Defined bacterial culture development for methane generation from lactose

The defined microbial cultures for methane generation from lactose were investigated. A mixed culture consisting of homolactic (Streptococcus lactis), homoacetic (Clostridium formicoaceticum), and acetate-utilizing methanogenic (Methanococcus mazei) bacteria was used to convert lactose and whey permeate to methane at mesophilic temperatures (35-37 degrees C) and a pH around 7.0. Lactose was first converted to lactic acid by S. lactis, then to acetic acid by C. formicoaceticum, and finally to methane and CO(2) by M. mazei. About 5.3 mol methane were obtained from each mole of lactose consumed, and the conversion of acetate to methane was the rate-limiting step for this mixed-culture fermentation.     

Metabolism of reduced methylated sulfur compounds in anaerobic sediments and by a pure culture of an estuarine methanogen

Addition of dimethylsulfide (DMS), dimethyldisulfide (DMDS), or methane thiol (MSH) to a diversity of anoxic aquatic sediments (e.g., fresh water, estuarine, alkaline/hypersaline) stimulated methane production. The yield of methane recovered from DMS was often 52 to 63%, although high concentrations of DMS (as well as MSH and DMDS) inhibited methanogenesis in some types of sediments. Production of methane from these reduced methylated sulfur compounds was blocked by 2-bromoethanesulfonic acid. Sulfate did not influence the metabolism of millimolar levels of DMS, DMDS, or MSH added to sediments. However, when DMS was added at approximately 2-muM levels as [C]DMS, metabolism by sediments resulted in a CH(4)/CO(2) ratio of only 0.06. Addition of molybdate increased the ratio to 1.8, while 2-bromoethanesulfonic acid decreased it to 0, but did not block CO(2) production. These results indicate the methanogens and sulfate reducers compete for DMS when it is present at low concentrations; however, at high concentrations, DMS is a "noncompetitive" substrate for methanogens. Metabolism of DMS by sediments resulted in the appearance of MSH as a transient intermediate. A pure culture of an obligately methylotrophic estuarine methanogen was isolated which was capable of growth on DMS. Metabolism of DMS by the culture also resulted in the transient appearance of MSH, but the organism could grow on neither MSH nor DMDS. The culture metabolized [C]-DMS to yield a CH(4)/CO(2) ratio of approximately 2.8. Reduced methylated sulfur compounds represent a new class of substrates for methanogens and may be potential precursors of methane in a variety of aquatic habitats.     

Continuous methane fermentation and the production of vitamin B12 in a fixed-bed reactor packed with loofah

A fixed-bed reactor with acclimated methanogens immobilized on a loofah support was studied on a laboratory scale to evaluate the system producing methane from the mixture of CO(2) and H(2) gas, with the production of vitamin B(12) as a by-product. Fermentation using CO(2)/H(2) acclimated methanogens was conducted in a jar fermentor with hydraulic retention times (HRTs) of three and six days. The performance of the reactor was mainly dependent on the HRT. With an HRT of three days, the methane production rate and the vitamin B(12) concentration in the culture broth were 6.18 l/l-reactor/h and 2.88 mg/l-culture liquid; these values were 11.96 l/l-reactor/h and 37.54 mg/l-culture liquid for an HRT of six days. A higher total cell mass of methanogens retained 42.5 g dry cell/l-culture liquid was achieved in the HRT of six days. The loofah carrier immobilized almost 95% of the methanogens, which led to a more effective bio-reaction. It was also observed that the fermentation system had a better ability to buffer pH, especially for an HRT of six days.     

Acetogens and acetoclastic methanosarcinales govern methane formation in abandoned coal mines

In abandoned coal mines, methanogenic archaea are responsible for the production of substantial amounts of methane. The present study aimed to directly unravel the active methanogens mediating methane release as well as active bacteria potentially involved in the trophic network. Therefore, the stable-isotope-labeled precursors of methane, [(13)C]acetate and H(2)-(13)CO(2), were fed to liquid cultures from hard coal and mine timber from a coal mine in Germany. Guided by methane production rates, samples for DNA stable-isotope probing (SIP) with subsequent quantitative PCR and denaturing gradient gel electrophoretic (DGGE) analyses were taken over 6 months. Surprisingly, the formation of [(13)C]methane was linked to acetoclastic methanogenesis in both the [(13)C]acetate- and the H(2)-(13)CO(2)-amended cultures of coal and timber. H(2)-(13)CO(2) was used mainly by acetogens related to Pelobacter acetylenicus and Clostridium species. Active methanogens, closely affiliated with Methanosarcina barkeri, utilized the readily available acetate rather than the thermodynamically more favorable hydrogen. Thus, the methanogenic microbial community appears to be highly adapted to the low-H(2) conditions found in coal mines.     

Methanogenic archaea: ecologically relevant differences in energy conservation

Most methanogenic archaea can reduce CO(2) with H(2) to methane, and it is generally assumed that the reactions and mechanisms of energy conservation that are involved are largely the same in all methanogens. However, this does not take into account the fact that methanogens with cytochromes have considerably higher growth yields and threshold concentrations for H(2) than methanogens without cytochromes. These and other differences can be explained by the proposal outlined in this Review that in methanogens with cytochromes, the first and last steps in methanogenesis from CO(2) are coupled chemiosmotically, whereas in methanogens without cytochromes, these steps are energetically coupled by a cytoplasmic enzyme complex that mediates flavin-based electron bifurcation.     

产甲烷菌研究进展

产甲烷菌是重要的环境微生物,在自然界的碳素循环中起重要作用。迄今已有5种产甲烷菌基因组测序完成。基因组信息使人们对产甲烷茵的细胞结构、进化、代谢及环境适应性有了更深的理解。目前已知的甲烷生物合成途径有3种,它们以乙酸、甲基化合物、氢/二氧化碳为起始,通过不同的反应途径都形成了甲基辅酶M,在甲基辅酶M还原酶的催化下最终形成甲烷。     

Hydrogenogenic CO conversion in a moderately thermophilic (55 degrees C) sulfate-fed gas lift reactor: competition for CO-derived H(2)

Thermophilic (55 degrees C) sulfate reduction in a gas lift reactor fed with CO gas as the sole electron donor was investigated. The reactor was inoculated with mesophilic granular sludge with a high activity of CO conversion to hydrogen and carbon dioxide at 55 degrees C. Strong competition for H(2) was observed between methanogens and sulfate reducers, while the homoacetogens present consumed only small amounts of H(2). The methanogens appeared to be more sensitive to pH and temperature shocks imposed to the reactor, but could not be completely eliminated. The fast growth rates of the methanogens (generation time of 4.5 h) enabled them to recover fast from shocks, and they rapidly consumed more than 90% of the CO-derived H(2). Nevertheless, steep increases in sulfide production in periods with low methane production suggests that once methanogenesis is eliminated, sulfate reduction with CO-rich gas as electron donor has great potential for thermophilic biodesulfurization.     

Thermophilic sulfate reduction and methanogenesis with methanol in a high rate anaerobic reactor

Sulfate reduction outcompeted methanogenesis at 65 degrees C and pH 7.5 in methanol and sulfate-fed expanded granular sludge bed reactors operated at hydraulic retention times (HRT) of 14 and 3.5 h, both under methanol-limiting and methanol-overloading conditions. After 100 and 50 days for the reactors operated at 14 and 3.5 h, respectively, sulfide production accounted for 80% of the methanol-COD consumed by the sludge. The specific methanogenic activity on methanol of the sludge from a reactor operated at HRTs of down to 3.5 h for a period of 4 months gradually decreased from 0. 83 gCOD. gVSS(-1). day(-1) at the start to a value of less than 0.05 gCOD. gVSS(-1). day(-1), showing that the relative number of methanogens decreased and eventually became very low. By contrast, the increase of the specific sulfidogenic activity of sludge from 0. 22 gCOD. gVSS(-1). day(-1) to a final value of 1.05 gCOD. gVSS(-1). day(-1) showed that sulfate reducing bacteria were enriched. Methanol degradation by a methanogenic culture obtained from a reactor by serial dilution of the sludge was inhibited in the presence of vancomycin, indicating that methanogenesis directly from methanol was not important. H(2)/CO(2) and formate, but not acetate, were degraded to methane in the presence of vancomycin. These results indicated that methanol degradation to methane occurs via the intermediates H(2)/CO(2) and formate. The high and low specific methanogenic activity of sludge on H(2)/CO(2) and formate, respectively, indicated that the former substrate probably acts as the main electron donor for the methanogens during methanol degradation. As sulfate reduction in the sludge was also strongly supported by hydrogen, competition between sulfate reducing bacteria and methanogens in the sludge seemed to be mainly for this substrate. Sulfate elimination rates of up to 15 gSO(4)(2-)/L per day were achieved in the reactors. Biomass retention limited the sulfate elimination rate.     

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.     

Formic acid production from H2 and bicarbonate by a formateutilizing methanogen

Summary Resting cells of a formate-utilizing methanogen (strain HU) were used as a biocatalyser for formic acid production from H₂ and bicarbonate. In the presence of methyl viologen, a potent inhibitor of methanogenesis, the cells could accumulate formic acid in the reaction mixture, whereas methane was produced in the absence of the inhibitor. Under optimal conditions (pH 8.0, 32°C, 100% H₂ gas phase), 674 mmol·l^-1 of formic acid (31 g·l^-1 yield; 33% conversion of bicarbonate) was obtained at about 0.7 atm of gas pressure. When the reaction was carried out in an ultrafiltration vessel with a hydrogen pressure of 3 atm, a final product concentration of 1.02 mol·l^-1 (47 g·l^-1 yield, 51% conversion) could be obtained, indicating that the hydrogen lyase system of this methanogen has a high tolerance to formic acid, bicarbonate and hydrogen. The results obtained in this work open a new perspective for the utilization of methanogens for processes other than methane production.     

Modeling of anaerobic formate kinetics in mixed biofilm culture using dynamic membrane mass spectrometric measurement

The dynamics of the anaerobic conversion of formate in a microbial mixed culture taken from an anaerobic fluidized bed reactor was studied using a new stirred micro reactor equipped with a membrane mass spectrometer. The microreactor with a toroidally shaped bottom and pitched blade turbine and a cylindrical flow guide was thermostated and additionally equipped with a pH electrode and pH control. During fed-batch experiments using formate, the dissolved gases (methane, hydrogen, and carbon dioxide), as well as the acid consumption rates for pH control were monitored continuously. Initially and at the end of each experiment, organic acids were analyzed using ion chromatography (IC). It was found that about 50% of the formate was converted to methane via hydrogen and carbon dioxide, 40% gave methane either directly or via acetate. This was calculated from experiments using H(13)CO(3) (-) pulses and measurement of (12)CH(4) and (13)CH(4) production rates. About 10% of the formate was converted to lactate, acetate, and propionate, thereby increasing the measured CO(2)/CH(4) production ratio. The nondissociated formic acid was shown to be rate determining. From the relatively high K(s) value of 2.5 mmol m(-3), it was concluded that formate cannot play an important role in electron transfer. During dynamic feeding of formate, hydrogen concentration always increased to a maximum before decreasing again. This peak was found to be very discriminative during modeling. From the various models set up, only those with two-stage degradation and double Monod kinetics, both for CO(2) and hydrogen, were able to describe the experimental data adequately. Additional discrimination was possible with the IC measurement of organic acids. (c) 1995 John Wiley & Sons, Inc.     

Feasibility of methanogenic digestion applied to a low pH acetic acid solution

This paper discusses the methanogenic digestion of a synthetic acetic acid wastewater inoculated using a mixed culture obtained from an anaerobic digester at a municipal wastewater treatment facility. Experiments were conducted in 500mL batch reactors containing an unbuffered acetic acid solution. Test conditions compared methane production and acetic acid degradation at both acidic (pH 4.5) and neutral (pH 7.0) initial system conditions. Results showed that methane production increased by 30% when the initial pH was decreased from 7.0 to 4.5.     

Acetate production from hydrogen and [13C]carbon dioxide by the microflora of human feces

Fecal suspensions from humans were incubated with 13CO2 and H2. The suspensions were from subjects who harbored 10(8) and 10(10) methanogens per g (dry weight) of feces, respectively, and from a subject who did not harbor methanogens. Quantitative nuclear magnetic resonance spectroscopy showed that acetate labeled in both the methyl and carboxyl groups was formed by suspensions from the subject without methanogens and the subject with the lower concentrations of methanogens. The amounts of labeled acetate formed were in agreement with the amounts expected based on measurements of H2 utilization. No labeled acetate was formed by suspensions from the subject with the higher concentrations of methanogens, and essentially all of the H2 used was accounted for by CH4 production. Suspensions from the subject with lower concentrations of methanogens produced both methane and acetate from H2 and CO2. The results indicate that reduction of CO2 to acetate may be a major pathway for microbial production of acetate in the human colon except when very high concentrations of methanogens (ca. 10(10) per g [dry weight] of feces) are present. Double-labeled acetate was also formed from H2 and 13CO2 by fecal suspensions from nonmethanogenic and moderately methanogenic rats.     

以pH为扰动因子探究电化学厌氧消化产甲烷代谢通量与微生物的关系

【背景】电化学厌氧消化(electrochemical anaerobic digestion,EAD)系统的代谢途径由具备不同功能的微生物所主导,其代谢通量与功能微生物丰度、活性及群落结构相关。【目的】探究EAD产甲烷代谢通量与微生物的关系。【方法】采用代谢通量分析(metabolic flux analysis,MFA)方法,以pH为扰动因子得到微生物群落与产甲烷通量的响应关系。【结果】pH 7.5扰动时产甲烷通量最大为0.398 4±0.029 3,较对照组(pH 6.9)的0.297 4±0.012 7和扰动组(pH 6.3)的0.136 5±0.012 0分别提高了25%和65%。另外,平均有33.8%±3.1%的氢气(通量)用于还原二氧化碳产甲烷和乙酸,平均有21.0%±2.6%的乙酸(通量)转化为甲烷。此外,产甲烷通量与Mariniphaga、Methanosaeta和Desulfomicrobium的丰度呈正相关,与Sedimentibacter的丰度呈负相关且影响显著。【结论】在EAD产甲烷体系中产甲烷菌和产酸菌共存时,pH值略大于7.0的环境有利于甲烷的生成,改变E...     

Acetate production from hydrogen and [13C]carbon dioxide by the microflora of human feces.

Fecal suspensions from humans were incubated with 13CO2 and H2. The suspensions were from subjects who harbored 10(8) and 10(10) methanogens per g (dry weight) of feces, respectively, and from a subject who did not harbor methanogens. Quantitative nuclear magnetic resonance spectroscopy showed that acetate labeled in both the methyl and carboxyl groups was formed by suspensions from the subject without methanogens and the subject with the lower concentrations of methanogens. The amounts of labeled acetate formed were in agreement with the amounts expected based on measurements of H2 utilization. No labeled acetate was formed by suspensions from the subject with the higher concentrations of methanogens, and essentially all of the H2 used was accounted for by CH4 production. Suspensions from the subject with lower concentrations of methanogens produced both methane and acetate from H2 and CO2. The results indicate that reduction of CO2 to acetate may be a major pathway for microbial production of acetate in the human colon except when very high concentrations of methanogens (ca. 10(10) per g [dry weight] of feces) are present. Double-labeled acetate was also formed from H2 and 13CO2 by fecal suspensions from nonmethanogenic and moderately methanogenic rats.     

Fermentation of cellulose and cellobiose by Clostridium thermocellum in the absence of Methanobacterium thermoautotrophicum.

The fermentation of cellulose and cellobiose by Clostridium thermocellum monocultures and C. thermocellum/Methanobacterium thermoautotrophicum cocultures was studied. All cultures were grown under anaerobic conditions in batch culture at 60 degrees C. When grown on cellulose, the coculture exhibited a shorter lag before initiation and growth and celluloysis than did the monoculture. Cellulase activity appeared earlier in the coculture than in the monoculture; however, after growth had ceased, cellulase activity was greater in the monoculture. Monocultures produced primarily ethanol, acetic acid, H2 and CO2. Cocultures produced more H2 and acetic acid and less ethanol than did the monoculture. In the coculture, conversion of H2 to methane was usually complete, and most of the methane produced was derived from CO2 reduction rather than from acetate conversion. Agents of fermentation stoppage were found to be low pH and high concentrations of ethanol in the monoculture and low pH in the coculture. Fermentation of cellobiose was more rapid than that of cellulose. In cellobiose medium, the methanogen caused only slight changes in the fermentation balance of the Clostridium, and free H2 was produced.     

Trace methane oxidation studied in several Euryarchaeota under diverse conditions

We used (13)C-labeled methane to document the extent of trace methane oxidation by Archaeoglobus fulgidus, Archaeoglobus lithotrophicus, Archaeoglobus profundus, Methanobacterium thermoautotrophicum, Methanosarcina barkeri and Methanosarcina acetivorans. The results indicate trace methane oxidation during growth varied among different species and among methanogen cultures grown on different substrates. The extent of trace methane oxidation by Mb. thermoautotrophicum (0.05 +/- 0.04%, +/- 2 standard deviations of the methane produced during growth) was less than that by M. barkeri (0.15 +/- 0.04%), grown under similar conditions with H(2) and CO(2). Methanosarcina acetivorans oxidized more methane during growth on trimethylamine (0.36 +/- 0.05%) than during growth on methanol (0.07 +/- 0.03%). This may indicate that, in M. acetivorans, either a methyltransferase related to growth on trimethylamine plays a role in methane oxidation, or that methanol is an intermediate of methane oxidation. Addition of possible electron acceptors (O(2), NO(3) (-), SO(4) (2-), SO(3) (2-)) or H(2) to the headspace did not substantially enhance or diminish methane oxidation in M. acetivorans cultures. Separate growth experiments with FAD and NAD(+) showed that inclusion of these electron carriers also did not enhance methane oxidation. Our results suggest trace methane oxidized during methanogenesis cannot be coupled to the reduction of these electron acceptors in pure cultures, and that the mechanism by which methane is oxidized in methanogens is independent of H(2) concentration. In contrast to the methanogens, species of the sulfate-reducing genus Archaeoglobus did not significantly oxidize methane during growth (oxidizing 0.003 +/- 0.01% of the methane provided to A. fulgidus, 0.002 +/- 0.009% to A. lithotrophicus and 0.003 +/- 0.02% to A. profundus). Lack of observable methane oxidation in the three Archaeoglobus species examined may indicate that methyl-coenzyme M reductase, which is not present in this genus, is required for the anaerobic oxidation of methane, consistent with the "reverse methanogenesis" hypothesis.     

The γ-irradiation of aqueous acetic acid-clay suspensions

-radiolysis of 0.8 mol dm^–3 aqueous, oxygen-free acetic acid solutions was investigated in the presence or absence of Na-montmorillonite (1–3 g per 10 cm^–3). The systems were irradiated at their natural pH (3.5), and 25 °C in a dose range from 0.01 to 500 kGy. H₂, CH₄, CO, CO₂, and a variety of polycarboxylic acids were formed in all systems. The major features of the radiolysis in the presence of clays were: (1) More solute molecules were decomposed; (2) Carbon dioxide was produced in higher yield; (3) The yield of methane was unaffected; and (4) 44% less polycarboxylic acids were formed. Three possible mechanisms that could account for the observed changes are suggested. The results are important in understanding heterogeneous processes in radiation catalysis and might be significant to prebiotic chemistry.     

In situ methane enrichment in anaerobic digestion

A major cost consideration in the use of anaerobic digestion to convert biomass and waste to utility-grade gas is the expense of separating CO(2) from the product gas. Anaerobic digestion has a number of inherent properties that can be exploited to increase the methane content of the gas directly produced by the digester, the most important of which is the high solubility of CO(2)(40-60 times that of methane) in water under digestion conditions. The methane enrichment concept examined in this study involved the recirculation of a liquid stream from the digester through a CO(2) desorption process and the return of the liquid stream back to the digester for absorption of additional CO(2) produced by the conversion of organic materials. A steady-state equilibrium model predicted that a digester gas methane content exceeding 94% could be achieved with this scheme using modest recirculation rates provided a desorption process could be designed to achieve a 60+% CO(2) removal efficiency in the degassing of the liquid recycle stream. Using fixed-film laboratory digesters operated on synthetic feedstocks, the technique of methane enrichment was tested under pressurized and unpressurized conditions. A 93 + 2% methane gas stream was produced from a volatile-acid-fed bench-scale digester simulating the methanogenic stage of two-phase digestion under conditions of (1) a pH swing achieved without caustic addition that allowed digestion at pH 7. 5 and air stripping at pH 6. 5-7. 0, (2) digester pressurization to 30 psig, and (3) a recycle rate of 0. 33 L/L reactor/day. Significant but lower levels of methane enrichment were achieved with the single-stage digester at the low experimental recycle rate. However, the narrow range among all experiments of CO(2) desorption efficiencies achieved in air stripping the recycle stream (35-60% CO(2) removal) suggests that comparable methane enrichment-may be achieved with unpressurized single-stage digestion using greater recycle rates. A materials balance analysis of data from an unpressurized, single-stage digester employing no chemical addition and using laboratory degassing efficiencies indicated that 94% methane could be produced at recycle rates of less than 1. 4 L/L reactor/day with a methane loss of less than 2%.     

The effect of pectin, corn and wheat starch, inulin and pH on in vitro production of methane, short chain fatty acids and on the microbial community composition in rumen fluid

Methane emission from livestock, ruminants in particular, contributes to the build up of greenhouse gases in the atmosphere. Therefore the focus on methane emission from ruminants has increased. The objective of this study was to investigate mechanisms for methanogenesis in a rumen fluid-based in vitro fermentation system as a consequence of carbohydrate source (pectin, wheat and corn starch and inulin) and pH (ranging from 5.5 to 7.0). Effects were evaluated with respect to methane and short chain fatty acid (SCFA) production, and changes in the microbial community in the ruminal fluid as assessed by terminal-restriction fragment length polymorphism (T-RFLP) analysis. Fermentation of pectin resulted in significantly lower methane production rates during the first 10 h of fermentation compared to the other substrates (P = 0.001), although total methane production was unaffected by carbohydrate source (P = 0.531). Total acetic acid production was highest for pectin and lowest for inulin (P < 0.001) and vice versa for butyric acid production from pectin and inulin (P < 0.001). Total propionic acid production was unaffected by the carbohydrate source (P = 0.791). Methane production rates were significantly lower for fermentations at pH 5.5 and 7.0 (P = 0.005), sustained as a trend after 48 h (P = 0.059), indicating that there was a general optimum for methanogenic activity in the pH range from 6.0 to 6.5. Decreasing pH from 7.0 to 5.5 significantly favored total butyric acid production (P < 0.001). Principle component analysis of T-RFLP patterns revealed that both pectin and pH 5.5 resulted in pronounced changes in the microbial community composition. This study demonstrates that both carbohydrate source and pH affect methane and SCFA production patterns, and the microbial community composition in rumen fluid.