Hydrocarbon-degrading Microbial Communities Research Articles

Methanogenesis coupled hydrocarbon biodegradation enhanced by ferric and sulphate ions.

Bioremediation provides an environmentally sound solution for hydrocarbon removal. Although bioremediation under anoxic conditions is slow, it can be coupled with methanogenesis and is suitable for energy recovery. By altering conditions and supplementing alternative terminal electron acceptors to the system to induce syntrophic partners of the methanogens, this process can be enhanced. In this study, we investigated a hydrocarbon-degrading microbial community derived from chronically contaminated soil. Various hydrocarbon mixtures were used during our experiments in the presence of different electron acceptors. In addition, we performed whole metagenome sequencing to identify the main actors of hydrocarbon biodegradation in the samples. Our results showed that the addition of ferric ions or sulphate increased the methane yield. Furthermore, the addition of CO2, ferric ion or sulphate enhanced the biodegradation of alkanes. A significant increase in biodegradation was observed in the presence of ferric ions or sulphate in the case of all aromatic components, while naphthalene and phenanthrene degradation was also enhanced by CO2. Metagenome analysis revealed that Cellulomonas sp. is the most abundant in the presence of alkanes, while Ruminococcus and Faecalibacterium spp. are prevalent in aromatics-supplemented samples. From the recovery of 25 genomes, it was concluded that the main pathway of hydrocarbon activation was fumarate addition in both Cellulomonas, Ruminococcus and Faecalibacterium. Chloroflexota bacteria can utilise the central metabolites of aromatics biodegradation via ATP-independent benzoyl-CoA reduction. KEY POINTS: • Methanogenesis and hydrocarbon biodegradation were enhanced by Fe3+ or SO42- • Cellulomonas, Ruminococcus and Faecalibacterium can be candidates for the main hydrocarbon degraders • Chloroflexota bacteria can utilise the central metabolites of aromatics degradation.

Interactions between plants and bacterial communities for phytoremediation of petroleum-contaminated soil.

Plants can stimulate the microbes to degrade ubiquitous petroleum hydrocarbons (PHCs), which has prompted a novel view on rhizoremediation. In the present study, the degradation rateof PHCs was investigated and 16S rRNA gene analysis was performed to investigate the PHC-degrading bacteria in petroleum-contaminated soil with different plants. Mirabilis jalapa (M. jalapa) has a higher PHC degradation rate than Lolium perenne (L. perenne) under petroleum contamination. The bacterial diversity in rhizospheric soil was decreased but the relative abundance of Actinobacteriota, Proteobacteria, and Candidatus Saccharibacteria were significant increased on 45days petroleum-contaminated rhizospheric soil. In addition, the relative expression of PHC degradation-related genes, the content ofmalic acid and citric acid of the root exudates in the two plants was significantly increased in response to petroleum stress. The content ofcitric acid increased 11.9 times in M. jalapa and 3.4 times in L. perenne, respectively, in response to petroleum stress. These results indicate that M. jalapa changes the hydrocarbon-degrading microbial community to enhance the degradation of PHCs by root exudates and phytostimulation.

A full-scale bioremediation study of diesel fuel-contaminated soil: the effect of plant species and soil amendments

This article presents a comprehensive on-site bioremediation of an excavated diesel fuel-contaminated soil aided by various soil amendments and plant species. In the first 125 days, the excavated soil was placed in a land treatment unit, mixed with compost, and seeded with white clover (Trifolium repens). In the next 155 days, the land treatment unit was re-established and divided into four experimental plots. Experimental plots 2 and 3 were seeded with a mixture of plant seeds (Trifolium alexandrinum, Fagopyrum esculentum Moench, Trifolium incarnatum, Phacelia tanacetifolia, Sinapis alba, Trifolium repens), while experimental plots 1 and 4 were left without seeded vegetation. Moreover, a zeolite and an Ascophyllum nodosum-based biostimulant were added to experimental plots 3 and 4. The objectives were to select the most beneficial plant species for the plant-based bioremediation process, to evaluate applicability of different soil amendments for an enhanced hydrocarbon biodegradation, and to study their effects on the microbial community in soil. Our results showed an overall reduction in hydrocarbon pollution by more than 95% within 280 days. The rates of hydrocarbon degradation and changes in the microbial population were not affected by the presence of the zeolite and the biostimulant. In addition, the use of different plant species did not have a statistically significant effect on hydrocarbon degradation but affected microbial population dynamics, confirming stabile and diverse indigenous hydrocarbon-degrading microbial community in the native soil.

A Review and Bibliometric Analysis on Applications of Microbial Degradation of Hydrocarbon Contaminants in Arctic Marine Environment at Metagenomic and Enzymatic Levels.

The globe is presently reliant on natural resources, fossil fuels, and crude oil to support the world’s energy requirements. Human exploration for oil resources is always associated with irreversible effects. Primary sources of hydrocarbon pollution are instigated through oil exploration, extraction, and transportation in the Arctic region. To address the state of pollution, it is necessary to understand the mechanisms and processes of the bioremediation of hydrocarbons. The application of various microbial communities originated from the Arctic can provide a better interpretation on the mechanisms of specific microbes in the biodegradation process. The composition of oil and consequences of hydrocarbon pollutants to the various marine environments are also discussed in this paper. An overview of emerging trends on literature or research publications published in the last decade was compiled via bibliometric analysis in relation to the topic of interest, which is the microbial community present in the Arctic and Antarctic marine environments. This review also presents the hydrocarbon-degrading microbial community present in the Arctic, biodegradation metabolic pathways (enzymatic level), and capacity of microbial degradation from the perspective of metagenomics. The limitations are stated and recommendations are proposed for future research prospects on biodegradation of oil contaminants by microbial community at the low temperature regions of the Arctic.

Effect of Temperature on Acetate Mineralization Kinetics and Microbial Community Composition in a Hydrocarbon-Affected Microbial Community During a Shift From Oxic to Sulfidogenic Conditions.

Aquifer thermal energy storage (ATES) allows for the seasonal storage and extraction of heat in the subsurface thus reducing reliance on fossil fuels and supporting decarbonization of the heating and cooling sector. However, the impacts of higher temperatures toward biodiversity and ecosystem services in the subsurface environment remain unclear. Here, we conducted a laboratory microcosm study comprising a hydrocarbon-degrading microbial community from a sulfidic hydrocarbon-contaminated aquifer spiked with 13C-labeled acetate and incubated at temperatures between 12 and 80°C to evaluate (i) the extent and rates of acetate mineralization and (ii) the resultant temperature-induced shifts in the microbial community structure. We observed biphasic mineralization curves at 12, 25, 38, and 45°C, arising from immediate and fast aerobic mineralization due to an initial oxygen exposure, followed by slower mineralization at sulfidogenic conditions. At 60°C and several replicates at 45°C, acetate was only aerobically mineralized. At 80°C, no mineralization was observed within 178 days. Rates of acetate mineralization coupled to sulfate reduction at 25 and 38°C were six times faster than at 12°C. Distinct microbial communities developed in oxic and strictly anoxic phases of mineralization as well as at different temperatures. Members of the Alphaproteobacteria were dominant in the oxic mineralization phase at 12–38°C, succeeded by a more diverse community in the anoxic phase composed of Deltaproteobacteria, Clostridia, Spirochaetia, Gammaproteobacteria and Anaerolinea, with varying abundances dependent on the temperature. In the oxic phases at 45 and 60°C, phylotypes affiliated to spore-forming Bacilli developed. In conclusion, temperatures up to 38°C allowed aerobic and anaerobic acetate mineralization albeit at varying rates, while mineralization occurred mainly aerobically between 45 and 60°C; thermophilic sulfate reducers being active at temperatures > 45°C were not detected. Hence, temperature may affect dissolved organic carbon mineralization rates in ATES while the variability in the microbial community composition during the transition from micro-oxic to sulfidogenic conditions highlights the crucial role of electron acceptor availability when combining ATES with bioremediation.

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.

An underappreciated DIET for anaerobic petroleum hydrocarbon-degrading microbial communities.

Direct interspecies electron transfer (DIET) via electrically conductive minerals can play a role in the anaerobic oxidation of petroleum hydrocarbons in contaminated sites and can be exploited for the development of new, more effective bioremediation approaches.

Structure and Functions of Hydrocarbon-Degrading Microbial Communities in Bioelectrochemical Systems

Bioelectrochemical systems (BESs) exploit the interaction between microbes and electrodes. A field of application thereof is bioelectrochemical remediation, an effective strategy in environments where the absence of suitable electron acceptors limits classic bioremediation approaches. Understanding the microbial community structure and genetic potential of anode biofilms is of great interest to interpret the mechanisms occurring in BESs. In this study, by using a whole metagenome sequencing approach, taxonomic and functional diversity patterns in the inoculum and on the anodes of three continuous-flow BES for the removal of phenol, toluene, and BTEX were obtained. The genus Geobacter was highly enriched on the anodes and two reconstructed genomes were taxonomically related to the Geobacteraceae family. To functionally characterize the microbial community, the genes coding for the anaerobic degradation of toluene, ethylbenzene, and phenol were selected as genetic markers for the anaerobic degradation of the pollutants. The genes related with direct extracellular electron transfer (EET) were also analyzed. The inoculum carried the genetic baggage for the degradation of aromatics but lacked the capacity of EET while anodic bacterial communities were able to pursue both processes. The metagenomic approach provided useful insights into the ecology and complex functions within hydrocarbon-degrading electrogenic biofilms.

Hydrocarbon-Degrading Microbial Communities Are Site Specific, and Their Activity Is Limited by Synergies in Temperature and Nutrient Availability in Surface Ocean Waters.

The objective of this study was to quantify the potential for hydrocarbon biodegradation in surface waters of three sites, representing geographic regions of major oil exploration (Beaufort Sea in the Arctic, northern Gulf of Mexico [GOM], and southern GOM), in a systematic experimental design that incorporated gradients in temperature and the availability of major nutrients. Surface seawater was amended in microcosms with Macondo surrogate oil to simulate an oil slick, and microcosms were incubated, with or without nutrient amendment, at temperatures ranging from 4 to 38ºC. Using respiration rate as a proxy, distinct temperature responses were observed in surface seawater microcosms based on geographic origin; biodegradation was nearly always more rapid in the Arctic site samples than in the GOM samples. Nutrient amendment enhanced respiration rates by a factor of approximately 6, stimulated microbial growth, and generally elevated the taxonomic diversity of microbial communities within the optimal temperature range for activity at each site, while diversity remained the same or was lower at temperatures deviating from optimal conditions. Taken together, our results advance the understanding of how bacterioplankton communities from different geographic regions respond to oil perturbation. A pulsed disturbance of oil is proposed to favor copiotrophic r-strategists that are adapted to pointed seasonal inputs of phytoplankton carbon, displaying carbon and nutrient limitations, rather than oil exposure history. Further understanding of the ecological mechanisms underpinning the complex environmental controls of hydrocarbon degradation is required for improvement of predictive models of the fate and transport of spilled oil in marine environments.IMPORTANCE The risk of an oil spill accident in pristine regions of the world's oceans is increasing due to the development and transport of crude oil resources, especially in the Arctic region, as a result of the opening of ice-free transportation routes, and there is currently no consensus regarding the complex interplay among the environmental controls of petroleum hydrocarbon biodegradation for predictive modeling. We examined the hydrocarbon biodegradation potential of bacterioplankton from three representative geographic regions of oil exploration. Our results showed that rates of aerobic respiration coupled to hydrocarbon degradation in surface ocean waters are controlled to a large extent by effects of temperature and nutrient limitation; hydrocarbon exposure history did not appear to have a major impact. Further, the relationship between temperature and biodegradation rates is linked to microbial community structure, which is specific to the geographic origin.

Benzene and Naphthalene Degrading Bacterial Communities in an Oil Sands Tailings Pond.

Oil sands process-affected water (OSPW), produced by surface-mining of oil sands in Canada, is alkaline and contains high concentrations of salts, metals, naphthenic acids, and polycyclic aromatic compounds (PAHs). Residual hydrocarbon biodegradation occurs naturally, but little is known about the hydrocarbon-degrading microbial communities present in OSPW. In this study, aerobic oxidation of benzene and naphthalene in the surface layer of an oil sands tailings pond were measured. The potential oxidation rates were 4.3 μmol L−1 OSPW d−1 for benzene and 21.4 μmol L−1 OSPW d−1 for naphthalene. To identify benzene and naphthalene-degrading microbial communities, metagenomics was combined with stable isotope probing (SIP), high-throughput sequencing of 16S rRNA gene amplicons, and isolation of microbial strains. SIP using 13C-benzene and 13C-naphthalene detected strains of the genera Methyloversatilis and Zavarzinia as the main benzene degraders, while strains belonging to the family Chromatiaceae and the genus Thauera were the main naphthalene degraders. Metagenomic analysis revealed a diversity of genes encoding oxygenases active against aromatic compounds. Although these genes apparently belonged to many phylogenetically diverse taxa, only a few of these taxa were predominant in the SIP experiments. This suggested that many members of the community are adapted to consuming other aromatic compounds, or are active only under specific conditions. 16S rRNA gene sequence datasets have been submitted to the Sequence Read Archive (SRA) under accession number SRP109130. The Gold Study and Project submission ID number in Joint Genome Institute IMG/M for the metagenome is Gs0047444 and Gp0055765.

Variation of Oxygenation Conditions on a Hydrocarbonoclastic Microbial Community Reveals Alcanivorax and Cycloclasticus Ecotypes.

Deciphering the ecology of marine obligate hydrocarbonoclastic bacteria (MOHCB) is of crucial importance for understanding their success in occupying distinct niches in hydrocarbon-contaminated marine environments after oil spills. In marine coastal sediments, MOHCB are particularly subjected to extreme fluctuating conditions due to redox oscillations several times a day as a result of mechanical (tide, waves and currents) and biological (bioturbation) reworking of the sediment. The adaptation of MOHCB to the redox oscillations was investigated by an experimental ecology approach, subjecting a hydrocarbon-degrading microbial community to contrasting oxygenation regimes including permanent anoxic conditions, anoxic/oxic oscillations and permanent oxic conditions. The most ubiquitous MOHCB, Alcanivorax and Cycloclasticus, showed different behaviors, especially under anoxic/oxic oscillation conditions, which were more favorable for Alcanivorax than for Cycloclasticus. The micro-diversity of 16S rRNA gene transcripts from these genera revealed specific ecotypes for different oxygenation conditions and their dynamics. It is likely that such ecotypes allow the colonization of distinct ecological niches that may explain the success of Alcanivorax and Cycloclasticus in hydrocarbon-contaminated coastal sediments during oil-spills.

Corexit 9500 Enhances Oil Biodegradation and Changes Active Bacterial Community Structure of Oil-Enriched Microcosms.

To better understand the impacts of Corexit 9500 on the structure and activity levels of hydrocarbon-degrading microbial communities, we analyzed next-generation 16S rRNA gene sequencing libraries of hydrocarbon enrichments grown at 5 and 25°C using both DNA and RNA extracts as the sequencing templates. Oil biodegradation patterns in both 5 and 25°C enrichments were consistent with those reported in the literature (i.e., aliphatics were degraded faster than aromatics). Slight increases in biodegradation were observed in the presence of Corexit at both temperatures. Differences in community structure were observed between treatment conditions in the DNA-based libraries. The 25°C consortia were dominated by Vibrio, Idiomarina, Marinobacter, Alcanivorax, and Thalassospira species, while the 5°C consortia were dominated by several species of the genera Flavobacterium, Alcanivorax, and Oleispira Most of these genera have been linked to hydrocarbon degradation and have been observed after oil spills. Colwellia and Cycloclasticus, known aromatic degraders, were also found in these enrichments. The addition of Corexit did not have an effect on the active bacterial community structure of the 5°C consortia, while at 25°C, a decrease in the relative abundance of Marinobacter was observed. At 25°C, Thalassospira, Marinobacter, and Idiomarina were present at higher relative abundances in the RNA than DNA libraries, suggesting that they were active in degradation. Similarly, Oleispira was greatly stimulated by the addition of oil at 5°C.IMPORTANCE While dispersants such as Corexit 9500 can be used to treat oil spills, there is still debate on the effectiveness on enhancing oil biodegradation and its potential toxic effect on oil-degrading microbial communities. The results of this study provide some insights on the microbial dynamics of hydrocarbon-degrading bacterial populations in the presence of Corexit 9500. Operational taxonomic unit (OTU) analyses indicated that several OTUs were inhibited by the addition of Corexit. Conversely, a number of OTUs were stimulated by the addition of the dispersant, many of which were identified as known hydrocarbon-degrading bacteria. The results highlight the value of using RNA-based methods to further understand the impact of dispersant on the overall activity of different hydrocarbon-degrading bacterial groups.

RNA-TGGE, a Tool for Assessing the Potential for Bioremediation in Impacted Marine Ecosystems

Cultivation-independent genomic approaches have greatly advanced our understanding of the ecology and diversity of microbial communities involved in biodegradation processes. However, much still needs to be resolved in terms of the structure, composition and dynamics of the microbial community in impacted ecosystems. Here we report on the RNA activity of the microbial community during the bioremediation process using RNA Temperature Gradient Gel Electrophoresis (RNA-TGGE). Dendrograms constructed from similarity matching data produced from the TGGE profiles separated a community exhibiting high remediation potential. Overall, increased Shannon Weaver Diversity indices (1–2.4) were observed in the high potential remediation treatment samples. The functionality of the microbial community was compared, with the microbial community showing the greatest organisation also showing the highest levels of hydrocarbon degradation. Subsequent sequencing of excised bands from the microbial community identified the presence of Gammaproteobacteria together with a number of uncultured bacteria. The data shows that RNA TGGE represents a simple, reproducible and effective tool for use in the assessment of a commercial bioremediation event, in terms of monitoring either the natural or augmented hydrocarbon-degrading microbial community.

Estuarine sediment hydrocarbon-degrading microbial communities demonstrate resilience to nanosilver

Little is currently known about the potential impact of silver nanoparticles (AgNPs) on estuarine microbial communities. The Colne estuary, UK, is susceptible to oil pollution through boat traffic, and there is the potential for AgNP exposure via effluent discharged from a sewage treatment works located in close proximity. This study examined the effects of uncapped AgNPs (uAgNPs), capped AgNPs (cAgNPs) and dissolved Ag2SO4, on hydrocarbon-degrading microbial communities in estuarine sediments. The uAgNPs, cAgNPs and Ag2SO4 (up to 50 mg L−1) had no significant impact on hydrocarbon biodegradation (80–92% hydrocarbons were biodegraded by day 7 in all samples). Although total and active cell counts in oil-amended sediments were unaffected by silver exposure; total cell counts in non-oiled sediments decreased from 1.66 to 0.84 × 107 g−1 dry weight sediment (dws) with 50 mg L−1 cAgNPs and from 1.66 to 0.66 × 107 g−1 dws with 0.5 mg L−1 Ag2SO4 by day 14. All silver-exposed sediments also underwent significant shifts in bacterial community structure, and one DGGE band corresponding to a member of Bacteroidetes was more prominent in non-oiled microcosms exposed to 50 mg L−1 Ag2SO4 compared to non-silver controls. In conclusion, AgNPs do not appear to affect microbial hydrocarbon-degradation but do impact on bacterial community diversity, which may have potential implications for other important microbial-mediated processes in estuaries.

Abundance and diversity of soil petroleum hydrocarbon-degrading microbial communities in oil exploring areas.

Alkanes and polycyclic aromatic hydrocarbons (PAHs) are the commonly detected petroleum hydrocarbon contaminants in soils in oil exploring areas. Hydrocarbon-degrading genes are useful biomarks for estimation of the bioremediation potential of contaminated sites. However, the links between environmental factors and the distribution of alkane and PAH metabolic genes still remain largely unclear. The present study investigated the abundances and diversities of soil n-alkane and PAH-degrading bacterial communities targeting both alkB and nah genes in two oil exploring areas at different geographic regions. A large variation in the abundances and diversities of alkB and nah genes occurred in the studied soil samples. Various environmental variables regulated the spatial distribution of soil alkane and PAH metabolic genes, dependent on geographic location. The soil alkane-degrading bacterial communities in oil exploring areas mainly consisted of Pedobacter, Mycobacterium, and unknown alkB-harboring microorganisms. Moreover, the novel PAH-degraders predominated in nah gene clone libraries from soils of the two oil exploring areas. This work could provide some new insights towards the distribution of hydrocarbon-degrading microorganisms and their biodegradation potential in soil ecosystems.

Microbial responses to the Deepwater Horizon oil spill: from coastal wetlands to the deep sea.

The Deepwater Horizon oil spill in the northern Gulf of Mexico represents the largest marine accidental oil spill in history. It is distinguished from past spills in that it occurred at the greatest depth (1,500 m), the amount of hydrocarbon gas (mostly methane) lost was equivalent to the mass of crude oil released, and dispersants were used for the first time in the deep sea in an attempt to remediate the spill. The spill is also unique in that it has been characterized with an unprecedented level of resolution using next-generation sequencing technologies, especially for the ubiquitous hydrocarbon-degrading microbial communities that appeared largely to consume the gases and to degrade a significant fraction of the petroleum. Results have shown an unexpectedly rapid response of deep-sea Gammaproteobacteria to oil and gas and documented a distinct succession correlated with the control of the oil flow and well shut-in. Similar successional events, also involving Gammaproteobacteria, have been observed in nearshore systems as well.

The metabolic pathways and environmental controls of hydrocarbon biodegradation in marine ecosystems.

The metabolic pathways and environmental controls of hydrocarbon biodegradation in marine ecosystems.

Limitations of microbial hydrocarbon degradation at the Amon mud volcano (Nile deep-sea fan)

Abstract. The Amon mud volcano (MV), located at 1250 m water depth on the Nile deep-sea fan, is known for its active emission of methane and non-methane hydrocarbons into the hydrosphere. Previous investigations showed a low efficiency of hydrocarbon-degrading anaerobic microbial communities inhabiting the Amon MV center in the presence of sulfate and hydrocarbons in the seeping subsurface fluids. By comparing spatial and temporal patterns of in situ biogeochemical fluxes, temperature gradients, pore water composition, and microbial activities over 3 yr, we investigated why the activity of anaerobic hydrocarbon degraders can be low despite high energy supplies. We found that the central dome of the Amon MV, as well as a lateral mud flow at its base, showed signs of recent exposure of hot subsurface muds lacking active hydrocarbon degrading communities. In these highly disturbed areas, anaerobic degradation of methane was less than 2% of the methane flux. Rather high oxygen consumption rates compared to low sulfide production suggest a faster development of more rapidly growing aerobic hydrocarbon degraders in highly disturbed areas. In contrast, the more stabilized muds surrounding the central gas and fluid conduits hosted active anaerobic hydrocarbon-degrading microbial communities. The low microbial activity in the hydrocarbon-vented areas of Amon MV is thus a consequence of kinetic limitations by heat and mud expulsion, whereas most of the outer MV area is limited by hydrocarbon transport.

Biodegradation of Crude Oil in Contaminated Soils by Free and Immobilized Microorganisms

The efficiencies of free and immobilized bacterial cultures of petroleum hydrocarbon degraders were evaluated and compared in this study. Hydrocarbon-degrading microbial communities with high tolerance to and high degrading ability of crude oil were obtained from the soil contaminated with crude oil in the Yellow River Delta. Then, the microbial cells were immobilized in sodium alginate (SA) beads and sodium alginate-diatomite (SAD) beads. The biodegradation of crude oil in soil by immobilized cells was compared with that by free cells at three inoculation concentrations, 1 × 104 colony forming units (cfu) kg−1 (low concentration, L), 5 × 104 cfu kg−1 (medium concentration, M), and 1 × 105 cfu kg−1 (high concentration, H). At 20 d after inoculation, the maximum degradation rate in the immobilized systems reached 29.8% (SAD-M), significantly higher (P < 0.05) than that of the free cells (21.1%), and the SAD beads showed greater degradation than the SA beads. Moreover, both microbial populations and total microbial activity reached significantly higher level (P < 0.05) in the immobilized systems than free cell systems at a same initial inoculation amount. The scanning electronic microscope (SEM) images also confirmed the advantages of the immobilized microstructure of SAD beads. The enhanced degradation and bacterial growth in the SAD beads indicated the high potential of SAD beads as an effective option for bioremediation of crude oil-contaminated soils in the Yellow River Delta.

Detection of anaerobic toluene and hydrocarbon degraders in contaminated aquifers using benzylsuccinate synthase (bssA) genes as a functional marker

Benzylsuccinate synthase (Bss) is the key enzyme of anaerobic toluene degradation and has been found in all anaerobic toluene degrading bacterial isolates tested. However, only a few pure cultures capable of anaerobic toluene oxidation are available to date, and it is important to understand the relevance of these model organisms for in situ bioremediation of hydrocarbon-contaminated aquifers. Due to their phylogenetic dispersal, it is not possible to specifically target anaerobic toluene degraders using marker rRNA genes. We therefore established an assay targeting a approximately 794 bp fragment within the Bss alpha-subunit (bssA) gene, which allows for the specific detection and affiliation of both known and unknown anaerobic degraders. Three distinct tar-oil-contaminated aquifer sites were screened for intrinsic bssA gene pools in order to identify and compare the diversity of hydrocarbon degraders present at these selected sites. We were able to show that local diversity patterns of degraders were entirely distinct, apparently highly specialized and well-adapted to local biogeochemical settings. Discovered at one of the sites were bssA genes closely related to that of Geobacter spp., which provides evidence for an importance of iron reduction for toluene degradation in these sediments. Retrieved from the other two sites, dominated by sulfate reduction, were previously unidentified bssA genes and also deeply branching putative bssA homologues. We provide evidence for a previously unrecognized diversity of anaerobic toluene degraders and also of other hydrocarbon degraders using fumarate-adding key reactions in contaminated aquifers. These findings enhance our current understanding of intrinsic hydrocarbon-degrading microbial communities in perturbed aquifers and may have potential for the future assessment and prediction of natural attenuation based on degradation genes.