Anaerobic Hydrocarbon Biodegradation Research Articles

Enrichment of Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Strictly Anaerobic Sulfate-Reducing Cultures from Contaminated Soil and Sediment.

Sulfate-reducing bacteria (SRB) are crucial players in global biogeochemical cycling and some have been implicated in the anaerobic biodegradation of organic pollutants, including recalcitrant and hazardous polycyclic aromatic hydrocarbons (PAHs). Obtaining PAH-degrading SRB cultures for laboratories is of paramount importance in the development of the young field of anaerobic biodegradation of PAHs. SRB grow exceptionally slowly on PAH substrates and are highly sensitive to oxygen. Consequently, enrichment and maintenance of PAH-degrading SRB cultures and characterization of the biodegradation process remain a tedious and formidable task, especially for new researchers. To address these technical constraints, we have developed robust and effective protocols for obtaining and characterizing PAH-degrading SRB cultures. In this set of protocols, we describe step-by-step procedures for preparing inocula from contaminated soil or sediment, preparing anoxic medium, establishing enrichment cultures with PAHs as substrates under completely anaerobic sulfate-reducing conditions, successive culture transfers to obtain highly enriched cultures, rapid verification of the viability of SRB in slow-growing cultures, assessment of PAH degradation by extracting residuals using organic solvent and subsequent analysis by gas chromatography-mass spectrometry, and spectrophotometric determination of sulfate and sulfide in miniaturized, medium-throughput format. These protocols are expected to serve as a comprehensive manual for obtaining and characterizing PAH-degrading sulfate-reducing cultures. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Obtaining PAH-degrading strictly anaerobic sulfate-reducing enrichment cultures from contaminated soil and sediment Support Protocol 1: Operation and maintenance of an anaerobic workstation Support Protocol 2: Setup of gas purging systems for preparing anoxic solutions Support Protocol 3: Verification of viability in slow-growing SRB enrichment cultures Support Protocol 4: Extraction of genomic DNA from low-biomass cultures Basic Protocol 2: Extraction of residual PAH from liquid culture and analysis by GC-MS Basic Protocol 3: Spectrophotometric determination of sulfate concentration in SRB cultures Basic Protocol 4: Spectrophotometric determination of sulfide concentrations in SRB cultures by the methylene blue method Alternate Protocol: Spectrophotometric determination of sulfide concentrations in SRB cultures by the colloidal copper sulfide method.

A combination of microbial electrolysis cells and bioaugmentation can effectively treat synthetic wastewater containing polycyclic aromatic hydrocarbon.

The anaerobic biodegradation of polycyclic aromatic hydrocarbons (PAHs) is challenging due to its toxic effect on the microbes. Microbial electrolysis cells (MECs), with their excellent characteristics of anodic and cathodic biofilms, can be a viable way to enhance the biodegradation of PAHs. This work assessed different cathode materials (carbon brush and nickel foam) combined with bioaugmentation on typical PAHs-naphthalene biodegradation and analyzed the inhibition amendment mechanism of microbial biofilms in MECs. Compared with the control, the degradation efficiency of naphthalene with the nickel foam cathode supplied with bioaugmentation dosage realized a maximum removal rate of 94.5 ± 3.2%. The highest daily recovered methane yield (227 ± 2 mL/gCOD) was also found in the nickel foam cathode supplied with bioaugmentation. Moreover, the microbial analysis demonstrated the significant switch of predominant PAH-degrading microorganisms from Pseudomonas in control to norank_f_Prolixibacteraceae in MECs. Furthermore, hydrogentrophic methanogenesis prevailed in MEC reactors, which is responsible for methane production. This study proved that MEC combined with bioaugmentation could effectively alleviate the inhibition of PAH, with the nickel foam cathode obtaining the fastest recovery rate in terms of methane yield.

Uncovering Anaerobic Hydrocarbon Biodegradation Pathways in Oil Sands Tailings from Two Different Tailings Ponds via Metabolite and Functional Gene Analyses.

Oil sands tailings, a slurry of alkaline water, silt, clay, unrecovered bitumen, and residual hydrocarbons generated during bitumen extraction, are contained in ponds. Indigenous microbes metabolize hydrocarbons and emit greenhouse gases from the tailings. Metabolism of hydrocarbons in tailings ponds of two operators, namely, Canadian Natural Upgrading Limited (CNUL) and Canadian Natural Resources Limited (CNRL), has not been comprehensively investigated. Previous reports have revealed sequential and preferential hydrocarbon degradation of alkanes in primary cultures established from CNUL and CNRL tailings amended separately with mixtures of hydrocarbons (n-alkanes, iso-alkanes, paraffinic solvent, or naphtha). In this study, activation pathway of hydrocarbon biodegradation in these primary cultures was investigated. The functional gene analysis revealed that fumarate addition was potentially the primary activation pathway of alkanes in all cultures. However, the metabolite analysis only detected transient succinylated 2-methylpentane and 2-methylbutane metabolites during initial methanogenic biodegradation of iso-alkanes and paraffinic solvent in all CNUL and CNRL cultures amended with iso-alkanes and paraffinic solvent. Under sulfidogenic conditions (prepared only with CNUL tailings amended with iso-alkanes), succinylated 2-methylpentane persisted throughout incubation period of ~ 1100days, implying dead-end nature of the metabolite. Though no metabolite was detected in n-alkanes- and naphtha-amended cultures during incubation, assA/masD genes related to Peptococcaceae were amplified in all CNUL and CNRL primary cultures. The findings of this present study suggest that microbial communities in different tailings ponds can biodegrade hydrocarbons through fumarate addition as activation pathway under methanogenic and sulfidogenic conditions.

The discrepant metabolic pathways of PAHs by facultative anaerobic bacteria under aerobic and nitrate-reducing conditions

Studies regarding the facultative anaerobic biodegradation of polycyclic aromatic hydrocarbons (PAHs) were still in the initial stage. In this study, a facultative anaerobe which was identified as Bacillus Firmus and named as PheN7 was firstly isolated from the mixed petroleum-polluted soil samples using phenanthrene and nitrate as the solo carbon resource and electron acceptor under anaerobic condition. The degradation rates of PheN7 towards phenanthrene were detected as 33.17 μM/d, 13.81 μM/d and 7.11 μM/d at the initial phenanthrene concentration of 250.17 μM with oxygen, nitrate and sulfate as the electron acceptor, respectively. The metabolic pathways toward phenanthrene by PheN7 were deduced combining the metagenome analysis of PheN7 and intermediate metabolites of phenanthrene under aerobic and nitrate-reducing conditions. Dioxygenation and carboxylation were inferred as the initial activation reactions of phenanthrene degradation in these two pathways. This study highlighted the significance of facultative anaerobic bacteria in natural PAHs biodegradation, revealing the discrepant metabolic fates of PAHs by one solo bacteria under aerobic and anaerobic environments.

Anaerobic hydrocarbon biodegradation by alkylotrophic methanogens in deep oil reservoirs.

In subsurface biodegraded oil reservoirs, methanogenic biodegradation of crude oil is a common process. This process was previously assigned to the syntrophy of hydrocarbon-degrading bacteria and methanogenic archaea. Recent studies showed that archaea of the Candidatus Methanoliparum named as alkylotrophic methanogens couple hydrocarbon degradation and methane production in a single archaeon. To assess the geochemical role of Ca. Methanoliparum, we analyzed the chemical and microbial composition and metabolites of 209 samples from 15 subsurface oil reservoirs across China. Gas chromatography-mass spectrometry analysis revealed that 92% of the tested samples were substantially degraded. Molecular analysis showed that 85% of the tested samples contained Ca. Methanoliparum, and 52% of the tested samples harbored multiple alkyl-coenzyme M derivatives, the intercellular metabolites of alkylotrophic archaea. According to metagenomic and metatranscriptomic analyses, Ca. Methanoliparum dominates hydrocarbon degradation in biodegraded samples from the Changqing, Jiangsu, and Shengli (SL) oilfields, and it is persistently present as shown in a 15-year-long sampling effort at the Shengli oilfield. Together, these findings demonstrate that Ca. Methanoliparum is a widely distributed oil degrader in reservoirs of China, suggesting that alkylotrophic methanogenesis by archaea plays a key role in the alteration of oil reservoirs, thereby expanding our understanding of biogeochemical process in the deep biosphere.

Evidence for the anaerobic biodegradation of higher molecular weight hydrocarbons in the Guaymas Basin

The hydrothermal sediments of the Guaymas Basin are rich in chemically complex petroleum components, including gaseous hydrocarbons, higher molecular weight n-alkanes and aromatic compounds. Studies of indigenous deep-sea microorganisms obtained from these sediments led to novel insights on the fundamental mechanisms for the anaerobic biodegradation of gaseous hydrocarbons. However, the metabolic fate of other hydrocarbons and the requisite organisms involved in the cycling of petroleum components remain largely unexplored. Metagenomic sequencing and laboratory enrichments were used to garner evidence for the anaerobic biodegradation of liquid hydrocarbons in hydrothermal sediments and mineral concretions from three sites in the Guaymas Basin (“Megamat II”, Rebecca's Roost and Octopus Mound). Elevated sulfate consumption by microbial enrichments from the Megamat II and Rebecca's Roost sites was observed at 55 °C when sediment incubations were amended with liquid n-alkanes (C6–C12; C16–C18) or with crude oil as potential electron donors. The biodegradation of the same hydrocarbons, linked to sulfate reduction, was also evident at 31 °C by microbial consortia enriched from the “Megamat II” and Octopus Mound sites. Notably, methane was concurrently produced in enrichments from the Octopus Mound site that was covered by Ampharetid methane-oxidizing worm mats. Metagenomic sequencing revealed that Deltaproteobacteria were predominant at the three sites (up to 47.48% of the microbial community). Moreover, the genes responsible for the activation of alkanes (Ass, alkylsuccinate synthase) and aromatic hydrocarbons (Bss, benzylsuccinate synthase) were identified from all three sites. The reconstruction of high-quality draft genomes from the Desulfobacterales further confirmed their metabolic potential to degrade alkanes via fumarate addition in Guaymas Basin sediments. Collectively, our findings demonstrate that mesophilic and thermophilic microbial consortia from hydrothermal sediments and mineral concretions in different regions of the Guaymas Basin are capable of anaerobically biodegrading C6–C12- and C16–C18n-alkanes as well as crude oil under the ambient temperature regimes. Our findings highlight the importance of integrating multiple approaches to better understand the ecological functioning of microbial communities in hydrocarbon-rich hydrothermal systems.

Enhancing the Anaerobic Biodegradation of Petroleum Hydrocarbons in Soils with Electrically Conductive Materials.

Anaerobic bioremediation is a relevant process in the management of sites contaminated by petroleum hydrocarbons. Recently, interspecies electron transfer processes mediated by conductive minerals or particles have been proposed as mechanisms through which microbial species within a community share reducing equivalents to drive the syntrophic degradation of organic substrates, including hydrocarbons. Here, a microcosm study was set up to investigate the effect of different electrically conductive materials (ECMs) in enhancing the anaerobic biodegradation of hydrocarbons in historically contaminated soil. The results of a comprehensive suite of chemical and microbiological analyses evidenced that supplementing the soil with (5% w/w) magnetite nanoparticles or biochar particles is an effective strategy to accelerate the removal of selected hydrocarbons. In particular, in microcosms supplemented with ECMs, the removal of total petroleum hydrocarbons was enhanced by up to 50% relative to unamended controls. However, chemical analyses suggested that only a partial bioconversion of contaminants occurred and that longer treatment times would have probably been required to drive the biodegradation process to completion. On the other hand, biomolecular analyses confirmed the presence of several microorganisms and functional genes likely involved in hydrocarbon degradation. Furthermore, the selective enrichment of known electroactive bacteria (i.e., Geobacter and Geothrix) in microcosms amended with ECMs, clearly pointed to a possible role of DIET (Diet Interspecies Electron Transfer) processes in the observed removal of contaminants.

CO2 improves the anaerobic biodegradation intensity and selectivity of heterocyclic hydrocarbons in heavy oil

Heterocyclic hydrocarbons pollution generated by oil spills and oilfield wastewater discharges threatens the ecological environment and human health. Here we described a strategy that combines the greenhouse gas CO2 reduction with microbial remediation. In the presence of nitrate, CO2 can improve the biodegradation efficiency of the resins and asphaltenes in heavy oil, particularly the biodegradation selectivity of the polar heterocyclic compounds by the newly isolated Klebsiella michiganensis. This strain encoded 80 genes for the xenobiotic biodegradation and metabolism, and can efficiently utilize CO2 when degrading heavy oil. The total abundance of resins and asphaltenes decreased significantly with CO2, from 40.816% to 26.909%, to 28.873% with O2, and to 36.985% with N2. The transcripts per million (TPM) value of accA gene was 57.81 under CO2 condition, while respectively 8.86 and 21.23 under O2 and N2 conditions. Under CO2 condition, the total relative percentage of N1-type heterocyclic compounds was selectively decreased from 32.25% to 22.78%, resulting in the heavy oil viscosity decreased by 46.29%. These results demonstrated a novel anaerobic degradation mechanism that CO2 can promote the anaerobic biodegradation of heterocyclic hydrocarbons in heavy oil, which provides a promising biotreatment technology for the oil-contaminated water.

Anaerobic biodegradation of polycyclic aromatic hydrocarbons (PAHs) by fungi isolated from anaerobic coal-associated sediments at 2.5km below the seafloor.

Fungi represent the dominant eukaryotic group in the deep biosphere and well-populated in the anaerobic coal-bearing sediments up to ∼2.5km below seafloor (kmbsf). But whether fungi are able to degrade and utilize coal to sustain growth in the anaerobic sub-seafloor environment remains unknown. Based on biodegradation investigation, we found that fungi isolated from sub-seafloor sediments at depths of ∼1.3-∼2.5 kmbsf showed a broad range of polycyclic aromatic hydrocarbons (PAHs) anaerobic degradation rates (3-25%). Among them, the white-rot fungus Schizophyllium commune 20R-7-F01 exhibited the highest degradation, 25%, 18% and 13%, of phenanthrene (Phe), pyrene (Pyr) and benzo[a]pyrene (BaP); respectively, after 10 days of anaerobic incubation. Phe was utilized well and about 40.4% was degraded by the fungus, after 20 days of anaerobic incubation. Moreover, the ability of fungi to degrade PAHs was positively correlated with the anaerobic growth of fungi, indicating that fungi can use PAHs as a sole carbon source under anoxic conditions. In addition, fungal degradation of PAHs was found to be related to the activity of carboxylases, but little or nothing to do with the activity of lignin modifying enzymes such as laccase (Lac), manganese peroxidase (MnP) and lignin peroxidase (LiP). These results suggest that sub-seafloor fungi possess a special mechanism to degrade and utilize PAHs as a carbon and energy source under anaerobic conditions. Furthermore, fungi living in sub-seafloor sediments may not only play an important role in carbon cycle in the anaerobic environments of the deep biosphere, but also be able to persist in deep sediment below seafloor for millions of years by using PAHs or related compounds as carbon and energy source. This anaerobic biodegradation ability could make these fungi suitable candidates for bioremediation of toxic pollutants such as PAHs from anoxic environments.

Anaerobic Biodegradation of Polycyclic Aromatic Hydrocarbons (Pahs) by Fungi Isolated from Anaerobic Coal-Associated Sediments at 2.5 Km Below the Seafloor

Anaerobic Biodegradation of Polycyclic Aromatic Hydrocarbons (Pahs) by Fungi Isolated from Anaerobic Coal-Associated Sediments at 2.5 Km Below the Seafloor

Unraveling the Metabolic Potential of Asgardarchaeota in a Sediment from the Mediterranean Hydrocarbon-Contaminated Water Basin Mar Piccolo (Taranto, Italy).

Increasing number of metagenome sequencing studies have proposed a central metabolic role of still understudied Archaeal members in natural and artificial ecosystems. However, their role in hydrocarbon cycling, particularly in the anaerobic biodegradation of aliphatic and aromatic hydrocarbons, is still mostly unknown in both marine and terrestrial environments. In this work, we focused our study on the metagenomic characterization of the archaeal community inhabiting the Mar Piccolo (Taranto, Italy, central Mediterranean) sediments heavily contaminated by petroleum hydrocarbons and polychlorinated biphenyls (PCB). Among metagenomic bins reconstructed from Mar Piccolo microbial community, we have identified members of the Asgardarchaeota superphylum that has been recently proposed to play a central role in hydrocarbon cycling in natural ecosystems under anoxic conditions. In particular, we found members affiliated with Thorarchaeota, Heimdallarchaeota, and Lokiarchaeota phyla and analyzed their genomic potential involved in central metabolism and hydrocarbon biodegradation. Metabolic prediction based on metagenomic analysis identified the malonyl-CoA and benzoyl-CoA routes as the pathways involved in aliphatic and aromatic biodegradation in these Asgardarchaeota members. This is the first study to give insight into the archaeal community functionality and connection to hydrocarbon degradation in marine sediment historically contaminated by hydrocarbons.

Damage of anodic biofilms by high salinity deteriorates PAHs degradation in single-chamber microbial electrolysis cell reactor.

The anaerobic biodegradation of polycyclic aromatic hydrocarbons (PAHs) in high salinity wastewater is rather hard due to the inhibition of microorganisms by complex and high dosage of salts. Microbial electrolysis cell (MEC), with its excellent characteristic of anodic biofilms, can be an effective way to enhance the PAHs biodegradation. This work evaluated the impact of NaCl concentrations (0g/L, 10g/L, 30g/L, and 60g/L) on naphthalene biodegradation and analyzed the damage protection mechanism of anodic biofilms in batching MECs. Compared with the open circuit, the degradation efficiency of naphthalene under the closed circuit with 10g/L NaCl concentration reached the maximum of 95.17% within 5days. Even when NaCl concentration reached 60g/L, the degradation efficiency only decreased by 10.02%, compared with the MEC without additional NaCl. Confocal scanning laser microscope (CSLM) proved the superiority of the biofilm states of MEC anode under high salinity in terms of thicker biofilms and higher proportion of live/dead bacteria cells. The highest dehydrogenase activity (DHA) was found in the MEC with 10g/L NaCl concentration. Moreover, microbial diversity analysis demonstrated the classical electroactive microorganisms Geobacter and Pseudomonas were found on the anodic biofilms of MECs, which have both PAHs degradability and the electrochemical activity. Therefore, this study proved that high salinity had adverse effects on the anodic biofilms, but MEC alleviated the damage caused by high salinity.

Release of carboxylic acids into the exometabolome during anaerobic growth of a denitrifying bacterium with single substrates or crude oil

Anaerobic biodegradation of hydrocarbons mainly related to petroleum can be observed in numerous natural and anthropogenically overprinted environments. While microbiological and chemical tracers are used to study the development of such habitats, not much is known about the impact of particular bacteria on the system. Therefore, we used Aromatoleum sp. HxN1, an n-alkane-utilizing betaproteobacterium capable of degrading crude oil constituents under strictly anoxic conditions, as a model organism to examine its potential to release carboxylic acid metabolites into the surrounding environment. We conducted cultivation experiments with pure substrates, as well as with crude oil, and demonstrated that intermediates of catabolic pathways were highly abundant in the exometabolome. Strikingly, the metabolites found in highest concentrations in the cell-free culture supernatant of strain HxN1 grown with crude oil were associated with the transformation of potentially toxic aromatic hydrocarbons, which do not support growth of this particular bacterium. Additionally, a deficiency in coenzyme A caused by a highly productive substrate transformation may induce the excretion of a surplus of metabolites in order to regain coenzyme A. Under natural conditions, i.e., in the presence of mixed communities, such transformation products may be further degraded by other organisms, ultimately leading to mineralization.

New Frontiers of Anaerobic Hydrocarbon Biodegradation in the Multi-Omics Era.

The accumulation of petroleum hydrocarbons in the environment substantially endangers terrestrial and aquatic ecosystems. Many microbial strains have been recognized to utilize aliphatic and aromatic hydrocarbons under aerobic conditions. Nevertheless, most of these pollutants are transferred by natural processes, including rain, into the underground anaerobic zones where their degradation is much more problematic. In oxic zones, anaerobic microenvironments can be formed as a consequence of the intensive respiratory activities of (facultative) aerobic microbes. Even though aerobic bioremediation has been well-characterized over the past few decades, ample research is yet to be done in the field of anaerobic hydrocarbon biodegradation. With the emergence of high-throughput techniques, known as omics (e.g., genomics and metagenomics), the individual biodegraders, hydrocarbon-degrading microbial communities and metabolic pathways, interactions can be described at a contaminated site. Omics approaches provide the opportunity to examine single microorganisms or microbial communities at the system level and elucidate the metabolic networks, interspecies interactions during hydrocarbon mineralization. Metatranscriptomics and metaproteomics, for example, can shed light on the active genes and proteins and functional importance of the less abundant species. Moreover, novel unculturable hydrocarbon-degrading strains and enzymes can be discovered and fit into the metabolic networks of the community. Our objective is to review the anaerobic hydrocarbon biodegradation processes, the most important hydrocarbon degraders and their diverse metabolic pathways, including the use of various terminal electron acceptors and various electron transfer processes. The review primarily focuses on the achievements obtained by the current high-throughput (multi-omics) techniques which opened new perspectives in understanding the processes at the system level including the metabolic routes of individual strains, metabolic/electric interaction of the members of microbial communities. Based on the multi-omics techniques, novel metabolic blocks can be designed and used for the construction of microbial strains/consortia for efficient removal of hydrocarbons in anaerobic zones.

Combined Use of Diagnostic Fumarate Addition Metabolites and Genes Provides Evidence for Anaerobic Hydrocarbon Biodegradation in Contaminated Groundwater.

The widespread use of hydrocarbon-based fuels has led to the contamination of many natural environments due to accidental spills or leaks. While anaerobic microorganisms indigenous to many fuel-contaminated groundwater sites can play a role in site remediation (e.g., monitored natural attenuation, MNA) via hydrocarbon biodegradation, multiple lines of evidence in support of such bioremediation are required. In this study, we investigated two fuel-contaminated groundwater sites for their potential to be managed by MNA. Microbial community composition, biogeochemical indicators, fumarate addition metabolites, and genes diagnostic of both alkane and alkyl-monoaromatic hydrocarbon activation were assessed. Fumarate addition metabolites and catabolic genes were detected for both classes of hydrocarbon biodegradation at both sites, providing strong evidence for in situ anaerobic hydrocarbon biodegradation. However, relevant metabolites and genes did not consistently co-occur within all groundwater samples. Using newly designed mixtures of quantitative polymerase chain reaction (qPCR) primers to target diverse assA and bssA genes, we measured assA gene abundances ranging from 105–108 copies/L, and bssA gene abundances ranging from 105–1010 copies/L at the sites. Overall, this study demonstrates the value of investigating fuel-contaminated sites using both metabolites and genes diagnostic of anaerobic hydrocarbon biodegradation for different classes of hydrocarbons to help assess field sites for management by MNA.

Oil Hydrocarbon Degradation by Caspian Sea Microbial Communities.

The Caspian Sea, which is the largest landlocked body of water on the planet, receives substantial annual hydrocarbon input from anthropogenic sources (e.g., industry, agriculture, oil exploration, and extraction) and natural sources (e.g., mud volcanoes and oil seeps). The Caspian Sea also receives substantial amounts of runoff from agricultural and municipal sources, containing nutrients that have caused eutrophication and subsequent hypoxia in the deep, cold waters. The effect of decreasing oxygen saturation and cold temperatures on oil hydrocarbon biodegradation by a microbial community is not well characterized. The purpose of this study was to investigate the effect of oxic and anoxic conditions on oil hydrocarbon biodegradation at cold temperatures by microbial communities derived from the Caspian Sea. Water samples were collected from the Caspian Sea for study in experimental microcosms. Major taxonomic orders observed in the ambient water samples included Flavobacteriales, Actinomycetales, and Oceanospirillales. Microcosms were inoculated with microbial communities from the deepest waters and amended with oil hydrocarbons for 17 days. Hydrocarbon degradation and shifts in microbial community structure were measured. Surprisingly, oil hydrocarbon biodegradation under anoxic conditions exceeded that under oxic conditions; this was particularly evident in the degradation of aromatic hydrocarbons. Important microbial taxa associated with the anoxic microcosms included known oil degraders such as Oceanospirillaceae. This study provides knowledge about the ambient community structure of the Caspian Sea, which serves as an important reference point for future studies. Furthermore, this may be the first report in which anaerobic biodegradation of oil hydrocarbons exceeds aerobic biodegradation.

Methanogenic biodegradation of crude oil storage tank sludge enhances bio-corrosion of mild steel

Methanogenic biodegradation of crude oil sludge was investigated using chemical and molecular approaches. 16S rRNA gene sequences recovered from the samples revealed significant presence of Marinobacterium (63%), Pseudomonas (3%), acetotrophic Methanosaeta (16%) and hydrogenotrophic Methanobacterium (5%). The resident microbial community was able to reduce the gravimetric weight of residual oil by 65.5% (with complete degradation of C5–C17 n-alkane fractions) in non-amended samples and 94.13% (with complete degradation of C5–C25 n-alkane fractions) in substrate-amended samples during the 60-day incubation period. As biodegradation progressed, acetotrophs consume acetate at the rate of 0.41 mM/day−1 while hydrogenotrophs consume hydrogen at the rate of 0.59 mM/day−1. Respective volume of methane produced and corrosion rates observed were higher in highly biodegraded samples (3.60 mmol/0.084 mm/year) than lesser biodegraded samples (1.64 mmol/0.018 mm/year). Our results showed that the resident methanogenic archaea were largely responsible for the anaerobic biodegradation of hydrocarbons in crude oil sludge and biodegradation was enhanced with substrate amendment, which further accelerated the corrosion rates of mild steel coupons. Considering the relatively high number of facultatively anaerobic Marinobacterium and significant presence of Pseudomonas in the sequenced data, we speculate that the bacteria were at least partially responsible for biodegradation of crude oil components potentially acting as syntotrophic organisms with methanogens to convert crude oil to methane and subsequently enhance corrosion rates of mild steel coupons.

Enzymes and sugars: Challenges and opportunities for biocatalytic process development

Anaerobic hydrocarbon biodegradation not only diminishes fuel quality, but also exacerbates the biocorrosion of the metallic infrastructure. While successional events in marine microbial ecosystems impacted by petroleum are well documented, far less is known about the response of communities chronically exposed to hydrocarbons. Shipboard oily wastewater was used to assess the biotransformation of different diesel fuels and their propensity to impact carbon steel corrosion. When amended with sulfate and an F76 military diesel fuel, the sulfate removal rate in the assay mixtures was elevated (26.8 μM/d) relative to incubations receiving a hydroprocessed biofuel (16.1 μM/d) or a fuel-unamended control (17.8 μM/d). Microbial community analysis revealed the predominance of Anaerolineae and Deltaproteobacteria in F76-amended incubations, in contrast to the Beta- and Gammaproteobacteria in the original wastewater. The dominant Smithella-like sequences suggested the potential for syntrophic hydrocarbon metabolism. The general corrosion rate was relatively low (0.83 − 1.29 ± 0.12 mpy) and independent of the particular fuel, but pitting corrosion was more pronounced in F76-amended incubations. Desulfovibrionaceae constituted 50–77% of the sessile organisms on carbon steel coupons. Thus, chronically exposed microflora in oily wastewater were differentially acclimated to the syntrophic metabolism of traditional hydrocarbons but tended to resist isoalkane-laden biofuels.

Anaerobic Biodegradation of Polyaromatic Hydrocarbons by a Sulfate Reducing Bacteria C1Fd Strain

Anaerobic Biodegradation of Polyaromatic Hydrocarbons by a Sulfate Reducing Bacteria C1Fd Strain

Comprehensive two-dimensional gas chromatography-mass spectrometry of complex mixtures of anaerobic bacterial metabolites of petroleum hydrocarbons

Anaerobic biotransformation of petroleum hydrocarbons is an important alteration mechanism, both subsurface in geological reservoirs, in aquifers and in anoxic deep sea environments. Here we report the resolution and identification, by comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC–MS), of complex mixtures of aromatic acid and diacid metabolites of the anaerobic biodegradation of many crude oil hydrocarbons. An extended range of metabolites, including alkylbenzyl, alkylindanyl, alkyltetralinyl, alkylnaphthyl succinic acids and alkyltetralin, alkylnaphthoic and phenanthrene carboxylic acids, is reported in samples from experiments conducted under sulfate-reducing conditions in a microcosm over two years. The range of metabolites identified shows that the fumarate addition mechanism applies to the alteration of hydrocarbons with up to C8 alkylation in monoaromatics and that functionalisation of up to three ring aromatic hydrocarbons with at least C1 alkylation occurs. The GC×GC–MS method might now be applied to the identification of complex mixtures of metabolites in samples from real environmental oil spills.