An approach to improve methanogenesis through the use of mixed cultures isolated from biogas digester

Biogas production has been shown to be inhibited by branched chain fatty acids (isobutyric, isovaleric) produced in the digester by cellulolytic organisms. Performance of these mixed cellulolytic cultures isolated at 25°C (C₂₅) and at 35° (C₃₅) in a batch digester using cattle manure confirmed that C₃₅, which forms mainly straight chain fatty acids from cellulose was more suitable for use as an inoculum than C₂₅ which formed predominantly branched chain fatty acids. Reconstitution of cellulolytic culture C₃₅ and mixed methanogens M₃₅ almost doubled both the amount and rate of methane production. Cellulolytic culture was useful in pretreatment of water hyacinth prior to its use as a substrate for methane generation A method for preservation and transportation of mixed cellulolytic culture for use as an inoculum in the digester is described.     

Mesophilic and thermophilic biotreatment of BTEX-polluted air in reactors

This study compares the removal of a mixture of benzene, toluene, ethylbenzene, and all three xylene isomers (BTEX) in mesophilic and thermophilic (50 degrees C) bioreactors. In the mesophilic reactor fungi became dominant after long-term operation, while bacteria dominated in the thermophilic unit. Microbial acclimation was achieved by exposing the biofilters to initial BTEX loads of 2-15 g m(-3) h(-1), at an empty bed residence time of 96 s. After adaptation, the elimination capacities ranged from 3 to 188 g m(-3) h(-1), depending on the inlet load, for the mesophilic biofilter with removal efficiencies reaching 96%. On the other hand, in the thermophilic reactor the average removal efficiency was 83% with a maximum elimination capacity of 218 g m(-3) h(-1). There was a clear positive relationship between temperature gradients as well as CO(2) production and elimination capacities across the biofilters. The gas phase was sampled at different depths along the reactors observing that the percentage pollutant removal in each section was strongly dependant on the load applied. The fate of individual alkylbenzene compounds was checked, showing the unusually high biodegradation rate of benzene at high loads under thermophilic conditions (100%) compared to its very low removal in the mesophilic reactor at such load (<10%). Such difference was less pronounced for the other pollutants. After 210 days of operation, the dry biomass content for the mesophilic and thermophilic reactors were 0.300 and 0.114 g g(-1) (support), respectively, reaching higher removals under thermophilic conditions with a lower biomass accumulation, that is, lower pressure drop.     

Granulation in thermophilic upflow anaerobic sludge blanket (UASB) reactors

The state of the art for thermophilic UASB reactors is discussed focusing on the start-up of UASB reactors, the influence of the waste water composition and temperature on the development and maintenance of thermophilic granules, and the microbial composition and structure of thermophilic granules.     

Inactivation of virus during anaerobic digestion of manure in laboratory scale biogas reactors

Reduction of porcine parvovirus, bovine enterovirus and faecal enterococci were measured in biogas reactors continuously run on manure and manure supplemented with household waste at 35°C and 55°C and in batch test run at 70°C. The aim of the experiments was to study the sanitation effect of anaerobic digestion and to evaluate the use of faecal enterococci as an indicator of sanitation. Parallel studies on the reduction of virus and faecal enterococci were done in physiological saline solution. Heat ínactivation was found to be an important factor in thermophilic biogas plants and the overall dominant factor at 70°C. However, other environmental factors with a substantial virucidal and bactericidal effect were involved in inactivation. The death rates for faecal enterococci were generally higher than for porcine parvovirus and lower than for bovine enterovirus. For faecal enterococci, a logarithmic reduction of 4 (corresponding to the recommended minimum guaranteed retention time) was obtained after 300 hours at 35°C and after 1–2 hours at 55°C. In continuously-fed reactors, a high reduction rate was initially seen for the virus tested, followed by a reduction in the rate. For porcine parvovirus, a minimum guaranteed retention time of 11–12 hours is necessary at 55°C in the initial phase (0–4 hours) and 54 hours hereafter (4–48 h). Correspondingly, for bovine enterovirus a MGRT of 23 hours was necessary at 35°C and < 0.5 hours at 55°C. The data indicate that faecal enterococci measurements give a good indication of inactivation of enterovirus and other more heat sensitive virus, especially under thermophilic conditions. Parvovirus is very suitable for comparative investigations on inactivation in the temperature range of 50–80°C, due to the extreme thermal resistance of this virus. However, in stipulating sanitation demands for biogas reactors it seems more reasonable to use less resistant virus, such as a reovirus or picornavirus, which better represents the pathogenic animal virus.Abbreviations BEV bovine enterovirus - CFU colony forming unit - FE faecal enterococci - HRT hydraulic retention time - MGRT minimum guaranteed retention time - ND not detected - PPV porcine parvovirus - TCID₅₀ tissue cell infective dose 50 % - VFA volatile fatty acids - VS volatile solids     

Biochemistry of Methanogenesis

Abstract

Methane is a product of the energy-yielding pathways of the largest and most phylogenetically diverse group in the Archaea. These organisms have evolved three pathways that entail a novel and remarkable biochemistry. All of the pathways have in common a reduction of the methyl group of methyl-coenzyme M (CH₃-S-CoM) to CH₄. Seminal studies on the CO₂-reduction pathway have revealed new cofactors and enzymes that catalyze the reduction of CO₂ to the methyl level (CH₃-S-CoM) with electrons from H₂ or formate. Most of the methane produced in nature originates from the methyl group of acetate. CO dehydrogenase is a key enzyme catalyzing the decarbonylation of acetyl-CoA; the resulting methyl group is transferred to CH₃-S-CoM, followed by reduction to methane using electrons derived from oxidation of the carbonyl group to CO₂ by the CO dehydrogenase. Some organisms transfer the methyl group of methanol and methylamines to CH₃-S-CoM; electrons for reduction of CH₃-S-CoM to CH₄ are provided by the oxidation of methyl groups to CO₂.     

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。     

Analysis of the methane production in thermophilic anaerobic reactors: use of autofluorescence microscopy

Methanogenic activity in thermophilic, anaerobic reactors was determined by comparing the amount of methane generated in single- and two-stage systems with the size of the methanogenic population, as determined by microscopy. The methanogenic activities were 2.71 × 10^–9 ml methane cell^–1 d^–1 and 1.10 × 10^–9 ml methane cell^–1 d^–1 for 10 and 4 days of the hydraulic retention time (HRT), in the single-stage system. In the two-stage system, 7.49 × 10^–9 ml methane cell^–1 d^–1 in the acidogenic reactor and 1.56 × 10^–9 ml methane cell^–1 d^–1 in the methanogenic reactor for 4 days of the HRT. A high correlation was evident between the methane production and methanogenic population [0.1354 ln(x) – 2.1375](R ² 0.8619).     

常温环境中产嗜热蛋白酶菌资源

通过计数、分离与筛选,对常温环境嗜热菌和产嗜热蛋白酶菌的分布及资源状况进行了研究。结果表明,常温环境中存在着一定数量的嗜热菌和产嗜热蛋白酶菌。土壤与水体相比,其嗜热菌资源相对丰富,且耕作肥沃的土壤中产嗜热蛋白酶菌多于贫瘠土壤;在水环境中,无论湖水、江水还是处理中的废水,在常温条件下均有一定比例的嗜热菌和产嗜热蛋白酶菌。在啤酒废水曝气阶段,产嗜热蛋白酶菌占嗜热菌的比例较大,达45％。本研究筛选的1株嗜热菌其产嗜热蛋白酶活性较高,该菌株在pH7．6、温度68℃条件下其蛋白酶活力达到642U·ml^-1。该项研究为开发产嗜热蛋白酶菌资源,在工业和环境治理等方面的应用提供重要科学依据。     

Methanogenesis in Arizona,USA dryland streams

Methanogenesis was studied in five streams of central and southern Arizona by examining the distribution of methane in interstitial water and evasion of methane in three subsystems (hyporheic, parafluvial and bank sediments). In Sycamore Creek, the primary study site (studied during summer and early autumn), methane content of interstitial water exhibited a distinct spatial pattern. In hyporheic (sediments beneath the wetted channel) and parfluvial zones (active channel sediments lateral to the wetted channel), which were well oxygenated due to high hydrologic exchange with the surface stream and had little particulate organic matter (POM), interstitial methane concentration averaged only 0.03 mgCH₄-C/L. Bank sediments (interface between the active channel and riparian zone), in contrast, which were typically vegetated, had high POM, low hydrologic exchange and concomitantly low dissolved oxygen levels, had interstitial concentration averaging 1.5 mgCH4-C/L. Methane emission from Sycamore Creek, similar to methane concentration, averaged only 3.7 mgCH₄-C·m⁻²·d⁻¹ from hyporheic and parafluvial zones as opposed to 170 mgCH₄-C·m⁻²·d⁻¹ from anoxic bank sediments. Methane in four additional streams sampled (one sampling date during late winter) was low and exhibited little spatial variation most likely due to cooler stream temperatures. Interstitial methane in parafluvial and bank sediments of all four streams ranged from only 0.005 to 0.1 mgCH₄-C/L. Similarly methane evasion was also low from these streams varying from 0 to 5.7 mgCH₄-C·m⁻²·d⁻¹. The effects of organic matter and temperature on methanogenesis were further examined by experimentally manipulating POM and temperature in stoppered flasks filled with hyporheic sediments and stream water. Methane production significantly increased with all independent variables. Methane production is greatest in bank sediments that are relatively isolated hydrologically and lowest in hyporheic and parafluvial sediments that are interactive with the surface stream.     

嗜热菌的耐热分子机制

对嗜热菌耐热机制在其细胞表层结构、ＤＮＡ螺旋的热稳定性和嗜热菌酶耐热性等方面的研究作一综述。     

Diversity of thermophilic populations during thermophilic aerobic digestion of potato peel slurry

Aims:  To study the diversity of thermophiles during thermophilic aerobic digestion (TAD) of agro-food waste slurries under conditions similar to full-scale processes.
Methods and Results:  Population diversity and development in TAD were studied by standard microbiological techniques and the processes monitored by standard fermentation procedures. Facultative thermophiles were identified as Bacillus coagulans and B. licheniformis, while obligate thermophiles were identified as B. stearothermophilus . They developed rapidly to peaks of 10⁷ to 10⁸ in ≤48 h. Stability of obligate thermophiles increased with process temperatures. Thermophiles were unstable at process pH above or below neutral, but developed rapidly at all aeration rates. Peak populations were higher in the median than at extremes of aeration rates. Obligate thermophiles were unstable at low aeration rates. Process self-heating was higher at lower than at higher aeration rate. Beyond 96 h most thermophiles were present as spores.
Conclusions:  Limited range of indigenous thermophiles drives TAD of slurry. They develop rapidly and are stable at most digestion conditions.
Significance and Impact of the Study:  Development and stability of thermophiles in TAD suggest that the process may be operated in a wide range of conditions; and even at short HRT in continuous processes without compromising efficiency.     

Thermophilic Archaeal Diversity and Methanogenesis from El Tatio Geyser Field,Chile

The hydrothermal fluid chemistry at El Tatio Geyser Field (ETGF) in northern Chile suggests that biogenic CO₂–CH₄ cycling may play an important role in water chemistry, and relatively low sulfate (0.6–1 mM) and high molecular hydrogen (H₂) concentrations (67–363 nM) suggest that methanogenic Archaea are present in ETGF microbial mats. In this study, δ¹³C analysis of dissolved inorganic carbon and methane was not indicative of biogenic methane production (δ¹³C_CH4 values ranging from ?15‰ to ?5.3‰); however, methanogenic Archaea were successfully cultured from each of the hydrothermal sites sampled. Sanger sequencing using universal Archaea primers identified putative methanogenic orders with varying metabolic capabilities, including Methanobacteriales, Methanomicrobiales and Methanosarcinales.     

Effect of the clay mineral bentonite on ammonia inhibition of anaerobic thermophilic reactors degrading animal waste

Addition of bentonite or the waste product bentonite-bound oil counteracted to some extent the inhibitory effect of ammonia during thermophilic anaerobic digestion of cattle manure. In continuously-fed reactor experiments, addition of bentonite or bentonite-bound oil delayed the onset of the inhibition and aided process recovery after initial inhibition. The effect was observed only when the ammonia concentration was increased gradually, indicating that the major effect of bentonite and BBO was not through a direct antagonistic effect towards ammonia but through an increased process resistance to toxic compounds. In batch experiments bentonite had a similar stimulatory effect leading to a decreased lag phase and increased methane production rate in ammonia inhibited reactors.     

Strategies for recovering inhibition caused by long chain fatty acids on anaerobic thermophilic biogas reactors

Long chain fatty acids (LCFA) concentrations over 1.0 g L⁻¹ were inhibiting manure thermophilic digestion, in batch and semi-continuous experiments, resulting in a temporary cease of the biogas production. The aim of the work was to test and evaluate several recovery actions, such as reactor feeding patterns, dilution and addition of adsorbents, in order to determine the most appropriate strategy for fast recovery of the reactor activity in manure based plants inhibited by LCFA. Dilution with active inoculum for increasing the biomass/LCFA ratio, or addition of adsorbents for adsorbing the LCFA and reducing the bioavailable LCFA concentration, were found to be the best recovery strategies, improving the recovery time from 10 to 2 days, in semi-continuously fed systems. Moreover, acclimatization was introduced by repeated inhibition and process recovery. The subsequent exposure of the anaerobic biomass to an inhibitory concentration of LCFA improved the recovery ability of the system, indicated as increasing degradation rates from 0.04 to 0.16 g COD_CH₄/g VS day. The incubation time between subsequent pulses, or discontinuous LCFA pulses, seems to be a decisive process parameter to tackle LCFA inhibition in manure anaerobic co-digestion.     

Growth kinetics of thermophilic Methanosarcina spp. isolated from full-scale biogas plants treating animal manures

This study determines the growth kinetics of thermophilic strains of Methanosarcina spp. from full-scale thermophilic biogas plants. The complete set of kinetic parameters, including maximum specific growth rate μ(max), half saturation constant K(S), acetate threshold concentration and cell growth yield Y(X/S), were determined for six Methanosarcina strains newly isolated from full-scale reactors and the type strain Methanosarcina thermophila TM-1(T). The kinetic experiments were performed in media supplemented with acetate and activated carbon at the optimum growth temperatures of the individual strains, 50-55 degrees C. The μ(max) values of the isolates were in the range of 0.044-0.064 h(-1), the K(S) ranged from 6.5 to 24.7 mM acetate and the threshold for acetate utilization from 0.11 to 0.40 mM. The cell growth yields of the strains were between 0.78 and 2.97 g dry weight cells mol(-1) acetate. The six isolates exhibited significantly higher μ(max) and had higher affinity to acetate than the type strain M. thermophila TM-1(T). Generally, the affinities of thermophilic Methanosarcina strains tested in this study cover a similar range to those reported in the literature for mesophilic Methanosarcina spp. with acetate as substrate. The strains isolated from plants treating mixtures of animal manures and industrial organic wastes had higher affinity for acetate and lower thresholds than strains isolated from reactors operating solely on manures.     

Immobilized plant cell reactors

The use of plant cells for the production of biochemicals represents a new area of biotechnological exploration. The techniques envisioned for industrial processes are related to those developed for microorganisms and a strong emphasis should be placed on immobilized cell systems. This review examines the spectrum of products that are synthesized by higher plants and the immobilization techniques that are suited to entrap plant cells from suspension culture. Different reactor configurations are described. Both packed-bed reactors with alginate-entrapped cells and hollow-fibre cartridges with sequestered cells have utility for the continuous production of biochemicals.     

Limitations of thermophilic anaerobic wastewater treatment and the consequences for process design

Thermophilic anaerobic digestion offers an attractive alternative for the treatment of medium- and high-strength wastewaters. However, literature reports reveal that thermophilic wastewater treatment systems are often more sensitive to environmental changes than the well-defined high-rate reactors at the mesophilic temperature range. Also, in many cases a poorer effluent quality is experienced while the carry over of suspended solids in the effluent is relatively high. In this paper recent achievements are discussed regarding the process stability of thermophilic anaerobic wastewater treatment systems. Laboratory experiments reveal a relatively low sensitivity to temperature changes if high-rate reactors with immobilized biomass are used. Other results show that if a staged process is applied, thermophilic reactors can be operated for prolonged periods of time under extreme loading conditions (80–100 kg chemical oxygen demand.m^-3.day^-1), while the concentrations of volatile fatty acids in the effluent remain at a low level.     

Responses of the biogas process to pulses of oleate in reactors treating mixtures of cattle and pig manure

The effect of oleate on the anaerobic digestion process was investigated. Two thermophilic continuously stirred tank reactors (CSTR) were fed with mixtures of cattle and pig manure with different total solid (TS) and volatile solid (VS) content. The reactors were subjected to increasing pulses of oleate. Following pulses of 0.5 and 1.0 g oleate/L, the most distinct increase in volatile fatty acid (VFA) concentrations were observed in the reactor with the lowest TS/VS content. This suggests a higher adsorption of oleate on the surfaces of biofibers in the reactor with the highest TS/VS and a less pronounced inhibition of the anaerobic digestion process. On the other hand, addition of 2.0 g oleate/L severely inhibited the process in both reactors, and a significant increase in all VFA concentrations combined with an immediate drop in methane production was noticed. However, 20 days after the reactors had been exposed to oleate both reactors showed a lower VFA concentration along with a higher methane production than before the pulses. This indicates that oleate had a stimulating effect on the overall process. The improved acetogenic and methanogenic activity in the reactors was confirmed in batch activity tests. In addition to this, toxicity tests revealed that the oleate pulses induced an increase in the tolerance level of acetotrophic methanogens towards oleate. When evaluating the usability of different process parameters (i.e., VFA and methane production) as indicators of process recovery, following the inhibition by oleate, propionate was found to be most suitable.     

Methanogenesis and microbial lipid synthesis in anoxic salt marsh sediments

In anoxic salt marsh sediments of Sapelo Island, GA, USA, the vertical distribution of CH₄ production was measured in the upper 20 cm of surface sediments in ten locations. In one section of high marsh sediments, the concentration and oxidation of acetate in sediment porewaters and the rate and amount of¹⁴C acetate and¹⁴CO₂ incorporation into cellular lipids of the microbial population were investigated. CH₄ production rates ranged from <1 to 493 nM CH₄ gram sediment⁻¹ day⁻¹ from intact subcores incubated under nitrogen. Replacement with H₂ stimulated the rate of methane release up to nine fold relative to N₂ incubations. Rates of lipid synthesis from CO₂ averaged 39.2 ×10⁻²nanomoles lipid carbon cm³ sediment⁻¹ hr⁻¹, suggesting that CO₂ may be an important carbon precursor for microbial membrane synthesis in marsh sediments under anoxic conditions. Qualitative measurements of lipid synthesis rates from acetate were found to average 8.7 × 10⁻² nanomoles. Phospholipids were the dominant lipids synthesized by both substrates in sediment cores, accounting for an average of 76.6% of all lipid radioactivity. Small amounts of ether lipids indicative of methanogenic bacteria were observed in cores incubated for 7 days, with similar rates of synthesis for both CO₂ and acetate. The low rate of ether lipid synthesis suggests that either methanogen lipid biosynthesis is very slow or that methanogens represent a small component of total microbial lipid synthesis in anoxic sediments. present address: The University of Maryland,, Chesapeake Biological Laboratory, Box 38, Solomons, MD 20688, USA