Electron acceptor availability alters carbon and energy metabolism in a thermoacidophile

The thermoacidophilic Acidianus strain DS80 displays versatility in its energy metabolism and can grow autotrophically and heterotrophically with elemental sulfur (S°), ferric iron (Fe³⁺) or oxygen (O₂) as electron acceptors. Here, we show that autotrophic and heterotrophic growth with S° as the electron acceptor is obligately dependent on hydrogen (H₂) as electron donor; organic substrates such as acetate can only serve as a carbon source. In contrast, organic substrates such as acetate can serve as electron donor and carbon source for Fe³⁺ or O₂ grown cells. During growth on S° or Fe³⁺ with H₂ as an electron donor, the amount of CO₂ assimilated into biomass decreased when cultures were provided with acetate. The addition of CO₂ to cultures decreased the amount of acetate mineralized and assimilated and increased cell production in H₂/Fe³⁺ grown cells but had no effect on H₂/S° grown cells. In acetate/Fe³⁺ grown cells, the presence of H₂ decreased the amount of acetate mineralized as CO₂ in cultures compared to those without H₂. These results indicate that electron acceptor availability constrains the variety of carbon sources used by this strain. Addition of H₂ to cultures overcomes this limitation and alters heterotrophic metabolism.     

Isolation and physiological characterization of Syntrophus buswellii strain GA from a syntrophic benzoate-degrading,strictly anaerobic coculture

A stable, syntrophic benzoate-degrading bacterial consortium was enriched from sewage sludge. It oxidized benzoate or 3-phenylpropionate to acetate, H₂ and CO₂. As hydrogen scavengers Methanospirillum hungatei and Desulfovibrio sp. were present. The benzoate-degrading bacteria of this syntrophic culture and of Syntrophus buswelli were able to grow with benzoate/crotonate or crotonate alone in the absence of a hydrogen-utilizing partner organism. If crotonate was the only substrate, acetate and butyrate were produced, while during growth on benzoate or 3-phenylpropionate crotonate served as a reducible co-substrate and was exclusively converted to butyrate. In the presence of crotonate interspecies hydrogen transfer was not necessary as a hydrogen sink. The benzoate degrader was isolated as a pure culture with crotonate as the only carbon source. The pure culture could also grow with benzoate/crotonate or 3-phenylpropionate/crotonate. The effect of high concentrations of crotonate and of acetate or butyrate on growth of the benzoate degrader was investigated. The benzoate degrader was compared with S. buswellii for its morphology, physiology and DNA base composition. Except for the fact that S. buswellii was also able to grow on cinnamate, no differences between the two organisms were detected. The isolate is named S. buswelli, strain GA.     

Neutrophilic, nitrate-dependent, Fe(II) oxidation by a Dechloromonas species

A species of Dechloromonas, strain UWNR4, was isolated from a nitrate-reducing, enrichment culture obtained from Wisconsin River (USA) sediments. This strain was characterized for anaerobic oxidation of both aqueous and chelated Fe(II) coupled to nitrate reduction at circumneutral pH. Dechloromonas sp. UWNR4 was incubated in anoxic batch reactors in a defined medium containing 4.5–5 mM NO₃ ^?, 6 mM Fe²⁺ and 1–1.8 mM acetate. Strain UWNR4 efficiently oxidized Fe²⁺ with 90 % oxidation of Fe²⁺ after 3 days of incubation. However, oxidation of Fe²⁺ resulted in Fe(III)-hydroxide-encrusted cells and loss of metabolic activity, suggested by inability of the cells to utilize further additions of acetate. In similar experiments with chelated iron (Fe(II)-EDTA), encrusted cells were not produced and further additions of acetate and Fe(II)-EDTA could be oxidized. Although members of the genus Dechloromonas are primarily known as perchlorate and nitrate reducers, our findings suggest that some species could be members of microbial communities influencing iron redox cycling in anoxic, freshwater sediments. Our work using Fe(II)-EDTA also demonstrates that Fe(II) oxidation was microbially catalyzed rather than a result of abiotic oxidation by biogenic NO₂ ^?.     

Molecular Assessment of Inoculated and Indigenous Bacteria in Biofilms from a Pilot-Scale Perchlorate-Reducing Bioreactor

Bioremediation of perchlorate-contaminated groundwater can occur via bacterial reduction of perchlorate to chloride. Although perchlorate reduction has been demonstrated in bacterial pure cultures, little is known about the efficacy of using perchlorate-reducing bacteria as inoculants for bioremediation in the field. A pilot-scale, fixed-bed bioreactor containing plastic support medium was used to treat perchlorate-contaminated groundwater at a site in Southern California. The bioreactor was inoculated with a field-grown suspension of the perchlorate-respiring bacterium Dechlorosoma sp. strain KJ and fed groundwater containing indigenous bacteria and a carbon source amendment. Because the reactor was flushed weekly to remove accumulated biomass, only bacteria capable of growing in biofilms in the reactor were expected to survive. After 26 days of operation, perchlorate was not detected in bioreactor effluent. Perchlorate remained undetected by ion chromatography (detection limit 4 μg L⁻¹) during 6 months of operation, after which the reactor was drained. Plastic medium was subsampled from top, middle, and bottom locations of the reactor for shipment on blue ice and storage at −80°C prior to analysis. Microbial community DNA was extracted from successive washes of thawed biofilm material for PCR-based community profiling by 16S-23S ribosomal intergenic spacer analysis (RISA). No DNA sequences characteristic of strain KJ were recovered from any RISA bands. The most intense bands yielded DNA sequences with high similarities to Dechloromonas spp., a closely related but different genus of perchlorate-respiring bacteria. Additional sequences from RISA profiles indicated presence of representatives of the low G+C gram-positive bacteria and the Cytophaga–Flavobacterium–Bacteroides group. Confocal scanning laser microscopy and fluorescence in situ hybridization (FISH) were also used to examine biofilms using genus-specific 16S ribosomal RNA probes. FISH was more sensitive than RISA profiling in detecting possible survivors from the initial inoculum. FISH revealed that bacteria hybridizing to Dechlorosoma probes constituted <1% of all cells in the biofilms examined, except in the deepest portions where they represented 3–5%. Numbers of bacteria hybridizing to Dechloromonas probes decreased as biofilm depth increased, and they were most abundant at the biofilm surface (23% of all cells). These spatial distribution differences suggested persistence of low numbers of the inoculated strain Dechlorosoma sp. KJ in parts of the biofilm nearest to the plastic medium, concomitant with active colonization or growth by indigenous Dechloromonas spp. in the biofilm exterior. This study demonstrated the feasibility of post hoc analysis of frozen biofilms following completion of field remediation studies.     

化能自养硫氧化细菌Halothiobacillus sp.LS2介导的以乙炔为电子受体的硫氧化反应

【目的】探究化能自养硫氧化细菌Halothiobacillus sp. LS2介导的以乙炔为电子受体的厌氧硫氧化反应。【方法】稀释涂布法测定细胞生长情况,离子色谱仪测试硫氧化动力学中SO_4~(2–)和S_2O_3~(2–)以及基于相对荧光定量法的基因表达分析。【结果】尽管菌株LS2在以氧气为电子受体时的最大反应速率V_(max)更高,但在厌氧条件下且以乙炔为电子受体时,菌株LS2的生长量是氧气为电子受体时的2倍,且硫氧化酶基因soxB的表达量显著高于氧气作为电子受体时。【结论】菌株LS2不仅可以以乙炔为电子受体完成厌氧硫氧化反应,且这一代谢过程的产能效率较有氧硫氧化过程更高。本研究首次发现了微生物介导的以乙炔为电子受体的厌氧硫氧化反应,对丰富硫的生物地球化学循环理论有积极意义。     

Sulfide-controlled continuous culture of sulfate-reducing bacteria

In continuous culture set-up for sulfate-reducing bacteria a sulfide electrode (made from silver wire) is used to control the electron donor supply and the medium pump. The sulfied concentration of the medium is kept at a low level by continuosly flushing out H₂S and replacing it with CO₂. The pH is controlled automatically by regulating the CO₂ content of the gas mixture flushed through the medium. With the sulfide-controlled set-up sulfate-reducing bacteria can be grown in chemostat culture under electron donor as well as electron acceptor limitation. Furthermore, by continuously washing out the culture to a preselected residual sulfide concentration, cells can be grown in sulfidostat culture under non-limiting conditions at maximal growth rate. Growth yields of Desulfotmaculum orientis, when growth in this system with hydrogen as electron donor, were considerably higher than previously reported.     

Evidence for a tungsten-stimulated aldehyde dehydrogenase activity of Desulfovibrio simplex that oxidizes aliphatic and aromatic aldehydes with flavins as coenzymes

The aldehyde dehydrogenase activity of the sulfate-reducing bacterium Desulfovibrio simplex strain DSM 4141 was characterized in cell-free extracts. Oxygen-sensitive, constitutive aldehyde dehydrogenase activity was found in cells grown on l(+)-lactate, hydrogen, or vanillin with sulfate as the electron acceptor. A 1.83- to 2.6-fold higher specific activity was obtained in cells grown in media supplemented with 1 μM WO₄ ^2–. The aldehyde dehydrogenase in cell-free extracts catalyzed the oxidation of aliphatic (K _m < 20 μM) and aromatic aldehydes (K _m < 0.32 mM) using methyl viologen as the electron acceptor. Flavins (FMN and FAD) were also active and are proposed to be the natural cofactors, while no activity was obtained with NAD⁺ or NADP⁺. ¹⁸⁵WO₄ ^2– was incorporated in vivo into D. simplex; it was found exclusively in the soluble fraction (≥ 98%). Anionic-exchange chromatography demonstrated coelution of ¹⁸⁵W with two distinct peaks, the first one containing hydrogenase and formate dehydrogenase activities, and the second one aldehyde dehydrogenase activity. Received: 7 February 1997 / Accepted: 6 June 1997     

Hydrogen as an energy source for the human pathogen <Emphasis Type="Italic">Bilophila wadsworthia</Emphasis>

The gram-negative anaerobic gut bacterium Bilophila wadsworthia is the third most common isolate in perforated and gangrenous appendicitis, being also found in a variety of other infections. This organism performs a unique kind of anaerobic respiration in which taurine, a major organic solute in mammals, is used as a source of sulphite that serves as terminal acceptor for the electron transport chain. We show here that molecular hydrogen, one of the major products of fermentative bacteria in the colon, is an excellent growth substrate for B. wadsworthia. We have quantified the enzymatic activities associated with the oxidation of H₂, formate and pyruvate for cells obtained in different growth conditions. The cell extracts present high levels of hydrogenase activity, and up to five different hydrogenases can be expressed by this organism. One of the hydrogenases appears to be constitutive, whereas the others show differential expression in different growth conditions. Two of the hydrogenases are soluble and are recognised by antibodies against a [FeFe] hydrogenase of a sulphate reducing bacterium. One of these hydrogenases is specifically induced during fermentative growth on pyruvate. Another two hydrogenases are membrane-bound and show increased expression in cells grown with hydrogen. Further work should be carried out to reveal whether oxidation of hydrogen contributes to the virulence of B. wadsworthia.     

Effect of bacterial inoculation of strains of pseudomonas aeruginosa,alcaligenes feacalis and bacillus subtilis on germination,growth and heavy metal (cd,cr, and ni) uptake of brassica juncea

Bacterial inoculation may influence Brassica juncea growth and heavy metal (Ni, Cr, and Cd) accumulation. Three metal tolerant bacterial isolates (BCr3, BCd33, and BNi11) recovered from mine tailings, identified as Pseudomonas aeruginosa KP717554, Alcaligenes feacalis KP717561, and Bacillus subtilis KP717559 were used. The isolates exhibited multiple plant growth beneficial characteristics including the production of indole-3-acetic acid, hydrogen cyanide, ammonia, insoluble phosphate solubilization together with the potential to protect plants against fungal pathogens. Bacterial inoculation improved seeds germination of B. juncea plant in the presence of 0.1 mM Cr, Cd, and Ni, as compared to the control treatment. Compared with control treatment, soil inoculation with bacterial isolates significantly increased the amount of soluble heavy metals in soil by 51% (Cr), 50% (Cd), and 44% (Ni) respectively. Pot experiment of B. juncea grown in soil spiked with 100 mg kg^?1 of NiCl₂, 100 mg kg^?1 of CdCl₂, and 150 mg kg^?1 of K₂Cr₂O₇, revealed that inoculation with metal tolerant bacteria not only protected plants against the toxic effects of heavy metals, but also increased growth and metal accumulation of plants significantly. These findings suggest that such metal tolerant, plant growth promoting bacteria are valuable tools which could be used to develop bio-inoculants for enhancing the efficiency of phytoextraction.     

Environmental Stress Determines the Quality of Bacterial Lysate and Its Utilization Efficiency in a Simple Microbial Loop

Heterotrophic bacteria provide the critical link in the microbial loop by converting dissolved organic matter (DOM) into particulate form. In this study, DOM was prepared from recently isolated estuarine bacterial strain Vibrio sp. (DSM14379) grown at different salinities [0.2%, 0.5%, 3%, 5%, or 10% (w/v)], washed, concentrated, and lysed by autoclaving. The corresponding lysate-containing media were designated LM_0.2, LM_0.5, LM₃, LM₅, and LM₁₀. Vibrio sp. cells grown at different salinities had similar C/N/P ratios, but different C/S ratios, different trace element composition, and different 2D gel electrophoresis protein profiles. Pseudoalteromonas sp. (DSM06238) isolated from a similar environment was able to grow on all lysates, and its biomass production was dependent on lysate type. The highest growth rate and biomass production of Pseudoalteromonas sp. at saturation lysate concentrations were observed in LM₃. The biomass production at saturation lysate concentrations was about 3-fold higher as compared to LM_0.2 and LM₁₀. The initial respiration rate, intracellular adenosine triphosphate (ATP) levels, and ³H-Leu and ³H-TdR incorporation rates were lowest in LM₃. On the other hand, in LM_0.2 or LM₁₀ lysates the situation was reversed, the growth rates and biomass production were lowest, whereas ³H-Leu and ³H-TdR incorporation, respiration rates, as well as ATP levels, were highest. These results imply uncoupling of catabolism from growth in either high- or low-salinity lysates. The results also suggest that differences in organic carbon quality generated during Vibrio sp. growth at different NaCl concentrations were propagated through the simple microbial loop, which may have important ecological implications for higher trophic levels that depend on microbial grazing.     

Growth yields of Desulfotomaculum orientis with hydrogen in chemostat culture

Desulfotomaculum orientis (strain Singapore 1) was grown autotrophically with H₂+CO₂ and sulfate, thiosulfate or sulfite as electron acceptor in sulfide- and pH-controlled continuous culture. Under sulfate-limiting conditions real growth yields of up to 9.7 g cell dry mass per mol sulfate were obtained. Electron acceptor limitation resulted in the excretion of up to 14.5 mmol acetate per liter, formed by reduction of CO₂ with H₂. Acetate production was not coupled to an increase of growth yields: under hydrogen-limiting conditions only 1.6 mmol acetate per liter was produced, and even higher growth yields of up to 12,4 g cell dry mass per mol sulfate were obtained. With thiosulfate or sulfite as electron acceptor growth yields increased up to 17.9 g cell dry mass per mol electron acceptor. Growth yields were not simply correlated with the growth rate, and did not allow the determination of maintenance coefficients and the extrapolation to maximal yields at infinite growth rate (Y ^max). The maximal growth rates (_max) with sulfate and thiosulfate were 0.090 and 0.109 h^-1, respectively, if cells were grown continuously in sulfidostat culture under nonlimiting conditions.The net energy yield of sulfate reduction and the energy requirement for the activation of sulfate by Desulfotomaculum orientis are discussed.     

THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM (DINOPHYCEAE) REQUIRES MARINE BACTERIA FOR GROWTH1

Interactions with the bacterial community are increasingly considered to have a significant influence on marine phytoplankton populations. Here we used a simplified dinoflagellate‐bacterium experimental culture model to conclusively demonstrate that the toxic dinoflagellate Gymnodinium catenatum H. W. Graham requires growth‐stimulatory marine bacteria for postgermination survival and growth, from the point of resting cyst germination through to vegetative growth at bloom concentrations (10³ cells · mL^?1). Cysts of G. catenatum were germinated and grown in unibacterial coculture with antibiotic‐resistant or antibiotic‐sensitive Marinobacter sp. DG879 or Brachybacterium sp., and with mixtures of these two bacteria. Addition of antibiotics to cultures grown with antibiotic‐sensitive strains of bacteria resulted in death of the dinoflagellate culture, whereas cultures grown with antibiotic‐resistant bacteria survived antibiotic addition and continued to grow beyond the 21 d experiment. Removal of either bacterial type from mixed‐bacterial dinoflagellate cultures (using an antibiotic) resulted in cessation of dinoflagellate growth until bacterial concentration recovered to preaddition concentrations, suggesting that the bacterial growth factors are used for dinoflagellate growth or are labile. Examination of published reports of axenic dinoflagellate culture indicate that a requirement for bacteria is not universal among dinoflagellates, but rather that species may vary in their relative reliance on, and relationship with, the bacterial community. The experimental model approach described here solves a number of inherent and logical problems plaguing studies of algal‐bacterium interactions and provides a flexible and tractable tool that can be extended to examine bacterial interactions with other phytoplankton species.     

EFFECT OF IRON NUTRITION ON THE MARINE CYANOBACTERIUM SYNECHOCOCCUS GROWN ON DIFFERENT N SOURCES AND IRRADIANCES1

The effects of Fe deficiency on the marine cyanobacterium Synechococcus sp. were examined in batch cultures grown on nitrate or ammonium as a sole nitrogen source under two different irradiances. Fe-stressed cells showed lower chlorophyll a content and cellular C and N quotas. Light limitation increased the critical iron concentration below which both suppression of growth rate and changes in cellular composition were observed. At a limiting irradiance (26 μmol.m⁻².s⁻¹), this critical value was ∼10 nM, a 10 times increase compared to high-light cultures. Moreover, at low light the cellular chlorophyll a concentration was higher than at saturating light (110 μmol.m⁻².s⁻¹), this difference being most pronounced under Fe-stressed conditions. Cells grown on ammonium showed a lower half-saturation constant for Fe (K_s) compared to cells grown on nitrate, indicating Synechococcus sp. has the ability to grow faster on ammonium than on nitrate in a low Fe environment at high light. Consequently, in high-nutrient and low-chlorophyll regions where Fe limits new production, cyanobacteria most likely grow on regenerated ammonium, which requires less energy for assimilation. The K_s for growth on Fe at low light was significantly higher than at high light compared with the cells grown on the same N source, suggesting the cells require more Fe at low light. Therefore, if cells that are already Fe-limited also become light-limited, their iron stress level will increase even more. For cyanobacteria this is the first report of a study combining the interactions of Fe limitation, light limitation, and nitrogen source (NO₃⁻ vs. NH₄⁺).     

Incorporation of [14C]Methanol and [14C]Bicarbonate by Hyphomicrobium sp. 53-49 under Various Growth Conditions: Aerobic,Denitrifying and Autotrophic

A study was made of the incorporation of methanol and bicarbonate into the cell constituents of denitrifying or aerobic methanol grown and autotrophic H₂–O₂–CO₂ grown Hyphomicrobium sp. 53-49. Cells were incubated with [¹⁴C]methanol or [¹⁴C]bicarbonate, and the distribution of the radioactivity in the nonvolatile constituents of ethanol extracts of cells was examined. When denitrifying grown cells were incubated with [¹⁴C]methanol, the major part of the radioactivity was fixed to serine as the first stable compound. Aerobic methanol grown cells also fixed [¹⁴C]methanol mainly to serine. These results suggest that methanol grown cells assimilate methanol by the serine pathway. When denitrifying or aerobic methanol grown cells were incubated with [¹⁴C]bicarbonate, malate was mainly observed as a nonvolatile compound in the initial period of the incubation. Autotrophic grown cells also fixed the major part of [¹⁴C]bicarbonate to malate. In this case, phosphoglyceric acid was found in the phosphorylated compounds area.     

Chemolithotrophic growth ofDesulfovibrio desulfuricans with hydrogen coupled to ammonification of nitrate or nitrite

Two of nine sulfate reducing bacteria tested,Desulfobulbus propionicus andDesulfovibrio desulfuricans (strain Essex 6), were able to grow with nitrate as terminal electron acceptor, which was reduced to ammonia. Desulfovibrio desulfuricans was grown in chemostat culture with hydrogen plus limiting concentrations of nitrate, nitrite or sulfate as sole energy source. Growth yields up to 13.1, 8.8 or 9.7 g cell dry mass were obtained per mol nitrate, nitrite or sulfate reduced, respectively. The apparent half saturation constants (K _s) were below the detection limits of 200, 3 or 100 mol/l for nitrate, nitrite of sulfate, respectively. The maximum growth rates {ie63-1} raised from 0.124 h^-1 with sulfate and 0.150 h^-1 with nitrate to 0.193 h^-1 with nitrite as electron acceptor. Regardless of the electron acceptor in the culture medium, cell extracts exhibited absorption maxima corresponding to cytochromec and desulfoviridin. Nitrate reductase was found to be inducible by nitrate or nitrite, whereas nitrite reductase was synthesized constitutively. The activities of nitrate and nitrite reductases with hydrogen as electron donor were 0.2 and 0.3 mol/min·mg protein, respectively. If limiting amounts of hydrogen were added to culture bottles with nitrate as electron acceptor, part of the nitrate was only reduced to the level of nitrite. In media containing nitrate plus sulfate or nitrite plus sulfate, sulfate reduction was suppressed.The results demonstrate that the ammonification of nitrate or nitrite can function as sole energy conserving process in some sulfate-reducing bacteria.     

Description of the novel perchlorate-reducing bacteria Dechlorobacter hydrogenophilus gen. nov., sp. nov. and Propionivibrio militaris, sp. nov.

Novel dissimilatory perchlorate-reducing bacteria (DPRB) were isolated from enrichments conducted under conditions different from those of all previously described DPRB. Strain LT-1^T was enriched using medium buffered at pH 6.6 with 2-(N-morpholino)ethanesulfonic acid (MES) and had only 95% 16S rRNA gene identity with its closest relative, Azonexus caeni. Strain MP^T was enriched in the cathodic chamber of a perchlorate-reducing bioelectrical reactor (BER) and together with an additional strain, CR (99% 16S rRNA gene identity), had 97% 16S rRNA gene identity with Propionivibrio limicola. The use of perchlorate and other electron acceptors distinguished strains MP^T and CR from P. limicola physiologically. Strain LT-1^T had differences in electron donor utilization and optimum growth temperatures from A. caeni. Strains LT-1^T and MP^T are the first DPRB to be described in the Betaproteobacteria outside of the Dechloromonas and Azospira genera. On the basis of phylogenetic and physiological features, strain LT-1^T represents a novel genus in the Rhodocyclaceae; strain MP^T represents a novel species within the genus Propionivibrio. The names Dechlorobacter hydrogenophilus gen. nov., sp. nov and Propionivibrio militaris sp. nov. are proposed.     

Growth yields and energy generation by Campylobacter sputorum subspecies bubulus during growth in continuous culture with different hydrogen acceptors

Campylobacter sputorum subspecies bubulus was grown in continuous culture with excess of l-lactate or formate, and growth-limiting amounts of oxygen, fumarate, nitrate or nitrite. l-Lactate was oxidized to acetate, fumarate was reduced to succinate, and nitrate and nitrite were reduced to ammonia. The Y _lactate values (g dry weight bacteria/g mol lactate) for the respective hydrogen acceptors were much higher than the Y _formate values. Steady state cultures on formate and nitrite could only be obtained at a low dilution rate and low nitrite concentrations in the growth medium. In H⁺/2e measurements with lactate-grown cells proton ejections were observed with lactate or pyruvate as a hydrogen donor, and oxygen or hydrogen peroxide as a hydrogen acceptor. Proton ejection was also observed with pyruvate and nitrate. Proton ejection did not occur with lactate and nitrate, neither with lactate or pyruvate and fumarate or nitrite. With formate as a hydrogen donor acidification occurred with all hydrogen acceptors mentioned. It has been concluded that during growth on lactate and fumarate or nitrite substrate level phosphorylation at acetate formation is the sole ATP-generating system. Growth on formate and fumarate or nitrite is explained by a proton gradient generated as a result of oxidation of formate at the periplasmic side of the cytoplasmic membrane. With oxygen and nitrate additional ATP is formed by electron transport-linked phosphorylation. The low molar growth yields with formate are explained by the observation that formate-grown cells had a great permeability to protons.Abbreviations H⁺/2e value number of protons ejected per electron pair transported in the respiratory system - P/2e value mol of ATP formed per electron pair transported in the respiratory system - CCCP carbonyl cyanide m-chlorophenyl-hydrazone     

Influence of electron acceptor,carbon, nitrogen,and phosphorus on polyhydroxyalkanoate (PHA) production by <Emphasis Type="Italic">Brachymonas</Emphasis> sp. P12

Polyhydroxyalkanoates (PHAs), intracellular carbon and energy reserve compounds in many bacteria, have been used extensively in biodegradable plastics. PHA formation is influenced by nutrient limitations and growth conditions. To characterize the PHA accumulation in a new denitrifying phosphorus-removing bacterium Brachymonas sp. P12, batch experiments were conducted in which the electron acceptor (oxygen or nitrate) was varied and different concentrations of carbon (acetate), nitrogen (NH₄Cl), and phosphorus (KH₂PO₄) were used. Polyhydroxybutyrate (PHB) was the dominant product during PHA formation when acetate was the sole carbon source. The PHB content of aerobically growing cells increased from 431 to 636 mg PHB g⁻¹ biomass, but the PHB concentration of an anoxic culture decreased (−218 mg PHB g⁻¹ biomass), when PHB was utilized simultaneously with acetate as an electron donor for anoxic denitrification. The specific PHB production rate of the carbon-limited batch, 158.2 mg PHB g⁻¹ biomass h⁻¹, was much greater than that of batches with normal or excess carbon. The effects of phosphorus and nitrogen concentrations on PHB accumulation were clearly less than the effect of carbon concentration. According to the correlation between the specific PHB production rate and the specific cell growth rate, PHB accumulation by Brachymonas sp. P12 is enhanced by nutrient limitation, is growth-associated, and provides additional energy for the biosynthesis of non-PHB cell constituents to increase the cell growth rate beyond the usual level.     

Isolation,Characterization and Kinetics of Perchlorate Reducing Magnetospirillum Species

Perchlorate reducing bacteria reduce perchlorate to chlorate (ClO₃?), which, in turn, is reduced to chlorite (ClO₂?) and ultimately to chloride (Cl^?). Magnetospirillum strains are reported to use chlorate/perchlorate as electron acceptors. This study describes the perchlorate reducing property of strain VITRJS5, a Magnetopsirillum isolated from freshwater sediment collected from Chelur freshwater lake, Kerala, India. The strain was microaerophile and was phylogenetically related to a Magnetospirillum sp., a member of the α-subclass of the class Proteobacteria. The placement of the isolate in the genus Magnetospirillum has further confirmed the presence of four key magnetosome membrane genes. PCR amplification and phylogenetic analysis of central metabolic genes such as nifH (nitrogenase) and cbbM (type II RubisCo) displayed the highest similarity (97% and 81%, respectively) with Magnetospirillum sp. BB-1 The growth kinetic parameters of the isolate were studied with acetate as the electron donor in batch experiments. Monod's substrate utilization model has been established with oxygen, nitrate and perchlorate as electron acceptors separately. The maximum specific growth rate (µ_max) and half-saturation constant (k_s^conc) for the bacterium varied while utilizing different electron acceptors. The maximum specific growth rate was 0.226, 0.190 and 0.096 per hour and half-velocity constant K_s was 25.09, 33.36 and 65.37 mg acetate/l for oxygen, nitrate and perchlorate, respectively. The reduction of perchlorate has been analyzed using kinetic studies of the substrate uptake by the bacteria and the half-velocity constant K_s was found to be 52.8 mg/l. The results indicate that the strain VITRJS5 effectively reduces perchlorate by using it as an electron acceptor.     

Metabolism of 2,4,6-trinitrotoluene (TNT) by Desulfovibrio sp. (B strain)

A sulfate-reducing bacterium, Desulfovibrio sp. (B strain) isolated from an anaerobic reactor treating furfural-containing waste-water was studied for its ability to metabolize trinitrotoluene (TNT). The result showed that this isolate could transform 100 ppm TNT within 7 to 10 days of incubation at 37°C, when grown with 30 mm pyruvate as the primary carbon source and 20 mm sulfate as electron acceptor. Under these conditions, the main intermediate produced was 2,4-diamino-6-nitrotoluene. Under culture conditions where TNT served as the sole source of nitrogen for growth with pyruvate as electron donor and sulfate as electron acceptor, TNT was first converted to 2,4-diamino-6-nitrotoluene within 10 days of incubation. This intermediate was further converted to toluene by a reductive deamination process via triaminotoluene. Apart from pyruvate, various other carbon sources such as ethanol, lactate, formate and H₂ + CO₂ were also studied as potential electron donors for TNT metabolism. The rate of TNT biotransformation by Desulfovibrio sp. (B strain) was compared with other sulfate-reducing bacteria and the results were evaluated. This new strain may be useful in decontaminating TNT-contaminated soil and water under anaerobic conditions in conjunction with toluene-degrading denitrifiers (Pseudomonas spp.) or toluene-degrading sulfate reducers in a mixed culture system. Correspondence to: R. Boopathy