Different Ionic Specificities of ATP Synthesis in Extremely Alkaliphilic Sulfate-Reducing and Acetogenic Bacteria

Ionic specificity of oxidative phosphorylation was studied in Natroniella acetigenaand Desulfonatronum lacustre, which are new alkaliphilic anaerobes that were isolated from soda lakes and have a pH growth optimum of 9.5–9.7. The ability of their cells to synthesize ATP in response to the imposition of artificial pH⁺and pNa⁺gradients was studied. As distinct from other marine and freshwater sulfate reducers and extremely alkaliphilic anaerobes, D. lacustreuses a Na⁺-translocating ATPase for ATP synthesis. The alkaliphilic acetogen N. acetigena, which develops at a much higher Na⁺concentration in the medium, generated primary pH⁺for ATP synthesis. Thus, the high Na⁺concentrations and alkaline pH values typical of soda lakes do not predetermine the type of bioenergetics of their inhabitants.     

Comparative Study of the Energy Metabolism of Anaerobic Alkaliphiles from Soda Lakes

We investigated the influence of inhibitors of energy metabolism and ionophores on the growth and formation of metabolic products in alkaliphilic anaerobes characterized by various catabolism types. It was shown that blockage of oxidative phosphorylation by the addition of N,N-dicyclohexylcarbodiimide (DCCD), an inhibitor of F₁F₀ ATP synthase, resulted in a complete arrest of the growth of the acetogenic bacterium Tindallia magadiensis with arginine as an electron acceptor. In the presence of pyruvate, substrate-level phosphorylation occurred. The methylotrophic methanogenic archaebacterium Methanosalsus zhilinae did not grow with DCCD and vanadate, an inhibitor of ₁₂ ATPase, suggesting the presence of two ATPase types in this species. In the saccharolytic alkaliphiles Halonatronum saccharophilum, Amphibacillus tropicus, and Spirochaeta alkalica (which are characterized by different pH optima), the contribution of the H⁺ gradient to the energy metabolism and, presumably, to the maintenance of the intracellular pH level decreased with an increase in the degree of alkaliphily. Based on the data of an inhibitor assay using protonophores, monensin, and amiloride, we suggest that all of the bacteria tested depend on H⁺and Na⁺ gradients. The Na⁺/H⁺ antiport appears to be a universal mechanism of regulating the intracellular pH level and the interaction between the Na⁺ and H⁺ cycles in bacterial cells cultivated under alkaline conditions.     

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) degradation by <Emphasis Type="Italic">Acetobacterium paludosum</Emphasis>

Substrates and nutrients are often added to contaminated soil or groundwater to enhance bioremediation. Nevertheless, this practice may be counterproductive in some cases where nutrient addition might relieve selective pressure for pollutant biodegradation. Batch experiments with a homoacetogenic pure culture of Acetobacterium paludosum showed that anaerobic RDX degradation is the fastest when auxiliary growth substrates (yeast extract plus fructose) and nitrogen sources (ammonium) are not added. This bacterium degraded RDX faster under autotrophic (H₂-fed) than under heterotrophic conditions, even though heterotrophic growth was faster. The inhibitory effect of ammonium is postulated to be due to the repression of enzymes that initiate RDX degradation by reducing its nitro groups, based on the known fact that ammonia represses nitrate and nitrite reductases. This observation suggests that the absence of easily assimilated nitrogen sources, such as ammonium, enhances RDX degradation. Although specific end products of RDX degradation were not determined, the production of nitrous oxide (N₂O) suggests that A. paludosum cleaved the triazine ring.     

Using electron balances and molecular techniques to assess trichoroethene-induced shifts to a dechlorinating microbial community

This study demonstrated the utility in correlating performance and community structure of a trichloroethene (TCE)-dechlorinating microbial consortium; specifically dechlorinators, fermenters, homoacetogens, and methanogens. Two complementary approaches were applied: predicting trends in the microbial community structure based on an electron balance analysis and experimentally assessing the community structure via pyrosequencing and quantitative polymerase chain reaction (qPCR). Fill-and-draw reactors inoculated with the DehaloR^2 consortium were operated at five TCE-pulsing rates between 14 and 168 μmol/10-day-SRT, amended with TCE every 2 days to give peak concentrations between 0.047 and 0.56 mM (6-74 ppm) and supplied lactate and methanol as sources of e(-) donor and carbon. The complementary approaches demonstrated the same trends: increasing abundance of Dehalococcoides and Geobacter and decreasing abundance of Firmicutes with increasing TCE pulsing rate, except for the highest pulsing rate. Based on qPCR, the abundance of Geobacter and Dehalococcoides decreased for the highest TCE pulsing rate, and pyrosequencing showed this same trend for the latter. This deviation suggested decoupling of Dehalococcoides growth from dechlorination. At pseudo steady-state, methanogenesis was minimal for all TCE pulsing rates. Pyrosequencing and qPCR showed suppression of the homoacetogenic genera Acetobacterium at the two highest pulsing rates, and it was corroborated by a decreased production of acetate from lactate fermentation and increased propionate production. Suppression of Acetobacterium, which can provide growth factors to Dehalococcoides, may have contributed to the decoupling for the highest TCE-pulsing rate.     

Thermodynamics of methylenetetrahydrofolate reduction to methyltetrahydrofolate and its implications for the energy metabolism of homoacetogenic bacteria

The thermodynamics of the methylenetetrahydrofolate reduction to 5-methyltetrahydrofolate was studied with the methylenetetrahydrofolate reductase purified from the homoacetogenic bacterium Peptostreptococcus productus. The equilibrium constants were determined for the forward and backward reactions of methylenetetrahydrofolate reduction with NADH or acetylpyridine adenine dinucleotide (APADH), respectively, as the electron donors. From the equilibrium constants and the known standard redox potentials at pH 7 (E ^o ) of the couples NAD⁺/NADH or APAD⁺/APADH the E ^o of the couple methylene-/methyltetrahydrofolate was determined to be about-200mV. This value is different from values reported before for this couple. The implications for the mechanism of energy conservation of homoacetogens is discussed.Abbreviations FH₄ tetrahydrofolate - CH₂=FH₄ 5,10-methylenetrahydrofolate - CH₃-FH₄ 5-methyltetrahydrofolate - K _eq equilibrium constant - G ^o Gibb's free energy change under standard conditions (all concentrations of reactants = 1 M) - G ^o G ^o at pH 7 ([H⁺]=10^-7 M) - E ^o standard redox potential - G ^o standard redox potential difference of two reactants - E ^o E ^o at pH 7 - R gas constant - F Faraday constant - APAD acetylpyridine adenine dinucleotide (NAD⁺-analogue)     

Effect of cobalt on the anaerobic thermophilic conversion of methanol

The importance of cobalt on the anaerobic conversion of methanol under thermophilic conditions was studied in three parallel lab-scale UASB-reactors and in cobalt-limited enriched cultures. Reactors R1, R2, and R3 were fed with methanol in a bicarbonate-buffered medium, supplied with iron and macronutrients: in R1 all metals were supplied (control), R2 was cobalt deprived, and in R3 all metals were deprived. In the 136 days of continuous experiment, a drop in performance was observed over the last 30 days. Particularly in R3, both methanol removal and methane formation dropped by 7.1% and 13.7%, respectively, compared to the control reactor, R1. When the medium was cobalt-deprived, acetate was not produced and, as a consequence, the enriched consortium lost its capacity to degrade acetate, indicating that the acetotrophic microorganisms were washed out. The addition of 0.5 microM of cobalt to a cobalt-deprived enrichment culture led to acetate accumulation. The results obtained in this study indicate that the mixed consortium requires a proper amount of cobalt, and its addition to a concentration of 0.1 microM leads to the highest methanol conversion rate, with methane as the sole end product from methanol.     

Elemental Composition of Extremely Alkaliphilic Anaerobic Bacteria

The contents of several chemical elements were assessed in the haloalkaliphilic acetogenic bacterium Natroniella acetigena and the alkaliphilic sulfate-reducing bacterium Desulfonatronum lacustre using X-ray microanalysis, stereoscanning microscopy, and mass spectrometry. The organisms were found to differ significantly in their relative contents of S, K, P, and Cl. The P/S ratio in cells of the alkaliphilic bacteria incubated in mineral media at different pH was pH-dependent. With a pH increase from 9 to 10, potassium extrusion from cells was observed, suggesting that secondary K⁺/H⁺ antiport activity accounts for the homeostasis of cytosolic pH. Deenergization of bacterial cells in the presence of inhibitors and ionophores results in specific changes in the P/S ratio, which may be considered an indicator of the cell energetic state. In Natroniella acetigena, the content of intracellular Cl was directly proportional to the NaCl concentration in the medium. Some metals were shown to be necessary for the N. acetigena viability; the requirement for Ni and Co was absolute. Although little demand for Mg was characteristic of the bacteria studied, their growth was stimulated by an increase in Mg concentration, and the cell resistance to lysis was enhanced.