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.     

Anaerobic degradation of monoaromatic hydrocarbons

Over the last two decades significant advances have been made in our understanding of the anaerobic biodegradability of monoaromatic hydrocarbons. It is now known that compounds such as benzene, toluene, ethylbenzene, and all three xylene isomers can be biodegraded in the absence of oxygen by a broad diversity of organisms. These compounds have been shown to serve as carbon and energy sources for bacteria growing phototrophically, or respiratorily with nitrate, manganese, ferric iron, sulfate, or carbon dioxide as the sole electron acceptor. In addition, it has also been recently shown that complete degradation of monoaromatic hydrocarbons can also be coupled to the respiration of oxyanions of chlorine such as perchlorate or chlorate, or to the reduction of the quinone moieties of humic substances. Many pure cultures of hydrocarbon-degrading anaerobes now exist and some novel biochemical and genetic pathways have been identified. In general, a fumarate addition reaction is used as the initial activation step of the catabolic process of the corresponding monoaromatic hydrocarbon compounds. However, other reactions may alternatively be involved depending on the electron acceptor utilized or the compound being degraded. In the case of toluene, fumarate addition to the methyl group mediated by benzylsuccinate synthase appears to be the universal mechanism of activation and is now known to be utilized by anoxygenic phototrophs, nitrate-reducing, Fe(III)-reducing, sulfate-reducing, and methanogenic cultures. Many of these biochemical pathways produce unique extracellular intermediates that can be utilized as biomarkers for the monitoring of hydrocarbon degradation in anaerobic natural environments.     

Bioenergetics of sulphate-reducing bacteria in relation to their environmental impact

The cellular physiology of the sulphate-reducing bacteria, and of other sulphidogenic species, is determined by the energetic requirements consequent upon their respiratory mode of metabolism with sulphate and other oxyanions of sulphur as terminal electron acceptors. As a further consequence of their, relatively, restricted catabolic activities and their requirement for conditions of anaerobiosis, sulphidogenic bacteria are almost invariably found in nature as component organisms within microbial consortia. The capacity to generate significant quantities of sulphide influences the overall metabolic activity and species diversity of these consortia, and is the root cause of the environmental impact of the sulphidogenic species: corrosion, pollution and the souring of hydrocarbon reservoirs.     

Protein degradation during anaerobic wastewater treatment: derivation of stoichiometry

The stoichiometry of reactions that describe protein degradation in anaerobic treatment systems were investigated. A methodology was developed to describe protein degradation to organic acids using a single reaction step. The reactions for individual amino acid fermentation and their mediating organisms were reviewed. The dominant fermentation pathways were selected based on a number of assumptions. Using the amino acid content of a model protein, it was then possible to determine stoichiometric coefficients for each major organic acid product in the overall degradation of the protein. The theoretical coefficients were then compared to those determined from two experimental runs on a continuously-fed, well-mixed, laboratory-scale anaerobic wastewater treatment system. In general, the coefficients compared well thus validating the use of a single reaction step for the overall catabolic reaction of protein degradation to organic acids. Furthermore, even when the protein concentration in feed or the feed flow rate was doubled, the amino acid fermentation pathways were found to occur predominantly by only one pathway. Although the choice of Stickland reactions over uncoupled degradation provided good comparisons, an electron balance showed that only about 40% of the amino acids could have proceeded coupled to other amino acid reactions. Uncoupled degradation of the remaining amino acids must have relied on the uptake of hydrogen produced from these reactions by hydrogen-consuming methane bacteria.     

石油烃生物降解过程的研究进展

石油烃污染物属于难降解混合物,生物修复已经成为石油烃污染环境的主要修复方法。文中简述了微生物对石油烃的间期适应过程和转运过程,并通过对部分典型石油烃成分的微生物降解机理和代谢路径的梳理和综述,阐释了石油烃生物降解过程中的菌株、基因、代谢路径等研究进展。此外,利用基因工程和代谢工程等手段,可对野生型石油烃降解菌进行改造,进一步提升其对石油烃污染环境的生物修复能力。最后,从石油烃降解菌的代谢途径改造、人工混菌体系的设计构建等角度,结合合成生物学和代谢工程的手段,提出了对石油烃降解的研究展望,以期提升对石油烃污染物的生物修复效果。     

A refinery sludge deposition site: presence of nahH and alkJ genes and crude oil biodegradation ability of bacterial isolates

204 bacterial isolates from four Greek refinery sludge deposition sites were investigated for the presence of nahH and alkJ genes encoding key enzymes of both aromatic and aliphatic hydrocarbon degradation pathways by PCR and DNA hybridisation. Members of Pseudomonas, Acinetobacter, Bacillus, Rhodococcus and Arthrobacter play important role in bioremediation processes in sandy/loam soil contaminated with oil and nahH and alkJ genes were present in the 73% of the isolates. Consortia of bacterial isolates that were used for biodegradation of aliphatic and aromatic hydrocarbons in crude oil using liquid cultures exhibited rates from 35% to 48% within 10 days of incubation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular diversity of dinoflagellate symbionts of Cnidaria: the psbA minicircle of Symbiodinium

Dinoflagellate algae of the genus Symbiodinium are important symbionts within corals and other benthic marine animals. The molecular diversity of Symbiodinium has been described mainly by use of ribosomal DNA sequence data. We tested whether minicircle sequences, which appear to form the chloroplast genome in many dinoflagellates, could be used as a marker for molecular diversity among symbionts found in corals and sea anemones. Partial and full-length sequences for psbA were obtained from environmental samples of coral and sea anemones of wide-ranging geographical distribution. Phylogenetic trees constructed with partial psbA sequences were consistent with the known phylotypes of the isolates. Further sequencing suggested that the psbA gene is present on a minicircle in all Symbiodinium phylotypes. The length and DNA sequence of the non-coding portion of the minicircles varied considerably among Symbiodinium phylotypes. In two Symbiodinium isolates from different phylotypes an elaborate pattern of repeat sequences of unknown function was found in the non-coding region. Phylogenetic analysis of the non-coding region of the psbA minicircle indicates that minicircle sequences could be a useful chloroplast-derived marker for differentiating both closely related and distantly related Symbiodinium isolates.     

Isolation and molecular characterization of thiosulfate-oxidizing bacteria from an Italian rice field soil

In rice paddy soils an active cycling of sulfur compounds takes place. To elucidate the diversity of thiosulfate-oxidizing bacteria these organisms were enriched from bulk soil and rice roots by the most probable number method in liquid medium. From the MPN enrichment cultures 21 bacterial strains were isolated on solid mineral medium, and could be further shown to produce sulfate from thiosulfate. These strains were characterized by 16S rDNA analyses. The isolates were affiliated to seven different phylogenetic groups within the alpha- and beta-subclass of Proteobacteria. Two of these phylotypes were already described as S-oxidizers in this environment (Xanthobacter sp. and Bosea sp. related strains), but five groups represented new S-oxidizers in rice field soil. These isolates were closely related to Mesorhizobium loti, to Hydrogenophaga sp., to Delftia sp., to Pandoraea sp. or showed sequence similarity to a strain of Achromobacter sp.     

Biodegradation of aliphatic hydrocarbon by indigenous fungi isolated from used motor oil contaminated sites

Eighteen indigenous fungal isolates has been successfully isolated from samples of used motor oil, top five centimetres of soil and drainage water contaminated with used motor oil. All of the pure fungal isolates obtained were identified, characterized and subjected to preliminary screening by evaluating the average growth rate of each fungal isolates on minimal media containing 1% (v/v) used motor oil. Trichoderma asperellum strain TUB F-1067 (SA4), Trichoderma asperellum strain Tr48 (SA5), Trichoderma asperellum strain TUB F-756 (SA6), Penicillium species (P1), and Aspergillus species (P9) were further selected for their hydrocarbon biodegradation potential. Among these five fungal isolates selected, P1 strain presented a significant degree of degradation by degrading almost all of the n-alkanes (n-C-₁₅ to n-C-₂₃ range) present in the used motor oil, thus of greater potential in degrading the aliphatic hydrocarbon compounds of used motor oil. The authors would like to certify that the work have not been sent/considered to be published in other journals.     

Characterization of diverse heterocyclic amine-degrading denitrifying bacteria from various environments

Although, there have been many published bacterial strains aerobically degrading the heterocyclic amine compounds, only one strain to date has been reported to degrade pyrrolidine under denitrifying conditions. In this study, denitrifying bacteria degrading pyrrolidine and piperidine were isolated from diverse geological and ecological origins through selective enrichment procedures. Based on the comparative sequence results of 16S rRNA genes, 30 heterocyclic amine-degrading isolates were grouped into ten distinct phylotypes belonging to the genera Thauera, Castellaniella, Rhizobium, or Paracoccus of the phylum Proteobacteria. The representative isolates of individual phylotypes were characterized by phylogenetic, phenotypic and chemotaxonomical traits, and dissimilatory nitrite reductase gene (nirK and nirS). All isolates completely degraded pyrrolidine and piperidine under both aerobic and anaerobic conditions. The anaerobic degradations were coupled to nitrate reduction. A metabolic pathway for the anaerobic degradation of pyrrolidine was proposed on the basis of enzyme activities implicated in pyrrolidine metabolism from three isolates. The three key pyrrolidine-metabolizing enzymes pyrrolidine dehydrogenase, γ-aminobutyrate/α-ketoglutarate aminotransferase, and succinic semialdehyde dehydrogenase, were induced by heterocyclic amines under denitrifying conditions. They were also induced in cells grown aerobically on heterocyclic amines, suggesting that the anaerobic degradation of pyrrolidine shares the pathway with aerobic degradation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Utilization of Drilling Fluid Base Oil Hydrocarbons by Microorganisms Isolated from Diesel-Polluted Soil

Hydrocarbon-degrading microorganisms identified as Pseudomonas luteola, Pseudomonas alcaligenes, Pseudomonas aeruginosa, and Actinomyces sp. were isolated from diesel oil-polluted soils using an enrichment culture technique. The isolates grew luxuriantly on hydrocarbons, including crude oil, diesel, kerosene, engine oil, cyclohexane, and dodecanol. Naphthalene and pyrene were poorly utilized, while there was no growth on benzene. The organisms utilized drilling fluid base oil as the sole source of carbon and energy, with rapid exponential growth at a rate ranging from 0.015 to 0.094 h^?1. The concomitant doubling time was between 7.4 and 45.5 h. Gas chromatographic analyses of the culture revealed reduction in the height of the n-alkane peaks, confirming biodegradation of the compounds. Among the isolates, P. alcaligenes had the highest (99.4%) percentage hydrocarbon degradation. Remarkable (99.2% and 98.7%) hydrocarbon removal was also noted for P. luteola and P. aeruginosa, while the lowest (92.3%) value was recorded in Actinomyces sp. These bacteria with high degradative capacity for hydrocarbons in oil-based drilling fluids would be useful in bioremediation of a tropical environment, polluted with spent drilling mud and drill cuttings.     

Microbial Community Succession in an Unvegetated,Recently Deglaciated Soil

Primary succession is a fundamental process in macroecosystems; however, if and how soil development influences microbial community structure is poorly understood. Thus, we investigated changes in the bacterial community along a chronosequence of three unvegetated, early successional soils (∼20-year age gradient) from a receding glacier in southeastern Peru using molecular phylogenetic techniques. We found that evenness, phylogenetic diversity, and the number of phylotypes were lowest in the youngest soils, increased in the intermediate aged soils, and plateaued in the oldest soils. This increase in diversity was commensurate with an increase in the number of sequences related to common soil bacteria in the older soils, including members of the divisions Acidobacteria, Bacteroidetes, and Verrucomicrobia. Sequences related to the Comamonadaceae clade of the Betaproteobacteria were dominant in the youngest soil, decreased in abundance in the intermediate age soil, and were not detected in the oldest soil. These sequences are closely related to culturable heterotrophs from rock and ice environments, suggesting that they originated from organisms living within or below the glacier. Sequences related to a variety of nitrogen (N)-fixing clades within the Cyanobacteria were abundant along the chronosequence, comprising 6–40% of phylotypes along the age gradient. Although there was no obvious change in the overall abundance of cyanobacterial sequences along the chronosequence, there was a dramatic shift in the abundance of specific cyanobacterial phylotypes, with the intermediate aged soils containing the greatest diversity of these sequences. Most soil biogeochemical characteristics showed little change along this ∼20-year soil age gradient; however, soil N pools significantly increased with soil age, perhaps as a result of the activity of the N-fixing Cyanobacteria. Our results suggest that, like macrobial communities, soil microbial communities are structured by substrate age, and that they, too, undergo predictable changes through time.     

Synergistic utilization of dichloroethylene as sole carbon source by bacterial consortia isolated from contaminated sites in Africa

The widespread use and distribution of chloroethylene organic compounds is of serious concern owing to their carcinogenicity and toxicity to humans and wildlife. In an effort to develop active bacterial consortia that could be useful for bioremediation of chloroethylenecontaminated sites in Africa, 16 combinations of 5 dichloroethylene (DCE)-utilizing bacteria, isolated from South Africa and Nigeria, were assessed for their ability to degradecis- andtrans-DCEs as the sole carbon source. Three combinations of these isolates were able to remove up to 72% of the compounds within 7 days. Specific growth rate constants of the bacterial consortia ranged between 0.465 and 0.716 d⁻¹ while the degradation rate constants ranged between 0.184 and 0.205 d⁻¹, with 86.36–93.53 and 87.47–97.12% of the stoichiometric-expected chloride released during growth of the bacterial consortia, incis- andtrans-DCE, respectively. Succession studies of the individual isolates present in the consortium revealed that the biodegradation process was initially dominated byAchromobacter xylosoxidans and subsequently byAcinetobacter sp. andBacillus sp., respectively. The results of this study suggest that consortia of bacteria are more efficient than monocultures in the aerobic biodegradation of DCEs, degrading the compounds to levels that are up to 60% below the maximum allowable limits in drinking water.     

Bioremediation of petroleum hydrocarbon contaminated soil: Effects of strategies and microbial community shift

Biodegradation of petroleum hydrocarbon oil (14,000 mg kg⁻¹) were investigated in six biopiles batches, differing in the remediation strategy: bioaugmentation (selected consortium and kitchen waste were introduced), biostimulation (added with rhamnolipid, high-level, or low-level nutrient), and bioaugmentation plus biostimulation (added both with rhamnolipid and bacterial consortia). After the 140-day operation, the kitchen waste (KW) and the low-level nutrient (NEL) batches achieved the highest total petroleum hydrocarbon degradation efficiency (>80%). The result of the hydrocarbon analysis revealed that the bioaugmentation approaches were the most effective ones in removing aromatic component (64% and 68%), and KW and NEL were the only two approaches that can remove the polar component with positive efficiency, 11% and 21%, respectively. The terminal-restriction fragment length polymorphism percentage (T-RFLP) abundance applied with nonmetric multidimensional scaling indicated a similarity of the bacterial communities during the early fastest remediation stage. The results of the oligonucleotide array targeting the ribosomal internal transcribed spacer (ITS) region, along with the hydrocarbon fractional analysis, indicated a successive degradation completed by the bacterial-fungi consortia. Before Day 70, the bacterial community was dominant in decomposing the saturated and partially aromatic hydrocarbons. After Day 70, the fungal community found to be dynamic and responsible for degradation of the polar hydrocarbons composing of recalcitrant metabolites.     

Molecular identification of diazotroph microbial consortia in mountain soil

Nitrogen fixing microbial consortia from soil samples taken from five altitudinal vegetation zones (alpine, subalpine, coniferous, beech, Maleia flood plain) of Parang Massif, Romania, were isolated and identified. Molecular characterisation of nitrogen fixing consortia was carried out by PCR and nested PCR with 7 primer sets specific to nifH genes. All nifH genes are specific to nitrogen fixation and are found within phylogenetically related organisms which have the nitrogenase enzyme complex. These molecular studies allowed the assessment of nifH gene diversity within these nitrogen fixing microbial consortia from different type of soils. At high altitude, a consortium of nitrogen fixing bacteria dominated by Azotobacter chroococcum and Azospirillum brasilense was found. Clostridium, Rhizobiales, Herbaspirillum, Frankia species were also found in different rations depending on the altitudinal vegetation zone.     

Adding handles to unhandy substrates: anaerobic hydrocarbon activation mechanisms

In spite of their chemical inertness, hydrocarbons are degraded by microorganisms in the complete absence of oxygen. As all known aerobic hydrocarbon degradation pathways start with oxygen-dependent reactions, hydrocarbon catabolism in anaerobes must be initiated by novel biochemical reactions. In recent years, the enzymes catalyzing oxygen-independent activation of several hydrocarbons have been identified. Surprisingly, a variety of reactions seems to be employed to overcome the activation barrier of different hydrocarbons. This review presents the current understanding on some of these reactions and the associated degradation pathways: oxygen-independent hydroxylation as employed in ethylbenzene metabolism, fumarate addition to methyl or methylene carbons in toluene or alkane degradation, and only recently discovered reactions such as methylation of naphthalene or anaerobic methane oxidation via reverse methanogenesis.     

Diversity of the ring-cleaving dioxygenase gene pcaH in a salt marsh bacterial community.

Degradation of lignin-related aromatic compounds is an important ecological process in the highly productive salt marshes of the southeastern United States, yet little is known about the mediating organisms or their catabolic pathways. Here we report the diversity of a gene encoding a key ring-cleaving enzyme of the beta-ketoadipate pathway, pcaH, amplified from bacterial communities associated with decaying Spartina alterniflora, the salt marsh grass that dominates these coastal systems, as well as from enrichment cultures with aromatic substrates (p-hydroxybenzoate, anthranilate, vanillate, and dehydroabietate). Sequence analysis of 149 pcaH clones revealed 85 unique sequences. Thirteen of the 53 amino acid residues compared were invariant in the PcaH proteins, suggesting that these residues have a required catalytic or structural function. Fifty-eight percent of the clones matched sequences amplified from a collection of 36 bacterial isolates obtained from seawater, marine sediments, or senescent Spartina. Fifty-two percent of the pcaH clones could be assigned to the roseobacter group, a marine lineage of the class alpha-Proteobacteria abundant in coastal ecosystems. Another 6% of the clones matched genes retrieved from isolates belonging to the genera Acinetobacter, Bacillus, and Stappia, and 42% of the clones could not be assigned to a cultured bacterium based on sequence identity. These results suggest that the diversity of the genes encoding a single step in aromatic compound degradation in the coastal marsh examined is high.     

Natural microbial diversity in superficial sediments of Milazzo Harbor (Sicily) and community successions during microcosm enrichment with various hydrocarbons

Hydrocarbon-contaminated superficial sediments collected from the Harbor of Milazzo (Tirrenean Sea, northern Sicily), a zone strongly affected by anthropogenic activities, were examined for in situ biodegradative capacities. A culture-independent molecular phylogenetic approach was used to study the influence of hydrocarbon and nutrient addition on the activity and diversity of the indigenous microbiota during a microcosm evaluation. The autochthonous microbial community in non-polluted sediments was represented by eubacterial phylotypes grouped within Proteobacteria, CFB and Firmicutes. The archaeal domain was represented by members of Marine Group I of Crenarchaeota. The majority of recovered sequences was affiliated with heterotrophic genera Clostridium and Vibrio, typical members of eutrophic coastal environments. Amendments of hydrocarbons and mineral nutrients to microcosms dramatically changed the initial diversity of the microbial community. Only bacterial phylotypes affiliated with Proteobacteria and CFB division were detected. The decrease in diversity observed in several microcosms could be explained by the strong selection for microorganisms belonging to group of marine hydrocarbonoclastic gamma-Proteobacteria, namely Alcanivorax, Cycloclasticus, Marinobacter, Marinobacterium/Neptunomonas and Thalassolituus. This study demonstrated that nutrient amendment to hydrocarbon-contaminated superficial sediments enhanced the indigenous microbial biodegradation activity and that highly specialized marine hydrocarbonoclastic bacteria, representing a minor fraction in the natural microbial community, play an important role in the biodegradation of petroleum hydrocarbons accidentally entering the coastal environment.     

Molecular typing and identification of Bdellovibrio-and-like organisms isolated from seawater shrimp ponds and adjacent coastal waters

Aims:  To apply and compare two PCR-based methods for typing saltwater Bdellovibrio- and-like organisms (BALOs) and to understand ecological and phylogenetic aspects of the BALOs isolated from shrimp mariculture systems.
Methods and Results:  Using double-layer agar technique, the numbers of culturable BALOs that lyse Vibrio alginolyticus were found to be 10–10³ PFU ml⁻¹ in the surface water samples. A total of 130 BALOs isolates were differentiated into five phylotypes by denaturing gradient gel electrophoresis targeting the 16S rDNA V3 region and four phylotypes by amplified rDNA restriction analysis of the Bacteriovoracaceae -specific 16S rDNA fragment respectively. Phylogenetic analysis of representative isolates showed that all of them were identified as Bacteriovorax spp., but affiliated with four different clusters in the family Bacteriovoracaceae.
Conclusions:  The two PCR-based methods both can be chosen to rapidly type the saltwater BALOs at cluster level. And the relatively large numbers of BALOs with various phylotypes recovered from the same habitats suggested that these predators might play important ecological role in shrimp mariculture environments.
Significance and Impact of the Study:  We proposed two effective methods to distinguish rapidly large numbers of BALOs isolates and our results would be helpful to understand the diversity and function of BALOs in mariculture environments.