Decreasing ammonia inhibition in thermophilic methanogenic bioreactors using carbon fiber textiles

Ammonia accumulation is one of the main causes of the loss of methane production observed during fermentation. We investigated the effect of addition of carbon fiber textiles (CFT) to thermophilic methanogenic bioreactors with respect to ammonia tolerance during the process of degradation of artificial garbage slurry, by comparing the performance of the reactors containing CFT with the performance of reactors without CFT. Under total ammonia-N concentrations of 3,000 mg L⁻¹, the reactors containing CFT were found to mediate stable removal of organic compounds and methane production. Under these conditions, high levels of methanogenic archaea were retained at the CFT, as determined by 16S rRNA gene analysis for methanogenic archaea. In addition, Methanobacterium sp. was found to be dominant in the suspended fraction, and Methanosarcina sp. was dominant in the retained fraction of the reactors with CFT. However, the reactors without CFT had lower rates of removal of organic compounds and production of methane under total ammonia-N concentrations of 1,500 mg L⁻¹. Under this ammonia concentration, a significant accumulation of acetate was observed in the reactors without CFT (130.0 mM), relative to the reactors with CFT (4.2 mM). Only Methanobacterium sp. was identified in the reactors without CFT. These results suggest that CFT enables stable proliferation of aceticlastic methanogens by preventing ammonia inhibition. This improves the process of stable garbage degradation and production of methane in thermophilic bioreactors that include high levels of ammonia.     

Co-cultivation of Thermoanaerobacter strains with a methanogenic partner enhances glycerol conversion

Glycerol-rich waste streams produced by the biodiesel, bioethanol and oleochemical industries can be treated and valorized by anaerobic microbial communities to produce methane. As current knowledge of the microorganisms involved in thermophilic glycerol conversion to methane is scarce, thermophilic glycerol-degrading methanogenic communities were enriched. A co-culture of Thermoanaerobacter and Methanothermobacter species was obtained, pointing to a non-obligately syntrophic glycerol degradation. This hypothesis was further studied by incubating Thermoanaerobacter brockii subsp. finnii and T. wiegelii with glycerol (10 mM) in pure culture and with different hydrogenotrophic methanogens. The presence of the methanogen accelerated glycerol fermentation by the two Thermoanaerobacter strains up to 3.3 mM day⁻¹, corresponding to 12 times higher volumetric glycerol depletion rates in the methanogenic co-cultures than in the pure bacterial cultures. The catabolic pathways of glycerol conversion were identified by genome analysis of the two Thermoanaerobacter strains. NADH and reduced ferredoxin formed in the pathway are linked to proton reduction, which becomes thermodynamically favourable when the hydrogen partial pressure is kept low by the hydrogenotrophic methanogenic partner.     

The effect of dietary concentrate and soya oil inclusion on microbial diversity in the rumen of cattle

Aims: Methane emissions from ruminants are a significant contributor to global greenhouse gas production. The aim of this study was to examine the effect of diet on microbial communities in the rumen of steers. Methods and Results: The effects of dietary alteration (50 : 50 vs 90 : 10 concentrate–forage ratio, and inclusion of soya oil) on methanogenic and bacterial communities in the rumen of steers were examined using molecular fingerprinting techniques (T‐RFLP and automated ribosomal intergenic spacer analysis) and real‐time PCR. Bacterial diversity was greatly affected by diet, whereas methanogen diversity was not. However, methanogen abundance was significantly reduced (P = 0·009) in high concentrate–forage diets and in the presence of soya oil (6%). In a parallel study, reduced methane emissions were observed with these diets. Conclusions: The greater effect of dietary alteration on bacterial community in the rumen compared with the methanogen community may reflect the impact of substrate availability on the rumen bacterial community. This resulted in altered rumen volatile fatty acid profiles and had a downstream effect on methanogen abundance, but not diversity. Significance and Impact of the Study: Understanding how rumen microbial communities contribute to methane production and how these microbes are influenced by diet is essential for the rational design of methane mitigation strategies from livestock.     

Diversity and abundance of the rumen and fecal methanogens in Altay sheep native to Xinjiang and the influence of diversity on methane emissions

This study aims to investigate the influence of diet roughage proportion on the methanogenic communities from the rumen and fecal samples in Altay local sheep native to Xinjiang and better understand the association of methanogenic diversity or abundance with methane emissions of the ruminants. In this study, the high roughage diet was found to cause more methane emissions for either maintenance or ad-lib group, but the total methanogenic abundance was not influenced by roughage proportion and showed no significant difference between groups. Furthermore, the denaturing gradient gel electrophoresis was conducted to reveal the difference in methanogenic diversity. Phylogenetic analysis showed that the sequences obtained were divided into three groups, affiliated to the genus of Methanobrevibacter, Methanocorpusculum and an unidentified methanogenic-like group. Of these sequences, the predominant diversity from the genus of Methanobrevibacter and the unidentified methanogenic-like archaeons in the rumen was found to be significantly induced by the high roughage diet, implying that the variation of diversity at the species or strain level might have an effect on methane emissions from the rumen. Further analysis showed that five methangenic sequences from the rumen were possibly associated with the differential methane emissions.     

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.     

一种厌氧真菌共培养甲烷菌株的分离及其甲烷生产特性解析

【背景】开发生物甲烷资源是减轻化石燃料供求紧张的有效措施,而秸秆类原料的预处理及甲烷生产方法需要不断创新,从而进一步满足可持续发展。厌氧真菌与甲烷菌共培养能够通过假根侵入及纤维降解酶双重预处理秸秆并生产甲烷,但目前全世界被报道的骆驼胃肠道来源的厌氧真菌分离培养物仅有1株。【目的】从新疆准噶尔双峰驼瘤胃内容物中分离出新型厌氧真菌和甲烷菌共培养物,研究其在降解秸秆并联合生产生物甲烷方面的应用潜力。【方法】采用Hungate滚管纯化技术将从骆驼胃肠道中分离的厌氧真菌和甲烷菌共培养,对其进行形态学及分子学鉴定,随后厌氧发酵5种底物(稻秸、芦苇、构树叶、苜蓿秆和草木樨),研究产甲烷量、降解效果及主要代谢产物等方面的特性。【结果】筛选到的共培养物中的厌氧真菌为Oontomyces sp. CR1,甲烷菌为Methanobrevibacter sp. CR1。其在降解稻秸时表现出最高的木聚糖酶酶活力(21.64 IU/mL)及甲烷产量(143.39 mL/g-DM),甲烷生产特性较分离自其他动物宿主的厌氧真菌共培养物更优。【结论】共培养厌氧真菌与甲烷菌菌株CR1是一种新型高效降解菌株资源,其在利用木质纤维素生物质生产生物甲烷方面具有良好的应用前景。     

A bioelectrochemical reactor containing carbon fiber textiles enables efficient methane fermentation from garbage slurry

A packed-bed system includes supporting materials to retain microorganisms and a bioelectrochemical system influences the microbial metabolism. In our study, carbon fiber textiles (CFT) as a supporting material was attached onto a carbon working electrode in a bioelectrochemical reactor (BER) that degrades garbage slurry to methane, in order to investigate the effect of combining electrochemical regulation and packing CFT. The potential on the working electrode in the BER containing CFT was set to −1.0 V or −0.8 V (vs. Ag/AgCl). BERs containing CFT exhibited higher methane production, elimination of dichromate chemical oxygen demand, and the ratio of methanogens in the suspended fraction than reactors containing CFT without electrochemical regulation at an organic loading rate (OLR) of 27.8 gCODcr/L/day. In addition, BERs containing CFT exhibited higher reactor performances than BERs without CFT at this OLR. Our results revealed that the new design that combined electrochemical regulation and packing CFT was effective.     

Efficient treatment of garbage slurry in methanogenic bioreactor packed by fibrous sponge with high porosity

Adding a supporting material to a methanogenic bioreactor treating garbage slurry can improve efficiency of methane production. However, little is known on how characteristics (e.g., porosity and hydrophobicity) of the supporting material affect the bioreactor degrading garbage slurry. We describe the reactor performances and microbial communities in bioreactors containing hydrophilic or hydrophobic sheets, or fibrous hydrophilic or hydrophobic sponges. The porosity affected the efficiency of methane production and solid waste removal more than the hydrophilic or hydrophobic nature of the supporting material. When the terminal restriction fragment length polymorphism technique was used at a lower organic loading rate (OLR), microbial diversities in the suspended fraction were retained on the hydrophobic, but not the hydrophilic, sheets. Moreover, real-time quantitative polymerase chain reaction (PCR) performed at a higher OLR revealed that the excellent performance of reactors containing fibrous sponges with high porosity (98%) was supported by a clear increase in the numbers of methanogens on these sponges, resulting in larger total numbers of methanogens in the reactors. In addition, the bacterial communities in fractions retained on both the hydrophobic and hydrophilic fibrous sponges differed from those in the suspended fraction, thus increasing bacterial diversity in the reactor. Thus, higher porosity of the supporting material improves the bioreactor performance by increasing the amount of methanogens and bacterial diversity; surface hydrophobicity contributes to maintaining the suspended microbial community.     

Latitudinal distribution of microbial communities in anaerobic biological stabilization ponds: effect of the mean annual temperature

Considering wide utilization and high methane fluxes from anaerobic biological stabilization ponds (ABSPs), understanding the methanogenesis in ABSPs is of fundamental importance. Here we investigated the variation and impact factors of methanogenesis in seven ABSPs that spanned from the north to the south of China. Results showed that methanogen abundance (7.7 × 10⁹–8.7 × 10¹⁰ copies g^?1 dry sediment) and methanogenic activities (2.2–21.2 μmol CH₄ g^?1 dry sediment h^?1) were considerable for all sediments. Statistical analysis demonstrated that compared with other factors (ammonium, pH, COD and TOC), mean annual temperature (MAT) showed the lowest P value and thus was the most important influencing factor for the methanogenic process. Besides, with the increasing MAT, methanogenic activity was enhanced mainly due to the shift of the dominant methanogenic pathway from acetoclastic (49.8–70.7%) in low MAT areas to hydrogenotrophic (42.0–54.6%) in high MAT areas. This shift of methanogenic pathway was also paralleled with changes in composition of bacterial communities. These results suggested that future global warming may reshape the composition of methanogen communities and lead to an increasing methane emission from ABSPs. Therefore, further research is urgently needed to globally estimate methane emissions from ABSPs and re‐examine the role of ABSPs in wastewater treatment.     

Colonization of rice roots with methanogenic archaea controls photosynthesis‐derived methane emission

The methane emitted from rice fields originates to a large part (up to 60%) from plant photosynthesis and is formed on the rice roots by methanogenic archaea. To investigate to which extent root colonization controls methane (CH₄) emission, we pulse‐labeled rice microcosms with ¹³CO₂ to determine the rates of ¹³CH₄ emission exclusively derived from photosynthates. We also measured emission of total CH₄ (¹²⁺¹³CH₄), which was largely produced in the soil. The total abundances of archaea and methanogens on the roots and in the soil were analysed by quantitative polymerase chain reaction of the archaeal 16S rRNA gene and the mcrA gene coding for a subunit of the methyl coenzyme M reductase respectively. The composition of archaeal and methanogenic communities was determined with terminal restriction fragment length polymorphism (T‐RFLP). During the vegetative growth stages, emission rates of ¹³CH₄ linearly increased with the abundance of methanogenic archaea on the roots and then decreased during the last plant growth stage. Rates of ¹³CH₄ emission and the abundance of methanogenic archaea were lower when the rice was grown in quartz‐vermiculite with only 10% rice soil. Rates of total CH₄ emission were not systematically related to the abundance of methanogenic archaea in soil plus roots. The composition of the archaeal communities was similar under all conditions; however, the analysis of mcrA genes indicated that the methanogens differed between the soil and root. Our results support the hypothesis that rates of photosynthesis‐driven CH₄ emission are limited by the abundance of methanogens on the roots.     

DiSC (direct saccharification of culms) process for bioethanol production from rice straw

A simple process (the direct-saccharification-of-culms (DiSC) process) to produce ethanol from rice straw culms, accumulating significant amounts of soft carbohydrates (SCs: glucose, fructose, sucrose, starch and β-1,3-1,4-glucan) was developed. This study focused on fully mature culms of cv. Leafstar, containing 69.2% (w/w of dried culms) hexoses from SCs and cellulose. Commercially-available wind-separation equipment successfully prepared a culm-rich fraction with a SC recovery of 83.1% (w/w) from rice straw flakes (54.1% of total weight of rice straw). The fraction was suspended in water (20%, w/w) for starch liquefaction, and the suspension was subjected to a simultaneous saccharification and fermentation with yeast, yielding 5.6% (w/v) ethanol (86% of the theoretical yield from whole hexoses in the fraction) after 24 h fermentation. Thus, the DiSC process produced highly-concentrated ethanol from rice straw in a one vat process without any harsh thermo-chemical pretreatments.     

Isolation and characterization of a motile hydrogenotrophic methanogen from rice paddy field soil in Japan

A hydrogenotrophic motile methanogen was isolated from flooded Japanese paddy field soil. Anaerobic incubation of the paddy soil on H(2)-CO(2) at 20 degrees C led to the enrichment of symmetrically curved motile autofluorescent rods. The methanogenic strain TM20-1 isolated from the culture was halotolerant and utilized H(2)-CO(2), 2-propanol-CO(2), or formate as a sole methanogenic substrate. Based on the 16S rRNA gene sequence similarity (94.8%) with Methanospirillum hungateii, and on the physiological and phenotypic characteristics, TM20-1 was suggested to be a newly identified species belonging to the genus Methanospirillum. This is the first report of isolation of the genus Methanospirillum strain from a rice paddy field.     

Diversity, abundance and novel 16S rRNA gene sequences of methanogens in rumen liquid, solid and epithelium fractions of Jinnan cattle

Three methanogen 16S rRNA gene clone libraries were constructed from liquid (LM), solid (SM) and epithelium (EM) fractions taken from the rumen of Jinnan cattle in China. After the amplification by PCR using methanogen-specific primers Met86F and Met1340R, equal quantities of PCR products from the same fractions from each of the four cattle were mixed together and used to construct the three libraries. Sequence analysis showed that the 268 LM clones were divided into 35 phylotypes with 18 sequences of phylotypes affiliated with the genus Methanobrevibacter (84.3% of clones). The 135 SM clones were divided into 19 phylotypes with 11 phylotypes affiliated with the genus Methanobrevibacter (77.8%). The 267 EM clones were divided into 33 phylotypes with 15 phylotypes affiliated with the genus Methanobrevibacter (77.2%). Clones closely related to Methanomicrobium mobile and Methanobrevibacter wolinii were only found in the LM library, and those to Methanobrevibacter ruminantium and Methanobrevibacter gottschalkii only in the SM library. LM library comprised 12.4% unidentified euryarchaeal clones, SM library 23.7% and EM library 25.5%, respectively. Five phylotypes (accession number: EF055528 and EF055531-EF055534) did not belong to the Euryarchaeota sequences we had known. One possible new genus (represented by phylotype E17, accession number EF055528) belonging to Methanobacteriaceae was identified from EM library. Quantitative real-time PCR for the first time revealed that epithelium fraction had significantly higher density of methanogens, with methanogenic mcrA gene copies (9.95 log 10 (copies per gram of wet weight)) than solid (9.26, P < 0.01) and the liquid (8.44, P < 0.001). The three clone libraries also appeared different in Shannon index (EM library 2.12, LM library 2.05 and SM library 1.73). Our results showed that there were apparent differences in the methanogenic diversity and abundance in the three different fractions within the rumen of Jinnan cattle, with Methanobrevibacter species predominant in all the three libraries and with epithelium fraction having more unknown species and higher density of methanogens.     

Genomic suppression subtractive hybridization as a tool to identify differences in mycorrhizal fungal genomes

Understanding the ecology of methanogens in natural and engineered environments is a prerequisite to predicting or managing methane emissions. In this study, a novel high-throughput fingerprint method was developed for determining methanogen diversity and relative abundance within environmental samples. The method described here, designated amplicon length heterogeneity PCR of the mcrA gene (LH-mcrA), is based on the natural length variation in the mcrA gene. The mcrA gene encodes the alpha-subunit of the methyl-coenzyme M reductase, which is involved in the terminal step of methane production by methanogens. The methanogenic communities from stored swine and dairy manures were distinct from each other. To validate the method, methanogenic communities in a plug flow-type bioreactor (PFBR) treating swine manure were characterized using LH-mcrA method and correlated to mcrA gene clone libraries. The diversity and relative abundance of the methanogenic groups were assessed. Methanobrevibacter, Methanosarcinaceae, Methanoculleus, Methanogenium, Methanocorpusculum and one unidentified group were assigned to particular LH-mcrA amplicons. Particular phylotypes related to Methanoculleus were predominant in the last compartment of the PFBR where the bulk of methane was produced. LH-mcrA method was found to be a reliable, fast and cost-effective alternative for diversity assessment of methanogenic communities in microbial systems.     

Hydrogen cycling in a three-tiered food web growing on the methanogenic conversion of 3-chlorobenzoate

Abstract A defined 3-chlorobenzoate-degrading methanogenic consortium was constructed by recombining key organisms isolated from a 3-chlorobenzoate-degrading methanogenic sludge enrichment. The organisms comprise a three-tiered food chain which includes: (1) reductive dechlorination of 3-chlorobenzoate; (2) oxidation of benzoate to acetate, H₂ and CO₂; (3) removal of H₂ plus CO₂ by conversion into methane. The defined consortium, consisting of a dechlorinating organism (DCB-1), a benzoate degrader (BZ-1) and a lithotrophic methanogen ( Methanospirillum strain PM-1) grew well in a basal salts medium supplemented with 3-chlorobenzoate (3.2 mM) as the sole energy source. The chlorine released from the aromatic ringe was recovered in stoichiometric amounts as the chloride ion. The reducing power required for reductive dechlorination was obtained from the hydrogen produced in the acetogenic oxidation of benzoate. One-third of the benzoate-derived hydrogen was recycled via the reductive dechlorination of 3-chlorobenzoate, indicating that the consortium operated as a food web rather than a food chain.     

Effect of sulfur source on the performance and metal retention of methanol-fed UASB reactors

The effect of a sulfur source on the performance and metal retention of methanol-fed upflow anaerobic sludge bed (UASB) reactors was investigated. For this purpose, two UASB reactors were operated with cobalt preloaded granular sludge (1 mM CoCl2; 30 degrees C; 24 h) at an organic loading rate (OLR) of 5 g COD.L reactor(-1).d(-1). One UASB reactor (R1) was operated without a sulfur source in the influent during the first 37 days. In this period the methanol conversion to methane remained very poor, apparently due to the absence of a sulfur source, because once cysteine, a sulfur-containing amino acid, was added to the influent of R1 (day 37) a full conversion of methanol to methane occurred within 6 days. The second reactor (R2) was operated with sulfate (0.41 mM) in the influent during the first 86 days of operation, during which no limitation in the methanol conversion to methane manifested. Cobalt washed out from the sludge at similar rates in both reactors. The leaching of cobalt occurred at two distinct rates, first at a high rate of 22 microg.g TSS(-1).d(-1), which proceeded mainly from the exchangeable and carbonate fraction and later at a relatively slow rate of 9 mug.g TSS(-1).d(-1) from the organic/sulfide fraction. This study showed that the supply of the sulfur source L-cysteine has a pronounced positive effect on the methanogenic activity and the retention of metals such as iron, zinc and molybdenum.     

Some studies on UASB bioreactors for the stabilization of low strength industrial effluents

The suitability of two stage biomethanation process using upflow anaerobic sludge blanket (UASB) bioreactors was studied for the treatment of low strength industrial effluents like rice mill wastewater. Maximum VFA yield was 0.75 mg (as acetic acid) per mg of COD consumed at a flow rate of 25 ml/min. Hydraulic retention time (HRT) of 1 hr was found suitable for acidification process. In the methanogenic reactor, the overall BOD and COD reductions were 89% and 78% respectively at loading rate of 3 kg COD m^х d^у, and HRT of 30 hrs. Gas yield in methanogenic reactor was 0.56 lits. per kg COD consumed which contains 62% v/v methane.     

Kinetics of rice straw methane fermentation

Summary The kinetics of anaerobic fermentation of rice straw to methane were studied. Rice straw was the only carbon source at influent volatile solid concentrations of 18.9 and 37.8 g/l. Semicontinous runs were carried out at 37°C in laboratory scale perfectly mixed reactors. The Contois' kinetic model constants were calculated from the experimental data. Arefrac tory coefficient was measured (R=0.374) to account for the nonbiodegradable portion of the organic matter of rice straw and incorporated into the kinetic equations. The predicted values of effluent substrate concentration, volumetric methane yield, volumetric methane production rate, and biodegradable conversion efficiency fit well with those measured experi mentally.Percent destruction values of feed constituents were measured.     

Biodegradability and toxicity of styrene in the anaerobic digestion process

Start-up and operation of an Upflow Anaerobic Sludge Blanket (UASB) reactor fed with an industrial effluent from a polymer synthesis plant containing 6 mg styrene l^–1 was unstable. In batch assays with 200 mg styrene l^–1, 74% of styrene was degraded at a rate of 7 ml methane g^–1 volatile suspended solids.day, without a lag phase. The toxicity limit (IC₅₀) of styrene was 1.4 mM for the acetoclastic activity, 0.45 and 1.6 mM for the methanogenic activity in the presence of 30 mM of propionate and ethanol respectively. Instability of UASB operation was attributed to other compounds such as acrylates or detergents present in the industrial effluent.