Shi-Zhong Yang

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.

Effects of Different Amino Acids in Culture Media on Surfactin Variants Produced by Bacillus subtilis TD7

Surfactin produced by Bacillus subtilis has different variants, which are affected by the composition of substrate available. To demonstrate the effects of amino acids on surfactin variants, B. subtilis TD7 was cultivated under the same conditions but with different amino acids supplied in media, respectively, and the type as well as the proportion of surfactin variants produced was analyzed with electrospray ionization mass spectrometry and gas chromatography–mass spectrometry. The result shows that the addition of different amino acids significantly influences the proportion of surfactin variants with different fatty acids. When Arg, Gln, or Val was added to the culture medium of B. subtilis TD7, the proportion of produced surfactin variants with even β-hydroxy fatty acids significantly increased, while the addition of Cys, His, Ile, Leu, Met, Ser, or Thr enhanced the proportion of surfactin variants with odd β-hydroxy fatty acids markedly. This result may be of some reference value in enhancing the production of specific surfactin variants as well as in the research on the relationship between culture media and the corresponding products of a certain bacterium.

Chemical Structure, Property and Potential Applications of Biosurfactants Produced by Bacillus subtilis in Petroleum Recovery and Spill Mitigation

Lipopeptides produced by microorganisms are one of the five major classes of biosurfactants known and they have received much attention from scientific and industrial communities due to their powerful interfacial and biological activities as well as environmentally friendly characteristics. Microbially produced lipopeptides are a series of chemical structural analogues of different families and, among them, 26 families covering about 90 lipopeptide compounds have been reported in the last two decades. This paper reviews the chemical structural characteristics and molecular behaviors of surfactin, one of the representative lipopeptides of the 26 families. In particular, two novel surfactin molecules isolated from cell-free cultures of Bacillus subtilis HSO121 are presented. Surfactins exhibit strong self-assembly ability to form sphere-like micelles and larger aggregates at very low concentrations. The amphipathic and surface properties of surfactins are related to the existence of the minor polar and major hydrophobic domains in the three 3-D conformations. In addition, the application potential of surfactin in bioremediation of oil spills and oil contaminants, and microbial enhanced oil recovery are discussed.

High Frequency of Thermodesulfovibrio spp. and Anaerolineaceae in Association with Methanoculleus spp. in a Long-Term Incubation of n-Alkanes-Degrading Methanogenic Enrichment Culture

In the present study, the microbial community and functional gene composition of a long-term active alkane-degrading methanogenic culture was established after two successive enrichment culture transfers and incubated for a total period of 1750 days. Molecular analysis was conducted after the second transfer (incubated for 750 days) for both the active alkanes-degrading methanogenic enrichment cultures (T2-AE) and the background control (T2-BC). A net increase of methane as the end product was detected in the headspace of the enrichment cultures amended with long-chain n-alkanes and intermediate metabolites, including octadecanoate, hexadecanoate, isocaprylate, butyrate, isobutyrate, propionate, acetate, and formate were measured in the liquid cultures. The composition of microbial community shifted through the successive transfers over time of incubation. Sequences of bacterial and archaeal 16S rRNA gene (16S rDNA) and mcrA functional gene indicated that bacterial sequences affiliated to Thermodesulfovibrio spp. and Anaerolineaceae and archaeal sequences falling within the genus Methanoculleus were the most frequently encountered and thus represented the dominant members performing the anaerobic degradation of long-chain n-alkanes and methanogenesis. In addition, the presence of assA functional genes encoding the alkylsuccinate synthase α subunit indicated that fumarate addition mechanism could be considered as a possible initial activation step of n-alkanes in the present study. The succession pattern of microbial communities indicates that Thermodesulfovibrio spp. could be a generalist participating in the metabolism of intermediates, while Anaerolineaceae plays a key role in the initial activation of long-chain n-alkane biodegradation.

The biofilm property and its correlationship with high-molecular-weight polyacrylamide degradation in a water injection pipeline of Daqing oilfield

Biofilms increase dragging force for liquid transportation, cause power consumption, and result in equipment corrosion in polymer-flooding oilfields. To reveal the responsible microorganisms for biofilm formation and stability of high-molecular-weight polyacrylamide (PAM), a biofilm, developed on the sieve of a piston plunger pump in a water transport and injection pipeline with partial hydrolyzed polyacrylamide (HPAM) in Daqing Oilfield, was collected and analyzed by molecular microbiology, chemical and physical methods. Diverse bacterial groups (11 families) were detected in the biofilm, including Pseudomonadaceae, Rhodocyclaceae, Desulfobulbaceae, Alcaligenaceae, Comamonadaceae, Oxalobacteraceae, Bacteriovoracaceae, Campylobacteraceae, Flavobacteriaceae, Clostridiales Incertae Sedis XIII and Moraxellaceae. Three archaeal orders of methanogens including Methanomicrobiales, Methanosarcinales and Thermoplasmatales were also detected separately. HPAM was degraded into lower molecular weight polymers and organic fragments with its amide groups hydrolyzed into carboxylic groups by the microorganisms. The microenvironment of the biofilm contained diverse bacterial and archaeal communities, correlating with the extracellular polymeric substance (EPS) and HPAM biodegradation. The results are helpful to provide information for biofilm control in oil fields.

The Rebirth of Waste Cooking Oil to Novel Bio-based Surfactants

Waste cooking oil (WCO) is a kind of non-edible oil with enormous quantities and its unreasonable dispose may generate negative impact on human life and environment. However, WCO is certainly a renewable feedstock of bio-based materials. To get the rebirth of WCO, we have established a facile and high-yield method to convert WCO to bio-based zwitterionic surfactants with excellent surface and interfacial properties. The interfacial tension between crude oil and water could reach ultra-low value as 0.0016 mN m−1 at a low dosage as 0.100 g L−1 of this bio-based surfactant without the aid of extra alkali, which shows a strong interfacial activity and the great potential application in many industrial fields, in particular, the application in enhanced oil recovery in oilfields in place of petroleum-based surfactants.

Diversity and Composition of Sulfate-Reducing Microbial Communities Based on Genomic DNA and RNA Transcription in Production Water of High Temperature and Corrosive Oil Reservoir

Deep subsurface petroleum reservoir ecosystems harbor a high diversity of microorganisms, and microbial influenced corrosion is a major problem for the petroleum industry. Here, we used high-throughput sequencing to explore the microbial communities based on genomic 16S rDNA and metabolically active 16S rRNA analyses of production water samples with different extents of corrosion from a high-temperature oil reservoir. Results showed that Desulfotignum and Roseovarius were the most abundant genera in both genomic and active bacterial communities of all the samples. Both genomic and active archaeal communities were mainly composed of Archaeoglobus and Methanolobus. Within both bacteria and archaea, the active and genomic communities were compositionally distinct from one another across the different oil wells (bacteria p = 0.002; archaea p = 0.01). In addition, the sulfate-reducing microorganisms (SRMs) were specifically assessed by Sanger sequencing of functional genes aprA and dsrA encoding the enzymes adenosine-5′-phosphosulfate reductase and dissimilatory sulfite reductase, respectively. Functional gene analysis indicated that potentially active Archaeoglobus, Desulfotignum, Desulfovibrio, and Thermodesulforhabdus were frequently detected, with Archaeoglobus as the most abundant and active sulfate-reducing group. Canonical correspondence analysis revealed that the SRM communities in petroleum reservoir system were closely related to pH of the production water and sulfate concentration. This study highlights the importance of distinguishing the metabolically active microorganisms from the genomic community and extends our knowledge on the active SRM communities in corrosive petroleum reservoirs.

Structural Diversity of the Microbial Surfactin Derivatives from Selective Esterification Approach

Surfactin originated from genus Bacillus is composed of a heptapeptide moiety bonded to the carboxyl and hydroxyl groups of a β-hydroxy fatty acid and it can be chemically modified to prepare the derivatives with different structures, owing to the existence of two free carboxyl groups in its peptide loop. This article presents the chemical modification of surfactin esterified with three different alcohols, and nine novel surfactin derivatives have been separated from products by the high performance liquid chromatography (HPLC). The novel derivatives, identified with Fourier transform infrared spectroscopy (FT-IR) and electrospray ionization mass spectrometry (ESI-MS), are the mono-hexyl-surfactin C14 ester, mono-hexyl-surfactin C15 ester, mono-2-methoxy-ethyl-surfactin C14 ester, di-hexyl-surfactin C14 ester, di-hexyl-surfactin ester C15, di-2-methoxy-ethyl-surfactin ester C14, di-2-methoxy-ethyl-surfactin ester C15, di-6-hydoxyl-hexyl-surfactin C14 ester and, di-6-hydoxyl-hexyl-surfactin C15 ester. The reaction conditions for esterification were optimized and the dependence of yields on different alcohols and catalysts were discussed. This study shows that esterification is one of the most efficient ways of chemical modification for surfactin and it can be used to prepare more derivatives to meet the needs of study in biological and interfacial activities.

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 anaerobic hydrocarbon degradation by microbial activity. To the best of our knowledge, mass spectrometry information for 2-(1-methylalkyl) 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-methylalkyl)succinic 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 spectrometry conditions, informative fragments of various molecular masses have been obtained. Results confirmed characteristic differences 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 + 14n)]+ 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.

Formate-Dependent Microbial Conversion of CO2 and the Dominant Pathways of Methanogenesis in Production Water of High-temperature Oil Reservoirs Amended with Bicarbonate

CO2 sequestration in deep-subsurface formations including oil reservoirs is a potential measure to reduce the CO2 concentration in the atmosphere. However, the fate of the CO2 and the ecological influences in carbon dioxide capture and storage (CDCS) facilities is not understood clearly. In the current study, the fate of CO2 (in bicarbonate form; 0∼90 mM) with 10 mM of formate as electron donor and carbon source was investigated with high-temperature production water from oilfield in China. The isotope data showed that bicarbonate could be reduced to methane by methanogens and major pathway of methanogenesis could be syntrophic formate oxidation coupled with CO2 reduction and formate methanogenesis under the anaerobic conditions. The bicarbonate addition induced the shift of microbial community. Addition of bicarbonate and formate was associated with a decrease of Methanosarcinales, but promotion of Methanobacteriales in all treatments. Thermodesulfovibrio was the major group in all the samples and Thermacetogenium dominated in the high bicarbonate treatments. The results indicated that CO2 from CDCS could be transformed to methane and the possibility of microbial CO2 conversion for enhanced microbial energy recovery in oil reservoirs.