Pseudomonas marginalis: its degradative capability on organic nitriles and amides

Pseudomonas marginalis, capable of utilizing acetonitrile as the sole source of carbon and nitrogen, was isolated from an industrial waste site. P. marginalis metabolized acetonitrile into ammonia and acetate. The minimal inhibitory concentration values of different nitriles and amides for P. marginalis were in the range 5–300 mM. The bacterium was able to transform high-molecular-mass nitrile compounds and their respective amides into ammonia. The data from substrate-dependent kinetics showed that the K _m and V _max values of P. marginalis for acetonitrile were 33 mM and 67 nmol oxygen consumed min^–1 (ml cell suspension)^–1 respectively. The study with [¹⁴C]acetonitrile indicated that nearly 66% of the carbon was released as ¹⁴CO₂ and 12% was associated with the biomass. The enzyme system involved in the hydrolysis of acetonitrile was shown to be intracellular and inducible. The specific activities of the enzymes nitrile aminohydrolase and amidase were determined in the cell-free extracts of P. marginalis. Both the enzymes could hydrolyze a wide range of nitriles and amides. The present study suggests that the biodegradation of organic nitriles and the bioproduction of organic acids may be achieved with the cells of P. marginalis.     

Proteomic Analysis of the Effect of Cyanide on Klebsiella oxytoca

Cyanide has been proved to be degraded by Klebsiella oxytoca. In order to examine the physiological responses of cyanide degradation by this bacterium, two-dimensional (2-DE) electrophoresis approach and MALDI–TOF–MS allow us to identify 106 proteins spots that were significantly altered in the presence of 1 mM cyanide in relative to that in 1 mM ammonia when K. oxytoca grown at the late-log phase. Among them, 27 proteins were successfully identified. These proteins were involved in carbohydrate metabolism, nucleotide metabolism, amino acid metabolism, nitrogen metabolism, stress responses, oxidation–reduction reactions, transporters, and miscellaneous function. Some proteins related with regulation of nitrogen assimilation pathways (glutamine synthetase), oxidative stress repairing (catalase), and protection (neutral trehalase and glycosyltransferase) could improve the effectiveness of cyanide biodegradation. Although the nitrogenase was suggested to participate in cyanide degradation in our previous study, this enzyme induction was not observed as expected. These findings could provide new insights into the inducible mechanisms underlying the capacity of K. oxytoca to tolerate cyanide stress.     

Utilization of aliphatic nitrile by Paracoccus sp. SKG isolated from chemical waste samples

A bacterial strain Paracoccus sp. SKG capable of utilizing acetonitrile as a sole source of carbon and nitrogen was isolated from the chemical waste samples. The molecular phylogram generated using the complete sequence of 16S rDNA of the strain SKG showed close links to the bacteria grouped under Brucellaceae family, that belongs to the class alphaproteobacteria. Specifically, the 16S rDNA sequence of strain SKG has shown 99% similarity to Paracoccus sp. This bacterium has also shown impressive growth on aliphatic nitriles like acetonitrile, propionitrile, acrylonitrile, valeronitrile and their corresponding amides. The nitriles degradation has led to the accumulation of ammonia and respective carboxylic acids. The acetonitrile grown cells showed the release of ammonia that contributes to the increase in pH of the medium. However, glucose grown cells failed to produce ammonia, thus indicating the inducible nature of acetonitrile degrading enzymes in Paracoccus sp. SKG. Nitrile hydratase and amidase are the two key enzymes involved in the degradation of acetonitrile. Degradation of acetonitrile in Paracoccus sp. SKG follows the bi-enzymatic pathway. Further, this strain is capable of degrading acetonitrile in the presence of other organic solvents such as methanol, ethanol and dimethylformamide. Therefore, this strain is efficiently used for the treatment of HPLC waste stream containing acetonitrile in the presence of other organic solvents.     

Utilization of nitriles by yeasts isolated from a Brazilian gold mine

Yeast strains from the genera Candida, Debaryomyces, Aureobasidium, Geotrichum, Pichia, Rhodotorula, Tremella, Hanseniaspora, and Cryptococcus were isolated from samples of a gold mine from liquid extraction circuit. These strains were tested for their ability to utilize acetonitrile at 12 mM as the sole nitrogen source. The yeasts that grew using acetonitrile at 12 mM were tested in the presence of acetonitrile, isobutyronitrile, methacrylnitrile, and propionitrile at concentrations of 12, 24, 48, 97, and 120 mM. One strain was selected for each nitrile and the concentration of nitrile in which the best growth occurred. Cryptococcus sp. strain UFMG-Y28 had a better growth on 120 mM propionitrile and 97 mM acetonitrile, Rhodotorula glutinis strain UFMG-Y5 on 48 mM methacrylnitrile, and Cryptococcus flavus strain UFMG-Y61 on 120 mM isobutyronitrile. The utilization of different nitriles and amides by yeast strains involves hydrolysis in a two-step reaction mediated by both inducible and intracellular nitrile hydratase and amidase.     

Studies on utilization of acetonitrile by Rhodococcus erythropolis A10

A petrochemical wastewater isolate, capable of utilizing high concentrations of acetonitrile and acetamide as the sole source of carbon and nitrogen was identified as Rhodococcus erythropolis A10. Cell-free extracts of acetonitrile-grown cells exhibited activities corresponding to nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4), which mediate the two-step breakdown of acetonitrile into acetic acid and ammonia. Studies indicated that both these enzymes in R. erythropolis A10 are intracellular, inducible and capable of hydrolysing a wide range of nitriles, including simple (acetonitrile, propionitrile), branched-chain (isobutyronitrile) and dinitrile (succinonitrile). The specific activity of the amidase was found to be several-fold higher than nitrile hydratase.     

Degradation of Acetonitrile by Pseudomonas putida

A bacterium capable of utilizing high concentrations of acetonitrile as the sole source of carbon and nitrogen was isolated from soil and identified as Pseudomonas putida. This bacterium could also utilize butyronitrile, glutaronitrile, isobutyronitrile, methacrylonitrile, propionitrile, succinonitrile, valeronitrile, and some of their corresponding amides, such as acetamide, butyramide, isobutyramide, methacrylamide, propionamide, and succinamide as growth substrates. Acetonitrile-grown cells oxidized acetonitrile with a K_m of 40.61 mM. Mass balance studies with [¹⁴C]acetonitrile indicated that nearly 66% of carbon of acetonitrile was released as ¹⁴CO₂ and 14% was associated with the biomass. Metabolites of acetonitrile in the culture medium were acetic acid and ammonia. The acetate formed in the early stages of growth completely disappeared in the later stages. Cell extracts of acetonitrile-grown cells contained activities corresponding to nitrile hydratase and amidase, which mediate the breakdown of actonitrile into acetic acid and ammonia. Both enzymes were intracellular and inducible and hydrolyzed a wide range of substrates. The specific activity of amidase was at least 150-fold higher than the activity of the enzyme nitrile hydratase.     

Metabolism of acetonitrile and propionitrile by Nocardia rhodochrous LL100-21.

Six nitrile compounds and two amide derivatives were degraded by Nocardia rhodochrous LL100-21. Acetonitrile, hydroacrylonitrile, and propionitrile were the best sources of carbon and nitrogen for growth, whereas butenenitrile, succinonitrile, and acetamide supported less growth. Acrylonitrile and acrylamide supported growth but only as a source of nitrogen. Gas chromatography of the culture medium revealed a decrease in acetonitrile with the sequential formation of acetamide and acetic acid. Ammonia was also detected by colorimetric procedures. The enzyme system responsible for the hydrolysis of acetonitrile was shown to be intracellular and inducible. The breakdown of acetonitrile by a crude bacterial extract was a two-step enzymatic hydrolysis with acetamide as the intermediate product and acetic acid and ammonia as the final products. Product formation was stoichiometric with substrate disappearance. When propionitrile was the growth substrate, there was complete conversion of the nitrile to propionic acid and ammonia as the major products. The enzymatic breakdown of the propionitrile, although slower than acetonitrile, yielded the corresponding carboxylic acid and ammonia. Propionamide was produced in very small amounts as an intermediate product.     

Enrichment strategies for nitrile-hydrolysing bacteria

A series of enrichments with different nitriles as sole source of nitrogen was performed in order to obtain a relationship between the selective nitrogen source and (i) the enzyme systems that are synthesized by the isolates and (ii) the enzyme specificities for the utilization of the nitriles. Bacteria were enriched with 2-phenylpropionitrile, 2-(2-methoxyphenyl)propionitrile, 2-phenylbutyronitrile, ibuprofen nitrile, naproxen nitrile, ketoprofen nitrile, ketoprofen amide, benzonitrile, or naphthalenecarbonitrile as sole nitrogen source and succinate as sole source of carbon and energy. 2-Phenylpropionitrile as nitrogen source resulted predominantly in the enrichment of gram-negative bacteria, which harboured nitrilase and in some cases also amidase activity. In contrast, with the other nitriles used, a substantial majority of gram-positive strains, mainly of the genus Rhodococcus, were isolated. These strains contained predominantly a nitrile hydratase/amidase system. The nitrilases and nitrile hydratases showed R or S selectivity with generally poor optical yields. In contrast, the amidases were almost exclusively S-selective, often forming the optically pure acids with an enantiomeric excess above 99%. The conversion of different nitriles by the isolates was compared. The nitrile-hydrolysing systems of the new isolates usually showed high activity against those nitriles that were used for the enrichment of the bacteria. Received: 13 November 1996 / Received revision: 4 February 1997 / Accepted: 10 February 1997     

ESR Spectral Studies on the Free Radical Formed by the Reaction of Dehydroascorbic Acid with Amino Acid

Acrylonitrile-hydrating activity was found in various bacteria belonging to the genera Arthrobacter, Corynebacterium, Microbacterium, Nocardia, Pseudomonas and Rhodococcus. A strain, N-774, isolated by acetonitrile enrichment culture from soil showed the highest activity. Taxonomic studies indicated that strain N-774 belonged to the genus Rhodococcus. The acrylonitrile-hydrating enzyme of strain N-774 was constitutively formed in the cells. Besides acrylonitrile, many nitriles were hydrated to the corresponding amides. «-Butyronitrile, suc- cinonitrile and chloroacetonitrille served as suitable substrates. This bacterium could utilize many aliphatic nitriles and amides as a sole source of nitrogen but hardly utilized malononitrile, acrylonitrile or acrylamide. Cells having high nitrile hydratase activity (about 50units/mg of dry cells) could be easily prepared by cultivation at 30°C for 40 hr in a malt extract and yeast extract medium.     

Optimized intermolecular potential for nitriles based on Anisotropic United Atoms model

An extension of the anisotropic united atoms intermolecular potential model is proposed for nitriles. The electrostatic part of the intermolecular potential is calculated using atomic charges obtained by a simple Mulliken population analysis. The repulsion-dispersion interaction parameters for methyl and methylene groups are taken from transferable AUA4 literature parameters [Ungerer et al., J. Chem. Phys., 2000, 112, 5499]. Non-bonding Lennard-Jones intermolecular potential parameters are regressed for the carbon and nitrogen atoms of the nitrile group (–C≡N) from experimental vapor-liquid equilibrium data of acetonitrile. Gibbs Ensemble Monte Carlo simulations and experimental data agreement is very good for acetonitrile, and better than previous molecular potential proposed by Hloucha et al. [J. Chem. Phys., 2000, 113, 5401]. The transferability of the resulting potential is then successfully tested, without any further readjustment, to predict vapor-liquid phase equilibrium of propionitrile and n-butyronitrile. Figure Saturated vapour pressure of nitriles calculated in this work by molecular simulation compared to experimental data: a) for acetonitrile and b) for both propionitrile and butyronitrile     

Nitrile- and Amide-hydrolysing Activity in Kluyveromyces thermotolerans MGBY 37

Summary Forty yeast strains were screened for nitrile-hydrolysing activity. Among them Kluyveromyces thermotolerans MGBY 37 exhibited highest nitrile-hydrolysing activity (0.030 μmol/h/mg dry cell weight). This yeast contained a two-enzyme system i.e. nitrile hydratase (NHase, EC 4.2.1.84) and amidase (EC 3.5.1.4) for the hydrolysis of nitriles/amides to corresponding acids and ammonia. However, these enzymes had more affinity for N-heterocyclic aromatic and aromatic nitriles/amides rather than unsaturated and saturated aliphatic nitriles/amides. The NHase–amidase activity was constitutively produced by K. thermotolerence MGBY 37. Addition of acetonitrile in the medium enhanced the production of this activity while other nitriles and amides lowered the production of NHase–amidase activity. This organism thus exhibited two types of amidase i.e. a constitutive amidase having affinity for N-heterocyclic aromatic, unsaturated and saturated aliphatic amides and another inducible amidase with affinity for aromatic amides. Formamide proved to be the best inducer of the latter amidase activity. This is the first report on nitrile- and amide-hydrolysing activity in Kluyveromyces.     

Aneuploidy in Drosophila, IV. Inhalation studies on the induction of aneuploidy by nitriles

The Drosophila ZESTE system was used to monitor the induction of sex chromosome aneuploidy following inhalation exposure of adult females to four nitriles: acetonitrile, propionitrile, acrylonitrile and fumaronitrile. Acetonitrile and propionitrile were highly effective aneuploidogens, inducing both chromosome loss and chromosome gain following brief exposures to low concentrations of these chemicals, and these nitriles also induced rapid paralysis. Acrylonitrile-induced chromosome loss only but did not induce paralysis. Fumaronitrile, in contrast with the results reported in yeast, was ineffective in inducing chromosome loss or gain. Virtually all exceptional offspring induced by acetonitrile and propionitrile were recovered in the first sampled eggs, corresponding to treated mature oocytes. Additionally, the time interval between treatment and sampling was shown to be important, suggesting rapid loss or detoxification of the nitriles. Genetic analysis demonstrated that most aneuploids resulted from induced segregation errors during the first division of meiosis. Cold treatments were found to be ineffective in enhancing the effects of acetonitrile, suggesting important differences between the Drosophila and yeast aneuploidy detection systems. Possible mechanisms by which nitriles may disrupt chromosome segregation in Drosophila oocytes are considered.     

Biodegradation of cyanide compounds by a Pseudomonas species (S1).

A Pseudomonas sp. (S1), isolated from soil by an enrichment technique was tested for its potential to degrade different cyanide compounds. Further, biodegradation/biotransformation of binary mixtures of the cyanide compounds by the culture was also studied. The results indicated that the culture could grow on the following nitriles by using them as carbon and nitrogen sources: acetonitrile, butyronitrile, acrylonitrile, adiponitrile, benzonitrile, glutaronitrile, phenylacetonitrile, and succinonitrile. Studies on the biodegradation of these cyanide compounds in binary mixtures showed that the presence of acrylonitrile or KCN delayed the degradation of acetonitrile in a mixture, while none of the other cyanide compounds affected the degradation of one another. The transformation products of the nitriles were their corresponding acids, and similarly, KCN was also directly transformed to formic acid. Studies on the transformation of these cyanide compounds showed that the rate of transformation of nitriles to their corresponding carboxylic acids was acrylonitrile > acetonitrile > adiponitrile > benzonitrile > KCN. This culture has the unique characteristic of transforming representatives of saturated aliphatic, aliphatic olefinic, aromatic, and aralkyl nitriles, as well as alkali cyanide, to their corresponding carboxylic acids.     

Elimination of carbon catabolite repression in Klebsiella oxytoca for efficient 2,3-butanediol production from glucose-xylose mixtures

Microbial preference for glucose implies incomplete and/or slow utilization of lignocellulose hydrolysates, which is caused by the regulatory mechanism named carbon catabolite repression (CCR). In this study, a 2,3-butanediol (2,3-BD) producing Klebsiella oxytoca strain was engineered to eliminate glucose repression of xylose utilization. The crp(in) gene, encoding the mutant cyclic adenosine monophosphate (cAMP) receptor protein CRP(in), which does not require cAMP for functioning, was characterized and overexpressed in K. oxytoca. The engineered recombinant could utilize a mixture of glucose and xylose simultaneously, without CCR. The profiles of sugar consumption and 2,3-BD production by the engineered recombinant, in glucose and xylose mixtures, were examined and showed that glucose and xylose could be consumed simultaneously to produce 2,3-BD. This study offers a metabolic engineering strategy to achieve highly efficient utilization of sugar mixtures derived from the lignocellulosic biomass for the production of bio-based chemicals using enteric bacteria.     

Isolation and Characterization of a Nitrile-Hydrolysing Bacterium Isoptericola variabilis RGT01

A nitrile-hydrolysing bacterium, identified as Isoptericola variabilis RGT01, was isolated from industrial effluent through enrichment culture technique using acrylonitrile as the carbon source. Whole cells of this microorganism exhibited a broad range of nitrile-hydrolysing activity as they hydrolysed five aliphatic nitriles (acetonitrile, acrylonitrile, propionitrile, butyronitrile and valeronitrile), two aromatic nitriles (benzonitrile and m-Tolunitrile) and two arylacetonitriles (4-Methoxyphenyl acetonitrile and phenoxyacetonitrile). The nitrile-hydrolysing activity was inducible in nature and acetonitrile proved to be the most efficient inducer. Minimal salt medium supplemented with 50 mM acetonitrile, an incubation temperature of 30 °C with 2 % v/v inoculum, at 200 rpm and incubation of 48 h were found to be the optimal conditions for maximum production (2.64 ± 0.12 U/mg) of nitrile-hydrolysing activity. This activity was stable at 30 °C as it retained around 86 % activity after 4 h at this temperature, but was thermolabile with a half-life of 120 min and 45 min at 40 °C and 50 °C respectively.     

Interaction of <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> and <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> in Dual-Species Batch Cultures and Biofilms as a Function of Growth Rate and Substrate Concentration

Dual-species microbial interactions have been extensively reported for batch and continuous culture environments. However, little research has been performed on dual-species interaction in a biofilm. This research examined the effects of growth rate and substrate concentration on dual-species population densities in batch and biofilm reactors. In addition, the feasibility of using batch reactor kinetics to describe dual-species biofilm interactions was explored. The scope of the research was directed toward creating a dual-species biofilm for the biodegradation of trichloroethylene, but the findings are a significant contribution to the study of dual-species interactions in general. The two bacterial species used were Burkholderia cepacia PR1-pTOM_31c, an aerobic organism capable of constitutively mineralizing trichloroethylene (TCE), and Klebsiella oxytoca, a highly mucoid, facultative anaerobic organism. The substrate concentrations used were different dilutions of a nutrient-rich medium resulting in dissolved organic carbon (DOC) concentrations on the order of 30, 70, and 700 mg/L. Presented herein are single- and dual-species population densities and growth rates for these two organisms grown in batch and continuous-flow biofilm reactors. In batch reactors, planktonic growth rates predicted dual-species planktonic species dominance, with the faster-growing organism (K. oxytoca) outcompeting the slower-growing organism (B. cepacia). In a dual-species biofilm, however, dual-species planktonic growth rates did not predict which organism would have the higher dual-species biofilm population density. The relative fraction of each organism in a dual-species biofilm did correlate with substrate concentration, with B. cepacia having a greater proportional density in the dual-species culture with K. oxytoca at low (30 and 70 mg/L DOC) substrate concentrations and K. oxytoca having a greater dual-species population density at a high (700 mg/L DOC) substrate concentration. Results from this research demonstrate the effectiveness of using substrate concentration to control population density in this dual-species biofilm.     

A plate method for screening of bacteria capable of degrading aliphatic nitriles

A novel indicator plate method was developed for screening of aliphatic-nitrile-degrading bacteria. Isolated bacteria were tested for utilization of acetonitrile as sole source of carbon and nitrogen with the release of ammonia. The released ammonia causes increase of the pH of the medium. Phenol red indicator is used for detection of ammonia based on colour change of the indicator dye from red to pink. The liberation of ammonia from aliphatic-nitrile-utilizing bacteria is also studied in plates containing other indicators such as bromothymol blue and phenolphthalein. The usefulness of the indicator plate is demonstrated for bacteria that degrade certain aliphatic nitriles. Bacteria degrading nitriles as a nitrogen source can also be isolated with a medium containing additional carbon source. This plate method would be useful in isolation and screening of bacteria for degradation of aliphatic nitriles and also for production of nitrile-hydrolyzing enzymes.     

Isolation of a novelAgrobacteriun spp capable of degrading a range of nitrile compounds

Summary Using soil enrichment techniques we have isolated micro-organisms capable of degrading simple nitrile compounds. One species identified as anAgrobacterium spp. was examined in detail. This isolate was capable of utilising a range of aliphatic and aromatic nitriles as sole sources of carbon and nitrogen. Assays for enzymes involved in nitrile degradation and growth/production studies with this organism growing on acetonitrile indicated that breakdown occurred via a two step mechanism firstly to the amide and then via the production of the corresponding acid with the subsequent release of ammonia. Our studies indicate that the system of nitrile breakdown is inducible and levels of the amidase are 170 times that of the nitrile hydratase in crude extracts.     

Biodegradation of Nitriles in Shale Oil

Enrichment cultures were obtained, after prolonged incubation on a shale oil as the sole source of nitrogen, that selectively degraded nitriles. Capillary gas chromatographic analyses showed that the mixed microbial populations in the enrichments degraded the homologous series of aliphatic nitriles but not the aliphatic hydrocarbons, aromatic hydrocarbons, or heterocyclic-nitrogen compounds found in this oil. Time course studies showed that lighter nitriles were removed more rapidly than higher-molecular-weight nitriles. A Pseudomonas fluorescens strain isolated from an enrichment, which was able to completely utilize the individual nitriles undecyl cyanide and undecanenitrile as sole sources of carbon and nitrogen, was unable to attack stearonitrile when provided alone as the growth substrate. A P. aeruginosa strain, also isolated from one of the enrichments, used nitriles but not aliphatic or aromatic hydrocarbons when the oil was used as a sole nitrogen source. However, when the shale oil was used as the sole source of carbon, aliphatic hydrocarbons in addition to nitriles were degraded but aromatic hydrocarbons were still not attacked by this P. aeruginosa strain.     

Metabolism of benzonitrile and butyronitrile by Klebsiella pneumoniae.

A strain of Klebsiella pneumoniae that used aliphatic nitriles as the sole source of nitrogen was adapted to benzonitrile as the sole source of carbon and nitrogen. Gas chromatographic and mass spectral analyses of culture filtrates indicated that K. pneumoniae metabolized 8.4 mM benzonitrile to 4.0 mM benzoic acid and 2.7 mM ammonia. In addition, butyronitrile was metabolized to butyramide and ammonia. The isolate also degraded mixtures of benzonitrile and aliphatic nitriles. Cell extracts contained nitrile hydratase and amidase activities. The enzyme activities were higher with butyronitrile and butyramide than with benzonitrile and benzamide, and amidase activities were twofold higher than nitrile hydratase activities. K. pneumoniae appears promising for the bioremediation of sites contaminated with aliphatic and aromatic nitriles.