association with ciliates

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1 Grain and artificial stimulation of the rumen change the abundance and diversity of methanogens and their association with ciliates by Claus Thagaard Christophersen Candidatus Agronomiae (M.Sc.) This thesis is presented for the degree of Doctor of Philosophy of The University of Western Australia Animal Science School of Animal Biology Faculty of Natural and Agricultural Sciences December, 2007

2 We create the future by citing the past (Unknown) ii

3 Declaration Declaration The work presented in this thesis is my own work except where stated below. This work was carried out in the School of Animal Biology at the University of Western Australia and at CSIRO Livestock Industries at Floreat Park. The material presented in this thesis has not been presented for any other degree. The volatile fatty acids analysed were by the Department of Agriculture, WA. Claus Thagaard Christophersen, December, iii

4 Publications Publications arising from this thesis Refereed publications: - Christophersen C.T., Wright, A-D.G. and Vercoe, P.E. (2007) Methane emission and acetate/propionate ratio decreased when artificial stimulation of the rumen wall are combined with increasing grain diets. J. Anim. Sci. in press Chapter 4 in this thesis is identical to the paper published in Journal of Animal Science except that the abstract has been removed. - Christophersen, C. T., Wright, A-D.G. and Vercoe, P. E. (2004). Examining diversity of free-living methanogens and those associated with protozoa in the rumen. J. Anim. Feed Sci. 13: Conference abstracts - Christophersen, C. T., Wright, A-D. G. and Vercoe, P. E. (2004). Does dietary manipulation change the diversity of methanogens and protozoa that interact within the rumen? 4 th joint INRA-RRI Symposium, Gut Microbiology Concerns and Responses to food safety, Health and Environmental issues, June 2004, Clermont-Ferrand, France. J. Repro. Nutri. Develop. 44: 52. iv

5 Acknowledgements Acknowledgements Here at the very end of my PhD I would like to thank several people, who have helped me through and been there when times were tough. Without the financial support from The University of Western Australia (IPRS) and the Danish Research Agency, it would never have been possible for me to undertake my PhD candidature. I am very grateful for being granted such a possibility. I was also awarded a University of Western Australia travel grant to attend the INRA-RRI 2004 gut microbes conference. For this I am very grateful. Huge thanks to my supervisors, Dr. Philip E. Vercoe (UWA) and Dr. Andre-Denis G. Wright, for not just being supervisors but also becoming great friends. They have both strongly supported my scientific research intellectually, and they are excellent supervisors and have wonderful personalities. Their encouragement and understanding have been invaluable. I also would like to thank Mr. John Beesley for his great support and guidance. John was always happy to share his 30+ years of experience working with animals, and he would also be the first to put his hand up when samples had to be collected. The measurements of liquid and particulate matters retention time had not been possible without him. Dr. Clare Engelke, thank you for being such a good friend and for taking samples and looking after the animals while I was away, you are a true life saver. My unofficial co-supervisors Dr. Lucy C. Skillman and Dr. Richard Cookson I also owe great thanks for taking the time to listen, discuss and answer all my questions. Numerous people have provided help, advice, encouragement and friendship during my PhD and it is impossible to mention all of them. However, I would like in particular to v

6 Acknowledgements thank: Mr. Andrew Toovey, Mr. Peter Hutton, Dr. Ian Williams, Dr. Suzanne Rea, Mrs. Carolyn Pimm, Mr. Andrew Williams, Dr. Zoey Durmic and Mrs. Margaret Blackberry. Thanks to my parents, Birthe and Ove Thagaard Nielsen, for supporting me in achieving my goals and for visiting us here down-under. I would also like to thank my parentsin-law, especially my mother-in-law for visiting us in Perth so many times, and for all the washing and cleaning and baby sitting, making life with two PhDs and two boys much easier for us. The love, encouragement and support from my lovely wife, Helle Martha Christophersen, have been outstanding. Words just cannot express how grateful I am for that. Claus Thagaard Christophersen, December, vi

7 Abstract Abstract In Australia, there is pressure to reduce the amount of methane produced by ruminant livestock because they are the single largest source of methane emitted from anthropogenic sources, accounting for 70.7% of agricultural methane emissions. In addition, methane production represents a loss of gross energy intake to the animal. The organisms that are responsible for methane production in the animal gut are a distinct group of Archaea called methanogens. Methanogens occupy three different niches within the rumen. Some live freely in the rumen digesta (planktonic), others are attached to the outer surface of the rumen ciliates (ectosymbiotic), and some reside within the ciliates (endosymbiotic). The types and number of methanogens, as well as rumen ciliates and their symbiotic interactions, influence the amount of methane produced from the rumen. These factors in turn are affected by many factors, including diet and ruminal retention time. In this thesis, I tested the general hypothesis that increasing the amount of grain in the diet and reducing the retention time would affect the abundance and diversity of methanogens in their different niches, including their association with ruminal ciliates. Twenty-four fistulated sheep were used in a complete factorial design with the sheep randomly divided into four groups. The sheep had a 2-wk acclimatization period on an oaten-chaff diet, followed by three, 3-wk diet phases. In diet phase 1 all sheep were given the same oaten-chaff diet. Two of the four groups were maintained on the oaten-chaff diet for the duration of the experiment with pot scrubbers added to the rumen of one of the two groups. The remaining two groups were offered a low grain diet (35% grain) in the second diet phase followed by a high grain diet (70% grain) in the third diet phase. Pot scrubbers were also added to the rumen of one of these two groups of grain-fed sheep. The pot scrubbers were inserted with the intention of vii

8 Abstract increasing rumen stimulation without changing the diet composition. Ruminal ph, volatile fatty acids was measured to monitor rumen fermentation. In addition, methane production (in vitro) and ruminal retention time (in vivo) were measured in the 4 treatment groups at the end of each diet phase. A robust real-time PCR assay was developed to quantify methanogens and a denaturing gradient gel electrophoresis (DGGE) assay was developed to examine the diversity of methanogens. Rumen ciliates were also examined using the same methods but with already published protocols. The DGGE gels were analysed using Gelcompar II and the resulting DGGE banding patterns were analysed using a multivariate statistical software program (PRIMER). The Shannon index for each treatment group was also calculated based on the banding patterns to indicate the direction of diversity changes. The addition of grain and pot scrubbers changed rumen parameters. Methane production in vitro was reduced on a low grain diet in sheep with pot scrubbers. Methane production was reduced in sheep fed the high grain diet, with or without pot scrubbers. Acetate/propionate ratios were also lower in sheep fed the high grain diet. The total abundance of methanogens and ciliates in diet phase 3 was not different between treatments. However, the abundance of methanogens associated endosymbiotically with rumen ciliates was significantly higher in high grain-fed sheep with and without pot scrubbers. Fifteen of the most dominant methanogen DGGE bands were sequenced and identified using a BLAST search. Ten of the 15 different bands had >98% identity to Methanobrevibacter spp., whereas the other five bands were found to be between 82% and 96% similar to Methanobrevibacter spp. The diversity of methanogens and ciliates were also found to vary between treatments. The change in DGGE banding patterns and Shannon indices when sheep were fed grain indicated that the types of methanogens viii

9 Abstract changed when sheep were fed low and high grain diets, but their diversity did not. In contrast, the diversity of rumen ciliates decreased when sheep were fed a high grain diet. A total of 18 bands from the DGGE analysis of the ciliates were sequenced. All except one, which was 98% similar to Cycloposthium sp. not found previously in the rumen, matched the sequences for previously identified rumen ciliates. Some of the rumen ciliates identified were not present in sheep fed the high grain diet. On a high grain diet, methanogens associate endosymbiotically with rumen ciliates to get better access to hydrogen. It appears that the association between methanogens and rumen ciliates is dictated by the availability of hydrogen in the rumen and not the generic composition of the ciliate population. Furthermore, endosymbiotic methanogens appear to produce less methane than methanogens in other niches. The pot scrubbers did not change ruminal retention time but they did reduce the acetate/propionate measurements observed in sheep on the high grain treatment. The reason why pot scrubbers had this effect remains unknown, but it is interesting to consider that some physical interaction has occurred between the pot scrubbers, the grain and the sheep that has improved the fermentation parameters in sheep fed a high grain diet. The results from this study have advanced our understanding of the interaction between methanogens and ruminal ciliates, and methanogenesis in the rumen in response to dietary changes and mechanical challenges. Extending this work to look more specifically at the species of methanogens that are most closely linked to high methane production and how they interact with the ruminal ciliates will be critical for manipulating enteric greenhouse gas emissions. ix

10 Table of content Table of content Chapter 1: General introduction...1 Chapter 2: Literature review Introduction Taxonomy and characteristics of rumen methanogens...6 Taxonomy...7 Pathways of methanogenesis...8 Substrate range...9 Ecology Taxonomy and characteristics of rumen ciliates The entodiniomorphid ciliates...13 Digestion and metabolism of dietary components The vestibuliferid ciliates...14 Digestion and metabolism of dietary components Interrelationships between species of rumen ciliates Interaction between methanogens and rumen ciliates...17 Niches occupied by methanogens in the rumen...17 Why is there an interaction between ciliates and methanogens?...17 Methanogens and ciliate species that associate...19 Time after feeding and the interaction between ciliates and methanogens Grain diets and ruminal retention times influence hydrogen availability, methanogens, rumen ciliates and their association...22 Hydrogen availability...22 Methanogens, rumen ciliates and their interaction Value and limitations of two molecular methods for examining methanogens and ciliates in the rumen Denaturing gradient gel electrophoresis (DGGE) Quantitative real-time PCR (real-time PCR) Summary...36 x

11 Table of content Chapter 3: Materials and methods Introduction Experimental design...39 Chapter 4: In vitro methane emission and acetate/propionate ratio are decreased when artificial stimulation of the rumen wall is combined with increasing grain diets in sheep Introduction Materials and methods...43 Experimental design...43 Estimation of mean retention time of liquid and particulate matter...43 Volatile fatty acids, ph and in vitro methane production...45 Statistical analyses Results...46 Effect of diet...46 Effect of pot scrubbers...49 Combined effect of diet and pot scrubbers Discussion...50 Combined effect of diet and pot scrubbers...50 Effect of diet...52 Effect of pot scrubbers...53 Chapter 5: Grain and artificial stimulation of the rumen wall changes the association between methanogens and rumen ciliates Introduction Materials and Methods...58 Experimental design...58 Rumen sampling...58 DNA extraction and quantification...59 Denaturing Gradient Gel Electrophoresis (DGGE)...60 Phylogenetic analysis...62 Quantitative real-time PCR...63 xi

12 Table of content Statistical analysis and diversity index...66 Nucleotide sequence accession number Results...66 Effect of diet...66 Combined effect of diet and pot scrubbers...68 Identification of DGGE bands and their phylogenetic relationship...71 Validation of real-time PCR assay Discussion...75 Validation of real-time PCR and DGGE...81 Chapter 6: Grain changes the diversity of rumen ciliates but not their abundance Introduction Materials and methods...86 Experimental design...86 Rumen sampling, extraction and quantification of DNA...86 Denaturing gradient gel electrophoresis (DGGE)...87 Real-time PCR...88 Statistical analysis and diversity index...89 Nucleotide sequence accession number Results...90 Effect of treatments...90 Identification of DGGE bands Discussion...94 Chapter 7: General discussion...99 Future studies Conclusion References xii

13 Chapter 1: General introduction CHAPTER 1 General introduction 1

14 Chapter 1: General introduction Methane is the second most important greenhouse gas emitted from anthropogenic sources and has a global warming potential 23 times more potent than carbon dioxide (Wuebbles and Hayhoe, 2002). Globally, methane production from ruminants accounts for about 28% of all methane produced from anthropogenic sources (Food and Agriculture Organization of the United Nations, 2000). In Australia, ruminant livestock are the single largest source of methane emissions, accounting for 70.7% of agricultural methane emissions (Australian Greenhouse Office, 2007). Methane is formed in the rumen during fermentation of feed by methanogenic Archaea (methanogens), expired via the lungs and exhaled at the nose and mouth. The production of methane represents an energy loss to the animal, which has been estimated to be between 2 15% of the animal s gross energy intake (Johnson and Johnson, 1995; McAllister et al., 1996; Van Nevel and Demeyer, 1996). The rumen methanogens occupy three different niches within the rumen. Some live freely in the rumen (free-living), others are attached to the outer surface of the rumen ciliates (ectosymbiotic), and some reside within the rumen ciliates (endosymbiotic) (Vogels et al., 1980; Stumm et al., 1982). The percentage of methane produced by methanogens living in or on the rumen ciliates has been estimated to be between 9-37% (Finlay et al., 1994; Newbold et al., 1995). Therefore, rumen ciliates have a significant role in methane production from ruminants (Krumholz et al., 1983; Finlay et al., 1994; Newbold et al., 1995). There is also evidence that changes in the abundance of rumen ciliates can affect methanogenesis, as Krumholz et al. (1983) found that the methanogenic activity in rumen fluid was highest in fractions containing high numbers of protozoa. Furthermore, a change in the generic composition of the rumen ciliates can also lead to a change in methane production (Itabashi et al., 1994). 2

15 Chapter 1: General introduction The amount of methane emitted from ruminants depends on the conditions in the rumen, which are controlled by factors including diet and rumen retention time (McAllister et al., 1996). For example, methane production decreases from ruminants when fed high levels of grain (Russell, 1998). This is thought to occur because of an increased competition for hydrogen between the methanogens and the hydrogenutilising propionate producing bacteria. One reason methanogens associate with ciliates is to get access to hydrogen and it is likely that when sheep are fed a high grain diet the competition for hydrogen would affect this association. However, how changing these factors affect the numbers and diversity of methanogens, rumen ciliates, and especially their association is largely unknown. It would be beneficial to explore this gap in knowledge to help reduce methane emissions from ruminant livestock. Therefore, in this thesis I have examined the effect that increasing the grain content of the diet and decreasing ruminal retention time has on the number and diversity of rumen methanogens and rumen ciliates, and how this affects their association. The general hypothesis tested in this thesis was that increasing the amount of grain in the diet and reducing the retention time would affect the abundance and diversity of methanogens in their different niches, as well as their association with ruminal ciliates. 3

16 Chapter 2: Literature review CHAPTER 2 Literature review 4

17 Chapter 2: Literature review 2.1 Introduction The rumen contains a microbial population made up of Archaea (methanogens) (Woese et al., 1990), bacteria, ciliates and fungi (Hungate, 1966). These microorganisms ferment the food that a ruminant consumes to produce energy. The microorganisms in the rumen also function via complex interactions with each other. Rumen microbes have developed different strategies to survive in a highly competitive environment where a change of feed source is likely to make significant changes to the structure of the microbial ecosystem. Due to the complexity of the rumen environment this Chapter is limited to a review of the methanogens and the ciliates living in the rumen. Understanding how different rumen manipulations, in particular diet and retention time, affect methanogens and their association with the rumen ciliates would be beneficial for reducing methanogenesis from ruminant livestock. In order to understand why change in diet and decreased retention time in the rumen may change the diversity and the numbers of methanogens, ciliates and the close association between them, four specific fields of literature need to be examined in more detail: The first two areas are a description of the taxonomy and characteristics of rumen methanogens and ciliates. Then the association between methanogens and rumen ciliates will be explained, and finally the value and limitations of molecular techniques, denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR, used to study rumen microbes will be covered. 5

18 Chapter 2: Literature review 2.2 Taxonomy and characteristics of rumen methanogens The organisms that produce methane are a distinct group of Archaea (Woese et al., 1990) called methanogens. Methanogens are a normal component of the microbial population in the rumen, but are also found in a wide range of other environments (Miller and Wolin, 1986). Classification of methanogens was initially based on a wide range of characteristics because they were considered to be bacteria. The minimal standards for classifying methanogens was monoculture, morphology, Gram staining, electron microscopy, susceptibility to lysis, motility, colony morphology, nutritional spectrum, end products, growth rates, growth conditions, G + C content of the DNA, lipid analysis, cell wall structure, protein analysis and antigenic fingerprinting (Boone and Whitman, 1988). However, classification is now based almost solely on DNA (16S rrna) analysis and it has been determined that Archaea belong to their own phylogenetic kingdom (Woese et al., 1990). Balch (1979) reorganized the taxonomy of the methanogens based upon these phylogenetic relationships. The 16S rrna gene has been very useful because it is conserved in all known species of methanogens, but is different from the 16S rrna gene found in other Archaea and bacteria. In this section I will concentrate on the taxonomy of methanogens, pathway of methanogenesis, their substrate range and their ecology. These areas are central in the understanding of methanogenesis from ruminants, as different methanogens have different affinity for hydrogen (Zinder, 1993). Therefore, a change in the methanogens population can potentially result in a changed methane production from the rumen. 6

19 Chapter 2: Literature review Taxonomy Based on the above characteristics, Archaea are classified into four phyla within the domain Archaea: Crenarchaeota, Euryarchaeota, Korarchaeota and Nanoarchaeota (Barns et al., 1994; 1996; Burggraf et al., 1997a; 1997b; Huber et al., 2002). To date, the only Archaea identified in the rumen are methanogens belonging to the phylum Euryarchaeota. Within this phylum rumen methanogens have been identified in two classes (Methanobacteria and Methanomicrobia). Methanobacteria and Methanomicrobia consist of three orders, Methanobacteriales, Methanomicrobiales and Methanosarcinales, which are described in more detail because they contain rumen methanogens (Ferry et al., 1974; Bryant and Boone, 1987; Whitman et al., 1991; Garcia et al., 2000). The order Methanobacteriales, comprises non-motile methanogens, is divided into two families: Family I, Methanobacteriaceae, contains four genera (Methanobacterium, Methanobrevibacter, Methanosphaera and Methanothermus). For this thesis the three most important genera in this family are Methanobacterium, Methanobrevibacter and Methanosphaera, because they are the only genera that contain methanogens observed in the rumen (Miller and Wolin, 1985). Methanobrevibacter is in fact the major archaeal genus found in the rumen and contains species like Mbr. ruminantium and Mbr. smithii (Smith and Hungate, 1958; Miller and Wolin, 1986). Family II, Methanothermaceae, contains one genus (Methanothermus), but it does not contain any methanogens that have been observed in the rumen. The order Methanomicrobiales comprises three families (Methanomicrobiaceae, Methanocorpusculaceae and Methanospirillaceae) and nine genera (Methanocorpusculum, Methanoculleus, Methanofollis, Methanogenium, Methanolacinia, Methanomicrobium, Methanoplanus, Methanospirillum and 7

20 Chapter 2: Literature review Methanocalculus). Only methanogens belonging to the genera Methanomicrobium and Methanospirillum have been identified from the rumen (Ferry et al., 1974). The order Methanosarcinales is divided into two families (Methanosarcinaceae and Methanosaetaceae) and nine genera (Methanosaeta, Methanimicrococcus, Methanococcoides, Methanohalobium, Methanohalophilus, Methanolobus, Methanomethylovorans, Metthanosalsum and Methanosarcina). The family Methanosarcinaceae contains methanogens living in the rumen. These methanogens belong to the genus Methanosarcina and are acetotrophic (Bryant and Boone, 1987). Recently, the existence of a novel group of methanogens has been suggested as a new order, but cultivars have to be characterised before a new order can be accepted (Wright et al., 2004, 2006, 2007). It consists of uncultured archaeal sequences from diverse anaerobic environments, which are distantly related (>20%) based on 16S rrna sequence similarity to Thermoplasma. Pathways of methanogenesis The production of methane gas is the major source of energy for growth of methanogens. It might be expected that the reduction of primarily C-1 compounds would be a simple reaction, but because of the structure and synthesis of many of the coenzymes involved in methanogenesis, biochemically, it is a complex process (Whitman et al., 1991). The pathway of methanogenesis is slightly different depending on the substrate. The best-known synthesis of methane is the reduction of CO 2 to CH 4, which has seven steps that involve a series of co-enzymes of which many are unique to methanogens (Rouviere and Wolfe, 1988) (Figure 2.1). The source of electrons is either H 2 or formate (Whitman et al., 1991). The cycle starts with the activation of CO 2, which requires energy in the form of ATP (Figure 2.1). The energy for the activation is derived from the final step of methane synthesis. After the initial step, several co-enzymes are 8

21 Chapter 2: Literature review involved before CH 4 is formed (Figure 2.1). The key intermediate in this process is methyl-co-enzyme M, which is formed after the sixth step and is required in the methyl reductase system that represents the completion of the cycle with the release of CH 4 and activation of CO 2 for the next cycle (Figure 2.1). Methyl-co-enzyme M is important because it is also required for methanogenesis from substrates other than CO 2 (Whitman et al., 1991). Figure 2.1: The pathway of methane formation from acetate, methanol and CO 2 goes through seven steps. The numbers refer to the seven steps of the cycle. MFR: Methanofuran, HS-HTP: 7- Mercaptoheptanoylthreonine phosphate, H 4 MPT: Tetrahydromethanopterin, F 420 : Coenzyme F 420, HS- CoM: Coenzyme M, F 430 : Coenzyme F 430 [adapted from Rouviere and Wolfe (1988)]. Substrate range The substrate range of methanogens is limited despite the large phylogenetic diversity. Methanogens are divided into three main nutritional categories, on the basis that some of them can use more than one substrate for methanogenesis, which means that these methanogens cannot be placed in a single category. The three categories are: i) hydrogenotrophic methanogens (e.g. Methanobrevibacter), which oxidize H 2 and reduce CO 2. This category also includes the utilization of formate, certain alcohols and has the 9

22 Chapter 2: Literature review highest energy conversion during methanogenesis (Table 2.1); ii) methylotrops (e.g. Methanosphaera), which utilise methyl compounds such as methylamines, methanol or dimethylsulfide; and iii) acetotrophic methanogens (e.g. Methanosarcina), which can produce methane from acetate (Whitman et al., 1991; Garcia et al., 2000). Table 2.1: Overview of the most common methanogenic reactions and their energy yield (Whitman et al., 1991). Reactants Products G `(kj/mol CH 4 ) 4 H 2 + HCO H + CH H 2 O HCO H + + H 2 O - CH HCO CH 3 CH 2 OH + HCO 3 CH CH 3 COO - + H + + H 2 O CH 3 COO - + H 2 O - CH 4 + HCO CH 3 OH 3 CH 4 + HCO H + + H 2 O CH 3 OH + H 2 CH 4 + H 2 O (CH 3 ) 3 -NH+ + 9 H 2 O 9 CH HCO NH H (CH 3 ) 2 -S + 3 H 2 O 3 CH 4 + HCO H 2 S + H Other short chain alcohols, methylated amines and methyl mercaptan are utilized. The most widespread catabolic reaction performed by methanogens is the reduction of CO 2 to CH 4 using H 2 as a reductant (Table 2.1). This is particularly relevant to the rumen because many other ruminal microbes produce H 2 as a major fermentation end-product (Zinder, 1993). Formate is also used by hydrogenotrophic methanogens but it can be hard to detect because it is rapidly converted to H 2 and CO 2 (Garcia et al., 2000). When short chain alcohols are used in methanogenesis, the alcohols are often oxidized to volatile fatty acid (VFA) (Garcia et al., 2000) (Table 2.1). In the rumen, all three categories of methanogens are represented, but the majority of rumen methanogens belong to the hydrogenotrophic methanogens. 10

23 Chapter 2: Literature review Ecology Methanogens normally compete for hydrogen with three other major groups of bacteria. The three competitors are sulphate reducing bacteria, acetogens and ferric iron (Fe 3+ ) reducers. In natural environments where substrates (electron donors) are limited, there is a hierarchy of hydrogen utilisers. Ferric iron reducers are at the top of the hierarchy followed by sulphate reducing bacteria, methanogens and then acetogens provided that the respective electron acceptors are present (Zinder, 1993). This hierarchy is in accordance with the energy yield from the reactions where Fe 3+ reducing bacteria have the highest G (Zinder, 1993). However, it has been shown that acetogens can compete with methanogens in vitro (Joblin, 1999), but this observation needs to be validated under in vivo conditions. In the rumen, levels of hydrogen vary depending on factors like diet, retention time and ph, and certain ruminal conditions can favour specific microorganisms, while suppressing others. For example a high grain diet will enhance the abundance of propionate producing bacteria and therefore propionate production, which requires hydrogen, and thereby reduce methane production (Moss et al., 1995; Lana et al., 1998; Tajima et al., 2001a). However, this competition between hydrogen utilisers may be different in vivo as opposed to in vitro, due to the interaction between microorganisms. Methanogens have been found to have a close association with ciliates in the rumen (Vogels et al., 1980; Stumm et al., 1982; Stumm and Zwart, 1986) and this association may change the hierarchy of competitors, as methanogens have been found associated with hydrogen producing organelles (i.e. hydrogenosomes) in ciliates (Van Hoek et al., 2000). 11

24 Chapter 2: Literature review 2.3 Taxonomy and characteristics of rumen ciliates The main focus of this section relates to the digestion of other ruminal microorganisms, carbohydrates and metabolism of dietary compounds by ciliates and the interrelationships between species of rumen ciliates. This forms the foundation for the coming section centred around the association between methanogens and ciliates. The metabolism of rumen ciliates is an important factor when examining rumen methane production, because there is evidence that both a change in the abundance of rumen ciliates and a change in the generic composition can lead to a change in methane production (Krumholz et al., 1983; Itabashi et al., 1994). The largest of the rumen microorganisms are the ciliates. They are divided into groups within the subclass Trichostomatia. These are the vestibuliferids (order Vestibuliferida), and the entodiniomorphids (order Entodiniomorphida) (Williams and Coleman, 1992). Analysis of the 18S rrna genes confirmed that the entodiniomorphid and vestibuliferids ciliates belong to two different orders of the class Litostomatea (Wright et al., 1997; Wright and Lynn, 1997a; Wright and Lynn, 1997b). The two groups are quite different and will be considered separately in this section. Traditionally, ciliate taxonomy has relied on morphology and cellular ultrastructures. Most rumen ciliates are difficult to identify because of their small size and limited morphology, and most of the described species have never been cultured (Williams and Coleman, 1997). Another problem with identifying rumen ciliates is that the original classifications were based on both internal and external characteristics, but unless samples are taken from an animal that has been starved for 24 hours, the ciliates are full of starch and internal structures are difficult to observe (Williams and Coleman, 1997). Therefore, new molecular tools have been developed to examine rumen ciliate 12

25 Chapter 2: Literature review diversity and abundance using the 18S rrna gene (Regensbogenova et al., 2004b; Sylvester et al., 2005; Skillman et al., 2006b) The entodiniomorphid ciliates Digestion and metabolism of dietary components Bacteria - Bacteria are believed to be the most important single source of nitrogenous compounds for the rumen ciliates. There is no consistent pattern across the ciliates regarding their preference for certain bacteria, even though the bacteria Selenomonas ruminantium and Butyrivibrio fibrisolvens are almost always taken up faster or at the same rate as other bacteria. In contrast, bacterial species like Escherichia coli and Prevotella ruminicola are not engulfed regularly or are taken up very slowly (Williams and Coleman, 1997). The rate at which ciliates take up bacteria has been compared in two ways: (a) the rate of uptake from an infinitely dense suspension, which measures the rate at which ciliates can pass bacteria down the oesophagus and form food vesicles, and (b) clearance-rate of bacteria from an infinitely dilute suspension, which measures the ability of ciliates to find and capture prey. For the conditions in the rumen (a) would be the most relevant (Williams and Coleman, 1997). It has been found that the uptake of bacteria is relatively unaffected by changes in salt concentrations, whereas ph plays a vital role, with ph 6 being the optimum, 75% uptake at ph 7 and no uptake at ph 5 (Coleman and Sandford, 1979). It is noteworthy that the uptake of the yeast Saccharomyces fragilis is almost independent of population density as they are digested at a steady rate (Williams and Coleman, 1992). After engulfment different bacteria are digested at different rates. The rate of digestion depends on their cell wall. Bacteria with a cell wall that is comparatively resistant to lysozyme have their cell contents digested 13

26 Chapter 2: Literature review before there is extensive digestion of their cell wall. In contrast, Gram-negative bacteria, like Escherichia coli, are digested very rapidly (Coleman and Hall, 1972). Rumen fungi - There is evidence that fungal rhizoids, zoospores and sporangia are all engulfed by ciliates, as there appears to be an inverse relationship between the population densities in the rumen of ciliates and fungi (Williams and Coleman, 1997). However, Newbold and Hillman (1990) believed that ciliates play a greater role in the turnover of bacterial protein than in the turnover of fungal protein. Carbohydrates - The uptake rate of starch grains varies between rumen ciliates. Entodinium spp. engulf starch grains very rapidly initially and then much more slowly. Epidinium spp. behave similarly, but at slower rates. In contrast, the larger entodiniomorphid ciliates engulf starch grains more slowly, but at a constant rate for several hours (Coleman, 1992). The fermentation of starch grains in ciliates are similar and the principal products are hydrogen, carbon dioxide, acetic acid, butyric acid and glycerol, depending on the oxygen and carbon dioxide in the gas phase (Ellis et al., 1991a, b). The starch is digested to maltose and then glucose, which is phosphorylated to glucose-6-phosphate, which can be stored or metabolised to produce energy (Williams and Coleman, 1997) The vestibuliferid ciliates Digestion and metabolism of dietary components Metabolic and biochemical studies of the vestibuliferid ciliates have been undertaken only with Dasytricha ruminantium and the two species of Isotricha (Isotricha prostoma and I. intestinalis) (Williams and Coleman, 1997). Carbohydrates - The vestibuliferids are believed to be involved in the utilisation of soluble sugars and non-structural polysaccharides. They are able to utilise fructose, 14

27 Chapter 2: Literature review glucose and galactose and certain soluble oligomers and polysaccharides containing one or more of these sugars; fructose containing carbohydrates are utilized most rapidly. Furthermore, the range of carbohydrates metabolised is genus dependent (Williams and Coleman, 1997). The rate at which sugars are taken up is affected by the nature and concentration of the sugar and by the rumen ph and temperature (Williams and Harfoot, 1976). The vestibuliferid ciliates have also been found to have a chemotaxic attraction to sources of sugar (Orpin and Letcher, 1978). These ciliates are able to maintain close contact to carbohydrate sources by colonising plant tissue (Orpin and Letcher, 1978). Despite differences in the range of disaccharides fermented by Dasytricha and Isotricha their enzyme profiles are identical, but the activities of the different enzymes are different (Williams and Coleman, 1997). The ciliates belonging to the order Vestibuliferida are also able to store polysaccharides as a branched homoglucan, similar to amylopectin, and it is believed that initially 75-80% of the sugar taken up is converted into this storage polymer (Williams and Coleman, 1997). Metabolites formed during carbohydrate fermentation by Dasytricha ruminantium and Isotricha spp. are lactic acid, butyric acid, acetic acid, hydrogen, carbon dioxide, storage polysaccharides and small amounts of propionic acid and alanine (Ellis et al., 1991b). The metabolite formation is affected by diet, nutrient status of the ciliate, rumen ph, temperature, presence of oxygen, headspace gas composition, and metabolic interactions with other microbial groups (e.g. methanogens) (Williams and Coleman, 1997) Interrelationships between species of rumen ciliates The different species of rumen ciliates are not all present in the rumen at the same time because certain species of ciliates feed on other ciliate species. This means that different population types have been identified and they have been designated Types A, B, O and 15

28 Chapter 2: Literature review K (Table 2.2) (Eadie, 1962). The A-type population is characterised by the presence of Polyplastron multivesiculatum (Table 2.2). The B-type population is characterised by Epidinium spp. and/or Eudiplodinium maggii (Table 2.2). The K-type (found in cattle only) is characterised by the presence of Elytroplastron bubali and O-type contains only the vestibuliferids, Dasytricha and Isotricha (i.e. no entodiniomorpids) (Table 2.2). However, within these groups individual ciliate species can appear and disappear for no apparent reason (Williams and Coleman, 1992). The different population types are not equally common. A- and B-type populations are dominant in sheep and in a flock of sheep it would be likely to find approximately equal numbers of A- and B-type populations. However, in New Zealand, no B population has been found in their animals (Williams and Coleman, 1992). Table 2.2: A classification scheme of different ciliate population types found in cattle, sheep and goats. Table was taken from Williams and Coleman (1992). Ciliates Type A Type B Type O Type K* Entodinium X X X Isotricha X X X X Dasytricha X X X X Diplodinium X X X Eremoplastron X X X Diploplastron affine X X Eodinium X X Enoploplastron X X Ostracodinium X X Ophryoscolex X Polyplastron multivesiculatum X Metadinium X Epidinium X Eudioplodinium maggii X Elytroplastron bubali X *Cattle only 16

29 Chapter 2: Literature review 2.4 Interaction between methanogens and rumen ciliates A close symbiosis between methanogens and ciliates has been observed in the rumen (Vogels et al., 1980; Stumm and Zwart, 1986; Embley and Finlay, 1993), and various species of methanogens have been found to associate with rumen ciliates (Vogels et al., 1980; Stumm et al., 1982; Finlay et al., 1994; Newbold et al., 1995; Tokura et al., 1997; Sharp et al., 1998; Chagan et al., 1999; Tokura et al., 1999; Schonhusen et al., 2003; Irbis and Ushida, 2004; Regensbogenova et al., 2004a). In this part of the literature review I outline the reasons why the interaction is thought to exist and identify the species of methanogens and ciliates that have been found to associate together. Niches occupied by methanogens in the rumen Methanogens occupy three different niches within the rumen. Some live freely in the rumen digesta (planktonic), some are attached to the outer surface of the rumen ciliates (ectosymbiotic), and some reside within the ciliates (endosymbiotic) (Vogels et al., 1980; Stumm et al., 1982; Stumm and Zwart, 1986; Finlay et al., 1994; Newbold et al., 1995; Tokura et al., 1997; Sharp et al., 1998; Chagan et al., 1999; Tokura et al., 1999; Schonhusen et al., 2003; Irbis and Ushida, 2004; Regensbogenova et al., 2004a). However, some methanogens can attach and detach themselves to ciliates depending on the conditions in the rumen (Stumm et al., 1982; Tokura et al., 1997), which means that they can be part of the free-living and ectosymbiotic pools. Why is there an interaction between ciliates and methanogens? Both ciliates and methanogens are thought to benefit from their association. The advantages for the ciliates are thought to be that the methanogens keep the H 2 concentration low, which enhances the energy yield per mol glucose converted by the 17

30 Chapter 2: Literature review ciliates (Hino, 1982; Stumm and Zwart, 1986). At low hydrogen concentrations, reoxidation of reduced coenzymes via ferrodoxin-linked hydrogenase is favoured over reoxidation by fermentation reactions because of the higher energy yields (Stumm and Zwart, 1986). The endosymbiotic methanogens have access to substrates for methanogenesis from the ciliate s metabolism. Stumm et al. (1982) proposed that the attachment of methanogens to the ciliates was dependent on the H 2 pressure in the surroundings. However, according to results obtained by Ushida et al. (1997), it is more likely that methanogens use ciliates as an easy way to access substrates for methanogenesis, for example H 2 or formate. Methanogens most likely use formate produced by the ciliates, as well as hydrogen, based on evidence provided by Hutten et al. (1980), Finlay and Fenchel (1992) and Ushida et al. (1997). Hydrogen is considered to be the main substrate and of greatest value to the endosymbionts because they have been observed in close assemblages or association with hydrogenosomes. Hydrogenosomes are membrane-bound organelles, like mitochondria, that produce ATP. They have only been found in anaerobic organisms that cannot use oxygen as an electron acceptor, but reduce protons to molecular hydrogen instead and are involved in terminal steps of anaerobic energy metabolism (Van Hoek et al., 2000). Endosymbiotic methanogens also have the advantage of living in a protected, oxygen-free, environment inside the ciliates. Methanogens can actually tolerate small amounts of oxygen (Scott et al., 1983), but their oxygen tolerance increases in the presence of rumen ciliates even if the methanogens are not endosymbiotic, due to the use of oxygen by the hydrogenosomes (Hillman et al., 1988). The ectosymbiotic methanogens are thought to attach themselves to the surface of the ciliates to get easy access to substrates for methanogenesis via interspecies 18

31 Chapter 2: Literature review hydrogen transfer (Stumm and Zwart, 1986; Ushida et al., 1997). Hydrogenosomes are generally located near the cell surface and hydrogen from the hydrogenosomes diffuses to the cell surface where it is absorbed by methanogens (Van Hoek et al., 2000). The ability of some methanogens to associate with rumen ciliates and not others is not understood. Even though a broad range of methanogens have been identified to associate with ciliates, there is a requirement for a better understanding of how diet affects the ecto- and endosymbiotic association between methanogens and ciliates in the rumen. Methanogens and ciliate species that associate Recently, studies have been conducted to examine the species of methanogens that associate with rumen ciliates using molecular analyses. In most of these studies the most abundant methanogens associating with rumen ciliates were Methanobrevibacter smithii and Methanobrevibacter gottschalkii-like (>99% similarity), within the order Methanobacteriales (Sharp et al., 1998; Chagan et al., 1999; Tokura et al., 1999; Irbis and Ushida, 2004). Regensbogenova et al. (2004a) also found that the main methanogens associated with the ciliates belonged to the order Methanomicrobiales, but also the order Methanosarcinales. In all of these experiments the samples examined came from a single animal fed different diets and limited data were available in relation to the overall diversity of methanogens in the samples. With the exception of Sharp et al. (1998), who examined the crude rumen fluid as well as the ciliate fraction isolated from the rumen fluid, both the crude rumen fluid and the ciliate fraction was examined using hybridisation probes. They found methanogens from the orders Methanobacteriales, Methanomicrobiales and Methanosarcinales in both the ciliate fraction and the rumen fluid, but there was only a very low representation of Methanomicrobiales in the ciliate fraction. This indicates that it may not be a specific 19

32 Chapter 2: Literature review group of methanogens that associate with the rumen ciliates, but a general representation of the methanogens found in the rumen fluid. Furthermore, Regensbogenova et al. (2004a) found no preference from any ciliates to associate with specific groups of methanogens. Nevertheless, these studies were performed on rumen fluid from very few animals, which could also explain the differences observed between the studies. This also means that there is a paucity of knowledge about what effect a change in diet and/or ruminal retention time has on the different methanogen populations and their association with the rumen ciliates. Using traditional microscopy, the rumen ciliates that associate with methanogens have been reported to belong to the order of Entodiniomorphida (Vogels et al., 1980), but in later studies using molecular techniques the vestibuliferid ciliates have been found to interact also with methanogens (Irbis and Ushida, 2004). This demonstrates that traditional methods may be limited in terms of giving a complete description of the methanogen and ciliate association, when compared to molecular tools. Time after feeding and the interaction between ciliates and methanogens It has been proposed by Stumm et al. (1982) that at least the ectosymbiotic association between methanogens and rumen ciliates is controlled by the surrounding physicochemical conditions such as the hydrogen partial pressure, with more methanogens associated with ciliates at low hydrogen pressure. Using microscopy, Smolenski and Robinson (1988) determined that the association between methanogens and ciliates decreases from 65% before feeding to 30% an hour after feeding and that hydrogen levels increase in the rumen at that time. In contrast, Tokura et al. (1997) found, using molecular techniques, that the number of methanogens per ciliate increased shortly (1h) after feeding and then decreased. The difference between these two studies may be due 20

33 Chapter 2: Literature review to the different methodologies that were used, as the microscopic counting would not include endosymbiotic methanogens. The difference may also have been due to the difference in diet between the two studies (Hegarty, 1999). In both studies, methane production was found to be highest an hour after feeding (Stumm et al., 1982; Tokura et al., 1997). 21

34 Chapter 2: Literature review 2.5 Grain diets and ruminal retention times influence hydrogen availability, methanogens, rumen ciliates and their association It is well established that the diversity of methanogens and rumen ciliates can be influenced by diet (Williams and Coleman, 1992; Wright et al., 2004; 2006; 2007) and rumen parameters also change depending on factors like ruminal retention time (Faichney et al., 1999). I focus now on the effect of diet and retention time on hydrogen availability, methanogens, ciliates and their interaction in the rumen. Hydrogen availability The overall microbial ecology in the rumen is influenced by increased grain content in the diet. One reason for this is thought to be the changes in the availability of hydrogen in the rumen. As established earlier, the main substrate for methanogenesis in the rumen is hydrogen. This is supported by Van Nevel et al. (1969), who found that inhibiting methanogens in sheep using chloral hydrate resulted in an accumulation of hydrogen and an increase in propionate production. The changes in propionate concentrations on a high grain diet have also been found in a number of other studies as its production requires hydrogen and the ratio of acetate to propionate in the rumen has an inverse relationship with methanogenesis (Van Kessel and Russell, 1996; Lana et al., 1998; Russell, 1998). This increase in propionate is the main indicator of a shift in rumen fermentation. It has been suggested that the decrease in methanogenesis when propionate production is increased in the rumen could be because, for diets high in grain, propionate is used as a sink for metabolic hydrogen by organisms that grow faster than methanogens under these conditions (Baker, 1997; Russell, 1998). One of these organisms could be Selenomonas ruminantium, which has been found previously to produce propionate (Scheifinger and Wolin, 1973), and have more than a two-fold 22

35 Chapter 2: Literature review increase in abundance after an extended period of feeding a high grain diet (Tajima et al., 2001a). Feeding a high grain diet also increases lactate production in the rumen (Counotte et al., 1983) as well as concentrations of lactate producing and utilising bacteria (Goad et al., 1998). Therefore, bacteria like Megasphaera elsdenii would contribute to the increase in propionate, as it has the ability to ferment lactate to butyrate and propionate. This process has been examined in Eubacterium hallii, which was found to utilise hydrogen (Duncan et al., 2004) and limit the amount of hydrogen available for methanogenesis. Methanogens, rumen ciliates and their interaction Diets and ruminal retention time are very closely linked; a change in diet, for example from pasture to grain, changes the retention time in the rumen. The addition of grain to diets changes rumen fermentation by changing a wide range of parameters such as decreasing ph and acetate/propionate ratios, as well as methane production (Moss et al., 1995; Baker, 1997; Lana et al., 1998). Evans (1981a,b) observed that, as the percentage of grain in the diet increased, the retention time also increased. This enables the methanogens and ciliates to stay in the rumen for longer. Ciliates are frequently attached to plant material in the rumen because it allows them to remain in the rumen when the liquid retention time is less than their growth rate (Bauchop and Clarke, 1976; Williams and Coleman, 1992). Methanogens, on the other hand, are either free-living or closely associated with the rumen ciliates, which mean that if retention time is decreased, then some free-living methanogens are likely to be washed out of the rumen because the turnover in the rumen is likely to be higher than the generation time of the methanogens. The generation time for methanogens grown in vitro varies between 2.25 hours under optimal conditions for Methanobacterium spp. and 73 hours or more for an acetate-fermenting strain of Methanosarcina on a basal medium (Mah et al., 1978; 23