Enzymatic dehalogenation of pentachlorophenol by Pseudomonas fluorescens of the microbial community from tannery effluent

Four different bacterial isolates obtained from a stable bacterial consortium were capable of utilizing pentachlorophenol (PCP) as sole carbon and energy source. The consortium was developed by continuous enrichment in the chemostat. The degradation of PCP by bacterial strain was preceded through an oxidative route as indicated by accumulation of tetrachloro-rho-hydroquinone and dichlorohydroquinone as determined by high performance liquid chromatography (HPLC). Among the four isolates, Pseudomonas fluorescens exhibited maximum degradation capability and enzyme production. PCP-monooxygenase enzyme was extracted from culture extract and fractionated by DEAE-cellulose ion exchange chromatography. The molecular weight of the enzyme, purified from Pseudomonas fluorescens, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography was found to be 24000 Da.     

Involvement of plasmid in degradation of pentachlorophenol by Pseudomonas sp. from a chemostat

Pseudomonas sp. strain IST103 obtained from a stable bacterial consortium was capable of utilizing pentachlorophenol (PCP) as sole carbon and energy source. The consortium was developed by continuous enrichment in a chemostat. The degradation of PCP by bacterial strain proceeded through an oxidative route as indicated by accumulation of tetrachloro-p-hydroquinone and chlorohydroquinone determined by high performance liquid chromatography (HPLC), and chloride molecules released in culture medium. Two different molecular size plasmids, of approximately 80 and 4 kilobase, were found to be responsible for carrying genes for degradation of PCP. This was evidenced by mutants produced by curing of plasmid by treatment of ethidium bromide. The derivatives were not able to utilize PCP, however, transformation of low molecular size plasmid of Pseudomonas sp. strain 103 into E. coli JM109 utilized PCP, indicated a possible involvement of plasmid in degradation of pentachlorophenol.     

Effect of rhamnolipid on biodegradation of hydrocarbons in non-aqueous-phase liquid (NAPL)

Aliphatic and aromatic hydrocarbons are environmental pollutants of serious concern. Their bioavailability is the major limiting factor that makes the bioremediation process slow. Therefore, the present study focuses on biodegradation of non-aqueous-phase liquids (NAPL) by a halophilic consortium (Pseudomonas aeruginosa and Escherichia fergusonii) in presence of rhamnolipid as well as a rhamnolipid-producing Pseudomonas aeruginosa AMB AS7. The study was performed in microcosms, and the residual hydrocarbons after degradation were estimated by gas chromatography. It was found that the degradation of hydrocarbons in NAPL was more in presence of rhamnolipid in comparison with their biotic controls. However, among NAPL, the degradation of phenanthrene (37.5%) and octadecane (47.8%) was found to be more by co-culture of halophilic consortium and rhamnolipid-producing P. aeruginosa AMB AS7. Denaturing gradient gel electrophoresis was performed to determine the viability of different bacterial strains (halophilic and rhamnolipid-producing bacterial strain). Besides, the results also revealed that during NAPL degradation, the cell surface hydrophobicity (CSH) of halophilic consortium increased from 9.12% to 69.55% when added with 100 mg/L of rhamnolipid, whereas CSH of rhamnolipid-producing P. aeruginosa AMB AS7 was constant at 31.9%, even though it produced about 271.8 mg/L of rhamnolipid.     

Enrichment and characterization of a microbial community from tannery effluent for degradation of pentachlorophenol

A bacterial community obtained by continuous enrichment from the microbial population of tannery effluent using pentachlorophenol (PCP) as sole source of carbon and energy, contained four different bacterial species including Serratia marcescens (three isolates, TE₁, TE₂ and TE₄) and Pseudomonas fluorescens (one isolate, TE₃). The members of the community grew separately on various chlorinated compounds, carbon and nitrogen sources and exhibited a remarkable ability to utilize PCP. Biodegradation studies revealed a time-dependent disappearance of PCP and its intermediary metabolites, tetrachloro-p-hydroquinone and chlorohydroquinone, and indicated the individual role of members of the community in the degradation of PCP.     

Biodegradation of 2-Nitrotoluene by <Emphasis Type="Italic">Micrococcus</Emphasis> sp. strain SMN-1

A bacterial consortium capable of degrading nitroaromatic compounds was isolated from pesticide-contaminated soil samples by selective enrichment on 2-nitrotoluene as a sole source of carbon and energy. The three different bacterial isolates obtained from bacterial consortium were identified as Bacillus sp. (A and C), Bacillus flexus (B) and Micrococcus sp. (D) on the basis of their morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. The pathway for the degradation of 2-nitrotoluene by Micrococcus sp. strain SMN-1 was elucidated by the isolation and identification of metabolites, growth and enzymatic studies. The organism degraded 2-nitrotoluene through 3-methylcatechol by a meta-cleavage pathway, with release of nitrite.     

Evaluation of pentachlorophenol-degrading potentiality of Pseudomonas sp. in a soil microcosm

Pseudomonas sp. strain IST103 obtained from a stable consortium was capable of degrading pentachlorophenol (PCP) as sole carbon and energy source. The PCP-degrading potentiality of the strain was determined by growth of bacteria in culture medium, utilization of PCP by high performance liquid chromatography (HPLC), chloride release and ring cleavage. The strain was applied in two set of soil microcosms containing 20 and 40% moisture, each having different concentrations, 0, 10, 100, 500, and 1000 mg/l, of PCP. The result showed significant utilization of PCP (77% in 45 days) and higher growth of bacterial strain when PCP was applied in 100 mg/l concentration at 40% moisture. Inhibitory effects on the growth of bacterial strain were seen in 500 and 1000 mg/l concentration.     

Dehalogenation of 4 — Chlorobenzoic Acid by <Emphasis Type="Italic">Pseudomonas</Emphasis> isolates

Twenty three bacterial isolates either pure or consortium were initially screened on the basis of their ability to degrade as well as dechlorinate 4 — chlorobenzoic acid (4-CBA). Based on comparative growth response, three pure isolates Pseudomonas putida GVS-4, Pseudomonas aeruginosa GVS-18 and Pseudomonas aeruginosa GWS-19 and a consortium SW-2 was finally selected for further studies. The enzyme studies performed with cell free extracts revealed that dehalogenase activity was substrate specific with maximum activity at 300 μgml⁻¹ substrate concentration. Catechol 1,2 dioxygenase activity was found to be present in cell free extracts suggesting that 4 — chlorobenzoic acid (4-CBA) is catabolized by ortho-ring cleavage pathway. The dehalogenase enzyme profile showed single enzyme band in case of GVS-4 (Rm 0.76), GVS-18 (Rm 0.84), GWS −19 (Rm 0.85) and two bands in SW-2 (Rm 0.71 & 0.10).     

Molecular cloning and characterization of pentachlorophenol-degrading monooxygenase genes of Pseudomonas sp. from the chemostat.

Pseudomonas sp. strain IST 103 (PCP103) capable of utilizing pentachlorophenol (PCP) was determined by utilization of a carbon source and release of the hydroxylating enzyme PCP-4 monooxygenase. The metabolites were extracted from the culture medium and analyzed by high-performance liquid chromatography. The enzyme purified to apparent homogeneity from an extract of PCP-grown cells indicated that a fraction of DEAE-cellulose ion exchange chromatography of molecular size of 30,000 kDa determined by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis was responsible for dechlorination of PCP. The plasmid isolated from the bacterium was subjected to Shotgun cloning by restriction digestion by BamHI, HindIII, and SalI, ligated to pUC19 vector, and transformed into Escherichia coli XLBlue1alpha. The recombinant clones having higher potentiality to degrade PCP were selected by utilization of a carbon source and release of intermediary metabolites during degradation of PCP as the sole source of carbon and energy. The recombinant clones, which contained an insert of 3.0 kb of SalI and HindIII sites, were sequenced and compared with gene sequences deposited in GenBank by BLAST search; this indicated homology with the thdf gene of monooxygenase of thiophene and furan. Southern blot analysis performed by developing gene probes indicated the presence of the PCP monooxygenase gene in plasmids of the bacterium.     

Biodegradation of chlorpyrifos by bacterial consortium isolated from agriculture soil

Organophosphorous pesticides are widely used in agriculture to control major insect pests. Chlorpyrifos is one of the major organophosphorous pesticides which is used to control insects including termites, beetles. The widespread use of these pesticides is hazardous to the environment and also toxic to mammals, thus it is essential to remove the same from the environment. From the chlorpyrifos contaminated soil nine morphologically different bacterial strains, one actinomycete and two fungal strains were isolated. Among those isolates four bacterial strains which were more efficient were developed as consortium. The four bacterial isolates namely Pseudomonas putida (NII 1117), Klebsiella sp., (NII 1118), Pseudomonas stutzeri (NII 1119), Pseudomonas aeruginosa (NII 1120) present in the consortia were identified on the basis of 16S rDNA analysis. The intracellular fractions of the consortium exhibited more organophosphorus hydrolase activity (0.171 ± 0.003 U/mL/min). The degradation studies were carried out at neutral pH and temperature 37°C with chlorpyrifos concentration 500 mg L⁻¹. LC-mass spectral analysis showed the presence of metabolites chlopyrifos-oxon and Diethylphosphorothioate. These results highlight an important potential use of this consortium for the cleanup of chlorpyrifos contaminated pesticide waste in the environment.     

Enhancement of phenol degradation by free and immobilized mixed culture of Providencia stuartii PL4 and Pseudomonas aeruginosa PDM isolated from activated sludge

Biodegradation of phenol has been investigated using a bacterial consortium consisting of two bacterial isolates; one of them used for the first time in phenol biodegradation. This consortium was isolated from activated sludge and identified as Providencia stuartii PL4 and Pseudomonas aeruginosa PDM (accession numbers KY848366 and MF445102, respectively). The degradation of phenol by this consortium was optimal at pH 7 with using 1500?mg?l^?1 ammonium chloride as a nitrogen source. Interestingly, after optimizing the biodegradation conditions, this consortium was able to degrade phenol completely up to 1500?mg?l^?1 within 58?h. The immobilization of this consortium on various supporting materials indicated that polyvinyl alcohol (PVA)-alginate beads and polyurethane foam (PUF) were more suitable for biodegradation process. The freely suspended cells could degrade only 6% (150?mg?l^?1) of 2500?mg?l^?1 phenol, whereas, the immobilized PVA-alginate beads and the immobilized PUF degraded this concentration completely within 120?h of incubation with degradation rates (q) 0.4839 and 0.5368 (1/h) respectively. Thus, the immobilized consortium of P. stuartii PL4 and P. aeruginosa PDM can be considered very promising in the treatment of effluents containing phenol.     

Purification and Characterization of a Phytase from <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> MOK1

A phytase (EC 3.1.3.8) from Pseudomonas syringae MOK1 was purified to apparent homogeneity in two steps employing cation and an anion exchange chromatography. The molecular weight of the purified enzyme was estimated to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The optimal activity occurred at pH 5.5 and 40°C. The Michaelis constant (K _m ) and maximum reaction rate (V_max) for sodium phytate were 0.38 mM and 769 U/mg of protein, respectively. The enzyme was strongly inhibited by Cu²⁺, Cd²⁺, Mn²⁺, and ethylenediaminetetraacetic acid (EDTA). It showed a high substrate specificity for sodium phytate with little or no activity on other phosphate conjugates. The enzyme efficiently released orthophosphate from wheat bran and soybean meal.Received: 9 September 2002 / Accepted: 6 December 2002     

Biodegradation potential of oily sludge by pure and mixed bacterial cultures

The biodegradation capacity of aliphatic and aromatic hydrocarbons of petrochemical oily sludge in liquid medium by a bacterial consortium and five pure bacterial cultures was analyzed. Three bacteria isolated from petrochemical oily sludge, identified as Stenotrophomonas acidaminiphila, Bacillus megaterium and Bacillus cibi, and two bacteria isolated from a soil contaminated by petrochemical waste, identified as Pseudomonas aeruginosa and Bacillus cereus demonstrated efficiency in oily sludge degradation when cultivated during 40 days. The bacterial consortium demonstrated an excellent oily sludge degradation capacity, reducing 90.7% of the aliphatic fraction and 51.8% of the aromatic fraction, as well as biosurfactant production capacity, achieving 39.4% reduction of surface tension of the culture medium and an emulsifying activity of 55.1%. The results indicated that the bacterial consortium has potential to be applied in bioremediation of petrochemical oily sludge contaminated environments, favoring the reduction of environmental passives and increasing industrial productivity.     

A suite of complementary biocontrol traits allows a native consortium of root‐associated bacteria to protect their host plant from a fungal sudden‐wilt disease

The beneficial effects of plant‐–bacterial interactions in controlling plant pests have been extensively studied with single bacterial isolates. However, in nature, bacteria interact with plants in multitaxa consortia, systems which remain poorly understood. Previously, we demonstrated that a consortium of five native bacterial isolates protected their host plant Nicotiana attenuata from a sudden wilt disease. Here we explore the mechanisms behind the protection effect against the native pathosystem. Three members of the consortium, Pseudomonas azotoformans A70, P. frederiksbergensis A176 and Arthrobacter nitroguajacolicus E46, form biofilms when grown individually in vitro, and the amount of biofilm increased synergistically in the five‐membered consortium, including two Bacillus species, B. megaterium and B. mojavensis. Fluorescence in situ hybridization and scanning electron microscopy in planta imaging techniques confirmed biofilm formation and revealed locally distinct distributions of the five bacterial strains colonizing different areas on the plant‐root surface. One of the five isolates, K1 B. mojavensis produces the antifungal compound surfactin, under in vitro and in vivo conditions, clearly inhibiting fungal growth. Furthermore, isolates A70 and A176 produce siderophores under in vitro conditions. Based on these results we infer that the consortium of five bacterial isolates protects its host against fungal phytopathogens via complementary traits. The study should encourage researchers to create synthetic communities from native strains of different genera to improve bioprotection against wilting diseases.     

Detection and characterisation of catechol 2,3-dioxygenase in an indigenous soil <Emphasis Type="Italic">Pseudomonad</Emphasis> by MALDI-TOF MS using a column separation

The key enzyme catalyzing the second step in the phenol degradation meta-cleavage pathway (C23O) has been purified to homogeneity from a new bacterial strain, which belongs to genus Pseudomonas. The species was growing on phenol as carbon source. The C23O was detected and identified by absorption spectroscopy. The protein was isolated using sucrose density centrifugation and anion exchange chromatography. The purified protein showed a molecular mass of 32 kDa to sodium dodecyl sulfate polyacrylamid gel electrophoresis and an isoelectric point of 5 estimated by analytical isoelectrical focusing. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and peptide mapping was attempted for the identification of the isolated protein and proteins involved in the metabolic pathway.     

嗜盐菌群对酸性金黄G的脱色特性和机理

【目的】为了获得能够在高盐环境下脱色偶氮染料的嗜盐菌群及其降解机理。【方法】采用富集驯化的方法获得一个嗜盐菌群,采用Illumina HiSeq2500测序平台对其群落结构进行测定;采用分光光度法测定了其降解特性;采用GC-MS和红外图谱分析了其降解机理;采用微核实验的方法比较了偶氮染料降解前后的毒性。【结果】该菌群在10%的盐度下,使100mg/L的酸性金黄G在8h内脱色。菌群主要由Zobellella、Rheinheimera、Exiguobacterium和Marinobacterium组成。最适宜的脱色条件是:pH=6,酵母粉为碳源,蛋白胨或硝酸钾作为氮源,盐度为1%–10%。酸性金黄G降解产物的毒性比降解前降低。酸性金黄G主要的降解产物是对氨基二苯胺和二苯胺。此外,该菌群还能使酸性大红GR和直接湖蓝5B等多种偶氮染料脱色,具有较好的脱色广谱性。【结论】获得了快速降解偶氮染料的嗜盐菌群及降解机理,为该嗜盐菌群应用于高盐印染废水的处理提供菌种资源和理论支持。     

Synergistic biodegradation of pentachlorophenol by <Emphasis Type="Italic">Bacillus cereus</Emphasis> (DQ002384), <Emphasis Type="Italic">Serratia marcescens</Emphasis> (AY927692) and <Emphasis Type="Italic">Serratia marcescens</Emphasis> (DQ002385)

The consortium of Bacillus cereus (DQ002384), Serratia marcescens (AY927692) and Serratia marcescens (DQ002385) were used for pentachlorophenol (PCP) degradation. The consortia showed better overall removal efficiencies than single strains by utilization of PCP as a carbon and energy source confirmed by pH dependent dye indicator bromocresol purple (BCP) in mineral salt media (MSM). Mixed culture was found to degrade up to 93% of PCP (300 mg/l) as compared to single strains (62.75–90.33%), at optimized conditions (30 ± 1°C, pH 7 ± 0.2, 120 rpm) at 168 h incubation. PCP degradation was also recorded at 20°C (62.75%) and 37°C (83.33%); pH 6 (70%) and pH 9 (75.16%); 50 rpm (73.33%) and 200 rpm (91.63%). The simultaneous release of chloride ion up to 90.8 mg/l emphasized the bacterial dechlorination in the medium. GC–MS analysis revealed the formation of low molecular weight compound, i.e., 6-chlorohydroxyquinol, 2,3,4,6-tetrachlorophenol and tetrachlorohydroquinone, from degraded sample as compared to control.     

Biodegradation of methyl <Emphasis Type="Italic">tert</Emphasis>-butyl ether using bacterial strains

Prospective methyl tert-butyl ether (MTBE) degrading bacterial strains and/or consortia were identified. The potential for aerobic degradation of MTBE was examined using bacterial isolates from contaminated soils and groundwater. Using the 16S rDNA protocol, two isolates capable of degrading MTBE (Rhodococcus pyridinivorans 4A and Achromobacter xylosoxidans 6A) were identified. The most efficient consortium of microorganisms was acquired from contaminated groundwater. The growth of both strains and the consortium on MTBE was supported by various organic substrates, and monitored using Bioscreen®. The biochemical oxygen demand of the cultures was measured using OxiTop®, and their MTBE concentrations were estimated by gas chromatography. After 3 weeks of aerobic cultivation using n-alkanes as cosubstrate, the concentration of MTBE in R. pyridinivorans 4A was reduced to 62.4 % of its initial amount (50 ppm).     

Biodegradation of 1,4-dioxane by a <Emphasis Type="Italic">Flavobacterium</Emphasis>

A dioxane-degrading consortium was enriched from soil obtained from a contaminated groundwater plume. The enriched consortium did not use dioxane as the sole source of carbon and energy but co-metabolized dioxane in the presence of tetrahydrofuran (THF). THF and dioxane concentrations up to 1000 ppm were degraded by the enriched consortium in about 2 weeks with a longer lag phase observable at 1000 ppm. Three colonies from the enriched consortium were then obtained on agar plates containing basal salts and glucose as the carbon source. Only one of the three colonies was capable of dioxane degradation. Further enrichment of this colony in liquid media led to a pure culture that grew on glucose and co-metabolically degraded dioxane after THF degradation. The rate and extent of dioxane degradation of this isolate increased with increasing THF concentration. This isolate was subsequently identified as a Flavobacterium by 16S rDNA sequencing. Using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analysis of microbial populations, Flavobacterium was determined to be the dominant species in the enriched consortium and was distinct from the two other colonies that did not degrade dioxane. This is the first report of a dioxane-degrading Flavobacterium which is phylogenetically distinct from any previously identified dioxane degrader.     

Biodegradation of Methyl <Emphasis Type="Italic">tert</Emphasis>-Butyl Ether by Enriched Bacterial Culture

Degradation of methyl tert-butyl ether (MTBE) as a sole carbon and energy source was investigated utilizing an enriched bacterial consortium derived from an old environmental MTBE spill. This enriched culture grew on MTBE with concentration up to 500 mg/l, reducing the MTBE in medium to undetectable concentrations in 23 days. Traces of tert-butyl alcohol were detected during MTBE degradation. The degradation was not affected by additional cobalt ions, whereas low concentration of glucose enhanced the rate of degradation. The bacterial community consisted of numerous bacterial genera, the majority being members of the phylum Acidobacteria and genus Terrimonas. The alkane 1-monooxygenase (alk) gene was detected in this consortium. Our findings suggest that environmental degradation of MTBE proceeds along the previously proposed pathway.     

Investigation of the biodegradation of pentachlorophenol by the predominant bacterial strains in a mixed culture

A pentachlorophenol (PCP) degrading mixed culture contained three predominant strains identified as Flavobacterium gleum, Agrobacterium radiobacter and Pseudomonas sp. The relative abilities of the three strains to degrade PCP were tested individually and in combination. Rates of PCP degradation by individual isolates were lower than that observed for the three isolates combined. Of the individual strains, Flavobacterium gleum manifested highest PCP degradation ability. A biodegradation medium inoculated with a combination of the three isolates exhibited PCP degradation patterns similar to the original mixed culture. Varying low amounts of tetrachlorophenol were found in degradation medium inoculated with individual isolates, but this intermediate was absent from media inoculated with the mixed culture.