Inhibitory effects of free propionic and butyric acids on the activities of hydrogenotrophic methanogens in mesophilic mixed culture fermentation.

The aim of this work was to study the inhibitory of free propionic acid (FPA) and free butyric acid (FBA) on enriched hydrogenotrophic methanogens. It demonstrated that concentrations of FPA and FBA were correlated well with the specific methanogenic activity. Coenzyme M concentrations also agreed well with the trends of FPA and FBA. Two fators of C50% (concentration at 50% inhibition) and CRC (recoverable concentration from inhibition) were used to quantitively analyze the inhibitory order using the former result of free acetic acid (FAA) and the results of FBA and FPA. The order according to C50% was FAA (5.2 mM) > FBA (8.3 mM) > FPA (8.5 mM), while for CRC it was FPA (9.3 mM) > FAA = FBA (13.5 mM). After comparing with literatue, it suggests that the toxicities of these three organic acids are similar. Thus, accumulating free organic acid offers a cost-effective method to inhibit methanogenesis.

Nitrate decreases ruminal methane production with slight changes to ruminal methanogen composition of nitrate-adapted steers.

BACKGROUND: This study was conducted to examine effects of nitrate on ruminal methane production, methanogen abundance, and composition. Six rumen-fistulated Limousin×Jinnan steers were fed diets supplemented with either 0% (0NR), 1% (1NR), or 2% (2NR) nitrate (dry matter basis) regimens in succession. Rumen fluid was taken after two-week adaptation for evaluation of in vitro methane production, methanogen abundance, and composition measurements. RESULTS: Results showed that nitrate significantly decreased in vitro ruminal methane production at 6 h, 12 h, and 24 h (P < 0.01; P < 0.01; P = 0.01). The 1NR and 2NR regimens numerically reduced the methanogen population by 4.47% and 25.82% respectively. However, there was no significant difference observed between treatments. The alpha and beta diversity of the methanogen community was not significantly changed by nitrate either. However, the relative abundance of the methanogen genera was greatly changed. Methanosphaera (PL = 0.0033) and Methanimicrococcus (PL = 0.0113) abundance increased linearly commensurate with increasing nitration levels, while Methanoplanus abundance was significantly decreased (PL = 0.0013). The population of Methanoculleus, the least frequently identified genus in this study, exhibited quadratic growth from 0% to 2% when nitrate was added (PQ = 0.0140). CONCLUSIONS: Correlation analysis found that methane reduction was significantly related to Methanobrevibacter and Methanoplanus abundance, and negatively correlated with Methanosphaera and Methanimicrococcus abundance.

Assessment of Bacterial Community Composition of Anaerobic Granular Sludge in Response to Short-Term Uranium Exposure.

The effect of 10-50 µM uranium (U(VI)) on the bacterial community of anaerobic granular sludge was investigated by 24-h exposure tests, after which the bacterial community was analyzed by high-throughput sequencing. The specific U(VI) reducing activity of the anaerobic granular sludge ranged between 3.1 to 19.7 µM U(VI) g-1(VSS) h-1, independently of the initial U(VI) concentration. Alpha diversity revealed that microbial richness and diversity was the highest for anaerobic granular sludge upon 10 µM uranium exposure. Compared with the original biomass, the phylum of Euryarchaeota was significantly affected, whereas the Bacteroidetes, Firmicutes, and Synergistetes phyla were only slightly affected. However, the abundance of Chloroflexi and Proteobacteria phyla clearly increased after 24 h uranium exposure. Based on the genus level analysis, significant differences appeared in the bacterial abundance after uranium exposure. The proportions of Pseudomonas, Acinetobacter, Parabacteroides, Brevundimonas, Sulfurovum, and Trichococcus increased significantly, while the abundance of Paludibacter and Erysipelotrichaceae incertae sedis decreased dramatically. This study shows a dynamic diversification of the bacterial composition as a response to a short time (24 h) U(VI) exposure (10-50 µM).

Effects of nicotine on the biosynthesis of carotenoids in halophilic Archaea (class Halobacteria): an HPLC and Raman spectroscopy study.

Nicotine has a profound influence on the carotenoid metabolism in halophilic Archaea of the class Halobacteria. In a study of Halobacterium salinarum, Haloarcula marismortui and Halorubrum sodomense, using different analytical techniques to monitor the production of different carotenoids as a function of the presence of nicotine, we showed that the formation of α-bacterioruberin was inhibited in all. In Hbt. salinarum, addition of nicotine led to a significant change in the color of the culture due to the accumulation of lycopene, in addition to the formation of bisanhydrobacterioruberin which does not differ in color from α-bacterioruberin. Very little or no lycopene was formed in Har. marismortui and in Hrr. sodomense; instead bisanhydrobacterioruberin was the only major carotenoid found in nicotine-amended cultures. The findings are discussed in the framework of the recently elucidated biochemical pathway for the formation of the different carotenoid pigments encountered in the Halobacteria.

Transport, fate, and long-term impacts of metal oxide nanoparticles on the stability of an anaerobic methanogenic system with anaerobic granular sludge.

The fate and long-term effect of different metal oxide (TiO2, CuO and ZnO) nanoparticles (NPs) on anaerobic granular sludge (AGS) was evaluated in an anaerobic methanogenic system. Operation stability and structural characteristics of the granules were compared, the metabolism changes in the microbial community were quantified, and NPs fate were investigated. CuO NPs had greatest toxic effect on AGS after extended exposure, whereas ZnO NPs benefited methanogenesis temporarily (no more than 5d). The inhibition on AGS caused by NPs varied due to the unique structure of AGS and different toxic mechanism. Structural changes of AGS provided new evidence that tested NPs have different toxicity.

Dietary pea fiber increases diversity of colonic methanogens of pigs with a shift from Methanobrevibacter to Methanomassiliicoccus-like genus and change in numbers of three hydrogenotrophs.

BACKGROUND: Pea fiber (PF) is a potential fibrous supplement in swine production. The influence of dietary PF on microbial community in the colon of pigs remains largely unexplored. Methanogens in the hindgut of monogastric animals play important roles in degradation of dietary fibers and efficient removal of microbial metabolic end product H2. Understanding the impact of dietary PF on the structure of colonic methanogens may help understand the mechanisms of microbe-mediated physiological functions of PF. This study investigated the influence of PF on the diversity and quantity and/or activity of colonic methanongens of piglets and finishing pigs. Four archaeal 16S rRNA clone libraries were constructed for piglets and finishers fed with control (Piglet-C and Finisher-C) or PF diet (Piglet-P and Finisher-P). RESULTS: There were 195, 190, 194 and 196 clones obtained from the library Piglet-C, Piglet-P, Finisher-C and Finisher-P, respectively, with corresponding 12, 11, 11 and 16 OTUs (operational taxonomic units). Significant differences of Shannon Index among the four libraries were found (P < 0.05). Libshuff analysis showed that the archaeal community structure among the four libraries were significantly different (P < 0.0001). The predominant methanogens shifted from Methanobrevibacter to Methanobrevibacter and Methanomassiliicoccus-like genus as a result of dietary PF. Supplementation of PF significantly increased the copy numbers of mcrA and dsrA genes (P < 0.05). CONCLUSIONS: Alteration of methanogenic community structure may lead to functional transition from utilization of H2/CO2 to employment of both H2/CO2 and methanol/CO2. Quantification of three functional genes (mcrA, dsrA and fhs) of methanogens, sulfate-reducing bacteria (SRB) and acetogens revealed that dietary PF also increased the activity of methanogens and SRB,probably associated with increased proportion of Methanomassiliicoccus luminyensis-species. Further study is required to examine the interaction between specific methanogens and SRB during fermentation of dietary PF.

Bioactive fractions from the pasture legume Biserrula pelecinus L. have an anti-methanogenic effect against key rumen methanogens.

Methanogenic archaea (methanogens) are common inhabitants of the mammalian intestinal tract. In ruminants, they are responsible for producing abundant amounts of methane during digestion of food, but selected bioactive plants and compounds may inhibit this activity. Recently, we have identified that, Biserrula pelecinus L. (biserrula) is one such plant and the current study investigated the specific anti-methanogenic activity of the plant. Bioassay-guided extraction and fractionation, coupled with in vitro fermentation batch culture were used to select the most bioactive fractions of biserrula. The four fractions were then tested against five species of methanogens grown in pure culture. Fraction bioactivity was assessed by measuring methane production and amplification of the methanogen mcrA gene. Treatments that showed bioactivity were subcultured in fresh broth without the bioactive fraction to distinguish between static and cidal effects. All four fractions were active against pure cultures, but the F2 fraction was the most consistent inhibitor of both methane production and cell growth, affecting four species of methanogens and also producing equivocal-cidal effects on the methanogens. Other fractions had selective activity affecting only some methanogens, or reducing either methane production or methanogenic cell growth. In conclusion, the anti-methanogenic activity of biserrula can be linked to compounds contained in selected bioactive fractions, with the F2 fraction strongly affecting key rumen methanogens. Further study is required to identify the specific plant compounds in biserrula that are responsible for the anti-methanogenic activity. These findings will help devise novel strategies to control methanogen populations and activity in the rumen, and consequently contribute in reducing greenhouse gas emissions from ruminants.

Effects and mode of action of chitosan and ivy fruit saponins on the microbiome, fermentation and methanogenesis in the rumen simulation technique.

This study investigates the effects of supplementing a control diet (CON) with chitosan (CHI) or ivy fruit saponins (IVY) as natural feed additives. Both additives had similar abilities to decrease rumen methanogenesis (-42% and -40%, respectively) using different mechanisms: due to its antimicrobial and nutritional properties CHI promoted a shift in the fermentation pattern towards propionate production which explained about two thirds of the decrease in methanogenesis. This shift was achieved by a simplification of the structure in the bacterial community and a substitution of fibrolytic (Firmicutes and Fibrobacteres) by amylolytic bacteria (Bacteroidetes and Proteobacteria) which led to greater amylase activity, lactate and microbial protein yield with no detrimental effect on feed digestibility. Contrarily, IVY had negligible nutritional properties promoting minor changes in the fermentation pattern and on the bacterial community. Instead, IVY modified the structure of the methanogen community and decreased its diversity. This specific antimicrobial effect of IVY against methanogens was considered its main antimethanogenic mechanism. IVY had however a negative impact on microbial protein synthesis. Therefore, CHI and IVY should be further investigated in vivo to determine the optimum doses which maintain low methanogenesis but prevent negative effects on the rumen fermentation and animal metabolism.

Bromochloromethane, a Methane Analogue, Affects the Microbiota and Metabolic Profiles of the Rat Gastrointestinal Tract.

Bromochloromethane (BCM), an inhibitor of methanogenesis, has been used in animal production. However, little is known about its impact on the intestinal microbiota and metabolic patterns. The present study aimed to investigate the effect of BCM on the colonic bacterial community and metabolism by establishing a Wistar rat model. Twenty male Wistar rats were randomly divided into two groups (control and treated with BCM) and raised for 6 weeks. Bacterial fermentation products in the cecum were determined, and colonic methanogens and sulfate-reducing bacteria (SRB) were quantified. The colonic microbiota was analyzed by pyrosequencing of the 16S rRNA genes, and metabolites were profiled by gas chromatography and mass spectrometry. The results showed that BCM did not affect body weight and feed intake, but it did significantly change the intestinal metabolic profiles. Cecal protein fermentation was enhanced by BCM, as methylamine, putrescine, phenylethylamine, tyramine, and skatole were significantly increased. Colonic fatty acid and carbohydrate concentrations were significantly decreased, indicating the perturbation of lipid and carbohydrate metabolism by BCM. BCM treatment decreased the abundance of methanogen populations, while SRB were increased in the colon. BCM did not affect the total colonic bacterial counts but significantly altered the bacterial community composition by decreasing the abundance of actinobacteria, acidobacteria, and proteobacteria. The results demonstrated that BCM treatment significantly altered the microbiotic and metabolite profiles in the intestines, which may provide further information on the use of BCM in animal production.

Characterization of a halotolerant acetoclastic methanogen highly enriched from marine sediment and its application in removal of acetate.

A marine sediment collected from Hiroshima Bay was cultured in artificial seawater, containing 0.51 M NaCl and 60 mM acetate and was found to exhibit active methane production at 37°C. Following four successive serial dilutions of cultures in medium containing 0.51 M NaCl, 60 mM acetate, and antibiotics, the well-acclimated methanogen was found to exhibit growth over a range of NaCl concentration (between 0 M and 2.06 M). The specific growth rates of the highly enriched methanogen, termed strain HA, in the absence of NaCl and in the presence of 1.54 M NaCl were estimated to be 0.037 h(-1) and 0.027 h(-1), respectively. The pH and temperature for optimum growth were determined to be 7.0-8.8 and 37°C, respectively. Although cells that had morphology similar to Methanosaeta sp. became dominant in the culture, methane production was still detected in the medium containing 0.51 M NaCl and other substrates such as methanol, formate, and methylamine, indicating contamination with other methanogens. The phylogenetic tree based on 16S rRNA gene sequences revealed that the strain HA was closely related to Methanosaeta harundinacea 6Ac and 8Ac(T), with sequence similarity of 98% and 97%, respectively. The continuous removal of acetate with upflow anaerobic filter reactor for industrial use of strain HA determined a methane production rate of 70 mM/d under condition of 0.51 M NaCl and successful methane production even under 1.54 M NaCl.

Propidium monoazide treatment to distinguish between live and dead methanogens in pure cultures and environmental samples.

In clinical trials investigating human health and in the analysis of microbial communities in cultures and natural environments, it is a substantial challenge to differentiate between living, potentially active communities and dead cells. The DNA-intercalating dye propidium monoazide (PMA) enables the selective masking of DNA from dead, membrane-compromised cells immediately before DNA extraction. In the present study, we evaluated for the first time a PMA treatment for methanogenic archaea in cultures and particle-rich environmental samples. Using microscopic analyses, we confirmed the applicability of the LIVE/DEAD(®) BacLight™ kit to methanogenic archaea and demonstrated the maintenance of intact cell membranes of methanogens in the presence of PMA. Although strain-specific differences in the efficiency of PMA treatment to methanogenic archaea were observed, we developed an optimal procedure using 130 µM PMA and 5min of photo-activation with blue LED light. The results showed that the effectiveness of the PMA treatment strongly depends on the texture of the sediment/soil: silt and clay-rich sediments represent a challenge at all concentrations, whereas successful suppression of DNA from dead cells with compromised membranes was possible for low particle loads of sandy soil (total suspended solids (TSS)≤200 mg mL(-1)). Conclusively, we present two strategies to overcome the problem of insufficient light activation of PMA caused by the turbidity effect (shielding) in particle-rich environmental samples by (i) dilution of the particle-rich sample and (ii) detachment of the cells and the free DNA from the sediment prior to a PMA treatment. Both strategies promise to be usable options for distinguishing living cells and free DNA in complex environmental samples.

Methanogenic toxicity evaluation of chlortetracycline hydrochloride

Background Anaerobic digestion is a technology applied successfully to converting organic matter into biogas. However, the presence of inhibitory compounds such as antibiotics can adversely affect methane production. The aim of this study is to evaluate the toxic effect of chlortetracycline hydrochloride (CLOR) on the methanogenic bacteria. In order to study the methanogenic toxicity of CLOR, different concentrations of CLOR (10, 50, 100, 200 mg L- 1) were evaluated by methanogenic toxicity assays using three feedings. Results Maximum methane production was obtained for the assays with 10 mg CLOR L- 1, the values obtained were 277 ± 4.07; 193 ± 11.31 and 166 ± 7.07 mL for the first, second and third feedings, respectively. The average values for acetic, propionic and butyric acid at start of the experiments were 2104 ± 139; 632 ± 7.6; 544 ± 26 mg L- 1, respectively. The VFA values obtained finally of the experiment were dependent on the evaluated antibiotic concentrations, indicating that the efficiency of methanogenesis is directly affected by the CLOR concentration. Conclusions CLOR is an effective methanogenic bacteria inhibitor. Moreover, the results show that CLOR has a bactericidal effect on methanogenic activity given that methane production did not recover during the third feeding. This study shows that the 50% inhibitory concentration (IC50) for methanogenic bacteria in 10 mg L- 1.

Salinity regulation of the interaction of halovirus SNJ1 with its host and alteration of the halovirus replication strategy to adapt to the variable ecosystem.

Halovirus is a major force that affects the evolution of extreme halophiles and the biogeochemistry of hypersaline environments. However, until now, the systematic studies on the halovirus ecology and the effects of salt concentration on virus-host systems are lacking. To provide more valuable information for understanding ecological strategies of a virus-host system in the hypersaline ecosystem, we studied the interaction between halovirus SNJ1 and its host Natrinema sp.J7-2 under various NaCl concentrations. We found that the adsorption rate and lytic rate increased with salt concentration, demonstrating that a higher salt concentration promoted viral adsorption and proliferation. Contrary to the lytic rate, the lysogenic rate decreased as the salt concentration increased. Our results also demonstrated that cells incubated at a high salt concentration prior to infection increased the ability of the virus to adsorb and lyse its host cells; therefore, the physiological status of host cells also affected the virus-host interaction. In conclusion, SNJ1 acted as a predator, lysing host cells and releasing progeny viruses in hypersaline environments; in low salt environments, viruses lysogenized host cells to escape the damage from low salinity.

Application of real-time PCR to determination of combined effect of antibiotics on Bacteria, Methanogenic Archaea, Archaea in anaerobic sequencing batch reactors.

This study evaluated the long-term effects of erythromycin-tetracycline-sulfamethoxazole (ETS) and sulfamethoxazole-tetracycline (ST) antibiotic combinations on the microbial community and examined the ways in which these antimicrobials impact the performance of anaerobic reactors. Quantitative real-time PCR was used to determine the effect that different antibiotic combinations had on the total and active Bacteria, Archae and Methanogenic Archae. Three primer sets that targeted metabolic genes encoding formylterahydrofolate synthetase, methyl-coenzyme M reductase and acetyl-coA synthetase were also used to determine the inhibition level on the mRNA expression of the homoacetogens, methanogens and specifically acetoclastic methanogens, respectively. These microorganisms play a vital role in the anaerobic degradation of organic waste and targeting these gene expressions offers operators or someone at a treatment plant the potential to control and the improve the anaerobic system. The results of the investigation revealed that acetogens have a competitive advantage over Archaea in the presence of ETS and ST combinations. Although the efficiency with which methane production takes place and the quantification of microbial populations in both the ETS and ST reactors decreased as antibiotic concentrations increased, the ETS batch reactor performed better than the ST batch reactor. According to the expression of genes results, the syntrophic interaction of acetogens and methanogens is critical to the performance of the ETS and ST reactors. Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors.

Accelerated glycerol fermentation in Escherichia coli using methanogenic formate consumption.

Escherichia coli can ferment glycerol anaerobically only under very defined restrictive conditions. Hence, it was the aim of this study to overcome this limitation via a co-cultivation approach. Anaerobic glycerol fermentation by a pure E. coli culture was compared to a co-culture that also contained the formate-oxidizing methanogen Methanobacterium formicicum. Co-cultivation of the two strains led to a more than 11-fold increased glycerol consumption. Furthermore, it supported a constantly neutral pH and a shift from ethanol to succinate production. Moreover, M. formicicum was analyzed for its ability to grow on different standard media and a surprising versatility could be demonstrated.

Inhibitory effects of ammonia on methanogen mcrA transcripts in anaerobic digester sludge.

Methanogens in anaerobic ammonia-rich digesters show differential responses to ammonia stress. The mechanism for this is poorly understood. In the present study, we determined the rates of methane production, the composition of methanogen mcrA (the gene coding for the alpha subunit of methyl-coenzyme M reductase) and their transcripts in response to ammonium addition in the anaerobic sludge retrieved from a full-scale digester treating swine manure. The rate of CH4 production substantially reduced with increased addition of ammonium. The analysis of natural (13)C abundances of CH4 and CO2 indicated that the aceticlastic methanogenesis was more sensitive than hydrogenotrophic methanogenesis. Quantitative PCR analysis revealed that mcrA copy number decreased by one order of magnitude in the treatment with a large amount of ammonium (10 g NH4+-N L(-1)) but did not change much with treatments of smaller amounts (3 and 7 g NH4+-N L(-1)) compared with the control. T-RFLP analysis of mcrA compositions showed that the structure of the methanogen community remained highly stable, with Methanosaetaceae dominating the methanogen community in all incubations. The composition of mcrA transcripts, however, showed a substantial response to the addition of ammonium. The relative abundance of Methanosaetaceae transcripts declined with increasing amounts of ammonium, whereas the transcript level of Methanobacteriales mcrA was relatively resistant. The differential responses corresponded to the shift of methanogenic pathway inferred from (13)C isotope fractionation. Our study suggests that methanogens in anaerobic sludge have a strong mcrA transcriptional response to ammonia stress without a change in the community structure.

Methylotrophic methanogenic Thermoplasmata implicated in reduced methane emissions from bovine rumen.

Rumen methanogens are major sources of anthropogenic methane emissions, and these archaea are targets in strategies aimed at reducing methane emissions. Here we show that the poorly characterised Thermoplasmata archaea in bovine rumen are methylotrophic methanogens and that they are reduced upon dietary supplementation with rapeseed oil in lactating cows. In a metatranscriptomic survey, Thermoplasmata 16S rRNA and methyl-coenzyme M reductase (mcr) transcripts decreased concomitantly with mRNAs of enzymes involved in methanogenesis from methylamines that were among the most abundant archaeal transcripts, indicating that these Thermoplasmata degrade methylamines. Their methylotrophic methanogenic lifestyle was corroborated by in vitro incubations, showing enhanced growth of these organisms upon methylamine supplementation paralleled by elevated methane production. The Thermoplasmata have a high potential as target in future strategies to mitigate methane emissions from ruminant livestock. Our findings and the findings of others also indicate a wider distribution of methanogens than previously anticipated.

The kinetics of process dependent ammonia inhibition of methanogenesis from acetic acid.

Advanced anaerobic digestion processes aimed at improving the methanization of sewage sludge may be potentially impaired by the production of inhibitory compounds (e.g. free ammonia). The result of methanogenic inhibition is relatively high effluent concentrations of acetic acid and other soluble organics, as well as reduced methane yields. An extreme example of such an advanced process is the thermal hydrolytic pretreatment of sludge prior to high solids digestion (THD). Compared to a conventional mesophilic anaerobic digestion process (MAD), THD operates in a state of constant inhibition driven by high free ammonia concentrations, and elevated pH values. As such, previous investigations of the kinetics of methanogenesis from acetic acid under uninhibited conditions do not necessarily apply well to the modeling of extreme processes such as THD. By conducting batch ammonia toxicity assays using biomass from THD and MAD reactors, we compared the response of these communities over a broad range of ammonia inhibition. For both processes, increased inhibitor concentrations resulted in a reduction of biomass growth rate (r(max) = µ(max)∙X) and a resulting decrease in the substrate half saturation coefficient (K(S)). These two parameters exhibited a high degree of correlation, suggesting that for a constant transport limited system, the K(S) was mostly a linear function of the growth rate. After correcting for reactor pH and temperature, we found that the THD and MAD biomass were both able to perform methanogenesis from acetate at high free ammonia concentrations (equivalent to 3-5 g/L total ammonia nitrogen), albeit at less than 30% of their respective maximum rates. The reduction in methane production was slightly less pronounced for the THD biomass than for MAD, suggesting that the long term exposure to ammonia had selected for a methanogenic pathway less dependent on those organisms most sensitive to ammonia inhibition (i.e. aceticlastic methanogens).

Nanosilver impact on methanogenesis and biogas production from municipal solid waste.

Silver nanoparticles (AgNPs, nanosilver) released from industrial activities and consumer products may be disposed directly or indirectly in sanitary landfills. To determine the impact of AgNPs on anaerobic digestion of landfill waste, municipal solid waste (MSW) was loaded in identical landfill bioreactors (9L volume each) and exposed to AgNPs (average particle size=21nm) at the final concentrations of 0, 1, and 10mgAg/kg solids. The landfill anaerobic digestion was carried out for more than 250 days, during which time the cumulative biogas production was recorded automatically and the chemical property changes of leachates were analyzed. There were no significant differences in the cumulative biogas volume or gas production rate between the groups of control and 1mgAg/kg. However, landfill solids exposed to AgNPs at 10mg/kg resulted in the reduced biogas production, the accumulation of volatile fatty acids (including acetic acid), and the prolonged period of low leachate pH (between 5 and 6). Quantitative PCR results after day 100 indicated that the total copy numbers of 16S rRNA gene of methanogens in the groups of control and 1mgAgNPs/kg were 1.97±0.21×10(7) and 0.90±0.03×10(7), respectively. These numbers were significantly reduced to 5.79±2.83×10(5)(copies/mL) in the bioreactor treated with 10mgAgNPs/kg. The results suggest that AgNPs at the concentration of 1mg/kg solids have minimal impact on landfill anaerobic digestion, but a concentration at 10mg/kg or higher inhibit methanogenesis and biogas production from MSW.

Effects of methanogenic inhibitors on methane production and abundances of methanogens and cellulolytic bacteria in in vitro ruminal cultures.

The objective of this study was to systematically evaluate and compare the effects of select antimethanogen compounds on methane production, feed digestion and fermentation, and populations of ruminal bacteria and methanogens using in vitro cultures. Seven compounds, including 2-bromoethanesulphonate (BES), propynoic acid (PA), nitroethane (NE), ethyl trans-2-butenoate (ETB), 2-nitroethanol (2NEOH), sodium nitrate (SN), and ethyl-2-butynote (EB), were tested at a final concentration of 12 mM. Ground alfalfa hay was included as the only substrate to simulate daily forage intake. Compared to no-inhibitor controls, PA, 2NEOH, and SN greatly reduced the production of methane (70 to 99%), volatile fatty acids (VFAs; 46 to 66%), acetate (30 to 60%), and propionate (79 to 82%), with 2NEOH reducing the most. EB reduced methane production by 23% without a significant effect on total VFAs, acetate, or propionate. BES significantly reduced the propionate concentration but not the production of methane, total VFAs, or acetate. ETB or NE had no significant effect on any of the above-mentioned measurements. Specific quantitative-PCR (qPCR) assays showed that none of the inhibitors significantly affected total bacterial populations but that they did reduce the Fibrobacter succinogenes population. SN reduced the Ruminococcus albus population, while PA and 2NEOH increased the populations of both R. albus and Ruminococcus flavefaciens. Archaeon-specific PCR-denaturing gradient gel electrophoresis (DGGE) showed that all the inhibitors affected the methanogen population structure, while archaeon-specific qPCR revealed a significant decrease in methanogen population in all treatments. These results showed that EB, ETB, NE, and BES can effectively reduce the total population of methanogens but that they reduce methane production to a lesser extent. The results may guide future in vivo studies to develop effective mitigation of methane emission from ruminants.