Magnetite accelerates syntrophic acetate oxidation in methanogenic systems with high ammonia concentrations

Ammonia accumulation is a major inhibitory substance causing anaerobic digestion upset and failure in CH₄ production. At high ammonia levels, CH₄ production through syntrophic acetate oxidization (SAO) pathways is more tolerant to ammonia toxicity than the acetoclastic methanogenesis pathway, but the low CH₄ production rate through SAO constitutes the main reason for the low efficiency of energy recovery in anaerobic digesters treating ammonia‐rich substrates. In this study, we showed that acetate fermentation to CH₄ and CO₂ occurred through SAO pathway in the anaerobic reactors containing a high ammonia concentration (5.0 g l^?1 NH₄⁺–N), and the magnetite nanoparticles supplementation increased the CH₄ production rates from acetate by 36–58%, compared with the anaerobic reactors without magnetite under the same ammonia level. The mechanism of facilitated methanogenesis was proposed to be the establishment of direct interspecies electron transfer (DIET) for SAO, in which magnetite facilitated DIET between syntrophic acetate oxidizing bacteria and methanogens. High‐throughput 16S rRNA gene sequencing analysis revealed that the bacterial Geobacteraceae and the archaeal Methanosarcinaceae and Methanobacteriaceae might be involved in magnetite‐mediated DIET for SAO and CH₄ production. This study demonstrated that magnetite supplementation might provide an effective approach to accelerate CH₄ production rates in the anaerobic reactors treating wastewater containing high ammonia.     

The nexus of syntrophy‐associated microbiota in anaerobic digestion revealed by long‐term enrichment and community survey

Anaerobic digestion (AD) processes are known to effectively convert organic waste to CO₂ and CH₄, but much of the microbial ecology remains unclear. Specifically, we have limited insights into symbiotic syntroph and methanogen (‘syntrophy’) acid degradation, although they are essential for preventing process deterioration. Also, we often observed many uncharacterized or uncultivated organisms, but poorly understood their role(s) in relation to syntrophy. To define syntrophy‐associated populations, this study enriched methanogenic communities with propionate, butyrate, benzoate, acetate, formate and H₂ from two different inocula over 3 years. 16S pyrotag analysis revealed core populations of known syntrophs (six clades) and methanogens (nine clades) associated with acid degradation, and evidence for substrate‐ and/or inoculum‐dependent specificity in syntrophic partnerships. Based on comprehensive re‐evaluation of publically available microbial community data for AD, the known syntrophs and methanogens identified were clearly representatives of the AD‐associated syntrophs and methanogens. In addition, uncultivated clades related to Bacteroidetes, Firmicutes, Actinobacteria and Chloroflexi were ubiquitously found in AD and enrichments. These organisms may be universally involved in AD syntrophic degradation, but only represented <23% of the yet‐to‐be‐cultivated organisms (89 of 390 clades). Thus, the contribution of these uncultured organisms in AD remains unclear and warrants further investigation.     

Effects of elevated ozone concentration on methane emission from a rice paddy in Yangtze River Delta,China

Few investigations have been made on the impact of elevated ozone (O₃) concentration on methane (CH₄) emission from rice paddies. Using open‐top chambers in situ with different O₃ treatments, CH₄ emissions were measured in a rice paddy in Yangtze River Delta, China in 2007 and 2008. There were four treatments applied: charcoal‐filtered air (CF), nonfiltered air (NF), and charcoal‐filtered air with different O₃ additions (O₃‐1 and O₃‐2). The mean O₃ concentrations during the O₃ fumigation were 19.7, 22.6, 69.6 and 118.6 ppb in 2007 and 7.0, 17.4, 82.2 and 138.3 ppb in 2008 for treatments CF, NF, O₃‐1 and O₃‐2, respectively. The rice yields, as compared with CF, were reduced by 32.8% and 37.1%, 58.3% and 52.1% in treatments O₃‐1 and O₃‐2 in 2007 and 2008, respectively. The diurnal patterns of CH₄ emission varied temporally with treatments and there was inconsistence in diurnal variations in CH₄ emissions from the paddy field. The daily mean CH₄ emissions were significantly lower in treatments O₃‐1 and O₃‐2 than those in treatments CF and NF. Compared with CF treatment, CH₄ emissions from the paddy field were decreased to 46.5% and 38.3%, 50.6% and 46.8% under treatments O₃‐1 and O₃‐2 in the whole growing seasons of 2007 and 2008, respectively. The seasonal mean CH₄ emissions were negatively related with AOT40 (accumulative O₃ concentration above 40 ppb; P < 0.01 in both years), but positively related to the relative rice yield (reference to CF; P < 0.01 in 2007 and P < 0.001 in 2008), aboveground biomass (P < 0.01 in both years) and underground biomass (P < 0.01 in 2007 and P < 0.05 in 2008). The decreased CH₄ emission from the rice paddy due to an increased O₃ exposure might partially mitigate the global warming potential induced by soil carbon loss under elevated O₃ concentrations.     

Interactions in Syntrophic Associations of Endospore-Forming, Butyrate-Degrading Bacteria and H2-Consuming Bacteria

Butyrate is an important intermediate in the anaerobic degradation of organic matter. In sulfate-depleted environments butyrate is oxidized to acetate and hydrogen by obligate proton reducers, in syntrophic association with hydrogen-consuming methanogens. This paper describes two enrichments of endospore-forming bacteria degrading butyrate in consortia with methanogens. The isolates are readily established in coculture with H₂-consuming, sulfate-reducing bacteria by pasteurizing the culture. The two original enrichments differed in that one grew to an optically dense culture while the second grew in clumps. Examination by scanning electron microscopy showed that clumping resulted from the production of large amounts of extracellular polymer. Several H₂-consuming methanogens were identified in the enrichments. Some of them grew closely associated to the butyrate degraders. This attachment to the hydrogen producer may permit some methanogens to compete for the growth substrate against other bacteria having higher substrate affinity.     

Multiple syntrophic interactions in a terephthalate-degrading methanogenic consortium

Terephthalate (TA) is one of the top 50 chemicals produced worldwide. Its production results in a TA-containing wastewater that is treated by anaerobic processes through a poorly understood methanogenic syntrophy. Using metagenomics, we characterized the methanogenic consortium inside a hyper-mesophilic (that is, between mesophilic and thermophilic), TA-degrading bioreactor. We identified genes belonging to dominant Pelotomaculum species presumably involved in TA degradation through decarboxylation, dearomatization, and modified β-oxidation to H₂/CO₂ and acetate. These intermediates are converted to CH₄/CO₂ by three novel hyper-mesophilic methanogens. Additional secondary syntrophic interactions were predicted in Thermotogae, Syntrophus and candidate phyla OP5 and WWE1 populations. The OP5 encodes genes capable of anaerobic autotrophic butyrate production and Thermotogae, Syntrophus and WWE1 have the genetic potential to oxidize butyrate to CO₂/H₂ and acetate. These observations suggest that the TA-degrading consortium consists of additional syntrophic interactions beyond the standard H₂-producing syntroph–methanogen partnership that may serve to improve community stability.     

Methane Metabolism in a Temperate Swamp

Comparisons between in situ CH₄ concentration and potential factors controlling its net production were made in a temperate swamp. Seasonal measurements of water table level and depth profiles of pH, dissolved CH₄, CO₂, O₂, SO₄^2-, NO₃^-, formate, acetate, propionate, and butyrate were made at two adjacent sites 1.5 to 2 m apart. Dissolved CH₄ was inversely correlated to O₂ and, in general, to NO₃^- and SO₄^2-, potential inhibitors of methanogenesis. At low water table levels (August 1992), maximal CH₄ (2 to 4 μM) occurred below 30 cm, whereas at high water table levels (October 1992) or under flooded conditions (May 1993), CH₄ maxima (4 to 55 μM) occurred in the top 10 to 20 cm. Higher CH₄ concentrations were likely supported by inputs of fresh organic matter from decaying leaf litter, as suggested by high acetate and propionate concentrations (25 to 100 μM) in one of the sites in fall and spring. Measurements of potential CH₄ production (and consumption) showed that the highest rates generally occurred in the top 10 cm of soil. Soil slurry incubations confirmed the importance of organic matter to CH₄ production but also showed that competition for substrates by nonmethanogenic microorganisms could greatly attenuate its effect.     

Degradation of Cinnamate via β-Oxidation to Benzoate by a Defined,Syntrophic Consortium of Anaerobic Bacteria

A syntrophic consortium was enriched in a basal medium containing cinnamate as the carbon and energy source. It was found to consist of three morphologically distinct microbes, viz., a short, rod-shaped, non-motile bacterium with distinctly pointed ends, Papillibacter cinnamivorans; a rod-shaped, motile bacterium with rounded ends, Syntrophus sp.; and a methanoarchaeon, Methanobacterium sp. This methanogen was then replaced by a collection strain of Methanobacterium formicicum. A syntrophic interdependency of the three partners of the consortium was observed during growth on cinnamate. In the presence of bromoethanesulfonic acid (BESA), cinnamate was transformed to benzoate, whereas under methanogenic conditions without BESA, cinnamate was first transformed to benzoate via β-oxidation and subsequently completely degraded into acetate, CH₄, and CO₂. Papillibacter cinnamivorans was responsible for benzoate production from cinnamate, whereas a syntrophic association between Syntrophus sp. and the methanogen degraded benzoate to acetate, CH₄, and CO₂. A new anaerobic degradation pathway of cinnamate into benzoate via β-oxidation by a pure culture of P. cinnamivorans is proposed. Received: 27 December 2001 / Accepted: 28 March 2002     

Variation of carbon isotope fractionation in hydrogenotrophic methanogenic microbial cultures and environmental samples at different energy status

Methane is a major product of anaerobic degradation of organic matter and an important greenhouse gas. Its stable carbon isotope composition can be used to reveal active methanogenic pathways, if associated isotope fractionation factors are known. To clarify the causes that lead to the wide variation of fractionation factors of methanogenesis from H₂ plus CO₂ (), pure cultures and various cocultures were grown under different thermodynamic conditions. In syntrophic and obligate syntrophic cocultures thriving on different carbohydrate substrates, fermentative bacteria were coupled to three different species of hydrogenotrophic methanogens of the families Methanobacteriaceae and Methanomicrobiaceae. We found that C‐isotope fractionation was correlated to the Gibbs free energy change (ΔG) of CH₄ formation from H₂ plus CO₂ and that the relation can be described by a semi‐Gauss curve. The derived relationship was used to quantify the average ΔG that is available to hydrogenotrophic methanogenic archaea in their habitat, thus avoiding the problems encountered with measurement of low H₂ concentrations on a microscale. Boreal peat, rice field soil, and rumen fluid, which represent major sources of atmospheric CH₄, exhibited increasingly smaller , indicating that thermodynamic conditions for hydrogenotrophic methanogens became increasingly more favourable. Vice versa, we hypothesize that environments with similar energetic conditions will also exhibit similar isotope fractionation. Our results, thus, provide a mechanistic constraint for modelling the ¹³C flux from microbial sources of atmospheric CH₄.     

Evidence for the coexistence of direct and riboflavin-mediated interspecies electron transfer in Geobacter co-culture

Geobacter species can secrete free redox-active flavins, but the role of these flavins in the interspecies electron transfer (IET) of Geobacter direct interspecies electron transfer (DIET) co-culture is unknown. Here, we report the presence of a new riboflavin-mediated interspecies electron transfer (RMIET) process in a traditional Geobacter DIET co-culture; in this process, riboflavin contributes to IET by acting as a free-form electron shuttle between free Geobacter species and serving as a bound cofactor of some cytochromes in Geobacter co-culture aggregates. Multiple lines of evidence indicate that RMIET facilitates the primary initiation of syntrophic growth between Geobacter species before establishing the DIET co-culture and provides additional ways alongside the DIET to transfer electrons to achieve electric syntrophy between Geobacter species. Redox kinetic analysis of riboflavin on either Geobacter species demonstrated that the Gmet_2896 cytochrome acts as the key riboflavin reduction site, while riboflavin oxidation by Geobacter sulfurreducens is the rate-limiting step in RMIET, and the RMIET makes only a minor contribution to IET in Geobacter DIET co-culture. The discovery of a new RMIET process in Geobacter DIET co-culture suggests the complexity of IET in syntrophic bacterial communities and provides suggestions for the careful examination of the IET of other syntrophic co-cultures.     

Selective distortion of the planar group CαC'(O)O to a chiral flat tetrahedron in the amino acid alanine

Based on a Cambridge Structural Database (CSD) search, a meta‐analysis of 116 structures of alanine H₃NC_αH(CH₃)C′(O)O and its derivatives H₃NC_αH(CH₃)C′(O)O(H/R/M), protonated, esterified, or coordinated at the carboxylic group, shows that in the first step of a chirality chain, the L configuration at C_α induces (M) and (P) conformations with respect to rotation around the central C′─C_α bond. In the second step, the (M) and (P) conformations selectively distort the planar carboxylic group C_αC'(O_cis)O_trans to asymmetric flat (R) and (S) tetrahedra. High diastereoselectivities are caused by the two players attraction N…O_cis and repulsion O_trans…C_Me, which work together in (L,M,R) configurations but against each other in (L,P,S) configurations.     

冬季水分管理和水稻覆膜栽培对川中丘陵地区冬水田CH4排放的影响

采用静态箱-气相色谱法观测冬季水分管理和水稻覆膜栽培对川中丘陵地区冬水田全年的CH_4排放通量。试验设置持续淹水(CF)、冬季直接落干+稻季淹水(TF)与冬季覆膜落干+稻季覆膜(PM)3个处理。结果表明,冬季休闲期,CF、TF和PM处理CH_4排放分别为16.1、1.4 g/m~2和2.7 g/m~2;水稻生长期,CF、TF和PM处理CH_4排放分别为57.7、27.7 g/m~2和13.5 g/m~2。相较于CF处理,TF与PM处理分别减少其全年CH_4排放60.6%和78.0%。TF与PM处理水稻生长期CH_4排放峰值分别较CF处理低33.0%和56.1%。休闲期,TF、PM处理厢面与厢沟区域CH_4排放与土壤温度显著正相关(P0.05),与土壤氧化还原电位(土壤Eh)显著负相关(P0.05),而CF处理CH_4排放仅与土壤温度显著正相关(P0.05)。水稻生长期,CF处理CH_4排放与土壤温度显著正相关(P0.05),与土壤Eh显著负相关(P0.05),TF处理CH_4排放仅与土壤Eh显著负相关(P0.05),PM处理厢沟CH_4排放与土壤Eh显著正相关(P0.05)。各处理水稻生长期土壤可溶性有机碳含量(DOC)与微生物生物量碳含量(MBC)显著高于休闲期(P0.05)。研究结果为进一步研究冬水田全年CH_4排放规律及寻求有效的减排措施提供数据支撑和科学依据。     

Circadian methane oxidation in the root zone of rice plants

In the root zone of rice plants aerobic methanotrophic bacteria catalyze the oxidation of CH₄ to CO₂, thereby reducing CH₄ emissions from paddy soils to the atmosphere. However, methods for in situ quantification of microbial processes in paddy soils are scarce. Here we adapted the push–pull tracer-test (PPT) method to quantify CH₄ oxidation in the root zone of potted rice plants. During a PPT, a test solution containing CH₄ ± O₂ as reactant(s), Cl^? and Ar as nonreactive tracers, and BES as an inhibitor of CH₄ production was injected into the root zone at different times throughout the circadian cycle (daytime, early nighttime, late nighttime). After a 2-h incubation phase, the test solution/pore-water mixture was extracted from the same location and rates of CH₄ oxidation were calculated from the ratio of measured reactant and nonreactive tracer concentrations. In separate rice pots, O₂ concentrations in the vicinity of rice roots were measured throughout the circadian cycle using a fiber-optic sensor. Results indicated highly variable CH₄ oxidation rates following a circadian pattern. Mean rates at daytime and early nighttime varied from 62 up to 451 μmol l^?1 h^?1, whereas at late nighttime CH₄ oxidation rates were low, ranging from 13 to 37 μmol l^?1 h^?1. Similarly, daytime O₂ concentration in the vicinity of rice roots increased to up to 250% air saturation, while nighttime O₂ concentration dropped to below detection (<0.15% air saturation). Our results suggest a functional link between root-zone CH₄ oxidation and photosynthetic O₂ supply.     

Carbon nanotubes accelerate methane production in pure cultures of methanogens and in a syntrophic coculture

Carbon materials have been reported to facilitate direct interspecies electron transfer (DIET) between bacteria and methanogens improving methane production in anaerobic processes. In this work, the effect of increasing concentrations of carbon nanotubes (CNT) on the activity of pure cultures of methanogens and on typical fatty acid‐degrading syntrophic methanogenic coculture was evaluated. CNT affected methane production by methanogenic cultures, although acceleration was higher for hydrogenotrophic methanogens than for acetoclastic methanogens or syntrophic coculture. Interestingly, the initial methane production rate (IMPR) by Methanobacterium formicicum cultures increased 17 times with 5 g·L^?1 CNT. Butyrate conversion to methane by Syntrophomonas wolfei and Methanospirillum hungatei was enhanced (～1.5 times) in the presence of CNT (5 g·L^?1), but indications of DIET were not obtained. Increasing CNT concentrations resulted in more negative redox potentials in the anaerobic microcosms. Remarkably, without a reducing agent but in the presence of CNT, the IMPR was higher than in incubations with reducing agent. No growth was observed without reducing agent and without CNT. This finding is important to re‐frame discussions and re‐interpret data on the role of conductive materials as mediators of DIET in anaerobic communities. It also opens new challenges to improve methane production in engineered methanogenic processes.     

Effects of insecticide profenophos on germination,early growth,meiotic behavior and chlorophyll mutation of barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

The genotoxicity of different concentrations of insecticide, profenophos (O-4-bromo-2-chlorophenyl O-ethyl S-propyl phosphorothioate) was evaluated at various stages of cell cycle (G₁, S and G₂) by using the seeds of barley (Hordeum vulgare L.). The aim of this work was to investigate the effects of insecticide profenophos at various stages of cell cycle on germination, seedling height and meiotic behavior in M₁ and chlorophyll mutations in M₂ generation. From the present study, it can be concluded that the stages of cell cycle were sensitive for the treatments of chemicals and it also showed that the S-phase of cell cycle is more sensitive than other phases of cell cycle.     

Formate and glucose stimulation of methane and hydrogen production in rumen liquor

Membrane-inlet mass spectrometry was used to investigate the effects of increasing the concentration of the rumen metabolites, formate and glucose, upon CH₄ and H₂ production during fermentation by unfractionated rumen liquor. Additions of formate up to 3.6 mM stimulated CH₄ and then excess H₂ production. Each addition caused a large accumulation of H₂ (>40 µM), which returned to in situ concentrations after periods of more than 1 h. Glucose additions up to 2.0 mM gave linear increases in CH₄ and H₂ production. The conversion of substrate carbon into CH₄ was found to decrease from 34% to 9% for formate, as concentrations were increased (1.6–3.6 mM); approximately 13.5% of the glucose carbon was converted to CH₄.     

Revising a process-based biogeochemistry model (DNDC) to simulate methane emission from rice paddy fields under various residue management and fertilizer regimes

A comprehensive biogeochemistry model, DNDC, was revised to simulate crop growth and soil processes more explicitly and improve its ability to estimate methane (CH₄) emission from rice paddy fields under a wide range of climatic and agronomic conditions. The revised model simulates rice growth by tracking photosynthesis, respiration, C allocation, tillering, and release of organic C and O₂ from roots. For anaerobic soil processes, it quantifies the production of electron donors [H₂ and dissolved organic carbon (DOC)] by decomposition and rice root exudation, and simulates CH₄ production and other reductive reactions based on the availability of electron donors and acceptors (NO₃^?, Mn⁴⁺, Fe³⁺, and SO₄^2?). Methane emission through rice is simulated by a diffusion routine based on the conductance of tillers and the CH₄ concentration in soil water. The revised DNDC was tested against observations at three rice paddy sites in Japan and China with varying rice residue management and fertilization, and produced estimates consistent with observations for the variation in CH₄ emission as a function of residue management. It also successfully predicted the negative effect of (NH₄)₂SO₄ on CH₄ emission, which the current model missed. Predicted CH₄ emission was highly sensitive to the content of reducible soil Fe³⁺, which is the dominant electron acceptor in anaerobic soils. The revised DNDC generally gave acceptable predictions of seasonal CH₄ emission, but not of daily CH₄ fluxes, suggesting the model's immaturity in describing soil heterogeneity or rice cultivar‐specific characteristics of CH₄ transport. It also overestimated CH₄ emission at one site in a year with low temperatures, suggesting uncertainty in root biomass estimates due to the model's failure to consider the temperature dependence of leaf area development. Nevertheless, the revised DNDC explicitly reflects the effects of soil electron donors and acceptors, and can be used to quantitatively estimate CH₄ emissions from rice fields under a range of conditions.     

Significant inhibition of hybridoma cells by exogenous application of ganglioside G M3, a possible modulator of cell growthin vitro

Gangliosides of the mouse-rat hybridoma cell line 187.1, which secretes an antibody against -light chain of mouse IgG, were isolated and structurally characterized by biochemical and immunological methods (overlay technique), and fast atom bombardment-mass spectrometry. Exclusively G _M3, substituted with C₂₄₁ and C₁₆₀ fatty acid and C₁₈₁ sphingosine, was found in this B cell derived cell line. A G _M3 (NeuGc) to G _M3(NeuAc) ratio (80 to 20), was characteristic for 187.1 cells, and absolute G _M3 amounts of about 0.3 mg 10^–9 viable cells were determined. Exogenous application of G _M3, which has been isolated from large cell preparations, to 187.1 cells showed growth inhibition in a concentration dependent manner. Using the MTT-assay and the [³H]thymidine incorporation assay, the cells exhibited a strong reduction in metabolic and proliferative activity, respectively, after exposure of cells to G _M3. G _M3 was applied in concentrations between 3M and 30M, giving evidence for strong inhibitory effects at 30M G _M3 and less but significant suppression after application of G _M3 concentrations lower than 20M. No cellular response was observed at the lowest concentration (3M) used in this study. Hybridoma cells as well as other cell types like fibroblasts, muscle cells and endothelial cells, are in general characterized by high expression of the G _M3 ganglioside, which is known to act as a modulator of cellular growth in monolayer cultures of adherent cells. Since gangliosides are released to the culture medium by cell lysis, i.e. cell death, and/or by active membrane shedding, the results obtained in this study suggest a growth regulatory role of G _M3 in high density hybridoma cell cultures.Abbreviations DMB 1,2-diamino-4,5-methylenedioxybenzene - FAB-MS fast atom bombardment-mass spectrometry - GSL(s) glycosphingolipid(s) - HPLC high performance liquid chromatography - HPTLC high performance thin layer chromatography - MTT 3,(4,5 dimethylthiazol-2-yl)2,5 diphenyl tetrazolium bromide - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - PBS phosphate buffered saline The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations (1977) and the nomenclature of Svennerholm (1963). Lactosylceramide or LacCer, Galß1–4Glcß1-1Cer; gangliotriaosylceramide or GgOse₃Cer; GalNAcß1–4Galß1–4Glcß1-1 Cer; gangliotetraosylceramide or GgOse₄Cer, Galß1–3GalNAcß1–4Galß1–4Glcß1-1Cer; G _M3(NeuAc), II³NeuAc-LacCer; G _M3(NeuGc), II³NeuGc-LacCer; G _M2(NeuGc), II³NeuGc-GgOse₃Cer; G _M1 or G _M1a, II³NeuAc-GgOse₄Cer; G _M1b, IV³NeuAc-GgOse₄Cer.