Functional genes (dsr) approach reveals similar sulphidogenic prokaryotes diversity but different structure in saline waters from corroding high temperature petroleum reservoirs

Oil reservoirs and production facilities are generally contaminated with H₂S resulting from the activity of sulphidogenic prokaryotes (SRP). Sulphidogenesis plays a major role in reservoir souring and microbial influenced corrosion in oil production systems. In the present study, sulphidogenic microbial diversity and composition in saline production fluids retrieved from three blocks of corroding high temperature (79?~?95 °C) oil reservoirs with high sulfate concentrations were investigated by phylogenetic analyses of gene fragments of the dissimilatory sulfite reductase (dsr). Analysis of dsr gene fragments revealed the presence of several clusters of sulphidogenic prokaryotes that cover the orders Desulfovibrionales (Desulfovibrio, Desulfomicrobium thermophilum), Desulfobacterales (Desulfobacterium, Desulfosarcina, Desulfococcus, Desulfotignum, Desulfobotulus, Desulfobulbus), Syntrophobacterales (Desulfacinum, Thermodesulforhabdus, Desulforhabdus), Clostridiales (Desulfotomaculum) and Archaeoglobales (Archaeoglobus); among which sequences affiliated to members of Desulfomicrobium, Desulfotomaculum and Desulfovibrio appeared to be the most encountered genera within the three blocks. Collectively, phylogenetic and non-metric multidimensional scaling analyses indicated similar but structurally different sulphidogenic prokaryotes communities within the waters retrieved from the three Blocks. This study show the diversity and composition of sulphidogenic prokaryotes that may play a role in the souring mediated corrosion of the oilfield and also provides a fundamental basis for further investigation to control oil reservoir souring and corrosion of pipelines and topside installations.     

The influence of nitrate on microbial processes in oil industry production waters

Sulfide accumulation due to bacterial sulfate reduction is responsible for a number of serious problems in the oil industry. Among the strategies to control the activity of sulfate-reducing bacteria (SRB) is the use of nitrate, which can exhibit a variety of effects. We investigated the relevance of this approach to souring oil fields in Oklahoma and Alberta in which water flooding is used to enhance oil recovery. SRB and nitrate-reducing bacteria (NRB) were enumerated in produced waters from both oil fields. In the Oklahoma field, the rates of sulfate reduction ranged from 0.05 to 0.16 μM S day⁻¹ at the wellheads, and an order of magnitude higher at the oil–water separator. Sulfide production was greatest in the water storage tanks in the Alberta field. Microbial counts alone did not accurately reflect the potential for microbial activities. The majority of the sulfide production appeared to occur after the oil was pumped aboveground, rather than in the reservoir. Laboratory experiments showed that adding 5 and 10 mM nitrate to produced waters from the Oklahoma and Alberta oil fields, respectively, decreased the sulfide content to negligible levels and increased the numbers of NRB. This work suggests that sulfate reduction control measures can be concentrated on aboveground facilities, which will decrease the amount of sulfide reinjected into reservoirs during the disposal of oil field production waters. Journal of Industrial Microbiology & Biotechnology (2001) 27, 80–86. Received 30 January 2001/ Accepted in revised form 30 June 2001     

Characterization of microbial souring in Berea‐sand porous medium with a North Sea oil field inoculum

Microbial souring (H₂S production) in porous medium was investigated in an anaerobic upflow porous medium reactor at 60°C using produced waters obtained from the North Sea Ninian oilfield as the inoculum. Multiple carbon sources commonly found in oil field waters (formate, acetate, propionate, iso‐ and n‐butyrates) with inorganic sulfate as the electron acceptor were used as the substrates. Stoichiometry and the rate of souring in the reactor column were calculated. A large proportion of H₂S was trapped in the column as FeS and possibly as a gas phase. Concentration gradients for the substrates (organic acids and sulfate) and H₂S were generated along the column. At steady state, the highest volumetric substrate consumption and H₂S production were found at the front part (inlet) of the reactor column. The average volumetric sulfate reduction rate after H₂S production had stabilized was calculated to be 203 ± 51 mg sulfate‐S.l^‐1.d^‐1. Comparison of the results with the authors’ previous work on the Alaska Kuparuk oilfield waters indicates that the two different microbial inocula (produced waters) exhibited the same experimental trends (rates and location) for souring in the experimental reactor system. This indicates that abiotic factors, as well as microbial parameters, may play an important role for microbial souring in the system.     

Biological souring and mitigation in oil reservoirs

Souring in oilfield systems is most commonly due to the action of sulfate-reducing prokaryotes, a diverse group of anaerobic microorganisms that respire sulfate and produce sulfide (the key souring agent) while oxidizing diverse electron donors. Such biological sulfide production is a detrimental, widespread phenomenon in the petroleum industry, occurring within oil reservoirs or in topside processing facilities, under low- and high-temperature conditions, and in onshore or offshore operations. Sulfate reducers can exist either indigenously in deep subsurface reservoirs or can be “inoculated” into a reservoir system during oilfield development (e.g., via drilling operations) or during the oil production phase. In the latter, souring most commonly occurs during water flooding, a secondary recovery strategy wherein water is injected to re-pressurize the reservoir and sweep the oil towards production wells to extend the production life of an oilfield. The water source and type of production operation can provide multiple components such as sulfate, labile carbon sources, and sulfate-reducing communities that influence whether oilfield souring occurs. Souring can be controlled by biocides, which can non-specifically suppress microbial populations, and by the addition of nitrate (and/or nitrite) that directly impacts the sulfate-reducing population by numerous competitive or inhibitory mechanisms. In this review, we report on the diversity of sulfate reducers associated with oil reservoirs, approaches for determining their presence and effects, the factors that control souring, and the approaches (along with the current understanding of their underlying mechanisms) that may be used to successfully mitigate souring in low-temperature and high-temperature oilfield operations.     

Low temperature,autotrophic microbial denitrification using thiosulfate or thiocyanate as electron donor

Wastewaters generated during mining and processing of metal sulfide ores are often acidic (pH < 3) and can contain significant concentrations of nitrate, nitrite, and ammonium from nitrogen based explosives. In addition, wastewaters from sulfide ore treatment plants and tailings ponds typically contain large amounts of inorganic sulfur compounds, such as thiosulfate and tetrathionate. Release of these wastewaters can lead to environmental acidification as well as an increase in nutrients (eutrophication) and compounds that are potentially toxic to humans and animals. Waters from cyanidation plants for gold extraction will often conjointly include toxic, sulfur containing thiocyanate. More stringent regulatory limits on the release of mining wastes containing compounds such as inorganic sulfur compounds, nitrate, and thiocyanate, along the need to increase production from sulfide mineral mining calls for low cost techniques to remove these pollutants under ambient temperatures (approximately 8 °C). In this study, we used both aerobic and anaerobic continuous cultures to successfully couple inorganic sulfur compound (i.e. thiosulfate and thiocyanate) oxidation for the removal of nitrogenous compounds under neutral to acidic pH at the low temperatures typical for boreal climates. Furthermore, the development of the respective microbial communities was identified over time by DNA sequencing, and found to contain a consortium including populations aligning within Flavobacterium, Thiobacillus, and Comamonadaceae lineages. This is the first study to remediate mining waste waters by coupling autotrophic thiocyanate oxidation to nitrate reduction at low temperatures and acidic pH by means of an identified microbial community.     

Acetate Production from Oil under Sulfate-Reducing Conditions in Bioreactors Injected with Sulfate and Nitrate

Oil production by water injection can cause souring in which sulfate in the injection water is reduced to sulfide by resident sulfate-reducing bacteria (SRB). Sulfate (2 mM) in medium injected at a rate of 1 pore volume per day into upflow bioreactors containing residual heavy oil from the Medicine Hat Glauconitic C field was nearly completely reduced to sulfide, and this was associated with the generation of 3 to 4 mM acetate. Inclusion of 4 mM nitrate inhibited souring for 60 days, after which complete sulfate reduction and associated acetate production were once again observed. Sulfate reduction was permanently inhibited when 100 mM nitrate was injected by the nitrite formed under these conditions. Pulsed injection of 4 or 100 mM nitrate inhibited sulfate reduction temporarily. Sulfate reduction resumed once nitrate injection was stopped and was associated with the production of acetate in all cases. The stoichiometry of acetate formation (3 to 4 mM formed per 2 mM sulfate reduced) is consistent with a mechanism in which oil alkanes and water are metabolized to acetate and hydrogen by fermentative and syntrophic bacteria (K. Zengler et al., Nature 401:266–269, 1999), with the hydrogen being used by SRB to reduce sulfate to sulfide. In support of this model, microbial community analyses by pyrosequencing indicated SRB of the genus Desulfovibrio, which use hydrogen but not acetate as an electron donor for sulfate reduction, to be a major community component. The model explains the high concentrations of acetate that are sometimes found in waters produced from water-injected oil fields.     

Effect of nitrate and nitrite on sulfide production by two thermophilic,sulfate-reducing enrichments from an oil field in the North Sea

Thermophilic sulfate-reducing bacteria (tSRB) can be major contributors to the production of H₂S (souring) in oil reservoirs. Two tSRB enrichments from a North Sea oil field, NS-tSRB1 and NS-tSRB2, were obtained at 58°C with acetate-propionate-butyrate and with lactate as the electron donor, respectively. Analysis by rDNA sequencing indicated the presence of Thermodesulforhabdus norvegicus in NS-tSRB1 and of Archaeoglobus fulgidus in NS-tSRB2. Nitrate (10 mM) had no effect on H₂S production by mid-log phase cultures of NS-tSRB1 and NS-tSRB2, whereas nitrite (0.25 mM or higher) inhibited sulfate reduction. NS-tSRB1 did not recover from inhibition, whereas sulfate reduction activity of NS-tSRB2 recovered after 500 h. Nitrite was also effective in souring inhibition and H₂S removal in upflow bioreactors, whereas nitrate was similarly ineffective. Hence, nitrite may be preferable for souring prevention in some high-temperature oil fields because it reacts directly with sulfide and provides long-lasting inhibition of sulfate reduction.     

Anoxygenic phototrophic bacteria from microbial communities of Goryachinsk thermal spring (Baikal Area,Russia)

Species composition of anoxygenic phototrophic bacteria in microbial mats of the Goryachinsk thermal spring was investigated along the temperature gradient. The spring belonging to nitrogenous alkaline hydrotherms is located at the shore of Lake Baikal 188 km north-east from Ulan-Ude. The water is of the sulfate-sodium type, contains trace amounts of sulfide, and salinity does not exceed 0.64 g/L, pH 9.5. The temperature at the outlet of the spring may reach 54°C. The cultures of filamentous anoxygenic phototrophic bacteria, nonsulfur and sulfur purple bacteria, and aerobic anoxygenic phototrophic bacteria were identified using the pufLM molecular marker. The fmoA marker was used for identification of green sulfur bacteria. Filamentous cyanobacteria predominated in the mats, with anoxygenic phototrophs comprising a minor component of the phototrophic communities. Thermophilic bacteria Chloroflexus aurantiacus were detected in the samples from both the thermophilic and mesophilic mats. Cultures of nonsulfur purple bacteria similar to Blastochloris sulfoviridis and Rhodomicrobium vannielii were isolated from the mats developed at high (50.6–49.4°C) and low temperatures (45–20°C). Purple sulfur bacteria Allochromatium sp. and Thiocapsa sp., as well as green sulfur bacteria Chlorobium sp., were revealed in low-temperature mats. Truly thermophilic purple and green sulfur bacteria were not found in the spring. Anoxygenic phototrophic bacteria found in the spring were typical of the sulfur communities, for which the sulfur cycle is mandatory. The presence of aerobic bacteriochlorophyll a-containing bacteria identified as Agrobacterium (Rhizobium) tumifaciens in the mesophilic (20°C) mat is of interest.     

Production-related petroleum microbiology: progress and prospects

Microbial activity in oil reservoirs is common. Methanogenic consortia hydrolyze low molecular weight components to methane and CO2, transforming light oil to heavy oil to bitumen. The presence of sulfate in injection water causes sulfate-reducing bacteria to produce sulfide. This souring can be reversed by nitrate, stimulating nitrate-reducing bacteria. Removing biogenic sulfide is important, because it contributes to pitting corrosion and resulting pipeline failures. Increased water production eventually makes oil production uneconomic. Microbial fermentation products can lower oil viscosity or interfacial tension and produced biomass can block undesired flow paths to produce more oil. These biotechnologies benefit from increased understanding of reservoir microbial ecology through new sequence technologies and help to decrease the environmental impact of oil production.     

Kinetic investigation of microbial souring in porous media using microbial consortia from oil reservoirs

Microbial souring (H2S production) in porous media was investigated in an anaerobic upflow porous media reactor at 60 degrees C using microbial consortia obtained from oil reservoirs. Multiple carbon sources (formate, acetate, propionate, iso- and n-butyrates) found in reservoir waters as well as sulfate as the electron acceptor was used. Kinetics and rates of souring in the reactor system were analyzed. Higher volumetric substrate consumption rates (organic acids and sulfate) and a higher volumetric H(2)S production rate were found at the from part of the reactor column after H(2)S production had stabilized. Concentration gradients for the substrates (organic acids and sulfate) and H(2)S were generated along the column. Biomass accumulation throughout the entire column was observed. The average specific sulfate reduction rate (H(2)S production rate) in the present reactor after H(2)S production had stabilized was calculated to be 11062 +/-2.22 mg sulfate-S/day g biomass. (c) 1994 John Wiley & Sons, Inc.     

Biological souring of crude oil under anaerobic conditions

Seawater injection into oil reservoirs for purposes of secondary oil recovery is frequently accompanied by souring (increased sulfide concentrations). Production of hydrogen sulfide causes various problems, such as microbiologically influenced corrosion (MIC) and deterioration of crude oil. Sulfate-reducing bacteria (SRB) are considered to be major players in souring. Volatile fatty acids (VFAs) in oil-field water are believed to be produced by microbial degradation of crude oil. The objective of this research was to investigate mechanisms of souring, focusing specifically on VFA production via crude oil biodegradation. To this end, a microbial consortium collected from an oil–water separator was suspended in seawater; crude oil or liquid n-alkane mixture was added to the culture medium as the sole carbon source, and the culture was incubated under anaerobic conditions for 190 days. Physicochemical analysis showed that preferential toluene degradation and sulfate reduction occurred concomitantly in the culture containing crude oil. Sulfide concentrations were much lower in the alkane-supplemented culture than in the crude oil-supplemented culture. These observations suggest that SRB are related to the toluene activation and VFA consumption steps of crude oil degradation. Therefore, the electron donors for SRB are not only VFA, but many components of crude oil, especially toluene. Alkanes were also degraded by microorganisms, but did not contribute to reservoir souring.     

Effect of temperature on the rate of oxidation of pyrrhotite-rich sulfide ore flotation concentrate and the structure of the acidophilic chemolithotrophic microbial community

Oxidation of flotation concentrate of a pyrrhotite-rich sulfide ore by acidophilic chemolithoautotrophic microbial communities at 35, 40, and 45°C was investigated. According to the physicochemical parameters of the liquid phase of the pulp, as well as the results of analysis of the solid residue after biooxidation and cyanidation, the community developed at 40°C exhibited the highest rate of oxidation. The degree of gold recovery at 35, 40, and 45°C was 89.34, 94.59, and 83.25%, respectively. At 40°C, the highest number of microbial cells (6.01 × 10⁹ cells/mL) was observed. While temperature had very little effect on the species composition of microbial communities (except for the absence of Leptospirillum ferriphilum at 35°C), the shares of individual species in the communities varied with temperature. Relatively high numbers of Sulfobacillus thermosulfidooxidans, the organism oxidizing iron and elemental sulfur at higher rates than other acidophilic chemolithotrophic species, were observed at 40°C.     

34S tracer study of pollutant sulfate behaviour in a lowland peatland

Field experiments were carried out in order to assess the practicality and application of ³⁴SO₄ ^2? as a tracer of the physical and geochemical fate of aerially derived sulfur in peat. Six enclosures were isolated in a lowland peat with high historical acid sulfate inputs at Thorne Moors, UK, and treated with regular additions of 99.9% pure ³⁴SO₄ ^2? for 12 months. The total ³⁴S sulfate addition resulted in negligible change to the sulfate concentration, but allowed unequivocal change to the isotopic composition of sulfate inputs relative to pre-experiment control data set. Migration and biogeochemical transformations of the ³⁴S tracer were monitored via depth-specific sampling of surface and pore-waters every 6 weeks, and sacrificial sampling of solid peat at 12, 24, and 48 week intervals. Tracer incorporation into the various sulfur forms within the surface and pore-waters, vegetation, organic and inorganic fractions of the peat was apparent through strong positive deviation of δ³⁴S from natural values (in comparison with 18 months control data set for the same site). Consistency within enclosures is good and a detailed model of sulfur cycling within each enclosure can be established but natural variability in the control data and differences between replicate enclosures prevents more quantitative assessment. The ³⁴S tracer was initially rapidly removed from surface waters. The majority of uptake was by living vegetation (5.7–33% of tracer added, mean 17.6%), or through transformation to the organic fraction of the upper peat (25 cm) after rapid bacterial reduction of sulfate to sulfide. Despite penetration of ³⁴S labelled sulfate to deeper pore-waters over time, there was no significant reduction to sulfide or subsequent incorporation into organic or inorganic fraction at these depths (>25 cm); organic and inorganic sulfur, and pore-water sulfide retained their initial unlabelled isotopic compositions. This limitation on sulfur cycling at relatively shallow depth may be attributed to diminished labile organic matter inhibiting the activity of sulfate reducing bacteria or poisoning of sulfate reducers by high dissolved sulfide, after long-term sulfur pollution of this ecosystem.     

(Per)chlorate reduction at high temperature: Physiological study of Archaeoglobus fulgidus and potential implications for novel souring mitigation strategies

The recent finding that Archaeoglobus fulgidus is able to couple (per)chlorate reduction to growth expanded this trait to the hyperthermophilic range of life. This sulfate-reducing archaeon is considered to be one of the major contributors to souring in hot oil reservoirs. Therefore, it is important to study its physiology in depth, particularly in view of novel souring mitigation strategies. A. fulgidus does not possess the classical (per)chlorate reduction pathway, as it lacks the key enzyme chlorite dismutase. Rather, the microorganism seems to couple (per)chlorate reduction to sulfur metabolism. Growth experiments show the strict necessity of sulfur compounds to sustain perchlorate reduction. Furthermore, the chemical formation of elemental sulfur was observed during perchlorate reduction, a compound that is biologically reduced again. Additional experiments showed that tetrathionate, but not elemental sulfur and polysulfide, serves as an electron acceptor for growth by A. fulgidus. Taken together these results provide further evidence for the importance of chemical and biological redox reactions involving sulfur compounds during (per)chlorate reduction. In non-reduced media also, nitrate could be reduced by A. fulgidus, though not coupled to growth. This observation and the fact that A. fulgidus had prolonged adaptation phases on sulfate after long-lasting growth on perchlorate are of interest in the development of new souring mitigation strategies using nitrate and/or (per)chlorate.     

Oil Field Souring Control by Nitrate-Reducing Sulfurospirillum spp. That Outcompete Sulfate-Reducing Bacteria for Organic Electron Donors

Nitrate injection into oil reservoirs can prevent and remediate souring, the production of hydrogen sulfide by sulfate-reducing bacteria (SRB). Nitrate stimulates nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB) and heterotrophic nitrate-reducing bacteria (hNRB) that compete with SRB for degradable oil organics. Up-flow, packed-bed bioreactors inoculated with water produced from an oil field and injected with lactate, sulfate, and nitrate served as sources for isolating several NRB, including Sulfurospirillum and Thauera spp. The former coupled reduction of nitrate to nitrite and ammonia with oxidation of either lactate (hNRB activity) or sulfide (NR-SOB activity). Souring control in a bioreactor receiving 12.5 mM lactate and 6, 2, 0.75, or 0.013 mM sulfate always required injection of 10 mM nitrate, irrespective of the sulfate concentration. Community analysis revealed that at all but the lowest sulfate concentration (0.013 mM), significant SRB were present. At 0.013 mM sulfate, direct hNRB-mediated oxidation of lactate by nitrate appeared to be the dominant mechanism. The absence of significant SRB indicated that sulfur cycling does not occur at such low sulfate concentrations. The metabolically versatile Sulfurospirillum spp. were dominant when nitrate was present in the bioreactor. Analysis of cocultures of Desulfovibrio sp. strain Lac3, Lac6, or Lac15 and Sulfurospirillum sp. strain KW indicated its hNRB activity and ability to produce inhibitory concentrations of nitrite to be key factors for it to successfully outcompete oil field SRB.     

Microbiological Processes in a High-Temperature Oil Field

Thermophilic sulfate-reducing bacteria (SRB) oxidizing lactate, butyrate, and C₁₂–C₁₆ n-alkanes of oil at a temperature of 90°C were isolated from samples of water and oil originating from oil reservoirs of the White Tiger high-temperature oil field (Vietnam). At the same time, no thermophiles were detected in the injected seawater, which contained mesophilic microorganisms and was the site of low-temperature processes of sulfate reduction and methanogenesis. Thermophilic SRB were also found in samples of liquid taken from various engineering reservoirs used for oil storage, treatment, and transportation. These samples also contained mesophilic SRB, methanogens, aerobic oil-oxidizing bacteria, and heterotrophs. Rates of bacterial production of hydrogen sulfide varied from 0.11 to 2069.63 at 30°C and from 1.18 to 173.86 at 70°C g S/(l day); and those of methane production, varied from 58.4 to 100 629.8 nl CH₄/(l day) (at 30°C). The sulfur isotopic compositions of sulfates contained in reservoir waters and of hydrogen sulfide of the accompanying gas indicate that bacterial sulfate reduction might be effective in the depth of the oil field.     

Thermoacidophilic microbial community oxidizing the gold-bearing flotation concentrate of a pyrite-arsenopyrite ore

An aboriginal community of thermophilic acidophilic chemolithotrophic microorganisms (ACM) was isolated from a sample of pyrite gold-bearing flotation concentrate at 45–47°C and pH 1.8–2.0. Compared to an experimental thermoacidophilic microbial consortium formed in the course of cultivation in parallel bioreactors, it had lower rates of iron leaching and oxidation, while its rate of sulfur oxidation was higher. A new thermophilic acidophilic microbial community was obtained by mutual enrichment with the microorganisms from the experimental and aboriginal communities during the oxidation of sulfide ore flotation concentrate at 47°C. The dominant bacteria of this new ACM community were Acidithiobacillus caldus (the most active sulfur oxidize) and Sulfobacillus thermotolerans (active oxidizer of both iron and sulfur), while iron-oxidizing archaea of the family Ferroplasmaceae and heterotrophic bacteria Alicyclobacillus tolerans were the minor components. The new ACM community showed promise for leaching/oxidation of sulfides from flotation concentrate at high pulp density (S : L = 1 : 4).     

Design features of offshore oil production platforms influence their susceptibility to biocorrosion

Offshore oil-producing platforms are designed for efficient and cost-effective separation of oil from water. However, design features and operating practices may create conditions that promote the proliferation and spread of biocorrosive microorganisms. The microbial communities and their potential for metal corrosion were characterized for three oil production platforms that varied in their oil-water separation processes, fluid recycling practices, and history of microbially influenced corrosion (MIC). Microbial diversity was evaluated by 16S rRNA gene sequencing, and numbers of total bacteria, archaea, and sulfate-reducing bacteria (SRB) were estimated by qPCR. The rates of ³⁵S sulfate reduction assay (SRA) were measured as a proxy for metal biocorrosion potential. A variety of microorganisms common to oil production facilities were found, but distinct communities were associated with the design of the platform and varied with different locations in the processing stream. Stagnant, lower temperature (<37 °C) sites in all platforms had more SRB and higher SRA compared to samples from sites with higher temperatures and flow rates. However, high (5 mmol L^?1) levels of hydrogen sulfide and high numbers (10⁷ mL^?1) of SRB were found in only one platform. This platform alone contained large separation tanks with long retention times and recycled fluids from stagnant sites to the beginning of the oil separation train, thus promoting distribution of biocorrosive microorganisms. These findings tell us that tracking microbial sulfate-reducing activity and community composition on off-shore oil production platforms can be used to identify operational practices that inadvertently promote the proliferation, distribution, and activity of biocorrosive microorganisms.     

Microbial diversity in Los Azufres geothermal field (Michoacán,Mexico) and isolation of representative sulfate and sulfur reducers

Los Azufres spa consists of a hydrothermal spring system in the Mexican Volcanic Axis. Five samples (two microbial mats, two mud pools and one cenote water), characterized by high acidity (pH between 1 and 3) and temperatures varying from 27 to 87 °C, were investigated for their microbial diversity by Terminal-Restriction Fragment Length Polymorphism (T-RFLP) and 16S rRNA gene library analyses. These data are the first to describe microbial diversity from Los Azufres geothermal belt. The data obtained from both approaches suggested a low bacterial diversity in all five samples. Despite their proximity, the sampling points differed by their physico-chemical conditions (mainly temperature and matrix type) and thus exhibited different dominant bacterial populations: anoxygenic phototrophs related to the genus Rhodobacter in the biomats, colorless sulfur oxidizers Acidithiobacillus sp. in the warm mud and water samples, and Lyzobacter sp.-related populations in the hot mud sample (87 °C). Molecular data also allowed the detection of sulfate and sulfur reducers related to Thermodesulfobium and Desulfurella genera. Several strains affiliated to both genera were enriched or isolated from the mesophilic mud sample. A feature common to all samples was the dominance of bacteria involved in sulfur and iron biogeochemical cycles (Rhodobacter, Acidithiobacillus, Thiomonas, Desulfurella and Thermodesulfobium genera).     

Temperature impacts on anaerobic biotransformation of LNAPL and concurrent shifts in microbial community structure

Thermally-enhanced bioremediation is a promising treatment approach for petroleum contamination; however, studies examining temperature effects on anaerobic biodegradation in zones containing light non-aqueous phase liquids (LNAPLs) are lacking. Herein, laboratory microcosm studies were conducted for a former refinery to evaluate LNAPL transformation, sulfate reduction, and methane generation over a one-year period for temperatures ranging from 4 to 40 °C, and microbial community shifts were characterized. Temperatures of 22 and 30 °C significantly increased total biogas generation compared to lower (4 and 9 °C) and higher temperatures (35 and 40 °C; p < 0.1). Additionally, at 22 and 30 °C methane generation commenced ~6 months earlier than for 35 and 40 °C. Statistically significant biodegradation of benzene, toluene and xylenes was observed at elevated temperatures but not at lower temperatures (p < 0.1). Additionally, a novel differential chromatogram approach was developed to overcome challenges associated with resolving losses in complex mixtures of hydrocarbons, and application of this method revealed greater losses of hydrocarbons at 22 and 30 °C as compared to lower and higher temperatures. Finally, molecular biology assays revealed that the composition and activity of microbial communities shifted in a temperature-dependent manner. Collectively, results demonstrated that anaerobic biodegradation processes can be enhanced by increasing the temperature of LNAPL-containing soils, but biodegradation does not simply increase as temperature increases likely due to a lack of microorganisms that thrive at temperatures well above the historical high temperatures for a site. Rather, optimal degradation is achieved by holding soils at the high end of, or slightly higher than, their natural range.