Prokaryotic communities of the north-eastern Mongolian soda lakes

We have studied the activity and composition of several geochemically significant physiological groups of bacteria in more than twenty alkaline salt lakes of the north-east Mongolia steppe with water salinity from 3 to 390 g l^?1 and pH values ranging from 9.0 to 10.6. Active and diverse microbial communities have been found in most of the lakes. The methanotrophic bacteria were represented by the Type I members. Among the culturable forms of sulfur-oxidizing bacteria obligately chemolithoautotrophic and haloalkaliphilic representatives of the genera Thioalkalimicrobium and Thioalkalivibrio were detected in the sediments at high numbers (up to 10⁶ cells ml^?1). The largest population of anaerobic phototrophic bacteria was represented by purple sulfur bacteria of the Ectothiorhodospiraceae family. Salinity was the key factor in determining the activity and the composition of the microbial communities. The most diverse and active prokaryotic populations, including aerobic and anaerobic phototrophic, methanogenic, methanotrophic, sulfur-oxidizing, sulfate-reducing and nitrifying bacteria, were found in lakes with salinity less than 60 g l^?1. In hypersaline lakes with a salinity >100 g l^?1, the sulfur cycle remained active due to the activity of extremely halotolerant and alkaliphilic sulfur bacteria, while other important functional groups responsible for nitrification and methane oxidation processes were not detected. Overall, the prokaryotic communities of the Mongolian alkaline salt lakes represent an interesting new example of a diverse community of haloalkaliphilic bacteria well adopted to a broad salinity range.     

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.     

Microbial Ecology in Anaerobic Digestion at Agitated and Non-Agitated Conditions

To investigate the distribution and dynamics of microbial community in anaerobic digestion at agitated and non-agitated condition, 454 pyrosequencing of 16s rRNA was conducted. It revealed the distinct community compositions between the two digesters and their progressive shifting over time. Methanogens and syntrophic bacteria were found much less abundant in the agitated digester, which was mainly attributed to the presence of bacterial genera Acetanaerobacterium and Ruminococcus with relatively high abundance. The characterization of the microbial community corroborated the digestion performance affected at the agitated condition, where lower methane yield and delayed methane production rate were observed. This was further verified by the accumulation of propionic acid in the agitated digester.     

Biomethanation of white rotted and brown rotted rice straw

Biomethanation of white rotted and brown rotted rice straw was taken for the present investigation and their efficiency on biomethanation has been tested. Rice straw was treated with white rot fungus Phanerochaete chrysosporium (P_C) and brown rot fungus Polyporus ostreiformis (P_O). Biogas and methane production was increased by about 34.73% and 46.19% in P_C-treated straw and 21.12% and 31.94% in P_O-treated straw respectively. VFA production has also been increased in P_C and P_O treated straw compared to control straw which were 76.73% and 30.69% respectively. Reduction of COD has also been found during biomethanation. The rate of reduction of COD during the initial period of digestion was 59.01%, 55.55% and 26.00% in P_C-treated, P_O-treated and control straw respectively after 21 days of digestion.     

Seasonal variation in methane flux from rice paddies associated with methane concentration in soil water,rice biomass and temperature,and its modelling

To attempt to develop physicochemical and physiological modelling for methane transport from the rhizosphere to the atmosphere through rice plants, methane flux, methane concentration in the soil water, and the biomass of rice were measured in lysimeter rice paddies (2.5 × 4 m, depth 2.0 m) once per week throughout the entire growing season in 1992 at Tsukuba, Japan. The addition of exogenous organic matter (rice straw) or soil amendments with the presence or absence of vegetation were also examined for their influence on methane emissions. The total methane emission over the growing season varied from 3.2 g CH₄ m^-2 y^-1 without the addition of rice straw to 49.7 g CH₄ m^-2 y^-1 with rice straw and microbiological amendment. In the unvegetated plot with the addition of rice straw, there was much ebullition of gas bubbles, particularly in the summer. The annual methane emission due to the ebullition of gas bubbles,from the unvegetated plot with the addition of rice straw was estimated to be almost the same as that from the vegetated site with the addition of rice straw. In the early growth stage, the methane flux can be analyzed by the diffusion model (Flux=Methane concentration × Conductance of rice body) using parameters for methane concentration in the soil water as a difference in concentration between the atmosphere and the rhizosphere, and for the biomass of rice as a conductance of rice body. On the other hand, although the diffusion model was inapplicable to a large extent from the middle to late growth stage, methane flux could be estimated by air temperature and concentration in the soil water. Thus, methane transport from the rhizosphere to the atmosphere through rice plants consisted of two phases: one was an explainable small part by diffusion in rice body; the other was a large part strongly, governed by air temperature. The existence of gas bubbles in the soil may be related to the transition between the two phases     

Treatment of coffee waste by slurry-state anaerobic digestion

Slurries containing 20% (w/v) coffee waste solids were treated anaerobically in one- and two-phase thermophilic methane fermentation systems (53°C) with or without pH control. In one-phase methane fermentation using a roller bottle reactor, the maximum gas evolution rate of 0.87 l/l·d was achieved during treatment for 91 d. However, this one-phase methane fermentation did not yield reproducible data. In a two-phase methane fermentation system consisting of a completely stirred tank reactor type (CSTR-type) liquefaction reactor without pH control and an anaerobic fluidized bed type gasification reactor, three-repetitions of treatment were conducted. Each treatment was very stable and the average gas evolution rate per volume of the gasification reactor was about 2.4 l/l·d. Two-repetitions of treatment were then done while controlling pH in the liquefaction at more than 6. The average gas evolution rate per volume of gasification reactor was found to have increased to 10.2 l/l·d, a value which corresponded to 0.84 l/l·d per total volume, including the liquefaction reactor. It was observed that treatment in a two-phase methane fermentation could be repeated in a stable fashion even in the closed system without discharging anything but the coffee waste residues.     

Inhibition of hydrogen sulfide, methane, and total gas production and sulfate-reducing bacteria in in vitro swine manure by tannins, with focus on condensed quebracho tannins

Management practices from large-scale swine production facilities have resulted in the increased collection and storage of manure for off-season fertilization use. Odor and emissions produced during storage have increased the tension among rural neighbors and among urban and rural residents. Production of these compounds from stored manure is the result of microbial activity of the anaerobic bacteria populations during storage. In the current study, the inhibitory effects of condensed quebracho tannins on in vitro swine manure for reduction of microbial activity and reduced production of gaseous emissions, including the toxic odorant hydrogen sulfide produced by sulfate-reducing bacteria (SRB), was examined. Swine manure was collected from a local swine facility, diluted in anaerobic buffer, and mixed with 1 %?w/v fresh feces. This slurry was combined with quebracho tannins, and total gas and hydrogen sulfide production was monitored over time. Aliquots were removed periodically for isolation of DNA to measure the SRB populations using quantitative PCR. Addition of tannins reduced overall gas, hydrogen sulfide, and methane production by greater than 90 % after 7 days of treatment and continued to at least 28 days. SRB population was also significantly decreased by tannin addition. qRT-PCR of 16S rDNA bacteria genes showed that the total bacterial population was also decreased in these incubations. These results indicate that the tannins elicited a collective effect on the bacterial population and also suggest a reduction in the population of methanogenic microorganisms as demonstrated by reduced methane production in these experiments. Such a generalized effect could be extrapolated to a reduction in other odor-associated emissions during manure storage.     

秸秆蓝藻混合发酵产沼气过程中微生物群落分析

采用实验室自制秸秆蓝藻混合厌氧反应装置进行沼气发酵实验,利用16S rRNA基因克隆文库的方法,对不同产气阶段的细菌和古菌的优势菌群进行多样性研究。结果表明:(1)不同产气阶段细菌优势种类存在差异,供试秸秆沼气反应器阶段细菌种类较为丰富,分属于6个门:产气初始阶段优势菌群为变形菌门(Proteobacteria),相对丰度为51.76%;产气高峰阶段优势菌群为厚壁菌门(Firmicutes),相对丰度为47.13%;产气结束阶段优势菌群为厚壁菌门(Firmicutes),相对丰度为28.89%;此外,还包括绿弯菌门、螺旋体门、绿菌门的细菌。(2)古菌种类明显少于细菌,均属于甲烷微菌纲(Methanomicrobia)、甲烷杆菌纲(Methanobacteria)和热原体纲(Thermoplasmata)。秸秆蓝藻沼气系统微生物群落结构的阐明具有一定的意义,可为秸秆沼气工程调控提供科学依据。     

Methane emissions associated with a green manure amendment to flooded rice in California

The goals of sustainable food production and mitigation of greenhouse gas emissions may be in conflict when green manures are used in flooded rice systems. A field study was initiated in early spring 1992 near Sacramento, California to quantify the potential for enhanced methane emissions following a green manure amendment to rice. Replicate flux measurements were made twice a day every 3–4 days throughout the growing season in four treatment plots: burned rice straw, spring incorporated rice straw, burned straw plus purple vetch and spring incorporated straw plus vetch. Seasonal methane emissions ranged from 66–136 g CH₄ m⁻² and were 1.5 to 1.8 times higher from the straw plus vetch treatments relative to the straw only treatments. No significant differences in emissions were found between the two straw only treatments or the straw plus vetch treatments. Methane fluxes were exponentially related to soil temperature, but no effect of redox potential or floodwater depth were observed. The potential impact of these results on the global methane budget is discussed.     

Liquefaction and gasification during anaerobic digestion of coffee waste by two-phase methane fermentation with slurry-state liquefaction

In order to improve the gas evolution rate during anaerobic digestion of coffee waste by two-phase methane fermentation with slurry-state liquefaction, the liquefaction and gasification processes were separately investigated. In the liquefaction process (including the acidification process), treatment at a pH above 6 had no major effects on the liquefaction and acidification rates. However, the VFA production rates were 880 and 320 mg/l·d during mesophilic (37°C) and thermophilic (53°C) liquefaction, respectively. Mesophilic conditions were superior to thermophilic conditions in the liquefaction. With respect to the gasification process, a high TOC volumetric loading rate of 21 g/l·d was achieved during thermophilic gasification. However, the mesophilic gasification did not yield stable data, even at a low TOC volumetric loading rate of 2 g/l·d. The gas yield was 1.7 l/g TOC consumed during thermophilic gasification. The thermophilic liquefaction and thermophilic gasification reactors were connected in series and a two-phase experiment was conducted with the reactors at various volumetric ratios. The maximum gas evolution rate of 1.43 l/l·d was achieved with a combination of a gasification reactor with a 0.45l working volume and liquefaction reactor with a 2l working volume. This rate was 1.7 times higher than the rate obtained in a previous study.     

Biochemical and Molecular Characterization of Potential Phosphate-Solubilizing Bacteria in Acid Sulfate Soils and Their Beneficial Effects on Rice Growth

A study was conducted to determine the total microbial population, the occurrence of growth promoting bacteria and their beneficial traits in acid sulfate soils. The mechanisms by which the bacteria enhance rice seedlings grown under high Al and low pH stress were investigated. Soils and rice root samples were randomly collected from four sites in the study area (Kelantan, Malaysia). The topsoil pH and exchangeable Al ranged from 3.3 to 4.7 and 1.24 to 4.25 cmol_c kg⁻¹, respectively, which are considered unsuitable for rice production. Total bacterial and actinomycetes population in the acidic soils were found to be higher than fungal populations. A total of 21 phosphate-solubilizing bacteria (PSB) including 19 N₂-fixing strains were isolated from the acid sulfate soil. Using 16S rRNA gene sequence analysis, three potential PSB strains based on their beneficial characteristics were identified (Burkholderia thailandensis, Sphingomonas pituitosa and Burkholderia seminalis). The isolated strains were capable of producing indoleacetic acid (IAA) and organic acids that were able to reduce Al availability via a chelation process. These PSB isolates solubilized P (43.65%) existing in the growth media within 72 hours of incubation. Seedling of rice variety, MR 219, grown at pH 4, and with different concentrations of Al (0, 50 and 100 µM) was inoculated with these PSB strains. Results showed that the bacteria increased the pH with a concomitant reduction in Al concentration, which translated into better rice growth. The improved root volume and seedling dry weight of the inoculated plants indicated the potential of these isolates to be used in a bio-fertilizer formulation for rice cultivation on acid sulfate soils.     

Effect of ducks on CH4 emission from paddy soils and its mechanism research in the rice-duck ecosystem

The effect of ducks on CH₄ emission from paddy soils and its mechanism were probed in order to decide the optimum number of ducks in the rice-duck ecosystem. Methane emission fluxes from paddy soils were measured by the static box technique. The correlations between methane emission and soil physical and chemical characteristics were also analyzed. The results showed that significant differences (p < 0.01) existed in the dissolved oxygen content of water body in the treatment fields, and the more the ducks, the higher the dissolved oxygen content. Secondly, the soil redox matter content and methanogenic bacteria population of the rice-duck ecosystem reduced more sharply than those of the no-duck rice farming, resulting in a lower methane production. Thirdly, the amount of methane emission differed between the treatments—the more the ducks, the less the methane emission. Other related analyses showed that the negative correlation was significant (p < 0.001) between the methane emission flux and dissolved oxygen content of water body. However, CH4 emission flux had significantly positive correlation (p < 0.01) with the soil redox matter content and rice field methanogenic population.     

芦岭煤田微生物群落结构和生物成因气的产甲烷类型研究

【目的】揭示芦岭煤田微生物群落组成,并分析其潜在的产甲烷类型及产甲烷途径。【方法】采集芦岭煤田的煤层气样品和产出水样品,分别分析样品的地球化学性质特征;利用Illumina HiSeq高通量测序技术分析产出水中的微生物群落结构;采用添加不同底物的厌氧培养实验进一步证实芦岭煤田生物成因气的产甲烷类型。【结果】该地区煤层气为生物成因和热成因的混合成因气;古菌16S rRNA基因分析表明在产出水中含有乙酸营养型、氢营养型和甲基营养型的产甲烷菌。丰度较高的细菌具有降解煤中芳香族和纤维素衍生化合物的潜力。厌氧富集培养结果表明,添加乙酸盐、甲酸盐、H2+CO2为底物的矿井水样均有明显的甲烷产生。【结论】芦岭煤田具有丰富的生物多样性,该地区同时存在三种产甲烷类型。本研究为利用微生物技术提高煤层气的采收率,实现煤层气的可持续开采提供科学依据。     

Polluting macrophytes Colombian lake Fúquene used as substrate by edible fungus Pleurotus ostreatus

Invasive aquatic plants from Lake Fúquene (Cundinamarca, Colombia), water hyacinth (Eichhornia crassipes C. Mart.) and Brazilian elodea (Egeria densa Planch.) have been removed mechanically from the lake and can be used for edible mushrooms production. The growth of the oyster mushroom (Pleurotus ostreatus) on these aquatic macrophytes was investigated in order to evaluate the possible use of fruiting bodies and spent biomass in food production for human and animal nutrition, respectively. Treatments included: water hyacinth, Brazilian elodea, sawdust, rice hulls and their combinations, inoculated with P. ostreatus at 3 %. Water hyacinth mixed with sawdust stimulated significantly fruiting bodies production (P = 3.3 × 10^?7) with 71 % biological efficacy, followed by water hyacinth with rice husk (55 %) and elodea with rice husk (48 %), all of these have protein contents between 26 and 47 %. Loss of lignin (0.9–21.6 %), cellulose (3.7–58.3 %) and hemicellulose (1.9–53.8 %) and increment in vitro digestibility (16.7–139.3 %) and reducing sugars (73.4–838.4 %) were observed in most treatments. Treatments spent biomass presented Relative Forage Values (RFV) from 46.1 to 232.4 %. The results demonstrated the fungus degrading ability and its potential use in aquatic macrophytes conversion biomass into digestible ruminant feed as added value to the fruiting bodies production for human nutrition.     

添加剂对水稻秸秆实地混合厌氧发酵产甲烷系统的作用

为提高水稻秸秆利用效率,改进水稻秸秆实地混合厌氧发酵产甲烷技术,本研究开展了添加剂在混合厌氧发酵系统的应用研究。试验选取水稻秸秆和猪粪作为发酵原料,通过分别添加不同的浓度的吐温20和腐植酸,测定甲烷的产气量和浓度、秸秆的降解和土壤肥力的变化,以揭示添加剂类型及其浓度对水稻田实地甲烷生产系统的影响。结果表明:添加剂的掺入并未影响产气的动态变化趋势,但显著地促进产气和提高产气浓度,整体效果由高到低为腐植酸吐温20对照,其中经腐植酸处理的产气量和产气浓度对照相比分别提高了50. 73%和24. 55%。添加剂的掺入有利于水稻秸秆纤维素和半纤维素的降解,但对木质素没有显著影响;其中以0. 15 g/L腐植酸和0. 30 g/L吐温20的降解率最高,相较于对照其纤维素降解率均提高了22. 11%,半纤维素降解率分别提高了107. 13%和98. 39%。添加剂的掺入能显著增加土壤肥力,以0. 15 g/L腐植酸和0. 30 g/L吐温20处理水平的效果最优,相较于对照,其土壤有机质分别增加了29. 63%和23. 72%,全氮分别增加了52. 32%和42. 38%,全磷分别增加了83. 33%和57. 14%。     

Inhibitory Effects of the Divalent Metal Ions on Biomethanation by Isolated Mesophilic Methanogen in AC21 Medium in Presence or Absence of Juices from Water Hyacinth

The effect of divalent metal ions on biomethanation was studied using pre-reduced methanogenic AC21 medium in absence or in presence of leaf or root extracts of Eichornia grassipes (water hyacinth) in vitro. The metal ions like Cu²⁺, Fe²⁺, Ni²⁺, and Co²⁺ were found to be effective in increasing methane production in vitro at low concentrations, but toxic inhibitory effects were observed at relatively high concentrations. Zn²⁺ ion was inhibitory in character with exceptions. The bioavailability of metal ions in AC21 medium alone or with juices from leaf or root extracts of plant (water hyacinth in the present case) was limited due to complex formation of metal ions with different constituents in the medium, precipitation, etc. However, the results of the microscopic experiments (in vials) with metal ions for methane production by isolated methanogens in the methanogenic medium should not be compared with the conversion of biomass to methane in biodigesters. In such cases, not only a large number of methanogenic bacteria of different strains but also a consortium of bacteria of different specificities is usually involved. Though the biomass may contain sufficient concentrations of metal ions, it is advisable to add metal ions like Cu²⁺, Fe²⁺ , Zn ²⁺ ,Co²⁺, etc. in the biomass, but the concentrations of the metal ions should be limited to 10–20 µM of the biomass to make methane generation successful.     

Viable methanotrophic bacteria enriched from air and rain can oxidize methane at cloud-like conditions

Atmospheric methane is degraded by both photooxidation and, in topsoils, by methanotrophic bacteria, but this may not totally account for the global sink of this greenhouse gas. Topsoils are a prominent source of airborne bacteria, which can degrade some organic atmospheric compounds at rates similar to photooxidation. Although airborne methanotrophs would have direct access to atmospheric methane, their presence and activity in the atmosphere has not been investigated so far. We enriched airborne methanotrophs from air and rainwater and showed that they oxidized methane at atmospheric concentration. The majority of seven OTUs, detected using pmoA gene clone libraries, were affiliated to the type II methanotrophic genera Methylocystis and Methylosinus. Furthermore, 16S rRNA gene clone libraries revealed the presence of OTUs affiliated with the genera Hyphomicrobium and Variovorax, members of which can stimulate methane oxidation by yet unidentified mechanisms. Simulating cloud-like conditions revealed that although both low pH and the presence of common cloud-borne organics negatively affected methane oxidation, airborne methanotrophs were able to degrade atmospheric methane in most cases. We demonstrate here for the first time that viable methanotrophic bacteria are present in air and rain and thus expand our knowledge on the global distribution of methanotrophs to include the atmosphere. The fact that they can degrade methane to below atmospheric concentrations when inoculated into artificial cloud water leads to an important possible effect of these organisms: the atmosphere may not only function as a medium for microbial dissemination, but also as a site of active microbial methane turnover.     

Microbial conversion of distillery waste to bioenergy: Effects of media enrichment with low chain fatty acids and Candida sp.

During microbial methanogensis of diluted distillery waste or spent wash (initial COD 25,000 mg/l) in the presence of sodium acetate, sodium propionate, or sodium butyrate at the concentration of 2000 mg/l, biogas containing 22.0% to 39.4% methane was produced in 20 days in a semicontinuous fermentation system. The analysis of the volatile fatty acid spectrum of the effluent showed accumulation of 46.3% branched chain fatty acids. When the fermentation medium was supplemented with modified Smith and Mah (SM) medium containing electrolytes and 1% sodium acetate, production of methane went up to 59.0% in 18 days. With the addition of a strain of Candida sp. the coculture produced about the same volume of methane (61%) but the time required was reduced (14 days). COD (16600.8 mg/l) was reduced and the effluent contained only 3% branched chain fatty acids adn 96% straight chain fatty acids. Fortification with the sodium salts of three branched-chain fatty acids as sources of carbon in SM medium (alone) reduced methane production. Only 0.0% to 14.2% methane was recorded. By combining these acids with straight chain fatty acids in SM medium, methane production increased significantly (about 4-fold) in proportion to the concentration of straight chain fatty acids added.     

Isobutene production by Rhodotorula minuta

Summary Isobutene production by Rhodotorula minuta IFO 1102 was studied. It was confirmed that the gas species produced by this yeast was isobutene from the result of analysis with a gas chromatograph mass spectrometer. Oxygen supply was essential to the microbial production of isobutene. The optimum pH was found to be approximately pH 6.0 and optimum temperature 25°–27° C. Isobutene production rate was maximal when l-leucine and l-phenylalanine in the medium were being uptaken by the yeast.The results from an investigation of the role of l-leucine and l-phenylalanine suggested that l-leucine was the precursor of isobutene and l-phenylalanine the inducer for the enzyme concerned with isobutene production.     

Saccharification of gamma-ray and alkali pretreated lignocellulosics

Enzymic saccharification of gamma ray and alkali pretreated sawdust, rice straw, and sugar cane bagasse showed higher release of reducing sugar from pretreated substrates. By gamma ray treatment alone (500 kGy) reducing sugar release of 2.8, 9.2, and 10 g/l was obtained from 7.5% (w/v) sawdust, rice straw, and bagasse and the same substrates showed reducing sugar release of 4.2, 30, and 20 g/l respectively when treated with alkali (0.1 g/g). Combination of gamma ray with alkali treatment further increased the reducing sugar release to 10.2, 33, and 36 g/l from sawdust, rice straw, and bagasse respectively. The effects of gamma ray and alkali treatment on saccharification varied with the nature of the substrate.