Response of Isovalerate-Degrading Methanogenic Microbial Community to Inhibitors.

Isovalerate is one of the key intermediates during anaerobic digestion treating protein-containing waste/wastewater. Investigating the effect of different kinds of inhibitors on isovalerate-degrading microbial community is necessary to develop measures for improving the effectiveness of the treatment plants. In the present study, dynamic changes in the isovalerate-degrading microbial community in presence of inhibitors (ammonium, sulfide, mixed ammonium and sulfide, and chlortetracycline (CTC)) were investigated using high-throughput sequencing of 16S rRNA gene. Our observations showed that the isovalerate-degrading microbial community responded differently to different inhibitors and that the isovalerate degradation and gas production were strongly repressed by each inhibitor. We found that sulfide inhibited both isovalerate oxidation followed by methanogenesis, while ammonium, mixed ammonium and sulfide, and CTC mainly inhibited isovalerate oxidation. Genera classified into Proteobacteria and Chloroflexi were less sensitive to inhibitors. The two dominant genera, which are potential syntrophic isovalerate oxidizers, exhibited different responses to inhibitors that the unclassified_Peptococcaceae_3 was more sensitive to inhibitors than the unclassified_Syntrophaceae. Upon comparison to acetoclastic methanogen Methanosaeta, hydrogenotrophic methanogens Methanoculleus and Methanobacterium were less sensitive to inhibitors.

Effects of conductive carbon materials on dry anaerobic digestion of sewage sludge: Process and mechanism.

Dry anaerobic digestion of sewage sludge (SS-DAD) is often inhibited by excessive acidification due to low water content and high organic loading. The effects of conductive carbon materials including powdered activated carbon (PAC) and powdered graphite (PG) on SS-DAD under mesophilic condition (35℃) were investigated. The results demonstrated that the addition of PAC increased methane production of SS-DAD. The methane yield of PAC50% reactor (dosage of PAC is 50% of the volatile solids) amounted to 210 mL·gVSadded-1, which is 49% higher than that of control. PAC addition significantly enhanced the biodegradation process, as the reduction rate of total solids (TS) and volatile solids (VS) were increased by 36.4% and 34.1%, respectively, compared to the control. Inhibitory substrate adsorption experiments showed that PAC has significant adsorption (13.6 mg g-1) for VFAs, while PG showed almost no adsorption (0.81 mg g-1). Microbial community structure analysis showed hydrogenotrophic methanogens (Methanobrevibacter and Methanosphaera) were reduced in the PAC50% reactor, while methanogens (Methanobacterium) which can also use formate as electron donor were increased. PAC amendment reshaped the microbial community in the SS-DAD system which may result in shifting of the major electron carrier from hydrogen to formate and increasing electron transfer efficiency of the SS-DAD system.

Effects of titanium dioxide and zinc oxide nanoparticles on methane production from anaerobic co-digestion of primary and excess sludge.

Anaerobic co-digestion of primary and excess sludge is regarded as an efficient way to reuse sludge organic matter to produce methane. In this study, short-term and long-term exposure experiments were conducted to investigate the possible effects of titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles (NPs) on methane production from anaerobic co-digestion of primary and excess sludge. The data showed that TiO2 NPs had no measurable impact on methane production, even at a high concentration (150 mg/g total suspended solids (TSS)). However, short-term (8 days) exposure to 30 or 150 mg/g-TSS of ZnO NPs significantly decreased methane production. More importantly, these negative effects of ZnO NPs on anaerobic sludge co-digestion were not alleviated by increasing the adaptation time to 105 days. Further studies indicated that the presence of ZnO NPs substantially decreased the abundance of methanogenic archaea, which reduced methane production. Meanwhile, the activities of some key enzymes involved in methane production, such as protease, acetate kinase, and coenzyme F420, were remarkably inhibited by the presence of ZnO NPs, which was also an important reason for the decreased methane production. These results provide a better understanding of the potential risks of TiO2 and ZnO NPs to methane production from anaerobic sludge co-digestion.

Lovastatin in Aspergillus terreus: fermented rice straw extracts interferes with methane production and gene expression in Methanobrevibacter smithii.

Lovastatin, a natural byproduct of some fungi, is able to inhibit HMG-CoA (3-hydroxy-3 methyl glutaryl CoA) reductase. This is a key enzyme involved in isoprenoid synthesis and essential for cell membrane formation in methanogenic Archaea. In this paper, experiments were designed to test the hypothesis that lovastatin secreted by Aspergillus terreus in fermented rice straw extracts (FRSE) can inhibit growth and CH4 production in Methanobrevibacter smithii (a test methanogen). By HPLC analysis, 75% of the total lovastatin in FRSE was in the active hydroxyacid form, and in vitro studies confirmed that this had a stronger effect in reducing both growth and CH4 production in M. smithii compared to commercial lovastatin. Transmission electron micrographs revealed distorted morphological divisions of lovastatin- and FRSE-treated M. smithii cells, supporting its role in blocking normal cell membrane synthesis. Real-time PCR confirmed that both commercial lovastatin and FRSE increased (P < 0.01) the expression of HMG-CoA reductase gene (hmg). In addition, expressions of other gene transcripts in M. smithii. with a key involvement in methanogenesis were also affected. Experimental confirmation that CH4 production is inhibited by lovastatin in A. terreus-fermented rice straw paves the way for its evaluation as a feed additive for mitigating CH4 production in ruminants.

Effects of nitrite concentration and exposure time on sulfide and methane production in sewer systems.

Nitrite dosing is a promising technology to prevent sulfide and methane formation in sewers, due to the known inhibitory/toxic effect of nitrite on sulfate-reducing bacteria (SRB) and methanogenic Archaea (MA). The dependency of nitrite-induced inhibition on sulfide and methane producing activities of anaerobic sewer biofilms on nitrite levels and exposure time is investigated using a range of nitrite concentrations (40, 80, 120 mg-N/L) and exposure time up to 24 days. The recovery of these activities after the 24-day nitrite dosage was also monitored for more than two months. The inhibition level was found to be dependent on both nitrite concentration and exposure time, with stronger inhibition observed at higher nitrite concentrations and/or longer exposure time. However, the time required for achieving 50% recovery of both sulfate-reducing and methanogenic activities after the cessation of nitrite dosage only marginally depended on nitrite concentration. Model-based analysis of the recovery data showed that the recovery was likely due to the regrowth of SRB and methanogens. The lab studies and mathematical analysis supported the development of an intermittent dosing strategy, which was tested in a 1-km long rising main sewer. The field trial confirmed that intermittent dosing of nitrite can effectively reduce/prevent the formation of both sulfide and methane.

A mathematical model for the kinetics of Methanobacterium bryantii M.o.H. considering hydrogen thresholds.

We develop a kinetic model that builds on the foundation of classic Monod kinetics, but incorporates new phenomena such as substrate thresholds and survival mode observed in experiments with the H2-oxidizing methanogen Methanobacterium bryantii M.o.H. We apply our model to the experimental data presented in our companion paper on H2 thresholds. The model accurately describes H2 consumption, CH4 generation, biomass growth, substrate thresholds, and survival state during batch experiments. Methane formation stops when its Gibbs free energy is equal zero, although this does not interrupt H2 oxidation. The thermodynamic threshold for H2 oxidation occurs when the free energy for oxidizing H2 and transferring electrons to biomass is no longer negative, at approximately 0.4 nM. This threshold is not controlled by the Gibbs free energy equation of methanogenesis from H2 + HCO3- as we show in our companion paper. Beyond this threshold, the microorganisms shift to a low-maintenance metabolism called "the survival state" in response to extended H2 starvation; adding the starvation response as another new feature of the kinetic model. A kinetic threshold (or S (min)), a natural feature of the Monod kinetics, is also captured by the model at H2 concentration of around approximately 2,400 nM. S (min) is the minimum substrate concentration to maintain steady-state biomass concentration. Our model will be useful for interpreting threshold results and designing new studies to understand thresholds and their ecological implications.

Thermodynamic and kinetic analysis of the H2 threshold for Methanobacterium bryantii M.o.H.

H2 thresholds, concentrations below which H2 consumption by a microbial group stops, have been associated with microbial respiratory processes such as dechlorination, denitrification, sulfate reduction, and methanogenesis. Researchers have proposed that observed H2 thresholds occur when the available Gibbs free energy is minimal (DeltaG approximately 0) for a specific respiratory reaction. Others suggest that microbial kinetics also may play a role in controlling the thresholds. Here, we comprehensively evaluate H2 thresholds in light of microbial thermodynamic and kinetic principles. We show that a thermodynamic H2 threshold for Methanobacterium bryantii M.o.H. is not controlled by DeltaG for methane production from H2 + HCO3-. We repeatedly attain a H2 threshold near 0.4 nM, with a range of 0.2-1 nM, and DeltaG for methanogenesis from H2 + HCO3- is positive, +5 to +7 kJ/mol-H2, at the threshold in most cases. We postulate that the H2 threshold is controlled by a separate reaction other than methane production. The electrons from H2 oxidation are transferred to an electron sink that is a solid-phase component of the cells. We also show that a kinetic threshold (S(min)) occurs at a theoretically computed H2 concentration of about 2400 nM at which biomass growth shifts from positive to negative.

[Aerotolerance of strictly anaerobic microorganisms and factors of defense against oxidative stress: a review].

Effects of aerobic conditions on strictly anaerobic microorganisms belonging to diverse taxa (clostridia, acetogenic bacteria, lactic acid bacteria, bacteroids, sulfate-reducing bacteria, and methanogenic archaea) and differing considerably in their oxygen resistance have been reviewed, with emphasis on the role of aerotolerance in the ecology of anaerobes. Consideration is given to components of nutritive media for anaerobe culturing, which decrease the toxic effects of oxygen and there by contribute significantly to maintenance and storage of industrial cultures of strictly anaerobic microorganisms. Physiological and biochemical factors are described, accounting for the relative resistance of many strict anaerobes to oxygen and products of incomplete reduction thereof. Specific attention is given to regulation of enzymes of antioxidative defense, operating in the cells of strict anaerobes under the conditions of oxidative stress caused by oxygen, superoxide anion, or hydrogen peroxide.

Carbonic anhydrase inhibitors. Inhibition of the beta-class enzyme from the methanoarchaeon Methanobacterium thermoautotrophicum (Cab) with anions.

The first inhibition study of the beta-class carbonic anhydrase (CA, EC 4.2.1.1) from the methanoarchaeon Methanobacterium thermoautotrophicum (Cab) with anions is reported here. Inhibition data of the alpha-class human isozymes hCA I and hCA II (cytosolic) as well as the membrane-bound isozyme hCA IV and the gamma-class enzyme from another archaeon, Methanosarcina thermophila (Cam) with a large number of anionic species such as halides, pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrosulfide, bisulfite, sulfate, etc., are also provided for comparison. The best Cab anion inhibitors were thiocyanate and hydrogen sulfide, with inhibition constants in the range of 0.52-0.70 mM, whereas cyanate, iodide, carbonate, and nitrate were weaker inhibitors (Ki's in the range of 7.8-13.2 mM). Fluoride, chloride, and sulfate do not inhibit this enzyme appreciably, whereas the CA substrate bicarbonate, or other anions, such as bromide, nitrite, bisulfite, or sulfamate behave as weak inhibitors (Ki in the range of 40-45 mM). It is interesting to note that the metal poison, coordinating anions cyanide and azide are also rather weak Cab inhibitors (Ki in the range of 27-55 mM), whereas sulfamide is a very weak Cab inhibitor (Ki of 103 mM), although it strongly inhibits Cam (Ki of 70 microM). Surprisingly, phenylboronic and phenylarsonic acids, which have been investigated for the inhibition of all these CAs for the first time, showed very weak activity against the alpha-CA isozymes, but were effective Cab and Cam inhibitors. The best Cab inhibitors were just these two compounds (Ki's of 0.20-0.33 mM), whereas the best Cam inhibitor was sulfamic acid (Ki of 96 nM). These major differences of behavior between the diverse CAs investigated here toward anion inhibitors can be difficultly explained considering the convergent evolution of so diverse enzymes for the binding and turnover of small molecules such as carbon dioxide and anions.

Activation of methyl-SCoM reductase to high specific activity after treatment of whole cells with sodium sulfide.

Here, we report a method to generate the active form of methyl-SCoM reductase (MCR) from Methanosarcina thermophila. The protocol involves adding sodium sulfide to a growing cell culture prior to harvest to yield a "ready" (MCRox1) state of the enzyme. This method can also generate a ready state of the Methanobacterium thermoautotrophicum (strain Marburg) MCR. Experiments using sodium 35S-labeled sulfide indicate the ready state that is generated involves a Ni-S adduct. As was shown earlier for the Mb. thermoautotrophicum MCRox1 [Goubeaud, M., Schreiner, G. and Thauer, R. K. (1997) Eur. J. Biochem. 17, 2374-2377], this ready state is converted to the highly active MCRred1 form by reductive activation with Ti(III) citrate. The reduction of MCRox1 to MCRred1 with concomitant increase in activity demonstrated that MCRred1 is the active form of MCR from Ms. thermophila. We also observed the loss of the 35S-sulfide label from the enzyme when MCRox1 was converted to MCRred1. Other states of MCR could be generated in the whole cells by adding different potential ligands to the cell medium; for example, the MCRox2 state was generated by treating cells with sodium sulfite or sodium dithionite.

A study of the Na+/H+ antiport in the archaeon Methanobacterium thermoautotrophicum strain delta H.

The ability of the cells of Mb. thermoautotrophicum strain delta H to generate a proton gradient (driven by a concentration gradient of sodium ions) at pH 6.8 as well as at pH 8 was demonstrated. The electrogenic Na+/H+ antiport responsible for this process was shown to be inhibited by EIPA and also by DCCD. Artificially increasing of intracellular concentration of Ca2+ in these cells enhanced the Na+/H+ antiport activity, while the lowering of external Ca2+ by EGTA significantly decreased this activity. The apparent K(m) values for Na+ about 14 and 3 mM at pH 6.8 and 8, respectively, and Vmax about 214 (pH 6.8) and 155 (pH 8) delta Q/min per mg of cell proteins, respectively, were calculated. It is concluded that the described processes are mediated by the Na+/H+ antiporter which might be a clue to the adaptive bioenergetic behaviour of the cells of Mb. thermoautotrophicum strain delta H under the different physiological conditions.

Medium-reductant directed expression of methyl coenzyme M reductase isoenzymes in Methanobacterium thermoautotrophicum (strain deltaH).

Methanobacterium thermoautotrophicum was grown in a chemostat under various controlled conditions in the presence of either sodium sulfide or sodium thiosulfate. After establishment of the steady state, cells were taken and examined for expression of the mRNA transcripts coding for the different forms of methyl coenzyme M reductase (MCR) and methylene tetrahydomethanopterin dehydrogenase (MDH). MCR isoenzyme II expression varied most markedly. Expression was found not only to depend on known parameters temperature, pH and gassing rate, but also on the medium composition, especially the reductant present.

Hydrogen regulation of growth, growth yields, and methane gene transcription in Methanobacterium thermoautotrophicum deltaH.

Changes in growth rate, methanogenesis, growth yield (Y(CH4)), and methane gene transcription have been correlated with changes in the supply of H2 to Methanobacterium thermoautotrophicum deltaH cells growing on H2 plus CO2 in fed-batch cultures. Under conditions of excess H2, biomass and methanogenesis increased exponentially and in parallel, resulting in cultures with a constant Y(CH4) and transcription of the mth and mrt genes that encode the H2-dependent N5,N10-methenyltetrahydromethanopterin (methenyl-H4MPT) reductase (MTH) and methyl coenzyme M reductase II (MRII), respectively. Reducing the H2 supply, by decreasing the percentage of H2 in the input gas mixture or by reducing the mixing speed of the fermentor impeller, decreased the growth rate and resulted in lower and constant rates of methanogenesis. Under such H2-limited growth conditions, cultures grew with a continuously increasing Y(CH4) and the mtd and mcr genes that encode the reduced coenzyme F420-dependent N5,N10-methenyl-H4MPT reductase (MTD) and methyl coenzyme M reductase I (MRI), respectively, were transcribed. Changes in the kinetics of growth, methanogenesis, and methane gene transcription directed by reducing the H2 supply could be reversed by restoring a high H2 supply. Methane production continued, but at a low and constant rate, and only mcr transcripts could be detected when the H2 supply was reduced to a level insufficient for growth. ftsA transcripts, which encode coenzyme F390 synthetase, were most abundant in cells growing with high H2 availability, consistent with coenzyme F390 synthesis signaling a high exogenous supply of reductant.

Isolation and characterization of a neomycin-resistant mutant of Methanobacterium thermoautotrophicum with a lesion in Na+-translocating ATPase (synthase).

A mutant of Methanobacterium thermoautotrophicum with a lesion in membrane Na+-translocating ATPase (synthase) was isolated. The total ATPase activity in permeabilized cells of this mutant was elevated three-fold as compared with the wild-type strain. In contrast to wild-type cells, mutant ATPase was neither inhibited by DCCD nor stimulated by Na+ ions. The methane formation orate of the mutant cells at pH 7.5 under non-growing conditions was nearly twice that of the wild-type strain and was stimulated by sodium ions. On the other hand, the ATP synthesis driven by methanogenesis under the same conditions was lower that of the wild-type under the same conditions, and contrary to the wild-type was not stimulated by Na+ ions. ATP synthesis driven by a potassium diffusion potential in the presence of sodium ions was markedly diminished in the mutant cells. The membrane potential values of the wild-type and the mutant cells in the presence of 10 mM NaCl at pH 7.0 were comparable at energized conditions (-223 mV and -230 mV respectively). The Mg2+-dependent ATPase activity of the 10(5) x g supernatant of broken cells from the mutant cells was 30% higher than in the wild-type. On the other hand, two bands with Mg2+-dependent ATPase activity were identified by native PAGE in this fraction in both wild-type as well as in mutant. These data suggest that the binding of Na+-translocating ATPase (synthase) to the membrane spanning part is changed in the mutant strain.

Halotolerance of Methanobacterium thermoautotrophicum delta H and Marburg.

Methanobacterium thermoautotrophicum delta H and Marburg were adapted to grow in medium containing up to 0.65 M NaCl. From 0.01 to 0.5 M NaCl, there was a lag before cell growth which increased with increasing external NaCl. The effect of NaCl on methane production was not significant once the cells began to grow. Intracellular solutes were monitored by nuclear magnetic resonance (NMR) spectroscopy as a function of osmotic stress. In the delta H strain, the major intracellular small organic solutes, cyclic-2,3-diphosphoglycerate and glutamate, increased at most twofold between 0.01 and 0.4 M NaCl and decreased when the external NaCl was 0.5 M. M. thermoautotrophicum Marburg similarly showed a decrease in solute (cyclic-2,3-diphosphoglycerate, 1,3,4,6-tetracarboxyhexane, and L-alpha-glutamate) concentrations for cells grown in medium containing > 0.5 M NaCl. At 0.65 M NaCl, a new organic solute, which was visible in only trace amounts at the lower NaCl concentrations, became the dominant solute. Intracellular potassium in the delta H strain, detected by atomic absorption and 39K NMR, was roughly constant between 0.01 and 0.4 M and then decreased as the external NaCl increased further. The high intracellular K+ was balanced by the negative charges of the organic osmolytes. At the higher external salt concentrations, it is suggested that Na+ and possibly Cl- ions are internalized to provide osmotic balance. A striking difference of strain Marburg from strain delta H was that yeast extract facilitated growth in high-NaCl-containing medium. The yeast extract supplied only trace NMR-detectable solutes (e.g., betaine) but had a large effect on endogenous glutamate levels, which were significantly decreased. Exogenous choline and glycine, instead of yeast extract, also aided growth in NaCl-containing media. Both solutes were internalized with the choline converted to betaine; the contribution to osmotic balance of these species was 20 to 25% of the total small-molecule pool. These results indicate that M. thermoautotrophicum shows little changes in its internal solutes over a wide range of external NaCl. Furthermore, they illustrate the considerable differences in physiology in the delta H and Marburg strains of this organism.

Effects of acetate, propionate, and butyrate on the thermophilic anaerobic degradation of propionate by methanogenic sludge and defined cultures.

The effects of acetate, propionate, and butyrate on the anaerobic thermophilic conversion of propionate by methanogenic sludge and by enriched propionate-oxidizing bacteria in syntrophy with Methanobacterium thermoautotrophicum delta H were studied. The methanogenic sludge was cultivated in an upflow anaerobic sludge bed (UASB) reactor fed with propionate (35 mM) as the sole substrate for a period of 80 days. Propionate degradation was shown to be severely inhibited by the addition of 50 mM acetate to the influent of the UASB reactor. The inhibitory effect remained even when the acetate concentration in the effluent was below the level of detection. Recovery of propionate oxidation occurred only when acetate was omitted from the influent medium. Propionate degradation by the methanogenic sludge in the UASB reactor was not affected by the addition of an equimolar concentration (35 mM) of butyrate to the influent. However, butyrate had a strong inhibitory effect on the growth of the propionate-oxidizing enrichment culture. In that case, the conversion of propionate was almost completely inhibited at a butyrate concentration of 10 mM. However, addition of a butyrate-oxidizing enrichment culture abolished the inhibitory effect, and propionate oxidation was even stimulated. All experiments were conducted at pH 7.0 to 7.7. The thermophilic syntrophic culture showed a sensitivity to acetate and propionate similar to that of mesophilic cultures described in the literature. Additions of butyrate or acetate to the propionate medium had no effect on the hydrogen partial pressure in the biogas of an UASB reactor, nor was the hydrogen partial pressure in propionate-degrading cultures affected by the two acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of pure cultures of methanogens by benzene ring compounds.

The inhibition of methane production by Methanosaeta concilii GP6, Methanospirillum hungatei GP1, Methanobacterium espanolae GP9, and Methanobacterium bryantii M.o.H. during short-term (6-h) exposure to eight benzene ring compounds was studied. The concentration that caused 50% inhibition of the methane production rate (IC50) was dependent on the species and the toxicant. Pentachlorophenol was the most toxic of the tested compounds, with an IC50 of less than 8 mg/liter for all species except M. hungatei. Abietic acid was the next most toxic compound for all the species, with an IC50 in the range of 21.4 to 203 mg/liter. Sodium benzoate was generally the least toxic, with an IC50 in the range of 1,225 to 32,400 mg/liter. 3-Chlorobenzoate was substantially more toxic (IC50, 450 to 1,460 mg/liter) than benzoate. The inhibition by benzene, phenol, vanillic acid, and toluene was intermediate to that of pentachlorophenol and benzoate. Long-term incubation (days) studies to determine effect on growth indicated that all eight compounds were usually much more toxic than predicted from the short-term data. In these latter studies, there was generally a good correlation in the observed inhibition as determined from growth and methane production.