Isolation and characterization of acetonitrile utilizing bacteria

Summary Bacteria utilizing high concentrations of acetonitrile as the sole carbon source were isolated and identified asChromobacterium sp. andPseudomonas aeruginosa. Maximum growth was attained after 96 h of incubation andP. aeruginosa grew slightly faster thanChromobacterium sp. The strains were able to grow and oxidize acetonitrile at concentrations as high as 600 mM. However, higher concentrations inhibited growth and oxygen uptake. Degradation studies with (¹⁴C)acetonitrile indicated 57% of acetonitrile was degraded byPseudomonas aeruginosa as compared to 43% byChromobacterium. The isolates utilized different nitrile compounds as carbon substrates.     

Microbial utilization of the industrial wastewater pollutants 2-ethylhexylthioglycolic acid and <Emphasis Type="Italic">iso</Emphasis>-octylthioglycolic acid by aerobic Gram-negative bacteria

Industrial wastewater from the production of sulfur containing esters and the resulting products of this synthesis, 2-ethylhexylthioglycolic acid (EHTG) and iso-octylthioglycolic acid (IOTG), were deployed in this study to enrich novel bacterial strains, since no wastewater and EHTG or IOTG degrading microorganisms were hitherto described or available. In addition, nothing is known about the biodegradation of these thiochemicals. The effect of this specific wastewater on the growth behaviour of microorganisms was investigated using three well-known Gram-negative bacteria (Escherichia coli, Pseudomonas putida, and Ralstonia eutropha). Concentrations of 5% (v/v) wastewater in complex media completely inhibited growth of these three bacterial strains. Six bacterial strains were successfully isolated, characterized and identified by sequencing their 16S rRNA genes. Two isolates referred to as Achromobacter sp. strain MT-E3 and Pseudomonas sp. strain MT-I1 used EHTG or IOTG, respectively, as well as the wastewater as sole source of carbon and energy for weak growth. More notably, both isolates removed these sulfur containing esters in remarkable amounts from the cultures supernatant. One further isolate was referred to as Klebsiella sp. strain 58 and exhibited an unusual high tolerance against the wastewater’s toxicity without utilizing the contaminative compounds. If cultivated with gluconic acid as additional carbon source, the strain grew even in presence of more than 40% (v/v) wastewater. Three other isolates belonging to the genera Bordetella and Pseudomonas tolerated these organic sulfur compounds but showed no degradation abilities.     

Biodegradation of 4-chlorobenzoic acid by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PA01 NC

A bacterial consortium capable of degrading chloroaromatic compounds was isolated from pulp and paper mill effluents by selective enrichment on 4-chlorobenzoic acid as sole source of carbon and energy. The four different bacterial isolates obtained from bacterial consortium were identified as Pseudomonas aeruginosa AY792969 (A), P. aeruginosa PA01 NC (B), Pseudomonas sp. ZZ5 DQ113452 (C) and Pseudomonas sp. AY762360 (D) based on their morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. These bacterial isolates were found to be versatile in degrading a variety of chloroaromatic compounds including fluoro- and iodobenzoic acids. P. aeruginosa PA01 NC utilized 4-chlorobenzoic acid at 2 g/l as growth substrate. Biodegradation studies have revealed that this organism degraded 4-chlorobenzoic acid through 4-chlorocatechol which was further metabolized by ortho-cleavage pathway and the dechlorination occurred after the ring-cleavage.     

Degradation of Aroclor 1221 and survival of strains in soil microcosms

Three bacterial strains able to use different aromatic compounds as the sole carbon and energy source were tested for their potential to degrade Aroclor 1221 in soil microcosms when present in mixed culture. Disappearance of polychlorinated biphenyls (PCBs), occurrence of metabolites, release of chloride, and survival of the laboratory-selected strains were investigated under different conditions. In principle, complete mineralization of various congeners of Aroclor 1221, a technical mixture of PCBs, by the mixed culture was possible. The autochthonous microflora negatively affected the degradation due to formation of a toxic compound from 4-chlorobenzoate. 4-Chlorobenzoate was produced by one of the added strains, Pseudomonas sp. JHK, during degradation of 4-chlorobiphenyl. The unknown metabolite of 4-chlorobenzoate led to a rapid decrease in viable counts of the laboratory-selected strains in the soil microcosm.Correspondence to: J. Havel     

Polyhydroxyalkanoate (PHA) accumulating bacteria from the gut of higher termite <Emphasis Type="Italic">Macrotermes carbonarius</Emphasis> (Blattodea: Termitidae)

The continuous quest for bacterial strains capable of accumulating polyhydroxyalkanoate (PHA) utilizing cheaper and renewable carbon source prompted us to explore newer and diverse environments like the gut of termites. Among the bacterial strains isolated from the gut of higher termite Macrotermes carbonarius, three strains were found to accumulate PHA, as observed by microscopic studies and PHA production experiments. Among them, strain MC1 with rapid growth and higher PHA accumulation was selected for further studies. API kit-50 CHB and 16S rRNA gene sequence analysis results indicated the strain to have 99% homology with Bacillus megaterium and Bacillus flexus. Bacillus sp. MC1 was able to accumulate PHA during the growth phase utilizing different carbon sources like glucose, fructose, sodium acetate, sodium valerate and 1,4-butanediol. Gas chromatography analysis of the polymer has shown it to be basically composed of poly (3-hydroxybutyrate) (PHB). Growth associated PHB biosynthesis was best in the presence of sodium acetate with 39 wt% after 16 h of cultivation. Though previous studies provided evidence confirming the presence of PHA producing bacteria in termite gut, isolation and characterization of these strains in pure culture has not been documented yet. Presence of other morphotypes in the termite gut with PHA like granular inclusions was evident from the transmission electron microscopy studies. This is a novel report and shows the feasibility of using potent strains capable of utilizing lignocellulosic degradation products as a renewable carbon source for the production of PHA in the future.     

Enzymatic Dehalogenation of Pentachlorophenol by <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> 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-ρ-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 24,000 Da. Received: 22 July 2002 / Accepted: 23 September 2002     

Utilization of homoserine lactone as a sole source of carbon and energy by soil Arthrobacter and Burkholderia species

Homoserine lactone (HSL) is a ubiquitous product of metabolism. It is generated by all known biota during the editing of certain mischarged aminoacyl-tRNA reactions, and is also released as a product of quorum signal degradation by bacterial species expressing acyl-HSL acylases. Little is known about its environmental fate over long or short periods of time. The mammalian enzyme paraoxonase, which has no known homologs in bacteria, has been reported to degrade HSL via a lactonase mechanism. Certain strains of Variovorax and Arthrobacter utilize HSL as a sole source of nitrogen, but not as a sole source of carbon or energy. In this study, the enrichment and isolation of four strains of soil bacteria capable of utilizing HSL as a carbon and energy source are described. Phylogenetic analysis of these isolates indicates that three are distinct members of the genus Arthrobacter, whereas the fourth clusters within the non-clinical Burkholderia. The optimal pH for growth of the isolates ranged from 6.0 to 6.5, at which their HSL-dependent doubling times ranged from 1.4 to 4 h. The biodegradation of HSL by these 4 isolates far outpaced its chemical decay. HSL degradation by soil bacteria has implications for the consortial mineralization of acyl-homoserine lactones by bacteria associated with quorum sensing populations.     

Isolation and characterization of bacteria from soil contaminated with diesel oil and the possible use of these in autochthonous bioaugmentation

Two bacterial species (isolates N and O) were isolated from a paddy soil microcosm that had been artificially contaminated with diesel oil to which extrinsic Pseudomonas aeruginosa strain WatG, had been added exogenously. One bacterial species (isolate J) was isolated from a similar soil microcosm that had been biostimulated with Luria–Bertani (LB) medium. Isolates N and O, which were tentatively identified as Stenotrophomonas sp. and Ochromonas sp., respectively, by sequencing of their 16 S rRNA genes had no ability to degrade diesel oil on their own in any liquid medium. When each strain was cocultivated with P. aeruginosa strain WatG in liquid mineral salts medium (MSM) containing 1% diesel oil, isolate N enhanced the degradation of diesel oil by P. aeruginosa strain WatG, but isolate O inhibited it. In contrast, isolate J, which was tentatively identified as a Rhodococcus sp., degraded diesel oil contained not only in liquid LB and MSM, but also in paddy soil microcosms supplemented with LB medium. The bioaugmentation capacity of isolate J in soil microcosms contaminated with diesel oil was much higher than that of P. aeruginosa strain WatG. The possibility of using isolate J for autochthonous bioaugmentation is discussed.     

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.     

Utilization of the herbicide phosphinothricin as a nitrogen source by soil bacteria

Summary Rhodococcus sp., Pseudomonas paucimobilis and five other bacterial soil isolates were found to be capable of utilizing the l-enantiomer of the herbicide phosphinothricin (2-amino-4-(methylphosphinyl)-butanoic acid) as a nitrogen source. This deamination yielded the structural analogue, 2-oxo-4-(methylphosphinyl)-butanoic acid as the main catabolite of phosphinothricin. On prolonged incubation, slow decarboxylation of the main catabolite occurred with the formation of the stable degradation product 3-methylphosphinico-propanoic acid.     

Biodegradation of tributyl phosphate by novel bacteria isolated from enrichment cultures

Tributyl phosphate (TBP) is an organophosphorous compound, used extensively (3000–5000 tonnes/annum) as a solvent for nuclear fuel processing and as a base stock in the formulation of fire-resistant aircraft hydraulic fluids and other applications. Because of its wide applications and relative stability in the natural environment TBP poses the problem of pollution and health hazards. In the present study, fifteen potent bacterial strains capable of using tributyl phosphate (TBP) as sole carbon and phosphorus source were isolated from enrichment cultures. These isolates were identified on the basis of biochemical and morphological characteristics and 16S rRNA gene sequence analysis. Phylogenetic analysis of 16S rRNA gene sequences revealed that two isolates belonged to class Bacilli and thirteen to β and γ-Proteobacteria. All these isolates were found to be members of genera Alcaligenes, Providencia, Delftia, Ralstonia, and Bacillus. These isolates were able to tolerate and degrade up to 5 mM TBP, the highest concentration reported to date. The GC–MS method was developed to monitor TBP degradation. Two strains, Providencia sp. BGW4 and Delftia sp. BGW1 showed respectively, 61.0 ± 2.8% and 57.0 ± 2.0% TBP degradation within 4 days. The degradation rate constants, calculated by first order kinetic model were between 0.0024 and 0.0099 h⁻¹. These bacterial strains are novel for TBP degradation and could be used as an important bioresource for efficient decontamination of TBP polluted waste streams.     

Microbial Degradation of Isopropyl-N-3-Chlorophenylcarbamate and 2-Chloroethyl-N-3-Chlorophenylcarbamate

Microbial degradation of isopropyl-N-3-chlorophenylcarbamate (CIPC) and 2-chloroethyl-N-3-chlorophenylcarbamate (CEPC) was observed in a soil perfusion system. Degradation in perfused soils, and by pure cultures of effective bacterial isolates, was demonstrated by the production of 3-chloroaniline and the subsequent liberation of free chloride ion. Identified isolates effective in degrading and utilizing CIPC as a sole source of carbon included Pseudomonas striata Chester, a Flavobacterium sp., an Agrobacterium sp., and an Achromobacter sp. Identified isolates, effective in degrading and utilizing CEPC as a sole source of carbon, included an Achromobacter sp. and an Arthrobacter sp. CIPC-effective isolates degraded CEPC more slowly than CIPC, whereas CEPC-effective isolates degraded CIPC more rapidly than CEPC. Both CIPC- and CEPC-effective isolates degraded isopropyl N-phenylcarbamate (IPC) more rapidly than either CIPC or CEPC.     

Biodegradation of Crude Oil by Constructed Bacterial Consortia and the Constituent Single Bacteria Isolated From Malaysia

Three bacterial isolates identified as Pseudomonas aeruginosa (UKMP-8T), Rhodococcus sp. M15-2 (UKMP-5T), and Rhodococcus sp. ZH8 (UKMP-7T) based on biochemical, physiological, and morphological characteristics and on 16S rDNA sequences were isolated from groundwater of a crude oil refinery plant. From these three isolates, four bacterial consortia were designed by mixing the single bacterial cultures in the following ratios: (P. aeruginosa:Rhodococcus sp. M15-2, 1:1), (P. aeruginosa:Rhodococcus sp. ZH8, 1:1), (Rhodococcus sp. M15-2: Rhodococcus sp. ZH8, 1:1), and (P. aeruginosa: Rhodococcus sp. ZH8:Rhodococcus sp. M15-2, 1:1:1), respectively. Bacterial isolates and consortia showed differing preferences for nitrogen source (0.01% ammonium chloride, 0.10% yeast extract, or 0.50% peptone) to reach optimum growth. When fortified with the preferred nitrogen sources and grown in minimal salt medium, within 7 days all three single isolates and the four bacterial consortia biodegraded 97.6-99.9% of Tapis Massa oil without any significant differences.     

Isolation of acetonitrile-utilizing bacteria

Corynebacterium sp. 3 B and three unidentified bacterial strains, all utilizing acetonitrile as a sole carbon and nitrogen source, were isolated from soil by the enrichment culture technique.Agrobacterium radiobacter 8/4/1, also capable of growing on acetonitrile as a sole C and N source, was obtained from a bromoxynil-metabolizing strainA. radiobacter 8/4 using the same method. Resting cells of strains BSA 1 and 3 B produced acetamide and acetic acid during incubation with acetonitrile, whileA. radiobacter 8/4/1 produced acetic acid only.     

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.     

Isolation and characterization of four di-<Emphasis Type="Italic">n</Emphasis>-butyl phthalate (DBP)-degrading <Emphasis Type="Italic">Gordonia</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> strains and cloning the 3,4-phthalate dioxygenase gene

Four aerobic bacterial strains capable of utilizing di-n-butyl phthalate (DBP) as the sole source of carbon and energy were isolated from river sediments. Based on the morphology, biochemical characterization, and 16S rRNA gene sequence analysis, they were identified as Gordonia sp. The optimal conditions for DBP degradation by these strains were found to be pH 7.0, 30°C, and stirring at 175 rpm. These four strains could degrade, respectively, 96, 98, 98, and 78% of DBP (400 mg l⁻¹) as well as dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-octyl phthalate (DOP), di-isooctyl phthalate (DIOP), and di-isononyl phthalate (DINP). Furthermore, partial sequences of the gene for 3,4-phthalate dioxygenase were obtained from all four strains. To our knowledge, this is the first time that the 3,4-phthalate dioxygenase gene has been successfully cloned from Gordonia sp.     

Glyphosate utilization byPseudomonas sp. andAlcaligenes sp. isolated from environmental sources

A total of 21 bacterial cultures were isolated that could utilize glyphosate (N-phosphonomethyl glycine) as a sole source of phosphorus in a mineral salts medium. Sources of inocula for enrichment cultures included aerobic digester liquid, raw sewage, trickling filter effluent, pesticide disposal pit liquid, and soil. Eleven cultures were identified asPseudomonas sp., one asPseudomonas stutzeri, and nine asAlcaligenes sp. Aminomethylphosphonic acid, the major metabolic intermediate of glyphosate degradation in soil, could also serve as a sole phosphorus source for all 21 isolates. Neither glyphosate nor aminomethylphosphonic acid could serve as carbon sources in mineral salts media. Experiments withPseudomonas sp. SG-1 (isolated from aerobic digester liquid) suggested that enzymatic activity responsible for glyphosate degradation was intracellular, inducible, and required the cofactors pyruvate and pyridoxal phosphate. The degradation pathway for glyphosate in this culture may be similar to that previously reported for aminoethylphosphonic acid.     

Bacterial Degraders of Polycyclic Aromatic Hydrocarbons Isolated from Salt-Contaminated Soils and Bottom Sediments in Salt Mining Areas

Fifteen bacterial strains capable of utilizing naphthalene, phenanthrene, and biphenyl as the sole sources of carbon and energy were isolated from soils and bottom sediments contaminated with waste products generated by chemical- and salt-producing plants. Based on cultural, morphological, and chemotaxonomic characteristics, ten of these strains were identified as belonging to the genera Rhodococcus, Arthrobacter, Bacillus, and Pseudomonas. All ten strains were found to be halotolerant bacteria capable of growing in nutrient-rich media at NaCl concentrations of 1–1.5 M. With naphthalene as the sole source of carbon and energy, the strains could grow in a mineral medium with 1 M NaCl. Apart from being able to grow on naphthalene, six of the ten strains were able to grow on phenanthrene; three strains, on biphenyl; three strains, on octane; and one strain, on phenol. All of the strains were plasmid-bearing. The plasmids of the Pseudomonas sp. strains SN11, SN101, and G51 are conjugative, contain genes responsible for the degradation of naphthalene and salicylate, and are characterized by the same restriction fragment maps. The transconjugants that gained the plasmid from strain SN11 acquired the ability to grow at elevated NaCl concentrations. Microbial associations isolated from the same samples were able to grow at a NaCl concentration of 2.5 M.     

Synchronous anaerobic and aerobic degradation of DDT by an immobilized mixed culture system

Summary For the investigation of a mixed anaerobic and aerobic degradation of xenobiotics the reductive dechlorination of 1,1,1-trichloro-2,2-bis (4-chlorophenyl)ethane (DDT) to 1,1-dichloro-2,2-bis (4-chlorophenyl)ethane (DDD) and the oxidative degradation of the DDT-conversion product 4,4-dichlorodiphenylmethane (DDM) were studied. Enrichments from digested sewage sludge led to the isolation of an Enterobacter cloacae-strain which is able to reductive dechlorination of DDT during the fermentation of lactose. From fresh sewage sludge 11 bacterial strains were isolated in batch-culture and in continuous culture utilizing diphenylmethane, a non chlorinated structural analogon of DDM, as sole source of carbon and energy. One of these isolates, Alcaliaenes sp. cometabolizes DDM during the aerobic growth with diphenylmethane. By coimmobilization of Alcaligenes sp. and Enterobacter cloacae in Ca-alginate a system could be established, in which the reductive dechlorination of DDT and the oxidative degradation of DDM and diphenylmethane proceeds simultaneously in one reactor vessel.     

Biodegradation of naphthalene-2-sulfonic acid present in tannery wastewater by bacterial isolates <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. 2AC and <Emphasis Type="Italic">Comamonas</Emphasis> sp. 4BC

Two bacterial strains, 2AC and 4BC, both capable of utilizing naphthalene-2-sulfonic acid (2-NSA) as a sole source of carbon, were isolated from activated sludges previously exposed to tannery wastewater. Enrichments were carried out in mineral salt medium (MSM) with 2-NSA as the sole carbon source. 16S rDNA sequencing analysis indicated that 2AC is an Arthrobacter sp. and 4BC is a Comamonas sp. Within 33 h, both isolates degraded 100% of 2-NSA in MSM and also 2-NSA in non-sterile tannery wastewater. The yield coefficient was 0.33 g biomass dry weight per gram of 2-NSA. A conceptual model, which describes the aerobic transformation of organic matter, was used for interpreting the biodegradation kinetics of 2-NSA. The half-lives for 2-NSA, at initial concentrations of 100 and 500 mg/l in MSM, ranged from 20 h (2AC) to 26 h (4BC) with lag-phases of 8 h (2AC) and 12 h (4BC). The carbon balance indicates that 75–90% of the initial TOC (total organic carbon) was mineralized, 5–20% remained as DOC (dissolved organic carbon) and 3–10% was biomass carbon. The principal metabolite of 2-NSA biodegradation (in both MSM and tannery wastewater) produced by Comamonas sp. 4BC had a MW of 174 and accounted for the residual DOC (7.0–19.0% of the initial TOC and 66% of the remaining TOC). Three to ten percent of the initial TOC (33% of the remaining TOC) was associated with biomass. The metabolite was not detected when Arthrobacter sp. 2AC was used, and a lower residual DOC and biomass carbon were recorded. This suggests that the two strains may use different catabolic pathways for 2-NSA degradation. The rapid biodegradation of 2-NSA (100 mg/l) added to non-sterile tannery wastewater (total 2-NSA, 105 mg/l) when inoculated with eitherArthrobacter 2AC or Comamonas 4BC showed that both strains were able to compete with the indigenous microorganisms and degrade 2-NSA even in the presence of alternate carbon sources (DOC in tannery wastewater = 91 mg/l). The results provide information useful for the rational design of bioreactors for tannery wastewater treatment.