TY - JOUR
AU - Callaway, T. R.
AB - ABSTRACT Robert Hungate, considered the father of rumen microbiology, was the first to initiate a systematic exploration of the microbial ecosystem of the rumen, but he was not alone. The techniques he developed to isolate and identify cellulose-digesting bacteria from the rumen have had a major impact not only in delineating the complex ecosystem of the rumen but also in clinical microbiology and in the exploration of a number of other anaerobic ecosystems, including the human hindgut. Rumen microbiology has pioneered our understanding of much of microbial ecology and has broadened our knowledge of ecology in general, as well as improved the ability to feed ruminants more efficiently. The discovery of anaerobic fungi as a component of the ruminal flora disproved the central dogma in microbiology that all fungi are aerobic organisms. Further novel interactions between bacterial species such as nutrient cross feeding and interspecies H2 transfer were first described in ruminal microorganisms. The complexity and diversity present in the rumen make it an ideal testing ground for microbial theories (e.g., the effects of nutrient limitation and excess) and techniques (such as 16S rRNA), which have rewarded the investigators that have used this easily accessed ecosystem to understand larger truths. Our understanding of characteristics of the ruminal microbial population has opened new avenues of microbial ecology, such as the existence of hyperammonia-producing bacteria and how they can be used to improve N efficiency in ruminants. In this review, we examine some of the contributions to science that were first made in the rumen, which have not been recognized in a broader sense. INTRODUCTION When microbiology students are first introduced to the study of microbial ecology, their entry point to the microbial world often begins in the rumen of cattle (Hungate, 1966). Although much of the interest in microbial ecology in recent years has focused on microbiology of extreme environments and unusual ecosystems (Brock et al., 1994), such as Yellowstone black pools, electrogenic bacteria from ocean silt, and deep oceanic vents (Stahl et al., 1985; Bond et al., 2002; Flores et al., 2012; Lesniewski et al., 2012), the beginning of our understanding of microbial community dynamics is found in the relatively “tame” (at least compared with deep-sea lithotrophic environments) ecosystem of the rumen. This understanding of the ruminal ecosystem comes from the work of a chain of brilliant scientific pioneers who have pushed back the frontier of knowledge working in the rumen, which was viewed as a suitable model ecosystem because of its “constant and continuous nature” (Hungate, 1966). Not only has the knowledge gained on the ruminal microbial ecosystem had a significant impact in improving ruminant nutrition, but the techniques and theories that were initially developed in the rumen have been now applied to a wide variety of other microbial ecosystems. In this review, we will highlight the evolution of rumen microbiology as it emerged to lead the field of microbial ecology and pioneered new theories and techniques and a possible revival of the study of anaerobic microbiology, especially with new researchers using genome sequencing to examine ecological theories and the role of community structure in environmental function. RUMINANT PHYSIOLOGY AND RELATIONSHIP BETWEEN THE HOST AND MICROBIOME Historically, the relationship between the ruminant animal and its resident microbial population has been noted from ancient times, although it was not understood for its symbiotic nature. Leviticus (11:3–4) states “Whatsoever parteth the hoof, and is cloven-footed, and cheweth the cud, among the beasts, that shall ye eat”; thus, the hallmarks of the ruminant animal were noted early in the association between cattle and humans. Aristotle, the Greek philosopher, described the relationship between teeth, diet, and complexity of the gut: “when an animal is of large size and feeds on substances of so thorny and ligneous a character … it may in consequence have several stomachs” (Lennox, 2001). As the field of microbiology emerged during the 19th century, research began into the relationship between the rumen fluid, bacteria, and fiber degradation; this quest has not ceased and is responsible for some of the improvements in how cattle are fed today. Early research into the biochemical mechanism of this relationship (vis-à-vis fermentation pathways) involved Nobel Prize laureate A. I. Virtanen (Virtanen and Tarnanen, 1931; Virtanen and Pulkki, 1933). His research into the basis of fermentation eventually led him to investigate the rumen, where he demonstrated that cattle could produce milk using nonprotein nitrogen as a sole N source because of bacterial assimilation of ammonia (Virtanen, 1966). Although we tend to focus on the beneficial aspects of the symbiosis between host and microbiome, this relationship is not always positive in regard to the animal. Research into ruminant health-impacting conditions, such as bloat, nitrite poisoning, and acidosis, has furthered our understanding of rumen function and the host-microbiome relationship. As studies into these ruminal dysfunctions continued, research became increasingly focused on understanding how the rumen worked and how its function could be altered (Lewis, 1951; Hungate et al., 1955; Head, 1959; Dunlop and Hammond, 1965). In 1951, in an effort to address the most prominent ruminant concerns, a “bloat conference” was convened to bring the top researchers in the field together, and this conference still continues today, having changed focus over a half century from rumen function to gut function. However, many of the details about the rumen microbial ecosystem eluded elucidation until the ruminal bacteria could be grown in pure culture. Development of Anaerobic Techniques Before World War II, there were only sporadic reports of culturing bacteria in the absence of oxygen. Most bacterial culture and isolation procedures were applied directly from clinical, soil, or dairy microbiology, with little success in the anaerobic ruminal environment. Although facultative ruminal bacteria (e.g., Streptococcus bovis) were isolated, the truly strict anaerobic bacterial population eluded cultivation because of the difficulty in achieving anaerobiosis during cultivation. Elements of successful anaerobiosis were known before 1900, but all the factors were not applied together to grow anaerobic bacteria until the 1940s. For example, early attempts to grow cellulolytic bacteria from the rumen involved adding rumen fluid to a meat-extract medium; although these attempts were not successful in growing cellulolytic bacteria, fermentation acids were detected, which indicated the fermentation process occurred in the rumen (Van Tappeiner, 1884). Rumen microbial populations were estimated initially to be in the range of 106 cfu/mL, but this estimate was performed using nonreduced media (Johnson et al., 1944). Other researchers began to narrow down the requirements for growth of ruminal bacteria (Gall and Huhtanen, 1951; Huhtanen et al., 1952; Huhtanen and Gall, 1953a,b). Finally, a combination of anaerobic techniques and reducing agents was found to be most suitable (Hungate, 1944, 1950, 1952) to growing cellulose-digesting bacteria from the rumen of cattle by Robert Hungate (Chung and Bryan, 1997), a zoologist who became the leader in ruminal microbiology, thus earning the title of the father of rumen microbiology. Because Petri dishes could not be used to grow anaerobic cellulolytic bacteria and because of the difficulty in seeing the cellulose clearings in agar tubes (“shake tubes”), Dr. Hungate developed “roll tubes” to obtain thin layers of agar in test tubes in which cellulose digestion and colony morphology could more easily be seen. Even today, the “roll-tube technique” is used to culture obligate anaerobes from a variety of anaerobic habitats. Dr. Hungate modified the traditional Delft University (the Netherlands) approach of enriching for specific species and determined that including rumen fluid (an essential nutritional supplement) and a CO2 atmosphere with bicarbonate to simulate the natural rumen habitat and the salivary buffering system were central to the isolation of cellulolytic bacteria (Hungate, 1950, 1952; Bryant, 1972). The Hungate method could best be described from our perspective as “less is more” and focused on developing the most accurate simulation of a habitat or environmental niche to be able to isolate and grow members of a bacterial ecosystem. This method of growing anaerobic bacteria, called the Hungate technique, was based on the ingenious concept pioneered by the development of the Winogradsky column (used to study photosynthetic bacteria) to simulate the conditions of the natural habitat in a culture tube (Brock et al., 1994). The rumen environment simulation technique involved the use of butyl rubber stoppers to keep the tube anaerobic (less permeable to oxygen), resazurin as a redox indicator, a gas (N2or CO2) to displace air, boiling the medium to remove dissolved oxygen, and the addition of a reducing agent to reduce it before sterilization. This technique led to explorations of other anaerobic environments and enriched our understanding of many macro- and microbiological environments. The basic simplicity, adaptability, and utility of this technique, which seems so logical and obvious to our current perspective, were viewed contemporaneously as a revolutionary approach pioneering the exploration into the ruminal habitat. The anaerobic techniques of Dr. Hungate have been modified in many ways to adapt them to the requirements of the anaerobic ecosystem. The methods developed at the Anaerobe Laboratory at Virginia Polytechnique Institute (the VPI technique) were designed mainly to isolate and identify anaerobes of medical importance and for human fecal microbiome studies (Anaerobe Laboratory, 1975). These anaerobic techniques were used in the development of anaerobic glove boxes as the next generation of anaerobic techniques. As researchers adjusted the gas mixtures in these large chambers to increase H2 and reduce N2 concentrations, the first interactions involving H2 and methanogenesis were noted, and the implications of this will be discussed below. By initiating the growth of pure bacterial cultures, Dr. Hungate, along with other researchers such as A. J. Kluyver (Delft University, the Netherlands), was able to deduce the relationship between the ruminant and the rumen microbial population as mutualism. Over time, the mutualism was determined to be not only between each organism that filled specific roles in the ecosystem but also between the rumen microbial population and the host. However, despite this knowledge, studies comparing molecular vs. cultural microbiome profiles indicate that we can culture less than 20% of the ruminal or gastrointestinal microbiome (Nocker et al., 2007). Our recognition of the global nature of the microbial ecosystem composition and its interrelationship with and impact on its environment has been critical to further developments in ruminant nutrition and has even played a role in the current discussions surrounding global climate change, especially in regard to CH4 production and nitrogen runoff. On the basis of the quantitative experiments on the rumen fermentation and “0 time rate” studies, Dr. Hungate recognized that the rumen is a continuous culture system. The following is his description of the ruminant animal: “a small fermentation unit which gathers the raw material, transfers it to the fermentation chamber, and regulates its further passage, continuously absorbs the fermentation products, and transforms them into a few valuable substances such as meat and milk” (Hungate, 1950:46). He conceived and constructed a 2-stage fermentor with cycle times based on analysis of batch growth curves to help his colleague (Stuart Adams) study recovery of ethanol from yeast fermentation of cannery waste (Adams and Hungate, 1950). This was one of the first attempts at an industrial continuous culture and contributed to the theory and practice of chemostats. This led Dr. Hungate to speculate that industrial cellulose fermentation might be profitable if the amount of cellulose fermented could be increased by continuous removal of fermentation products, which is one strategy used in current efforts to produce ethanol from cellulosic materials (Hungate, 1950). Discovery of Anaerobic Fungi The rumen has a large number of fungi, which have been estimated to be around 10% of the microbial mass (though numbers vary widely on the basis of animals and diet), yet they remained unrecognized until about 35 yr ago (Orpin, 1975, 1977a,b,c; Yokoyama and Johnson, 1988). In contrast, the existence of protozoa and bacteria in the rumen was known for more than 100 yr. One of the reasons for the lack of detection was the dogma in microbiology that all fungi are aerobic organisms and therefore not likely to be a component of the “flora” (now microbiome) of the rumen, an anaerobic ecosystem. Another reason is likely attributable to the routine practice of rumen microbiologists to work with strained ruminal fluid for convenience and to discard the solid digesta containing most of the plant-fiber-associated fungal biomass. Rumen microbiologists had observed the existence of actively motile, uni- or polyflagellated cells in the rumen. These cells were considered to be flagellated protozoa. In 1966, A. C. I. Warner reported an observation of a 25-fold increase in the number of flagellated cells in the rumen of sheep within 1 h after feeding and suggested that the increase was due to the migration of the cells from the ruminal wall in response to chemotaxis (Warner, 1966). The sequestration of the cells was a logical explanation because the increase in the number of flagellated cells could not be due to binary division of protozoan cells. Orpin (1975) showed that the increase in flagellated protozoa occurred in vitro when ruminal digesta were treated with an extract of oats and in the absence of particulate digesta the oat extract failed to cause the increase in the number of cells. Orpin (1975) concluded that the flagellated protozoa were not sequestered in the ruminal wall but were, in fact, zoospores of a chytrid-like fungus released from sporangia associated with plant fragments in the digesta. Orpin identified 3 species of anaerobic fungi, Neocallimastix frontalis, Sphaeromonas (now named Caecomyces) communis, and Piromonas (now named Piromyces) communis in the rumen of sheep and described a 2-stage life cycle: a free-floating, motile zoospore stage and a nonmotile, vegetative mycelial structure (thallus) carrying a sporangium (Orpin, 1975). Subsequently, Orpin (1977c) showed that the cell walls of the fungi contained chitin, a polymer, which in microbes is only found in fungi. From that time, anaerobic fungi have been isolated from the foregut and hindgut of a wide range of herbivores, and these fungi can exist in an aerotolerant stage that can be transmitted between animals (Wubah et al., 1991; Davies et al., 1993). Interestingly, anaerobic fungi have only been recently found in an environment (landfills) where they were not gastrointestinal symbionts (McDonald et al., 2010). Although the overall contribution of anaerobic fungi to ruminal digestion has yet to be quantified, it is evident that, at least for low-quality, fibrous diets, plant fragments entering the rumen are rapidly and extensively colonized by the zoosporic fungi (Bauchop, 1979a,b). Ruminal fungi have fibrolytic activity similar to that of aerobic soil fungi and are currently used in industry and agriculture, particularly in relation to the use of abundant cellulosic (and other than fibrous) substrates. It is also suggested that zoosporic fungi are morphologically adapted to penetrate and disrupt plant tissues, which will facilitate bacterial access to plant biomass and enhance bacterial colonization and degradation (Theodorou and Lowe, 1988). Cooperation between Microorganisms Once the members of the microbial population were isolated and grown in pure cultures, the race to understand their role in the microbial ecology of the rumen was on (Bryant and Burkey, 1953; Bryant, 1959). However, it was quickly determined that no single organism was responsible for the complete degradation of complex substrates in the rumen, such as cellulose, starch, and protein (Bladen et al., 1961). Researchers identified that a complex succession of microorganisms took part in the cooperative catabolism of substrates in the rumen and in the production of fermentation end products (Wolin, 1975). Complex feedstuffs are colonized and broken into oligomers and finally to tri-, di-, or monomers of each component. The oligomers or shorter pieces can be transported and fermented by other members of the microbial population that in turn produce branched chain fatty acids, vitamins, or other cofactors for other bacteria responsible for degrading feedstuffs (Allison et al., 1961; Bryant, 1973). This community analysis approach to the breakdown of nutrients by a microbial consortium led to the discovery of the concept of cross feeding of nutrients across and within environmental niches and can support the overall health of the host animal. Production of B vitamins by ruminal bacteria is important to ensure bacterial growth and, particularly, fiber degradation and is also responsible for ensuring animal health and well-being (Scott and Dehority, 1965). For example, a strain of the prominent ruminal cellulolytic bacterium (Ruminococcus albus) was found to require phenylpropanoic acid (PPA) to digest cellulose (Stack and Hungate, 1984). Ruminal microbes produce PPA from the fermentation of phenolic compounds, and the lack of PPA in cultures meant that R. albus could not adhere to cellulose. The PPA was thought to act as a “glue” that holds the cellulolytic enzyme complex in a stable conformation, allowing for cellulose degradation (Stack and Hungate, 1984). As researchers delineated bacterial functions and nutritional requirements, it became clear that there are significant interactions between microbial populations, both within an environmental niche and between niches. Application of this concept spread into analysis of other microbial habitats (Schultz and Breznak, 1979) and has shaped our view of how microbes interact with the world around them. From the time of these early studies, we have discovered that quorum signals play a role in other natural microbial environments (Khan et al., 1995; Miller and Bassler, 2001;Sperandio et al., 2003) and that these biochemical messenger molecules can cause changes in microbial growth and fermentation. Furthermore, it now appears that a 2-way communication between the host and microbial population exists, meaning that the endocrine status of the host can have a direct impact on the gut microbial population (Sperandio et al., 2003; Lyte and Freestone, 2009; Freestone and Lyte, 2010; Bailey et al., 2011). This has, in turn, led to the developing concept of the “microbial organ” playing a critical role in animal production and health (Lyte, 2010) and the theory that probiotics or the ruminal microbial population could be used as a drug delivery vehicle to improve animal performance. Methanogens (Archaea) and Methanogenesis in the Rumen From ancient times, “combustible gas” has been known to seep from geological fissures in various areas of the world. The experiments of Alessandro Volta in 1776 that described bubbles emerging from the sediments in the shallow area of lakes that burned with a “beautiful blue flame” laid the foundation for the study of CH4 biogenesis. Although the relationship of methanogenesis to decaying plant material was noted by Volta, almost 100 yr later proof of the microbiological origin was obtained with studies on “intestinal” contents of ruminants incubated with plant material by Tappeiner in 1882 (Wolfe, 1993). The first isolation of an organism that “oxidized” H2 and reduced CO2 was reported in 1933 (Stephenson and Stickland, 1933). However, the development of the Hungate technique was the major impetus for the isolation and characterization of methanogens from the rumen and other habitats. Methanogens had earned the reputation as the most difficult organisms to grow. Paul Smith worked with Hungate to isolate Methanobrevibacter ruminantium from ruminal contents (Smith and Hungate, 1958), and the key to isolation was to employ a cultural procedure to simulate ruminal conditions. To make the medium more anaerobic, it was incubated with Escherichia coli to use all oxygen, and after killing the E. coli, ruminal fluid was inoculated with a syringe to obtain “lemon yellow colored” colonies of M. ruminantium. Syntrophic H2 Transfer During the catabolic process of fermentation H2 is produced by some microbes and used by others in a syntrophic process known as interspecies H2 transfer. Syntrophic H2 transfer was first hypothesized more than 45 yr ago (Hungate, 1966), and the interaction not only allows growth of methanogens and benefits the fermentative bacteria, protozoa, and fungi in that they obtain more energy for growth but also profoundly affects the balance of fermentation products produced in the habitat. Discovery of this major interaction began with the recognition that a culture of Methanobacillus omelianskii, widely used to study methanogenesis and known to produce CH4 from ethanol and CO2, was, in fact, a mixed culture of 2 distinct species. One was a curved rod called the S organism that was never formally named and was subsequently lost and that oxidized ethanol to acetate and H2, and the second, a methanogen (now called Methanobacterium bryantii), reduced CO2 with H2 to produce CH4. The 2 species would not grow alone but grew well together with ethanol and CO2 because the S species provided the methanogen with H2 to grow and the methanogen removed the H2 that inhibited the growth of the S species (Bryant et al., 1967; Reddy et al., 1972). Although production and subsequent eructation of CH4 are considered as energy loss for the ruminant (and an important greenhouse gas impacting global climate change), the use of H2 by methanogens in the rumen is an extremely important process because any interference, unless redirected to other H2-sink reactions, leads to accumulation of H2 and can slow down the fermentation rate and efficiency because of an accumulation of reducing equivalents. Consequently, the products formed by the fermentative organisms in the presence of H2 differ from those produced when H2 is used to form CH4. For example, Ruminococcus albus, a major cellulose-digesting species in the rumen, produces ethanol, acetate, H2, and CO2 in the absence of methanogen but produces more acetate and less or no ethanol in the presence of methanogens (Iannotti et al., 1973). Because acetate production results in ATP synthesis, the interaction results in increased growth and cellulose digestion. This startling concept demonstrated that although excessive ruminal CH4 production was negative from the perspective of the host animal and the environment, a certain level of CH4 production is necessary to remove reducing equivalents, thereby allowing the overall ruminal fermentation to proceed. As a result, rumen fermentations will likely always continue to produce at least some CH4. Until the recognition of interspecies H2 transfer, the perception of anaerobic fermentation of organic matter was that microbes form acetate, propionate, butyrate, H2, and CO2. Because the major precursors of methanogens are acetate, H2, and CO2, the question that arose was how propionate and butyrate would become CH4 in anaerobic ecosystems other than the rumen. The studies of Marvin Bryant and Meyer Wolin at the University of Illinois showed that propionate and butyrate are first converted to acetate and then to CH4 by syntrophic interaction between fatty-acid-oxidizing bacteria (H2 producers) and methanogens, although not necessarily in high abundance in the rumen (Bryant et al., 1967; Reddy et al., 1972). Interspecies H2 transfer is now considered to be a critical ecological component in nearly all methanogenic ecosystems and anaerobic digesters (Thiele and Zeikus, 1988; Ishii et al., 2005). Archaea and the Tree of Life The 1970s are generally viewed as the heyday of rumen microbiology, with rumen microbiologists being in the vanguard of developing new concepts and techniques. The discovery of interspecies H2 transfer by Wolin and Bryant led to prominent discoveries by their colleagues, Carl Woese and R. S. Wolfe, in the University of Illinois microbiology department. These researchers (among others) made discoveries that literally revolutionized the dogma surrounding the tree of life (Woese et al., 1990). Archaea, a new domain (taxonomic division above kingdom), was created to include a group of organisms that are known to have novel physiological features and are only distantly related to other bacteria. Before the recognition of Archaea as a distinct group, all bacteria were placed in a single prokaryotic kingdom at the same taxonomic level with the 4 eukaryotic kingdoms of animals, plants, fungi, and protozoa. The first group of organisms to be included in Archaea was the methanogens. Carl Woese and his colleagues analyzed oligonucleotide sequences of the small subunit ribosomal RNA (the 16S rRNA) from a variety of organisms, including ruminal microorganisms obtained from Dr. Bryant, to assess molecular relationships. Lying on the floor in his office with rumen microbiologists, examining rRNA sequences, Woese recognized that certain variable regions of rRNA could be used to trace genetic linkages and could serve as a biologic or evolutionary “clock.” By carefully analyzing sequences of hundreds of organisms, Woese discovered that methanogens are no more related to other prokaryotes than they are to eukaryotes. In addition to rRNA-derived phylogeny, methanogens are distinctly different from true bacteria by a number of characteristics, including a lack of typical peptidoglycan and possession of membrane lipids composed of ether linkages, rather than the ester linkage, to glycerol. Carl Woese coined the term archaebacteria (archae for antiquity) to reflect a primitive and ancient divergence from other lineages (Woese and Fox, 1977; Woese, 1987; Woese et al., 1990), and this term was later changed to archaea. The group also includes some extreme halophiles and thermophiles, commonly referred to as “extremophiles.” The 3-domain model of true bacteria (eubacteria), archaea, and eukaryotes has become a biological standard. This fundamental shift in the understanding of the most basic aspect of biology arose, at least in part, from insights gained from rumen microbiology. Demonstration of rRNA Oligonucleotide Probes in an Ecosystem The discovery of the importance of rRNA and its utility as a tool to determine phylogenetic relationships and the identity of bacterial populations has allowed us to study the ecology, even without the need to cultivate, of mixed microbial environments in situ (Pace et al., 1986; Amann and Ludwig, 2000). When rRNA oligonucleotide probes were first introduced, they were used to explore novel, extreme environments of relatively low diversity (Stahl et al., 1985; Amann and Ludwig, 2000). However, the use of these probes had to be validated within a “known” diverse microbial ecosystem. Naturally, the ecology that was known of the rumen, coupled with the density and diversity of bacterial, archaeal, protozoal, and fungal populations, quickly drew attention. The initial rumen probe validation studies by David Stahl and others demonstrated that 16S rRNA probes could indeed be used in the complex rumen microbial ecosystem to track changes in the microbial population caused by feed types and antimicrobial usage (Stahl et al., 1988). After this pioneering work, researchers found that oligonucleotide probes could be used to follow rumen bacterial populations and competition (Odenyo et al., 1994) and to elucidate differences in the microbial community structure of the gastrointestinal tract of many animals (Lin et al., 1997). Validation of probe usage in the rumen opened the door for exploration of other environments using this powerful new technique of oligonucleotide probes. The symbiotic relationships between protozoa, bacteria, fungi, and archaea were verified through the use of 16S rRNA probes, which confirmed the results of earlier autofluorescence work that protozoa harbor populations of symbionts that play a role in interdomain H2 transfer (Irbis and Ushida, 2004). Furthermore, the use of archaea-specific oligonucleotide probes allowed researchers to detect the presence of methanogenic endosymbionts within ruminal ciliated protozoa (Finlay et al., 1994; Hackstein, 2010), further clarifying the interlocked nature of the tree of life within the rumen microbial ecosystem. Ruminal Detoxification As the knowledge of the composition of the microbial ecology of the rumen grew, so did interest in their function at the animal level. Although the ability of the rumen microbial population to detoxify toxic components in forages was recognized, the involvement of the rumen microbial ecosystem was not elucidated until the 1980s. Leucaena is an arboreal legume that is rich in protein and is used as a forage throughout much of the tropics and subtropics (Allison et al., 1990). Unfortunately, Leucaena contains mimosine, an AA, at concentrations of up to 5%, which is toxic to nonruminants (Allison et al., 1990). Mimosine is toxic because of microbial conversion to 3-hydroxy-4-[1H]-pyridone (3,4-DHP), a goitrogen (Hegarty et al., 1976), yet ruminant animals can be adapted to tolerate mimosine because the ruminal microbial population can be “adapted” to detoxify it. Furthermore, researchers noted geographic variations in the ability of ruminants to tolerate Leucaena was related to ruminal microbes that could degrade the toxic 3,4-DHP (Jones, 1981). Later, researchers were able to confirm this via the transfer of ruminal fluid from Indonesia and Hawaii to Australian goats (Jones and Lowry, 1984; Jones and Megarrity, 1986). A ruminal bacterium, Synergesties jonesii, was isolated and found to degrade 3,4-DHP and has been used to inoculate ruminants in regions where Leucaena was not previously used (Allison et al., 1992; Andrew et al., 2000). This was the first time that a specific detoxifying bacterium was taken from 1 environment and added to another to remediate, a practice now common to biological remediation of environmental contamination. Obligate AA-Fermenting/Hyperammonia-Producing Bacteria Protein is an important and most expensive component of ruminant diets, and urinary nitrogen represents an economic cost to the producer and carries an environmental impact that is becoming increasingly crucial to the animal industry (Chen and Russell, 1991; Cherney et al., 1994). Researchers noted that the specific activity of ammonia production (mg ammonia·mg bacterial protein−1·min−1) of the individual proteolytic bacterial species of the rumen was lower than the specific rate of the whole-rumen contents (Russell, 1983; Russell et al., 1983). Jim Russell and his associates at Cornell University used methods similar to those of Robert Hungate to enrich and isolate ruminal bacteria that could grow solely on AA as a carbon and energy source (Russell et al., 1988; Chen and Russell, 1989). These obligate AA-fermenting bacteria (Clostridium aminophilum, Clostridium sticklandii, and Peptostreptococcus anaerobius) had very high specific activities of deamination; subsequently, other hyperammonia-producing bacteria from the rumen and other environments have been identified, although their impact and role in the rumen is still being fully elucidated (Attwood et al., 1998; Wallace et al., 2003). It has been suggested that because hyperammonia-producing bacteria use such large amounts of AA for growth functions, this represents a highly inefficient use of dietary protein, sequestering it from use by the animal. Furthermore, the excessive ruminal ammonia production from this process is eliminated in the urine and is a significant source of environmental pollution. Thus, methods that can control or reduce these species (and other as-yet-undiscovered microbes with high specific activities of deamination) may have profound impacts on animal production efficiency and environmental quality. With the elucidation that a group (or guild) of high-specific-activity bacteria plays a significant role in ecology, animal health, and the environment, rumen microbiology again was in the forefront of microbial ecology. This has led to the reevaluation of other microbial habitats for members critical to the function of their ecosystem that had previously escaped early detection and classification. Host Immunization to Improve Efficiency As we have increased our understanding of the function and interspecies interactions in the rumen, interest has increased in harnessing the power of the ruminant immune system to modify the microbial population to enhance production efficiency and animal health. Because lactic acidosis is an animal health issue related to rumen function, it became an early target for fermentation modifications (including the use of ionophores). Much of the development of lactic acidosis (acute, subacute, and chronic) has been linked to increasing populations of Lactobacillus spp. and Streptococcus bovis after increased availability of fermentable soluble starch in the rumen (Slyter, 1976; Russell and Hino, 1985; Plaizier et al., 2012). Vaccines were developed that targeted ruminal populations of Lactobacillus and Streptococcus bovis (Shu et al., 1999, 2000). Although initially promising, further research has been lacking in preparing a vaccine for market. Because protozoa can represent inefficiencies in terms of protein use in the rumen, researchers also developed vaccines that reduced protozoal populations, although the direct effect on the level of ruminal antibodies was not demonstrated (Williams et al., 2008). Interestingly, because of the relationship between methanogens and protozoa, it would be expected that reducing protozoa populations would reduce CH4 production albeit indirectly (Hook et al., 2010; Leahy et al., 2010; Martin et al., 2010; Attwood et al., 2011; Wright and Klieve, 2011). As researchers have become increasingly focused on the impact of CH4 production on the environment, studies have examined the role of ruminant animals in greenhouse gas emission and global climate change (U.S. Environmental Protection Agency, 2006; Hook et al., 2011;Wright and Klieve, 2011). A vaccine against specific members of the archaeal genus Methanobrevibacter has been developed (Wright et al., 2004). This vaccine induced a humoral immune response, showing specific IgG and IgA concentrations in plasma and saliva as well as IgG in the ruminal fluid (Williams et al., 2009). This resulted in a nearly 8% decrease in CH4 production/kg DMI (Wright et al., 2004). However, researchers found that there was not a tremendous amount of cross protection between archaeal species and that instead of elimination of methanogen populations, the composition of the archaeal population changed markedly (Williams et al., 2009). The change in the composition of archaeal populations after vaccination was accompanied by a significant increase in the diversity of the methanogen populations, and the use of a broader-based vaccination approach may be most effective in eliminating methanogens (Williams et al., 2009; Leahy et al., 2010). Our understanding of the ruminal population and interactions among archaea, protozoa, bacteria, and the host eukaryote was further enhanced by the experimental vaccination against a microbial commensal organism. Furthermore, the ability to target microbes that are not directly pathogenic to the host animal offers opportunities and a template to reduce ruminal foodborne pathogenic bacterial populations commonly associated with ruminants (e.g., Salmonella sp. and enterohemorrhagic E. coli). Contributions of Denis Krause In writing this review, it is hard to complete without acknowledging the contributions of Denis Krause in a career sadly cut short. Most of his contributions were on the cutting edge of current microbial and host research theory and are still being examined as ecological hypotheses, and their long-term impacts are not yet fully realized. The majority of the research of Dr. Krause focused on the relationship between the microbiome and animal performance, especially in cattle undergoing subacute ruminal acidosis (SARA), and how diet affects the microbiome-host relationship (Gozho et al., 2005; Khafipour et al., 2011; Li et al., 2011; Plaizier et al., 2012). Further studies of Dr. Krause examined the effects of probiotics on the microbial population of the gut and of dietary protein on the microbiome (Bhandari et al., 2010; Krause et al., 2010). Although these research streams will be carried forward by others because of his pioneering work, Denis will be missed by our animal science community as well as the larger scientific community. Present and Future of Rumen Microbiology The relatively recent changes in DNA technology have dramatically changed our approaches to ecology in general and microbial ecology in particular. With the advent of whole-genome sequencing (Morrison et al., 2003; Morrison et al., 2010) and, recently, pyrosequencing (Roesch et al., 2007), researchers have access to more data on the composition of individual members of an ecosystem than ever before.. These, along with newly developed and recently affordable techniques (e.g., genome sequencing, pyrosequencing, proteomics, transcriptomics), have allowed the construction of a metagenome of the rumen microbial environment, as well as a holistic approach to functional analysis of an ecosystem (Ransom-Jones et al., 2012). The metagenomic-proteomic approach to examination of the ruminant gut as a source of industrial proteins (Brulc et al., 2009; Iakiviak et al., 2011) and provides a better understanding of the complexity of the levels of interaction within the microbial community of the gastrointestinal tract (Dowd et al., 2008; Callaway et al., 2010). For many years researchers have focused on the use of genetic engineering to create “superbugs” that could ferment a wide variety of substrates and improve ruminant efficiency. Information elucidated from the FibRumBa database and other sequencing of a variety of ruminal microorganisms has demonstrated that the rumen is filled with specialist organisms that are adapted to fill their ecological niche (Leahy et al., 2010; Morrison et al., 2010; Ransom-Jones et al., 2012). This new discovery underscores the difficulty, if not impossibility, for a generalist (or specifist) engineered superbug to successfully colonize the ruminal microbial ecosystem, suggesting that new approaches need to be used to improve ruminant efficiency, animal health, and food safety. Methods of manipulating the ruminal fermentation and microbial population (e.g., ionophores, antimicrobials, colicins and bacteriocins, diet change) can now be examined in detail to understand the impact they have on the microbial ecosystem and how those changes can be replicated to produce the greatest gains in efficiency. By studying the microbial ecology of the rumen via metagenomics, it may be possible to design specific probiotics and competitive exclusion cultures for use in cattle to mimic a “core” healthy microbial population (Tap et al., 2009; Turnbaugh et al., 2009) that can be established to optimize efficiency or to counteract stress (feed lot entry or freshening), diet changes, or exposure to novel environments (Ghareeb et al., 2008; Freestone and Lyte, 2010; Uyeno et al., 2010). Furthermore, this established gastrointestinal core microbial population could then be further tweaked by the use of specifically targeted prebiotics to combat illnesses or production and environmental stresses faced by cattle (Ghareeb et al., 2008; Uyeno et al., 2010; Bailey et al., 2011). Further developments in vaccines for methanogens or even AA-fermenting bacteria offer further hope in improving ruminant nutritional efficiency. CONCLUSION The history of rumen microbiology is rich with significant impact, in the understanding not only of the digestive physiology of the ruminant but also of the ecology of the other anaerobic ecosystems. Overall, the series of snapshots in the history of ruminal microbiology presented in this review indicates the leading role that this discipline of animal science has played in the exploration of other microbial environments and habitats. Although the rumen has served a test bed role for other microbial environments and technology development and validation, studies of this ecosystem have benefitted animal production directly (e.g., use of monensin, control of acidosis, reduction of liver abscesses). Recent developments in microbial ecology have again been adapted and validated in the rumen, providing the ability to understand the gastrointestinal consortium at a level never dreamed of in the times of Hungate and Bryant. The recent development of pyrosequencing can potentially elucidate the bacterial interactions with the host and allow us to finally harness the power of the microbial diversity to enhance animal health, productivity, and food safety. LITERATURE CITED Adams S. L. Hungate R. E. 1950. Continuous fermentation cycle times-prediction from growth analysis. Ind. Eng. Chem.  42: 1815– 1818. Google Scholar CrossRef Search ADS   Allison M. J. Bryant M. P. Doetsch R. N. 1961. 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TI - Board-invited review: Rumen microbiology: Leading the way in microbial ecology,
JF - Journal of Animal Science
DO - 10.2527/jas.2012-5567
DA - 2013-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/board-invited-review-rumen-microbiology-leading-the-way-in-microbial-07yhHFBXNb
SP - 331
EP - 341
VL - 91
IS - 1
DP - DeepDyve
ER -