Bio-catalytic degradation of dibenzothiophene (DBT) in petroleum distillate (diesel) by Pseudomonas spp.

Biodesulfurization (BDS) was employed in this study to degrade dibenzothiophene (DBT) which accounts for 70% of the sulfur compounds in diesel using a synthetic and typical South African diesel in the aqueous and biphasic medium. Two Pseudomonas sp. bacteria namely Pseudomonas aeruginosa and Pseudomonas putida were used as biocatalysts. The desulfurization pathways of DBT by the two bacteria were determined by gas chromatography (GC)/mass spectrometry (MS) and High-Performance Liquid Chromatography (HPLC). Both organisms were found to produce 2-hydroxy biphenyl, the desulfurized product of DBT. Results showed BDS performance of 67.53% and 50.02%, by Pseudomonas aeruginosa and Pseudomonas putida, respectively for 500 ppm initial DBT concentration. In order to study the desulfurization of diesel oils obtained from an oil refinery, resting cells studies by Pseudomonas aeruginosa were carried out which showed a decrease of about 30% and 70.54% DBT removal for 5200 ppm in hydrodesulfurization (HDS) feed diesel and 120 ppm in HDS outlet diesel, respectively. Pseudomonas aeruginosa and Pseudomonas putida selectively degraded DBT to form 2-HBP. Application of these bacteria for the desulfurization of diesel showed promising potential for decreasing the sulfur content of South African diesel oil.

Bacterial diversity in aqueous/sludge phases within diesel fuel storage tanks.

Diesel fuel storage tanks are not hostile environments for microorganisms and tend to form sludges in the water deposited at the bottom of the tanks. The lack of nutrient, carbon and energy limitations within these habitats boost the abundance and the metabolic activity of microorganisms providing microbial hotspots with high growing rates of diesel degradation (0.10 ± 0.021 d-1). Five different Phyla (Thermotogae, Spirochaetes, Firmicutes, Bacteroidetes Proteobacteria) were identified within the aqueous/sludge phase from in situ diesel storage tanks, by cultured independent molecular surveys using the 16S rDNA gene fragment. The identified dominant strains were Geotoga aestuarianus, Flavobacterium ceti, Spirochaeta thermophila, Propionispira arboris, Sporobacterium olearium and Dysgonomonas genera. The altitude where the storage tanks are located and the organic carbon concentration within the aqueous/sludge phases affected the bacterial diversity. Therefore, the more diverse the microbial communities are, the more probability of the presence of bacteria with capacity to metabolized diesel and eliminate organic matter. Despite, only phosphate showed an effect on the bacterial distribution within the storage tanks, there was an apparent lack of deterministic process in structuring microbial communities. Consequently, preventative protocols are a priority to avoid the microbial growth within diesel fuel storage tanks. A new focus of this worldwide problem within the oil industry would be to explore deeply the wide range of metabolic and adaptive capacities of these microorganisms. These microbial consortia are potential tools with new specific services to apply in bioremediation among others.

Whole genome shotgun sequencing of POPs degrading bacterial community dwelling tannery effluents and petrol contaminated soil.

The present study involved identification of genes which are present in the genome of native bacteria to make them effective tools for bioremediation of persistent organic pollutants (POPs). During this study, forty-one POPs (naphthalene, toluene and petrol) metabolizing bacteria were isolated from tannery effluents and petrol contaminated soil samples by successive enrichment culturing. The taxonomic diversity and gene repertoire conferring POPs degradation ability to the isolated bacterial community were studied through whole genome shotgun sequencing of DNA consortium. The DNA consortium contained equimolar concentration of DNA extracted from each bacterial isolate using organic method. To add a double layer of confirmation the established DNA consortium was subjected to 16S rRNA metagenome sequencing and whole genome shotgun sequencing analysis. Biodiversity analysis revealed that the consortium was composed of phyla Firmicutes (80 %), Proteobacteria (12 %) and Actinobacteria (5%). Genera found included Bacillus (45 %), Burkholderia (25 %), Brevibacillus (9%) and Geobacillus (4%). Functional profiling of consortium helped us to identify genes associated with degradation pathways of a variety of organic compounds including toluene, naphthalene, caprolactam, benzoate, aminobenzoate, xylene, 4-hydroxyphenyl acetic acid, biphenyl, anthracene, aminobenzoate, chlorocyclohexane, chlorobenzene, n-phenylalkanoic acid, phenylpropanoid, salicylate, gentisate, central meta cleavage of aromatic compounds, cinnamic acid, catechol and procatechuate branch of ß-ketoadipate pathway, phenyl-acetyl CoA and homogentisate catabolic pathway. The information thus generated has ensured not only biodegradation potential but also revealed many possible future applications of the isolated bacteria.

A survey of microbial contamination in aviation fuel from aircraft fuel tanks.

Microbial contamination poses a great threat to aviation system security through mechanisms such as microbiologically influenced corrosion (MIC), fuel filter clogging, and fuel deterioration. In this study, a survey of microbial contamination in aviation fuel obtained from aircraft fuel tanks was performed to test the relationship between microbial contamination and aircraft service life. The contaminating microorganisms were counted, isolated, identified, and subjected to preliminary characterization. A low risk of microbial contamination in the selected samples was confirmed, and there was no significant difference in the counts between culturable bacteria and fungi (p > 0.05). Phylogenetic analysis tree indicated that the diversity of culturable microorganisms was rather low, with 17 bacterial isolates belonging to 13 genera and 12 fungal isolates belonging to 5 genera. No yeast was isolated. The growth characteristics of these isolates indicated that the aircraft fuel tanks harbored various microorganisms that were able to utilize the aviation fuel as a source of carbon and energy. Meanwhile, some isolates caused emulsification and produced acid. The conclusions of this study were that various hazardous microorganisms can root in aircraft aviation fuel tanks. There was no relationship between microbial contamination and aircraft service life (p > 0.05), and continuous good maintenance suppressed microbial proliferation.

Enhanced Degradation of Diesel Oil by Using Biofilms Formed by Indigenous Purple Photosynthetic Bacteria from Oil-Contaminated Coasts of Vietnam on Different Carriers.

Oil pollution in marine environment caused by oil spillage has been a main threat to the ecosystem including the ocean life and to the human being. In this research, three indigenous purple photosynthetic strains Rhodopseudomonas sp. DD4, DQ41, and FO2 were isolated from oil-contaminated coastal zones in Vietnam. The cells of these strains were immobilized on different carriers including cinder beads (CB), coconut fiber (CF), and polyurethane foam (PUF) for diesel oil removal from artificial seawater. The mixed biofilm formed by using CB, CF, and PUF as immobilization supports degraded 90, 91, and 95% of diesel oil (DO) with the initial concentration of 17.2 g/L, respectively, after 14 days of incubation. The adsorption of DO on different systems was accountable for the removal of 12-16% hydrocarbons for different carriers. To the best of our knowledge, this is the first report on diesel oil degradation by purple photosynthetic bacterial biofilms on different carriers. Moreover, using carriers attaching purple photosynthetic bacteria to remove diesel oil in large scale is considered as an essential method for the improvement of a cost-effective and efficient bioremediation manner. This study can be a promising approach to eliminate DO from oil-contaminated seawater.

Microaerobic conditions caused the overwhelming dominance of Acinetobacter spp. and the marginalization of Rhodococcus spp. in diesel fuel/crude oil mixture-amended enrichment cultures.

The aim of the present study was to reveal how different microbial communities evolve in diesel fuel/crude oil-contaminated environments under aerobic and microaerobic conditions. To investigate this question, aerobic and microaerobic bacterial enrichments amended with a diesel fuel/crude oil mixture were established and analysed. The representative aerobic enrichment community was dominated by Gammaproteobacteria (64.5%) with high an abundance of Betaproteobacteriales (36.5%), followed by Alphaproteobacteria (8.7%), Actinobacteria (5.6%), and Candidatus Saccharibacteria (4.5%). The most abundant alkane monooxygenase (alkB) genotypes in this enrichment could be linked to members of the genus Rhodococcus and to a novel Gammaproteobacterium, for which we generated a high-quality draft genome using genome-resolved metagenomics of the enrichment culture. Contrarily, in the microaerobic enrichment, Gammaproteobacteria (99%) overwhelmingly dominated the microbial community with a high abundance of the genera Acinetobacter (66.3%), Pseudomonas (11%) and Acidovorax (11%). Under microaerobic conditions, the vast majority of alkB gene sequences could be linked to Pseudomonas veronii. Consequently, results shed light on the fact that the excellent aliphatic hydrocarbon degrading Rhodococcus species favour clear aerobic conditions, while oxygen-limited conditions can facilitate the high abundance of Acinetobacter species in aliphatic hydrocarbon-contaminated subsurface environments.

Profiling the microbial contamination in aviation fuel from an airport.

Microbial contamination during fuel storage can cause fuel system fouling and corrosion. Characterizing microbial contamination is critical for preventing and solving these problems. In this study, culture-based combing with the culture-independent methods, were used to profile the microbial contamination in aviation fuel. High-throughput sequencing (HTS) modified by propidium monoazide (PMA) revealed a higher diversity of contaminating microorganisms in samples than the culture method. Proteobacteria (47%), Actinobacteria (21%) and Ascomycota (>99%, fungi) were the most abundant phyla, and the neglected archaea was also detected. Additionally, qPCR-based methods revealed all samples contained a heavy level of microbial contamination, which was more accurate than its culturable counterparts, and fungal contamination was still a problem in aviation fuel. The application of a PCR-based method gives deeper insight into microbial contamination in aviation fuel than the conventional culture method, thus using it for regular detection and accurate description of fuel contamination is strongly recommended in the case of explosive microbial growth.

Comparative genome sequencing and analyses of Mycobacterium cosmeticum reveal potential for biodesulfization of gasoline.

Mycobacterium cosmeticum is a nontuberculous Mycobacterium recovered from different water sources including household potable water and water collected at nail salon. Individual cases of this bacterium have been reported to be associated with gastrointestinal tract infections. Here we present the first whole-genome study and comparative analysis of two new clinically-derived Mycobacterium sp. UM_RHS (referred as UM_RHS after this) and Mycobacterium sp. UM_NYF (referred as UM_NYF after this) isolated from patients in Indonesia and Malaysia respectively to have a better understanding of the biological characteristic of these isolates. Both strains are likely Mycobacterium cosmeticum as supported by the evidence from molecular phylogenetic, comparative genomic and Average Nucleotide Identity (ANI) analyses. We found the presence of a considerably large number of putative virulence genes in the genomes of UM_RHS and UM_NYF. Interestingly, we also found a horizontally transferred genomic island carrying a putative dsz operon proposing that they may have potential to perform biodesulfization of dibenzothiophene (DBT) that may be effective in cost reduction and air pollution during fuel combustion. This comparative study may provide new insights into M. cosmeticum and serve as an important reference for future functional studies of this bacterial species.

Flavobacterium petrolei sp. nov., a novel psychrophilic, diesel-degrading bacterium isolated from oil-contaminated Arctic soil.

This study presents taxonomic description of two novel diesel-degrading, psychrophilic strains: Kopri-42T and Kopri-43, isolated during screening of oil-degrading psychrotrophs from oil-contaminated Arctic soil. A preliminary 16S rRNA gene sequence and phylogenetic tree analysis indicated that these Arctic strains belonged to the genus Flavobacterium, with the nearest relative being Flavobacterium psychrolimnae LMG 22018T (98.9% sequence similarity). The pairwise 16S rRNA gene sequence identity between strains Kopri-42T and Kopri-43 was 99.7%. The DNA-DNA hybridization value between strain Kopri-42T and Kopri-43 was 88.6 ± 2.1% indicating that Kopri-42T and Kopri-43 represents two strains of the same genomospecies. The average nucleotide identity and in silico DNA-DNA hybridization values between strain Kopri-42T and nearest relative F. psychrolimnae LMG 22018T were 92.4% and 47.9%, respectively. These values support the authenticity of the novel species and confirmed the strain Kopri-42T belonged to the genus Flavobacterium as a new member. The morphological, physiological, biochemical and chemotaxonomic data also distinguished strain Kopri-42T from its closest phylogenetic neighbors. Based on the polyphasic data, strains Kopri-42T and Kopri-43 represents a single novel species of the genus Flavobacterium, for which the name Flavobacterium petrolei sp. nov. is proposed. The type strain is Kopri-42T (=KEMB 9005-710T = KACC 19625T = NBRC 113374T).

Effect of copper on diesel degradation in Pseudomonas extremaustralis.

Environments co-contaminated with heavy metals and hydrocarbons have become an important problem worldwide, especially due to the effect of metals on hydrocarbon degrading microorganisms. Pseudomonas extremaustralis, a bacterium isolated from a pristine pond in Antarctica, showed high capabilities to cope with environmental stress and a very versatile metabolism that includes alkane degradation under microaerobic conditions. In this work, we analyzed P. extremaustralis' capability to resist high copper concentrations and the effect of copper presence in diesel biodegradation. We observed that P. extremaustralis resisted up to 4 mM CuSO4 in a rich medium such as LB. This copper resistance is sustained by the presence of the cus and cop operons together with other efflux systems and porins located in a single region in P. extremaustralis genome. When copper was present, diesel degradation was negatively affected, even though copper enhanced bacterial attachment to hydrocarbons. However, when a small amount of glucose (0.05% w/v) was added, the presence of CuSO4 enhanced alkane degradation. In addition, atomic force microscopy analysis showed that the presence of glucose decreased the negative effects produced by copper and diesel on the cell envelopes.

Thermophilic biodesulfurization and its application in oil desulfurization.

To reduce the harm caused to the environment by fuel combustion and meet the increasingly stringent emission standards, the sulfur content of fuels should be reduced. Dibenzothiophene, benzothiophene, and their derivatives are sulfur-containing components of fuels that are difficult to desulfurize and can therefore cause great environmental damage. Biodesulfurization is a desulfurization method that has the advantage of being able to remove dibenzothiophene and its derivatives removed easily under conditions that are relatively mild when compared with hydrodesulfurization. This paper introduces the advantages of thermophilic biodesulfurization compared with mesophilic biodesulfurization; analyzes the desulfurization mechanism, including the desulfurization pathways and enzymic systems of desulfurization bacteria; and discusses the application of biodesulfurization in oil desulfurization. The main problems existing in biodesulfurization and possible solutions are also analyzed in this paper. Biological desulfurization is a promising method for desulfurization; accordingly, more studies investigating biodesulfurization of actual oil are needed to enable the industrialized application of biodesulfurization.

Pseudallescheria boydii and Meyerozyma guilliermondii: behavior of deteriogenic fungi during simulated storage of diesel, biodiesel, and B10 blend in Brazil.

Due to their renewable and sustainable nature, biodiesel blends boost studies predicting their stability during storage. Besides chemical degradation, biodiesel is more susceptible to biodegradation due to its raw composition. The aim of this work was to evaluate the deteriogenic potential (growth and degradation) of Pseudallescheria boydii and Meyerozyma guilliermondii in degrading pure diesel (B0), pure biodiesel (B100), and a B10 blend in mineral medium during storage. The biodeterioration susceptibility at different fuel ratios and in BH minimal mineral medium were evaluated. The biomass measurements of P. boydii during 45 days indicated higher biomass production in the B10 blend. The growth curve of M. guilliermondii showed similar growth in B10 and B100. Although there was no significant production of biosurfactant, lipase production was detected in the tributyrin agar medium of both microorganisms. The main compounds identified in the aqueous phase by GC-MS were alcohols, esters, acids, sulfur, ketones, and phenols. The results showed that P. boydii grew at the expense of fuels, degrading biodiesel esters, and diesel hydrocarbons. M. guilliermondii grew in B100 and B10; however, degradation was not detected.

Paraburkholderia aromaticivorans sp. nov., an aromatic hydrocarbon-degrading bacterium, isolated from gasoline-contaminated soil.

A Gram-stain-negative, facultatively aerobic, aromatic hydrocarbon-degrading bacterium, designated strain BN5T, was isolated from gasoline-contaminated soil. Cells were motile and slightly curved rods with a single flagellum showing catalase and oxidase activities. Growth was observed at 20-37 °C (optimum, 25-30 °C), pH 3-7 (optimum, pH 5-6) and 0-2 % NaCl (optimum, 0 %). Ubiquinone-8 was the predominant respiratory quinone. The major fatty acids were C16 : 0, cyclo-C19 : 0ω8c and summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c). Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified phosphoamino lipid, three unidentified amino lipids and eight unidentified lipids were the identified polar lipids. The DNA G+C content was 62.93 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain BN5T formed a phylogenic lineage with members of the genus Paraburkholderia and showed the highest 16S rRNA gene sequence similarities to Paraburkholderia phytofirmans PsJNT (99.4 %), Paraburkholderia dipogonis DL7T (98.8 %) and Paraburkholderia insulsa PNG-AprilT (98.8 %). The average nucleotide identity and in silico DNA-DNA hybridization (DDH) values between strain BN5T and P. phytofirmans PsJNT were 88.5 and 36.5 %, respectively. The DDH values for strain BN5T with P. dipogonis LMG 28415T and P. insulsa DSM 28142T were 41.0±4.9 % (reciprocal, 33.0±4.3 %) and 47.1±6.6 % (reciprocal, 51.7±5.4 %), respectively. Based on its physiological, chemotaxonomic and phylogenetic features, we conclude that strain BN5T is a novel species of the genus Paraburkholderia, for which the name Paraburkholderia aromaticivorans sp. nov. is proposed. The type strain is BN5T (=KACC 19419T=JCM 32303T).

Impact of the Fenton-like treatment on the microbial community of a diesel-contaminated soil.

Fenton-like treatment (FLT) is an ISCO technique relying on the iron-induced H2O2 activation in the presence of additives aimed at increasing the oxidant lifetime and maximizing iron solubility under natural soil pH conditions. The efficacy of FLT in the clean-up of hydrocarbon-contaminated soils is well established at the field-scale. However, a better assessment of the impact of the FLT on density, diversity and activity of the indigenous soil microbiota, might provide further insights into an optimal combination between FLT and in-situ bioremediation (ISB). The aim of this work was to assess the impacts of FLT on the microbial community of a diesel-contaminated soil collected nearby a gasoline station. Different FLT conditions were tested by varying either the H2O2 concentrations (2 and 6%) or the oxidant application mode (single or double dosage). The impact of these treatments on the indigenous microbial community was assessed immediately after the Fenton-like treatment and after 30, 60 and 90 d and compared with enhanced natural attenuation (ENA). After FLT, a dramatic decrease in bacterial density, diversity and functionality was evident. Although in microcosms with double dosing at 2% H2O2 a delayed recovery of the indigenous microbiota was observed as compared to those subjected to single oxidant dose, after 60 d incubation the respiration rate increased from 0.036 to 0.256 µg CCO2 g-1soil h-1. Irrespective of the oxidant dose, best degradation results after 90 d incubation (around 80%) were observed with combined FLT, relying on double oxidant addition, and bioremediation.

Biodesulphurization of gasoline by Rhodococcus erythropolis supported on polyvinyl alcohol.

A new biodesulphurization (BDS) method has been considered using Rhodococcus erythropolis supported on polyvinyl alcohol (PVA) for BDS of thiophene as a gasoline sulphur model compound in n-hexane as the solvent, subsequently this biocatalyst has been applied to BDS of gasoline samples. The obtained results according to UV-Spectrophotometer analysis at 240 nm showed that 97·41% of thiophene at the optimum condition of primary concentration 80 mg l-1 , pH = 7, by 0·1 g of biocatalyst in 30°C and after 20 h of contact time has been degraded. These optimum conditions have been applied to gasoline BDS and the biodegradation of gasoline thiophenic compounds have been investigated by gas chromatography-mass spectrometry (GC-MS). According to GC-MS, thiophene and its 2-methyl, 3-methyl and 2- ethyl derivatives had acceptable biodegradation efficiencies of about 26·67, 21·03, 23·62% respectively. Also, benzothiophene that has been detected in a gasoline sample had 38·89% biodegradation efficiency at optimum conditions, so biomodification of PVA by R. erythropolis produces biocatalysts with an active metabolism that facilitates the interaction of bacterial strain with gasoline thiophenic compounds. The morphology and surface functional groups of supported R. erythropolis on PVA have been investigated by scanning electron microscope (SEM) and FT-IR spectroscopy respectively. SEM images suggest some regular layered shape for the supported bacteria. FT-IR spectra indicate a desirable interaction between bacterial cells and polymer supports. Also, the recovery of biocatalyst has been investigated and after three times of using in BDS activity, its biocatalytic ability had no significant decreases. SIGNIFICANCE AND IMPACT OF THE STUDY: The biomodification of polyvinyl alcohol by Rhodococcus erythropolis described herein produces a new biocatalyst which can be used for significantly reducing the thiophenic compounds of gasoline and other fossil fuels. The immobilization process is to increase the biodegradation efficiency of cells and accelerating the biodesulphurization process.

Optimization of Enterobacter cloacae (KU923381) for diesel oil degradation using response surface methodology (RSM).

Efficiency of Enterobacter cloacae KU923381 isolated from petroleum hydrocarbon contaminated soil was evaluated in batch culture and bioreactor mode. The isolate were screened for biofilm formation using qualitative and quantitative assays. Response surface methodology (RSM) was used to study the effect of pH, temperature, glucose concentration, and sodium chloride on diesel degradation. The predicted values for diesel oil degradation efficiency by the statistical designs are in a close agreement with experimental data (R 2 = 99.66%). Degradation efficiency is increased by 36.78% at pH = 7, temperature = 35°C, glucose = 5%, and sodium chloride concentration = 5%. Under the optimized conditions, the experiments were performed for diesel oil degradation by gas chromatographic mass spectrometric analysis (GC-MS). GC-MS analysis confirmed that E. cloacae had highly degrade hexadecane, heptadecane, tridecane, and docosane by 99.71%, 99.23%, 99.66%, and 98.34% respectively. This study shows that rapid bioremoval of hydrocarbons in diesel oil is acheived by E. cloacae with abet of biofilm formation. The potential use of the biofilms for preparing trickling filters (gravel particles) for the degradation of hydrocarbons from petroleum wastes before their disposal in the open environment is highly suggested. This is the first successful attempt for artificially establishing petroleum hydrocarbon degrading bacterial biofilm on solid substrates in bioreactor.

Co-production of acetone and ethanol with molar ratio control enables production of improved gasoline or jet fuel blends.

The fermentation of simple sugars to ethanol has been the most successful biofuel process to displace fossil fuel consumption worldwide thus far. However, the physical properties of ethanol and automotive components limit its application in most cases to 10-15 vol% blends with conventional gasoline. Fermentative co-production of ethanol and acetone coupled with a catalytic alkylation reaction could enable the production of gasoline blendstocks enriched in higher-chain oxygenates. Here we demonstrate a synthetic pathway for the production of acetone through the mevalonate precursor hydroxymethylglutaryl-CoA. Expression of this pathway in various strains of Escherichia coli resulted in the co-production of acetone and ethanol. Metabolic engineering and control of the environmental conditions for microbial growth resulted in controllable acetone and ethanol production with ethanol:acetone molar ratios ranging from 0.7:1 to 10.0:1. Specifically, use of gluconic acid as a substrate increased production of acetone and balanced the redox state of the system, predictively reducing the molar ethanol:acetone ratio. Increases in ethanol production and the molar ethanol:acetone ratio were achieved by co-expression of the aldehyde/alcohol dehydrogenase (AdhE) from E. coli MG1655 and by co-expression of pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (AdhB) from Z. mobilis. Controlling the fermentation aeration rate and pH in a bioreactor raised the acetone titer to 5.1 g L(-1) , similar to that obtained with wild-type Clostridium acetobutylicum. Optimizing the metabolic pathway, the selection of host strain, and the physiological conditions employed for host growth together improved acetone titers over 35-fold (0.14-5.1 g/L). Finally, chemical catalysis was used to upgrade the co-produced ethanol and acetone at both low and high molar ratios to higher-chain oxygenates for gasoline and jet fuel applications. Biotechnol. Bioeng. 2016;113: 2079-2087. © 2016 Wiley Periodicals, Inc.

Trichoderma longibrachiatum Evx1 is a fungal biocatalyst suitable for the remediation of soils contaminated with diesel fuel and polycyclic aromatic hydrocarbons.

Trichoderma sp. strain Evx1 was isolated from a semi-deciduous forest soil in Southern Italy. It decolorizes polynuclear organic dyes and tolerates high concentrations of phenanthrene, anthracene, fluoranthene, and pyrene. The ability of this ascomycete fungus to degrade polycyclic aromatic hydrocarbons was verified in vitro and confirmed by its strong phenoloxidase activity in the presence of gallic acid. Phylogenetic characterization of Trichoderma sp. Evx1 positioned this strain within the species Trichoderma longibrachiatum. The potential use of this species for the bioremediation of contaminated environmental matrices was tested by inoculating diesel-spiked soil with a dense mycelial suspension. The biodegradation percentage of the C12-40 hydrocarbon fraction in the inoculated soil rose to 54.2 ± 1.6 %, much higher than that in non-inoculated soil or soil managed solely by a combination of watering and aeration. The survival and persistence of T. longibrachiatum Evx1 throughout the bioremediation trial was monitored by PCR-DGGE analysis. The fungal strain was still present in the soil 30 days after bioaugmentation. These findings indicate that T. longibrachiatum Evx1 may be a suitable inoculum in bioremediation protocols for the reclamation of soils contaminated by complex mixtures of hydrocarbons.

Polyphasic analysis of an Azoarcus-Leptothrix-dominated bacterial biofilm developed on stainless steel surface in a gasoline-contaminated hypoxic groundwater.

Pump and treat systems are widely used for hydrocarbon-contaminated groundwater remediation. Although biofouling (formation of clogging biofilms on pump surfaces) is a common problem in these systems, scarce information is available regarding the phylogenetic and functional complexity of such biofilms. Extensive information about the taxa and species as well as metabolic potential of a bacterial biofilm developed on the stainless steel surface of a pump submerged in a gasoline-contaminated hypoxic groundwater is presented. Results shed light on a complex network of interconnected hydrocarbon-degrading chemoorganotrophic and chemolitotrophic bacteria. It was found that besides the well-known hydrocarbon-degrading aerobic/facultative anaerobic biofilm-forming organisms (e.g., Azoarcus, Leptothrix, Acidovorax, Thauera, Pseudomonas, etc.), representatives of Fe(2+)-and Mn(2+)-oxidizing (Thiobacillus, Sideroxydans, Gallionella, Rhodopseudomonas, etc.) as well as of Fe(3+)- and Mn(4+)-respiring (Rhodoferax, Geobacter, Magnetospirillum, Sulfurimonas, etc.) bacteria were present in the biofilm. The predominance of ß-Proteobacteria within the biofilm bacterial community in phylogenetic and functional point of view was revealed. Investigation of meta-cleavage dioxygenase and benzylsuccinate synthase (bssA) genes indicated that within the biofilm, Azoarcus, Leptothrix, Zoogloea, and Thauera species are most probably involved in intrinsic biodegradation of aromatic hydrocarbons. Polyphasic analysis of the biofilm shed light on the fact that subsurface microbial accretions might be reservoirs of novel putatively hydrocarbon-degrading bacterial species. Moreover, clogging biofilms besides their detrimental effects might supplement the efficiency of pump and treat systems.

Modular and selective biosynthesis of gasoline-range alkanes.

Typical renewable liquid fuel alternatives to gasoline are not entirely compatible with current infrastructure. We have engineered Escherichia coli to selectively produce alkanes found in gasoline (propane, butane, pentane, heptane, and nonane) from renewable substrates such as glucose or glycerol. Our modular pathway framework achieves carbon-chain extension by two different mechanisms. A fatty acid synthesis route is used to generate longer chains heptane and nonane, while a more energy efficient alternative, reverse-ß-oxidation, is used for synthesis of propane, butane, and pentane. We demonstrate that both upstream (thiolase) and intermediate (thioesterase) reactions can act as control points for chain-length specificity. Specific free fatty acids are subsequently converted to alkanes using a broad-specificity carboxylic acid reductase and a cyanobacterial aldehyde decarbonylase (AD). The selectivity obtained by different module pairings provides a foundation for tuning alkane product distribution for desired fuel properties. Alternate ADs that have greater activity on shorter substrates improve observed alkane titer. However, even in an engineered host strain that significantly reduces endogenous conversion of aldehyde intermediates to alcohol byproducts, AD activity is observed to be limiting for all chain lengths. Given these insights, we discuss guiding principles for pathway selection and potential opportunities for pathway improvement.