Rumen microbial degradation of bromoform from red seaweed(Asparagopsis taxiformis)and the impact on rumen fermentation and methanogenic archaea

Background The red macroalgae Asparagopsis is an effective methanogenesis inhibitor due to the presence of halogenated methane(CH_(4))analogues,primarily bromoform(CHBr_(3)).This study aimed to investigate the degradation process of CHBr3 from A taxiformis in the rumen and whether this process is diet-dependent.An in vitro batch culture system was used according to a 2×2 factorial design,assessing two A taxiformis inclusion rates[0(CTL)and 2%DM diet(AT)]and two diets[high-concentrate(HC)and high-forage diet(HF)].Incubations lasted for 72 h and samples of headspace and fermentation liquid were taken at 0,0.5,1,3,6,8,12,16,24,48 and 72 h to assess the pattern of degradation of CHBr_(3) into dibromomethane(CH_(2)Br_(2))and fermentation parameters.Additionally,an in vitro experiment with pure cultures of seven methanogens strains(Methanobrevibacter smithii,Methanobrevibacter ruminantium,Methanosphaera stadtmanae,Methanosarcina barkeri,Methanobrevibacter millerae,Methanorhermobacter wolfei and Methanobacterium mobile)was conducted to test the effects of increasing concentrations of CHBr3(0.4,2,10and 50μmol/L).Results The addition of AT significantly decreased CH_(4) production(P=0.002)and the acetate:propionate ratio(P=0.003)during a 72-h incubation.The concentrations of CHBr_(3) showed a rapid decrease with nearly 90%degraded within the first 3 h of incubation.On the contrary,CH_(2)Br_(2) concentration quickly increased during the first 6 h and then gradually decreased towards the end of the incubation.Neither CHBr_(3) degradation nor CH_(2)Br_(2) synthesis were affected by the type of diet used as substrate,suggesting that the fermentation rate is not a driving factor involved in CHBr_(3)degradation.The in vitro culture of methanogens showed a dose-response effect of CHBr3 by inhibiting the growth of M.smithii,M.ruminantium,M.stadtmanae,M.barkeri,M.millerae,M.wolfei,and M.mobile.Conclusions The present work demonstrated that CHBr_(3) from A.taxiformis is quickly degraded to CH_(2)Br_(2)in the rumen and that the fermentation rate promoted by different diets is not a driving factor involved in CHBr_(3)degradation.

Batch methanogenic fermentation experiments of wastewater from a brown coal low-temperature coke plant

Coke plant effluents with high contents of organic compounds are mainly treated by biological aerobic fermentation after physical pre-treatment. In this study, a brown coal condensate wastewater from a low temperature coking process was fermented under methanogenic conditions in discontinuous experiments. By this fermentation, acetate, propionate, and the main polyphenolic compounds （catechol, resorcinol and hydroquinone） were degraded to a level below the detection limit. The COD was reduced by 72% with a residual concentration of 2.1 g/L. This anaerobic fermented wastewater had a residual BOD5 of 0.66 g/L and 2.2 L CH4 were formed per litre of wastewater. An abiotic pre-treatment for this wastewater with air had a negative effect on the COD reduction and decrease of colour on the methanogenic fermentation due to the autoxidation of polyphenolic compounds to humic-like compounds. This study showed that methanogenic fermentations in the treatment sequence of brown coal coking wastewaters could reduce energy consumption for aeration in further treatment processes and had the potential for a better effluent quality due to a less formation of recalcitrant humic-like compounds.

Weaning methods affect ruminal methanogenic archaea composition and diversity in Holstein calves

The objective of the present study was to examine the effect of different weaning methods on the ruminal methanogenic archaea composition and diversity in Holstein calves.Thirty-six newborn Holstein bull calves were assigned to 1 of 3 treatments:(1)conventional weaning(d 56)and fed a high proportion of solid feed(CWS);(2)conventional weaning(d 56)and fed a high proportion of liquid feed(CWL);(3)early weaning(d 42)and fed with a high proportion of solid feed(EWS).High-throughput sequencing of the methyl coenzyme M reductase(mcr A)gene,which encodes theα-subunit of methyl coenzyme M reductase-the enzyme that catalyzes the final step in methanogenesis was used to determine the composition and diversity of rumen methanogens.No significant difference(P>0.05)was observed for operational taxonomic units(OTUs)or richness indices,but diversity indices increased(P<0.05)for calves fed high dietary solids.Predominant families across the three treatments were Methanobacteriaceae,Thermoplasmataceae and Methanomassiliicoccaceae.Calves in the EWS treatment had a higher(P<0.05)relative abundance of Methanobrevibacter sp.strain AbM4 and Methanosphaera stadtmanae,while calves in the CWL treatment had a higher(P<0.05)abundance of Methanosphaera sp.strain SM9.A positive(P<0.05)relationship was identified between butyrate and Methanobrevibacter sp.strain AbM4.In conclusion,the composition and diversity of methanogens in the rumen of Holstein calves varied under the different weaning methods.This study identified a positive relationship between butyrate and Methanobrevibacter sp.strain AbM4,potentially reflecting correlations between ruminal fermentation variables and methanogenesis function.These in-depth analyses provide further understanding of weaning methods for intensified production systems.

Effect of sulfate on the methanogenic activity of a bacterial culture from a brewery wastewater during glucose degradation

The maximum specific methanogenic activity （SMA） of a sludge originating from a brewery wastewater treatment plant on the degradation of glucose was investigated at various levels of sulfate on a specific loading basis. Batch experiments were conducted in serum bottles at pH 7 and 35℃. A comparison of the values indicates that the SMA of this mixed culture was increased and reached its highest level of 0.128 g CH4 gas COD/（g VSS.d） when biomass was in contact with sulfate at a ratio of 1：0.114 by weight.

Rumen methanogens and mitigation of methane emission by anti-methanogenic compounds and substances

Methanogenic archaea reside primarily in the rumen and the lower segments of the intestines of ruminants, where they utilize the reducing equivalents derived from rumen fermentation to reduce carbon dioxide, formic acid, or methylamines to methane（CH_4）. Research on methanogens in the rumen has attracted great interest in the last decade because CH_4 emission from ruminants contributes to global greenhouse gas emission and represents a loss of feed energy. Some DNA-based phylogenetic studies have depicted a diverse and dynamic community of methanogens in the rumen. In the past decade, researchers have focused on elucidating the underpinning that determines and affects the diversity, composition, structure, and dynamics of methanogen community of the rumen. Concurrently, many researchers have attempted to develop and evaluate interventions to mitigate enteric CH_4 emission. Although much work has been done using plant secondary metabolites, other approaches such as using nitrate and 3-nitrooxy propanol have also yielded promising results. Most of these antimethanogenic compounds or substances often show inconsistent results among studies and also lead to adverse effects on feed intake and digestion and other aspects of rumen fermentation when fed at doses high enough to achieve effective mitigation. This review provides a brief overview of the rumen methanogens and then an appraisal of most of the antimethanogenic compounds and substances that have been evaluated both in vitro and in vivo. Knowledge gaps and future research needs are also discussed with a focus on methanogens and methane mitigation.

Rapid Acclimation of Methanogenic Granular Sludge into Autotrophic Partial Denitrification Granules

Rapid formation of autotrophic partial denitrification(APD)granules is of practical interest to start up an expanded granular sludge bed reactor for wastewater treatment.This study demonstrates that methanogenic granules can be easily acclimated into autotrophic partial denitrification granules in one day,with the ability to remove 82%of 2.7 kg-S/(m^3·d)sulfide into S^0 and to convert 97%of 0.9 kg-N/(m^3·d)nitrate into nitrite,which can provide a promising feedstock for anaerobic ammonia oxidation process.Arcobacter sp.is essential for S^0 accumulation.Under high loadings,the abundance of Arcobacter sp.decreased,while on the contrary the abundance of unclassified_p_Firmicutes increased,leading to the deterioration of autotrophic partial denitrification performance.The granules performance could be recovered by adopting the strategies of properly reducing the influent loadings.

Comparison of Fecal Methanogenic Archaeal Community Between Erhualian and Landrace Pigs Using Denaturing Gradient Gel Electrophoresis and Real-Time PCR Analysis

Erhualian and Landrace breeds are typical genetically obese and lean pigs, respectively. To compare the fecal methanogenic Archaeal community between these two pig breeds, fecal samples from different growth phase pigs were collected and used for PCR-denaturing gradient gel electrophoresis （DGGE） with two primer pairs （344fGC/519r and 519f/915rGC） and real-time PCR analysis. Results showed that a better separation and higher quality of bands pattern were obtained in DGGE proifles using primers 344fGC/519r as compared with primers 519f/915rGC. Sequencing of DGGE bands showed that the predominant methanogens in the feces of Erhualian and Landrace pigs belonged to Methanobrevibacter spp. and Methanosphaera spp. Real-time PCR analysis revealed that there was no signiifcant difference in the numbers of fecal total methanogens between Erhualian and Landrace pigs;however, pig growth phase affected the numbers of 16S rRNA genes of total methanogens and Methanobrevibacter smithii. Dissociation curves of methyl coenzyme-M reductase subunit A （mcrA） gene fragments ampliifed with real-time PCR showed all samples possessed a single peak at 82＆#176;C, which might be associated with M. smithii. Samples from the same growth phase of each breed showed good replicative dissociation curves. The results suggest that the growth phase （including diet factor） other than genotype of pig may affect the fecal methanogenic Archaeal community of pigs.

Resilience and functional redundancy of methanogenic digestion microbiome safeguard recovery of methanogenesis activity under the stress induced by microplastics

Microplastics and nanoplastics are emerging pollutants that substantially influence biological element cycling in natural ecosystems.Plastics are also prevalent in sewage,and they accumulate in waste-activated sludge(WAS).However,the impacts of plastics on the methanogenic digestion of WAS and the underpinning microbiome remain underexplored,particularly during long-term operation.In this study,we found that short-term exposure to individual microplastics and nanoplastics(polyethylene,polyvinyl chloride,polystyrene,and polylactic acid)at a low concentration(10 particles/g sludge)slightly enhanced methanogenesis by 2.1%−9.0%,whereas higher levels(30−200 particles/g sludge)suppressed methanogenesis by 15.2%−30.1%.Notably,the coexistence of multiple plastics,particularly at low concentrations,showed synergistic suppression of methanogenesis.Unexpectedly,methanogenesis activity completely recovered after long-term exposure to plastics,despite obvious suppression of methanogenesis by initial plastic exposure.The inhibition of methanogenesis by plastics could be attributed to the stimulated generation of reactive oxygen species.The stress induced by plastics dramatically decreased the relative abundance of methanogens but showed marginal influence on putative hydrolytic and fermentation populations.Nonetheless,the digestion sludge microbiome exhibited resilience and functional redundancy,contributing to the recovery of methanogenesis during the long-term operation of digesters.Plastics also increased the complexity,modularity,and negative interaction ratios of digestion sludge microbiome networks,but their influence on community assembly varied.Interestingly,a unique plastisphere was observed,the networks and assembly of which were distinct from the sludge microbiome.Collectively,the comprehensive evaluation of the influence of microplastics and nanoplastics on methanogenic digestion,together with the novel ecological insights,contribute to better understanding and manipulating this engineered ecosystem in the face of increasing plastic pollution.

Effect of 2-butenal manufacture wastewater to methanogenic activity and microbial community

The synthesis of 2-butenal,which is a vital raw material for the production of sorbic acid as a food preservative,generates some toxic by-products,so it is urgent to seek better detoxification strategies for the treatment of 2-butenal manufacture wastewater.In this study,batch experiments were carried out to investigate the inhibition effect of wastewater on the methanogenic activity.To understand the wastewater toxicity to anaerobic granular sludge,variations of the specific methanogenic activity(SMA)and extracellular polymeric substance(EPS)constituents at various wastewater CODs were investigated.Ultraviolet-visible(UV-vis)spectra and Fourier transform infrared(FT-IR)spectra were employed to analyze the structure of the EPS.The results showed that the inhibitory ratio of 2-butenal manufacture wastewater was less than 8.4%on the anaerobic granular sludge when the CODs were less than 959 mg/L.However,the inhibitory ratio increased from 36.4%to 93.6% when CODs increased from 2396 mg/L to 9585 mg/L,with the SMA decreasing from 39.1 mL CH4/(gVSS·d)to 3.2 mL CH4/(gVSS·d).The diversity of the microbial community under various CODs was researched by Illumina 16S rRNA Miseq sequencing and the results demonstrated that Proteiniphilum、Petrimonas and Syntrophobacter were the dominant bacteria genera in all sample.Regarding archaea,Methanobacterium was the most dominated archaea genera,followed by the Methanosaeta group in all samples.Moreover,the bacterial communities had changed obviously with increasing CODs,which indicated high CODs played a negative impact on the richness and diversity of bacterial community in the sludge samples.

Methanogenic community dynamics in anaerobic co-digestion of fruit and vegetable waste and food waste

A lab-scale continuously-stirred tank reactor （CSTR）, used for anaerobic co-digestion of fruit and vegetable waste （FVW） and food waste （FW） at different mixture ratios, was operated for 178 days at the organic loading rate of 3 kg VS （volatile solids）/（m3.day）. The dynamics of the Archaeal community and the correlations between environmental variables and methanogenic community structure were analyzed by polymerase chain reactions - denaturing gradient gel electrophoresis （PCR-DGGE） and redundancy analysis （RDA）, respectively. PCR-DGGE results demonstrated that the mixture ratio of FVW to FW altered the community composition of Archaea. As the FVW]FW ratio increased, Methanoculleus, Methanosaeta and Methanosarcina became the predominant methanogens in the community. Redundancy analysis results indicated that the shift of the methanogenic community was significantly correlated with the composition of acidogenic products and methane production yield. Different mixture ratios of substrates led to different compositions of intermediate metabolites, which may affect the methanogenic community. These results suggested that the analysis of microbial communities could be used to diagnose anaerobic processes.

Long-term submergence of non-methanogenic oxic upland field soils helps to develop the methanogenic archaeal community as revealed by pot and field experiments

The community structure of methanogenic archaea is relatively stable,i.e.,it is sustained at a high abundance with minimal changes in composition,in paddy field soils irrespective of submergence and drainage.In contrast,the abundance in non-methanogenic oxic soils is much lower than that in paddy field soils.This study aimed to describe methanogenic archaeal community development following the long-term submergence of non-methanogenic oxic upland field soils in pot and field experiments.In the pot experiment,a soil sample obtained from an upland field was incubated under submerged conditions for 275 d.Soil samples periodically collected were subjected to culture-dependent most probable number(MPN)enumeration,polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE)analysis of archaeal 16 S r RNA gene,and quantitative PCR analysis of the methyl-coenzyme M reductase alpha subunit gene(mcr A)of methanogenic archaea.The abundance of methanogenic archaea increased from 102 to 103 cells g-1 dry soil and 104 to 107 copies of mcr A gene g-1 dry soil after submergence.Although no methanogenic archaeon was detected prior to incubation by the DGGE analysis,members from Methanocellales,Methanosarcinaceae,and Methanosaetaceae proliferated in the soils,and the community structure was relatively stable once established.In the field experiment,the number of viable methanogenic archaea in a rice paddy field converted from meadow(reclaimed paddy field)was monitored by MPN enumeration over five annual cycles of field operations.Viability was also determined simultaneously in a paddy field where the plow layer soil from a farmer’s paddy field was dressed onto the meadow(dressed paddy field)and an upland crop field converted from the meadow(reclaimed upland field).The number of viable methanogenic archaea in the reclaimed paddy field was below the detection limit before the first cultivation of rice and in the reclaimed upland field.Then,the number gradually increased over five years and finally reached 103–104 cells g-1 dry soil,which was comparable to that in the dressed paddy field.These findings showed that the low abundance of autochthonous methanogenic archaea in the non-methanogenic oxic upland field soils steadily proliferated,and the community structure was developed following repeated and long-term submergence.These results suggest that habitats suitable for methanogenic archaea were established in soil following repeated and long-term submergence.

Acid resistance of methanogenic bacteria in a two-stage anaerobic process treating high concentration methanol wastewater

In this study,the two-stage upflow anaerobic sludge blanket(UASB)system and batch experiments were employed to evaluate the performance of anaerobic digestion for the treatment of high concentration methanol wastewater.The acid resistance of granular sludge and methanogenic bacteria and their metabolizing activity were investigated.The results show that the pH of the first UASB changed from 4.9 to 5.8 and 5.5 to 6.2 for the second reactor.Apparently,these were not the advisable pH levels that common metha-nogenic bacteria could accept.The methanogenic bacteria of the system,viz.Methanosarcina barkeri,had some acid resis-tance and could still degrade methanol at pH 5.0.If the meth-anogenic bacteria were trained further,their acid resistance would be improved somewhat.Granular sludge of the system could protect the methanogenic bacteria within its body against the impact of the acidic environment and make them degrade methanol at pH 4.5.The performance of granular sludge was attributed to its structure,bacteria species,and the distribution of bacterium inside the granule.

Characterization of a phenanthrene-degrading methanogenic community

Polycyclic aromatic hydrocarbons(PAHs)often occur in oil-contaminated soil,coke wastewater and domestic sludge;however,associated PAH degraders in these environments are not clear.Here we evaluated phenanthrene degradation potential in the mixed samples of above environments,and obtained a methanogenic community with different microbial profile compared to those from sediments.Phenanthrene was efficiently degraded(1.26 mg/L/d)and nonstoichiometric amount of methane was produced simultaneously.16S rRNA gene sequencing demonstrated that bacterial populations were mainly associated with Comamonadaceae Nocardiaceae and Thermodesulfobiaceae,and that methanogenic archaea groups were dominated by Methanobacterium and Methanothermobacter.Substances such as hexane,hexadecane,benzene and glucose showed the most positive effects on phenanthrene degradation.Substrate utilization tests indicated that this culture could not utilize other PAHs.These analyses could offer us some suggestions on the putative phenanthrene-degrading microbes in such environments,and might help us develop strategies for the removal of PAHs from contaminated soil and sludge.

Methanogenic community structure in simultaneous methanogenesis and denitrification granular sludge

A laboratory scale up-flow anaerobic sludge bed （UASB） bioreactor ted with synthetic wastewater was operated with simultaneous methanogenesis and denitrification （SMD） granules for 235 days with a gradient decrease of C/N. Molecular cloning, qRT-PCR and T-RFLP were applied to study tile methanogenic community structures in SMD granules and their changes in response to changing influent C/N. The results indicate that when C/N was 20：1, the methane production rate was tastest, and Methanosaetaceae and Methanobacteriaceae were the primary methanogens within tile Archaea. The richness and evenness of methanogenic bacteria was best with the highest T-RFLP diversity index of 1.627 in the six granular sludge samples. When C/N was reduced from 20：1 to 5：1, tbe methanogenic activity of SMD granules decreased gradually, and the relative quantities of methanogens decreased from 36.5% to 10.9%. The abundance of Methanosaetaceae in Archaea increased from 64.5% to 84.2%, while that of Methanobacteriaceae decreased from 18.6% to 11.8%, and the richness and evenness of methanogens decreased along with the T-RFLP diversity index to 1.155, suggesting that the community structure reflected the succession to an unstable condition represented by high nitrate concentrations.

一种快速测定厌氧污泥活性的顶空气相色谱技术

将含有厌氧污泥的样品放入一顶空样品瓶内,在恒定的温度下平衡,并通过多次顶空抽提的方式对样品瓶中所产生的甲烷量随时间的变化进行气相色谱检测,从而实现厌氧污泥比产甲烷活性的检测。实验表明,多次顶空抽提-气相色谱测定厌氧污泥产甲烷活性的技术用约3h就可以得出厌氧污泥产甲烷的线性规律,其结果与对比试验的数据相近。因而该方法适用于厌氧污泥活性的快速预测,可大大提高相关厌氧污泥活性测定及废水处理领域研究的效率。

Effect of inorganic carbon on anaerobic ammonium oxidation enriched in sequencing batch reactor

The present lab-scale research reveals the enrichment of anaerobic ammonium oxidation microorganism from methanogenic anaerobic granular sludge and the effect of inorganic carbon(sodium bicarbonate)on anaerobic ammonium oxidation.The enrichment of anammox bacteria was carried out in a 7.0-L sequencing batch reactor(SBR)and the effect of bicarbonate on anammox was conducted in a 3.0-L SBR.Research results,especially the biomass,showed first signs of anammox activity after 54 d cultivation with synthetic wast...

Comparison between controlled landfill reactor and conditioned landfill bioreactor

Bioreactor landfills allow a more active landfill management that recognizes the biological, chemical and physical processes involved in a landfill environment. The laboratory-scale simulators of landfill reactors treating municipal solid wastes were studied, the effect of solid waste size, leachate recirculation, nutrient balance, pH value, moisture content and temperature on the rate of municipal solid waste(MSW) biodegradation were determined, and it indicated the optimum pH value, moisture content and temperature decomposing MSW. The results of waste biodegradation were compared with that of the leachate-recirculated landfill simulator and conservative sanitary landfill simulator. In the control experiment the antitheses of a decreasing trend of the organic load, measured as biological oxygen demand and chemical oxygen demand, was shown. An obvious enhancement of effective disposal from conservative sanitary landfill(CSL) simulator, to the leachate-recirculated landfill(LRL) simulator and to the conditioned bioreactor landfill(CBL) simulator would be noted, through displaying the compared results of solid waste settlement, heavy metal concentration in leachate, methane production rate, biogas composition, BOD and COD as well as their ratio.

Effect of different fertilizers on methane emission from a paddy field of Zhejiang, China

Methane emissions from Chinese paddy soil (Zhejiang Province) were measured over the rice growing seasons. Different fertilizers (organic and chemical) were applied, emissions of methane were high during two periods(5 days after peak tillering and 7 days after heading flowering stage) and significant effect of fertilizers was observed. Methanogenic activities in soils treated with organic manures were obviously higher than those with chemical fertilizers. Among the organic manured fields the maximum methane emission from green manure, biogas residue and beef manure treatment were 52, 20 and 19 times respectively of that given by control, and among chemical fertilizers it was NH\-4HCO\-3>CO(NH 2) 2>(NH 4) 2SO 4>NH 4Cl>NaNO 3 with 2\^4, 2, 1\^5,1\^3 and 0\^2 times respectively of that from control.

Microbiological Characteristics of Anaerobic Granular Sludge in Hybrid Anaerobic Baffled Reactor

Anaerobic granular sludge is of key importance for highly effective operation of hybrid anaerobic baffled reactor(HABR).An observation and analysis on the composition of anaerobic granular sludge in each separation compartment of HABR was conducted by using scanning electron microscope(SEM)and molecular biotechnology,and specific methanogenic activity(SMA)and coenzyme F420 content were determined.It was indicated that the disparity of microbial composition was significant among these separation compartments of HABR,and the HABR encouraged phase separation.The results show the understanding of microbiological characteristics of anaerobic granular sludge in HABR is helpful for cultivating granular sludge,which ensures the effective operation of the reactor.

Archaea and the human gut:New beginning of an old story

Methanogenic archaea are known as human gut inhabitants since more than 30 years ago through the detection of methane in the breath and isolation of two methanogenic species belonging to the order Methanobacteriales,Methanobrevibacter smithii and Methanosphaera stadtmanae.During the last decade,diversity of archaea encountered in the human gastrointestinal tract(GIT)has been extended by sequence identification and culturing of new strains.Here we provide an updated census of the archaeal diversity associatedwith the human GIT and their possible role in the gut physiology and health.We particularly focus on the still poorly characterized 7th order of methanogens,the Methanomassiliicoccales,associated to aged population.While also largely distributed in non-GIT environments,our actual knowledge on this novel order of methanogens has been mainly revealed through GIT inhabitants.They enlarge the number of final electron acceptors of the gut metabolites to mono-di-and trimethylamine.Trimethylamine is exclusively a microbiota-derived product of nutrients(lecithin,choline,TMAO,L-carnitine)from normal diet,from which seems originate two diseases,trimethylaminuria(or Fish-Odor Syndrome)and cardiovascular disease through the proatherogenic property of its oxidized liver-derived form.This therefore supports interest on these methanogenic species and its use as archaebiotics,a term coined from the notion of archaea-derived probiotics.