Ecological Aspects on Rumen Microbiome

Introduction

Ruminants are cloven-hoofed herbivores mammals of the order Artiodactyla and obtain their food by grazing on plant material (Hackmann and Spain 2010). The ruminants are distinguished from other mammals as they possess peculiarities for cud chewing, the process which is called rumination (Hungate 1966). The ruminant stomach consists of four compartments: abomasum, omasum, rumen, and reticulum. In adult animals, the average proportion in size of the rumen, reticulum, omasum, and abomasum of the complex stomach is about 80%, 5%, 7%, and 8%, respectively ( Fig. 16.1a). The abomasum mainly functions to secrete enzymes and gastric fluids (Hodgson 1971;Mcleay and Titcheng 1975). This compartment is Laboratory of Environmental Microbiology, Embrapa Environment, Rodovia SP340 Km 127.5, Jaguariuna, SP 13820-000, Brazil e-mail: rodrigo.mendes@embrapa.br comparable to the true stomach in nonruminant animals. When the ruminant swallows, there is a slight tendency of the concentrates and succulents portions of the diet to pass through the rumen more rapidly than roughages. After rumination, the material returns to the rumen, the reduction in particle size achieved during the chewing will determine whether any particle proceeds to the omasum. The rumen and reticulum occupy most of the left side of the abdomen (Balch 1950). The rumen is an anaerobic fermenter chamber, with a pH between 5.5 and 6.9, and temperature ~ 38-40 °C (Hungate 1966;Church 1969;Clarke 1977;Dehority 2003).

The rumen harbors a rich and diverse microbial community responsible for the degradation of proteins (Brooks et al. 2012), lipids (Kim et al. 2009), starch (Offner et al. 2003), cellulose, lignin, and hemicellulose (Koike and Kobayashi 2009). Rumen has its enormous space filled with digested and partially digested feed material. The feed is swallowed and repeatedly regurgitated from the rumen (Hungate 1966). The mechanical mastication and enzymatic reaction are responsible for breaking large molecules into small pieces increasing the contact surface for microorganisms on the fiber. The digestion is influenced by several factors, such as, retention time of feeds in the rumino-reticulum, mastication, and mixing of saliva, flow rate of digesta to omasum, and lower part of the digestive tract, the buffering action of the saliva and by the rate of metabolites removal, like volatile fatty acids (VFAs), ammonia, carbon dioxide, and methane. All the VFAs are absorbed by epithelium surface (Fig. 16.2) mainly by diffusion (Storm et al. 2012) and the gases CO 2 and CH 4 are removed during eructation through the mouth and nose ( Fig. 16.2) (Hungate 1966). Ross et al. 2013;Petri et al. 2013;Jami et al. 2013;Jami et al. 2014;Yáñez-Ruiz et al. 2015;Henderson et al. 2015) Hofmann (1973) claims that ruminants have achieved high levels of digestive efficiency at each evolutionary stage based on comparative morphological studies of the digestive system of several ruminants and they are classified according to their feeding behavior: concentrate selectors, opportunistic and mixed feeders. Recently, the concentrate selectors were divided in "browsers" and "frugivorous" that prefer eating fruits, shoots, and leaves (typically from shrubs, forbs, and trees); grazers, eaters of grass, and roughage and intermediate feeders that switch between browse and grass, usually depending on their seasonal availability (Hackmann and Spain 2010). Depending on the diet, ruminants have evolved different traits to digest, which might cause a selection on the microorganisms associated in the rumen (Hofmann 1989). Apart from different feeding behaviors, variations in rumen bacterial community composition in animals from different regions were observed, likely to be caused by differences in diet, climate, and farming practices (Henderson et al. 2015). Recently, a "core bacterial microbiome" was identified by Henderson et al. (2015), where Prevotella, Butyrivibrio, and Ruminococcus, as well as unclassified Lachnospiraceae, Ruminococcaceae, Bacteroidales, and Clostridiales are present in a large selection of ruminants, although in different abundances according to the animal species. These differences in abundance according to the animal species might have happened due to the coevolution of ruminants and their associated microorganisms. Kim et al. 2011;Jami and Mizrahi 2012;Petri et al. 2013;Jami et al. 2013;Jami et al. 2014;Sirohi et al. 2012;Lima et al. 2014;McCann et al. 2014;Kumar et al. 2015;Weimer 2015;Henderson et al. 2015), which affects microbial functions and consequently biomass degradation, resulting in the release of methane (CH 4 ) and carbon dioxide (CO 2 ) via eructation and volatile fatty acids that are absorbed by the epithelium

Coevolution Between Ruminants and Microorganisms

One of the most interesting features of the digestive system is the symbiotic relationship between the animal and the microorganisms that live in the digestive tract. This includes transient and autochthonous microorganisms that develop into relatively stable populations, characteristic of each species (Dubos 1966). In this interaction, the gene products (enzymes) of ruminal microorganisms take over tasks that are not developed by the host genome, such as degradation of nutritional components, for example, cellulose, expanding the spectrum of metabolic functions and capabilities of the host. On the other hand, the rumen environment provides growing conditions suitable for the development of microorganisms present there (Rosenberg et al. 2010).

It is believed that the interaction between ruminants and microorganisms had the origin about 55-36 million years ago, in the Eocene, second phase of the Paleogene period. The interaction occurred with the microorganisms affecting the evolution of ruminants, and ruminants affecting the evolution of microbial species sheltered by them, generating, thereafter, a relationship of interdependence (Troyer 1984). This process, called coevolution, is defined as "[…] the process of reciprocal evolutionary change that occurs between pairs of species or among groups of species as they interact with one another. The activity of each species that participates in the interaction applies selection pressure to the others" (Rafferty and Thompson 2016).

During the Eocene, the weather in North America and Eurasia was humid and tropical. Proto-ruminants had a diet consisting of low cellulose content based on plant structures still in the early stages of growth. However, with the beginning of the Oligocene, third phase of the Paleogene, the weather started to get colder and dry and the plants development more seasonal, and thus, the animals began to consume a diet rich in fiber during some periods of the year (Janis 1976). As the ruminants are unable to synthesize cellulolytic enzymes to degrade and enjoy the new diet rich in fibers, these animals started the evolution of their digestive systems, with the emergence of fermentation sites, and establishment of symbiotic relationships with microorganisms producers of cellulases (Janis 1976). Throughout the evolutionary process, ruminants have developed a huge fermentation chamber, the rumen, which gave them advantages such as greater utilization of microbial action on the fibrous part of the diet as the rumen is located prior to chemical digestion. Another advantage is no need to digest food in the place of consumption, avoiding excessive exposure to predators (Janis 1976;Alexander 2009).

Considering that, climate change during this period occurred gradually, as well as changes in the digestive system, over a long period much of the volume of food consumed by the animals was not well utilized, thus requiring the ingestion of large volumes of the diet. Moreover, with continued cooling climate, the animals needed to store more fat in order to better conserve body heat. Thus, there was also at this time a strong selective pressure for animal size increase (Janis 1976). The increase in body size, fibrous diet, and drier weather also led to a greater need to recycle nitrogen, which became increasingly scarce and unavailable. The presence of microorganisms able to break down urea allowing nitrogen recycling, resulted in reduction in water loss, since urea is excreted via urine (Janis 1976). It is believed that these factors led slowly to the initiation of microbial colonization in animals slowly inducing the formation of a chamber with ideal conditions for carrying out the microbial activity, as the pH near neutrality. The gradual increase in the fiber content of the diet might have led to a concomitant selection of cellulolytic microorganisms and urease producers, and, little by little, the rumen was emerging as is known today, adapted to the requirements of the C and N cycles (Janis 1976).

As the plant constituents of the diet of the protoruminants during the Eocene presented high amount of toxic substances, some authors, such as Foose (1974), believe that the growth and division of the stomach of ruminants first was the selection pressure on the necessity of the presence of detoxifying bacteria in the digestive system of animals.

From the microbial point of view, the evolution of this interaction occurred initially by water consumption by animals. Water naturally carries anaerobic microorganisms capable of degrading organic matter and their ingestion by animals started this association. With the diet enriched in fiber, a selection pressure acted on the microorganisms for decomposing the cellulolytic compounds, gradually increasing the potential for microbial degradation of the fibrous biomass. Following the evolution, some species were more efficient in certain metabolic pathways, losing features no longer needed, such as O 2 tolerance, developing an intricate system of cross-feeding, and metabolic expertise in this ecosystem, creating interdependence between microbial species and between the microbial community and the host (Hungate 1966).

The Rumen Microbiome

Ruminants and their microbiome have coevolved over millions of years and the functional plasticity of ruminal microbial communities makes ruminants highly adaptable to different diets (Morgavi et al. 2013). However, many other factors can influence the rumen microbial community, including animal age (Fonty et al. 1987(Fonty et al. , 2007, use of antibiotics (Kleen et al. 2003), host health (Kleen et al. 2003;Rustomo et al. 2006a), geographic location (Sundset et al. 2007), season (Orpin et al. 1985;Crater et al. 2007), photoperiod (McEwan et al. 2005, stress level (Uyeno et al. 2010), environment (Romero-Pérez et al. 2011), and feeding regimen (Rustomo et al. 2006b). In addition, the microbial species and activities have also been shown to be affected by feed intake levels (Crater et al. 2007) and frequency of feeding (Pulido et al. 2009), suggesting that it is possible to manipulate the composition of ruminal microbial community by diet and management. Recent studies revealed that the host may also have an effect on selecting rumen bacteria Hernandez-Sanabria et al. 2013), indicating that it may be possible to breed selected rumen microbial animals for better performance.

The rumen microbiome comprises a diverse symbiotic community of anaerobic bacteria, archaea, protozoa, bacteriophage, and fungi ( Fig. 16.1c) (Krause and Russell 1996), however, bacteria and protists together represent over 90% of the microbial biomass, but only 8% have been isolated (Weimer 2015). The symbiotic microbes present in the rumen produce an arsenal of enzymes that deconstruct plant polysaccharides providing essential nutrients, and detoxify harmful molecules, such as urea, H 2 , CO 2 , and CH 4 (Krause et al. 2003), protecting the host against invading pathogens and parasites, modulating development and immunity (Hooper et al. 2012). The ruminal microorganisms can be subdivided into four major subpopulations: (i) liquid-associated populations, which are composed of the planktonic microorganisms in the rumen liquid, including those detached from the feed particles and the ones consuming soluble feed components from the rumen liquid (McAllister et al. 1994), (ii) solid-associated populations, which include microorganisms that are loosely or tightly adhered to the feed particles and are fundamental in digesting the ingesta (McAllister et al. 1994), they account for up to 75% of the total rumen microorganism populations (Koike et al. 2003), (iii) epitheliumassociated populations, which attach to the rumen epithelium and counts for only 1% of the rumen populations (Czerkawski 1986), they are more diverse than the other subpopulations (Malmuthuge et al. 2012) and are considered to be more closely related to the host metabolic activities than the other subpopulations (Wallace et al. 1979); and (iv) eukaryote-associated populations, which are represented by the Bacteria and Archaea attached to the surface of protozoa or fungal sporangia ( Fig. 16.1b) (Miron et al. 2001).

Structure of the Rumen Bacterial Community

The rumen bacterial community is almost strictly composed by anaerobes, but some of the bacterial species are facultative anaerobes and may assist in maintaining anaerobic conditions. Phylogenetic analyses have revealed the presence of a core microbiome, dominated by the bacterial phyla Firmicutes and Bacteroidetes (Fig. 16.2), present in all ruminants across a wide geographic range but with a great deal of variation among individual animals in relative population sizes of different taxa (Henderson et al. 2015).

Bacteria are known to colonize the rumen soon after birth and contribute to carbohydrate metabolism through fermentation. The bacterial density in the rumen can be as high as 10 11 cells per gram of rumen content measured by direct counts (Mackie et al. 2000), and the bacterial community comprises of more than 200 species (McSweeney et al. 2005). The sequencing of bacterial 16S rRNA gene has shown that regardless of the type of diet, species or age, Bacteroidetes and Firmicutes phyla may represent approximately 80% of the bacterial population in ruminants (de Menezes et al. 2011;Henderson et al. 2013;Mohammed et al. 2014). The phyla Proteobacteria, Fibrobacter, Verrucomicrobia, Tenericutes, and Spirochaetes are present, however, in the minority (de Menezes et al. 2011).

Members of the phylum Bacteroidetes are skilled in the degradation of complex polysaccharides, including starch, xylan, pectin, galactomannan, and arabinogalactan (Martens et al. 2011). Also in Bacteroidetes, the Prevotella genus has been described as the most abundant in the rumen in various dietary conditions (Stevenson and Weimer 2007;Pitta et al. 2010). Ramsak et al. (2000) showed that Prevotella has highly varied genetic divergence and proposed that this genus in particular has a wide functional versatility. Prevotella has been described participating in the fermentation of structural polysaccharides (Matsui et al. 2000), and species of this genus are found in fibrous (Koike and Kobayashi 2009) and liquid (Pitta et al. 2010) fractions in the rumen. The phylum Firmicutes has species that can use starch, xylan, cellulose, hemicellulose, and galactomannan as energy source (Dassa et al. 2014). In Firmicutes, Ruminococcus, Clotridium, and Butyrivibrio have bacterial species with different attack mechanisms on the ruminal fiber (Kim et al. 2011). In particular, Ruminococcus genus representing ~10% of the bacterial population (Palmonari et al. 2010) and species with different adhesion mechanisms ruminal fiber (Morrison and Miron 2000).

Bacteria in the rumen are also classified according to their metabolic activity: fibrolytic (e.g., Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenes, and Butyrivibrio fibrisolvens), amylolytic (e.g., Selenomonas ruminantium, Streptococcus bovis), proteolytic (e.g., Prevotella spp.), lipolytic (e.g., Anaerovibrio lipolytica), lactate production (e.g., Streptococcus bovis and Selenomonas ruminantium), and lactate consumer (e.g., Megasphaera elsdenii) (Hungate 1966;McCann et al. 2014). The isolation of ruminal microorganisms, mainly bacteria, has been described for more than 50 years (Bryant et al. 1958;Hungate 1966). The most studied rumen bacteria are Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus, Butyrivibrio fibrisolvens, Prevotella ruminicola, Eubacterium cellulosolvens, and Eubacterium ruminantium (Stewart et al. 1997). These bacteria are characterized as fibrolytic, or participating in the depolymerization plant cell wall (Koike and Kobayashi 2001). Lopes et al. (2015) described the composition of bacterial community in the sheep (Ovis aries) rumen using 16S rRNA gene tag sequencing and revealed that the bacterial community was dominated by the Prevotellaceae family, with more than 30% of total classified 16S rRNA gene sequences.

Streptococcus bovis are spherical Gram-positive lactic acid bacteria and when large amounts of starch or sugars are present in the diet, they will grow explosively, as Streptococcus bovis produces large amounts of lactic acid and capsular polysaccharide causing acute ruminal and bloat, respectively (Russell and Rychlik 2001). On the other hand, Megasphaera elsdenii requires lactic acid to grow that helps to clean up the rumen a bit and raise rumen pH, sustaining the growth of the acidintolerant fiber digesters in the rumen (Kung and Hession 1995). Selenomonas ruminantium is capable of growing on tannic acid as a sole energy source and has been isolated from fecal matter of goats browsing on high tannin-containing acacia species (Skene and Brooker 1995).

Structure of the Archaeal Community in the Rumen

Previously, methanogens were grouped with the domain Bacteria, but based on peculiar cell wall structure and 16S rRNA sequence, they were classified in a separate domain Archaea (Woese and Fox 1977), though both Bacteria and Archaea share the same ancestors. Woese et al. (1990) suggested a three-domain system by grouping together all the eukaryotes into the domain Eukarya and by calling the other two major domains Bacteria and Archaea. Archaea have been found in many different habitats (Liu and Whitman 2008). This domain houses known methanogens taxa, which are an essential part of rumen microbiome as they keep a low H 2 pressure in the rumen (Janssen and Kirs 2008).

A recent study shows that methanogens can be detected in the ovine rumen 17 h after birth (Gagen et al. 2012). Methanogens are the essential part of rumen microbiome because they maintain steady-state fermentation in the rumen. During fermentation of monosaccharides in the rumen, hydrogen is generated that is used by the methanogens for methane production in the rumen (Janssen and Kirs 2008). The abundance of methanogens species in the rumen is dependent on the studied target gene (rrs or mcrA) and qPCR results report values between 10 7 and 10 8 number of copies per gram of rumen contents (Mosoni et al. 2011).

Three different groups are describe in the methanogens metabolism: (i) hydrogenotrophs (Methanobrevibacter, Methanomicrobium, and Methanobacterium spp.), which convert hydrogen and/or formate to CH 4 , (ii) methylotrophs (Methanosphaera spp. and members of the order Methanomassiliicoccales), which produce CH 4 from methyl compounds such as methanol and methylamines, and (iii) acetoclastic methanogens (Methanosarcina), which can utilize acetate to produce CH 4 in addition to the hydrogenotrophic and methylotrophic pathways (Janssen and Kirs 2008). Currently, genome sequences are available for four rumen methanogens including strains of Methanobrevibacter ruminantium (Leahy et al. 2010), Methanobrevibacter boviskoreani (Lee et al. 2013;Leahy et al. 2013), Methanobacterium formicicum (Kelly et al. 2014), and Methanosarcina barberi (Lambie et al. 2015).

Methanobrevibacter was predominant in protozoa-associated methanogens, whereas Methanomicrobium was predominant in free-living methanogens. Janssen and Kirs (2008), after reviewing global data, described rumen methanogen archaea can be classified into three genuses: Methanobrevibacter, Methanomicrobium, and a group of uncultured rumen archaea affiliated with Thermoplasmatales.

Methanogens can be closely associated with protozoa, and some can be freeliving, and the association is species specific. The community structure of freeliving methanogens and protozoa-associated methanogens have been analyzed by using 16S rRNA and methyl coenzyme M reductase (mcrA) genes (Tymensen et al. 2012). Both groups comprised of Methanobrevibacter, Methanomicrobium, and rumen cluster C (RCC), which is distantly related to Thermoplasmata.

Association of methanogens with anaerobic fungi has been established recently and a rumen fungus was collected from the goat rumen and subcultured to obtain uniform colonies (Jin et al. 2014). The same authors used the mcrA gene to confirm methanogens found with fungal cultures and they were classified as members of a novel RCC cluster.

The methanogen-associated fungus was identified as Candidatus Methanomethylophilus alvus, which represents a very small part of the RCC cluster. It is distantly related to Thermoplasmatales and was classified with a new order Methanoplasmatales (Paul et al. 2012). Poulsen et al. (2013) observed that Thermoplasmata are poorly characterized in bovine rumen. The same authors suggested that these methylotrophic methanogens can reduce methane emission in lactating cows.

These rumen microorganisms utilize the H 2 and CO 2 produced by the protozoa, fungi, and bacteria from the catabolism of hexoses to produce CH 4 and generate ATP (Albers et al. 2007;Ferry and Kastead 2007), which benefits the donors by providing an electron sink for reducing equivalents to minimize the partial pressure of H 2 (Wolin and Miller 1988;Lange et al. 2005) inside the rumen. These microorganisms are found in the liquid, adhered to the rumen fiber, on protozoa, and on rumen epithelium. The growth rate of methanogenic archaea communities varies according to the rumen fractions, animal, and the rate of passage through the system (Janssen and Kirs 2008). The appropriate selection of carbohydrates in the diet or ration and the proportion of nonfibrous or structural carbohydrates may help carbon dioxide reduction and hydrogen, which is the main methane production precursors in the rumen (Mitsumori and Sun 2008).

Methanogens are found in a symbiotic association with rumen bacteria (Wolin and Miller 1988) and protozoa (Lange et al. 2005). The establishment and maintenance of the stable population of methanogens are affected by the type of diet and level and frequency of feeding (Kumar et al. 2012(Kumar et al. , 2013aSirohi et al. 2013a). The symbiotic association of hydrophobic methanogens with H 2 producers is usually realized by the attachment or by floc formation (Thiele et al. 1988;Lange et al. 2005). The symbiotic relation between methanogens and ciliates may generate up to 37% of rumen CH 4 emission (Finlay et al. 1994). Methane emissions can vary according to the host and diet, Singhal et al. (2005) observed that contribution of crossbred cattle, indigenous cattle, buffalo, goat, and sheep in methane emission through enteric fermentation was different according to host species. Kempton et al. (1976) reported that gray kangaroos emitted less methane than sheep despite being kept under the same diet. This low methane emission was also observed in camelids compared with ruminant livestock (Pinares-Patino et al. 2003;Dittmann et al. 2014).

Methanogens are a unique group of microorganisms generating methane as a stoichiometric end product of their metabolism (Janssen and Kirs 2008). Although the production of enteric methane contributes to the production of greenhouse gas (GHG) emissions, it also leads to a 2-12% loss of energy intake in domesticruminants (Johnson and Johnson 1995), and decreasing methane emissions by livestock has become a priority and an integral part of climate control policies (Thorpe 2008).

The Role of Protozoa in the Rumen

Rumen protozoa can contribute ~50% of the biomass in the rumen. They engulf bacteria and feed particles and digest carbohydrates, proteins, and fats (Williams and Coleman 1992). Two groups, that is, holotrich and entodiniomorphid protozoa, have been studied inside the rumen. Morphological identification using optical microscopy has been used for studying ciliate species in the rumen (Williams and Coleman 1992;Imai 1998).

Entodinium genus is the most abundant representative protozoa in the rumen and are involved in control of bacterial populations (Nagaraja et al. 1992), helping to maintain rumen pH (Dehority 2005). Protozoa feed on carbohydrates and cellulose (Findley et al. 2011), and consequently release H 2 in the ecosystem, which is used by methanogenic (Skillman et al. 2006;Tymensen et al. 2012). Several studies have accessed the diversity of protozoa in the rumen via molecular techniques by designing specific primers for this group (Skillman et al. 2006;Tymensen et al. 2012). Sylvester et al. (2004) observed that diet directly affects the diversity of protozoa in the rumen and duodenum and identified the predominant species as Epidinium caudatum, Entodinium caudatum, and Isotricha prostoma. Other protozoa genera have been described as Dasytricha, Ostracodinium, Diplodinium, Diploplastron, Eudiplodinium, Epidinium, Ophryoscolex, and Polyplastron. These ciliates organisms play an important role in fiber digestion and the modulation of the fermentation profiles. The rumen protozoa produce fermentation end products similar to the bacteria, particularly acetate, butyrate, and H 2 . Rumen methane archaea actually attach and live on the surface of rumen protozoa for immediate access to H 2 . They utilize large amounts of starch at one time and can store it in their bodies (Williams and Coleman 1992). This engulfment helps to slow down the bacterial fermentation and production of acids that decrease pH, benefiting the rumen (Mackie et al. 1978).

Rumen protozoa multiply very slowly in the rumen as compared to bacteria. For this reason, the rumen protozoa hide out in the slower moving fiber mat of the rumen, so that these are not washed out before these have a chance to multiply. Diets with low concentration of roughage decrease the retention time of fiber in the rumen and influence the number of protozoa in ruminant. The rumen ciliates are also proteolytic producing ammonia and amino acids as metabolic end products (Warner 1956). Protozoa obtain protein by digestion promoted by the engulfed bacteria (Coleman 1975). Rumen ciliates have enzymes capable of digesting plant proteins (Coleman 1983), but is inefficient (Coleman 1975). The role of protozoa in rumen microbial ecosystem remains was considered unclear (Williams and Coleman 1992), and even recently, it has been difficult to clearly establish the role of ciliate protozoa in rumen fiber degradation (Newbold et al. 2015).

The Role of Anaerobic Fungi

Anaerobic fungi were firstly described in the rumen by Colin Orpin in the 1970s, they establish within 8-10 days of birth and constitute 5-8% of total rumen biomass. Initially, their presence in the rumen was confused with flagellate protozoa and then categorized with them (Liebetanz 1910;Braune 1913;Akin and Benner 1988). The first rumen fungus was identified as Neocallimastix frontalis in the rumen of sheep, which showed motile (zoospores) and nonmotile zoosporangium (Orpin 1975). According to the recent classification system (Hibbett et al. 2007), anaerobic fungi have been described in the phylum Neocallimastigomycota (Dagar et al. 2011;Sirohi et al. 2013b;Gruninger et al. 2014).

Classification and identification of anaerobic fungi have mainly been based on the pattern of thallus/rhizoid morphology and may be monocentric or polycentric (Ho and Barr 1995). These forms are determined in the earliest stages of growth, soon after zoosporogenesis. The zoospores may be posteriorly uniflagellated or polyflagellated in both forms. The life cycle of anaerobic fungi is asexual, and no sexual stage has been described, as only mitotic nuclear divisions have been observed (Heath et al. 1986). It alters between a motile zoospore, encysted zoospore, and vegetative zoosporangial stage. Once released from zoosporangium, the motile zoospores move by chemotaxis to colonize the plant fibrous material and shed its flagella to get transformed into cyst (encystment). The cyst germinates by producing a germ tube, which ultimately gives rise to a rhizoidal system. In monocentric forms, the development may further be classified as endogenous or exogenous (Sirohi et al. 2012). They attach through flagella, encyst, and develop a rhizoidal system, which penetrates the substrate with the help of polysaccharide-degrading enzymes. The attachment of zoospores is very fast usually within 15-30 min of incubation of feed in the rumen. Rumen fungi secrete an array of enzymes including esterases (feruloyl esterase, p-coumaryl esterase, and acetyl esterase), which break the ester bonds between hemicelluloses and lignin, thus releasing free celluloses and hemicelluloses for the other microbes to attack (Yue et al. 2009).

In rumen, anaerobic fungi is represented by the phylum Neocallimastigomycota, which houses the genera, Orpinomyces, Caecomyces, Anaeromyces, Cyllamyces, Piromyces, and Neocallimastix. Each of these genera has distinct morphological characteristics (Ho and Barr 1995;Ozkose et al. 2001;Griffith et al. 2009). The fungi in the rumen have organelles called hidrogenossomos that are coupled to the metabolism of glucose for energy without oxygen production. These organelles have features in common with mitochondria (van Der Giezen 2002)-possibly are derived from them (Embley et al. 1997;Voncken et al. 2002;Muller et al. 2012). The hydrogenossome contains the hydrogenase enzyme responsible for producing H 2 , CO 2 , lactate, and acetate as metabolic end products of fungal anaerobic fermentation of plant cell wall polysaccharides (Brul and Stumm 1994;Theodorou et al. 1996;Muller et al. 2012).

The genome of the strain of C1A Orpinomyces sp. was recently sequenced and provided valuable information about the diversity of active enzymes in the degradation of carbohydrates (Youssef et al. 2013). The cellulolytic anaerobic fungi machinery consists of free enzymes and of multienzyme complex (Wilson and Wood 1992), known as cellulosomes (Krause et al. 2003;Joblin et al. 2010). This characteristic has drawn attention for biotechnological applications as well as for microbial supplement for ruminant production as a means of improving the use of low-quality feeds. The inclusion of anaerobic fungi cultures in diets has been made and the result indicates improvement in feed intake, animal growth rate, feed efficiency, and increased milk production (Gao et al. 2013).

The Bacteriophages from Rumen

Bacteriophages are one of the most important members of the rumen microbial community and are present typically at >10 9 particles per mL (Tarakanov 2006). Phages are the most abundant organisms in the biosphere and they have been shown to be a driving factor in the evolution of microbial communities in various environments (Breitbart et al. 2002(Breitbart et al. , 2003Fierer et al. 2007;Dinsdale et al. 2008;Rohwer and Thurber 2009;Parsley et al. 2010;Reyes et al. 2010;Rodriguez-Brito et al. 2010). They play an important role in controlling the numbers of microbes in an ecosystem, naturally selecting phage-resistant microbes, and facilitating horizontal gene transfer (HGT) (Breitbart and Rohwer 2005;Rodriguez-Valera et al. 2009;Rohwer and Thurber 2009). HGT is a widespread and common phenomenon in microbial communities and contributes to the evolution of the microbes in those communities (Koonin and Wolf 2008;Aminov 2011).

Enteric methane mitigation strategies include bacteriophage therapy (Patra 2012) as bacteriophages can target and lyse specific unwanted bacteria (Klieve et al. 1999;Bach et al. 2002). McAllister and Newbold (2008) reported siphophages that can infect methanogens as Methanobacterium, Methanobrevibacter, and Methanococcus spp., revealing the potential of phage therapy to be used as a strategy for controlling methanogen populations and other populations, such as pathogens, in the rumen (Ackermann 2007;Patra 2012).