Effect of pH on aluminum-driven methanogenesis by Methanococcus thermolithotrophicus

Methanogenesis from various elemental metals as electron sources has been demonstrated before. In this study, we have examined the influence of pH on the methanogenic activity of Methanococcus thermolithotrophicus dependent on cathodic hydrogen produced by elemental aluminum wires. When grown on H₂+CO₂, M. thermolithotrophicus had an optimum pH of 6.2, but when all the H₂ was supplied from A1°, the pH optimum was 5.7, consistent with thermodynamic predictions. The results also indicated that aluminum is quite resistant to anaerobic corrosion when compared to iron, most likely due to adhesion of aluminum oxide or hydroxide layers on the surface of the wires. Correspondence to: R. Boopathy     

The effects of substrate composition, quantity, and diversity on microbial activity

Variation in organic matter inputs caused by differences in plant community composition has been shown to affect microbial activity, although the mechanisms controlling these effects are not entirely understood. In this study we determine the effects of variation in substrate composition, quantity, and diversity on soil extracellular enzyme activity and respiration in laboratory microcosms. Microbial respiration responded predictably to substrate composition and quantity and was maximized by the addition of labile substrates and greater substrate quantity. However, there was no effect of substrate diversity on respiration. Substrate composition significantly affected enzyme activity. Phosphatase activity was maximized with addition of C and N together, supporting the common notion that addition of limiting resources increases investment in enzymes to acquire other limiting nutrients. Chitinase activity was maximized with the addition of chitin, suggesting that some enzymes may be stimulated by the addition of the substrate they degrade. In contrast, activities of glucosidase and peptidase were maximized by the addition of the products of these enzymes, glucose and alanine, respectively, for reasons that are unclear. Substrate diversity and quantity also stimulated enzyme activity for three and four of the six enzymes assayed, respectively. We found evidence of complementary (i.e., non-additive) effects of additions of different substrates on activity for three of the six enzymes assayed; for the remaining enzymes, effects of adding a greater diversity of substrates appeared to arise from the substrate-specific effects of those substrates included in the high-diversity treatment. Finally, in a comparison of measures of microbial respiration and enzyme activity, we found that labile C and nutrient-acquiring enzymes, not those involved in the degradation of recalcitrant compounds, were the best predictors of respiration rates. These results suggest that while composition, quantity, and diversity of inputs to microbial communities all affect microbial enzyme activity, the mechanisms controlling these relationships are unique for each particular enzyme.     

Effect of substrate concentration,inorganic nitrogen,O2 concentration,temperature and pH on dehydrogenase activity in soil

Summary Dehydrogenase activity was measured in a sandy loam soil under a variety of incubation conditions using the reduction of 2-(p-iodophenyl-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to iodonitrotetrazolium formazan (INT-formazan). There was a high positive correlation between dehydrogenase activity and substrate concentration, incubation temperature, and soil pH. Dehydrogenase activity also displayed a high negative correlation with O₂ concentrations. Ammonium sulfate at concentrations from 40 to 120 g/g soil had no significant effect on dehydrogenase activity. However, at concentrations of 160 and 200 g/g, dehydrogenase activity was significantly reduced. Potassium nitrate at concentrations ranging from 40 to 200 g/g had no significant effect on soil dehydrogenase activity, whereas sodium nitrite significantly inhibited activity at concentrations of 120 and 160 g/g soil.     

The effect of pH on ruminal methanogenesis

Abstract: When a fistulated cow was fed an all forage diet, ruminal pH remained more or less constant (6.7 to 6.9). The ruminal pH of a concentrate-fed cow decreased dramatically in the period soon after feeding, and the pH was as low as 5.45. Mixed ruminal bacteria from the forage-fed cow converted CO₂ and H₂ to methane, but the ruminal fluid from the concentrate-fed cow did not produce methane. When the pH of the ruminal fluid from the concentrate-fed cow was adjusted to pH 7.0, methane was eventually detected, and the absolute rate constant of methane production was as high as the one observed with ruminal fluid from the forage fed cow (0.32 h⁻¹). Based on the zero-time intercepts of methane production, it appeared that the concentrate-fed cow had fewer methanogens than the forage-fed cow. When the mixed ruminal bacteria were incubated in a basal medium containing 100 mM acetate, methanogenesis was pH-dependent, and no methane was detected at pH values less than 6.0. Because the removal of acetic acid completely reversed the inhibition of methanogenesis, it appeared that volatile fatty acids were causing the pH-dependent inhibition. Based on these results, concentrate diets that lower ruminal pH may provide a practical means of decreasing ruminal methane production.     

Effect of sulfate on carbon and electron flow during microbial methanogenesis in freshwater sediments.

The effect of sulfate on methane production in Lake Mendota sediments was investigated to clarify the mechanism of sulfate inhibition of methanogenesis. Methanogenesis was shown to be inhibited by the addition of as little as 0.2 mM sulfate. Sulfate inhibition was reversed by the addition of either H2 or acetate. Methane evolved when inhibition was reversed by H2 additions was derived from 14CO2. Conversely, when acetate was added to overcome sulfate inhibition, the evolved methane was derived from [2-14C]acetate. A competition for available H2 and acetate was proposed as the mechanism by which sulfate inhibited methanogenesis. Acetate was shown to be metabolized even in the absence of methanogenic activity. In the presence of sulfate, the methyl position of acetate was converted to CO2. The addition of sulfate to sediments did not result in the accumulation of significant amounts of sulfide in the pore water. Sulfate additions did not inhibit methanogenesis unless greater than 100 mug of free sulfide per ml was present in the pore water. These results indicate that carbon and electron flow are altered when sulfate is added to sediments. Sulfate-reducing organisms appear to assume the role of methanogenic bacteria in sulfate-containing sediments by utilizing methanogenic precursors.     

Effects of urine on soil microbial biomass,methanogenesis, nitrification and denitrification in grassland soils

Urine was added under controlled conditions to intact turfs taken from long-term permanent pasture on clay loam and sandy loam soils in South West England. Methane exchanges were small (<+/−0.03 μg CH₄ m^-2 min^-1) and overall absorption equalled or exceeded emission in both soils. On the clay loam, wetting with water or urine increased soil microbial biomass C and N contents by about 20% but there was no specific effect of urine. Urine, however, caused an increase in soil respiration of >50% and the average increase was greater for cow's urine (30.8 mg CO₂ m^-2 min^-1) than for an artificial urine (20.1 mg CO₂ m^-2 min^-1). Emissions of nitric and nitrous oxides following urine application were substantial (on average 0.36 μg NO-N and 29 μg N₂O-N m^-2 min^-1) but short lived (<40 days). The high levels of ammonium found in the urine treated soils (>200 mg NH₄ ⁺-N kg^-1) were nitrified to nitrate over a period of 42 days. Qualitative changes in the soil microbial biomass were evidently not related to biomass size. Relationships between trace gas emissions and soil processes are discussed. ei]Section editor: R Merckx     

Performance of microbial fuel cell subjected to variation in pH, temperature, external load and substrate concentration

During field application, the microbial fuel cell (MFC) will be exposed to variations in operating parameters. Hence, the performance of MFC, exposed to variation in temperature, pH, external resistance and influent chemical oxygen demand (COD), was investigated in the terms of coulombic efficiency (CE) and COD removal efficiency, while treating a synthetic wastewater. The performance was analyzed under two temperature ranges such as 20-35 degrees C and 8-22 degrees C. Operation under higher temperature range favored higher COD removal efficiency of 90% and lower current (0.7 mA) and CE (1.5%). At lower temperature range, although the COD removal efficiency of MFC decreased (59%), it gave higher current (1.4 mA) and CE (5%). The highest current was generated at pH of 6.5 in the anodic chamber with CE of 4%. Higher pH difference between anodic and cathodic electrolyte favored higher current and voltage. Within the range of COD tested (100-600 mg/l), linear correlation was observed between the current and substrate removed.     

Influence of external resistance on electrogenesis, methanogenesis, and anode prokaryotic communities in microbial fuel cells

The external resistance (R(ext)) of microbial fuel cells (MFCs) regulates both the anode availability as an electron acceptor and the electron flux through the circuit. We evaluated the effects of R(ext) on MFCs using acetate or glucose. The average current densities (I) ranged from 40.5 mA/m(2) (9,800 Ω) to 284.5 mA/m(2) (150 Ω) for acetate-fed MFCs (acetate-fed reactors [ARs]), with a corresponding anode potential (E(an)) range of -188 to -4 mV (versus a standard hydrogen electrode [SHE]). For glucose-fed MFCs (glucose-fed reactors [GRs]), I ranged from 40.0 mA/m(2) (9,800 Ω) to 273.0 mA/m(2) (150 Ω), with a corresponding E(an) range of -189 to -7 mV. ARs produced higher Coulombic efficiencies and energy efficiencies than GRs over all tested R(ext) levels because of electron and potential losses from glucose fermentation. Biogas production accounted for 14 to 18% of electron flux in GRs but only 0 to 6% of that in ARs. GRs produced similar levels of methane, regardless of the R(ext). However, total methane production in ARs increased as R(ext) increased, suggesting that E(an) might influence the competition for substrates between exoelectrogens and methanogens in ARs. An increase of R(ext) to 9,800 Ω significantly changed the anode bacterial communities for both ARs and GRs, while operating at 970 Ω and 150 Ω had little effect. Deltaproteobacteria and Bacteroidetes were the major groups found in anode communities in ARs and GRs. Betaproteobacteria and Gammaproteobacteria were found only in ARs. Bacilli were abundant only in GRs. The anode-methanogenic communities were dominated by Methanosaetaceae, with significantly lower numbers of Methanomicrobiales. These results show that R(ext) affects not only the E(an) and current generation but also the anode biofilm community and methanogenesis.     

Influence of pH on the balance between methanogenesis and iron reduction

Methanogenesis and iron reduction play major roles in determining global fluxes of greenhouse gases. Despite their importance, environmental factors that influence their interactions are poorly known. Here, we present evidence that pH significantly influences the balance between each reaction in anoxic environments that contain ferric (oxyhydr)oxide minerals. In sediment bioreactors that contained goethite as a source of ferric iron, both iron reduction and methanogenesis occurred but the balance between them varied significantly with pH. Compared to bioreactors receiving acidic media (pH 6), electron donor oxidation was 85% lower for iron reduction and 61% higher for methanogenesis in bioreactors receiving alkaline media (pH 7.5). Thus, methanogenesis displaced iron reduction considerably at alkaline pH. Geochemistry data collected from U.S. aquifers demonstrate that a similar pattern also exists on a broad spatial scale in natural settings. In contrast, in bioreactors that were not augmented with goethite, clay minerals served as the source of ferric iron and the balance between each reaction did not vary significantly with pH. We therefore conclude that pH can regulate the relative contributions of microbial iron reduction and methanogenesis to carbon fluxes from terrestrial environments. We further propose that the availability of ferric (oxyhydr)oxide minerals influences the extent to which the balance between each reaction is sensitive to pH. The results of this study advance our understanding of environmental controls on microbial methane generation and provide a basis for using pH and the occurrence of ferric minerals to refine predictions of greenhouse gas fluxes.     

Effect of tea saponin on methanogenesis,microbial community structure and expression of mcrA gene,in cultures of rumen micro‐organisms

Aims: To determine the in‐vitro effect and mode of action of tea saponin on the rumen microbial community and methane production. Methods and Results: Saponin extracted from tea seeds was added to (1) an in‐vitro fermentation inoculated with rumen fluid and (2) a pure culture of Methanobrevibacter ruminantium. Methane production and expression of the methyl coenzyme‐M reductase subunit A (mcrA) were monitored in both cultures. Abundance of methanogens, protozoa, rumen fungi and cellulolytic bacteria were quantified using real‐time PCR, and bacterial diversity was observed using denaturing gradient gel electrophoresis. Addition of tea saponin significantly reduced methane production and mcrA gene expression in the ruminal fermentation but not with the pure culture of M. ruminantium. The abundance of protozoa and fungi were significantly decreased 50% and 79% respectively but methanogen numbers were not affected, and Fibrobacter succinogenes increased by 41%. Bacterial diversity was similar in cultures with or without tea saponin. Conclusions: Tea saponin appeared to reduce methane production by inhibiting protozoa and presumably lowering methanogenic activity of protozoal‐associated methanogens. Significance and Impact of the Study: Tea saponin may be useful as a supplement to indirectly inhibit methane production in ruminants without a deleterious effect on rumen function.     

Effect of explant orientation, pH, solidifying agent and wounding on initiation of soybean somatic embryos

Summary Several methods have been developed to obtain somatic embryos of soybean. We report here a new procedure that results in high frequency somatic embryo initiation in a short period of time. Somatic embryos were induced from immature cotyledons of the cultivars “Jack,” “Thorne,” “Resnik,” and “Chapman.” Immature cotyledons were cultured on a medium containing MS salts, B₅ vitamins, 6% sucrose, and 40 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Culture modifications included: orientation of the explants (adaxial or abaxial side of the cotyledon in contact with the medium), adjustment of medium pH (5.7 or 7.0), wounding of cotyledons with scalpel blades, inclusion of ethylene modulators, and use of Noble agar or Gelrite™ as the solidifying agent. The treatment that resulted in the highest embryo induction across the cultivars consisted of abaxial side of the explant facing the medium, pH 7.0 and 0.2% Gelrite™. “Jack” was the most responsive cultivar showing the first embryos as early as 14 d after culture. After 21 d, an average of 44 embryos per cotyledon was obtained with this cultivar. The inclusion of silver nitrate (AgNO₃) in the culture medium did not enhance the number of primary somatic embryos induced per cotyledon, but the addition of 15 μM AgNO₃ did result in a faster production of secondary embryos using the cultivar “Jack.” Wounding of the explants with a scalpel resulted in an earlier induction of somatic embryos. Embryo initials were first observed after only 7 d. Histological examination of cultured cotyledons indicated that the somatic embryos originated from the subepidermal tissues and were of multicellular origin. This somatic embryo induction procedure could be useful for direct transformation work and permits the production of embryogenic tissue within 2 wk.     

Effect of abscisic acid on endogenous IAA, auxin protector levels and peroxidase activity during adventitious root initiation in Vigna radiata cuttings

Endogenous levels of free and conjugated IAA, auxin protectors (Prs) and peroxidase (PER) activity and their relation to adventitious root initiation (ARI) were investigated at the potential sites of adventitious rooting in relation to exogenous application of 250 μM ABA during the first 120 h after treatment. Cuttings from 7-day-old mung bean [Vigna radiata (L.) Wilcz.] seedlings were treated with 125, 250, and 500 μM ABA for 24 h. ABA significantly stimulated ARI but extremely inhibited epicotyl growth as compared to control. Free and conjugated IAA were measured by reversed-phase high performance liquid chromatography while Prs and PER activities were measured spectrophotometrically. The present results also indicate that endogenous free IAA levels peaked later in ABA-treated cuttings than that in control, suggesting that ABA extended the length of the induction phase of rooting process in treated cuttings and that might explain the significant delay of the appearance of roots at the treated cuttings. Higher level of IAA conjugates was found in ABA-treated cuttings than that in untreated ones. Pr level also peaked later in ABA-treated cuttings than that in control, indicating that ABA extended the period of Pr activity. An initial temporary decrease of PER activity was found in associating with high levels of free IAA and Prs during most of the primary events, while the opposite occurred during the secondary events of adventitious rooting process in both treated and untreated cuttings. Thus, ABA may stimulate ARI in mung bean Vigna radiata cuttings by regulating the concentration and /or activities of endogenous IAA, Prs, and PER activity in favor of inducing a large number of adventitious roots at their potential sites of adventitious rooting.     

Effect of Kepone on estuarine microbial activity

In situ heterotrophic uptake of mixed¹⁴C-amino acids and direct viable cell (DVC) count of Chesapeake Bay water samples were not significantly affected by the insecticide Kepone at concentrations 0.01 mg/1. Maximum inhibition of heterotrophic uptake,ca. 85–90%, and DVC count, 45–97%, was evident at concentrations of Kepone exceeding 0.2 mg/1. A specific activity index (Metabolic Activity/DVC or Kepone-resistant DVC), heterotrophic uptake, and DVC count were found to be statistically correlated (a=0.05) to one another, but negatively correlated with concentration of Kepone. The direct viable cell count proved to be a rapid, simple method for estimating the effect of Kepone on in situ estuarine microbial activity.     

Effect of substrate and pH on the oxidative half-reaction of phenol hydroxylase

The oxidative half-reaction of phenol hydroxylase has been studied by stopped-flow spectrophotometry. Three flavin-oxygen intermediates can be detected when the substrate is thiophenol, or m-NH2, m-OH, m-CH3, m-Cl, or p-OH phenol. Intermediate I, the flavin C(4a)-hydroperoxide, has an absorbance maximum at 380-390 nm and an extinction coefficient approximately 10,000 M-1 cm-1. Intermediate III, the flavin C(4a)-hydroxide, has an absorbance maximum at 365-375 nm and an extinction coefficient approximately 10,000 M-1 cm-1. Intermediate II has absorbance maxima of 350-390 nm and extinction coefficients of 10,000-16,000 M-1 cm-1 depending on the substrate. A Hammett plot of the logarithm of the rates of the oxygen transfer step, the conversion of intermediate I to intermediate II, gives a straight line with a slope -0.5. Fluoride ion is a product of the enzymatic reaction when 2,3,5,6-tetrafluorophenol is the substrate. These results are consistent with an electrophilic substitution mechanism for oxygen transfer. The conversions of I to II and II to III are acid-catalyzed. A kinetic isotope effect of 8 was measured for the conversion of II to III using deuterated resorcinol as substrate. The conversion of III to oxidized enzyme is base-catalyzed, suggesting that the reaction depends on the removal of the flavin N(5) proton. Product release occurs at the same time as the formation of intermediate III, or rapidly thereafter. The results are interpreted according to the ring-opened model of Entsch et al. (Entsch, B., Ballou, D. P., and Massey, V. (1976) J. Biol. Chem. 251, 2550-2563).     

Genomic markers of ancient anaerobic microbial pathways: sulfate reduction,methanogenesis, and methane oxidation

Genomic markers for anaerobic microbial processes in marine sediments-sulfate reduction, methanogenesis, and anaerobic methane oxidation-reveal the structure of sulfate-reducing, methanogenic, and methane-oxidizing microbial communities (including uncultured members); they allow inferences about the evolution of these ancient microbial pathways; and they open genomic windows into extreme microbial habitats, such as deep subsurface sediments and hydrothermal vents, that are analogs for the early Earth and for extraterrestrial microbiota.     

不同pH缓冲液对由乙酸产甲烷菌群结构的影响

【目的】研究不同p H缓冲液对乙酸产甲烷过程及对细菌和古菌群落结构的影响。【方法】分别添加磷酸盐(PB)、4-羟乙基哌嗪乙磺酸(HEPES)、哌嗪-1,4-二乙磺酸(PIPES)和Na HCO3/CO2缓冲液到乙酸产甲烷菌系中,定期监测甲烷产生趋势,到稳定期后收集菌体,进行16S rRNA基因的末端限制性片段多态性分析(T-RFLP)。【结果】发现PB组的乙酸产甲烷菌系延滞期约为40d,显著高于其他组的20-24 d(P0.05);Na HCO3/CO2组乙酸转化为甲烷的比例为(88.3±0.5)%,显著高于其他组的77%-81%(P0.05);不同缓冲液组的最大甲烷比生长速率为0.46-0.57 d-1(P0.05);Na HCO3/CO2组的细菌群落变化最明显,主要是未培养细菌(unclassified bacteria)、螺旋菌科细菌(Spirochaetaceae)和未培养WWE1类群的丰度较其他组分别增加到(15.5±9.4)%、(7.3±4.6)%和(17.6±6.3)%,而互养菌科(Synergistaceae)的细菌丰度降低到(8.9±8.1)%。AC+PB组中的古菌类群发生了明显变化,以竹节状甲烷鬃毛菌(Methanosaeta harundinacea)相关的产甲烷古菌占主导(97±2%),而在HEPES、PIPES和Na HCO3/CO2组和不加缓冲液组中同时存在两类乙酸营养型产甲烷古菌M.harundinacea和联合鬃毛甲烷菌(Methanosaeta concilii),以及属于甲烷杆菌目(Methanobacteriales)的氢营养型产甲烷古菌。【结论】在乙酸产甲烷菌系中加入PB增加了甲烷产生的延滞期,加入Na HCO3/CO2增加了甲烷产量,但是添加p H缓冲液不会影响到菌系的最大甲烷比生长速率。加入PB和Na HCO3/CO2都会显著改变微生物的菌群结构。这些研究为设计适宜的产甲烷菌系生长条件提供了参考。