Biodegradation of asphaltenes by an indigenous bioemulsifier-producing Pseudomonas stutzeri YWX-1 from shale oil in the Ordos Basin: Biochemical characterization and complete genome analysis.

Crude oil pollution is environmentally ubiquitous and has become a global public concern about its impact on human health. Asphaltenes are the key components of heavy crude oil (HCO) that are underutilized due to their high viscosity and density, and yet, the associated information about biodegradation is extremely limited in the literature. In the present study, an indigenous bacterium with effective asphaltene-degrading activity was isolated from oil shale and identified as Pseudomonas stutzeri by a polyphasic taxonomic approach, named YWX-1. Supplemented with 75 g L-1 heavy crude oil as the sole carbon source for growth in basic mineral salts liquid medium (MSM), strain YWX-1 was able to remove 49% of asphaletene fractions within 14 days, when it was cultivated with an initial inoculation size of 1%. During the degradation process, the bioemulsifier produced by strain YWX-1 could emulsify HCO obviously into particles, as well as it had the ability to solubilize asphaletenes. The bioemulsifier was identified to be a mixture of polysaccharide and protein through Fourier transform infrared spectroscopy (FT-IR). The genome of strain YWX-1 contains one circular chromosome of 4488441 bp with 63.98% GC content and 4145 protein coding genes without any plasmid. Further genome annotation indicated that strain YWX-1 possesses a serial of genes involved in bio-emulsification and asphaltenes biodegradation. This work suggested that P. stutzeri YWX-1 could be a promising species for bioremediation of HCO and its genome analysis provided insight into the molecular basis of asphaltene biodegradation and bioemulsifier production.

Use of the ptxD gene as a portable selectable marker for chloroplast transformation in Chlamydomonas reinhardtii.

Synthetic biology and genetic engineering in algae offer an unprecedented opportunity to develop species with traits that can help solve the problems associated with food and energy supply in the 21st century. In the green alga Chlamydomonas reinhardtii, foreign genes can be expressed from the chloroplast genome for molecular farming and metabolic engineering to obtain commodities and high-value molecules. To introduce these genes, selectable markers, which rely mostly on the use of antibiotics, are needed. This has risen social concern associated with the potential risk of horizontal gene transfer across life kingdoms, which has led to a quest for antibiotic-free selectable markers. Phosphorus (P) is a scarce nutrient element that most organisms can only assimilate in its most oxidized form as phosphate (Pi); however, some organisms are able to oxidize phosphite (Phi) to Pi prior to incorporation into the central metabolism of P. As an alternative to the use of the two positive selectable makers already available for chloroplast transformation in C. reinhardtii, the aadA and the aphA-6 genes, that require the use of antibiotics, we investigated if a phosphite-based selection method could be used for the direct recovery of chloroplast transformed lines in this alga. Here we show that following bombardment with a vector carrying the ptxD gene from Pseudomonas stutzeri WM88, only cells that integrate and express the gene proliferate and form colonies using Phi as the sole P source. Our results demonstrate that a selectable marker based on the assimilation of Phi can be used for chloroplasts transformation in a biotechnologically relevant organism. The portable selectable marker we have developed is, in more than 18 years, the latest addition to the markers available for selection of chloroplast transformed cells in C. reinhardtii. The ptxD gene will contribute to the repertoire of tools available for synthetic biology and genetic engineering in the chloroplast of C. reinhardtii.

Comparative genomic, proteomic and exoproteomic analyses of three Pseudomonas strains reveals novel insights into the phosphorus scavenging capabilities of soil bacteria.

Bacteria that inhabit the rhizosphere of agricultural crops can have a beneficial effect on crop growth. One such mechanism is the microbial-driven solubilization and remineralization of complex forms of phosphorus (P). It is known that bacteria secrete various phosphatases in response to low P conditions. However, our understanding of their global proteomic response to P stress is limited. Here, exoproteomic analysis of Pseudomonas putida BIRD-1 (BIRD-1), Pseudomonas fluorescens SBW25 and Pseudomonas stutzeri DSM4166 was performed in unison with whole-cell proteomic analysis of BIRD-1 grown under phosphate (Pi) replete and Pi deplete conditions. Comparative exoproteomics revealed marked heterogeneity in the exoproteomes of each Pseudomonas strain in response to Pi depletion. In addition to well-characterized members of the PHO regulon such as alkaline phosphatases, several proteins, previously not associated with the response to Pi depletion, were also identified. These included putative nucleases, phosphotriesterases, putative phosphonate transporters and outer membrane proteins. Moreover, in BIRD-1, mutagenesis of the master regulator, phoBR, led us to confirm the addition of several novel PHO-dependent proteins. Our data expands knowledge of the Pseudomonas PHO regulon, including species that are frequently used as bioinoculants, opening up the potential for more efficient and complete use of soil complexed P.

Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium.

Three bacterial isolates identified as Alcanivorax borkumensis SK2, Rhodococcus erythropolis HS4 and Pseudomonas stutzeri SDM, based on 16S rRNA gene sequences, were isolated from crude oil enrichments of natural seawater. Single strains and four bacterial consortia designed by mixing the single bacterial cultures respectively in the following ratios: (Alcanivorax: Pseudomonas, 1:1), (Alcanivorax: Rhodococcus, 1:1), (Pseudomonas: Rhodococcus, 1:1), and (Alcanivorax: Pseudomonas: Rhodococcus, 1:1:1), were analyzed in order to evaluate their oil degrading capability. All experiments were carried out in microcosms systems containing seawater (with and without addition of inorganic nutrients) and crude oil (unique carbon source). Measures of total and live bacterial abundance, Card-FISH and quali-, quantitative analysis of hydrocarbons (GC-FID) were carried out in order to elucidate the co-operative action of mixed microbial populations in the process of biodegradation of crude oil. All data obtained confirmed the fundamental role of bacteria belonging to Alcanivorax genus in the degradation of linear hydrocarbons in oil polluted environments.

Complete genome sequence of the petroleum-emulsifying bacterium Pseudomonas stutzeri SLG510A3-8.

Pseudomonas stutzeri SLG510A3-8, isolated from oil-contaminated soil in Shengli Oilfield, China, has the potential to be applied for microbial enhanced oil recovery. Here, we reported the complete genome sequence of this bacterium. It has a 4,650,155bp circular chromosome encoding 4450 genes, and the genome consists of genes that are involved in denitrification, chemotaxis, benzoate degradation, molecule transportation, and other functions. The genome contains a complete set of genes for type I secretion system in comparison with sequences of other P. stutzeri strains.

Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium

Three bacterial isolates identified as Alcanivorax borkumensis SK2, Rhodococcus erythropolis HS4 and Pseudomonas stutzeri SDM, based on 16S rRNA gene sequences, were isolated from crude oil enrichments of natural seawater. Single strains and four bacterial consortia designed by mixing the single bacterial cultures respectively in the following ratios: (Alcanivorax: Pseudomonas, 1:1), (Alcanivorax: Rhodococcus, 1:1), (Pseudomonas: Rhodococcus, 1:1), and (Alcanivorax: Pseudomonas: Rhodococcus, 1:1:1), were analyzed in order to evaluate their oil degrading capability. All experiments were carried out in microcosms systems containing seawater (with and without addition of inorganic nutrients) and crude oil (unique carbon source). Measures of total and live bacterial abundance, Card-FISH and quali-, quantitative analysis of hydrocarbons (GC-FID) were carried out in order to elucidate the co-operative action of mixed microbial populations in the process of biodegradation of crude oil. All data obtained confirmed the fundamental role of bacteria belonging to Alcanivorax genus in the degradation of linear hydrocarbons in oil polluted environments.

Heterologous production of Pseudomonas aeruginosa rhamnolipid under anaerobic conditions for microbial enhanced oil recovery.

AIMS: The ex situ application of rhamnolipid to enhance oil recovery is costly and complex in terms of rhamnolipid production and transportation, while in situ production of rhamnolipid is restricted by the oxygen-deficient environments of oil reservoirs. To overcome the oxygen-limiting conditions and to circumvent the complex regulation of rhamnolipid biosynthesis in Pseudomonas aeruginosa, an engineered strain Pseudomonas stutzeri Rhl was constructed for heterologous production of rhamnolipid under anaerobic conditions. METHODS AND RESULTS: The rhlABRI genes for rhamnolipid biosynthesis were cloned into a facultative anaerobic strain Ps. stutzeri DQ1 to construct the engineered strain Rhl. Anaerobic production of rhamnolipid was confirmed by thin layer chromatography and Fourier transform infrared analysis. Rhamnolipid product reduced the air-water surface tension to 30.3 mN m(-1) and the oil-water interfacial tension to 0.169 mN m(-1). Rhl produced rhamnolipid of 1.61 g l(-1) using glycerol as the carbon source. Rhl anaerobic culture emulsified crude oil up to EI24 ≈ 74. An extra 9.8% of original crude oil was displaced by Rhl in the core flooding test. CONCLUSIONS: Strain Rhl achieved anaerobic production of rhamnolipid and worked well for enhanced oil recovery in the core flooding model. The rhamnolipid produced by Rhl was similar to that of the donor strain SQ6. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to achieve anaerobic and heterologous production of rhamnolipid. Results demonstrated the potential feasibility of Rhl as a promising strain to enhance oil recovery through anaerobic production of rhamnolipid.

Metagenomics of petroleum muck: revealing microbial diversity and depicting microbial syntrophy.

Present study attempts in revealing taxonomic and functional diversity of microorganism from petroleum muck using metagenomics approach. Using Ion Torrent Personal Genome Machine, total of 249 Mb raw data were obtained which was analysed using MG-RAST platform. The taxonomic analysis revealed predominance of Proteobacteria with Gammaproteobacteria as major class and Pseudomonas stutzeri as most abundant organism. Several enzymes involved in aliphatic and aromatic hydrocarbon degradation through both aerobic and anaerobic routes and proteins related to stress response were also present. Comparison of our metagenome with the existing metagenomes from oil-contaminated sites and wastewater treatment plant indicated uniqueness of this metagenome taxonomically and functionally. Based on these results a hypothetical community model showing survival and syntrophy of microorganisms in hydrocarbon-rich environment is proposed. Validation of the metagenome data was done in three tiers by validating major OTUs by isolating oil-degrading microbes, confirmation of key genes responsible for hydrocarbon degradation by Sanger sequencing and studying functional dynamics for degradation of the hydrocarbons by the muck meta-community using GC-MS.

Optimization of culture medium for anaerobic production of rhamnolipid by recombinant Pseudomonas stutzeri Rhl for microbial enhanced oil recovery.

UNLABELLED: Response surface methodology was employed to enhance the anaerobic production of rhamnolipid by recombinant Pseudomonas stutzeri Rhl. Glycerol is a promising carbon source used to anaerobically produce rhamnolipid. In a Plackett-Burman design, glycerol, KH2 PO4 and yeast extract were significant factors. The proposed optimized medium contained the following: 46·55 g l(-1) glycerol; 3 g l(-1) NaNO3 ; 5·25 g l(-1) K2 HPO4 ·3H2 O; 5·71 g l(-1) KH2 PO4 ; 0·40 g l(-1) MgSO4 ·7H2 O; 0·13 g l(-1) CaCl2 ; 1·0 g l(-1) KCl; 1·0 g l(-1) NaCl; and 2·69 g l(-1) yeast extract. Using this optimized medium, we obtained an anaerobic yield of rhamnolipid of 3·12 ± 0·11 g l(-1) with a 0·85-fold increase. Core flooding test results also revealed that Ps. stutzeri Rhl grown in an optimized medium enhanced the oil recovery efficiency by 15·7%, which was 6·6% higher than in the initial medium. Results suggested that the optimized medium is a promising nutrient source that could effectively mobilize oil by enhancing the in situ production of rhamnolipid. SIGNIFICANCE AND IMPACT OF THE STUDY: The ex situ application of rhamnolipid for microbial enhanced oil recovery (MEOR) is costly and complex in terms of rhamnolipid production, purification and transportation. Compared with ex situ applications, the in situ production of rhamnolipid in anaerobic oil reservoir is more advantageous for MEOR. This study is the first to report the anaerobic production optimization of rhamnolipid. Results showed that the optimized medium enhanced not only the anaerobic production of rhamnolipid but also crude oil recovery.

Production of melanin pigment from Pseudomonas stutzeri isolated from red seaweed Hypnea musciformis.

UNLABELLED: Hypnea musciformis red seaweed is popularly known to produce carrageenan was collected from the Gulf of Mannar, India. Strain HMGM-7 [MTCC 11712] was isolated from the surface of this seaweed, which was capable of producing an extracellular black-coloured polymeric pigment. Based on phenotypic characterization and 16S rDNA sequencing, the strain HMGM-7 was identified as Pseudomonas stutzeri. Biophysical characterization by UV-visible, FT-IR, EPR and XRD spectroscopic studies confirmed the pigment as melanin. Further chemical characterization showed that it was acid-resistant, alkali-soluble and alkali-insoluble in most of the organic solvents and distilled water. To our knowledge, this is a first report on a marine Pseudomonas stutzeri strain producing significant amounts of melanin of about 6·7 g l(-1) without L-tyrosine supplementation in the sea-water production medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This investigation reports a marine Pseudomonas stutzeri strain HMGM-7 [MTCC 11712] that produces significant quantities of melanin (6·7 g l(-1) ) in sea-water medium without the supplementation of L-tyrosine. The confirmation of the produced melanin was carried out by various chemical and physical characterization studies. The isolated melanin may find potential application for use in cosmetic and/or pharmaceutical industries.

Removal of 2,4,5-trichlorophenol by bacterial isolates from the secondary sludge of pulp and paper mill.

2,4,5-trichlorophenol (2,4,5-TCP) mineralizing bacteria were isolated from the secondary sludge of pulp and paper industry. These isolates used 2,4,5-TCP as a source of carbon and energy and were capable of degrading this compound, as indicated by stoichimetric release of chloride and biomass formation. Based on 16S rRNA sequence analysis, these bacteria were identified as Kocuria sp. (CL2), Bacillus pumillus (CL5), Pseudomonas stutzeri (CL7). HPLC analysis revealed that these isolates were able to degrade 2,4,5-TCP at higher concentrations (600 mg/l or 3.0 mM). A consortia of these isolates completely removed 2,4,5-TCP from the sludge obtained from pulp and paper mill within 2 weeks when supplemented at a rate of 100 mg l(-1) . Bacterial consortium also significantly reduced absorbable organic halogen (AOX) and extractable organic halogen (EOX) by 61% and 63%, respectively from the sludge. These isolates have high potential to remove 2,4,5-TCP and may be used for remediation of pulp paper mill waste containing 2,4,5-TCP.

Physiological role of NahW, the additional salicylate hydroxylase found in Pseudomonas stutzeri AN10.

The physiological role of NahW, the second salicylate hydroxylase of Pseudomonas stutzeri AN10, has been analysed by gene mutation and further complementation. When grown on naphthalene as a unique carbon and energy source, the nahW mutant showed a strong decrease in salicylate hydroxylase activity when compared with the wild-type strain, exhibited lower specific growth rates and accumulated salicylate in culture supernatants. Similarly, lower specific growth rates and salicylate accumulation were observed for the nahW mutant when growth on naphthalene supplemented with succinate or pyruvate. When P. stutzeri AN10 was grown in Luria-Bertani medium in the presence of salicylate, or was cultivated on minimal medium supplemented with salicylate as a unique carbon and energy source, an increase in the lag phase and a decrease in the specific growth rate were observed on increasing the salicylate concentrations, suggesting a plausible toxic effect. This toxic effect of salicylate was much more evident for the nahW mutant than for the wild-type strain. Complementation of the nahW mutant restored all growth parameters. These results indicate that NahW may have two functions in P. stutzeri AN10: (1) to improve its capacity to degrade naphthalene and (2) effectively convert the salicylate produced during naphthalene degradation to tricarboxylic acid cycle intermediates, preventing its toxic effect.

[Analysis of a new nitrogen fixation gene in Pseudomonas stutzeri A1501].

OBJECTIVE: We studied the role of the nitrogen fixation gene PST1305 located within the nitrogen fixation island of Pseudomonas stutzeri A1501. METHODS: We constructed the mutant strain (np1305) by homologous recombination and triparental conjugation, and determined the nitrogenase activity by the acetylene reduction test. Through RT-PCR, we analyzed the transcriptional units of PST1305 gene and its nearby genes. Real-Time PCR was applied to compare the expression level of PST1305 gene between optimal and non-nitrogen fixating conditions. RESULTS: Compared to the wild type, the nitrogenase activity in mutant strain (np1305) was partially decreased, however, functional complementary strain (np1305Comp) could restore nitrogenase activity close to wild type level. PST1305 gene was co-transcribed with its upstream gene (nifB and fdxN) and downstream gene (nifQ, PST1303 and PST1302). In contrast to the nitrogen excess conditions, expression of PST1305 under nitrogen-fixing conditions was significantly upregulated for 38.7-fold. CONCLUSION: Disruption of PST1305 exhibited a declined nitrogenase activity compared to the wild type A1501. PST1305 gene might participate in biological nitrogen fixation by involving in the electron transport or the oxygen protection mechanism of nitrogenase. These results suggested that PST1305 gene was a new gene required for optimal nitrogenase activity of Pseudomonas stutzeri A1501.

Pyridine-2,6-bis(thiocarboxylic acid) produced by Pseudomonas stutzeri KC reduces and precipitates selenium and tellurium oxyanions.

The siderophore of Pseudomonas stutzeri KC, pyridine-2,6-bis(thiocarboxylic acid) (pdtc), is shown to detoxify selenium and tellurium oxyanions in bacterial cultures. A mechanism for pdtc's detoxification of tellurite and selenite is proposed. The mechanism is based upon determination using mass spectrometry and energy-dispersive X-ray spectrometry of the chemical structures of compounds formed during initial reactions of tellurite and selenite with pdtc. Selenite and tellurite are reduced by pdtc or its hydrolysis product H(2)S, forming zero-valent pdtc selenides and pdtc tellurides that precipitate from solution. These insoluble compounds then hydrolyze, releasing nanometer-sized particles of elemental selenium or tellurium. Electron microscopy studies showed both extracellular precipitation and internal deposition of these metalloids by bacterial cells. The precipitates formed with synthetic pdtc were similar to those formed in pdtc-producing cultures of P. stutzeri KC. Culture filtrates of P. stutzeri KC containing pdtc were also active in removing selenite and precipitating elemental selenium and tellurium. The pdtc-producing wild-type strain KC conferred higher tolerance against selenite and tellurite toxicity than a pdtc-negative mutant strain, CTN1. These observations support the hypothesis that pdtc not only functions as a siderophore but also is involved in an initial line of defense against toxicity from various metals and metalloids.

Monitoring the spread of recombinant DNA from field plots with transgenic sugar beet plants by PCR and natural transformation of Pseudomonas stutzeri.

Previous studies had shown that recombinant DNA can be detected for several months in soil after the deposition of litter from transgenic (tg) plants. Here we show by PCR monitoring of field releases of tg sugar beet plants that during the growth of the plants the soil close to the plants and also plant material contains recombinant DNA, in the form of extracellular molecules. Surprisingly, the monitoring also revealed the presence of tg DNA in many field plots (30-70%) in which tg plants were never grown. These studies and the further monitoring during other tg sugar beet release experiments by PCR and a novel bioassay (measuring the transforming potential of recombinant DNA for Pseudomonas stutzeri) indicated that recombinant DNA was only detectable in the surface soil of field plots and their vicinity where flowering of the tg beet plants was allowed. Recombinant DNA was found in soil at a distance of 50 m from pollen-producing plants surrounded by a strip with hemp plants as a containment regime. It is concluded that recombinant DNA is deposited in soil during the growth of tg sugar beets and that a major mechanism of recombinant DNA spread in the environment is the dispersal of pollen which allows recombinant DNA to persist in the field plot for at least a year.