Genome Analysis and Physiological Comparison of Alicycliphilus denitrificans Strains BC and K601T

The genomes of the Betaproteobacteria Alicycliphilus denitrificans strains BC and K601^T have been sequenced to get insight into the physiology of the two strains. Strain BC degrades benzene with chlorate as electron acceptor. The cyclohexanol-degrading denitrifying strain K601^T is not able to use chlorate as electron acceptor, while strain BC cannot degrade cyclohexanol. The 16S rRNA sequences of strains BC and K601^T are identical and the fatty acid methyl ester patterns of the strains are similar. Basic Local Alignment Search Tool (BLAST) analysis of predicted open reading frames of both strains showed most hits with Acidovorax sp. JS42, a bacterium that degrades nitro-aromatics. The genomes include strain-specific plasmids (pAlide201 in strain K601^T and pAlide01 and pAlide02 in strain BC). Key genes of chlorate reduction in strain BC were located on a 120 kb megaplasmid (pAlide01), which was absent in strain K601^T. Genes involved in cyclohexanol degradation were only found in strain K601^T. Benzene and toluene are degraded via oxygenase-mediated pathways in both strains. Genes involved in the meta-cleavage pathway of catechol are present in the genomes of both strains. Strain BC also contains all genes of the ortho-cleavage pathway. The large number of mono- and dioxygenase genes in the genomes suggests that the two strains have a broader substrate range than known thus far.     

Physiological and phylogenetic characterization of a stable benzene-degrading, chlorate-reducing microbial community

A stable anoxic enrichment culture was obtained that degraded benzene with chlorate as an electron acceptor. The benzene degradation rate was 1.65 mM benzene per day, which is similar to reported aerobic benzene degradation rates but 20-1650 times higher than reported for anaerobic benzene degradation. Denaturing gradient gel electrophoresis of part of the 16S rRNA gene, cloning and sequencing showed that the culture had a stable composition after the seventh transfer. Five bacterial clones were further analyzed. Two clones corresponded to bacteria closely related to Alicycliphilus denitrificans K601. The three other clones corresponded to bacteria closely related to Zoogloea resiniphila PIV-3A2w, Mesorhizobium sp. WG and Stenotrophomonas acidaminiphila. DGGE analysis of cultures grown with different electron donors and acceptors indicated that the bacterium related to Alicycliphilus denitrificans K601 is able to degrade benzene coupled to chlorate reduction. The role of the other bacteria could not be conclusively determined. The bacterium related to Mesorhizobium sp. WG can be enriched with benzene and oxygen, but not with acetate and chlorate, while the bacterium related to Stenotrophomonas acidaminophila grows with acetate and chlorate, but not with benzene and oxygen. As oxygen is produced during chlorate reduction, an aerobic pathway of benzene degradation is most likely.     

Isolation and Characterization of Alicycliphilus denitrificans Strain BC, Which Grows on Benzene with Chlorate as the Electron Acceptor

A bacterium, strain BC, was isolated from a benzene-degrading chlorate-reducing enrichment culture. Strain BC degrades benzene in conjunction with chlorate reduction. Cells of strain BC are short rods that are 0.6 μm wide and 1 to 2 μm long, are motile, and stain gram negative. Strain BC grows on benzene and some other aromatic compounds with oxygen or in the absence of oxygen with chlorate as the electron acceptor. Strain BC is a denitrifying bacterium, but it is not able to grow on benzene with nitrate. The closest cultured relative is Alicycliphilus denitrificans type strain K601, a cyclohexanol-degrading nitrate-reducing betaproteobacterium. Chlorate reductase (0.4 U/mg protein) and chlorite dismutase (5.7 U/mg protein) activities in cell extracts of strain BC were determined. Gene sequences encoding a known chlorite dismutase (cld) were not detected in strain BC by using the PCR primers described in previous studies. As physiological and biochemical data indicated that there was oxygenation of benzene during growth with chlorate, a strategy was developed to detect genes encoding monooxygenase and dioxygenase enzymes potentially involved in benzene degradation in strain BC. Using primer sets designed to amplify members of distinct evolutionary branches in the catabolic families involved in benzene biodegradation, two oxygenase genes putatively encoding the enzymes performing the initial successive monooxygenations (BC-BMOa) and the cleavage of catechol (BC-C23O) were detected. Our findings suggest that oxygen formed by dismutation of chlorite can be used to attack organic molecules by means of oxygenases, as exemplified with benzene. Thus, aerobic pathways can be employed under conditions in which no external oxygen is supplied.     

Chlorate and nitrate reduction in the phototrophic bacteriaRhodobacter capsulatus andRhodobacter sphaeroides

Chlorate or trimethylamine-N-oxide (TMAO) added to phototrophic cultures ofRhodobacter sphaeroides DSM 158 increased both the growth rate and the growth yield although this stimulation was not observed in the presence of tungstate. This strain, exhibited basal activities of nitrate, chlorate, and TMAO reductases independently of the presence of these substrates in the culture medium, and nitrate reductase (NR) activity was competitively inhibited by chlorate. Phototrophic growth ofRhodobacter capsulatus B10, a strain devoid of NR activity, was inhibited only by 100 mM chlorate. However, growth of the nitrate-assimilatingR. capsulatus strains E1F1 and AD2 was sensitive to 10mm chlorate, and their NR activities were not inhibited by chlorate. Both NR and chlorate reductase (CR) activities of strain E1F1 were induced in the presence of nitrate or chlorate respectively, whereas strain AD2 showed basal levels of these activities in the absence of the substrates. A basal TMAO reductase (TR) activity was also observed when these strains ofR. capsulatus were cultured in the absence of this electron acceptor. These results suggest that chlorate and TMAO can be used as ancillary oxidants byRhodobacter strains and that a single enzyme could be responsible for nitrate and chlorate reduction inR. sphaeroides DSM 158, whereas these reactions are catalyzed by two different enzymes inR. capsulatus E1F1 and AD2.     

Anaerobic degradation of benzene, toluene, ethylbenzene, and xylene compounds by Dechloromonas strain RCB

Dechloromonas strain RCB has been shown to be capable of anaerobic degradation of benzene coupled to nitrate reduction. As a continuation of these studies, the metabolic versatility and hydrocarbon biodegradative capability of this organism were investigated. The results of these revealed that in addition to nitrate, strain RCB could alternatively degrade benzene both aerobically and anaerobically with perchlorate or chlorate [(per)chlorate] as a suitable electron acceptor. Furthermore, with nitrate as the electron acceptor, strain RCB could also utilize toluene, ethylbenzene, and all three isomers of xylene (ortho-, meta-, and para-) as electron donors. While toluene and ethylbenzene were completely mineralized to CO2, strain RCB did not completely mineralize para-xylene but rather transformed it to some as-yet-unidentified metabolite. Interestingly, with nitrate as the electron acceptor, strain RCB degraded benzene and toluene concurrently when the hydrocarbons were added as a mixture and almost 92 microM total hydrocarbons were oxidized within 15 days. The results of these studies emphasize the unique metabolic versatility of this organism, highlighting its potential applicability to bioremediative technologies.     

Physiological study and taxonomy of Alcaligenes species: A denitrificans, A. odorans and A faecalis

We have studied 43 strains of the species Alcaligenes dentrificans, A. odorans, and A. faecalis. Twenty-five of them were isolated by enrichment culture on minimal medium containing an organic acid (L-malate, succinate, tartrate, adipate, or itaconate) and N2O as a respiratory electron acceptor. These constitute a single phenon with the A. dentrificans strain type and 9 other strains isolated from clinical specimens. However, strain 4 differs from the other 34 strains in 12 nutritional characters, in its ability to effect a meta cleavage of diphenols, and by the absence of tetrathionate reductase. The percentages of G + C are the following: strains isolated from soil, 66.4 +/- 1.1; collection strains, 67.0 +/- 1.3. The 5 strains of A. odorans differ from the 34 strains of A. denitrificans (not including strain 4) in their inability to denitrify nitrate and use D-saccharate, adipate, pimelate, suberate, beta-hydroxy-beta-methylglutarate meso-tartrate, azelate, and itaconate. Their percentage of G + C is much lower: 56.1 +/- 0.4. From the nutritional point of view the 3 strains of A. faecalis resemble A. dentrificans. However, they differ from the latter by their inability to grow anaerobically on NO3-, NO2-, N2O, and by a slightly lower percentage of G+ C: 64.3 +/- 0.0. The 43 strains synthesize poly-beta-hydroxybutyric acid. None of them is chemolithotrophic.     

Aerobic and anaerobic bacterial respiration monitored by electrodes.

A technique is described by which both oxygen and nitrate (or nitrate or chlorate) levels were continuously monitored during bacterial respiration. Paracoccus (Micrococcus) denitrificans and Escherichia coli oxidizing succinate rapidly ceased to reduce nitrate when oxygen was available, and equally rapidly commenced nitrate reduction when all the oxygen had been consumed. By contrast, membrane vesicles isolated from P. denitrificans reduced oxygen and nitrate simultaneously. The respiratory nitrate reductase in intact cells of P. denitrificans appeared to be inacessible to chlorate present in the reaction medium, and it is suggested that the nitrate reductase is orientated on the plasma membrane so that nitrate gains access from the inner (cytosolic) face.     

Environmental factors influencing nitrate respiration in Escherichia coli K12 and Rhizobium trifolii.

The nitrate respiratory systems of both the facultative anaerobe Escherichia coli K12 strain W3350 and the aerobe Rhizobium trifolii strain T1 are regulated in a similar manner by a complex set of interactions involving H2, N2, CO2, glucose and nitrate. In addition, the nitrate respiratory system of strain T1 can be regulated by chlorate. A possible mechanism is presented to illustrate how these complex interactions might take place.     

Phenotypic and Genotypic Description of Sedimenticola selenatireducens Strain CUZ,a Marine (Per)Chlorate-Respiring Gammaproteobacterium,and Its Close Relative the Chlorate-Respiring Sedimenticola Strain NSS

Two (per)chlorate-reducing bacteria, strains CUZ and NSS, were isolated from marine sediments in Berkeley and San Diego, CA, respectively. Strain CUZ respired both perchlorate and chlorate [collectively designated (per)chlorate], while strain NSS respired only chlorate. Phylogenetic analysis classified both strains as close relatives of the gammaproteobacterium Sedimenticola selenatireducens. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) preparations showed the presence of rod-shaped, motile cells containing one polar flagellum. Optimum growth for strain CUZ was observed at 25 to 30°C, pH 7, and 4% NaCl, while strain NSS grew optimally at 37 to 42°C, pH 7.5 to 8, and 1.5 to 2.5% NaCl. Both strains oxidized hydrogen, sulfide, various organic acids, and aromatics, such as benzoate and phenylacetate, as electron donors coupled to oxygen, nitrate, and (per)chlorate or chlorate as electron acceptors. The draft genome of strain CUZ carried the requisite (per)chlorate reduction island (PRI) for (per)chlorate respiration, while that of strain NSS carried the composite chlorate reduction transposon responsible for chlorate metabolism. The PRI of strain CUZ encoded a perchlorate reductase (Pcr), which reduced both perchlorate and chlorate, while the genome of strain NSS included a gene for a distinct chlorate reductase (Clr) that reduced only chlorate. When both (per)chlorate and nitrate were present, (per)chlorate was preferentially utilized if the inoculum was pregrown on (per)chlorate. Historically, (per)chlorate-reducing bacteria (PRB) and chlorate-reducing bacteria (CRB) have been isolated primarily from freshwater, mesophilic environments. This study describes the isolation and characterization of two highly related marine halophiles, one a PRB and the other a CRB, and thus broadens the known phylogenetic and physiological diversity of these unusual metabolisms.     

Anaerobic respiration and energy conservation in Paracoccus denitrificans. Functioning of iron-sulfur centers and the uncoupling effect of nitrite.

1. Electron paramagnetic resonance spectra at 8-60 K of NADH-reduced membrane particles prepared from Paracoccus denitrificans grown anaerobically with nitrate as terminal electron acceptor show the presence of iron-sulfur centers 1-4 in the NADH-ubiquinone segment of the respiratory chain. In addition resonance lines at g = 2.058, g = 1.953 and g = 1.88 are detectable in the spectra of succinate-reduced membranes at 15 K, which are attributed to the iron-sulfur-containing nitrate reductase. 2. Sulphate-limited growth under anaerobic conditions does not affect the iron-sulfur pattern of NADH dehydrogenase or nitrate reductase. Furthermore respiratory chain-linked electron transport and its inhibition by rotenone are not influenced. These results contrast those observed for sulphate-limited growth of P. denitrificans under aerobic conditions [Eur. J. Biochem. (1977) 81, 267-275]. 3. Proton translocation studies of whole cells indicate that nitrite increases the proton conductance of the cytoplasmic membrane, resulting in a collapse of the proton gradient across the membrane. Nitrite accumulates under anaerobic growth conditions with nitrate as terminal electron acceptor; the extent of accumulation depends on the specific growth conditions. Thus the low efficiencies of respiratory chain-linked energy conservation observed during nitrate respiration [Arch. Microbiol. (1977) 112, 17-23] can be explained by the uncoupling action of nitrite.     

基于响应面法对一株好氧反硝化菌脱氮效能优化

【目的】水体富营养化是当今我国水环境面临的重大水域环境问题,氮素超标排放是主要的引发因素之一。好氧反硝化菌构建同步硝化反硝化工艺比传统脱氮工艺优势更大。获得高效的好氧反硝化菌株并通过生长因子优化使脱氮效率达到最高。【方法】经过序批式生物反应器(Sequencing batch reactor,SBR)的定向驯化,筛选获得高效好氧反硝化菌株,采用响应面法优化好氧反硝化过程影响总氮去除效率的关键因子(碳氮、溶解氧、pH、温度)。【结果】从运行稳定的SBR反应器中定向筛选高效好氧反硝化菌株Pseudomonas T13,采用响应面法对碳氮比、pH和溶解氧关键因子综合优化获得在18 h内最高硝酸盐去除率95%,总氮去除率90%。该菌株的高效反硝化效果的适宜温度范围为25?30 °C;最适pH为中性偏碱;适宜的COD/NO3?-N为4:1以上;最佳溶解氧浓度在2.5 mg/L。【结论】从长期稳定运行的SBR反应器中筛选获得一株高效好氧反硝化菌Pseudomonas T13,硝酸盐还原酶比例占脱氮酶基因的30%以上,通过运行条件优化获得硝氮去除率达到90%以上,对强化废水脱氮工艺具有良好应用价值。     

The periplasmic nitrate reductase in Pseudomonas sp. strain G-179 catalyzes the first step of denitrification

Both membrane-bound and periplasmic nitrate reductases have been found in denitrifying bacteria. Yet the role of periplasmic nitrate reductase in denitrification has not been clearly defined. To analyze the function of the periplasmic nitrate reductase in Pseudomonas sp. strain G-179, the nap gene cluster was identified and found to be linked to genes involved in reduction of nitrite and nitric oxide and anaerobic heme biosynthesis. Mutation in the nap region rendered the cells incapable of growing under anaerobic conditions with nitrate as the alternative electron acceptor. No nitrate reduction activity was detected in the Nap- mutant, but that activity could be restored by complementation with the nap region. Unlike the membrane-bound nitrate reductase, the nitrate reduction activity in strain G-179 was not inhibited by a low concentration of azide. Nor could it use NADH as the electron donor to reduce nitrate or use chlorate as the alternative substrate. These results suggest that the periplasmic nitrate reductase in this strain plays a primary role in dissimilatory nitrate reduction.     

Oxidative phosphorylation in intact chl-r mutants of Escherichia coli K 12.

The efficiency of oxidative phosphorylation was estimated in intact resting cells of Escherichia coli K 12, strain PA 601 (chl-s) and its chl-r mutants, all of them grown anaerobically in the presence of nitrate. The oxidation of endogenous NADH in intact chl-s cells was accompanied by the formation of ATP whatever the terminal electron acceptor, oxygen or nitrate, so that it was possible to conclude that the energy conservation sites are operating with either of the two acceptors in cells grown anaerobically in the presence of nitrate. For chl-r mutants oxidation of endogenous NADH correlated with ATP-production was found only with oxygen as electron acceptor. It is concluded that the energy-conservation sites are preserved in these mutants, the nitrate respiratory chain of which is altered. This assumption is corroborated by the effects of uncouplers of oxidative phosphorylation on ATP-synthesis.     

Isolation of the Acrylamide Denitrifying Bacteria from a Wastewater Treatment System Manufactured with Polyacrylonitrile Fiber

Acrylamide has carcinogenicity and toxicity, so its discharge to natural water and soil systems might have an adverse impact on water quality, endangering public health and welfare. The investigation attempts to isolate acrylamide denitrifying bacteria from a wastewater treatment system manufactured with polyacrylonitrile (PAN) fiber. The goal is to elucidate the effectiveness of isolated pure strain and PAN mixed strains in treating acrylamide from synthetic wastewater. The results reveal that Ralstonia eutropha TDM-3 was isolated from the wastewater treatment system manufactured with PAN fiber. The PAN mixed strains and R. eutropha TDM-3 can consume up to 1446 mg/L acrylamide to denitrify from synthetic wastewater. Complete acrylamide removal depended on the supply of sufficient electron acceptors (nitrate). Strain R. eutropha TDM-3, Azoarcus sp. pF6, Azoarcus sp. T, and Herbaspirillum sp. G8A1 are related closely, according to the phylogenetic analyses of 16S rDNA sequences.     

Anaerobic Degradation of Benzene, Toluene, Ethylbenzene, and Xylene Compounds by Dechloromonas Strain RCB

Dechloromonas strain RCB has been shown to be capable of anaerobic degradation of benzene coupled to nitrate reduction. As a continuation of these studies, the metabolic versatility and hydrocarbon biodegradative capability of this organism were investigated. The results of these revealed that in addition to nitrate, strain RCB could alternatively degrade benzene both aerobically and anaerobically with perchlorate or chlorate [(per)chlorate] as a suitable electron acceptor. Furthermore, with nitrate as the electron acceptor, strain RCB could also utilize toluene, ethylbenzene, and all three isomers of xylene (ortho-, meta-, and para-) as electron donors. While toluene and ethylbenzene were completely mineralized to CO₂, strain RCB did not completely mineralize para-xylene but rather transformed it to some as-yet-unidentified metabolite. Interestingly, with nitrate as the electron acceptor, strain RCB degraded benzene and toluene concurrently when the hydrocarbons were added as a mixture and almost 92 μM total hydrocarbons were oxidized within 15 days. The results of these studies emphasize the unique metabolic versatility of this organism, highlighting its potential applicability to bioremediative technologies.     

Enhanced Toluene Degradation Under Chlorate-Reducing Conditions by Bioaugmentation of Sand Columns with Chlorate- and Toluene-Degrading Enrichments

Chlorate was examined as a potential electron acceptor for enhancing toluene degradation. Most chlorate respiring bacteria (CRB) use nitrate as an electron acceptor, and toluene is known to be degraded under denitrifying conditions. Therefore, it was hypothesized that there would be bacteria that could degrade toluene using chlorate as an electron acceptor, and that chlorate could be used to stimulate toluene degradation. Repeated tests and different approaches in batch tests failed to produce an enrichment capable of toluene degradation supported by chlorate reduction. However, the addition of chlorate increased the overall rate of toluene degradation in bioaugmented columns that were fed chlorate vs. a control column. Toluene removal at an influent toluene concentration of 11 mg/L was 93±5%, which was larger by a factor of 1.95 than toluene removal in a nonbioaugmented control column. Following the discontinued feed of chlorate, toluene removal decreased to 69±4%, demonstrating that chlorate could be used to produce a 1.36-fold increase in toluene removal.     

Isolation and characterization of transposon Tn5 mutants of Rhodobacter sphaeroides deficient in both nitrate and chlorate reduction.

Abstract The wild-type strain Rhodobacter sphaeroides DSM 158 is a nitrate-reducing bacterium with a periplasmic nitrate reductase. Addition of chlorate to the culture medium causes a stimulation of the phototrophic growth, indicating that this strain is able to use chlorate as an ancillary oxidant. Several mutant strains of R. sphaeroides deficient in nitrate reductase activity were obtained by transposon Tn5 mutagenesis. Mutant strain NR45 exhibited high constitutive nitrate and chlorate reductase activities and phototrophic growth was also increased by the presence of chlorate. In contrast, the stimulation of growth by chlorate was not observed in mutant strains NR8 and NR13, in which transposon Tn5 insertion causes the simultaneous loss of both nitrate and chlorate reductase activities. Tn5 insertion probably does not affect molybdenum metabolism since NR8 and NR13 mutants exhibit both xanthine dehydrogenase and nitrogenase activities. These results that a single enzyme could reduce both nitrate and chlorate in R. sphaeroides DSM 158.     

Benzene Degradation at a Site Amended with Nitrate or Chlorate

Aromatic hydrocarbons are widespread in nature and often contribute to the pollution of soils, sediments, and groundwater. The contamination of soil with mobile aromatic compounds, generally termed BTEX (benzene, toluene, ethylbenzene, xylene) is observed at many industrial sites, especially those associated with the petrochemical industry. In situ bioremediation of sites that are contaminated with BTEX can be applied both aerobically and anaerobically. The use of anaerobic in situ bioremediation is advantageous because supply of oxygen is not needed. Nevertheless, anaerobic in situ bioremediation is less commonly used for BTEX contaminated sites. This paper describes push-pull experiments in order to stimulate the degradation of benzene by the addition of nitrate or chlorate. Deuterated benzene was subjected with nitrate-amended groundwater to the aquifer, and the mineralization was traced by the enrichment of deuterium in the groundwater. Nitrate can be used as electron acceptor, and the addition of nitrate at a site in The Netherlands resulted in partial degradation of benzene. This was demonstrated by comparing various push-pull experiments, benzene concentration measurements, stable isotope analyses of benzene and water, and modeling. Chlorate can be used for the in situ production of oxygen, followed by degradation of benzene with oxygen as electron acceptor. The addition of chlorate at the site resulted in the complete removal of benzene demonstrating a complete degradation within 4 weeks. A pull phase was not needed during this run.     

Purification and properties of a dissimilatory nitrate reductase from Haloferax denitrificans.

A membrane-bound nitrate reductase (nitrite:(acceptor) oxidoreductase, EC 1.7.99.4) from the extremely halophilic bacterium Haloferax denitrificans was solubilized by incubating membranes in buffer lacking NaCl and purified by DEAE, hydroxylapatite, and Sepharose 6B gel filtration chromatography. The purified nitrate reductase reduced chlorate and was inhibited by azide and cyanide. Preincubating the enzyme with cyanide increased the extent of inhibition which in turn was intensified when dithionite was present. Although cyanide was a noncompetitive inhibitor with respect to nitrate, nitrate protected against inhibition. The enzyme, as isolated, was composed of two subunits (Mr 116,000 and 60,000) and behaved as a dimer during gel filtration (Mr 380,000). Unlike other halobacterial enzymes, this nitrate reductase was most active, as well as stable, in the absence of salt.     

Nitrate-dependent degradation of acetone by Alicycliphilus and Paracoccus strains and comparison of acetone carboxylase enzymes

A novel acetone-degrading, nitrate-reducing bacterium, strain KN Bun08, was isolated from an enrichment culture with butanone and nitrate as the sole sources of carbon and energy. The cells were motile short rods, 0.5 to 1 by 1 to 2 μm in size, which gave Gram-positive staining results in the exponential growth phase and Gram-negative staining results in the stationary-growth phase. Based on 16S rRNA gene sequence analysis, the isolate was assigned to the genus Alicycliphilus. Besides butanone and acetone, the strain used numerous fatty acids as substrates. An ATP-dependent acetone-carboxylating enzyme was enriched from cell extracts of this bacterium and of Alicycliphilus denitrificans K601(T) by two subsequent DEAE Sepharose column procedures. For comparison, acetone carboxylases were enriched from two additional nitrate-reducing bacterial species, Paracoccus denitrificans and P. pantotrophus. The products of the carboxylase reaction were acetoacetate and AMP rather than ADP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of cell extracts and of the various enzyme preparations revealed bands corresponding to molecular masses of 85, 78, and 20 kDa, suggesting similarities to the acetone carboxylase enzymes described in detail for the aerobic bacterium Xanthobacter autotrophicus strain Py2 (85.3, 78.3, and 19.6 kDa) and the phototrophic bacterium Rhodobacter capsulatus. Protein bands were excised and compared by mass spectrometry with those of acetone carboxylases of aerobic bacteria. The results document the finding that the nitrate-reducing bacteria studied here use acetone-carboxylating enzymes similar to those of aerobic and phototrophic bacteria.