Effect of fumarate reducing bacteria on in vitro rumen fermentation,methane mitigation and microbial diversity

The metabolic pathways involved in hydrogen (H₂) production, utilization and the activity of methanogens are the important factors that should be considered in controlling methane (CH₄) emissions by ruminants. H₂ as one of the major substrate for CH₄ production is therefore should be controlled. One of the strategies on reducing CH₄ is through the use of hydrogenotrophic microorganisms such as fumarate reducing bacteria. This study determined the effect of fumarate reducing bacteria, Mitsuokella jalaludinii, supplementation on in vitro rumen fermentation, CH4 production, diversity and quantity. M. jalaludinii significantly reduced CH₄ at 48 and 72 h of incubation and significantly increased succinate at 24 h. Although not significantly different, propionate was found to be highest in treatment containing M. jalaludinii at 12 and 48 h of incubation. These results suggest that supplementation of fumarate reducing bacteria to ruminal fermentation reduces CH₄ production and quantity, increases succinate and changes the rumen microbial diversity.     

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.     

Quantitative evaluation of ruminal methane and carbon dioxide formation from formate through C-13 stable isotope analysis in a batch culture system

Methane produced from formate is one of the important methanogensis pathways in the rumen. However, quantitative information of CH₄ production from formate has been rarely reported. The aim of this study was to characterize the conversion rate (CR) of formic acid into CH₄ and CO₂ by rumen microorganisms. Ground lucerne hay was incubated with buffered ruminal fluid for 6, 12, 24 and 48 h. Before the incubation, ¹³C-labeled H¹³COOH was also supplied into the incubation bottle at a dose of 0, 1.5, 2.2 or 2.9 mg/g of DM substrate. There were no interactions (P>0.05) between dose and incubation time for all variables evaluated. When expressed as an absolute amount (ml in gas sample) or a relative CR (%), both ¹³CH₄ and ¹³CO₂ production quadratically increased (P<0.01) with the addition of H¹³COOH. The total ¹³C (¹³CH₄ and ¹³CO₂) CR was also quadratically increased (P<0.01) when H¹³COOH was added. Moreover, formate addition linearly decreased (P<0.031) the concentrations of NH₃-N, total and individual volatile fatty acids (acetate, propionate and butyrate), and quadratically decreased (P<0.014) the populations of protozoa, total methanogens, Methanosphaera stadtmanae, Methanobrevibacter ruminantium M1, Methanobrevibacter smithii and Methanosarcina barkeri. In summary, formate affects ruminal fermentation and methanogenesis, as well as the rumen microbiome, in particular microorganisms which are directly or indirectly involved in ruminal methanogenesis. This study provides quantitative verification for the rapid dissimilation of formate into CH₄ and CO₂ by rumen microorganisms.     

Electron transfer of dissolved organic matter and its potential significance for anaerobic respiration in a northern bog

We investigated electron transfer processes of dissolved organic matter (DOM) and their potential importance for anaerobic heterotrophic respiration in a northern peatland. Electron accepting and donating capacities (EAC, EDC) of DOM were quantified using dissolved H₂S and ferric iron as reactants. Carbon turnover rates were obtained from porewater profiles (CO₂, CH₄) and inverse modeling. Carbon dioxide was released at rates of 0.2–5.9 mmol m⁻² day⁻¹ below the water table. Methane (CH₄) formation contributed <10%, and oxygen consumption 2% to 40%, leaving a major fraction of CO₂ production unexplained. DOM oxidized H₂S to thiosulfate and was reduced by dissolved ferric iron. Reduction with H₂S increased the subsequently determined EDC compared to untreated controls, indicating a reversibility of the electron transfer. In situ redox capacities of DOM ranged from 0.2 to 6.1 mEq g⁻¹ C (EAC) and from 0.0 to 1.4 mEq g⁻¹ C (EDC), respectively. EAC generally decreased with depth and changed after a water table drawdown and rebound by 20 and −45 mEq m⁻², respectively. The change in EAC during the water table fluctuation was similar to CH₄ formation rates. In peatlands, electron transfer of DOM may thus significantly contribute to the oxidation of reduced organic substrates by anaerobic heterotrophic respiration, or by maintaining the respiratory activity of sulfate reducers via provision of thiosulfate. Part of the anaerobic electron flow in peat soils is thus potentially diverted from methanogenesis, decreasing its contribution to the total carbon emitted to the atmosphere.     

Screening of plants from diversified natural grasslands for their potential to combine high digestibility,and low methane and ammonia production

A total of 156 plant species from 35 botanical families collected from diversified grasslands in the French Massif Central were screened in vitro for their potential to combine high nutritive value for ruminants and a reduced impact on the environment. The vegetative part of plants were analyzed for their chemical composition and incubated in a batch system containing buffered rumen fluid at 39°C for 24 h. The gas production and composition were recorded, and the fermentation end-product concentrations in the incubation medium and the in vitro true organic matter digestibility (IVTOMD) were determined. The results were expressed relative to perennial ryegrass (PRG) values used as a reference. We observed that no relationship between methane (CH₄) and volatile fatty acids (VFA) was evidenced for 12 plants, the fermentation of these plants producing significantly less CH₄ for a similar level of VFA production. In all, 13 plants showed 50% less CH₄ production per unit of organic matter truly digested (OMTD) than PRG. Among these plants, two reduced CH₄ by more than 80% and four species had an IVTOMD higher than 80%. The underlying modes of action seem to be different among plants: some result in an accumulation of H₂ in the fermentation gas, but others do not. In terms of nitrogen (N) use efficiency, the fermentation of 37 plants halved the ratio between ammonia (N–NH₃) and plant N content compared with PRG, of which six showed a complete absence of N–NH₃ in the medium. Among these plants, four maintained the IVTDMO at values not significantly different from PRG (P>0.05). Considering the multi-criteria selection, 16 plants showed simultaneously a reduction of more than 80% in N–NH₃ production and 30% in CH₄ emission per unit of OMTD compared with PRG, including three with an IVTOMD higher than 80%. Overall, the botanical families that reduced simultaneously CH₄ and N–NH₃ most efficiently were the Rosaceae, Onagraceae, Polygonaceae and Dipsacaceae. The Onagraceae also gave high values for IVTOMD.     

Long-term effect of linseed plus nitrate fed to dairy cows on enteric methane emission and nitrate and nitrite residuals in milk

A previous study showed the additive methane (CH₄)-mitigating effect of nitrate and linseed fed to non-lactating cows. Before practical application, the use of this new strategy in dairy cows requires further investigation in terms of persistency of methanogenesis reduction and absence of residuals in milk products. The objective of this experiment was to study the long-term effect of linseed plus nitrate on enteric CH₄ emission and performance in dairy cows. We also assessed the effect of this feeding strategy on the presence of nitrate residuals in milk products, total tract digestibility, nitrogen (N) balance and rumen fermentation. A total of 16 lactating Holstein cows were allocated to two groups in a randomised design conducted in parallel for 17 weeks. Diets were on a dry matter (DM) basis: (1) control (54% maize silage, 6% hay and 40% concentrate; CON) or (2) control plus 3.5% added fat from linseed and 1.8% nitrate (LIN+NIT). Diets were equivalent in terms of CP (16%), starch (28%) and NDF (33%), and were offered twice daily. Cows were fed ad libitum, except during weeks 5, 16 and 17 in which feed was restricted to 95% of dry matter intake (DMI) to ensure complete consumption of meals during measurement periods. Milk production and DMI were measured weekly. Nitrate and nitrite concentrations in milk and milk products were determined monthly. Daily CH₄ emission was quantified in open circuit respiration chambers (weeks 5 and 16). Total tract apparent digestibility, N balance and rumen fermentation parameters were determined in week 17. Daily DMI tended to be lower with LIN+NIT from week 4 to 16 (−5.1 kg/day on average). The LIN+NIT diet decreased milk production during 6 non-consecutive weeks (−2.5 kg/day on average). Nitrate or nitrite residuals were not detected in milk and associated products. The LIN+NIT diet reduced CH₄ emission to a similar extent at the beginning and end of the trial (−47%, g/day; −30%, g/kg DMI; −33%, g/kg fat- and protein-corrected milk, on average). Diets did not affect N efficiency and nutrients digestibility. In the rumen, LIN+NIT did not affect protozoa number but reduced total volatile fatty acid (−12%) and propionate (−31%) concentrations. We concluded that linseed plus nitrate may have a long-term CH₄-mitigating effect in dairy cows and that consuming milk products from cows fed nitrate may be safe in terms of nitrate and nitrite residuals. Further work is required to optimise the doses of linseed plus nitrate to avoid reduced cows performance.     

Effects of the inclusion of Moringa oleifera seed on rumen fermentation and methane production in a beef cattle diet using the rumen simulation technique (Rusitec)

Moringa oleifera seeds are currently being used as a livestock feed across tropical regions of the world due to its availability and palatability. However, limited knowledge exists on the effects of the raw seeds on ruminant metabolism. As such, the rumen stimulation technique was used to evaluate the effects of substituting increasing concentrations of ground Moringa seeds (0, 100, 200 and 400 g/kg concentrate dry matter (DM)) in the diet on rumen fermentation and methane production. Two identical, Rusitec apparatuses, each with eight fermenters were used with the first 8 days used for adaptation and days 9 to 16 used for measurements. Fermenters were fed a total mixed ration with Urochloa brizantha as the forage. Disappearance of DM, CP, NDF and ADF linearly decreased (P<0.01) with increasing concentrations of Moringa seeds in the diet. Total volatile fatty acid production and the acetate to propionate ratio were also linearly decreased (P<0.01). However, only the 400 g/kg (concentrate DM basis) treatment differed (P<0.01) from the control. Methane production (%), total microbial incorporation of ¹⁵N and total production of microbial N linearly decreased (P<0.01) as the inclusion of Moringa seeds increased. Though the inclusion of Moringa seeds in the diet decreased CH₄ production, this arose from an unfavourable decrease in diet digestibility and rumen fermentation parameters.     

Changes in in vitro gas and methane production from rumen fluid from dairy cows during adaptation to feed additives in vivo

The adaptation of dairy cows to methane (CH₄)-mitigating feed additives was evaluated using the in vitro gas production (GP) technique. Nine rumen-fistulated lactating Holstein cows were grouped into three blocks and within blocks randomly assigned to one of three experimental diets: Control (CON; no feed additive), Agolin Ruminant^R (AR; 0.05 g/kg dry matter (DM)) or lauric acid (LA; 30 g/kg DM). Total mixed rations composed of maize silage, grass silage and concentrate were fed in a 40 : 30 : 30 ratio on DM basis. Rumen fluid was collected from each cow at days −4, 1, 4, 8, 15 and 22 relative to the introduction of the additives in the diets. On each of these days, a 48-h GP experiment was performed in which rumen fluid from each individual donor cow was incubated with each of the three substrates that reflected the treatment diets offered to the cows. DM intake was on average 19.8, 20.1 and 16.2 kg/day with an average fat- and protein-corrected milk production of 30.7, 31.7 and 26.2 kg/day with diet CON, AR and LA, respectively. In general, feed additives in the donor cow diet had a larger effect on gas and CH₄ production than the same additives in the incubation substrate. Incubation substrate affected asymptotic GP, half-time of asymptotic CH₄ production, total volatile fatty acid (VFA) concentration, molar proportions of propionate and butyrate and degradation of organic matter (OMD), but did not affect CH₄ production. No substrate×day interactions were observed. A significant diet×day interaction was observed for in vitro gas and CH₄ production, total VFA concentration, molar proportions of VFA and OMD. From day 4 onwards, the LA diet persistently reduced gas and CH₄ production, total VFA concentration, acetate molar proportion and OMD, and increased propionate molar proportion. In vitro CH₄ production was reduced by the AR diet on day 8, but not on days 15 and 22. In line with these findings, the molar proportion of propionate in fermentation fluid was greater, and that of acetate smaller, for the AR diet than for the CON diet on day 8, but not on days 15 and 22. Overall, the data indicate a short-term effect of AR on CH₄ production, whereas the CH₄-mitigating effect of LA persisted.     

Effects of Propionibacterium strains on ruminal fermentation,nutrient digestibility and methane emissions in beef cattle fed a corn grain finishing diet

Twenty ruminally cannulated beef heifers were fed a high corn grain diet in a randomized block design to determine the effect of three direct fed microbial (DFM) strains of Propionibacterium on ruminal fermentation, nutrient digestibility and methane (CH₄) emissions. The heifers were blocked in five groups on the basis of BW and used in five 28-day periods. Dietary treatments included (1) Control and three strains of Propionibacterium (2) P169, (3) P5, and (4) P54. Strains were administered directly into the rumen at 5×10⁹ CFU with 10 g of a maltodextrin carrier in a gel capsule; Control heifers received carrier only. All heifers were fed the basal diet (10 : 90 forage to concentrate, dry matter basis). Rumen contents were collected on days 15 and 18, ruminal pH was measured continuously between days 15 and 22, enteric CH₄ emissions were measured between days 19 and 22 and diet digestibility was measured from days 25 to 28. Mean ruminal pH was 5.91 and was not affected by treatments. Similarly, duration of time that pH<5.8 and 5.6 was not affected by treatment. Likewise, total and major volatile fatty acid profiles were similar among all treatments. No effects were observed on dry matter intake and total tract digestibility of nutrients. Total enteric CH₄ production (g/day) was not affected by Propionibacterium strains and averaged 139 g/day. Similarly, mean CH₄ yield (g CH₄/kg of dry matter intake) was similar for all the treatments. The relative abundance of total Propionibacteria in the rumen increased with administration of DFM and were greater 3 h post-dosing relative to Control, but returned to baseline levels before feeding. Populations of Propionibacterium P169 were higher at 3 and 9 h as compared with the levels at 0 h. In conclusion, moderate persistency of the inoculated strains within the ruminal microbiome and pre-existing high propionate production due to elevated levels of starch fermentation might have reduced the efficacy of Propionibacterium strains to increase molar proportion of propionate and subsequently reduce CH₄ emissions.     

Effects of protozoa on Methane production in rumen and hindgut of calves around time of weaning

Effects of the presence or absence of ciliate protozoa on methanogenesis in the rumen and hindgut were investigated in young calves during a 7-week period. Ten Holstein calves, aged 7 days, were divided in two groups (n = 5) and fed an increasing amount of a commercial milk replacer and small amounts of a calves starter. One group was inoculated with ciliate fauna on two occasions, week 5 and 6, while the second remained ciliate-free. The absence of protozoa in the rumen decreased rumen empty weight ( ? 23%, P < 0.01), and rumen pool size of N ( ? 36%, P < 0.01) and crude fat ( ? 37%, P < 0.05). Rumen bacteria of non-faunated calves contained a higher proportion of total amino acid-N per 16 g N ( + 3%, P < 0.01) and D-alanine-N per 16 g N ( + 13%, P < 0.05) compared to faunated calves. Further results contain a reference for a higher bacterial mass in the ciliate-free rumen with an increased number of bacteria adherent to rumen mucosa. The CH₄ production in the rumen increased exponentially with the increase in protozoa population size (R² = 0.68). In presence of 46 · 10⁴ protozoa per ml rumen fluid, the in vitro CH₄ production of rumen fluid per mol total VFA was about 34% higher in faunated than in non-faunated calves (P < 0.001). Hydrogen (2H) recovery of rumen fermentation was positively correlated (R² = 0.55) to the CH₄ production rate. Methanogens were attached on rumen mucosa. Methanogenesis, induced by rumen mucosa attached bacteria, was stimulated by ruminal protozoa. In the absence of protozoa in the rumen, the acetate - propionate ratio and butyrate proportion of VFA were reduced. In vivo in the absence of protozoa not only the whole animal CH₄ production ( ? 30%, P < 0.05) but also the digestibility of carbohydrates ( ? 4%, P < 0.05) was reduced. Thereby no difference was observed in the intake of ME per kg DM between the groups. In conclusion, the methanogenesis in the rumen, but not in hindgut, is associated with the development of the ruminal protozoa population. The level of methanogenesis (mol/mol VFA) in the hindgut amounts to 20% of the ruminal methanogenesis.     

Comparison of the effects of lanthanum,cerium and praseodymium on rumen fermentation,nutrient digestion and plasma biochemical parameters in beef cattle

The objectives of the trial were to compare the effects of supplementing rare earth elements (REE) lanthanum (La), cerium (Ce) and praseodymium (Pr) on rumen fermentation, nutrient digestion, methane (CH₄) production, nitrogen (N) balance and plasma biochemical parameters in beef cattle. Four Simmental male cattle, aged 12 months, with initial average liveweight of 333 ± 9 kg and fitted with rumen cannulas, were fed with a basal ration composed of concentrate mixture and maize silage. Animals received a basal ration without adding REE (Control) or three treatments, i.e. supplementing LaCl₃, CeCl₃ or PrCl₃ at 204 mg/kg DM to the basal ration, respectively, which were allocated in a 4 × 4 Latin square design. Each experimental period lasted 15 d, consisting of 12 d for pre-treatment and three subsequent days for sampling. Results showed that all tested levels of REE tended to increase neutral detergent fibre digestibility (p = 0.064) and tended to decrease rumen CH₄ production (p = 0.056). Supplementing LaCl₃ and CeCl₃ decreased total N excretion and urinary N excretion, increased N retention (p < 0.05), tended to increase total urinary purine derivatives (PD) (p = 0.053) and microbial N flow (p = 0.095), whereas supplementing PrCl₃ did not affect N retention, urinary PD and microbial N flow. No differences were found in the effects of nutrient digestibility, CH₄ production and plasma biochemical parameters among LaCl₃, CeCl₃ and PrCl₃. Further trials using graded levels of LaCl₃, CeCl₃ and PrCl₃ in a wide range are needed to obtain more pronounced results for comparing effects of La, Ce and Pr on rumen fermentation and nutrient digestion in beef cattle.     

Partitioning of CH4 and CO2 Production Originating from Rice Straw,Soil and Root Organic Carbon in Rice Microcosms

Flooded rice fields are an important source of the greenhouse gas CH₄. Possible carbon sources for CH₄ and CO₂ production in rice fields are soil organic matter (SOM), root organic carbon (ROC) and rice straw (RS), but partitioning of the flux between the different carbon sources is difficult. We conducted greenhouse experiments using soil microcosms planted with rice. The soil was amended with and without ¹³C-labeled RS, using two ¹³C-labeled RS treatments with equal RS (5 g kg⁻¹ soil) but different δ¹³C of RS. This procedure allowed to determine the carbon flux from each of the three sources (SOM, ROC, RS) by determining the δ¹³C of CH₄ and CO₂ in the different incubations and from the δ¹³C of RS. Partitioning of carbon flux indicated that the contribution of ROC to CH₄ production was 41% at tillering stage, increased with rice growth and was about 60% from the booting stage onwards. The contribution of ROC to CO₂ was 43% at tillering stage, increased to around 70% at booting stage and stayed relatively constant afterwards. The contribution of RS was determined to be in a range of 12–24% for CH₄ production and 11–31% for CO₂ production; while the contribution of SOM was calculated to be 23–35% for CH₄ production and 13–26% for CO₂ production. The results indicate that ROC was the major source of CH₄ though RS application greatly enhanced production and emission of CH₄ in rice field soil. Our results also suggest that data of CH₄ dissolved in rice field could be used as a proxy for the produced CH₄ after tillering stage.     

Oxygen effects on methane production and oxidation in humid tropical forest soils

We investigated the effects of oxygen (O₂) concentration on methane (CH₄) production and oxidation in two humid tropical forests that differ in long‐term, time‐averaged soil O₂ concentrations. We identified sources and sinks of CH₄ through the analysis of soil gas concentrations, surface emissions, and carbon isotope measurements. Isotope mass balance models were used to calculate the fraction of CH₄ oxidized in situ. Complementary laboratory experiments were conducted to determine the effects of O₂ concentration on gross and net rates of methanogenesis. Field and laboratory experiments indicated that high levels of CH₄ production occurred in soils that contained between 9±1.1% and 19±0.2% O₂. For example, we observed CH₄ concentrations in excess of 3% in soils with 9±1.1% O₂. CH₄ emissions from the lower O₂ sites were high (22–101 nmol CH₄ m^?2 s^?1), and were equal in magnitude to CH₄ emissions from natural wetlands. During peak periods of CH₄ efflux, carbon dioxide (CO₂) emissions became enriched in ¹³C because of high methanogenic activity. Gross CH₄ production was probably greater than flux measurements indicated, as isotope mass balance calculations suggested that 48–78% of the CH₄ produced was oxidized prior to atmospheric egress. O₂ availability influenced CH₄ oxidation more strongly than methanogenesis. Gross CH₄ production was relatively insensitive to O₂ concentrations in laboratory experiments. In contrast, methanotrophic bacteria oxidized a greater fraction of total CH₄ production with increasing O₂ concentration, shifting the δ¹³C composition of CH₄ to values that were more positive. Isotopic measurements suggested that CO₂ was an important source of carbon for methanogenesis in humid forests. The δ¹³C value of methanogenesis was between ?84‰ and ?98‰, which is well within the range of CH₄ produced from CO₂ reduction, and considerably more depleted in ¹³C than CH₄ formed from acetate.     

Effects of dehydrated lucerne and soya bean meal on milk production and composition,nutrient digestion,and methane and nitrogen losses in dairy cows receiving two different forages

Dehydrated lucerne is used as a protein source in dairy cow rations, but little is known about the effects of lucerne on greenhouse gas production by animals. Eight Holstein dairy cows (average weight: 582 kg) were used in a replicated 4×4 Latin square design. They received diets based on either maize silage (M) or grass silage (G) (45% of diet on dry matter (DM) basis), with either soya bean meal (15% of diet DM) completed with beet pulp (15% of diet DM) (SP) or dehydrated lucerne (L) (30% of diet DM) as protein sources; MSP, ML, GSP and GL diets were calculated to meet energy requirements for milk production by dairy cows and degradable protein for rumen microbes. Dry matter intake (DMI) did not differ among diets (18.0 kg/day DMI); milk production was higher with SP diets than with L diets (26.0 v. 24.1 kg/day), but milk production did not vary with forage type. Milk fatty-acid (FA) composition was modified by both forage and protein sources: L and G diets resulted in less saturated FA, less linoleic acid, more trans-monounsaturated FA, and more linolenic acid than SP and M diets, respectively. Enteric methane (CH₄) production, measured by the SF₆ tracer method, was higher for G diets than for M diets, but did not differ with protein source. The same effects were observed when CH₄ was expressed per kg milk. Minor effects of diets on rumen fermentation pattern were observed. Manure CH₄ emissions estimated from faecal organic matter were negatively related to diet digestibility and were thus higher for L than SP diets, and higher for M than G diets; the resulting difference in total CH₄ production was small. Owing to diet formulation constraints, N intake was higher for SP than for L diets; interaction between forage type and protein source was significant for N intake. The same statistical effects were found for N in milk. Faecal and urinary N losses were determined from total faeces and urine collection. Faecal N output was lower for M than for G diets but did not differ between protein sources. Urinary N output did not differ between forage types, but was lower for cows fed L diets than for cows fed SP diets, potentially resulting in lower ammonia emissions with L diets. Replacing soya bean meal plus beet pulp with dehydrated lucerne did not change CH₄ production, but resulted in more N in faeces and less N in urine.     

Predicting long‐term carbon mineralization and trace gas production from thawing permafrost of Northeast Siberia

The currently observed Arctic warming will increase permafrost degradation followed by mineralization of formerly frozen organic matter to carbon dioxide (CO₂) and methane (CH₄). Despite increasing awareness of permafrost carbon vulnerability, the potential long‐term formation of trace gases from thawing permafrost remains unclear. The objective of the current study is to quantify the potential long‐term release of trace gases from permafrost organic matter. Therefore, Holocene and Pleistocene permafrost deposits were sampled in the Lena River Delta, Northeast Siberia. The sampled permafrost contained between 0.6% and 12.4% organic carbon. CO₂ and CH₄ production was measured for 1200 days in aerobic and anaerobic incubations at 4 °C. The derived fluxes were used to estimate parameters of a two pool carbon degradation model. Total CO₂ production was similar in Holocene permafrost (1.3 ± 0.8 mg CO₂‐C gdw^?1 aerobically, 0.25 ± 0.13 mg CO₂‐C gdw^?1 anaerobically) as in 34 000–42 000‐year‐old Pleistocene permafrost (1.6 ± 1.2 mg CO₂‐C gdw^?1 aerobically, 0.26 ± 0.10 mg CO₂‐C gdw^?1 anaerobically). The main predictor for carbon mineralization was the content of organic matter. Anaerobic conditions strongly reduced carbon mineralization since only 25% of aerobically mineralized carbon was released as CO₂ and CH₄ in the absence of oxygen. CH₄ production was low or absent in most of the Pleistocene permafrost and always started after a significant delay. After 1200 days on average 3.1% of initial carbon was mineralized to CO₂ under aerobic conditions while without oxygen 0.55% were released as CO₂ and 0.28% as CH₄. The calibrated carbon degradation model predicted cumulative CO₂ production over a period of 100 years accounting for 15.1% (aerobic) and 1.8% (anaerobic) of initial organic carbon, which is significantly less than recent estimates. The multiyear time series from the incubation experiments helps to more reliably constrain projections of future trace gas fluxes from thawing permafrost landscapes.     

Partitioning pathways of CO2 production in peatlands with stable carbon isotopes

Although methanogenic pathways generally produce equimolar amounts of carbon dioxide and methane, CO₂ concentrations are often reported to be higher than CH₄ concentrations in both field and laboratory incubation studies of peat decomposition. In field settings, higher pore water concentrations of CO₂ may result from the loss of methane by: (1) ebullition due to the low solubility of methane in pore water and (2) vascular-plant transport. Higher CO₂ concentrations may also be caused by: (1) production of additional CO₂ by high-molecular weight (HMW) organic matter (OM) fermentation and/or (2) respiration from non-methanogenic pathways. In this study of a peatland where advection and transverse dispersion were the dominant pore water solute transport mechanisms, an isotope-mass balance approach was used to determine the proportions of CO₂ formed from non-fractionating OM respiration and HMW fermentation relative to CO₂ production from methanogenesis. This approach also allowed us to estimate the loss of CH₄ from the belowground system. The pathways of CO₂ production varied with depth and surface vegetation type. In a Carex-dominated fen, methane production initially produced 40 % of the total CO₂ and then increased to 90–100 % with increasing depth. In a Sphagnum-dominated bog, methanogenesis resulted in 60 % of total CO₂ production which increased to 100 % at depth. Both bogs and fens showed 85–100 % of methane loss from pore waters. Our results indicate that the isotopic composition of dissolved CO₂ is a powerful indicator to allow partitioning of the processes affecting peat remineralization and methane production.     

Individual variation and repeatability of methane production from dairy cows estimated by the CO2 method in automatic milking system

The objectives of this study were to investigate the individual variation, repeatability and correlation of methane (CH₄) production from dairy cows measured during 2 different years. A total of 21 dairy cows with an average BW of 619±14.2 kg and average milk production of 29.1±6.5 kg/day (mean±s.d.) were used in the 1^st year. During the 2^nd year, the same cows were used with an average BW of 640±8.0 kg and average milk production of 33.4±6.0 kg/day (mean±s.d.). The cows were housed in a loose housing system fitted with an automatic milking system (AMS). A total mixed ration was fed to the cows ad libitum in both years. In addition, they were offered concentrate in the AMS based on their daily milk yield. The CH₄ and CO₂ production levels of the cows were analysed using a Gasmet DX-4030. The estimated dry matter intake (EDMI) was 19.8±0.96 and 23.1±0.78 (mean±s.d.), and the energy-corrected milk (ECM) production was 30.8±8.03 and 33.7±5.25 kg/day (mean±s.d.) during the 1^st and 2^nd year, respectively. The EDMI and ECM had a significant influence (P<0.001) on the CH₄ (l/day) yield during both years. The daily CH₄ (l/day) production was significantly higher (P<0.05) during the 2^nd year compared with the 1^st year. The EDMI (described by the ECM) appeared to be the key factor in the variation of CH₄ release. A correlation (r=0.54) of CH₄ production was observed between the years. The CH₄ (l/day) production was strongly correlated (r=0.70) between the 2 years with an adjusted ECM production (30 kg/day). The diurnal variation of CH₄ (l/h) production showed significantly lower (P<0.05) emission during the night (0000 to 0800 h). The between-cows variation of CH₄ (l/day, l/kg EDMI and l/kg ECM) was lower compared with the within-cow variation for the 1^st and 2^nd years. The repeatability of CH₄ production (l/day) was 0.51 between 2 years. In conclusion, a higher EDMI (kg/day) followed by a higher ECM (kg/day) showed a higher CH₄ production (l/day) in the 2^nd year. The variations of CH₄ (l/day) among the cows were lower than the within-cow variations. The CH₄ (l/day) production was highly repeatable and, with an adjusted ECM production, was correlated between the years.     

Potential use of milk mid-infrared spectra to predict individual methane emission of dairy cows

This study investigates the feasibility to predict individual methane (CH₄) emissions from dairy cows using milk mid-infrared (MIR) spectra. To have a large variability of milk composition, two experiments were conducted on 11 lactating Holstein cows (two primiparous and nine multiparous). The first experiment aimed to induce a large variation in CH₄ emission by feeding two different diets: the first one was mainly composed of fresh grass and sugar beet pulp and the second one of maize silage and hay. The second experiment consisted of grass and corn silage with cracked corn, soybean meal and dried pulp. For each milking period, the milk yields were recorded twice daily and a milk sample of 50 ml was collected from each cow and analyzed by MIR spectrometry. Individual CH₄ emissions were measured daily using the sulfur hexafluoride method during a 7-day period. CH₄ daily emissions ranged from 10.2 to 47.1 g CH₄/kg of milk. The spectral data were transformed to represent an average daily milk spectrum (AMS), which was related to the recorded daily CH₄ data. By assuming a delay before the production of fermentation products in the rumen and their use to produce milk components, five different calculations were used: AMS at days 0, 0.5, 1, 1.5 and 2 compared with the CH₄ measurement. The equations were built using Partial Least Squares regression. From the calculated R²_cv, it appears that the accuracy of CH₄ prediction by MIR changed in function of the milking days. In our experimental conditions, the AMS at day 1.5 compared with the measure of CH₄ emissions gave the best results. The R² and s.e. of the cross-validation were equal to 0.79 and 5.14 g of CH₄/kg of milk. The multiple correlation analysis performed in this study showed the existence of a close relationship between milk fatty acid (FA) profile and CH₄ emission at day 1.5. The lower R² (R² = 0.76) obtained between FA profile and CH₄ emission compared with the one corresponding to the obtained calibration (R²_c = 0.87) shows the interest to apply directly the developed CH₄ equation instead of the use of correlations between FA and CH₄. In conclusion, our preliminary results suggest the feasibility of direct CH₄ prediction from milk MIR spectra. Additional research has the potential to improve the calibrations even further. This alternative method could be useful to predict the individual CH₄ emissions at farm level or at the regional scale and it also could be used to identify low-CH₄-emitting cows.     

Methane and soil CO2 production from current‐season photosynthates in a rice paddy exposed to elevated CO2 concentration and soil temperature

Quantification of rhizodeposition (root exudates and root turnover) represents a major challenge for understanding the links between above‐ground assimilation and below‐ground anoxic decomposition of organic carbon in rice paddy ecosystems. Free‐air CO₂ enrichment (FACE) fumigating depleted ¹³CO₂ in rice paddy resulted in a smaller ¹³C/¹²C ratio in plant‐assimilated carbon, providing a unique measure by which we partitioned the sources of decomposed gases (CO₂ and CH₄) into current‐season photosynthates (new C) and soil organic matter (old C). In addition, we imposed a soil‐warming treatment nested within the CO₂ treatments to assess whether the carbon source was sensitive to warming. Compared with the ambient CO₂ treatment, the FACE treatment decreased the ¹³C/¹²C ratio not only in the rice‐plant carbon but also in the soil CO₂ and CH₄. The estimated new C contribution to dissolved CO₂ was minor (ca. 20%) at the tillering stage, increased with rice growth and was about 50% from the panicle‐formation stage onwards. For CH₄, the contribution of new C was greater than for heterotrophic CO₂ production; ca. 40–60% of season‐total CH₄ production originated from new C with a tendency toward even larger new C contribution with soil warming, presumably because enhanced root decay provided substrates for greater CH₄ production. The results suggest a fast and close coupling between photosynthesis and anoxic decomposition in soil, and further indicate a positive feedback of global warming by enhanced CH₄ emission through greater rhizodeposition.     

Studies on potential effects of fumaric acid on rumen microbial fermentation,methane production and microbial community

The greenhouse gas methane (CH₄) contributes substantially to global climate change. As a potential approach to decrease ruminal methanogenesis, the effects of different dosages of fumaric acid (FA) on ruminal microbial metabolism and on the microbial community (archaea, bacteria) were studied using a rumen simulation technique (RUSITEC). FA acts as alternative hydrogen acceptor diverting 2H from methanogenesis of archaea towards propionate formation of bacteria. Three identical trials were conducted with 12 fermentation vessels over a period of 14 days. In each trial, four fermentation vessels were assigned to one of the three treatment groups differing in FA dosage: low fumaric acid (LFA), high fumaric acid (HFA) and without FA (control). FA was continuously infused with the buffer. Grass silage and concentrate served as substrate. FA led to decreases in pH and to higher production rates of total short chain fatty acids (SCFA) mediated by increases in propionate for LFA of 1.69 mmol d^?1 and in propionate and acetate production for HFA of 4.49 and 1.10 mmol d^?1, respectively. Concentrations of NH₃-N, microbial crude protein synthesis, their efficiency, degradation of crude nutrients and detergent fibre fraction were unchanged. Total gas and CH₄ production were not affected by FA. Effects of FA on structure of microbial community by means of single strand conformation polymorphism (SSCP) analyses could not be detected. Given the observed increase in propionate production and the unaffected CH₄ production it can be supposed that the availability of reduction equivalents like 2H was not limited by the addition of FA in this study. It has to be concluded from the present study that the application of FA is not an appropriate approach to decrease the ruminal CH₄ production.