Characteristics of microbiota, core sulfate-reducing taxa and corrosion rates in production water from five petroleum reservoirs in China.

Microbial diversity and activities in petroleum reservoir systems can be altered by water-flooding operation, but the current understanding of the mechanism for such changes in microbial composition characteristics and community is inadequate. In this study, microbial communities especially functional groups in production water from five petroleum reservoirs in China were investigated by chemical and molecular biological analyses. The dominant and core phyla in the five oil reservoirs were Proteobacteria, Deferribacterota, Firmicutes, Desulfobacterota, Euryarchaeota and Thermoplasmatota. At the genus level, the dominant taxa in each petroleum reservoir were different, and not all of the dominant genera were the core members across the five oil reservoirs. The microbiologically influenced corrosion (MIC) were investigated for the functional groups in each production water. The corrosion rates in production water were higher than controls with a positive correlation to the abundances of sulfate-reducing prokaryotes (SRP). The SRP diversity based on the aprA and dsrA gene analysis showed that obvious differences were evident between onshore (JS, SL, DQ and XJ) and offshore (BS) oilfields. The core SRP taxa in onshore oilfields were Desulfomicrobium and Desulfovibrio, also with Desulfotomaculum in medium/low-temperature oil reservoirs (DQ and XJ), but in high-temperature petroleum reservoirs (JS, BS and SL), Archaeoglobus, Thermodesulfobacterium and Thermodesulfovibrio were the core groups. Statistical analysis indicated that temperature, electron acceptors and donors showed significant influence on the SRP community. This research reveals the characteristics of microbial and functional community as well as their interaction mechanism on corrosion in petroleum reservoir environments, and will improve industrial bio-control and management of MIC in oilfields.

Bacterial and Archaeal Community Distribution in Oilfield Water Re-injection Facilities and the Influences from Microorganisms in Injected Water.

Water flooding is widely employed for oil production worldwide. However, there has never been a systematic investigation of the microbial communities occurring in oilfield water re-injection facilities. Here, we investigated the distribution of bacterial and archaeal communities in water re-injection facilities of an oilfield, and illustrated the combined influences of environmental variation and the microorganisms in injected water on the microbial communities. Bacterial communities from the surface injection facilities were dominated by aerobic or facultative anaerobic Betaproteobacteria, Alphaproteobacteria, and Flavobacteria, whereas Clostridia, Deltaproteobacteria, Anaerolineae, and Synergistia predominated in downhole of the injection wells, and Gammaproteobacteria, Betaproteobacteria, and Epsilonproteobacteria predominated in the production wells. Methanosaeta, Methanobacterium, and Methanolinea were dominant archaea in the injection facilities, while Methanosaeta, Methanomethylovorans, and Methanoculleus predominated in the production wells. This study also demonstrated that the microorganisms in injected water could be easily transferred from injection station to wellheads and downhole of injection wells, and environmental variation and diffusion-limited microbial transfer resulted from formation filtration were the main factors determining microbial community assembly in oil-bearing strata. The results provide novel information on the bacterial and archaeal communities and the underlying mechanisms occurring in oilfield water re-injection facilities, and benefit the development of effective microbiologically enhanced oil recovery and microbiologically prevented reservoir souring programs.

Stochastic assembly process dominates bacterial succession during a long-term microbial enhanced oil recovery.

Microbial enhanced oil recovery (MEOR) has been successfully used in oil exploitation to increase oil production. However, the mechanisms of microbial interactions and community assembly related to oil production performance along MEOR process are poorly understood. Here, we investigated the microbiome of an oil reservoir for a period of 5 years under three phases of different treatments with the injection of a mixture of microbes, nutrients, and air at different intensity. During the MEOR process, amplification of functional genes revealed an increase of genes related to hydrocarbon degradation linked to methanogenesis, supported by stable isotope analysis for confirmation of the methanogenesis activity. Meanwhile, a lower contribution of the ubiquitous/common taxa, closer and more positive associations, and lower modularity were observed in bacterial co-occurrence networks, with the rare taxa being the keystone taxa. The null model analysis and structural equation modeling revealed that the contribution of stochastic processes affected by functional groups and co-occurrence patterns to bacterial community increased significantly with the increase of oil production. This provides new insight that stochastic assembly in bacterial community increased along with MEOR process, and it is worthwhile paying attention to the uncertain consequences caused by random evolution since the treatment effect of MEOR is closely related to the in-situ community in oil reservoir.

Dredging alleviates cyanobacterial blooms by weakening diversity maintenance of bacterioplankton community.

Disentangling ecological mechanisms behind dredging is meaningful to implement environmental policy for improving water quality. However, environmental adaptation and community assembly processes of bacterioplankton in response to dredging disturbance are poorly understood. Based on Illumine MiSeq sequencing and multiple statistical analyses, we estimated interactions, functions, environmental breadths, phylogenetic signals, phylogenetic clustering, and ecological assembly processes of bacterioplankton community before and after dredging. We found distinct change in community composition, comparable decreases in diversity, functional redundancy and conflicting interaction, relatively low phylogenetic clustering, and relatively weak environmental adaptation after dredging. The bacterioplankton community assembly was affected by both stochastic and deterministic processes before dredging, but dominated by stochasticity after dredging. Sediment total phosphorus was a decisive factor in balancing determinism and stochasticity for bacterioplankton community assembly before and after dredging. Consequently, taxonomic and phylogenetic α-diversities of bacterioplankton exhibited higher contributions to the water trophic level represented by chlorophyl α before dredging than afterwards. Our results emphasized bacterioplankton in response to environmental changes caused by dredging, with nutrient loss and ecological drift playing important roles. These findings extend knowledge of contribution of bacterioplankton diversity to water trophic level and decipher mechanisms of bacterioplankton diversity maintenance in response to dredging, which is useful for guiding mitigation of cyanobacterial blooms.

The dynamics of phosphorus fractions and the factors driving phosphorus cycle in Zoige Plateau peatland soil.

Phosphorus (P) is an essential nutrient, limiting plant growth and microbial activity in many ecosystems. However, a few studies have been conducted to investigate P dynamics and the factors driving P dynamics in peatland soils. Therefore, this study chose Zoige Plateau peatland (the largest peatland in China) to reveal P dynamics and the possible driving factors through fractionating soil P and investigating a series of abiotic and biotic factors. It is found that season, peatland type, and soil depth could strongly affect P dynamics. H2O-P and NaHCO3-P (labile P) had lower content, while NaOH-P, HCl-P, Mix-P, and Residual-P (non-labile P) were the dominant fractions. Besides, the sum of P fractions was higher than the traditional measurement of total P, suggesting P storage might be underestimated in peatland soils. Moreover, it is observed that biotic factors affected P fractions more than abiotic factors, and fungi affected refractory P more than bacteria. This study provides essential information for understanding P cycling in peatland soils and emphasizes specific microbes related to P cycling, which should be paid more attention to in the future.

Highly efficient removal of phosphorus from agricultural runoff by a new akadama clay barrier-vegetated drainage ditch system (VDD) and its mechanism.

A vegetated drainage ditch (VDD) system is an effective management practice for removing excess phosphorus (P) from agricultural runoff. However, the maximization of P removing efficiency by VDD remains a challenge. In this study, new VDDs with akadama clay barriers (particle size of clay: 1-6 mm; height of barrier: 5-15 cm and length of barrier: 10-90 cm) were designed in lab scale, and the mechanism of phosphate removal by akadama clay was investigated. It was found that a new VDD with akadama clay barriers (particle size:1 mm; height:10 cm and length: 90 cm) exhibited the highest removal efficiency of total P (TP) (97.1%), particulate P(PP) (96.9%), and dissolved P (DP) (97.4%), respectively. The retained P was mainly adsorbed in akadama clay barrier sections, and a low concentration of P was observed in soil sections in the new VDD. The maximum adsorption capacity of phosphate to akadama clay was 5.06 mg/g at 298 K, and XPS analysis indicated that phosphate was adsorbed by the inner-sphere complexation formation with the metal elements (Al, Fe). This study indicates that the new VDD with akadama clay barriers is a promising technique to efficiently remove P from agricultural runoff and significantly minimize the risk of P release into streams through runoff.

Survival strategies of ammonia-oxidizing archaea (AOA) in a full-scale WWTP treating mixed landfill leachate containing copper ions and operating at low-intensity of aeration.

Recent studies indicate that ammonia-oxidizing archaea (AOA) may play an important role in nitrogen removal by wastewater treatment plants (WWTPs). However, our knowledge of the mechanisms employed by AOA for growth and survival in full-scale WWTPs is still limited. Here, metagenomic and metatranscriptomic analyses combined with a laboratory cultivation experiment revealed that three active AOAs (WS9, WS192, and WS208) belonging to family Nitrososphaeraceae were active in the deep oxidation ditch (DOD) of a full-scale WWTP treating landfill leachate, which is configured with three continuous aerobic-anoxic (OA) modules with low-intensity aeration (≤ 1.5 mg/L). AOA coexisted with AOB and complete ammonia oxidizers (Comammox), while the ammonia-oxidizing microbial (AOM) community was unexpectedly dominated by the novel AOA strain WS9. The low aeration, long retention time, and relatively high inputs of ammonium and copper might be responsible for the survival of AOA over AOB and Comammox, while the dominance of WS9, specifically may be enhanced by substrate preference and uniquely encoded retention strategies. The urease-negative WS9 is specifically adapted for ammonia acquisition as evidenced by the high expression of an ammonium transporter, whereas two metabolically versatile urease-positive AOA strains (WS192 and WS208) can likely supplement ammonia needs with urea. This study provides important information for the survival and application of the eutrophic Nitrososphaeraceae AOA and advances our understanding of archaea-dominated ammonia oxidation in a full-scale wastewater treatment system.

Enhanced reduction of sulfate and chromium under sulfate-reducing condition by synergism between extracellular polymeric substances and graphene oxide.

Microbial reduction of sulfate and metal were simultaneously enhanced in the presence of graphene oxide (GO)-like nanomaterials, however, the mechanism remained unclear. In this study, bio-reduction of Cr was compared between free-living bacterium BY7 and immobilized BY7 (BY-rGO) on reduced GO particles. The role of extracellular polymeric substances (EPS) and rGO material on reduction of sulfate and Cr was investigated. Cr(VI) was reduced to Cr(III) and elemental Cr by BY-rGO particles up to 51% and 28%, respectively. EPS produced by the bacterium BY7 mainly consisted of proteins, polysaccharides, nucleic acids and humic substances. Concentration of EPS was sharply increased (about 54%) with the addition of graphene oxide, while the composition of EPS components was strongly affected by the exposure to Cr. By removing surface EPS without breaking the cells, reduction activities of sulfate and chromium by both BY-rGO particles and free-living BY7 cells were decreased. In contrast, reduction of sulfate and Cr by the free-living BY7 cells was enhanced with external addition of extracted EPS. Based on electrochemical analysis, the reduction peak indicating enhanced electron transfer was lost after removing EPS. Moreover, the contribution of each EPS fractions on sulfate and Cr reduction followed an order of polysaccharides > proteins > humic substances. Therefore, microbial sulfate and Cr reduction processes in the presence of BY-rGO particles were enhanced by the increasing amounts of EPS, which likely mediated electron transfer during sulfate and Cr reduction, and relieved bacteria from metal toxicity. Nevertheless, the presence of rGO was crucially important for elemental Cr production under sulfate-reducing condition, which might contribute to lowering electric potential or reducing activation energy for Cr(III) reduction. This work provided direct evidences for enhancing sulfate and Cr reduction activities by supplement of EPS as an additive to increase treatment efficiency in environmental bioremediation.

Improved anaerobic co-digestion of food waste and domestic wastewater by copper supplementation - Microbial community change and enhanced effluent quality.

Anaerobic co-digesters are biorefineries for energy recovery from food waste and domestic wastewater via methane production. Nonetheless, the performance of this technology was not always satisfied due to the long chain fatty acids (LCFAs) generation from food waste. Micronutrient supplementation is an effective strategy that could be applied during the anaerobic (co-)digestion to further enhance the digestion efficiency while treating food waste. In this study, supplementing copper (as CuSO4 and CuCl2) at 10, 30, and 50 mg/L Cu2+ was selected to further enhance the methane production of anaerobic co-digester while treating food waste and domestic wastewater. Overall, with the supplementation of copper, the chemical oxygen demand (COD) removal efficiency was over 90%, while higher methane yields (0.260-0.325 L CH4/g COD removed) were obtained compared to the control without supplementation (0.175 L CH4/g COD removed). For the cumulative methane yield, the highest increment of 94.1% was obtained when 10 mg/L of Cu2+ were added. The results showed copper as a cofactor of many microbial enzymes and coenzymes involved in the methane production further improved both methane production and COD removal efficiency. Meanwhile, the microbial community analysis verified the copper supplementation significantly changed the bacterial communities but with the limited effect on the diversity of archaea. Furthermore, since the anaerobic co-digester was not that much efficient on the nutrients removal, the effluent from the upflow anaerobic sludge blanket (UASB) reactor was further treated by the anaerobic/anoxic/oxic (A2O) rector and the resulting effluent reached the satisfying quality in terms of COD, total nitrogen (TN), and NH3-N removal, meeting the regional effluent discharge limits.

Methanogenic degradation of branched alkanes in enrichment cultures of production water from a high-temperature petroleum reservoir.

Branched alkanes are important constituents of crude oil and are usually regarded as resistant to microbial degradation, resulting in little knowledge of biochemical processes involved in anaerobic branched alkanes biodegradation. Here, we initiated an incubation study by amendment of iso-C9 (2-methyl, 3-methyl, and 4-methyloctane) as substrates for methanogenic degradation in production water from a high-temperature petroleum reservoir. Over an incubation period of 367 days, significant methanogenesis was observed in samples amended with these branched alkanes. The strong methanogenic activity only observed in iso-C9 amendments suggested the presence of microbial transformation from iso-alkanes into methane. GC-MS-based examination of the original production water identified an intermediate tentatively to be iso-C9-like alkylsuccinate, but was not detected in the enrichment cultures, combined with the successful amplification of assA functional gene in inoculating samples, revealing the ability of anaerobic biodegradation of iso-C9 via fumarate addition pathway. Microorganisms affiliated with members of the Firmicutes, Synergistetes, and methanogens of genus Methanothermobacter spp. were highly enriched in samples amended with iso-C9. The co-occurrence of known syntrophic acetate oxidizers Thermoacetogenium spp. and Methanothermobacter spp. (known hydrogenotrophic methanogens) indicates a potential syntrophic acetate oxidation associated with the methanogenic biodegradation of iso-C9. These results provide some useful information on the potential biodegradation of branched alkanes via methanogenesis and also suggest that branched alkanes are likely activated via fumarate addition in high-temperature petroleum reservoirs.

Characterization of bacterial composition and diversity in a long-term petroleum contaminated soil and isolation of high-efficiency alkane-degrading strains using an improved medium.

Bacterial community and diversity in a long-term petroleum-contaminated soil of an oilfield were characterized using 16S rRNA gene-based Illumina MiSeq high-throughput sequencing. Results indicated that Proteobacteria (49.11%) and Actinobacteria (24.24%) were the most dominant phyla, and the most abundant genera were Pseudoxanthomonas (8.47%), Luteimonas (3.64%), Alkanindiges (9.76%), Acinetobacter (5.26%) and Agromyces (8.56%) in the soil. Meanwhile a series of cultivations were carried out for isolation of alkane degraders from petroleum-contaminated soil with gellan gum and agar as gelling agents. And the isolates were classified by their 16S rRNA genes. Nine of the isolates including Enterobacter, Pseudomonas,Acinetobacter, Rhizobium, Bacillus, Sphingomonas, Paenibacillus, Variovorax and Rhodococcus showed strong biodegradability of alkane mixture (C9-C30) in a wide range of chain-length, which could be potentially applied in enhancement of bioremediation.

Influence of Macrofaunal Burrows on Extracellular Enzyme Activity and Microbial Abundance in Subtropical Mangrove Sediment.

Bioturbation and bioirrigation induced by burrowing macrofauna are recognized as important processes in aquatic sediment since macrofaunal activities lead to the alteration of sediment characteristics. However, there is a lack of information on how macrofauna influence microbial abundance and extracellular enzyme activity in mangrove sediment. In this study, the environmental parameters, extracellular enzyme activities, and microbial abundance were determined and their relationships were explored. Sediment samples were taken from the surface (S) and lower layer (L) without burrow, as well as crab burrow wall (W) and bottom of crab burrow (B) located at the Mai Po Nature Reserve, Hong Kong. The results showed that the burrowing crabs could enhance the activities of oxidase and hydrolases. The highest activities of phenol oxidase and acid phosphatase were generally observed in B sediment, while the highest activity of N-acetyl-glucosaminidase was found in W sediment. The enzymatic stoichiometry indicated that the crab-affected sediment had similar microbial nitrogen (N) and phosphorous (P) availability relative to carbon (C), lower than S but higher than L sediment. Furthermore, it was found that the highest abundance of both bacteria and fungi was shown in S sediment, and B sediment presented the lowest abundance. Moreover, the concentrations of phosphorus and soluble phenolics in crab-affected sediment were almost higher than the non-affected sediment. The alterations of phenolics, C/P and N/P ratios as well as undetermined environmental factors by the activities of crabs might be the main reasons for the changes of enzyme activity and microbial abundance. Finally, due to the important role of phenol oxidase and hydrolases in sediment organic matter (SOM) decomposition, it is necessary to take macrofaunal activities into consideration when estimating the C budget in mangrove ecosystem in the future.

Iron oxides alter methanogenic pathways of acetate in production water of high-temperature petroleum reservoir.

Acetate is a key intermediate in anaerobic crude oil biodegradation and also a precursor for methanogenesis in petroleum reservoirs. The impact of iron oxides, viz. ß-FeOOH (akaganéite) and magnetite (Fe3O4), on the methanogenic acetate metabolism in production water of a high-temperature petroleum reservoir was investigated. Methane production was observed in all the treatments amended with acetate. In the microcosms amended with acetate solely about 30% of the acetate utilized was converted to methane, whereas methane production was stimulated in the presence of magnetite (Fe3O4) resulting in a 48.34% conversion to methane. Methane production in acetate-amended, ß-FeOOH (akaganéite)-supplemented microcosms was much faster and acetate consumption was greatly improved compared to the other conditions in which the stoichiometric expected amounts of methane were not produced. Microbial community analysis showed that Thermacetogenium spp. (known syntrophic acetate oxidizers) and hydrogenotrophic methanogens closely related to Methanothermobacter spp. were enriched in acetate and acetate/magnetite (Fe3O4) microcosms suggesting that methanogenic acetate metabolism was through hydrogenotrophic methanogenesis fueled by syntrophic acetate oxidizers. The acetate/ß-FeOOH (akaganéite) microcosms, however, differed by the dominance of archaea closely related to the acetoclastic Methanosaeta thermophila. These observations suggest that supplementation of ß-FeOOH (akaganéite) accelerated the production of methane further, driven the alteration of the methanogenic community, and changed the pathway of acetate methanogenesis from hydrogenotrophic methanogenesis fueled by syntrophic acetate oxidizers to acetoclastic.

Microbial extracellular enzymes in biogeochemical cycling of ecosystems.

Extracellular enzymes, primarily produced by microorganisms, affect ecosystem processes because of their essential roles in degradation, transformation and mineralization of organic matter. Extracellular enzymes involved in the cycling of carbon (C), nitrogen (N) and phosphorus (P) have been widely investigated in many different ecosystems, and several enzymes have been recognized as key components in regulating C storage and nutrient cycling. In this review, it was the first time to summarize the specific extracellular enzymes related to C storage and nutrient cycling for better understanding the important role of microbial extracellular enzymes in biogeochemical cycling of ecosystems. Subsequently, ecoenzymatic stoichiometry - the relative ratio of extracellular enzyme, has been reviewed and further provided a new perspective for understanding biogeochemical cycling of ecosystems. Finally, the new insights of using microbial extracellular enzyme in indicating biogeochemical cycling and then protecting ecosystems have been suggested.

Impact of nitrogen pollution/deposition on extracellular enzyme activity, microbial abundance and carbon storage in coastal mangrove sediment.

This study applied different concentration of NaNO3 solution to simulate the effect of inorganic nitrogen (N) deposition/pollution on carbon (C) storage in coastal mangrove sediment through observing the changes of enzyme activity and microbial abundance. Sediment collected from mangrove forest (MG) and intertidal zone (IZ) were incubated with different N rates (0 (control), 5 (low-N) and 20 (high-N) µg N g-1 dry sediment, respectively). After incubation, the activities of phenol oxidase (PHO) and acid phosphatase (ACP) were enhanced, but ß-glucosidase (GLU) and N-ß-acetyl-glucosaminidase (NAG) activities were reduced by N addition. The altered enzymatic stoichiometries by N input implied that microbial phosphorus (P) limitation was increased, whereas C and N limitation were alleviated. Besides, N input decreased the bacterial abundance but increased fungal abundance in both types of sediment. The increased pH and soluble phenolics along with the exacerbated P limitation by N addition might explain these changes. Furthermore, sediment with N addition (except high-N treated MG sediment) showed a trend of C sequestration, which might be largely caused by the decrease of bacterial abundance and GLU activity. However, MG sediment with high-N suggested a trend of C loss, and the possible reason for this discrepancy might be the relatively higher increase of PHO and ACP activity. To better understand the influence of N deposition/pollution on C cycling, the long-term N effect on microorganisms, enzymes, and thus C storage should be paid more attention in the future.

Activation of CO2-reducing methanogens in oil reservoir after addition of nutrient.

Nutrient addition as part of microbial enhanced oil recovery (MEOR) operations have important implications for more energy recovery from oil reservoirs, but very little is known about the in situ response of microorganisms after intervention. An analysis of two genes as biomarkers, mcrA encoding the key enzyme in methanogenesis and fthfs encoding the key enzyme in acetogenesis, was conducted during nutrient addition in oil reservoir. Clone library data showed that dominant mcrA sequences changed from acetoclastic (Methanosaetaceae) to CO2-reducing methanogens (Methanomicrobiales and Methanobacteriales), and the authentic acetogens affiliated to Firmicutes decreased after the intervention. Principal coordinates analysis (PCoA) and Jackknife environment clusters revealed evidence on the shift of the microbial community structure among the samples. Quantitative analysis of methanogens via qPCR showed that Methanobacteriales and Methanomicrobiales increased after nutrient addition, while acetoclastic methanogens (Methanosaetaceae) changed slightly. Nutrient treatment activated native CO2-reducing methanogens in oil reservoir. The high frequency of Methanobacteriales and Methanomicrobiales (CO2-reducers) after nutrient addition in this petroleum system suggested that CO2-reducing methanogenesis was involved in methane production. The nutrient addition could promote the methane production. The results will likely improve strategies of utilizing microorganisms in subsurface environments.

Relationship of proteomic variation and toxin synthesis in the dinoflagellate Alexandrium tamarense CI01 under phosphorus and inorganic nitrogen limitation.

Paralytic shellfish toxins (PSTs) are originated from cyanobacteria and dinoflagellates, including Alexandrium tamarense, the common dinoflagellate species. In this study, a toxic dinoflagellate strain of A. tamarense CI01 was selected for studying the PSTs' concentration and the related protein variation during the whole cell cycle under different nutrient conditions. High-performance liquid chromatography, 2-D DIGE and Western blotting were used collectively for protein profiling and identification. Results showed that the toxin content was suppressed under nitrogen limiting condition, but enhanced in phosphorous limiting medium. Based on the results of proteomics analysis, 7 proteins were discovered to be related to the PSTs biosynthesis of A. tamarense CI01, including S-adenosylhomocysteine hydrolase, ornithine cyclodeaminase, argininosuccinate synthase, methyluridine methyltransferase cystine ABC transporter, phosphoserine phosphatase, argininosuccinate synthase and acyl-CoA dehydrogenase, which corresponds to the metabolism of the methionine, cysteine, ornithine, arginine and proline. Moreover, some photosynthesis relating proteins also increased their expression during PST synthesis period in A. tamarense CI01, such as phosphoenolpyruvate carboxylase, chloroplast phosphoglycerate kinase, peridinin-chlorophyll α-binding protein, Mg(2+) transporter protein and chloroplast phosphoglycerate kinase. The above findings are in support of our hypothesis that these proteins are involved in toxin biosynthesis of A. tamarense CI01, but cause-and-effect mechanisms need to be investigated in further studies.

Insights into the Anaerobic Biodegradation Pathway of n-Alkanes in Oil Reservoirs by Detection of Signature Metabolites.

Anaerobic degradation of alkanes in hydrocarbon-rich environments has been documented and different degradation strategies proposed, of which the most encountered one is fumarate addition mechanism, generating alkylsuccinates as specific biomarkers. However, little is known about the mechanisms of anaerobic degradation of alkanes in oil reservoirs, due to low concentrations of signature metabolites and lack of mass spectral characteristics to allow identification. In this work, we used a multidisciplinary approach combining metabolite profiling and selective gene assays to establish the biodegradation mechanism of alkanes in oil reservoirs. A total of twelve production fluids from three different oil reservoirs were collected and treated with alkali; organic acids were extracted, derivatized with ethanol to form ethyl esters and determined using GC-MS analysis. Collectively, signature metabolite alkylsuccinates of parent compounds from C1 to C8 together with their (putative) downstream metabolites were detected from these samples. Additionally, metabolites indicative of the anaerobic degradation of mono- and poly-aromatic hydrocarbons (2-benzylsuccinate, naphthoate, 5,6,7,8-tetrahydro-naphthoate) were also observed. The detection of alkylsuccinates and genes encoding for alkylsuccinate synthase shows that anaerobic degradation of alkanes via fumarate addition occurs in oil reservoirs. This work provides strong evidence on the in situ anaerobic biodegradation mechanisms of hydrocarbons by fumarate addition.

Synthesis of anaerobic degradation biomarkers alkyl-, aryl- and cycloalkylsuccinic acids and their mass spectral characteristics.

Anaerobic biodegradation of petroleum hydrocarbons has been reported to proceed predominantly via fumarate addition to yield substituted succinate metabolites. These metabolites, commonly regarded as signature biomarkers, are specific indicators of anaero- bic hydrocarbon degradation by microbial activity. To the best of our knowledge, mass spectrometry information for 2-(1-methylalkylj succinic acids, 2-arylsuccinic acids, 2-cycloalkylsuccinic acids and/or their derivatives is still incomplete, especially for the analysis of environmental samples. Here, a novel approach is proposed for the successful synthesis of five hydrocarbon-derived succinic acids. The characteristic fragments of 2-[1-methylalkyllsuccinic acid diesters were investigated by four derivatization processes (methyl, ethyl, n-butyl and trimethylsilyl esterification], some of which are not available in official Libraries. Under electron ionization mass spec- trometry conditions, informative fragments of various molecular masses have been obtained. Results confirmed characteristic differ- ences among the derivatization processes of the chemically synthesized compounds. In the case of 2-[cyclo)alkylsuccinate esters, four intermediate fragments were observed at m/z 114 + 14n, 118 + 28n, [M - [17 + 14n1]]+ and [M - (59 + 14n)]+ (n = 1, 2 and 4 for methyl, ethyl and n-butyl ester]. However, for silylation the abundant fragment ions are at m/z 262, 217, 172, 147, 73 and [M - 15]+. These data provide information for the identification of hydrocarbon-derived succinic acids as anaerobic biodegradation intermediates in hydrocarbons- rich environments.

Comparison of bacterial community in aqueous and oil phases of water-flooded petroleum reservoirs using pyrosequencing and clone library approaches.

Bacterial communities in both aqueous and oil phases of water-flooded petroleum reservoirs were characterized by molecular analysis of bacterial 16S rRNA genes obtained from Shengli Oil Field using DNA pyrosequencing and gene clone library approaches. Metagenomic DNA was extracted from the aqueous and oil phases and subjected to polymerase chain reaction amplification with primers targeting the bacterial 16S rRNA genes. The analysis by these two methods showed that there was a large difference in bacterial diversity between the aqueous and oil phases of the reservoir fluids, especially in the reservoirs with lower water cut. At a high phylogenetic level, the predominant bacteria detected by these two approaches were identical. However, pyrosequencing allowed the detection of more rare bacterial species than the clone library method. Statistical analysis showed that the diversity of the bacterial community of the aqueous phase was lower than that of the oil phase. Phylogenetic analysis indicated that the vast majority of sequences detected in the water phase were from members of the genus Arcobacter within the Epsilonproteobacteria, which is capable of degrading the intermediates of hydrocarbon degradation such as acetate. The oil phase of reservoir fluid samples was dominated by members of the genus Pseudomonas within the Gammaproteobacteria and the genus Sphingomonas within the Alphaproteobacteria, which have the ability to degrade crude oil through adherence to hydrocarbons under aerobic conditions. In addition, many anaerobes that could degrade the component of crude oil were also found in the oil phase of reservoir fluids, mainly in the reservoir with lower water cut. These were represented by Desulfovibrio spp., Thermodesulfovibrio spp., Thermodesulforhabdus spp., Thermotoga spp., and Thermoanaerobacterium spp. This research suggested that simultaneous analysis of DNA extracted from both aqueous and oil phases can facilitate a better understanding of the bacterial communities in water-flooded petroleum reservoirs.