Effect of energy deprivation on metabolite release by anaerobic marine naphthalene-degrading sulfate-reducing bacteria.

The aromatic hydrocarbon naphthalene, which occurs in coal and oil, can be degraded by aerobic or anaerobic microorganisms. A wide-spread electron acceptor for the latter is sulfate. Evidence for in situ naphthalene degradation stems in particular from the detection of 2-naphthoate and [5,6,7,8]-tetrahydro-2-naphthoate in oil field samples. Because such intermediates are usually not detected in laboratory cultures with high sulfate concentrations, one may suppose that conditions in reservoirs, such as sulfate limitation, trigger metabolite release. Indeed, if naphthalene-grown cells of marine sulfate-reducing Deltaproteobacteria (strains NaphS2, NaphS3 and NaphS6) were transferred to sulfate-free medium, they released 2-naphthoate and [5,6,7,8]-tetrahydro-2-naphthoate while still consuming naphthalene. With 2-naphthoate as initial substrate, cells produced [5,6,7,8]-tetrahydro-2-naphthoate and the hydrocarbon, naphthalene, indicating reversibility of the initial naphthalene-metabolizing reaction. The reactions in the absence of sulfate were not coupled to observable growth. Excretion of naphthalene-derived metabolites was also achieved in sulfate-rich medium upon addition of the protonophore carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone or the ATPase inhibitor N,N'-dicyclohexylcarbodiimide. In conclusion, obstruction of electron flow and energy gain by sulfate limitation offers an explanation for the occurrence of naphthalene-derived metabolites in oil reservoirs, and provides a simple experimental tool for gaining insights into the anaerobic naphthalene oxidation pathway from an energetic perspective.

Biodesulfurization of Thiophenic Compounds by a 2-Hydroxybiphenyl-Resistant Gordonia sp. HS126-4N Carrying dszABC Genes.

Microorganisms can metabolize or transform a range of known chemical compounds present in fossil fuels by naturally having highly specific metabolic activities. In this context, the microbial desulfurization of fuels is an attractive and alternative process to the conventional hydrodesulfurization (HDS) process, since the thiophenic sulfur containing compounds such as dibenzothiophene (DBT) and benzothiophene (BT) cannot be removed by HDS. A DBT desulfurizing mesophilic bacterium, identified on the basis of 16S rRNA gene sequence as Gordonia sp. HS126-4N (source: periphery soil of a coal heap) has been evaluated for its biodesulfurization traits and potential to desulfurize the thiophenic compounds. The HPLC and LC/MS analyses of the metabolites produced from DBT desulfurization and PCR-based nucleotide sequence confirmation of the key desulfurizing genes (dszA/dszB/dszC) proved that HS126-4N could convert DBT to 2-hydroxybiphenyl (2-HBP) via the 4S pathway. The isolate could convert 0.2 mM of DBT to 2-HBP within 48 h and was reasonably tolerant against the inhibitory effect of 2-HBP (retained 70% of growth at 0.5 mM 2-HBP). The isolated biocatalyst desulfurized/degraded 100% of 0.2 mM of 4-methyl DBT, 2,8-dimethyl DBT, BT and 3-methyl BT within 108 h. The capabilities to survive and desulfurize a broad range of thiophenic sulfur containing substrates as well as less inhibition by the 2-HBP suggest that HS126-4N could be a potential candidate for improved biodesulfurization/organic sulfur removal from fossil fuels.

Metabolic and process engineering for biodesulfurization in Gram-negative bacteria.

Microbial desulfurization or biodesulfurization (BDS) is an attractive low-cost and environmentally friendly complementary technology to the hydrotreating chemical process based on the potential of certain bacteria to specifically remove sulfur from S-heterocyclic compounds of crude fuels that are recalcitrant to the chemical treatments. The 4S or Dsz sulfur specific pathway for dibenzothiophene (DBT) and alkyl-substituted DBTs, widely used as model S-heterocyclic compounds, has been extensively studied at the physiological, biochemical and genetic levels mainly in Gram-positive bacteria. Nevertheless, several Gram-negative bacteria have been also used in BDS because they are endowed with some properties, e.g., broad metabolic versatility and easy genetic and genomic manipulation, that make them suitable chassis for systems metabolic engineering strategies. A high number of recombinant bacteria, many of which are Pseudomonas strains, have been constructed to overcome the major bottlenecks of the desulfurization process, i.e., expression of the dsz operon, activity of the Dsz enzymes, retro-inhibition of the Dsz pathway, availability of reducing power, uptake-secretion of substrate and intermediates, tolerance to organic solvents and metals, and other host-specific limitations. However, to attain a BDS process with industrial applicability, it is necessary to apply all the knowledge and advances achieved at the genetic and metabolic levels to the process engineering level, i.e., kinetic modelling, scale-up of biphasic systems, enhancing mass transfer rates, biocatalyst separation, etc. The production of high-added value products derived from the organosulfur material present in oil can be regarded also as an economically viable process that has barely begun to be explored.

Microbial Biotechnology 2020; microbiology of fossil fuel resources.

This roadmap examines the future of microbiology research and technology in fossil fuel energy recovery. Globally, the human population will be reliant on fossil fuels for energy and chemical feedstocks for at least the medium term. Microbiology is already important in many areas relevant to both upstream and downstream activities in the oil industry. However, the discipline has struggled for recognition in a world dominated by geophysicists and engineers despite widely known but still poorly understood microbially mediated processes e.g. reservoir biodegradation, reservoir souring and control, microbial enhanced oil recovery. The role of microbiology is even less understood in developing industries such as shale gas recovery by fracking or carbon capture by geological storage. In the future, innovative biotechnologies may offer new routes to reduced emissions pathways especially when applied to the vast unconventional heavy oil resources formed, paradoxically, from microbial activities in the geological past. However, despite this potential, recent low oil prices may make industry funding hard to come by and recruitment of microbiologists by the oil and gas industry may not be a high priority. With regards to public funded research and the imperative for cheap secure energy for economic growth in a growing world population, there are signs of inherent conflicts between policies aimed at a low carbon future using renewable technologies and policies which encourage technologies which maximize recovery from our conventional and unconventional fossil fuel assets.

An Evaluation of Kinetic Models in the Biodesulfurization of Synthetic Oil by Rhodococcus erythropolis ATCC 4277.

Biodesulfurization is an eco-friendly technology applied in the removal of sulfur from fossil fuels. This technology is based on the use of microorganisms as biocatalysts to convert the recalcitrant sulfur compounds into others easily treatable, as sulfides. Despite it has been studied during the last decades, there are some unsolved questions, as per example the kinetic model which appropriately describes the biodesulfurization globally. In this work, different kinetic models were tested to a batch desulfurization process using dibenzothiophene (DBT) as a model compound, n-dodecane as organic solvent, and Rhodococcus erythropolis ATCC 4277 as biocatalyst. The models were solved by ODE45 function in the MATLAB. Monod model was capable to describe the biodesulfurization process predicting all experimental data with a very good fitting. The coefficients of determination achieved to organic phase concentrations of 20, 80, and 100 % (v/v) were 0.988, 0.995, and 0.990, respectively. R. erythropolis ATCC 4277 presented a good affinity with the substrate (DBT) since the coefficients of saturation obtained to reaction medium containing 20, 80, and 100 % (v/v) were 0.034, 0.07, and 0.116, respectively. This kinetic evaluation provides an improvement in the development of biodesulfurization technology because it showed that a simple model is capable to describe the throughout process.

Biohydrogen production: strategies to improve process efficiency through microbial routes.

The current fossil fuel-based generation of energy has led to large-scale industrial development. However, the reliance on fossil fuels leads to the significant depletion of natural resources of buried combustible geologic deposits and to negative effects on the global climate with emissions of greenhouse gases. Accordingly, enormous efforts are directed to transition from fossil fuels to nonpolluting and renewable energy sources. One potential alternative is biohydrogen (H2), a clean energy carrier with high-energy yields; upon the combustion of H2, H2O is the only major by-product. In recent decades, the attractive and renewable characteristics of H2 led us to develop a variety of biological routes for the production of H2. Based on the mode of H2 generation, the biological routes for H2 production are categorized into four groups: photobiological fermentation, anaerobic fermentation, enzymatic and microbial electrolysis, and a combination of these processes. Thus, this review primarily focuses on the evaluation of the biological routes for the production of H2. In particular, we assess the efficiency and feasibility of these bioprocesses with respect to the factors that affect operations, and we delineate the limitations. Additionally, alternative options such as bioaugmentation, multiple process integration, and microbial electrolysis to improve process efficiency are discussed to address industrial-level applications.

Issues for storing plant-based alternative fuels in marine environments.

Two coastal seawaters (Key West, FL, USA and the Persian Gulf, Bahrain, representing oligotrophic and eutrophic environments, respectively) were used to evaluate potential biodegradation and corrosion problems during exposure to alternative and conventional fuels. Uncoated carbon steel was exposed at the fuel/seawater interface and polarization resistance was monitored. Under typical marine storage conditions, dioxygen in natural seawater exposed to fuel and carbon steel was reduced to <0.1parts-per-million within 2d due to consumption by corrosion reactions and aerobic microbial respiration. Sulfides, produced by anaerobic sulfate-reducing bacteria, and chlorides were co-located in corrosion products. Transient dioxygen influenced both metabolic degradation pathways and resulting metabolites. Catechols, indicative of aerobic biodegradation, persisted after 90d exposures. Detection of catechols suggested that initial exposure to dioxygen resulted in the formation of aerobic metabolites that exacerbated subsequent corrosion processes.

From fields to fuels: recent advances in the microbial production of biofuels.

Amid grave concerns over global climate change and with increasingly strained access to fossil fuels, the synthetic biology community has stepped up to the challenge of developing microbial platforms for the production of advanced biofuels. The adoption of gasoline, diesel, and jet fuel alternatives derived from microbial sources has the potential to significantly limit net greenhouse gas emissions. In this effort, great strides have been made in recent years toward the engineering of microorganisms to produce transportation fuels derived from alcohol, fatty acid, and isoprenoid biosynthesis. We provide an overview of the biosynthetic pathways devised in the strain development of biofuel-producing microorganisms. We also highlight many of the commonly used and newly devised engineering strategies that have been employed to identify and overcome pathway bottlenecks and problems of toxicity to maximize production titers.

Culture-independent analysis of bacterial fuel contamination provides insight into the level of concordance with the standard industry practice of aerobic cultivation.

Bacterial diversity in contaminated fuels has not been systematically investigated using cultivation-independent methods. The fuel industry relies on phenotypic cultivation-based contaminant identification, which may lack accuracy and neglect difficult-to-culture taxa. By the use of industry practice aerobic cultivation, 16S rRNA gene sequencing, and strain genotyping, a collection of 152 unique contaminant isolates from 54 fuel samples was assembled, and a dominance of Pseudomonas (21%), Burkholderia (7%), and Bacillus (7%) was demonstrated. Denaturing gradient gel electrophoresis (DGGE) of 15 samples revealed Proteobacteria and Firmicutes to be the most abundant phyla. When 16S rRNA V6 gene pyrosequencing of four selected fuel samples (indicated by "JW") was performed, Betaproteobacteria (42.8%) and Gammaproteobacteria (30.6%) formed the largest proportion of reads; the most abundant genera were Marinobacter (15.4%; JW57), Achromobacter (41.6%; JW63), Burkholderia (80.7%; JW76), and Halomonas (66.2%; JW78), all of which were also observed by DGGE. However, the Clostridia (38.5%) and Deltaproteobacteria (11.1%) identified by pyrosequencing in sample JW57 were not observed by DGGE or aerobic culture. Genotyping revealed three instances where identical strains were found: (i) a Pseudomonas sp. strain recovered from 2 different diesel fuel tanks at a single industrial site; (ii) a Mangroveibacter sp. strain isolated from 3 biodiesel tanks at a single refinery site; and (iii) a Burkholderia vietnamiensis strain present in two unrelated automotive diesel samples. Overall, aerobic cultivation of fuel contaminants recovered isolates broadly representative of the phyla and classes present but lacked accuracy by overrepresenting members of certain groups such as Pseudomonas.

(Per)chlorate reduction by an acetogenic bacterium, Sporomusa sp., isolated from an underground gas storage.

A mesophilic bacterium, strain An4, was isolated from an underground gas storage reservoir with methanol as substrate and perchlorate as electron acceptor. Cells were Gram-negative, spore-forming, straight to curved rods, 0.5-0.8 microm in diameter, and 2-8 microm in length, growing as single cells or in pairs. The cells grew optimally at 37 degrees C, and the pH optimum was around 7. Strain An4 converted various alcohols, organic acids, fructose, acetoin, and H(2)/CO(2) to acetate, usually as the only product. Succinate was decarboxylated to propionate. The isolate was able to respire with (per)chlorate, nitrate, and CO(2). The G+C content of the DNA was 42.6 mol%. Based on the 16S rRNA gene sequence analysis, strain An4 was most closely related to Sporomusa ovata (98% similarity). The bacterium reduced perchlorate and chlorate completely to chloride. Key enzymes, perchlorate reductase and chlorite dismutase, were detected in cell-free extracts.

Characterization by culture and molecular analysis of the microbial diversity of a deep subsurface gas storage aquifer.

The bacterial diversity of a subsurface water sample collected from a gas storage aquifer in an Upper Jurassic calcareous formation was investigated by culture of microorganisms and construction of a 16S rRNA gene library. Both culture and molecular techniques showed that members of the phyla Firmicutes and class delta-proteobacteria dominated the bacterial community. The presence of hydrogen-utilizing autotrophic bacteria including sulfate reducers (e.g. Desulfovibrio aespoeensis) and homoacetogens (e.g. Acetobacterium carbinolicum) suggested that CO(2) and H(2) are the main carbon and energy sources sustaining a nutrient-limited subsurface lithoautotrophic microbial ecosystem (SLiME). Gram-positive SRB belonging to the genus Desulfotomaculum, frequently observed in subsurface environments, represented 25% of the clone library and 4 distinct phylotypes. No Archaea were detected by both experimental approaches. Water samples were collected in an area of the rauracian geological formation located outside the maximum seasonal extension of underground gas storage. Considering the observed microbial diversity, there is no evidence of any influence on the microbial ecology of the aquifer in the surroundings of maximum extension reached by the gas bubble of the underground storage, which should have resulted from the introduction of exogenous carbon and energy sources in a nutrient-limited ecosystem.

Biodesulfurization of refractory organic sulfur compounds in fossil fuels.

The stringent new regulations to lower sulfur content in fossil fuels require new economic and efficient methods for desulfurization of recalcitrant organic sulfur. Hydrodesulfurization of such compounds is very costly and requires high operating temperature and pressure. Biodesulfurization is a non-invasive approach that can specifically remove sulfur from refractory hydrocarbons under mild conditions and it can be potentially used in industrial desulfurization. Intensive research has been conducted in microbiology and molecular biology of the competent strains to increase their desulfurization activity; however, even the highest activity obtained is still insufficient to fulfill the industrial requirements. To improve the biodesulfurization efficiency, more work is needed in areas such as increasing specific desulfurization activity, hydrocarbon phase tolerance, sulfur removal at higher temperature, and isolating new strains for desulfurizing a broader range of sulfur compounds. This article comprehensively reviews and discusses key issues, advances and challenges for a competitive biodesulfurization process.

Environmental biocatalysis: from remediation with enzymes to novel green processes.

Modern biocatalysis is developing new and precise tools to improve a wide range of production processes, which reduce energy and raw material consumption and generate less waste and toxic side-products. Biocatalysis is also achieving new advances in environmental fields, from enzymatic bioremediation to the synthesis of renewable and clean energies and biochemical cleaning of 'dirty' fossil fuels. Despite the obvious benefits of biocatalysis, the major hurdles hindering the exploitation of the repertoire of enzymatic processes are, in many cases, the high production costs and the low yields obtained. This article will discuss these issues, pinpointing specific new advances in recombinant DNA techniques amenable to future biocatalyst development, in addition to drawing the attention of the biotechnology community to the active pursuit and development of environmental biocatalysis, from remediation with enzymes to novel green processes.

Isolation of iodide-oxidizing bacteria from iodide-rich natural gas brines and seawaters.

Iodide-oxidizing bacteria (IOB), which oxidize iodide (I-) to molecular iodine (I2), were isolated from iodide-rich (63 microM to 1.2 mM) natural gas brine waters collected from several locations. Agar media containing iodide and starch were prepared, and brine waters were spread directly on the media. The IOB, which appeared as purple colonies, were obtained from 28 of the 44 brine waters. The population sizes of IOB in the brines were 10(2) to 10(5) colony-forming units (CFU) mL(-1). However, IOB were not detected in natural seawaters and terrestrial soils (fewer than 10 CFU mL(-1) and 10(2) CFU g wet weight of soils(-1), respectively). Interestingly, after the enrichment with 1 mM iodide, IOB were found in 6 of the 8 seawaters with population sizes of 10(3) to 10(5) CFU mL(-1). 16S rDNA sequencing and phylogenetic analyses showed that the IOB strains are divided into two groups within the alpha-subclass of the Proteobacteria. One of the groups was phylogenetically most closely related to Roseovarius tolerans with sequence similarities between 94% and 98%. The other group was most closely related to Rhodothalassium salexigens, although the sequence similarities were relatively low (89% to 91%). The iodide-oxidizing reaction by IOB was mediated by an extracellular enzyme protein that requires oxygen. Radiotracer experiments showed that IOB produce not only I2 but also volatile organic iodine, which were identified as diiodomethane (CH2I2) and chloroiodomethane (CH2ClI). These results indicate that at least two types of IOB are distributed in the environment, and that they are preferentially isolated in environments in which iodide levels are very high. It is possible that IOB oxidize iodide in the natural environment, and they could significantly contribute to the biogeochemical cycling of iodine.

Isolation from oil reservoirs of novel thermophilic anaerobes phylogenetically related to Thermoanaerobacter subterraneus: reassignment of T. subterraneus, Thermoanaerobacter yonseiensis, Thermoanaerobacter tengcongensis and Carboxydibrachium pacificum to Caldanaerobacter subterraneus gen. nov., sp. nov., comb. nov. as four novel subspecies.

Novel thermophilic, anaerobic, Gram-positive, rod-shaped bacteria, strains SL9 and OCA1, were isolated from oilfields in France and Australia, respectively. Both strains, together with Thermoanaerobacter yonseiensis KB-1(T) (=DSM 13777(T)), Thermoanaerobacter tengcongensis MB4(T) (=DSM 15242(T)) and Carboxydibrachium pacificum JM(T) (=DSM 12653(T)), possessed genomic (DNA-DNA hybridization studies) and phylogenetic similarities with Thermoanaerobacter subterraneus SEBR 7858(T) (=DSM 13054(T)), which was isolated recently from an oilfield reservoir in south-west France. Marked phenotypic differences exist between the three oilfield isolates (T. subterraneus, strain OCA1 and strain SL9): they include temperature range for growth and substrates used. Differences were also observed in the DNA G+C contents of all organisms. Similarly to T. subterraneus, strains SL9 and OCA1, and also T. yonseiensis, T. tengcongensis and Carboxydibrachium pacificum, produced acetate and L-alanine as major end products of glucose metabolism [0.8-1.0 mol L-alanine produced (mol glucose consumed)(-1)] and reduced thiosulfate, but not sulfate, to sulfide. Because of these significant metabolic and phylogenetic differences between the oilfield isolates (T. subterraneus, strain OCA1 and strain SL9), T. yonseiensis, T. tengcongensis and Carboxydibrachium pacificum and other Thermoanaerobacter species, it is proposed to reassign them as a novel genus and species, Caldanaerobacter subterraneus gen. nov., sp. nov., comb. nov., with the creation of four novel subspecies, Caldanaerobacter subterraneus subsp. subterraneus subsp. nov., comb. nov., Caldanaerobacter subterraneus subsp. yonseiensis subsp. nov., comb. nov., Caldanaerobacter subterraneus subsp. tengcongensis subsp. nov., comb. nov. and Caldanaerobacter subterraneus subsp. pacificus subsp. nov., comb. nov.

Toluene-degrading Antarctic Pseudomonas strains from fuel-contaminated soil.

Two psychrotolerant toluene-degrading Pseudomonas spp. were isolated from JP8 jet-fuel-contaminated soils, Scott Base, Antarctica. Isolates metabolized meta-toluate as sole carbon source at temperatures ranging from 6 to 30 degrees C. Large plasmids (>64kb) were isolated from both isolates. Sequence analysis of PCR products amplified using xylB (the gene encoding benzyl alcohol dehydrogenase) primers revealed that isolates 7/167 and 8/46 were 100% and 92% homologous, respectively, to the xylB gene of the meta-cleavage toluene degradative pathway encoded by the TOL plasmid (pWWO) of Pseudomonas putida mt-2. Assays of cell-free extracts of 7/167 and 8/46 demonstrated activity of catechol 2,3-dioxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase, indicating that the isolates use the meta-cleavage pathway enzymes of toluene degradation typical of TOL type plasmids. As both isolates are able to grow at 6 degrees C ex situ it is feasible that they would be able to metabolize toluene in the Antarctic soils from where they were originally isolated.

Biodesulfurization of fossil fuels.

Biotechnological techniques enabling the specific removal of sulfur from fossil fuels have been developed. In the past three years there have been important advances in the elucidation of the mechanisms of biodesulfurization; some of the most significant relate to the role of a flavin reductase, DszD, in the enzymology of desulfurization, and to the use of new tools that enable enzyme enhancement via DNA manipulation to influence both the rate and the substrate range of Dsz. Also, a clearer understanding of the unique desulfinase step in the pathway has begun to emerge.