Isolation of microorganisms capable of degrading isoquinoline under aerobic conditions

Isoquinoline-degrading microbial cultures were isolated from oil- and creosote-contaminated soils. The establishment of initial enrichment cultures required the use of emulsified isoquinoline. Once growth on isoquinoline was established, isoquinoline emulsification was no longer required for utilization of isoquinoline as the sole source of carbon and nitrogen by these cultures. An isoquinoline-degrading Acinetobacter strain was isolated from one of the enrichment cultures. The degradation of isoquinoline was accompanied by the accumulation of a red cell-associated pigment and of 1-hydroxyisoquinoline, which was further degraded to unknown intermediary ring-cleavage products and carbon dioxide.     

New Obligate Methylotroph

A new and unique obligate methylotroph was isolated from enrichment cultures with methanol as the sole source of carbon and energy. The organism grows only on methanol and methylamine and not on methane. It does not have a complex intracellular membrane system. (14)C-acetate was assimilated by growing cultures and cell suspensions but was incorporated into only a limited number of cell constituents. (14)C-acetate incorporation was strictly dependent on the oxidation of methanol or methylamine as a source of energy. Extracts had relatively low levels of enzymes of the tricarboxylic acid cycle, and alpha-ketoglutarate dehydrogenase was not detected. Comparisons were made with a facultative methylotroph isolated from the same enrichment cultures. The new obligate methylotroph contained hexose phosphate synthetase, a key enzyme in the ribose phosphate cycle of methyl metabolism.     

Cross-induction of pyrene and phenanthrene in a Mycobacterium sp. isolated from polycyclic aromatic hydrocarbon contaminated river sediments.

A polycyclic aromatic hydrocarbon (PAH)-degrading culture enriched from contaminated river sediments and a Mycobacterium sp. isolated from the enrichment were tested to investigate the possible synergistic and antagonistic interactions affecting the degradation of pyrene in the presence of low molecular weight PAHs. The Mycobacterium sp. was able to mineralize 63% of the added pyrene when it was present as a sole source of carbon and energy. When the enrichment culture and the isolated bacterium were exposed to phenanthrene, de novo protein synthesis was not required for the rapid mineralization of pyrene, which reached 52% in chloramphenicol-treated cultures and 44% in the absence of the protein inhibitor. In the presence of chloramphenicol, < 1% of the added pyrene was mineralized by the mixed culture after exposure to anthracene and naphthalene. These compounds did not inhibit pyrene utilization when present at the same time as pyrene. Concurrent mineralization of pyrene and phenanthrene after exposure to either compound was observed. Cross-acclimation between ring classes of PAHs may be a potentially important interaction influencing the biodegradation of aromatic compounds in contaminated environments.     

Isolation of a methyl parathion-degrading Pseudomonas sp. that possesses DNA homologous to the opd gene from a Flavobacterium sp

Two mixed bacterial cultures isolated by soil enrichment were capable of utilizing methyl parathion (O,O-dimethyl O-p-nitrophenylphosphorothioate) and parathion (O,O-diethyl O-p-nitrophenylphosphorothioate) as a sole source of carbon. Four isolates from these mixed cultures lost their ability to utilize the pesticides independently in transfers subsequent to the initial isolation. One member of the mixed cultures, a Pseudomonas sp., however, hydrolyzed the pesticides to p-nitrophenol but required glucose or another carbon source for growth. The crude cell extracts prepared from this bacterium showed an optimum pH range from 7.5 to 9.5 for the enzymatic hydrolysis. Maximum enzymatic activity occurred between 35 and 40 degrees C. The enzyme activity was not inhibited by heavy metals, EDTA, or NaN3. Another isolate from the mixed cultures, a Flavobacterium sp., used p-nitrophenol for growth and degraded it to nitrite. Nitrite was assimilated into the cells under conditions during which the nitrogen source was excluded from the minimal growth medium. The hybridization data showed that the DNAs from a Pseudomonas sp. and from the mixed culture had homology with the opd (organophosphate degradation) gene from a previously reported parathion-hydrolyzing bacterium, Flavobacterium sp. The use of the opd gene as a probe may accelerate progress toward understanding the complex interactions of soil microorganisms with parathions.     

Isolation of a methyl parathion-degrading Pseudomonas sp. that possesses DNA homologous to the opd gene from a Flavobacterium sp.

Two mixed bacterial cultures isolated by soil enrichment were capable of utilizing methyl parathion (O,O-dimethyl O-p-nitrophenylphosphorothioate) and parathion (O,O-diethyl O-p-nitrophenylphosphorothioate) as a sole source of carbon. Four isolates from these mixed cultures lost their ability to utilize the pesticides independently in transfers subsequent to the initial isolation. One member of the mixed cultures, a Pseudomonas sp., however, hydrolyzed the pesticides to p-nitrophenol but required glucose or another carbon source for growth. The crude cell extracts prepared from this bacterium showed an optimum pH range from 7.5 to 9.5 for the enzymatic hydrolysis. Maximum enzymatic activity occurred between 35 and 40 degrees C. The enzyme activity was not inhibited by heavy metals, EDTA, or NaN3. Another isolate from the mixed cultures, a Flavobacterium sp., used p-nitrophenol for growth and degraded it to nitrite. Nitrite was assimilated into the cells under conditions during which the nitrogen source was excluded from the minimal growth medium. The hybridization data showed that the DNAs from a Pseudomonas sp. and from the mixed culture had homology with the opd (organophosphate degradation) gene from a previously reported parathion-hydrolyzing bacterium, Flavobacterium sp. The use of the opd gene as a probe may accelerate progress toward understanding the complex interactions of soil microorganisms with parathions.     

Denitrification with Methanol: a Selective Enrichment for Hyphomicrobium Species

Hyphomicrobium species were enriched in media with methanol as sole carbon source under conditions supporting denitrification. Pure cultures of Hyphomicrobium species were isolated which denitrified vigorously with methanol. Hyphomicrobium B522, isolated by aerobic enrichment, was adapted to anaerobic growth and denitrification. Hyphomicrobium B522 and a new isolate were surveyed for anaerobic growth and denitrification on a number of simple organic compounds. Cell suspensions were tested for denitrifying activity. Nitrogen production from nitrate and nitrite and carbon dioxide production from methanol were stoichiometric.     

Microbial metabolism of quinoline and related compounds. IV. Degradation of isoquinoline by Alcaligenes faecalis Pa and Pseudomonas diminuta 7

From sewage and soil isoquinoline-degrading organisms were enriched. Two strains could be isolated which were able to utilize isoquinoline as sole carbon source. The bacteria were tentatively identified as Alcaligenes faecalis and Pseudomonas diminuta with respect to their morphological and physiological characters. When growing on isoquinoline both strains excrete a metabolite into the medium which was identified as 1-oxo-1,2-dihydroisoquinoline. Alcaligenes faecalis was cultivated in continuous culture on 1-oxo-1,2-dihydroisoquinoline to improve growth on isoquinoline and degradative activity.     

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.     

Biodegradation of propoxur by Pseudomonas species

A bacterium capable of degrading propoxur (2-isopropoxyphenyl-N-methylcarbamate) was isolated from soil by enrichment cultures and was identified as a Pseudomonas species. The organism grew on propoxur at 2 g/l as sole source of carbon and nitrogen, and accumulated 2-isopropoxyphenol as metabolite in the culture medium. The cell free extract of Pseudomonas sp. grown on propoxur contained the activity of propoxur hydrolase. The results suggest that the organism degraded propoxur by hydrolysis to yield 2-isopropoxyphenol and methylamine, which was further utilized as carbon source.     

Possible initial steps in the catabolism of 1,2-diphenylethanone (deoxybenzoin) by Pseudomonas fluorescens DB-5.

A natural bacterial strain, identified as Pseudomonas fluorescens DB-5, was isolated in enrichment cultures containing 1,2-diphenylethanone as the only source of carbon and energy. On the basis of characteristic features observed in the mass spectra of degradation intermediates, it is proposed that metabolism of 1,2-diphenylethanone is initiated by two hydroxylations on the benzyl ring. Phenol, presumably arising from the benzoyl ring, was transiently detected as a catabolic intermediate.     

3-Nitrobenzenesulfonate, 3-Aminobenzenesulfonate, and 4-Aminobenzenesulfonate as Sole Carbon Sources for Bacteria

Aerobic, carbon-limited, enrichment cultures containing 3-aminobenzenesulfonate or 3-nitrobenzenesulfonate as the sole source of carbon and energy yielded growth and complete substrate disappearance. Pure cultures of putative pseudomonads were isolated which utilized these compounds quantitatively. Degradation was compared with that of 2- and 4-aminobenzenesulfonate.     

Microbial degradation of monoterpenes in the absence of molecular oxygen.

Anaerobic degradation of natural monoterpenes by microorganisms was evaluated by using Pseudomonas citronellolis DSM 50332 and enrichment cultures containing nitrate as an electron acceptor. P. citronellolis grew anaerobically on 3,7-dimethyl-1-octanol and citronellol but not on geraniol, nerol, and alicyclic monoterpenes. In contrast, several a-, mono-, and bicyclic monoterpenes supported microbial growth and denitrification in enrichment cultures. We found that consumption of linalool, menthol, menth-1-ene, alpha-phellandrene, limonene, 2-carene, alpha-pinene, and fenchone in enrichment cultures depended on the presence of living microorganisms and nitrate. In these experiments, the ratios of number of electrons derived from complete substrate oxidation to number of electrons derived from nitrate reduction ranged from 1.2:1 to 2.9:1. Microbial degradation was accompanied by the formation of small traces of monoterpenes, which were characterized by gas chromatography-mass spectroscopy. The formation of geraniol and geranial from linalool suggested that a 3,1-hydroxyl-delta 1-delta 2-mutase reaction initiates linalool degradation. Seven strains of motile, oval to rod-shaped, facultatively denitrifying bacteria were isolated on agar bottle plates by using linalool, menthol, menth-1-ene, alpha-phellandrene, 2-carene, eucalyptol, and alpha-pinene as sole carbon and energy sources.     

Reduction of sulfur by spirillum 5175 and syntrophism with Chlorobium.

A small spirillum, designated 5175, was isolated from an anaerobic enrichment culture for Desulfuromonas in which the major medium constituents were acetate and elemental sulfur. The organisms grew only under anaerobic or microaerophilic conditions. Elemental sulfur was formed anaerobically in a malate-sulfide medium, and cell densities of 10(8) cells/ml were obtained. Hydrogen and formate were actively oxidized as substrates for growth under anaerobic conditions; S0, S032-, or S2O32-, but not SO42-, served as electron acceptors and were stoichiometrically reduced to sulfide. Malate or fumarate likewise served as electron acceptors and were reduced to succinate. Nutritional requirements were simple, no vitamins or amino acids being required. For growth in inorganic media when carbon dioxide was the only carbon source, the addition of acetate was required as a source of cell carbon. The organism is gram negative. Cells had a diameter of 0.5 mum and a wavelength of 5.0 mum. Cell suspensions exhibited an absorption spectrum indicative of a cytochrome with peaks in the reduced form at 552, 523, and 416 nm. Well growing syntrophic cultures with Chlorobium were established with formate as the substrate.     

Isolation and characterization of soil strains producing glutaryl-7-aminocephalosporanic acid acylase

A search was undertaken to screen microorganisms that produce an enzyme capable of deacylating glutaryl-7-aminocephalosporanic acid to 7-aminocephalosporanic acid in soil samples. The screening was carried out by preparing enrichment cultures containing glutaryl-7ACA and cephalosporin C as selective carbon sources. A non-β-lactam model compound, glutaryl-p-nitroanilide, was synthesized as a substrate suitable for the rapid screening of microorganisms isolated from the enrichment cultures. Two isolates exhibiting acylase activity, designated BY7.4 and BY8.1, were identified as strains ofPseudomonas species.Pseudomonas BY8.1 showed higher acylase activity toward Gl-7ACA thanPseudomonas BY7.4. Environmental conditions for the optimal acylase activity ofPseudomonas BY8.1 were shown to be pH 9 and 30°C.     

Microbial oxidation of isoprene, a biogenic foliage volatile and of 1,3-butadiene, an anthropogenic gas

Nocardia strains that were able to degrade isoprene were isolated from several locations using enrichment cultures with isoprene or 1,3-butadiene as the sole carbon and energy source. Specific growth rates of representative isolates on isoprene and 1,3-butadiene ranged from 0.05 to 0.2 h⁻¹. The initial oxygenation of both 1,3-butadiene and isoprene was mediated by mono-oxygenases which converted these alkadienes into the respective epoxyalkanes.     

Degradation of some phenoxy acid herbicides by mixed cultures of bacteria isolated from soil treated with 2-(2-methyl-4-chloro)phenoxypropionic acid

Mixed bacterial cultures capable of using 2-methyl-4-chIorophenoxyacetic acid (MCPA) and 2, 4-dichlorophenoxyacetic acid (2, 4-D) as the sole source of carbon and energy were isolated from field soil treated with the herbicide (±)2-(2-methyl-4-chloro)phenoxypropionic acid (mecoprop). An enrichment technique with two aromatic compounds as sources of carbon was used. Effects of temperature and substrate concentration were studied. The mixed cultures retained their ability to degrade MCPA although the bacteria were grown for 3 months (32 successive passages) with glucose as the sole source of carbon and energy. With benzoic acid as co-substrate, one of the cultures was also able to degrade mecoprop and (±)2-(2, 4-dichloro)phenoxypropionic acid (dichlorprop). This ability was not maintained, however, over more than 10 passages.     

Microbial transformation of kepone.

Pseudomonas aeruginosa strain KO3 and a mixed aerobic enrichment culture, isolated from sewage sludge lagoon water, were found to aerobically transform the chlorinated insecticide Kepone, yielding monohydro-Kepone. Identification of the product was confirmed by gas chromatography and electron impact mass spectrometry. The mixed culture and P. aeruginosa strain KO3 produced about 4 and 16%, respectively, dihydro-Kepone, determined by cochromatography using authentic standards. Reduced amounts of monohydro-Kepone, compared with the mixed and pure cultures, were produced by James River sediment microorganisms. Kepone was not utilized as a sole carbon or energy source by any of the bacteria or mixed cultures examined in this study.     

Characterization of <Emphasis Type="Italic">Rhodococcus wratislaviensis</Emphasis> strain J3 that degrades 4-nitrocatechol and other nitroaromatic compounds

The bacterial strain J3 was isolated from soil by selective enrichment on mineral medium containing 4-nitrocatechol as the sole carbon and energy source. This strain was identified as Rhodococcus wratislaviensis on the basis of morphology, biochemical, physiological and chemotaxonomic characterization and complete sequencing of the 16S rDNA gene. The isolated bacterium could utilize 4-nitrocatechol, 3-nitrophenol and 5-nitroguaiacol as sole carbon and energy sources. Stoichiometric release of nitrites was measured during degradation of 4-nitrocatechol both in growing cultures and for stationary phase cells. The J3 strain was unable to degrade 4-nitroguaiacol, 2-nitrophenol, 4-nitrophenol, 2,4-dinitrobenzoic acid, 4,5-dimethoxy-2-nitrobenzoic acid and 2,3-difluoro-6-nitrophenol. The J3 strain is deposited in the Czech Collection of Microorganisms as CCM 4930.     

Production of single-cell protein with cellulomonas sp. on hempstalk wastes

Strains of Cellulomonas isolated from enrichment cultures with cellulose powder were cultivated with hempstalk wastes as the sole carbon source in a mineral medium. The yields of protein were not significantly different using the crude or alkali pretreated substrate in flasks experiments. Up to 12.5% protein was obtained on the untreated substrate with the best strain cultivated in a small fermenter.     

Yeast species utilizing uric acid,adenine,n-alkylamines or diamines as sole source of carbon and energy

Yeast strains utilizing uric acid, adenine, monoamines or diamines as sole source of carbon and energy were isolated from several soil samples by the enrichment culture method. The most common species wasTrichosporon cutaneum. Strains ofCandida catenulata, C. famata, C. parapsilosis, C. rugosa, Cryptococcus laurentii, Stephanoascus ciferrii andTr. adeninovorans were also isolated. All strains utilizing uric acid as sole carbon source utilized some primaryn-alkyl-l-amines hydroxyamines or diamines as well. The ascomycetous yeast strains showing these characteristics all belonged to species known to assimilate hydrocarbons. Type strains of hydrocarbon-positive yeast species which were not found in the enrichment cultures generally assimilated putrescine, some type strains also butylamine or pentylamine, but none assimilated uric acid. Methanol-positive species were not isolated. Type strains of methanol-positive and of hydrocarbon-negative species did not assimilate uric acid, butylamine or putrescine. Assimilation of putrescine as sole source of carbon and energy may be a valuable diagnostic criterion in yeast taxonomy.