Diversity and activity of enriched ruminal cultures of anaerobic fungi and methanogens grown together on lignocellulose in consecutive batch culture

Consecutive batch cultures (CBC), involving nine serial transfers at 3, 5 and 7 d intervals (21, 45 and 63 d, respectively) were established to enrich for plant fibre degrading co-cultures of anaerobic fungi and methanogens from rumen digesta. Microbial diversity and fermentation end-products were measured at appropriate intervals over each CBC time-course. While methanogenic populations remained diverse, anaerobic fungal diversity was related to transfer interval and appeared to decrease with increasing transfer number. Acetate was the principal aqueous fermentation end-product with minimal quantities of lactate and formate detected. Methane and carbon dioxide were detected in the gaseous head-space of all co-cultures and the total amounts of gas generated per transfer was greater with transfer intervals of 5 and 7 d compared with a 3 d interval, although the 3 d interval tended to be more efficient per unit time. In conclusion, rapidly growing, methane producing co-cultures of anaerobic fungi and methanogens from rumen digesta were easy to establish on lignocellulose (barley straw) and maintain over considerable time periods. These results suggest such co-cultures have potential in industrial scale anaerobic digestion (AD) of highly fibrous substrates, which are resistant to degradation in conventional AD plants.     

Enhancement of bioenergy production from organic wastes by two-stage anaerobic hydrogen and methane production process

The present study investigated a two-stage anaerobic hydrogen and methane process for increasing bioenergy production from organic wastes. A two-stage process with hydraulic retention time (HRT) 3 d for hydrogen reactor and 12 d for methane reactor, obtained 11% higher energy compared to a single-stage methanogenic process (HRT 15 d) under organic loading rate (OLR) 3 gVS/(L d). The two-stage process was still stable when the OLR was increased to 4.5 gVS/(L d), while the single-stage process failed. The study further revealed that by changing the HRT_hydrogen:HRT_methane ratio of the two-stage process from 3:12 to 1:14, 6.7%, more energy could be obtained. Microbial community analysis indicated that the dominant bacterial species were different in the hydrogen reactors (Thermoanaerobacterium thermosaccharolyticum-like species) and methane reactors (Clostridiumthermocellum-like species). The changes of substrates and HRT did not change the dominant species. The archaeal community structures in methane reactors were similar both in single- and two- stage reactors, with acetoclastic methanogens Methanosarcina acetivorans-like organisms as the dominant species.     

厌氧真菌和甲烷菌共培养的研究进展

厌氧真菌是自然界中降解植物纤维素类物质最高效的微生物之一.近年来,大量厌氧真菌和甲烷菌共培养菌株被分离.共培养中,甲烷菌通过对厌氧真菌代谢产物的利用显著提高厌氧真菌对木质纤维素的降解;厌氧真菌通过为甲烷菌提供能量和营养物质使甲烷菌快速生成大量甲烷.全面深入地了解共培养中两者的互作关系以及共培养降解木质纤维素产甲烷的特性...     

Performance and microbial community analysis of two-stage process with extreme thermophilic hydrogen and thermophilic methane production from hydrolysate in UASB reactors

The two-stage process for extreme thermophilic hydrogen and thermophilic methane production from wheat straw hydrolysate was investigated in up-flow anaerobic sludge bed (UASB) reactors. Specific hydrogen and methane yields of 89 ml-H₂/g-VS (190 ml-H₂/g-sugars) and 307 ml-CH₄/g-VS, respectively were achieved simultaneously with the overall VS removal efficiency of 81% by operating with total hydraulic retention time (HRT) of 4 days . The energy conversion efficiency was dramatically increased from only 7.5% in the hydrogen stage to 87.5% of the potential energy from hydrolysate, corresponding to total energy of 13.4 kJ/g-VS. Dominant hydrogen-producing bacteria in the H₂-UASB reactor were Thermoanaerobacter wiegelii, Caldanaerobacter subteraneus, and Caloramator fervidus. Meanwhile, the CH₄-UASB reactor was dominated with methanogens of Methanosarcina mazei and Methanothermobacter defluvii. The results from this study suggest the two stage anaerobic process can be effectively used for energy recovery and for stabilization of hydrolysate at anaerobic conditions.     

Characterization of wheat straw-degrading anaerobic alkali-tolerant mixed cultures from soda lake sediments by molecular and cultivation techniques

Alkaline pretreatment has the potential to enhance the anaerobic digestion of lignocellulosic biomass to biogas. However, the elevated pH of the substrate may require alkalitolerant microbial communities for an effective digestion. Three mixed anaerobic lignocellulolytic cultures were enriched from sediments from two soda lakes with wheat straw as substrate under alkaline (pH 9) mesophilic (37°C) and thermophilic (55°C) conditions. The gas production of the three cultures ceased after 4 to 5 weeks, and the produced gas was composed of carbon dioxide and methane. The main liquid intermediates were acetate and propionate. The physiological behavior of the cultures was stable even after several transfers. The enrichment process was also followed by molecular fingerprinting (terminal restriction fragment length polymorphism) of the bacterial 16S rRNA gene and of the mcrA/mrtA functional gene for methanogens. The main shift in the microbial community composition occurred between the sediment samples and the first enrichment, whereas the structure was stable in the following transfers. The bacterial communities mainly consisted of Sphingobacteriales, Clostridiales and Spirochaeta, but differed at genus level. Methanothermobacter and Methanosarcina genera and the order Methanomicrobiales were predominant methanogenes in the obtained cultures. Additionally, single cellulolytic microorganisms were isolated from enrichment cultures and identified as members of the alkaliphilic or alkalitolerant genera. The results show that anaerobic alkaline habitats harbor diverse microbial communities, which can degrade lignocellulose effectively and are therefore a potential resource for improving anaerobic digestion.     

Anaerobic digestion of slaughterhouse waste: main process limitations and microbial community interactions

Fresh pig/cattle slaughterhouse waste mixtures, with different lipid-protein ratios, were characterized and their anaerobic biodegradability assessed in batch tests. The resultant methane potentials were high (270-300 L_CH4 kg⁻¹_COD) making them interesting substrates for the anaerobic digestion process. However, when increasing substrate concentrations in consecutive batch tests, up to 15 g_COD kg⁻¹, a clear inhibitory process was monitored. Despite the reported severe inhibition, related to lipid content, the system was able to recover activity and successfully degrade the substrate. Furthermore, 16SrRNA gene-based DGGE results showed an enrichment of specialized microbial populations, such as β-oxidizing/proteolitic bacteria (Syntrophomonas sp., Coprothermobacter sp. and Anaerobaculum sp.), and syntrophic methanogens (Methanosarcina sp.). Consequently, the lipid concentration of substrate and the structure of the microbial community are the main limiting factors for a successful anaerobic treatment of fresh slaughterhouse waste.     

Effects of nitrate on methane production, fermentation, and microbial populations in in vitro ruminal cultures

The objective of this study was to examine the effects of nitrate on methane production, important fermentation characteristics, Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens, total bacteria, and methanogens using in vitro ruminal cultures. Potential adaptation of the above microbes and persistency of nitrate to mitigate CH₄ production were also evaluated. Methane production was reduced by 70% at 12 μmol ml⁻¹ and nearly completely at ?24 μmol ml⁻¹ nitrate. Production of volatile fatty acids (VFAs) was affected to different extents at different nitrate concentrations. Over a series of six consecutive cultures receiving 12 μmol ml⁻¹nitrate, production of CH₄ and VFA did not change significantly. R. albus and R. flavefaciens seemed to adapt to nitrate, while F. succinogenes and methanogens did not. Nitrate may be used in achieving persistent mitigation of CH4 production by ruminants.     

闽江口芦苇沼泽湿地土壤产甲烷菌群落结构的垂直分布

应用PCR-RFLP技术及测序分析对闽江口芦苇湿地土壤产甲烷菌群落结构的垂直分布特征进行了研究。在构建的6个克隆文库中,每个克隆文库随机挑选100个克隆进行菌落PCR验证,共得到591个阳性克隆。PCR产物经限制性内切酶MspⅠ进行RFLP分析后得到37个不同的分类操作单元(OTUs)。对37个克隆子进行了序列测定,与GenBank数据库中的序列进行比对,最近相似性在91%—99%之间。RFLP分析和系统发育分析表明,闽江口芦苇湿地土壤中产甲烷菌群落包括3大类群:甲烷杆菌目(Methanobacteriales)、甲烷微菌目(Methanomirobiales)和甲烷八叠球菌目(Methanosarcinales)。不同土壤深度中产甲烷菌群落的分布呈现出不同的特征。土壤表层(0—10 cm)优势产甲烷菌类群为Methanoregula,约占76%;10—20 cm土层主要的产甲烷菌类群为Methanolinea和Methanoregula,分别约占23%和29%;20—30cm土层优势的产甲烷菌类群为Methanolinea,约占66%。Shannon指数(H’)和Simpson多样性指教(D)表明,10—20cm土层产甲烷菌多样性高于土壤表层(0—10 cm)和20—30 cm土层。37个测序OTUs中有26个OTUs属于不可培养的产甲烷菌序列,表明闽江口芦苇湿地土壤中存在大量不可培养的产甲烷菌。     

Occurrence of Methanogenic Archaea in Highly Polluted Sediments of Tropical Santos–São Vicente Estuary (São Paulo, Brazil)

Little is known about the ability of methanogens to grow and produce methane in estuarine environments. In this study, traditional methods for cultivating strictly anaerobic microorganisms were combined with Fluorescence in situ hybridization (FISH) technique to enrich and identify methanogenic Archaea cultures occurring in highly polluted sediments of tropical Santos–São Vicente Estuary (São Paulo, Brazil). Sediment samples were enriched at 30°C under strict anaerobic and halophilic conditions, using a basal medium containing 2% of sodium chloride and amended with glucose, methanol, and sodium salts of acetate, formate and lactate. High methanogenic activity was detected, as evidenced by the biogas containing 11.5 mmol of methane at 20 days of incubation time and methane yield of 0.138-mmol CH₄/g organic matter/g volatile suspense solids. Cells of methanogenic Archaea were selected by serial dilution in medium amended separately with sodium acetate, sodium formate, or methanol. FISH analysis revealed the presence of Methanobacteriaceae and Methanosarcina sp. cells.     

Nutritional characterization of Mucuna pruiriens: 2. In vitro ruminal fluid fermentability of Mucuna pruriens,Mucunal-dopa and soybean meal incubated with or without l-dopa

Three in vitro experiments were conducted to determine the rumen fermentability of Mucuna (M) pruriens (24 g 3,4-dihydroxy-l-phenylalanine (l-dopa)/kg dry matter (DM) and soybean meal treated with (SBD) or without (SB) 138 g l-dopa/kg DM). Additional objectives were to determine if l-dopa inhibits rumen fermentation, and if ruminal microbes can adapt to l-dopa or M. In Experiment 1, ground (1 mm) substrates were incubated in triplicate at 38 °C in 9 ml nutrient media and 1 ml rumen fluid in a series of six, 48 h, consecutive batch cultures. The first culture was inoculated with rumen fluid from two donor cows. Subsequent cultures were inoculated with fluid (1 ml) from the previous culture. The DM digestibility (DMD, 616 g/kg vs. 540 g/kg; P<0.01) and gas production (51.7 ml/g vs. 44.2 ml/g DM; P<0.05) were higher from fermentation of M versus SB but similar for SB and SBD (540 g/kg vs. 554 g/kg and 44.2 ml/g DM vs. 43.5 ml/g DM, respectively). The slopes of the relationships between DMD (g/kg) or gas production (ml/g DM) and fermentation period were not reduced by fermenting M (−0.014 DMD slope; 2.28 gas production slope) or SBD (−0.014 DMD slope; 0.459 gas production slope), instead of SB (−0.002 DMD slope; 1.039 gas production slope), indicating microbial adaptation to M and SBD. Total volatile fatty acid concentration (VFA; 53.7, 54.9 and 54.9 mmol/l) and molar proportions of VFA were similar among substrates. Gas production kinetics of M versus SB (Experiment 2), and SB versus SBD (Experiment 3) were also measured after substrates were incubated in triplicate in buffered rumen fluid for 24 h using a non-linear exponential model to fit the data. Residual l-dopa was measured after separate fermentation of substrates in triplicate for 0, 4, 8, 16 and 24 h. Fermentation of M versus SB produced more (P<0.05) gas (250 ml/g vs. 100 ml/g DM) and total VFA (203 mmol/l vs. 180 mmol/l) and a lower (P<0.05) acetate:propionate ratio (1.35 vs. 1.87; P<0.05). Adding l-dopa to SB increased (P<0.01) gas production (92 ml/g DM vs. 200 ml/g DM), and total VFA concentration (132 mmol/l vs. 188 mmol/l), but reduced (P<0.05) gas production rate (0.08 ml/h vs. 0.05 ml/h). The concentration of l-dopa in fermented M and SBD decreased by 53 and 47%, respectively during fermentation. In conclusion, M was more fermented than SB and degradation of l-dopa during ruminal fermentation and microbial adaptation to l-dopa were confirmed. Adding l-dopa to SB did not impair ruminal fermentation.     

Biodegradation of phenanthrene and pyrene by Ganoderma lucidum

Biodegradation of two polycyclic aromatic hydrocarbons (PAHs), phenanthrene and pyrene, by a white rot fungus, Ganoderma lucidum, in broth cultures was investigated. It was found that the biomass of the organism decreased with the increase of PAH concentration in the cultures. In the cultures with 2 to 50 mg l⁻¹ PAHs, the degradation rate constants (k₁) increased with the PAH concentration, whereas, at the level of 100 mg l⁻¹, the degradation rate constants decreased. In the presence of 20 mg l⁻¹ PAHs, the highest degradation rates of both PAHs occurred in cultures with an initial pH of 4.0 at 30 °C. The addition of CuSO₄, citric acid, gallic acid, tartaric acid, veratryl alcohol, guaiacol, 2,2′-azino-bis-(3- ethylbenzothazoline-6-sulfonate) (ABTS) enhanced the degradation of both PAHs and laccase activities; whereas the supplement of oxalate, di-n-butyl phthalate (DBP), and nonylphenol (NP) decreased the degradation of both PAHs and inhibited laccase production. In conclusion, G. lucidum is a promising white rot fungus to degrade PAHs such as phenanthrene and pyrene in the environment.     

Effect of disodium fumarate on microbial abundance,ruminal fermentation and methane emission in goats under different forage : concentrate ratios

This study investigated the effects of disodium fumarate (DF) on methane emission, ruminal fermentation and microbial abundance in goats under different forage (F) : concentrate (C) ratios and fed according to maintenance requirements. Four ruminally fistulated, castrated male goats were used in a 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments and the main factors being the F : C ratios (41 : 59 or 58 : 42) and DF supplementation (0 or 10 g/day). DF reduced methane production (P < 0.05) on average by 11.9%, irrespective of the F : C ratio. The concentrations of total volatile fatty acids, acetate and propionate were greater in the rumen of goats supplemented with DF (P < 0.05), whereas the abundance of methanogens was lower (P < 0.05). In high-forage diets, the abundance of Selenomonas ruminantium, a fumarate-reducing bacterium, was greater in the rumen of goats supplemented with DF. The abundance of fungi, protozoa, Ruminococus flavefaciens and Fibrobacter succinogenes were not affected by the addition of DF. Variable F : C ratios affected the abundance of methanogens, fungi and R. flavefaciens (P < 0.05), but did not affect methane emission. The result implied that DF had a beneficial effect on the in vivo rumen fermentation of the goats fed diets with different F : C ratios and that this effect were not a direct action on anaerobic fungi, protozoa and fibrolytic bacteria, the generally recognized fiber-degrading and hydrogen-producing microorganisms, but due to the stimulation of fumarate-reducing bacteria and the depression of methanogens.     

Interactions of Taxol-producing endophytic fungus with its host (Taxus spp.) during Taxol accumulation

Endophytic fungi (Fusarium mairei) culture broth (EFCB) was added to cell suspension cultures of Taxus cuspidata. After 5 days, cultures of T. cuspidata given 4 ml of EFCB produced a maximal yield of 6.11 mg/l paclitaxel, with a release ratio of 75%, 2- and 6.8-fold, respectively, greater than the controls. The active element in EFCB is an exopolysaccharide of ∼79 kD. Endophytic fungi produced 0.19 mg/l of paclitaxel in its producing medium. However, when the supernatant of Taxus cell suspension cultures from day 20 was added to the paclitaxel-producing medium, the biomass of fungi decreased by 24% and the yield of paclitaxel by 45%. In a co-culture system of plant and fungus, the yield of paclitaxel (12.8 mg/l) was >2-fold higher than that in the EFCB-treatment system.     

Rapid start-up of thermophilic anaerobic digestion with the turf fraction of MSW as inoculum

This study aims to determine suitable start-up conditions and inoculum sources for thermophilic anaerobic digestion. Within days of incubation MSW at 55 °C, methane was produced at a high rate. In an attempt to narrow down which components of typical MSW contained the thermophilic methanogens, vacuum cleaner dust, banana peel, kitchen waste, and garden waste were tested as inoculum for thermophilic methanogenesis with acetate as the substrate. Results singled out grass turf as the key source of thermophilic acetate degrading methanogenic consortia. Within 4 days of anaerobic incubation (55 °C), anaerobically incubated grass turf samples produced methane accompanied by acetate degradation enabling successful start-up of thermophilic anaerobic digestion. Other essential start-up conditions are specified. Stirring of the culture was not conducive for successful start-up as it resulted specifically in propionate accumulation.     

Bioenergetics and anaerobic respiratory chains of aceticlastic methanogens

Methane-forming archaea are strictly anaerobic microbes and are essential for global carbon fluxes since they perform the terminal step in breakdown of organic matter in the absence of oxygen. Major part of methane produced in nature derives from the methyl group of acetate. Only members of the genera Methanosarcina and Methanosaeta are able to use this substrate for methane formation and growth. Since the free energy change coupled to methanogenesis from acetate is only − 36 kJ/mol CH₄, aceticlastic methanogens developed efficient energy-conserving systems to handle this thermodynamic limitation. The membrane bound electron transport system of aceticlastic methanogens is a complex branched respiratory chain that can accept electrons from hydrogen, reduced coenzyme F₄₂₀ or reduced ferredoxin. The terminal electron acceptor of this anaerobic respiration is a mixed disulfide composed of coenzyme M and coenzyme B. Reduced ferredoxin has an important function under aceticlastic growth conditions and novel and well-established membrane complexes oxidizing ferredoxin will be discussed in depth. Membrane bound electron transport is connected to energy conservation by proton or sodium ion translocating enzymes (F₄₂₀H₂ dehydrogenase, Rnf complex, Ech hydrogenase, methanophenazine-reducing hydrogenase and heterodisulfide reductase). The resulting electrochemical ion gradient constitutes the driving force for adenosine triphosphate synthesis. Methanogenesis, electron transport, and the structure of key enzymes are discussed in this review leading to a concept of how aceticlastic methanogens make a living. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Performance and spatial community succession of an anaerobic baffled reactor treating acetone-butanol-ethanol fermentation wastewater

An anaerobic baffled reactor with four compartments (C1-C4) was successfully used for treatment of acetone-butanol-ethanol fermentation wastewater and methane production. The chemical oxygen demand (COD) removal efficiency was 88.2% with a CH₄ yield of 0.25 L/(g COD_removed) when organic loading rate (OLR) was 5.4 kg COD m⁻³ d⁻¹. C1 played the most important role in solvents (acetone, butanol and ethanol) and COD removal. Community structure of C2 was similar to that in C1 at stage 3 with higher OLR, but was similar to those in C3 and C4 at stages 1-2 with lower OLR. This community variation in C2 was consistent with its increased role in COD and solvent removal at stage 3. During community succession from C1 to C4 at stage 3, abundance of Firmicutes (especially OTUs ABRB07 and ABRB10) and Methanoculleus decreased, while Bacteroidetes and Methanocorpusculum became dominant. Thus, ABRB07 coupled with Methanoculleus and/or acetogen (ABRB10) may be key species for solvents degradation.     

Effect of sludge age on methanogenic and glycogen accumulating organisms in an aerobic granular sludge process fed with methanol and acetate

The influence of sludge age on granular sludge formation and microbial population dynamics in a methanol- and acetate-fed aerobic granular sludge system operated at 35°C was investigated. During anaerobic feeding of the reactor, methanol was initially converted to methane by methylotrophic methanogens. These methanogens were able to withstand the relatively long aeration periods. Lowering the anaerobic solid retention time (SRT) from 17 to 8 days enabled selective removal of the methanogens and prevented unwanted methane formation. In absence of methanogens, methanol was converted aerobically, while granule formation remained stable. At high SRT values (51 days), γ-Proteobacteria were responsible for acetate removal through anaerobic uptake and subsequent aerobic growth on storage polymers formed [so called metabolism of glycogen-accumulating organisms (GAO)]. When lowering the SRT (24 days), Defluviicoccus-related organisms (cluster II) belonging to the α-Proteobacteria outcompeted acetate consuming γ-Proteobacteria at 35°C. DNA from the Defluviicoccus-related organisms in cluster II was not extracted by the standard DNA extraction method but with liquid nitrogen, which showed to be more effective. Remarkably, the two GAO types of organisms grew separately in two clearly different types of granules. This work further highlights the potential of aerobic granular sludge systems to effectively influence the microbial communities through sludge age control in order to optimize the wastewater treatment processes.     

Bioenergy potential of Ulva lactuca: biomass yield, methane production and combustion

The biomass production potential at temperate latitudes (56°N), and the quality of the biomass for energy production (anaerobic digestion to methane and direct combustion) were investigated for the green macroalgae, Ulva lactuca. The algae were cultivated in a land based facility demonstrating a production potential of 45 T (TS) ha⁻¹ y⁻¹. Biogas production from fresh and macerated U. lactuca yielded up to 271 ml CH₄ g⁻¹ VS, which is in the range of the methane production from cattle manure and land based energy crops, such as grass-clover. Drying of the biomass resulted in a 5-9-fold increase in weight specific methane production compared to wet biomass. Ash and alkali contents are the main challenges in the use of U. lactuca for direct combustion. Application of a bio-refinery concept could increase the economical value of the U. lactuca biomass as well as improve its suitability for production of bioenergy.     

Characteristics of hydrogen and methane production from cornstalks by an augmented two- or three-stage anaerobic fermentation process

This paper presents the co-production of hydrogen and methane from cornstalks by a two- or three-stage anaerobic fermentation process augmented with effective artificial microbial community. Two-stage fermentation by using the anaerobic sludge and DGGE analysis showed that effective and stable strains should be introduced into the system. We introduced Enterobacter aerogens or Clostridium paraputrificum into the hydrogen stage, and C. paraputrificum was proven to be more effective. In the three-stage process consisting of the improved hydrolysis, hydrogen and methane production stages, the highest soluble sugars (0.482 kg/kg cornstalks) were obtained after the introduction of Clostridium thermocellum in the hydrolysis stage, under the thermophilic (55 °C) and acidic (pH 5.0) conditions. Hydrolysates from 1 kg of cornstalks could produce 2.61 mol (63.7 l) hydrogen by augmentation with C. paraputrificum and 4.69 mol (114.6 l) methane by anaerobic granular sludge, corresponding to 54.1% energy recovery.