Enumeration, identification and characterisation of methanogens colonising pre-ruminant calves : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science at Massey University, Palmerston North, New Zealand

Methane-producing archaea, methanogens, in ruminant animals are a major source of anthropogenic methane. With a global warming potential 23 times greater than carbon dioxide, methane has been targeted for reduction under the Kyoto protocol. In New Zealand methane emissions from ruminant animals are major contributor to the national greenhouse gas inventory. For this reason agricultural industries are challenged with reducing methane emissions from ruminants. This investigation on methanogens in young dairy calves was carried out to obtain information on methanogen colonisation and establishment in the rumen because little is known about this process. In this study, methanogen colonisation occurred within two days after birth in four calves that were raised in the absence of cows. Anaerobic culture techniques were used to enumerate methanogens in gut samples and showed that methanogen numbers increased over time, but dropped below detection limits in two of four calves between six and 11 days after birth. Methanogens in these two calves then reappeared at day 13. By three weeks of age methanogen densities in all four calves were approximately 108 cells ml -1. These densities are similar to those found by other workers for 3-week old and mature ruminants. Colonies picked from anaerobic agar roll-tubes prepared from enumeration cultures yielded 31 methanogenic isolates and 28 isolates that utilised hydrogen but did not produce methane. Eleven of the 31 methanogenic isolates were selected for purification. Despite extensive efforts only four methanogens were able to be purified from the eleven isolates because of persistent non-methanogenic eubacteria also present in cultures. A phylogenetic analysis of 16S rRNA gene sequences from purified and partially-purified methanogen isolates was carried out and dendograms constructed to identify methanogens. Some phenotypic characteristics of purified methanogens were determined. This revealed a number of methanogen species previously not found in the rumen. The results showed Methanofollis liminatans (three isolates), Methanoculleus palmolei (three isolates) or Methanosarcina barkeri (one isolate) were the predominant culturable methanogens colonising the rumen two days after birth. The three isolates identified as M. liminatans were only 96.0% identical at the 16S rRNA gene level to the M. liminatans type strain, DSM 4140, and appear to be a new methanogen species. In gut samples collected 3-5 days after birth, Methanobacterium bryantii (three isolates) was found to be a predominant methanogen in some calves apparently replacing the first methanogens colonising the developing rumen. Twenty two days after birth Methanobrevibacter thaueri (one isolate) was identified as a predominant methanogen in one calf. These results are the first to suggest that there is a successional change in the methanogen populations as the rumen develops in young ruminants. Consideration of the colonising species showed that Mcl. palmolei were obtained from only two calves (calves 10 and 12) and that Mfl. liminatans-like isolates were obtained only from a different cohort of calves penned separately to calves 10 and 12. These methanogens, previously found only in terrestrial or aquatic environments, are probably the primary colonising methanogens because there were no mature ruminants to provide alternative inocula. It appears that the developing rumen of young calves provides a niche suitable for opportunistic hydrogenotrophic methanogens. A PCR investigation using targeted primers specific for seven groupings of methanogens was carried out on all rumen samples to obtain information not dependant on culturing. This analysis on DNA extracts showed methanogens belonging to the Methanobacteriales were present in almost every sample. Methanogens belonging to the Methanosarcinales and Methanomicrobiales were not detected in any sample. At the end of the trial (22 days), PCR analysis showed the presence of Methanobacterium spp. and Methanobrevibacter spp. in all four calves. Although there were some disagreements with results for isolates cultured, overall, PCR results confirmed the concept of successional changes in methanogen populations in pre-ruminant calves

Speciation and Mobilization of Toxic Heavy Metal Ions by Methanogenic Bacteria

HWRIC Project HWR 92-09

Quantitative analysis of ruminal methanogenic microbial populations in beef cattle divergent in phenotypic residual feed intake (RFI) offered contrasting diets

peer-reviewedBackground Methane (CH4) emissions in cattle are an undesirable end product of rumen methanogenic fermentative activity as they are associated not only with negative environmental impacts but also with reduced host feed efficiency. The aim of this study was to quantify total and specific rumen microbial methanogenic populations in beef cattle divergently selected for residual feed intake (RFI) while offered (i) a low energy high forage (HF) diet followed by (ii) a high energy low forage (LF) diet. Ruminal fluid was collected from 14 high (H) and 14 low (L) RFI animals across both dietary periods. Quantitative real time PCR (qRT-PCR) analysis was conducted to quantify the abundance of total and specific rumen methanogenic microbes. Spearman correlation analysis was used to investigate the association between the relative abundance of methanogens and animal performance, rumen fermentation variables and diet digestibility. Results Abundance of methanogens, did not differ between RFI phenotypes. However, relative abundance of total and specific methanogen species was affected (P < 0.05) by diet type, with greater abundance observed while animals were offered the LF compared to the HF diet. Conclusions These findings suggest that differences in abundance of specific rumen methanogen species may not contribute to variation in CH4 emissions between efficient and inefficient animals, however dietary manipulation can influence the abundance of total and specific methanogen species.Funding for the development and main work of this research was provided under the National Development Plan, through the Research Stimulus Fund, administered by the Department of Agriculture, Fisheries & Food, Ireland RSF 05 224

Comparative Analysis of Root Microbiomes of Rice Cultivars with High and Low Methane Emissions Reveals Differences in Abundance of Methanogenic Archaea and Putative Upstream Fermenters.

Rice cultivation worldwide accounts for ∼7 to 17% of global methane emissions. Methane cycling in rice paddies is a microbial process not only involving methane producers (methanogens) and methane metabolizers (methanotrophs) but also other microbial taxa that affect upstream processes related to methane metabolism. Rice cultivars vary in their rates of methane emissions, but the influence of rice genotypes on methane cycling microbiota has been poorly characterized. Here, we profiled the rhizosphere, rhizoplane, and endosphere microbiomes of a high-methane-emitting cultivar (Sabine) and a low-methane-emitting cultivar (CLXL745) throughout the growing season to identify variations in the archaeal and bacterial communities relating to methane emissions. The rhizosphere of the high-emitting cultivar was enriched in methanogens compared to that in the low emitter, whereas the relative abundances of methanotrophs between the cultivars were not significantly different. Further analysis of cultivar-sensitive taxa identified families enriched in the high emitter that are associated with methanogenesis-related processes. The high emitter had greater relative abundances of sulfate-reducing and iron-reducing taxa which peak earlier in the season than methanogens and are necessary to lower soil oxidation reduction potential before methanogenesis can occur. The high emitter also had a greater abundance of fermentative taxa which produce methanogenesis precursors (acetate, CO2, and H2). Furthermore, the high emitter was enriched in taxa related to acetogenesis which compete with methanogens for CO2 and H2 These taxa were enriched in a spatio-specific manner and reveal a complex network of microbial interactions on which plant genotype-dependent factors can act to affect methanogenesis and methane emissions.IMPORTANCE Rice cultivation is a major source of anthropogenic emissions of methane, a greenhouse gas with a potentially severe impact on climate change. Emission variation between rice cultivars suggests the feasibility of breeding low-emission rice, but there is a limited understanding of how genotypes affect the microbiota involved in methane cycling. Here, we show that the root microbiome of the high-emitting cultivar is enriched both in methanogens and in taxa associated with fermentation, iron, and sulfate reduction and acetogenesis, processes that support methanogenesis. Understanding how cultivars affect microbes with methanogenesis-related functions is vital for understanding the genetic basis for methane emission in rice and can aid in the development of breeding programs that reduce the environmental impact of rice cultivation

Relating Anaerobic Digestion Microbial Community and Process Function

Anaerobic digestion (AD) involves a consortium of microorganisms that convert substrates into biogas containing methane for renewable energy. The technology has suffered from the perception of being periodically unstable due to limited understanding of the relationship between microbial community structure and function. The emphasis of this review is to describe microbial communities in digesters and quantitative and qualitative relationships between community structure and digester function. Progress has been made in the past few decades to identify key microorganisms influencing AD. Yet, more work is required to realize robust, quantitative relationships between microbial community structure and functions such as methane production rate and resilience after perturbations. Other promising areas of research for improved AD may include methods to increase/control (1) hydrolysis rate, (2) direct interspecies electron transfer to methanogens, (3) community structure–function relationships of methanogens, (4) methanogenesis via acetate oxidation, and (5) bioaugmentation to study community–activity relationships or improve engineered bioprocesses

Chemical markers for rumen methanogens and methanogenesis

Peer reviewedPublisher PD

Temperature effects on methanogenesis and sulfidogenesis during anaerobic digestion of sulfur-rich macroalgal biomass in sequencing batch reactors

Methanogenesis and sulfidogenesis, the major microbial reduction reactions occurring in the anaerobic digestion (AD) process, compete for common substrates. Therefore, the balance between methanogenic and sulfidogenic activities is important for efficient biogas production. In this study, changes in methanogenic and sulfidogenic performances in response to changes in organic loading rate (OLR) were examined in two digesters treating sulfur-rich macroalgal waste under mesophilic and thermophilic conditions, respectively. Both methanogenesis and sulfidogenesis were largely suppressed under thermophilic relative to mesophilic conditions, regardless of OLR. However, the suppressive effect was even more significant for sulfidogenesis, which may suggest an option for H2S control. The reactor microbial communities developed totally differently according to reactor temperature, with the abundance of both methanogens and sulfate-reducing bacteria being significantly higher under mesophilic conditions. In both reactors, sulfidogenic activity increased with increasing OLR. The findings of this study help to understand how temperature affects sulfidogenesis and methanogenesis during AD

Influence of high gas production during thermophilic anaerobic digestion in pilot-scale and lab-scale reactors on survival of the thermotolerant pathogens Clostridium perfringens and Campylobacter jejuni in piggery wastewater

Safe reuse of animal wastes to capture energy and nutrients, through anaerobic digestion processes, is becoming an increasingly desirable solution to environmental pollution. Pathogen decay is the most important safety consideration and is in general, improved at elevated temperatures and longer hydraulic residence times. During routine sampling to assess pathogen decay in thermophilic digestion, an inversely proportional relationship between levels of Clostridium perfringens and gas production was observed. Further samples were collected from pilot-scale, bench-scale thermophilic reactors and batch scale vials to assess whether gas production (predominantly methane) could be a useful indicator of decay of the thermotolerant pathogens C. perfringens and Campylobacter jejuni. Pathogen levels did appear to be lower where gas production and levels of methanogens were higher. This was evident at each operating temperature (50, 57, 65 °C) in the pilot-scale thermophilic digesters, although higher temperatures also reduced the numbers of pathogens detected. When methane production was higher, either when feed rate was increased, or pH was lowered from 8.2 (piggery wastewater) to 6.5, lower numbers of pathogens were detected. Although a number of related factors are known to influence the amount and rate of methane production, it may be a useful indicator of the removal of the pathogens C. perfringens and C. jejuni

Microbial co-existence and stable equilibria in a mechanistic model of enteric methane production : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Mathematics at Massey University, Manawatū Campus, New Zealand

Globally, 14.5% of all anthropogenic greenhouse gases come from ruminants. One of these is methane, which is produced in the rumen of ruminant animals. Feed is degraded by microbes to produce volatile fatty acids (which are absorbed by the animal) and hydrogen (which is metabolized by methanogens to form methane). The dynamics of hydrogen production and metabolism are subject to thermodynamic control imposed by the hydrogen concentration. Existing models to estimate methane production are based on calculation of hydrogen balances without considering the presence of methanogens and do not include thermodynamic control. In this project, a model is developed based on glucose-hydrogenmethanogen dynamics to estimate methane production and illustrates a co-existence of microbes that employs different fermentation pathways competing for the same food source in the rumen. Glucose was chosen as an example of a fermentable feed component. A thermodynamic term was integrated into a Monod-type model to represent the thermodynamic control of hydrogen concentration on the rates of hydrogen generation and hydrogen metabolism. Results of this model suggest that the microbial community composition and the combination of the different pathways are determined by the rumen environment, biological parameters of the microbes and the feedback imposed by substrate and product concentrations. The mathematical enunciation of this model is therefore consistent with biological expectations. This model could be expanded to include plant polymer degradation rate, feeding level and feeding frequency to explore their effects on methane production. This model could also be integrated into models of whole rumen function to address more complex questions. It would also support experimentation with animals for understanding factors that control methane formation and to explore methane mitigation strategies