttatcc ttcggggtac ggcggacaca
1020ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag
1080cgcaaccctt gtctttagtt gccatcatta agttgggcac tctaaagaga ctgccgatga
1140caaatcggag gaaggtgggg atgacgtcaa atcatcatgc cctttatgtc ctgggctaca
1200cacgtgctac aatggtcggt acaacgagaa gcaagtcagc gatggcaagc aaatctctaa
1260aagccgatcc cagttcggat tgcaggctgc aactcgcctg catgaagtcg gagttgctag
1320taatcgcgga tcagaatgcc gcggtgaatg cgttcccggg tcttgtacac accgcccgtc
1380acaccatggg agt
1393671440DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Thauera
aromatica 67tggctcagat tgaacgctgg cggcatgctt tacacatgca agtcgaacgg
cagcgggggc 60ttcggcctgc cggcgagtgg cgaacgggtg agtaatgcat cggaacgtgc
ccatgtcgtg 120ggggataacg tatcgaaagg tacgctaata ccgcatacgt cctgagggag
aaagtggggg 180atcttcggac ctcacgctat cagatgagcc taggtcggat tagctagttg
gcgaggtaaa 240ggctcaccaa ggcgacgatc cgtagcgggt ctgagaggat gatccgccac
actgggactg 300agacacggcc cagactccta cgggaggcag cagtggggaa ttttggacaa
tgggcgcaag 360cctgatccag ccatgccgcg tgagtgaaga aggccttcgg gttgtaaagc
tctttcggcc 420gggaagaaat cgtggtctct aacataggcc atggatgacg gtaccggact
aagaagcacc 480ggctaactac gtgccagcag ccgcggtaat acgtagggtg cgagcgttaa
tcggaattac 540tgggcgtaaa gcgtgcgcag gcggttttgt aagacagatg tgaaatcccc
gggctcaacc 600tgggaactgc gtttgtgact gcaaggctag agtacggcag aagggggtgg
aattcctggt 660gtancantga aatgcgtaaa gatcaagagg aacaccgatg gcgaaagcag
ccccctgggc 720ctgtactgac cctcatgcac gaaagcgtgg ggagcaaaca agattaaata
ccctggtagt 780ccacgcccta aacgatgtcg actagtcgtt tggagcagca atgcactgag
tgacgcagct 840aacgcgtgaa gtcgaccgcc tggggagtac ggccgcaagg ttaaaactca
aaggaattga 900cggggacccg cacaagcggt ggatgatgtg gattaatttg atgcaacgcg
aaaaacctta 960cctacccttg acatgccagg aaccttgccg agaggcgagg gtgccttcgg
gagcctggac 1020acaggtgctg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa
gtcccgcaac 1080gagcgcaacc cttgtcacta gttgccatca tttggttggg cactctagtg
agactgccgg 1140tgacaaaccg gaggaaggtg gggatgacgt caagtcctca tggcccttat
gggtagggct 1200tcacacgtca tacaatggtc ggtacagagg gttgccaagc cgcgaggtgg
agccaatccc 1260ttagagccga tcgtagtccg gatcgtagtc tgcaactcga ctacgtgaag
tcggaatcgc 1320tagtaatcgc agatcagcat gctgcggtga atacgttccc gggtcttgta
cacaccgccc 1380gtcacaccat gggagtgggt ttcaccagaa gtaggtagct taaccttcgg
gagggcgctt 1440681452DNAunknownUnknown clone from enriched
environmental sample that by rDNA sequence analysis has highest
identity to Thauera aromatica 68tggctcagga cgaacgctga cggcgtgcct
aacacatgca agtcgagcgg agaatgcaga 60aatgtttaca tggaagtatt cttagcggcg
gacgggtgag taacacgtgg gtaacctgcc 120tcaaagtggg ggatagcctt ccgaaaggaa
gattaatacc gcataagcct acagtgccgc 180atggcacagc aggaaaagga gcaatccgct
ttgagatgga cccgcggcgc attagctagt 240tggtgaggta acggctcacc aaggcgacga
tgcgtagccg acctgagagg gtgatcggcc 300acattggaac tgagacacgg cccagactcc
tacgggaggc agcagtgggg aattttggac 360aatgggcgca agcctgatcc agccatgccg
cgtgagtgaa gaaggccttc gggttgtaaa 420gctctttcgg ccgggaagaa atcgtggtct
ctaacatagg ccatggatga cggtaccgga 480ctaagaagca ccggctaact acgtgccagc
agccgcggta atacgtaggg tgcgagcgtt 540aatcggaatt actgggcgta aagcgtgcgc
aggcggtttt gtaagacaga tgtgaaatcc 600ccgggctcaa cctgggaact gcgtttgtga
ctgcaaagct agagtacggc agaagggggt 660ggaattcctg gtgtagcagt gaaatgcgta
gagatcagga ggaacaccga tggcgaaggc 720agccccctgg ggcctgtact gacgctcatg
cacgaaagcg gggggagcaa acaggattag 780ataccctggt agtccacgcc ctaaacgatg
tcgactagtc gttcggagca gcaatgcact 840gagtgacgca gctaacgcgt gaagtcgacc
gcctggggag tacggccgca aggttaaaac 900tcaaaggaat tgacggggac ccgcacaagc
ggtggatgat gtggattaat tcgatgcaac 960gcgaaaaacc ttacctaccc ttgacatgcc
aggaaccttg ccgagaggcg agggtgcctt 1020cgggagcctg gacacaggtg ctgcatggct
gtcgtcagct cgtgtcgtga gatgttgggt 1080taagtcccgc aacgagcgca acccttgtca
ctagttgcca tcatttggtt gggcactcta 1140gtgagactgc cggtgacaaa ccggaggaag
gtggggatga cgtcaagtcc tcatggccct 1200tatgggtagg gcttcacacg tcatacaatg
gtcggtacag agggttgcca agccgcgagg 1260tggagccaat cccttaaagc cgatcgtagt
ccggatcgta gtctgcaact cgactacgtg 1320aagtcggaat cgctagtaat cgcagatcag
catgctgcgg tgaatacgtt cccgggtctt 1380gtacacaccg cccgtcacac catgggagtg
ggtttcacca gaagtaggta gcttaacctt 1440cgggagggcg ct
1452691440DNAunknownUnknown clone from
enriched environmental sample that by rDNA sequence analysis has
highest identity to Azoarcus sp. EH21 69tggctcagat tgaacgctgg
cggcatgctt tacacatgca agtcgaacgg cagcgggggc 60ttcggcctgc cggcgagtgg
cgaacgggtg agtaatgcat cggaacgtgc ccatgtcgtg 120ggggataacg tatcgaaagg
tacgctaata ccgcatacgt cctgagggag aaagcggggg 180atcttcggac ctcgcgcgat
tggagcggcc gatgtcggat tagctagtag gtgaggtaaa 240ggctcaccta ggcgacgatc
cgtagcgggt ctgagaggat gatccgccac actgtgactg 300agacacggcc cagactccta
cgggaggcag cagtggggaa ttttggacaa tgggcgcaag 360cctgatccag ccatgccgcg
tgagtgaaga aggccttcgg gttgtaaagc tctttcggcc 420gggaagaaat cgtggtctct
aacataggcc atggatgacg gtaccggact aagaagcacc 480ggctaactac gtgccagcag
ccgcggtaat acgtagggtg cgagcgttaa tcggaattac 540tgggcgtaaa gcgtgcgcag
gcggttttgt aagacagatg tgaaatcccc gggctcaacc 600tgggaactgc gtttgtgact
gcaaggctag agtacggcag aggggggtgg aattcctggt 660gtaacaatga aatgcgtaga
gatcaggagg aacacggatg gcgaaggcag ccccctgggc 720ctgtactgac gctcatgcac
gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 780ccacgccgta aacgatgtcg
actagccgtt gggatccttg agatcttagt ggcgcagcta 840acgcattaag tcgaccgcct
ggggagtacg gccgcaaggt taaaactcaa atgaattgac 900gggggcccgc acaagcggtg
gagcatgtgg tttaattcga agcaacgcga agaaccttac 960caggccttga catgcagaga
actttccaga gatggattgg tgccttcggg agctctgaca 1020caggtgctgc atggctgtcg
tcagctcgtg tcgtgagatg ttgggttaag tcccgtaacg 1080agcgcaaccc ttgtccttag
ttaccagcac gttaaggtgg gcactctaag gagactgccg 1140gtgacaaacc ggaggaaggt
ggggatgacg tcaagtcatc atggccctta cggcctgggc 1200tacacacgtg ctacaatggt
cggtacaaag ggttgccaag ccgcgaggtg gagctaatcc 1260cataaaaccg atcgtagtcc
ggatcgtagt ctgcaactcg actacgtgaa gtcggaatcg 1320ctagtaatcg cagatcagca
tgctgcggtg aatacgttcc cgggtcttgt acacaccgcc 1380cgtcacacca tgggagtggg
tttcaccaga agtaggtagc ttaaccttcg ggagggcgct 1440701438DNAunknownUnknown
clone from enriched environmental sample that by rDNA sequence
analysis has highest identity to Azotobacter beijerinckii
70tggctcagga cgaacgctgg cggcgtgcct aacacatgca agtcgagcga agtgactctg
60agagaaattt tcggatggat cgaagagtca tcttagcggc ggacgggtga gtaacgcgtg
120agaaacctgc ctttcacaaa gggatagcct cgggaaactg ggattaatac cttatgaaac
180tgaattaccg catggtagat cagtcaaagc gaataagcgg tgaaagatgg tctcgcgtcc
240tattagctag ttggtgaggt aacggctcac caaggcttcg ataggtagcc ggcctgagag
300ggtgaacggc cacactggaa ctgagacacg gtccagactc ctacgggagg cagcagtggg
360gaatattgca caatggagga aactctgatg cagcgacgcc gcgtgaatga tgaaggcctt
420cgggttgtaa agttctgtcc ttggggaaga taatgacggt acccaaggag gaagccccgg
480ctaactacgt gccagcagcc gcggtaatac gtagggtgcg agcgttaatc ggaattactg
540ggcgtaaagc gtgcgcaggc ggttttgtaa gacagatgtg aaatccccgg gctcaacctg
600ggaactgcgt ctgtgactgc aaggctagag tacggcagag gggggtggaa ttcctggtgt
660agcagtgaaa tgcgtacaga tcacgaggaa caccgatggc gaaggcagcc ccctggccct
720gtactgacgt tcatgcacaa aagcgtgggg agcaaacagg gattagatac cctggtagtc
780cacgccctaa acgatgttga ttagtcgttc ggagcagcaa tgcactgagt gacgcagcta
840acgcgtgaag tcgaccgcct ggggagtacg gccgcaaggt taaaactcaa aggaattgac
900ggggacccgc acaagcggtg gatgatgtgg attaattcga tgcaacgcga aaaaccttac
960ctacccttga catgccagga accttgccga gaggcgaggg tgccttcggg agcctggaca
1020caggtgctgc atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg
1080agcgcaaccc ttgtcactag ttgccatcat ttggttgggc actctagtga gactgccggt
1140gacaaaccgg aggaaggtgg ggatgacgtc aagtcctcat ggcccttatg ggtagggctt
1200cacacgtcat acaatggtcg gtacagaggg ttgccaagcc gcgaggtgga gccaatccct
1260taaagccgat cgtagtccgg atcgtagtct gcaactcgac tacgtgaagt cggaatcgct
1320agtaatcgca gatcagcatg ctgcggtgaa tacgttcccg ggtcttgtac acaccgcccg
1380tcacaccatg ggagtgggtt tcaccagaag taggtagctt aaccttcggg agggcgct
1438711437DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Azotobacter
beijerinckii 71tggctcagat tgaacgctgg cggcaggcct aacacatgca agtcgagcgg
atgagtggag 60cttgctccat gattcagcgg cggacgggtg agtaatgcct aggaatctgc
ctggtagtgg 120gggacaacgt ttcgaaagga acgctaatac cgcatacgtc ctacgggaga
aagtggggga 180tcttcggacc tcacgctatc agatgagcct aggtcggatt agctagttgg
tgaggtaaag 240gctcaccaag gcgacgatcc gtaactggtc tgagaggatg atcagtcaca
ctggaactga 300gacacggtcc agactcctac gggaggcagc agtggggaat attggacaat
gggcgaaagc 360ctgatccagc catgccgcgt gtgtgaagaa ggtcttcgga ttgtaaagca
ctttaagttg 420ggaggaaggg cagtaagtta ataccttgct gttttgacgt taccgacaga
ataagcaccg 480gctaacttcg tgccagcagc cgcggtaata cgaagggtgc aagcgttaat
cggaattact 540gggcgtaaag cgcgcgtagg tggttcgtta agttggatgt gaaagccccg
ggctcaacct 600gggaactgca tccaaaacta gcgagctaga gtatggcaga gggtggtgga
atttcctgtg 660tagcggtgaa atgcgtagat ataggaagga acaccagtgg cgaaggcgac
cacctggggt 720aatactgaca ctgaagtgcg aaagcggggg gagcaaacag gattagatac
cctggtattc 780cacgccgtaa acgatgtcga ctagccgttg ggatccttga gatcttagtg
gcgcagctaa 840cgcattaagt cgaccgcctg gggagtacgg ccgcaaggtt aaaactcaaa
ggaattgacg 900gggacccgca caagcggtgg atgatgtgga ttaattcgat gcaacgcgaa
aaaccttacc 960tacccttgac atgccaggaa ccttgccgag aggcgagggt gccttcggga
gcctggacac 1020aggtgctgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt
cccgcaacga 1080gcgcaaccct tgtcactagt tgccatcatt tggttgggca ctctagtgag
actgccggtg 1140acaaaccgga ggaaggtggg gatgacgtca agtcctcatg gcccttatgg
gtagggcttc 1200acacgtcata caatggtcgg tacagagggt tgccaagccg cgaggtggag
ccaatccctt 1260aaagccgatc gtagtccgga tcgtagtctg caactcgact acgtgaagtc
ggaatcgcta 1320gtaatcgcag atcagcatgc tgcggtgaat acgttcccgg gtcttgtaca
caccgcccgt 1380cacaccatgg gagtgggttt caccagaagt aggtagctta accttcggga
gggcgct 1437721452DNAunknownUnknown clone from enriched
environmental sample that by rDNA sequence analysis has highest
identity to Azotobacter beijerinckii 72agagtttgat tctggctcag
attgaacgct ggcggcatgc tttacacatg caagtcgaac 60ggcagcgggg gcttcggcct
gccggcgagt ggcgaacggg tgagtaatgc atcggaacgt 120gcccatgtcg tgggggataa
cgtatcgaaa ggtacgctaa taccgcatac gtcctgaggg 180agaaagcggg ggatcttcgg
acctcgcgcg attggagcgg ccgatgtcgg attagctagt 240aggtgaggta aaggctcacc
taggcgacga tccgtagcgg gtctgagagg atgatccgcc 300acactgggac tgagacacgg
cccagactcc tacgggaggc agcagtgggg aattttggac 360aatgggcgca agcctgatcc
agccatgccg cgtgagtgaa gaaggccttc gggttgtaaa 420gctctttcgg ccgggaagaa
atcgtggtct ctaacatagg ccatggttga cgttaccgac 480agattaagca ccggctaact
tcgtgccagc agccgcggta atacgaaggg tgcaagcgtt 540aatcggaatt actgggcgta
aagcgcgcgt aggtggttcg ttaagttgga tgtgaaagcc 600ccgggctcaa cctgggaact
gcatccaaaa ctggcgagct agagtatggc agagggtggt 660ggaatttcct gtgtagcggt
gaaatgcgta catataggaa ggaacaccag tggcgaaggc 720gaccacctgg gctaatactg
acactgaggt gcgaaagcgt ggggagcaaa caggattaga 780taccctggta gtccacgccg
taaacgatgt cgactagccg ttgggatcct tgagatctta 840gtggcgcagc taacgcatta
agtcgaccgc ctggggagta cggccgcaag gttaaaactc 900aaatgaattg acgggggccc
gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc 960gaagaacctt accaggcctt
gacatgctga gaacctgcca gagatggcgg ggtgccttcg 1020ggaactcaga cacaggtgct
gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta 1080agtcccgtaa cgagcgcaac
ccttgtcctt agttaccagc acgttatggt gggcactcta 1140aggagactgc cggtgacaaa
ccggaggaag gtggggatga cgtcaagtca tcatggccct 1200tacggcctgg gctacacacg
tgctacaatg gtcggtacaa agggttgcca agccgcgagg 1260tggagctaat cccataaaac
cgatcgtagt ccggatcgca gtctgcaact cgaccgcgtg 1320aagtcggaat cgctagtaat
cgtgaatcag aatgtcacgg tgaatacgtt cccgggcctt 1380gtacacaccg cccgtcacac
catgggggtg ggttgctcca gaagtagcta gtctaacctt 1440cggggggacg gt
145273893DNAunknownUnknown
clone from enriched environmental sample that by rDNA sequence
analysis has highest identity to Azotobacter beijerinckii
73cggttacctt gttacgactt caccctcctc gccggacgta ccttcggaac cgccccccct
60cgcgggttgg gctggcgact tcgggtaccc ccgactcgga tggtgtgacg ggcggtgtgt
120acaaggcccg ggaacgtatt caccgcgcca tgctgatgcg cgattactag cgattccaac
180ttcatggagt cgggttgcag actccaatcc gtactgggac cggctttaag ggattggctc
240cacctcgcgg cttggcaacc ctctgtaccg accattgtat gacgtgtgaa gccctaccca
300taagggccat gaggacttga cgtcatcccc accttcctcc ggtttgtcac cggcagtctc
360actagagtgc ccaaccaaat gatggcaact agtgacaagg gttgcgctcg ttgcgggact
420taacccaaca tctcacgaca cgagctgacg acagccatgc agcacctgtg tccaggctcc
480cgaaggcacc ctcgcctctc ggcaaggttc ctggcatgtc aagggtaggt aaggtttttc
540gcgttgcatc gaattaatcc acatcatcca ccgcttgtgc gggtccccgt caattccttt
600gagttttaac cttgcggccg tactccccag gcggtcgact tcacgcgtta gctgcgtcac
660tcagtgcatt gctgctccga acgactagtc gacatcgttt agggcgtgga ctaccagggt
720atctaatcct gtttgctccc cacgctttcg tgcatgagcg tcagtacagg cccagggggc
780tgccttcgcc atcggtgttc ctcctgatct ctacgcattt cactgctaca ccaggaattc
840cacccccctc tgccgtactc tagccttgca gtcacaaacg cagttcccag gtt
89374846DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Azotobacter
beijerinckii 74agcggtttgt agacagatgt gaatcccggc tcaactggac tgcgtttgat
gcaagctaga 60gtcgcagagg gggggaatct gtgtagcagt gaatgcgtag agatcagagg
acacgatgcg 120aagcagcccc tgggctgtac tgacgtcatg cacgaaagcg ggggagcaaa
caggattaga 180tacctggtag tcacgcctaa acgatgtcga ctagtcgtcg gagcagcaat
gcactgagtg 240acgcagctaa cgcgtgaagt cgaccgctgg ggagtacggc cgcaaggtta
aaactcaaag 300gaattgacgg ggacccgcac aagcggtgga tgatgtggat taattcgatg
caacgcgaaa 360aaccttacct acccttgaca tgccaggaac cttgccgaga ggcgagggtg
ccttcgggag 420cctggacaca ggtgctgcat ggctgtcgtc agctcgtgtc gtgagatgtt
gggttaagtc 480ccgcaacgag cgcaaccctt gtcactagtt gccatcattt ggttgggcac
tctagtgaga 540ctgccggtga caaaccggag gaaggtgggg atgacgtcaa gtcctcatgg
cccttatggg 600tagggcttca cacgtcatac aatggtcggt acagagggtt gccaagccgc
gaggtggagc 660caatccctta aagccgatcg tagtccggat cgtagtctgc aactcgacta
cgtgaagtcg 720gaatcgctag taatcgcaga tcagcatgct gcggtgaata cgttcccggg
tcttgtacac 780accgcccgtc acaccatggg agtgggtttc accagaagta ggtagcttaa
ccttcgggag 840ggcgct
846751426DNAunknownUnknown clone from enriched environmental
sample that by rDNA sequence analysis has highest identity to
Clostridium chartatabidium 75tggctcagga cgaacgctgg cggcgtgcct aacacatgca
agtcgagcgg agaatgcaga 60aatgtttaca tggaagtatt cttagcggcg gacgggtgag
taacacgtgg gtaacctgcc 120tcgaagtggg ggatagcctt ccgaaaggaa gattaatacc
gcataagcct acagtgccgc 180atggcacagc aggaaaagga gcaatccgct ttgagatgga
cccgcggcgc attagctagt 240tggtgaggta acggctcacc aaggcgacga tgcgtagccg
acctgagagg gtgatcggcc 300acattggaac tgagacacgg tccagactcc tacgggaggc
agcagtgggg aatattgcac 360aatgggcgaa agcctgatgc agcaacgccg cgtgagtgat
gaaggccttc gggtcgtaaa 420gctctttgat cagggatgat aatgacagta cctgaaaaac
aagccacggc taactacgtg 480ccagcagccg cggtaatacg taggtggcga gcgttgtccg
gaattactgg gcgtaaagga 540tgcgtaggtg gatacttaag tgggatgtga aatccccggg
ctcaacccgg gaactgcatt 600ccaaactggg tatctagagt gcaggagagg aaagcggaat
tcctagtgta gcggtgaaat 660gcgtagatat taggaggaac accagtggcg aaggcggctt
tctggactgt aactgacact 720gaggcatgaa agcgtgggta gcaaacagga ttagataccc
tggtagtcca cgccgtaaac 780gatgggtact aggtgtagga ggtatcgacc ccttctgtgc
cgttgttaac acaataagta 840ccccgcctgg ggagtacggt cgcaagacta aaactcaaag
gaattgacgg gggcccgcac 900aagcagcgga gcatgtggtt taattagaag caacgcgaaa
aaccttacct acccttgaca 960tgccaggaac cttgccgaga ggcgagggtg ccttcgggag
cctggacaca ggtgctgcat 1020ggctgtagtc agctcgtgtc gtgagatgtt gggttaagtc
ccgcaacgag cgcaaccctt 1080gtcactagtt gccatcattt ggttgggcac tctagtgaga
ctgccggtga caaaccggag 1140gaaggtgggg atgacgtcaa gtcctcatgg cccttatggg
tagggcttca cacgtcatac 1200aatggtcggt acagagggtt gccaagtcgt gaggtggagc
caatccctta aagccgatcg 1260tagtccggat cgtagtctgc aactcgacta cgtgaagtcg
gaatcgctag taatcgcaga 1320tcagcatggt gcggtgaata cgttcccggg tcttgtacac
accgcccgtc acaccatggg 1380agtgggtttc accagaagta ggtagcttaa ccttcgggag
ggcgct 1426761420DNAunknownUnknown clone from enriched
environmental sample that by rDNA sequence analysis has highest
identity to Clostridium aceticum 76tggctcagat tgaacgctgg cggcatgctt
tacacatgca agtcgaacgg cagcgggggc 60ttcggcctgc cggcgagtgg cgaacgggtg
agtaatgcat cggaacgtgc ccatgtcgtg 120ggggataacg tatcgaaagg tacgctaata
ccgcatacgt cctgagggag aaagcggggg 180atcttcggac ctcgcgcgat tggagcggcc
gatgtcggat tagctagtag gtgaggtaaa 240ggctaaccta ggcgacgatc cgtagcgggt
ctgagagggt gaacggccac actggaactg 300agacacggtc cagactccta cgggaggcag
cagtggggaa tattgcacaa tggaggaaac 360tctgatgcag cgacgccgcg tgaatgatga
aggccttcgg gttgtaaagt tctgtccttg 420gggaagataa tgacggtacc caaggaggaa
gccccggcta actacgtgcc agcagccgcg 480gtaatacgta gggggcgagc gttgtccgga
attattgggc gtaaagggtt cgcaggcggt 540ctgataagtc agatgtgaaa ggcgtaggct
caacctacgt aagcatttga aactgtcaga 600cttgagttaa ggagaggaaa gtggaattcc
tagtgtagcg gtgaaatgcg tagatattag 660gaggaatacc agtggcgaag gcgactttct
ggacttatac tgacgctgag gaacgaaagc 720gtggggagca aacaggatta gataccctgg
tagtccacgc cgtaaacgat gagtgctagg 780tgttgggggt caaacctcgg tgccgcagct
aacgcattaa gcactccgcc tggggagtac 840gtacgcaagt atgaaactca aaggaattga
cggggacccg cacaagcagc ggagcatgtg 900gtttaattcg aagcaacgcg aagaacctta
ccagggcttg acatgccgct gaccggttta 960gagatagatc tttacccttc ggggtacagc
ggacacaggt ggtgcatggt tgtcgtcagc 1020tcgtgtcgtg agatgttggg ttaagtcccg
caacgagcgc aacccttgtc tttagttgcc 1080atcattaagt tgggcactct aaagagactg
ccgatgacaa atcggaggaa ggtggggatg 1140acgtcaaatc atcatgccct ttatgtcctg
ggctacacac gtgctacaat ggtcggtaca 1200acgagaagca agtcagcgat ggcaagcaaa
tctctaaaag ccgatcccag ttcggattgc 1260aggctgcaac tcgcctgcat gaagtcggag
ttgctagtaa tcgcggatca gaatgccgcg 1320gtgaatgcgt tcccgggtct tgtacacacc
gcccgtcaca ccatgggagt tgtcaatacc 1380cgaagccagt gagctaacca taaaaggagg
cagctgtcga 1420771430DNAunknownUnknown clone from
enriched environmental sample that by rDNA sequence analysis has
highest identity to Deferribacter desulfuricans 77agagtttgat
tatggctcag aacgaacgct ggcggcgtgc ttaacacatg caagtcaagg 60agaaagtctc
ttcgggggcg agtaaactgg cgcacgggtg agtaacgcgt gaggaacctg 120cccatatgtc
tgggataacc tgctgaaaag cgggctaata ctggatatat tgtttaccgc 180atggtggaca
aggaaagttg gtgtaagcta acgcatatgg atggtctcgc gtctgattag 240ctagttggtg
gggtaaaggc tcaccaaggc aacgatcagt agcgggtctg agaggatgat 300ccgccacact
gggactgaga cacggcccag actcctacgg gaggcagcag tggggaatat 360tgcacaatgg
gcgaaagcct gatgcagcaa cgccgcgtga gtgatgaagg ccttcgggtc 420gtaaagctct
ttgatcaggg atgataatga cagtacctga aaaacaagcc acggctaact 480acgtgccagc
agccgcggta atacgtatgt ggcgagcgtt gtccggaatt attgggcgta 540aagggttcgc
aggcggtctg ataagtcaga tgtgaaaggc gtaggctcaa cctacgtaag 600catttgaaac
tgtcagactt gagttaagga gaggaaagtg gaattcctag tgtagcggtg 660aaatgcgtag
atattaggag gaataccagt ggcgaaggcg actttctgga cttatactga 720cgctgaggaa
cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccgt 780aaacgatgag
tgctaggtgt tgggggtcaa acctcggtgc cgcagctaac gcattaagca 840ctccgcctgg
ggagtacgta cgcaagtatg aaactcaaag gaattgacgg ggacccgcac 900aagcagcgga
gcatgtggtt taattcgaag caacgcgaag aaccttacca gggcttgaca 960tgccgctgac
cggtttagag atagatcttt acccttcggg gtacggcgga cacaggtggt 1020gcatggttgt
cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080ccttattttt
agttgctacc attcagttga gcactctaaa gagactgccc gggttaaccg 1140ggaggaaggt
ggggatgacg tcaaatcatc atgcccctta tgtctagggc tacacacgtg 1200ctacaatggt
cggtacagag ggttgccaag ccgcgaggtg gagccaatcc cttaaagccg 1260atcgtagtcc
ggatcgtagt ctgcaactcg actacgtgaa gtcggaatcg ctagtaatcg 1320cagatcagca
tgctgcggtg aatacgttcc cgggtcttgt acacaccgcc cgtcacacca 1380tgggagtggg
tttcaccaga agtaggtagc ttaaccttcg ggagggcgct
1430781436DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Bacteroides
sp. EH30 78tggctcagga tgaacgctag cggcaggctt aatacatgca agtcgaacgg
gattcgaggt 60agcaatactt tgatgagagt ggcgcacggg tgcgtaacgc gtatgcaacc
tacctttaac 120tgggagatag ccccgagaaa tcgggattaa taccccataa cattacgaat
tggcatcaat 180ttgtgattaa agctccggcg gttagagatg ggcatgcgtg acattagctg
gttggtgagg 240taacggctca ccaaggcaac gatgtctagg ggtcctgaga gggttatccc
ccacactggt 300actgagacac ggaccagact cctacgggag gcagcagtaa ggaatattgg
tcaatgggcg 360caagcctgaa ccagccatgc cgcgtgcagg aagacggccc tatgggttgt
aaactgcttt 420tatcagggaa taaacccccg ctcgtgagcg gggctgaagg tacctgagga
ataagcatcg 480gctaactccg tgccagcagc cgcggtaata cggaggatgc aagcgttatc
cggattcatt 540gggtttaaag ggtgcgcagg cggattggta agtcaggggt gaaatcccac
agctcaactg 600tggaactgcc tttgatactg tcagtctaga gtatagttga agttggcgga
atgtgtcatg 660tagcggtgaa atgcttagat atgacacaga acaccgatcg cgaaggcagc
tagctaagct 720ataactgacg ctcatgcacg aaagcgtggg gatcaaacag gattagatac
cctggtagtc 780cacgctgtaa acgatgatta ctcgatgttg gcgatacaca gtcagcgttt
gagcgaaagc 840aataagtaat ccacctgggg agtacggccg caaggttaaa actcaaatga
attgacgggg 900gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa
ccttaccagg 960ccttgacatg cagagaactt tccagagatg gattggtgcc ttcgggagct
ctgacacagg 1020tgctgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc
gtaacgagcg 1080caacccttgt ccttagttac cagcacgtta aggtgggcac tctaaggaga
ctgccggtga 1140caaaccggag gaaggtgggg atgacgtcaa gtcatcatgg cccttacggc
ctgggctaca 1200cacgtgctac aatggtcggt acaaagggtt gccaagccgc gaggtggagc
taatcccata 1260aaaccgatcg tagtccggat cgcagtctgc aactcgactg cgtgaagtcg
gaatcgctag 1320taatcgtgaa tcagaatgtc acggtgaata cgttcccggg ccttgtacac
accgcccgtc 1380acaccatggg agtgggtttc accagaagta ggtagcttaa ccttcgggag
ggcgct 1436791439DNAunknownUnknown clone from enriched
environmental sample that by rDNA sequence analysis has highest
identity to Finegoldia magna 79tggctcagga cgaacgctgg cggcgtgcct
aacacatgca agtcgagcga agtgactcgg 60agagaagttt tcggatggat cgaagagtca
tcttagcggc ggacgggtga gtaacgcgtg 120agaaacctgc ctttcacaaa gggatagcct
cgggaaactg ggattaatac cttatgaaac 180tgaattaccg catggtagat cagtcaaagc
gaataagcgg tgaaagatgg tctcgcgtcc 240tattagctag ttggtgaggt aacggctcac
caaggcttcg ataggtagcc ggcctgagag 300ggtgaacggc cacactggaa ctgagacacg
gtccagactc ctacgggagg cagcagtggg 360gaatattgca caatggagga aactctgatg
cagcgacgcc gcgtgaatga tgaaggcctt 420cgggttgtaa agttctgtcc ttggggaaga
taatgacggt acccaaggag gaagccccgg 480ctaactacgt gccagcagcc gcggtaatac
gtagggggcg agcgttgtcc ggaattattg 540ggcgtaaagg gttcgcaggc ggtctgataa
gtcagatgtg aaaggcgtag gctcaaccta 600cgtaagcatt tgaaactgtc agacttgagt
taaggagagg aaagtggaat tcctagtgta 660gcggtgaaat gcgtagatat taggaggaat
accagtggcg aaggcgactt tctggactta 720tactgacgct gaggaacgaa agcgtgggga
gcaaacagga ttagataccc tggtaattcc 780cgccgtaaac gatgagtgct aggtgttggg
ggtcaaacct cggtgccgca gctaacgcat 840taagcactcc gcctggggag tacgtacgca
agtatgaaac tcaaaggaat tgacggggac 900ccgcacaagc agcggagcat gtggtttaat
ttgaagcaac gcgaagaacc ttaccagggc 960ttgacatgcc gctgaccggt gcagagatgc
atctttatcc ttcggggtac agcggacaca 1020ggtggtgcat ggttgtcgtc agctcgtgtc
gtgagatgtt gggttaagtc ccgcaacgag 1080cgcaaccctt gtctttagtt gccatcatta
agttgggcac tctaaagaga ctgccgatga 1140caaatcggag gaaggtgggg atgacgtcaa
atcatcatgc cctttatgtc ctgggctaca 1200cacgtgctac aatggtcggt acaacgagaa
gcaagtcagc gatggcaagc aaatctctaa 1260aagccgatcc cagttcggat tgcagtctgc
aactcgactg catgaagtcg gaatcgctag 1320taatcgcagg tcagcaaaac tgcggtgaat
acgttcccgg gccttgtaca caccgcccgt 1380cacaccacgg gagtcggttg tacctgaagc
cggtggccca accgcaaggg gggagccgt 143980882DNAunknownUnknown clone from
enriched environmental sample that by rDNA sequence analysis has
highest identity to Pseudomonas putida 80tggctcagat tgaacgctgg
cggcatgctt tacacatgca agtcgaacgg cagcgggggc 60ttcggcctgc cggcgagtgg
cgaacgggtg agtaatgcct aggaatctgc ctggtagtgg 120gggacaacgt ttcgaaagga
acgctaatac cgcatacgtc ctacgggaga aagtggggga 180tcttcggacc tcacgctatc
agatgagcct aggtcggatt agctagttgg tgaggtaaag 240gctcaccaag gcgacgatcc
gtaactggtc tgagaggatg atcagtcaca ctggaactga 300gacacggtcc agactcctac
gggaggcagc agtggggaat attggacaat gggcgaaagc 360ctgatccagc catgccgcgt
gtgtgaagaa ggtcttcgga ttgtaaagca ctttaagttg 420ggaggaaggg cagtaagtta
ataccttgct gttttgacgt taccgacaga ataagcaccg 480gctaacttcg tgccagcagc
cgcggtaata cgaagggtgc aagcgttaat cggaattact 540gggcgtaaag cgcgcgtagg
tggttcgtta agttagatgt gaaagccccg ggctcaacct 600gggaactgca tccaaaactg
gcgagctaga gtatggcaga gggtggtgga atttcctgtg 660tagcggtgaa atgcgtagat
ataggaagga acaccagtgg cgaaggcgac cacctgggct 720aatactgaca ctgaggtgcg
aaagcgtggg gagcaaacag gattagatac cctggtagtc 780cacgccgtaa acgatgtcga
ctagccgttg ggatccttga gatcttagtg gcgcagctaa 840cgcattaagc gtaccgcctg
gggagtacgg ccgcaaggtt ga 882811442DNAunknownUnknown
clone from enriched environmental sample that by rDNA sequence
analysis has highest identity to Clostridium aceticum 81tggctcagga
tgaacgctgg cggcgtgcct aacacatgca agtcgagcgg tatatagtgg 60aatgaaactt
cggtcgagtg aagctataga gagcggcgga cgggtgagta acgcgtaggc 120aacctgcccc
atacagaggg atagcctcgg gaaaccggga ttaaaacctc ataacgcgag 180gagttcacat
ggactgctcg ccaaagattc atcggtatgg gatgggcctg cgtctgatta 240gctagttggt
gaggtaacgg ctcaccaagg cgacgatcag tagccgacct gagagggtaa 300tcggccacat
tggaactgag acacggtcca aactcctacg ggaggcagca gtggggaatt 360ttgcacaatg
ggggcaaccc tgatgcagcg acgccgcgtg aacgaggaag gccttcgggt 420cgtaaagttc
tttcgacggg gaagaaatgt tatacgagta actgcgtata atttgacggt 480acctgtagaa
gcagccccgg ctaactccgt gccagcagcc gcggtaatac ggagggggcg 540agcgttgttc
ggagttactg ggcgtaaagc gcacgtaggc ggtgcggtaa gtcaggggtt 600aaaggtcaca
gctcaactgt gataaggcct ttgatactat cgtgctagag tgtcagagag 660ggtagcggaa
ttcccggtgt agcggtgaaa tgcgtagata tcgggaggaa caccagtagc 720gaaggcggct
acctggctga taactgacgc tgaggtgcga gagcgtgggg agcaaacagg 780attagatacc
ctggtagtcc acgctgtaaa cgatggacgt taggtgttgg gggaaccgac 840cccctcagtg
ccgaagctaa cgcgttaaac gtcccgcctg gggagtacgg ccgcaaggtt 900gaaactcaaa
ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa 960gcaacgcgca
gaaccttacc agcccttgac ataccggtcg cggacacaga gatgtgtctt 1020tcagttcggc
tggaccggat acaggtgctg catggctgtc gtcagctcgt gccgtgagat 1080gttgggttaa
gtcccgcaac gagcgcaacc ctcgccctta gttgccagca tttagttggg 1140cactctaagg
ggactgccgg tgataagccg agaggaaggt ggggatgacg tcaagtcctc 1200atggccctta
cgggctgggc tacacacgtg ctacaatggt ggtgacagtg ggcagcgagc 1260acgcgagtgt
gagctaatct ccaaaagcca tctcagttcg gattgcactc tgcaactcga 1320gtgcatgaag
ttggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc 1380gggccttgta
cacaccgccc gtcacaccat gggagttggt tttacccgaa ggcgctgtgc 1440ta
144282861DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Anaerovorax
sp. EH34 82gtgaaaggca atagcttaac tattgtaagc cttgcgaact gtgtggcttg
agtgcaggag 60aggaaagtgg aattcctagt gtagcggtga aatgcgtaga tattaggagg
aacaccagtg 120gcgaaggcga ctttctggac tgtaactgac actgaggcac gaaagcgtgg
gagcaaacag 180gattagatac cctggtagtc cacgccgtaa acgatgagca ctaggtgtcg
gggtcgcaag 240acttcggtgc cgcagttaac gcattaagtg ctccgcctgg ggagtacgca
cgcaagtgtg 300aaactcaaag gaattgacgg ggacccgcac aagcagcgga gcatgtggtt
taattcgaag 360caacgcgaag aaccttacca gggcttgaca tccctctgac agtcccttaa
ccgggacctt 420cttcggacag aggagacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt
gagatgttgg 480gttaagtccc gcaacgagcg caacccttgt ctttagttgc catcattcag
ttgggcactc 540tagagagact gccgaggata actcggagga aggtggggat gacgtcaaat
catcatgccc 600cttatgccct gggctacaca cgtgctacaa tggctggtac aaagagacgc
aagaccgcga 660ggtggagcaa atctcaaaaa ccagtcccag ttcggattgc aggctgcaac
tcgcctgcat 720gaagttggag ttgctagtaa tcgcagatca gaatgctgcg gtgaatgcgt
tcccgggtct 780tgtacacacc gcccgtcaca ccatgggagt tgtcaatacc cgaagccagt
gagctaacca 840taaaaggagg cagctgtcga a
86183620DNAunknownUnknown clone from enriched environmental
sample that by rDNA sequence analysis has highest identity to
Pseudomonas putida 83tggctcagat tgaacgctgg cggcaggcct aacacatgca
agtcgagcgg atgaatggag 60cttgctccat gattcagcgg cggacgggtg agtaatgcct
aggaatctgc ctggtagtgg 120gggacaacgt ttcgaaagga acgctaatac cgcatacgtc
ctacgggaga aagtggggga 180tcttcggacc tcacgctatc agatgagcct aggtcggatt
agctagttgg cgaggtaaag 240gctcaccaag gcgacgatcc gtaactggtc tgagaggatg
atcagtcaca ctggaactga 300gacacggtcc agactcctac gggaggcagc agtggggaat
attggacaat gggcgaaagc 360ctgatccagc catgccgcgt gtgtgaagaa ggtcttcgga
ttgtaaagca ctttaagttg 420ggaggaaggg cagtaagtta ataccttgct gttttgacgt
taccgacaga ataagcaccg 480gctaacttcg tgccagcagc cgcggtaata cgtaaggtgc
gagcgttaat cggaattact 540gggcgtaaag cgtgcgcagg cggttttgta agacagatgt
gaaatccccg ggctcatcct 600gggaactgcg tctgtgactg
62084854DNAunknownUnknown clone from enriched
environmental sample that by rDNA sequence analysis has highest
identity to Azotobacter beijerinckii 84gccctgggct caacatgggc
atccatccag acaggcgagc tagagtatag cagaggggtg 60gtgtaatttc cagcgtagcg
atgaaatgag ttgagatagg aagccacacc agaagggaag 120cagaccacct gggataatca
tgacagtgag gtacgaaagc gtgcggagca aacaagataa 180catacccgtg cagtccatgc
agtaaatgat gtcgcctagc cgatgggatc catcagatcg 240gagcggcgca gctaatgcac
taagtgcacc gcgtggggag tacggccgca aggtttcaaa 300tcaaatgaat tggcggggga
ccgcacaagc ggcgcagcat gtggtttaat tcgaagcaac 360gagcagaacc ttaccaggcc
atcccatgca tagaactttc cagagaggga tcggggcctt 420ccggaggtgt gacaccggtg
gcgccaggcc gttgttaagt tgggtcctgg gatggtgggg 480taaattccgt aacgagggcc
aaccctgtct ttagttaccc acccgttaag gtgggcactc 540taaggagacc gccggggaca
aaccggagga aggtggggat gacgtcaagt catcatggcc 600cttacggcct gggctacaca
cgtgctacaa tggtcggtac aaagggttgc caagccgcga 660ggtggagcta atcccataaa
accgatcgta gtccggatcg cagtctgcaa ctcgactgcg 720tgaagtcgga atcgctagta
atcgtgaatc agaatgtcac ggtgaatacg ttcccgggcc 780ttgtacacac cgcccgtcac
accatgggag tgggttgctc cagaagtagc tagtctaacc 840ttcgggggga cggt
85485824DNAunknownUnknown
clone from enriched environmental sample that by rDNA sequence
analysis has highest identity to Azotobacter beijerinckii
85caaatctcgc ctcaactcgc gcttggtttg catgcagctt cgtagtgtga gcgagtggat
60cctgcttgca atgaatgagt caagtcagat ggagcacgga tggcagggat ctcctcgcgc
120atgtactacg tcttgcacgc aagagtgagg agcaaacaag caatagctac ctgttactcc
180tccccctcaa tgatgatgat tattagtcgt agcagcaaaa ctctggtgtc gaagctaata
240cggaagtctc acctggggag tactgcgcat tataaatact caaaggattt tggtgtcgcc
300ccccagcggg gatatgtgga ttaattagat gaaacgcgaa aaaccttccc tcccctcgac
360atacgacgaa ccctttgaga ggggagggtg cttttgggag cctggacaca ggtgccgcat
420gggtgtcgtc acctcgtgtc gtgagatgtt gggttatgtc tcgcaacgag cgcaacccct
480gtcactagcg ccatcatttg ggggggcact ctagtgagac cccggtgaca aaccggagga
540aggggggggg gacgtcaagt cctcatggcc cttatgggta gggcttccca cgtctcacaa
600tggtcggtac agagggggtc ccagcccccg agggggagcc aatccccaaa gccgatcgta
660gtccggatgg tagtttgcaa ctcgcctacg tgaagtcgga atcgtttgta attgcagatc
720accatggtgc ggggaatacc ttcccgggtt tggtaccccc cgcccctccc cccatggggg
780ggggtttccc cggaagtagg aagcttaccc ttcggggggg gggt
82486827DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to Azoarcus sp.
EH36 86tatggaagag agtcggttag tgcgtggagg agctgcgtat gtatctactt ctctccgctc
60tcctctctgc atgcgtgtca gacagccaga gggggcgggg tccccatcca tcgttgtctt
120actttatgca ttaatgatac aacaggagta aacccccctc ttctgtatat agcatcgcag
180tagcaacaac agctgttggg tggagcaggg gatgatttat catatgtcta aaacagcccg
240cgcgcacgct ttatgcacag tatttgattg aaactcgcac ccccctgtat atccccgggg
300tgccgcacac atagttaggg ggtgtttttt tttccggcac ccccaccccg cgcgtgttag
360agagaccgtg attttttttg gcggagagag agctttataa accgaagggt tttcactcac
420ccgcggcagg gggggatcag gcttgcgccc cttttcaaaa aattcccccc ggcccccccc
480cgaggggggg tggggccgtt tttcagtccc cagggggggg gggtatcctc ttttcacccc
540cccggattgt tgtggagggg gggggtttca ccccacggaa agatacaaac ccattaagcg
600ctccaatcgc gggaggtcgg aagatccccc gctttttccc tcaggaggtt tggggtatta
660gggtaccttt cgatacgttt tcccccccgc caggggcacg tttcgagcca ttattcaccc
720gtttgcccct tgccggcagg ccgaagcccc ccctcccttt ggaatggcat ttgtaaagca
780tgcccccagg gttcaatttg agccaaaata aaacttaaag ggggaat
82787349DNAunknownUnknown clone from enriched environmental sample
that by rDNA sequence analysis has highest identity to FLexistipes
sp. vp180 87ctattgtcag ttgccatcaa gtaaggtggg cactgtaaag agtccgctgg
ggataacccg 60gaggaaggtg gggacgatgt caagtcatca tggtcgtgat gtacagggtt
acgcacctga 120tacaatggtg catacagagg gcagtgagac accgacgtta agagaatacg
ttaaagtgca 180cctcacttcg gaatgcagta tgcaaatcga atgcatggtg ttggaattgt
tagtaattgc 240aggtcagcaa tagtggggtg attacgttcc cgggcatggt acacaccgcc
agtcacacca 300tgggagtcgg ttgtacatga agccggtggc ccaaccgcaa ggggggagc
349
User Contributions:
Comment about this patent or add new information about this topic: