Cometabolic isoterpinolene formation from isolimonene by denitrifying Alcaligenes defragrans

Alcaligenes defragrans strains denitrify on monoterpenes with an unsaturated hydrocarbon structure. A new cometabolic reaction, the formation of isoterpinolene from isolimonene, was detected in cultures that grew on a monoterpene. The biotransformation of isolimonene, a monocyclic monoterpene with a sp³-hybridized C1 atom of the menthane skeleton, contrasts with the complete mineralization of monoterpenes with a sp²-hybridized C1 atom. This selectivity indicates a demand for a sp²-hybridized C1 atom as structural property for monoterpenes that can be oxidized by A. defragrans.     

Anaerobic metabolism of cyclohexanol by denitrifying bacteria

Three strains of denitrifying bacteria were anaerobically enriched and isolated from oxic or anoxic habitats with cyclohexanol or cyclohexanone as sole electron donor and carbon source and with nitrate as electron acceptor. The bacteria were facultatively anaerobic, Gramnegative and metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as terminal electron acceptor. Cyclohexanol and cyclohexanone were degraded both anaerobically and aerobically. Aromatic compounds were oxidized in the presence of molecular oxygen only. One of the bacterial strains was further characterized. During anaerobic cyclohexanol degradation approximately 40% of the substrate was oxidized to phenol, which accumulated as dead-endproduct in the growth medium; 60% of cyclohexanol was completely oxidized to CO₂ and assimilated, respectively. In addition to phenol formation, transient accumulation of cyclohexanone, 2-cyclohexenone and 1,3-cyclohexanedione was observed. Based on these findings we propose a pathway for anaerobic cyclohexanol degradation involving these intermediates.     

Anaerobic degradation of 3-halobenzoates by a denitrifying bacterium

A denitrifying bacterium was isolated from a river sediment after enrichment on 3-chlorobenzoate under anoxic, denitrifying conditions. The bacterium, designated strain 3CB-1, degraded 3-chlorobenzoate, 3-bromobenzoate, and 3-iodobenzoate with stoichiometric release of halide under conditions supporting anaerobic growth by denitrification. The 3-halobenzoates and 3-hydroxybenzoate were used as growth substrates with nitrate as the terminal electron acceptor. The doubling time when growing on 3-halobenzoates ranged from 18 to 25 h. On agar plates with 1 mM 3-chlorobenzoate as the sole carbon source and 30 mM nitrate as the electron acceptor, strain 3CB-1 formed small colonies (1–2 mm in diameter) in 2 to 3 weeks. Anaerobic degradation of both 3-chlorobenzoate and 3-hydroxybenzoate was dependent on nitrate as an electron acceptor and resulted in nitrate reduction corresponding to the stoichiometric values for complete oxidation of the substrate to CO₂. 3-Chlorobenzoate was not degraded in the presence of oxygen. 3-Bromobenzoate and 3-iodobenzoate were also degraded under denitrifying conditions with stoichiometric release of halide, but 3-fluorobenzoate was not utilized by the bacterium. Utilization of 3-chlorobenzoate was inducible, while synthesis of enzymes for 3-hydroxybenzoate degradation was constitutively low, but inducible. Degradation was specific to the position of the halogen substituent, and strain 3CB-1 did not utilize 2- or 4-chlorobenzoate. Received: 6 November 1998 / Accepted: 19 January 1999     

Anaerobic degradation of toluene by pure cultures of denitrifying bacteria

Several denitrifying Pseudomonas spp., isolated with various aromatic compounds, were tested for the ability to degrade toluene in the absence of molecular oxygen. Four out of seven strains were able to degrade toluene in the presence of N₂O. More than 50% of the ¹⁴C from ring-labelled toluene was released as CO₂, and up to 37% was assimilated into cell material. Furthermore it was demonstrated for two strains that they were able to grow on toluene as the sole carbon and energy source in the presence of N₂O. Suspensions of cells pre-grown on toluene degraded toluene, benzaldehyde or benzoate without a lag phase and without accumulation of intermediates. p-Cresol, p-hydroxybenzylalcohol, p-hydroxybenzaldehyde or p-hydroxybenzoate was degraded much slower or only after distinct lag times. In the presence of fluoroacetate [¹⁴C]toluene was transformed to [¹⁴C]benzoate, which suggests that anaerobic toluene degradation proceeds through oxidation of the methyl side chain to benzoate.     

Isolation and characterization of novel halotolerant and/or halophilic denitrifying bacteria with versatile metabolic pathways for the degradation of trimethylamine

Four denitrifying bacteria capable of degrading trimethylamine under both aerobic and denitrifying conditions were newly isolated from coastal sediments and wastewater contaminated by marine water. All strains were in alpha-Proteobacteria. Strain GP43 was classified as a member of genus Paracoccus, and strain PH32, PH34 and GRP21 were novel organisms with remote phylogenetic position from other genus alpha-Proteobacteria. Among these four strains were the halophilic strains PH32, PH34 and GRP21, which did not grow in the absence of sodium chloride in culture medium. Cells grown under denitrifying conditions possessed trimethylamine dehydrogenase while cells grown aerobically possessed two different enzymes for oxidation of trimethylamine, trimethylamine dehydrogenase and trimethylamine monooxygenase. The newly isolated strain PH32, PH34 and GRP21 may be the first halophilic bacteria to degrade trimethylamine under denitrifying conditions.     

Enzyme polymorphisms influencing the metabolism of heterocyclic aromatic amines

Heterocyclic aromatic amines are dietary carcinogens possibly involved in human carcinogenesis, DNA-adduct formation being an obligatory step in this multistage process. Heterocyclic amine binding to DNA largely depends on the balance between metabolic activation and detoxification pathways and DNA repair efficiency. Several genes coding for metabolic enzymes are polymorphic, which affects gene expression and/or enzyme activity. This paper briefly reviews the effect of polymorphisms of activating/detoxifying enzymes on the metabolism of heterocyclic amines. Despite some epidemiological evidence of an association between genetic polymorphisms and susceptibility to cancer possibly resulting from dietary exposure to heterocyclic aromatic amines (HA), the genetic polymorphisms had only slight effects on biomarker levels, suggesting the existence of further unknown factors.     

Formation and analysis of heterocyclic aromatic amine-DNA adducts in vitro and in vivo

The detection and quantification of heterocyclic aromatic amine (HAA)-DNA adducts, critical biomarkers in interspecies extrapolation of toxicity data for human risk assessment, remains a challenging analytical problem. The two main analytical methods currently in use to screen for HAA-DNA adducts are the 32P-postlabeling assay and mass spectrometry, using either accelerated mass spectrometry (AMS) or liquid chromatography and electrospray ionization mass spectrometry (LC-ESI-MS). In this review, the principal methods to synthesize and characterize DNA adducts, and the methods applied to measure HAA-DNA adduct in vitro and vivo are discussed.     

Isolation and characterization of diverse halobenzoate-degrading denitrifying bacteria from soils and sediments

Denitrifying bacteria capable of degrading halobenzoates were isolated from various geographical and ecological sites. The strains were isolated after initial enrichment on one of the monofluoro-, monochloro-, or monobromo-benzoate isomers with nitrate as an electron acceptor, yielding a total of 33 strains isolated from the different halobenzoate-utilizing enrichment cultures. Each isolate could grow on the selected halobenzoate with nitrate as the terminal electron acceptor. The isolates obtained on 2-fluorobenzoate could use 2-fluorobenzoate under both aerobic and denitrifying conditions, but did not degrade other halobenzoates. In contrast, the 4-fluorobenzoate isolates degraded 4-fluorobenzoate under denitrifying conditions only, but utilized 2-fluorobenzoate under both aerobic and denitrifying conditions. The strains isolated on either 3-chlorobenzoate or 3-bromobenzoate could use 3-chlorobenzoate, 3-bromobenzoate, and 2- and 4-fluorobenzoates under denitrifying conditions. The isolates were identified and classified on the basis of 16S rRNA gene sequence analysis and their cellular fatty acid profiles. They were placed in nine genera belonging to either the alpha-, beta-, or gamma-branch of the Proteobacteria, namely, Acidovorax, Azoarcus, Bradyrhizobium, Ochrobactrum, Paracoccus, Pseudomonas, Mesorhizobium, Ensifer, and Thauera. These results indicate that the ability to utilize different halobenzoates under denitrifying conditions is ubiquitously distributed in the Proteobacteria and that these bacteria are widely distributed in soils and sediments.     

Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying pseudomonads

From various oxic or anoxic habitats several strains of bacteria were isolated which in the absence of molecular oxygen oxidized phenol to CO₂ with nitrate as the terminal electron acceptor. All strains grew in defined mineral salts medium; two of them were further characterized. The bacteria were facultatively anaerobic Gramnegative rods; metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as electron acceptor. The isolates were tentatively identified as pseudomonads. Besides phenol many other benzene derivatives like cresols or aromatic acids were anaerobically oxidized in the presence of nitrate. While benzoate or 4-hydroxybenzoate was degraded both anaerobically and aerobically, phenol was oxidized under anaerobic conditions only. Reduced alicyclic compounds were not degraded. Preliminary evidence is presented that the first reaction in anaerobic phenol oxidation is phenol carboxylation to 4-hydroxybenzoate.     

贝壳砂改良土壤中反硝化细菌的分析

【目的】通过对一处经过长期使用贝壳砂进行改良的土壤中的反硝化细菌的多样性和细菌分离分析,研究该土壤中反硝化细菌的组成特征。【方法】采用454焦磷酸测序的方法分析了土壤样品中微生物群落的组成,选用Giltay培养基培养、鉴定从土壤中挑选的分离物的反硝化能力,并对具有反硝化能力的微生物进行了16S rRNA基因鉴定。【结果】该土壤样品中占据优势地位的为Proteobacteria、Acidobacteria、Bacteroidetes、Chloroflexi等门的微生物,属的水平上则有近70%尚未确立分类地位。所分离的细菌中,共得到12株厌氧条件下具有较高硝酸盐去除效率的微生物,分属Pseudomonas、Aeromonas、Serratia和Acinetobacter,均为γ变形菌纲的微生物。【结论】该土壤中具有较高的微生物多样性,包括很多未知类型的微生物和众多类型的反硝化细菌;分离到了11株具有反硝化能力的菌株,可用于该土壤的反硝化过程的进一步研究。     

Initial reactions of anaerobic metabolism of alkylbenzenes in denitrifying and sulfate-reducing bacteria

The initial activation reactions of anaerobic oxidation of the aromatic hydrocarbons toluene and ethylbenzene were investigated in cell extracts of a toluene-degrading, sulfate-reducing bacterium, Desulfobacula toluolica, and in cell extracts of strain EbN1, a denitrifying bacterium capable of degrading toluene and ethylbenzene. Extracts of toluene-grown cells of both species catalysed the addition of fumarate to the methyl group of [phenyl-¹⁴C]-toluene and formed [¹⁴C]-labeled benzylsuccinate. Extracts of ethylbenzene-grown cells of strain EbN1 did not catalyse this reaction, but catalysed the formation of 1-phenylethanol and acetophenone from [methylene-¹⁴C]-ethylbenzene. Toluene-grown cells of D. toluolica and strain EbN1 synthesised highly induced polypeptides corresponding to the large subunits of benzylsuccinate synthase from Thauera aromatica. These polypeptides were absent in strain EbN1 after growth on ethylbenzene, although a number of different polypeptides were highly induced. Thus, formation of benzylsuccinate from toluene and fumarate appears to be the general initiating step in anaerobic toluene degradation by bacteria affiliated with the phylogenetically distinct β-subclass (strain EbN1 and T. aromatica) and δ-subclass (D. toluolica) of the Proteobacteria. Anaerobic ethylbenzene oxidation proceeds via a different pathway involving a two-step oxidation of the methylene group to an alcohol and an oxo group; these steps are most probably followed by a biotin-independent carboxylation reaction and thiolytic cleavage. Received: 16 March 1998 / Accepted: 27 June 1998     

The distribution of denitrifying bacteria in soils monitored by DNA-probing

Abstract DNA probes of dissimilatory NO₂⁻- and N₂O-reductases have been used to screen for the distribution of denitrifying bacteria in soils. In control experiments with known organisms, denitrifiers gave positive DNA-DNA hybridization signals in the dot-blot experiments whereas non-denitrifiers did not hybridize in almost all cases. Bacteria from soil of our institute garden were isolated, grown up and assayed for denitrification activity and for DNA-hybridizations with both gene probes. A correlation of about 75% was obtained between activities and signals. The remainder of the isolates gave a weak signal in the dot blots and only half of them expressed denitrification activities in the cultures. The confidence of about 75% was good enough to assay for the distribution of denitrifying bacteria in five different soil-types of the Düsseldorf area. In all cases, the percentage of bacteria which gave no hybridization signal was high in the plant-free, bulk soil, whereas bacteria with strong or very strong signals favourably associated with the roots of plants isolated from the different soils. The result that denitrifying bacteria predominantly occur at the surface of roots was obtained when the samples were taken both in June and November. The probes for either NO₂⁻- or N₂O-reductase gave essentially the same results. For comparison, a gene probe for nitrogenase was included in this investigation. N₂-fixing bacteria showed a tendency to associate with the roots of plants in some but not in all soils. The overall number of bacteria was much higher at the roots of the plants than in the bulk soil in any of the samples assayed in the present investigation.     

Anaerobic oxidation of the aromatic plant hydrocarbon p-cymene by newly isolated denitrifying bacteria

The capability of nitrate-reducing bacteria to degrade alkyltoluenes in the absence of molecular oxygen was investigated with the three isomers of xylene, ethyltoluene, and isopropyltoluene (cymene) in enrichment cultures inoculated with freshwater mud. Denitrifying enrichment cultures developed most readily (within 4 weeks) with p-cymene, a natural aromatic hydrocarbon occurring in plants, and with m-xylene (within 6 weeks). Enrichment of denitrifiers that utilized m-ethyltoluene and p-ethyltoluene was slow (within 8 and 12 weeks, respectively); no enrichment cultures were obtained with the other alkylbenzenes within 6 months. Anaerobic degradation of p-cymene, which has not been reported before, was studied in more detail. Two new types of denitrifying bacteria with oval cells, strains pCyN1 and pCyN2, were isolated; they grew on p-cymene (diluted in an inert carrier phase) and nitrate with doubling times of 12 and 16 h, respectively. Strain pCyN1, but not strain pCyN2, also utilized p-ethyltoluene and toluene. Both strains grew with some alkenoic monoterpenes structurally related to p-cymene, e.g., α-terpinene. In addition, the isolates utilized p-isopropylbenzoate, and mono- and dicarboxylic aliphatic acids. Determination of the degradation balance of p-cymene and growth with acetate and nitrate indicated the capacity for complete oxidation of organic substrates under anoxic conditions. Adaptation studies with cells of strain pCyN1 suggest the existence of at least two enzyme systems for anaerobic alkylbenzene utilization, one metabolizing p-cymene and p-ethyltoluene, and the other metabolizing toluene. Excretion of p-isopropylbenzoate during growth on p-cymene indicated that the methyl group is the site of initial enzymatic attack. Although both strains were facultatively aerobic, as revealed by growth on acetate under air, growth on p-cymene under oxic conditions was observed only with strain pCyN1. Strains pCyN1 and pCyN2 are closely related to members of the Azoarcus-Thauera cluster within the β-subclass of the Proteobacteria, as revealed by 16S rRNA gene sequence analysis. This cluster encompasses several described denitrifiers that oxidize toluene and other alkylbenzenes. Received: 15 July 1998 / Revision received: 29 July 1999 / Accepted: 2 August 1999     

Anaerobic mineralization of cholesterol by a novel type of denitrifying bacterium

A novel denitrifying bacterium, strain 72Chol, was enriched and isolated under strictly anoxic conditions on cholesterol as sole electron donor and carbon source. Strain 72Chol grew on cholesterol with oxygen or nitrate as electron acceptor. Strictly anaerobic growth in the absence of oxygen was demonstrated using chemically reduced culture media. During anaerobic growth, nitrate was initially reduced to nitrite. At low nitrate concentrations, nitrite was further reduced to nitrogen gas. Ammonia was assimilated. The degradation balance measured in cholesterol-limited cultures and the amounts of carbon dioxide, nitrite, and nitrogen gas formed during the microbial process indicated a complete oxidation of cholesterol to carbon dioxide. A phylogenetic comparison based on total 16S rDNA sequence analysis indicated that the isolated micro-organism, strain 72Chol, belongs to the β2-subgroup in the Proteobacteria and is related to Rhodocyclus, Thauera, and Azoarcus species. Received: 16 July 1996 / Accepted: 5 December 1996     

Thermophilic denitrifying bacteria: A survey of hot springs in Southwestern Iceland

Abstract Samples of water, sediment and bacterial mat from hot springs in Grændalur and Hveragerdi areas in southwestern Iceland were screened at 70°C and 80°C for thermophilic denitrifying bacteria by culturing in anaerobic media containing nitrate or N₂O as the terminal oxidant. The s springs ranged in temperature from 65–100°C and included both neutral (pH 7–8.5) and acidic (pH 2.5–4) types. Nitrate reducing bacteria (nitrate → nitrite) and denitrifiers (nitrate → N₂) were found that grew at 70°C but not at 80°C in nutrient media at pH 8. Samples from neutral springs that were cultured at pH 8 failed to yield a chemolithotrophic, sulfur-oxidizing and nitrate-reducing bacterium, and samples from acidic springs that were cultured at pH 3.5 seemed entirely to lack dissimilatory, nitrate-utilizing bacteria. No sample yielded an organism capable of growth solely by N₂O respiration. The denitrifiers appeared to be Bacillus . Two such Bacillus strains were examined in pure culture and found to exhibit the unusual denitrification phenotype described previously for the mesophile, Pseudomonas aeruginosa , and one other strain of thermophilic Bacillus . The phenotype is characterized by the ability to grow by reduction of nitrate to N₂ with N₂O as an intermediate but a virtual inability to reduce N₂O when N₂O was the sole oxidant.     

Fermentative degradation of putrescine by new strictly anaerobic bacteria

Three strains of new strictly anaerobic, Grampositive, non-sporeforming bacteria were isolated from various anoxic sediment samples with putrescine as sole carbon and energy source. Optimal growth in carbonate-buffered defined medium occurred at 37°C at pH 7.2–7.6. The DNA base ratio of strain NorPut1 was 29.6±1 mol% guanine plus cytosine. In addition to a surface layer and the peptidoglycan layer, the cell wall contained a second innermost layer with a periodic arrangement of subunits. All strains fermented putrescine to acetate, butyrate, and molecular hydrogen; the latter originated from both oxidative putrescine deamination and 4-aminobutyraldehyde oxidation. In defined mixed cultures with methanogens or homoacetogenic bacteria, methane or additional acetate were formed due to interspecies hydrogen transfer. Also 4-aminobutyrate and 4-hydroxybutyrate were fermented to acetate and butyrate, but no hydrogen was released from these substrates. No sugars, organic acids, other primary amines or amino acids were used as substrates. Neither sulfate, thiosulfate, sulfur, nitrate nor fumarate was reduced. Most of the enzymes involved in putrescine degradation could be demonstrated in cell-free extracts. A pathway of putrescine fermentation via 4-aminobutyrate and crotonyl-CoA with subsequent dismutation to acetate and butyrate is suggested.     

Thermophilic denitrifying bacteria: A survey of hot springs in Southwestern Iceland

Abstract Samples of water, sediment and bacterial mat from hot springs in Grændalur and Hveragerdi areas in southwestern Iceland were screened at 70°C and 80°C for thermophilic denitrifying bacteria by culturing in anaerobic media containing nitrate or N₂O as the terminal oxidant. The springs ranged in temperature from 65–100°C and included both neutral (pH 7–8.5) and acidic (pH 2.5–4) types. Nitrate reducing bacteria (nitrate → nitrite) and denitrifiers (nitrate → N₂) were found that grew at 70°C but not at 80°C in nutrient media at pH 8. Samples from neutral springs that were cultured at pH 8 failed to yield a chemolithotrophic, sulfur-oxidizing and nitrate-reducing bacterium, and samples from acidic springs that were cultured at pH 3.5 seemed entirely to lack dissimilatory, nitrate-utilizing bacteria. No sample yielded an organism capable of growth solely by N₂O respiration. The denitrifiers appeared to be Bacillus . Two such Bacillus strains were examined in pure culture and found to exhibit the unusual denitrification phenotype described previously for the mesophile, Pseudomonas aeruginosa , and one other strain of thermophilic Bacillus . The phenotype is characterized by the ability to grow by reduction of nitrate to N₂ with N₂O as an intermediate but a virtual inability to reduce N₂O when N₂O was the sole oxidant.     

Anaerobic degradation of ethylbenzene and other aromatic hydrocarbons by new denitrifying bacteria

Anaerobic degradation of alkylbenzenes with side chains longer than that of toluene was studied in freshwater mud samples in the presence of nitrate. Two new denitrifying strains, EbN1 and PbN1, were isolated on ethylbenzene and n-propylbenzene, respectively. For comparison, two further denitrifying strains, ToN1 and mXyN1, were isolated from the same mud with toluene and m-xylene, respectively. Sequencing of 16SrDNA revealed a close relationship of the new isolates to Thauera selenatis. The strains exhibited different specific capacities for degradation of alkylbenzenes. In addition to ethylbenzene, strain EbN1 utilized toluence, but not propylbenzene. In contrast, propylbenzene-degrading strain PbN1 did not grow on toluene, but was able to utilize ethylbenzene. Strain ToN1 used toluene as the only hydrocarbon substrate, whereas strain mXyN1 utilized both toluene and m-xylene. Measurement of the degradation balance demonstrated complete oxidation of ethylbenzene to CO₂ by strain EbN1. Further characteristic substrates of strains EbN1 and PbN1 were 1-phenylethanol and acetophenone. In contrast to the other isolates, strain mXyN1 did not grow on benzyl alcohol. Benzyl alcohol (also m-methylbenzyl alcohol) was even a specific inhibitor of toluene and m-xylene utilization by strain mXyN1. None of the strains was able to grow on any of the alkylbenzenes with oxygen as electron acceptor. However, polar aromatic compounds such as benzoate were utilized under both oxic and anoxic conditions. All four isolates grew anaerobically on crude oil. Gas chromatographic analysis of crude oil after growth of strain ToN1 revealed specific depletion of toluene.     

Anaerobic oxidation of alkanes by newly isolated denitrifying bacteria

The capacity of denitrifying bacteria for anaerobic utilization of saturated hydrocarbons (alkanes) was investigated with n-alkanes of various chain lengths and with crude oil in enrichment cultures containing nitrate as electron acceptor. Three distinct types of denitrifying bacteria were isolated in pure culture. A strain (HxN1) with oval-shaped, nonmotile cells originated from a denitrifying enrichment culture with crude oil and was isolated with n-hexane (C₆H₁₄). Another strain (OcN1) with slender, rod-shaped, motile cells was isolated from an enrichment culture with n-octane (C₈H₁₈). A third strain (HdN1) with oval, somewhat pleomorphic, partly motile cells originated from an enrichment culture with aliphatic mineral oil and was isolated with n-hexadecane (C₁₆H₃₄). Cells of hexane-utilizing strain HxN1 grew homogeneously in the growth medium and did not adhere to the alkane phase, in contrast to the two other strains. Quantification of substrate consumption and cell growth revealed the capacity for complete oxidation of alkanes under strictly anoxic conditions, with nitrate being reduced to dinitrogen. Received: 3 August / Accepted: 6 October 1999