Batch- and continuous-culture studies of a methane-utilizing mixed culture

A methane-utilizing mixed culture isolated from activated sludge by selective enrichment at 45°C was found to consist of three interacting species: a methaneutilizing bacterium, a citrate-utilizing bacterium, and a methanol-utilizing bacterium. All three species grew well at 45°C. Three different stable mixed cultures were reconstituted by various combinations of these pure cultures. The nutritional requirements and substrate ranges for each pure culture were determined. The nutritional requirements and substrate ranges for each pure culture were determined. The saturation constant for the methane-utilizing bacterium on methane (K) and for the methanol-utilizing bacterium on methanol (K) were 1.73 × 10^?6M and 4.51 × 10^?7M, respectively. The volumetric mass transfer coefficient for methane (K_L a) was determined to be 65.6 hr^?1.     

Effects of copper on expression of methane monooxygenases,trichloroethylene degradation,and community structure in methanotrophic consortia

Copper plays a key role in regulating the expression of enzymes that promote biodegradation of contaminants in methanotrophic consortia (MC). Here, we utilized MC isolated from landfill cover to investigate cometabolic degradation of trichloroethylene (TCE) at nine different copper (Cu²⁺) concentrations. The results demonstrated that an increase in Cu²⁺ concentration from 0 to 15 μM altered the specific first‐order rate constant k_1,TCE, the expression levels of methane monooxygenase (pmoA and mmoX) genes, and the specific activity of soluble methane monooxygenase (sMMO). High efficiency TCE degradation (95%) and the expression levels of methane monooxygenase (MMO) were detected at a Cu²⁺ concentration of 0.03 μM. Notably, sMMO‐specific activity ranged from 74.41 nmol/(mg_cell h) in 15 μM Cu²⁺ to 654.99 nmol/(mg_cell h) in 0.03 μM Cu²⁺, which contrasts with cultures of pure methanotrophs in which sMMO activity is depressed at high Cu²⁺ concentrations, indicating a special regulatory role for Cu²⁺ in MC. The results of MiSeq pyrosequencing indicated that higher Cu²⁺ concentrations stimulated the growth of methanotrophic microorganisms in MC. These findings have important implications for the elucidation of copper‐mediated regulatory mechanisms in MC.     

Lactobionic and cellobionic acid production profiles of the resting cells of acetic acid bacteria

Lactobionic acid was produced by acetic acid bacteria to oxidize lactose. Gluconobacter spp. and Gluconacetobacter spp. showed higher lactose-oxidizing activities than Acetobacter spp. Gluconobacter frateurii NBRC3285 produced the highest amount of lactobionic acid per cell, among the strains tested. This bacterium assimilated neither lactose nor lactobionic acid. At high lactose concentration (30%), resting cells of the bacterium showed sufficient oxidizing activity for efficient production of lactobionic acid. These properties may contribute to industrial production of lactobionic acid by the bacterium. The bacterium showed higher oxidizing activity on cellobiose than that on lactose and produced cellobionic acid.     

Oxidative degradation of Amaranth dye by a new genus Kerstersia sp.

A dye-decolorizing bacterium was isolated from a coconut coir sample and identified as a new genus Kerstersia sp. by various biochemical tests and 16S rRNA gene sequencing. This bacterium was capable of degrading sulfonated azo dye Amaranth aerobically at 40?°C and pH 7.0. Tests conducted on intracellular crude enzyme extract identified an oxygen insensitive azoreductase. The optimum dye-decolorizing activity at pH 7.0 and 40?°C for the decolorization of dye was 0.091?U mL^?1 (μ_max 0.522?mg h^?1). The K_s 104.51?μM^?1 has been evaluated by plotting Lineweaver–Burk plot for the Amaranth dye. The dye degraded products were extracted and characterized by TLC, diazotization and Carbylamines test, which indicated that Amaranth was biotransformed into non-toxic aromatic metabolite without amine group.     

An obligate methylotrophic, methane-oxidizing Methylomicrobium species from a highly alkaline environment

A new, obligately methylotrophic, methane-oxidizing bacterium, strain AMO 1, was isolated from a mixed sample of sediments from five highly alkaline soda lakes (Kenya). Based on its cell ultrastructure and high activity of the hexulose-6-phosphate synthase, the new isolate belongs to the type I methanotrophs. It differed, however, from the known neutrophilic methanotrophs by the ability to grow and oxidize methane at high pH values. The bacterium grew optimally with methane at pH 9–10. The oxidation of methane, methanol, and formaldehyde was optimal at pH 10, and cells were still active up to pH 11. AMO 1 was able to oxidize ammonia to nitrite at high pH. A maximal production of nitrite from ammonia in batch cultures at pH 10 was observed with 10% of CH₄ in the gas phase when nitrate was present as nitrogen source. Washed cells of AMO 1 oxidized ammonia most actively at pH 10–10.5 in the presence of limiting amounts of methanol or CH₄. The bacterium was also capable of oxidizing organic sulfur compounds at high pH. Washed cells grown with methane exhibited high activity of CS₂ oxidation and low, but detectable, levels of DMS and DMDS oxidation. The GC content of AMO 1 was 50.9 mol%. It showed only weak DNA homology with the previously described alkaliphilic methanotroph "Methylobacter alcaliphilus" strain 20 Z and with the neutrophilic species of the genera Methylobacter and Methylomonas. According to the 16S rRNA gene sequence analysis, strain AMO 1 was most closely related to a neutrophilic methanotroph, Methylomicrobium pelagicum (98.2% sequence similarity), within the gamma-Proteobacteria. Received: July 26, 1999 / Accepted: January 4, 2000     

Trichloroethylene Biodegradation by a Methane-Oxidizing Bacterium

Trichloroethylene (TCE), a common groundwater contaminant, is a suspected carcinogen that is highly resistant to aerobic biodegradation. An aerobic, methane-oxidizing bacterium was isolated that degrades TCE in pure culture at concentrations commonly observed in contaminated groundwater. Strain 46-1, a type I methanotrophic bacterium, degraded TCE if grown on methane or methanol, producing CO₂ and water-soluble products. Gas chromatography and ¹⁴C radiotracer techniques were used to determine the rate, methane dependence, and mechanism of TCE biodegradation. TCE biodegradation by strain 46-1 appears to be a cometabolic process that occurs when the organism is actively metabolizing a suitable growth substrate such as methane or methanol. It is proposed that TCE biodegradation by methanotrophs occurs by formation of TCE epoxide, which breaks down spontaneously in water to form dichloroacetic and glyoxylic acids and one-carbon products.     

Microbially enhanced dissolution of HgS in an acid mine drainage system in the California Coast Range

Mercury sulfides (cinnabar and metacinnabar) are the main ores of Hg and are relatively stable under oxic conditions (K_sp = 10^?54 and 10^?52, respectively). However, until now their stability in the presence of micro‐organisms inhabiting acid mine drainage (AMD) systems was unknown. We tested the effects of the AMD microbial community from the inoperative Hg mine at New Idria, CA, present in sediments of an AMD settling pond adjacent to the main waste pile and in a microbial biofilm on the surface of this pond, on the solubility of crystalline HgS. A 16S rRNA gene clone library revealed that the AMD microbial community was dominated by Fe‐oxidizing (orders Ferritrophicales and Gallionellas) and S‐oxidizing bacteria (Thiomonas sp.), with smaller amounts (≤6%) being comprised of the orders Xanthomondales and Rhodospirillales. Though the order Ferritrophicales dominate the 16S rRNA clones (>60%), qPCR results of the microbial community indicate that the Thiomonas sp. represents ~55% of the total micro‐organisms in the top 1 cm of the AMD microbial community. Although supersaturated with respect to cinnabar and metacinnabar, microcosms inoculated with the AMD microbial community were capable of releasing significantly more Hg into solution compared to inactivated or abiotic controls. Four different Hg‐containing materials were tested for bacterially enhanced HgS dissolution: pure cinnabar, pure metacinnabar, mine tailings, and calcine material (processed ore). In the microcosm with metacinnabar, the presence of the AMD microbial community resulted in an increase of dissolved Hg concentrations up to 500 μg L^‐1 during the first 30 days of incubation. In abiotic control microcosms, dissolved Hg concentrations did not increase above 100 ng L^?1. When Hg concentrations were below 50 μg L^‐1, the Fe‐oxidizing bacteria in the AMD microbial community were still capable of oxidizing Fe(II) to Fe(III) in the AMD solution, whereas concentrations above 50 μg L^?1 resulted in inhibition of microbial iron oxidation. Our experiments show that the AMD microbial community contributes to the dissolution of mercury sulfide minerals. These findings have major implications for risk assessment and future management of inoperative Hg mines worldwide.     

A three-year study of controls on methane emissions from two Michigan peatlands

We investigate temporal changes in methane emissions over a three-year period from two peatlands in Michigan. Mean daily fluxes ranged from 0.6–68.4 mg CH₄ m^–2d^–1 in plant communities dominated by Chamaedaphne calyculata, an eficaceous shrub, to 11.5–209 mg CH₄ m^–2d^–1 in areas dominated by plants with aerenchymatous tissues, such as Carex oligosperma and Scheuchzeria palustris. Correlations between methane flux and water table position were significant at all sites for one annual cycle when water table fluctuations ranged from 15 cm above to 50 cm below the peat surface. Correlations were not significant during the second and third annual periods with smaller water table fluctuations. Methane flux was strongly correlated with peat temperatures at –5 to –40 cm (r _s = 0.82 to 0.98) for all three years at sites with flora acting as conduits for methane transport. At shrub sites, the correlations between methane flux and peat temperature were weak to not significant during the first two years, but were strong in the third year.Low rates of methane consumption (–0.2 to –1.5 mg CH₄ m^–2 d^–1 ) were observed at shrub sites when the water table was below –20 cm, while sites with plants capable of methane transport always had positive net fluxes of methane. The methane oxidizing potential at both types of sites was confirmed by peat core experiments. The results of this study indicate that methane emissions occur at rates that cannot be explained by diffusion alone; plant communities play a significant role in altering methane flux from peatland ecosystems by directly transporting methane from anaerobic peat to the atmosphere.     

A novel identified Pseudomonas aeruginosa,which exhibited nitrate- and nitrite-dependent methane oxidation abilities,could alleviate the disadvantages caused by nitrate supplementation in rumen fluid fermentation

After the occurrence of nitrate-dependent anaerobic methane oxidation (AMO) in rumen fluid culture was proved, the organisms that perform the denitrifying anaerobic methane oxidizing (DAMO) process in the rumen of dairy goat were investigated by establishing two enrichment culture systems, which were supplied with methane as the sole carbon source and NaNO₃ or NaNO₂ as the electron acceptor. Several Operational Taxonomic Units (OTU) belonging to Proteobacteria became dominant in the two enrichment systems. The identified Pseudomonas aeruginosa, which was isolated from the NaNO₂ enrichment system, could individually perform a whole denitrifying anaerobic methane oxidizing process. Further in vitro rumen fermentation showed that supplementation with the isolated P. aeruginosa could reduce methane emissions, alleviate the nitrite accumulation and prevent the decrease in propionic acid product caused by nitrate supplementation.     

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.     

顺北典型油气藏上方土壤甲烷氧化菌菌群特征及其勘探意义

【目的】甲烷氧化细菌(MOB)长期以来一直被用作石油和天然气勘探的重要油气指示菌,其仅能利用甲烷作为唯一碳源。根据甲烷氧化菌菌群特征结合地质剖面可以较好地预测深部油气藏,为石油勘探提供良好的数据支撑。由于传统平板培养法只能针对可培养甲烷氧化菌,方法具有一定局限性。【方法】本文采用分子生物学技术结合地球化学烃类指标研究了顺北典型油气藏上方土壤中甲烷氧化菌的分布。【结果】研究结果显示,油气田上方pmoA基因拷贝数与酸解烃含量具有一定的正相关性,且油气区比背景区高0.5–2个数量级。16SrRNA基因高通量测序和pmoA基因的克隆文库结果显示顺北油藏上方土壤中甲烷氧化菌主要以I型为主,水平剖面中甲烷氧化菌随着离油田距离增加存在I型向II型演变的现象,且Methylomonassp.在背景区与油气区的丰度有较大差异,具有良好的油气指示潜力。【结论】综上所述,长期微渗透过程中甲烷氧化菌(MOB)的菌群特征对预测深层油藏具有一定的指示作用,结合地质剖面和地表烃类可以有效预测有利油藏区域。     

Radioisotopic tracing of carbon monoxide conversion by anaerobic thermophilic prokaryotes

The rate of CO conversion by a pure culture of a thermophilic CO-oxidizing, H₂-producing bacterium Carboxydocella sp. strain 1503 was determined by the radioisotopic method. The overall daily uptake of ¹⁴CO by the bacterium was estimated at 38–56 μmol CO per 1 ml of the culture. A radioisotopic method was developed to separate and quantitatively determine the products of anaerobic CO conversion by microbial communities in hot springs. The new method was first tested on the microbial community from a sample obtained from a hot spring in Kamchatka. The potential rate of CO conversion by the anaerobic microbial community was found to be 40.75 nmol CO/cm³ sediment per day. 85% of the utilized ¹⁴CO was oxidized to carbon dioxide; 14.5% was incorporated into dissolved organic matter, including 0.2% that went into volatile fatty acids; 0.5% was used for cell biomass production; and only just over 0.001% was converted to methane.     

增温对冻土区泥炭沼泽土壤孔隙水甲烷关联微生物和溶解性有机碳的影响

多年冻土区泥炭沼泽土壤孔隙水甲烷关联微生物及底物的研究有助于深入理解气候变化背景下寒区湿地生态系统甲烷循环过程。选取大兴安岭连续多年冻土区柴桦-泥炭藓和狭叶杜香-泥炭藓两种典型植被群落泥炭沼泽,设置开顶箱(Open Top Chamber,OTC)增温实验。于生长季(6月、7月、8月和9月)采集土壤孔隙水样品,对比分析OTC内外土壤孔隙水中产甲烷菌数量、甲烷氧化菌数量及溶解性有机碳(Dissolved Organic Carbon,DOC)浓度的动态变化特征,并探究土壤孔隙水甲烷关联微生物与DOC浓度的关系。结果表明:增温提高了生长季大兴安岭多年冻土区土壤孔隙水中产甲烷菌数量和DOC浓度,而对甲烷氧化菌数量的影响因月份而异。生长季柴桦-泥炭藓和狭叶杜香-泥炭藓泥炭沼泽土壤孔隙水中产甲烷菌数量的平均增加幅度分别为54.52%和44.97%,DOC浓度的平均增加幅度分别为34.16%和28.33%。增温使得生长季柴桦-泥炭藓和狭叶杜香-泥炭藓泥炭沼泽土壤孔隙水中甲烷氧化菌平均数量分别降低了46.20%和31.42%。一元线性回归分析结果表明,土壤孔隙水中DOC浓度可分别解释柴桦-泥炭藓和狭叶杜香-泥炭藓泥炭沼泽土壤孔隙水中产甲烷菌数量变化的29.00%和24.10%(P<0.01),而对两种植被群落下土壤孔隙水中甲烷氧化菌数量的影响并不显著(P>0.05)。     

Ecological observations on Heterotrophic,methane oxidizing and sulfate reducing bacteria in Pond

Numbers of heterotrophic, methane oxidizing and sulfate reducing bacteria were counted in Lake Vechten. A dynamic distribution pattern was found in the stratified lake. A maximum of heterotrophs (numbers of 10⁹ bact./l) occured in the deepest part of the lake in spring and in the metalimnion during summer-stratification. These bacteria use nearly all available oxygen in the hypolimnion. It was found that the concentration of available organic material and the oxygen tension caused the numbers of heterotrophs in the metalimnion to be high.The maximal numbers of methane oxidizers (numbers of 5.10⁵ bact./l) were found at a depth of maximal methane concentration: the de-oxygenated hypolimnion. Preliminary evidence indicated that these organisms were facultative methane oxidizers and must be regarded as micro-aerophyllics. By oxidizing methane they removed the residual oxygen under the metalimnion.The sulfate reducing bacteria could be observed in the hypolimnion only. Decreased SO _inf4 ^sup–2 concentration and increased numbers, of bacterai were found in the bottom water. An association between the methane oxidizers and the sulfate reducers could be deduced. It was assumed that favourable redox requirements for obligate anaerobic sulfate reducers were the results of the activities of the methane oxidizing bacteria.The dynamic distribution equilibrium of the investigated groups of bacteria was disturbed by the autumn turn-over. The heterotrophic and methane oxidizing bacteria decreased in number at that period and were equally distributed, no sulfate reducers could be detected in the free water of Lake Vechten.     

Anaerobic Degradation of Glycerol by Desulfovibrio fructosovorans and D. carbinolicus and Evidence for Glycerol-Dependent Utilization of 1,2-Propanediol

The degradation of glycerol by Desulfovibrio carbinolicus and Desulfovibrio fructosovorans was tested in pure culture with sulfate and in coculture with Methanospirillum hungatei. Desulfovibrio carbinolicus degraded glycerol into 3-hydroxypropionate with the formation of sulfide in pure culture and methane in the coculture. The maximum growth rates were 0.063 h⁻¹ in pure culture and 0.014 h⁻¹ in coculture (corresponding growth yields: 8.9 and 6.0 g dry weight/mol glycerol). With D. fructosovorans, the pathway of glycerol degradation depended upon the terminal electron acceptor. Acetate and sulfide were produced in the presence of sulfate, while 3-hydroxypropionate and methane were formed by the syntrophic association with M. hungatei. The maximum growth rates were 0.057 h⁻¹ in pure culture and 0.020 h⁻¹ in coculture (corresponding growth yields: 8.9 and 6.0 g dry weight/mol glycerol). In a medium containing both glycerol and 1,2-propanediol but no sulfate, D. carbinolicus and D. fructosovorans degraded both substrates. A drop in the concentration of 1,3-propanediol was observed, and propionate and n-propanol production was recorded. Putative biochemical pathways of 1,2-propanediol degradation by D. carbinolicus and D. fructosovorans indicated that the enzymes involved in this metabolism are present only when the strains are grown on a mixture of 1,2-propanediol and glycerol without sulfate. Received: 1 October 1997 / Accepted: 3 November 1997     

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.     

Phylogenetic diversity and activity of anaerobic microorganisms of high-temperature horizons of the Dagang oil field (P. R. China)

The number of microorganisms of major metabolic groups and the rates of sulfate reduction and methanogenesis processes in the formation waters of the high-temperature horizons of Dagang oil field have been determined. Using cultural methods, it was shown that the microbial community contained aerobic bacteria oxidizing crude oil, anaerobic fermentative bacteria, sulfate-reducing bacteria, and methanogens. Using cultural methods, the possibility of methane production from a mixture of hydrogen and carbon dioxide (H₂ + CO₂) and from acetate was established, and this result was confirmed by radioisotope methods involving NaH¹⁴CO₃ and ¹⁴CH₃COONa. Analysis of enrichment cultures 16S rDNA of methanogens demonstrated that these microorganisms belong to Methanothermobacter sp. (M. thermautotrophicus), which consumes hydrogen and carbon dioxide as basic substrates. The genes of acetate-utilizing bacteria were not revealed. Phylotypes of the representatives of Thermococcus spp. were found among archaeal 16S rDNA. 16S rRNA genes of bacterial clones belong to the orders Thermoanaerobacteriales (Thermoanaerobacter, Thermovenabulum, Thermacetogenium, and Coprothermobacter spp.), Thermotogales, Nitrospirales (Thermodesulfovibrio sp.) and Planctomycetales. 16S rDNA of a bacterium capable of oxidizing acetate in the course of syntrophic growth with H₂-utilizing methanogens was found in high-temperature petroleum reservoirs for the first time. These results provide further insight into the composition of microbial communities of high-temperature petroleum reservoirs, indicating that syntrophic processes play an important part in acetate degradation accompanied by methane production.     

Fractionation of sulfur isotopes during dissimilatory reduction of sulfate by a thermophilic gram-negative bacterium at 60 °C

Sulfur isotope (³⁴S/³²S) fractionation during reduction of dissolved sulfate was investigated with a growing batch culture of a thermophilic, gram-negative, sulfate-reducing bacterium (strain MT-96) at 60 °C. The completely oxidizing strain was isolated from geothermally heated sediments of a shallow-water hydrothermal vent in the Mediterranean Sea. The hydrogen sulfide produced in the experiments was enriched in ³²S by approximately 19‰ as compared to sulfate, which indicates that stable isotope discrimination by this thermophile is within the range found previously for mesophilic sulfate-reducing bacteria, and only slightly higher than that observed for the thermophilic gram-positive Desulfotomaculum nigrificans. Received: 1 December 1998 / Accepted: 25 May 1999     

Activity of a Methanotrophic Consortium Isolated from Landfill Cover Soil: Response to Temperature,pH, CO2, and Porous Adsorbent

A robust, naturally evolving methanotrophic community in landfill cover soil (LFCS) can be the simplest way to mitigate landfill methane emission. In this study, bacterial community composition in LFCS and methane oxidation potential of enriched methanotrophic consortium, in comparison to that of axenic Methylosinus sporium, was investigated. Growth and methane oxidation of the consortium was studied in liquid phase batch experiments under varying temperature (20–40°C), pH (5–10), headspace CO2, and in presence of porous adsorbent (1.3 cm³ sponge cubes). The 16S rRNA gene analysis revealed presence of both type-I and type-II methanotrophs along with few obligate methylotroph in LFCS. Though the optimal growth condition of the consortium was at 30°C and pH 7, it was more resilient in comparison to M. sporium. With increasing availability of porous adsorbent, methane consumption by the consortium was significantly improved (p < 0.001) reaching a maximum specific methane oxidation rate of 11.4 μmol mg^?1 biomass h^?1. Thus, inducing naturally thriving methanotrophs in LFCS is a better alternative to axenic methanotrophic culture in methane emission management.     

Emission of methane from chalk streams has potential implications for agricultural practices

1. The emission of biogenic gases, particularly methane, is usually associated with wetlands rather than clean streams. Here, we investigated methane production from a southern English chalk stream, where increased sedimentation, compounded by extensive macrophyte growth, may have altered ecosystem function. 2. Cover of the channel by the dominant macrophyte, Ranunculus penicillatus, peaked in August, when plant beds were associated with low water velocity and the accumulation of sediment (<2000 μm) dominated by the sand‐sized fraction (63–1000 μm). 3. Over spring and summer there was a marked increase in the silt/clay fraction of the sediment, a concomitant drop in mean particle size and, hence, inferred permeability. At the same time there was an increase in CH₄ transport through Ranunculus stems and an increase in water column CH₄ concentration, while the sediment CH₄ concentration increased 100‐fold between February and April. A marked seasonal enrichment in the δ¹⁵N of N₂ dissolved in the pore water correlated with CH₄ flux and, coupled to the shift in particle size, suggested a transient input of organic matter, possibly of terrestrial origin. 4. Potential areal methane production and measured efflux were similar to that from some U.K. peatlands and represent one of the first accounts of significant methanogenesis to be measured in a stream channel. Most (>90%) of the methane flux is transported to the atmosphere through the Ranunculus stems. 5. Although the total flux of methane from U.K. chalk streams is probably relatively modest (estimated at 3.2 × 10⁻⁶ Tg CH₄ year⁻¹), this phenomenon changes our perception of the health of these ecosystems and indicates another deleterious side effect of agriculture.