Biofilm lifestyle enhances diesel bioremediation and biosurfactant production in the Antarctic polyhydroxyalkanoate producer Pseudomonas extremaustralis

Diesel is a widely distributed pollutant. Bioremediation of this kind of compounds requires the use of microorganisms able to survive and adapt to contaminated environments. Pseudomonas extremaustralis is an Antarctic bacterium with a remarkable survival capability associated to polyhydroxyalkanoates (PHAs) production. This strain was used to investigate the effect of cell growth conditions--in biofilm versus shaken flask cultures--as well as the inocula characteristics associated with PHAs accumulation, on diesel degradation. Biofilms showed increased cell growth, biosurfactant production and diesel degradation compared with that obtained in shaken flask cultures. PHA accumulation decreased biofilm cell attachment and enhanced biosurfactant production. Degradation of long-chain and branched alkanes was observed in biofilms, while in shaken flasks only medium-chain length alkanes were degraded. This work shows that the PHA accumulating bacterium P. extremaustralis can be a good candidate to be used as hydrocarbon bioremediation agent, especially in extreme environments.     

Genome sequence of the polyhydroxybutyrate producer Pseudomonas extremaustralis, a highly stress-resistant Antarctic bacterium

Pseudomonas extremaustralis 14-3b presents genes involved in the synthesis of different polyhydroxyalkanoates, in tolerance and degradation of pollutants, and in microaerobic metabolism. Several genomic islands were detected. Genetic machinery could contribute to the adaptability to stressful conditions. This is the first genome sequence reported from a Pseudomonas isolated from cold environments.     

Effect of protein hydrolysate on the degradation of diesel fuel in soil*

The addition of protein hydrolysate solution to soil contaminated with diesel fuel was investigated for effects on diesel degradation. The application of protein hydrolysate solution led to an increase in the removal of diesel from the soil. At the end of the 21d experimental period the amount of diesel removed from the soil was 21% greater with the addition of protein hydrolysate solution when compared to a control system. This increased removal was linked to increases in both the number of hydrocarbon degrading micro-organisms, and an extension to their period of activity.     

Enhanced degradation of petrol (Slovene diesel) in an aqueous system by immobilized Pseudomonas fluorescens

The rate of degradation of n -alkanes C₁₂-C₁₈, in petrol (Slovene diesel) in an aqueous system, by free and immobilized Pseudomonas fluorescens in shaking flasks was investigated. Cells were immobilized to a biosupport, Biofix, and a biosorbant, Drizit. Analysis of cellular growth of the free and immobilized bacteria over 8 d of incubation with diesel as the sole carbon source, showed a reduction in the lag phase in the immobilized cultures in comparison to the free system. The free system degraded 52·3% of C₁₂ and 11·6% of C₁₃, but C₁₄-C₁₈ were not degraded. In comparison to the free system and diesel which had not been exposed to experimental conditions (unexposed), the immobilized systems degraded significantly more of C₁₃-C₁₈. Biofix-immobilized cells degraded 14·8% of C₁₂ and an average of 53·5% of C₁₃-C₁₈. Drizit-immobilized cells degraded 24·5% of C₁₂, 52·4% of C₁₃ and an average of 91·2% of C₁₄-C₁₈. This study shows the successful use of immobilized bacteria technology to enhance the degradation of diesel in an aqueous system.     

Effect of surfactants on pyrene degradation by Pseudomonas fluorescens 29L

The effect of surfactants on pyrene degradation in Pseudomonas fluorescens 29L was investigated. This strain produced 30.1 μM of rhamnolipid equivalents (RE) of biosurfactants on 50 mg of pyrene per liter of medium. The production of biosurfactants was significantly correlated with the water solubility (S _w) of the substrate and the growth rate on it. When chrysene, with a S _w of 2.8 × 10⁻³ mg per liter of water, was the carbon source, 13.1 μM of RE of biosurfactants were produced compared to 10.3 μM of RE of biosurfactants on acenaphthene with a S _w of 1.9 mg per liter of water. No biosurfactants were produced on salicylic acid, catechol, and citrate. All of the strain 29L mutants which grew on pyrene produced biosurfactants while among the mutants which grew on naphthalene, only 88.4% produced biosurfactants. The rhamnolipid mixture, JBR425, inhibited the growth of Strain 29L wild type (WT) and all of its mutants on pyrene. However, these mutants were able to grow in the presence of pyrene when the growth medium was supplemented with 10⁻⁶ mg of emulsan per milliliter of medium. This study implies biosurfactants are produced by Strain 29L as a physiological response to the hydrophobicity of pyrene. The combined use of indigenous biosurfactants and the added biosurfactant, emulsan, is a biotechnology to enhance pyrene degradation by Pseudomonas fluorescens 29L.     

Effect of copper on the degradation of phenanthrene by soil micro-organisms

AIMS: The effect of copper on the degradation by soil micro-organisms of phenanthrene, a polycyclic aromatic hydrocarbon, was investigated. METHODS AND RESULTS: Inert nylon filters were incubated in the soil for 28 days at 25 degrees C. Each filter was inoculated with a soil suspension, phenanthrene (400 ppm), copper (0, 70, 700 or 7000 ppm) and nitrogen/phosphorus sources. The filters were assessed for phenanthrene degradation, microbial respiration and colonization. Phenanthrene degradation proceeded even at toxic copper levels (700/7000 ppm), indicating the presence of phenanthrene-degrading, copper-resistant and/or -tolerant microbes. However, copper at these high levels reduced microbial activity (CO2 evolution). CONCLUSION: High levels of copper caused an incomplete mineralization of phenanthrene and possible accumulation of its metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of heavy metals in soils could seriously affect the bioremediation of PAH-polluted environments.     

Purine degradation in Pseudomonas aeruginosa and Pseudomonas testosteroni.

1. Adenine, hypoxanthine, xanthine and guanine are broken down in Pseudomonas aeruginosa and Pseudomonas testosteroni to allantoin by the concerted action of the enzymes adenine deaminase, guanine deaminase, NAD+-dependent xanthine dehydrogenase and uricase. 2. Uric acid is broken down by an unstable, membrane-bound uricase with an unusually low pH optimum. 3. In both strains adenine inhibits growth and xanthine dehydrogenase. A second type of inhibition is manifest only in Ps. testosteroni and concerns the regulation of the biosynthesis of amino acids of the aspartate family. Enzymic studies showed that in this strain aspartate kinase is inhibited by AMP.     

Oxygen-sensitive global regulator, Anr, is involved in the biosynthesis of poly(3-hydroxybutyrate) in Pseudomonas extremaustralis

We analyzed the influence of the redox global regulator Anr on the accumulation of poly(3-hydroxybutyrate) (PHB) in Pseudomonas extremaustralis. Anr regulates a set of genes in the aerobic-anaerobic transition including genes involved in nitrate reduction and arginine fermentation. An anr mutant was constructed using PCR-based strategies. The wild-type strain was able to grow in both microaerobic and anaerobic conditions using nitrate as the terminal electron acceptor while the mutant strain was unable to grow under anaerobic conditions. In bioreactor cultures, PHB content in the wild-type strain was higher in microaerobic and anaerobic cultures compared with highly aerated cultures. The mutant strain showed decreased PHB levels in both aerobic and microaerobic conditions compared with the wild-type strain. Inactivation of anr led to decreased expression of phaC and phaR genes as demonstrated in real-time RT-PCR experiments. Associated with the PHB gene region, two putative binding sites for Anr were found that, in line with the phenotype observed in bioreactor cultures, suggest a role of this regulator in PHB biosynthesis.     

Genes involved in copper resistance influence survival of Pseudomonas aeruginosa on copper surfaces

Aims:  To evaluate the killing of Pseudomonas aeruginosa PAO1 on copper cast alloys and the influence of genes on survival on copper containing medium and surfaces.
Methods and Results:  Different strains of P. aeruginosa were inoculated on copper containing medium or different copper cast alloys and the survival rate determined. The survival rates were compared with rates on copper-free medium and stainless steel as control. In addition, the effect of temperature on survival was examined.
Conclusions:  Copper cast alloys had been previously shown to be bactericidal to various bacteria, but the mechanism of copper-mediated killing is still not known. In this report, we demonstrate that P. aeruginosa PAO1 is rapidly killed on different copper cast alloys and that genes involved in conferring copper resistance in copper-containing medium also influenced survival on copper cast alloys. We also show that the rate of killing is influenced by temperature.
Significance and Impact of the Study:  To use copper surfaces more widely as bactericidal agents in various settings, it is important to understand how genes influence survival on these surfaces. Here we show that genes shown to be involved in copper resistance in P. aeruginosa PAO1 can have an impact on the length of survival time on copper cast alloys under certain conditions. This is an important first step for evaluation of future use of copper surfaces as bactericidal agents.     

Effect of dissolved oxygen regime on growth dynamics of Pseudomonas spp during benzene degradation

We investigated the effect of different oxygen regimes on growth patterns of Pseudomonas spp. during benzene degradation in microcosm batch studies. Benzene degradation was induced by limiting oxygen available for microbial activity, which consists of three initial-dissolved oxygen (DO) levels of oxic, hypoxic, and anoxic conditions. Batch experiments were performed for cell growth and benzene degradation by inoculating three strains of Pseudomonas spp. (Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida) in mineral salt medium containing aqueous benzene. Results showed that all strains were capable to grow and degrade benzene under all oxygen regimes but in a different manner. The highest cell growth of P. aeruginosa and P. fluorescens was achieved under oxic and anoxic condition, respectively, but there was no substantial difference on benzene degradation between the oxygen treatments with about 25% reduction for both strains. P. putida showed a facultative process for both cell growth and benzene degradation. This reveals that care should be taken in selection of microorganisms with regard to environmental studies since they exhibit different responses for given environmental conditions such as DO levels.     

Response to lethal UVA radiation in the Antarctic bacterium Pseudomonas extremaustralis: polyhydroxybutyrate and cold adaptation as protective factors

Extremophiles - Pseudomonas extremaustralis is an Antarctic bacterium with high stress resistance, able to grow under cold conditions. It is capable to produce polyhydroxyalkanoates (PHAs) mainly...     

Influence of oxygen on the degradation of diesel fuel in soil bioreactors

In fixed-bed bioreactors, the influence of the oxygen content in the inlet air on the biodegradation of diesel fuel in unsaturated soil/compost mixtures was analyzed at 30°C over a period of 7 weeks. Firstly, a wide range from 0 to 80 vol.% O₂ was investigated. Subsequently, the range below 5 vol.% O₂ was examined more closely. Over the whole test period of seven weeks, no significant influence of oxygen could be observed above 1 vol. % O₂ in the inlet air - either on the decrease of the total contaminants or on the total mineralization. Anaerobic conditions should be avoided for the degradation of diesel fuel. During the test period, the courses of CO₂ production varied significantly depending on oxygen supply. Furthermore, a model was developed to estimate the total mineralization as a function of oxygen supply. More investigations are recommended in order to test this model for practical application.     

Effect of chlorobenzoates on the degradation of polychlorinated biphenyls (PCB) by Pseudomonas stutzeri

Chlorobenzoic acids (CBA) are frequently dead-end products of partial aerobic biodegradation of polychlorinated biphenyls (PCB). When CBA produced from PCB accumulate in the growth medium, they can inhibit the bacterial growth and consequently, slow down PCB biodegradation. In this study, the effects of seven mono- and dichlorinated CBA on growth of Pseudomonas stutzeri on different substrates and on the PCB degradation by this strain in a liquid mineral medium were tested. 3-CBA was the strongest growth inhibitor for P. stutzeri growing on glucose, benzoate and biphenyl. It was found to inhibit heavily the elimination of some di- and trichlorinated biphenyls. In contrast, its influence on the elimination of more chlorinated congeners was much less significant.The authors are with the Department of Biochemical Technology, Faculty of Chemical Technology, Slovak Technical University, 812 37 Bratislava, Slovakia.     

Pentachlorophenol degradation by Pseudomonas aeruginosa

Five Pseudomonas species were tested for ability to degrade pentachlorophenol (PCP). Pseudomonas aeruginosa completely degraded PCP up to 800 mg/l in 6 days with glucose as co-substrate. With 1000 mg PCP/l, 53% was degraded. NH₄ ⁺ salts were better at enhancing degradation than organic nitrogen sources and shake-cultures promoted PCP degradation compared with surface cultures. Degradation was maximal at pH 7.6 to 8.0 and at 30 to 37°C. Only PCP induced enzymes that degraded PCP and chloramphenicol inhibited this process. The PCP was degraded to CO₂, with release of Cl^-.The authors are with the Bacteriology Laboratory, Central Leather Research Institute, Madras-600 020, India.     

Fluoranthene degradation in Pseudomonas alcaligenes PA-10

Pseudomonas alcaligenes strain PA-10 degrades thefour-ring polycyclic aromatic hydrocarbon fluoranthene, co-metabolically. HPLC analysisof the growth medium identified four intermediates, 9-fluorenone-1-carboxylicacid; 9-hydroxy-1-fluorene carboxylic acid; 9-fluorenone and 9-fluorenol, formedduring fluoranthene degradation. Pre-exposure of PA-10 to 9-fluorenone-1-carboxylic acidand 9-hydroxy-1-fluorene-carboxylic acid resulted inincreases in fluoranthene removal, while pre-exposure to9-fluorenone and 9-fluorenol resulted in a decrease influoranthene degradation. The rate of indole transformation was similarly affected by pre-exposureto these metabolic intermediates, indicating a link between fluoranthenedegradation and indigo formation in this strain.     

近海柴油降解菌群的构建及其对柴油的降解特性

【目的】实施近海柴油污染的生物治理。【方法】以柴油为唯一碳源,从深圳港口海域富集筛选柴油降解菌;采用复配、正交试验等方式构建混合菌群;通过单因素试验研究环境因素对菌群降解柴油的影响;使用气相色谱-氢火焰检测器(GC-FID)分析降解前后柴油各组分的变化;通过生理生化试验和16S rRNA基因序列分析对菌株进行鉴定。【结果】获得了16株柴油降解菌,7 d内对柴油的降解率最高达40.8%;选择菌株C1-8、C2-10、C3-13构建了混合菌群CQ1,投加量分别为0.5%、2.0%和1.0%,CQ1对柴油去除率比单菌提高了10%以上;CQ1的最适环境条件为：温度30 °C、pH 7.6、摇床转速220 r/min、柴油浓度20 g/L,优化后9 d内对柴油去除率达60%以上;GC-FID结果显示,菌群CQ1可降解大部分C11?C27的正构烷烃,对C21?C27的烷烃降解可达100%。经鉴定,菌株C1-8、C2-10和C3-13分别为微杆菌(Microbacterium sp.)、剑菌(Ensifer sp.)和变异棒杆菌(Corynebacterium variabile)。【结论】CQ1在近海柴油污染的生物修复中具有良好的应用前景。