Microbial Rumen Fermentation

Microbial Rumen Fermentation JAMES B. RUSSELL 1"4 and R O B E R T B. HESPELL 2'3 Departments of Animal Science and Dairy Science University of Illinois Urbana 61801

INTRODUCTION

With the ruminant animal, we have essentially two ecosystems, namely, the microbial ecosystem within the rumen and the animal's external environment. In dealing with the microbial ecosystem, we have made significant strides in the last few decades toward accomplishing the overall goal of ecology, which is to understand the relationships of organisms to their environments. As pointed out by Hungate (47), a complete ecological analysis of any natural habitat requires an elaboration of: 1 ) kinds and numbers of organisms, 2) activities of the organisms, and 3) extent to which their activities are expressed. Within the last 25 yr, much information has been gained in the first two aspects of the ecological analysis. Currently we are just beginning to address the third aspect. The rumen is an ideal fermentation site. In most ruminant species, the rumen is approximately one-seventh of the mass of the animal, is maintained at a relatively constant temperature (39°C), is buffered well by salivary secretions, and compared to many other microbial ecosystems is well supplied with nutrients. End-products of the fermentation (e.g., volatile fatty acids), which can be toxic to microbial metabolism, are removed across the rumen wail. The microflora inhabiting the rumen is dense and contains approximately 10 l° to 1011 bacterial and 106 protozoal cells per milliliter. Diversity within this population is extensive, and approximately 200 species of bacteria and 20 species of protozoa have been isolated (13). Although a few of these bacteria may be "casual passengers brought in with the food" and thus not be "authentic rumen bacteria", Received August 29, 1980. 1 Department of Animal Science. 2 Department of Dairy Science. 3 To whom reprint requests should be addressed. 4 Department of Animal Science, Cornell University, Ithaca, NY 14850. 1981 J Dairy Sci 64:1153-1169

the complexity of ruminal bacteria is great (13). During rumen fermentation, short chain fatty acids and microbial ceils are formed from feedstuffs, and these products serve as sources of energy and protein, respectively, to the animal. Methane, heat, and ammonia are evolved as well, and these products can represent a loss of energy and nitrogen for the animal. The efficiency of nutrient utilization by ruminants is determined largely by the balance of these fermentation products, and this balance ultimately is controlled by the types of microorganisms in the rumen. C U L T I V A T I O N OF R U M I N A L M I C R O O R G A N I S M S

The rumen environment is one of extreme anaerobiosis, and, as expected, inhabiting microorganisms are strict anaerobes sensitive to oxygen. It was not until development by Hungate (46) of techniques that prevented exposure to oxygen at all times that definitive studies on ruminal bacteria could be begun in earnest. Although basic concepts and manipulations remain the same, these anaerobic techniques have been modified (15, 40, 49) and must be employed in any meaningful studies involving ruminal microorganisms. Within the last decade, a major development in cultivating strict anaerobes has been the refinement and usage of plastic anaerobic gloveboxes and serum-capped vessels. These newer techniques have not supplanted but rather are used in conjunction with the Hungate techniques and allow one to do such manipulations as routine Petri dish plating or replica plating (41, 56) and cultivation of methanogens on a large scale (5). Beginning in the 1950's and extending through the 1960's, many successful studies were undertaken to develop appropriate cultivation media and to isolate, enumerate, and characterize various bacterial species. Through the perseverance of these researchers, primarily M. P. Bryant and his colleagues, we now know

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many (if not most) of the major bacterial species and have a reasonable understanding of their general functional roles in the rumen (Table 1 ). The majority of ruminal bacterial species can be grown on relatively simple media that includes one or more carbohydrates (e.g., cellulose, cellobiose, starch, xylan, glucose), ammonia and Trypticase, b-vitamins, heme, vitamin K derivatives, mineral salts, and a reducing agent such as sodium sulfide and L-cysteine. Many species also require (or their growth is stimulated by) short straight- or branch-chained volatile fatty acids and carbon dioxide. For nonselective isolation and enumeration, clarified rumen fluid often is included in the media as a source of nonspecific factors (19); however, rumen fluid can be replaced by other components (21). Within the last several years, media have been developed that have been selective for enumeration of carbohydrateutilizing subgroups (but not to the species level) from ruminal contents (3, 34, 56). Specific details on the nutrition and t a x o n o m y of ruminal bacteria have been discussed elsewhere (13, 26, 48). With respect to cultivation and studies with ruminal protozoa, microbiological effort has not been as successful as with bacteria, probably because of a combination of factors, including the inability of investigators to grow protozoa in the absence of bacteria or other protozoal species, that protozoa are not essential to the host animal, and the general scarcity of protozoologists competent in anaerobic techniques. Given these limitations, however, accomplishments have been significant since the early cultivation attempts by Margolin (62) and Hungate (45). There have been a number of studies, primarily by G. S. Coleman and his colleagues, in which various species of ciliate protozoa have been cultivated with bacteria in vitro for as long as 5 yr (28). Cultivation media have included a complex mixture of basal salts, clarified rumen fluid, and carbohydrate energy sources such as whole-grain flour, dried grass, rice starch, or washed bran. The use of these particulate carbohydrates along with inclusion of one or two antibiotics in the media allows for proliferation of protozoa without having overgrowth of bacteria. Because of the undefined nature of cultivation media and presence of bacteria, we do not have accurate Journal of Dairy Science Vol. 64, No. 6, 1981

information on nutritional requirements of ruminal protozoa. As shown by numerous studies (28, 29, 82), some protozoa prefer to use soluble carbohydrates whereas others engulf particulate carbohydrates. Engulfed bacterial cells can serve as the major nitrogen source for most species, but other nitrogenous sources such as particulate proteins, amino acids, peptides, and ammonia also are utilized to varying extent, depending upon the particular species. Bacterial cells also serve as a source of precursors for synthesis of protozoal nucleic acids (30 and references therein), and it is logical to assume they serve as sources of many other nutritional factors such as vitamins, minerals, etc. The ruminal protozoal population is predominated by ciliates, although a few species of flagellates can be present. Flagellates are often numerous in calves prior to development of the ciliate population. Shortly after feeding in the adult animal, large increases in the flagellate Neocallirnastix frontalis can take place (74). The ciliate ruminal protozoa are composed of 20 or so species that can be divided into two groups - the holotrichs and oligotrichs. The holotrichs possess a simple morphology, superficially resemble paramecia, and are members of the genera Isotricba or Dasytricha. In contrast, the oligotrichs are morphologically complex, with various bands of cilia, skeletal plates, and surface projections such as spines. Species of Entodinum, Epidinium, Diplodium, and Opbyroscolex are in varying numbers in the rumen. An extensive description of the ruminal protozoa and their morphologies may be found in Hungate's book (48). BIOCHEMICAL SIGNIFICANCES OF R U M I N A L M I C R O O R G A N I S M S

Overall Fermentation

Within the rumen, an intensive microbial degradation of foodstuffs takes place. Plants primarily are composed of carbohydrate polymers, and these are hydrolyzed to small saccharides that in turn are fermented to numerous products (Figure 1). Although numerous intermediates may be formed, the final fermentation products that accumulate within the rumen are acetate, propionate,

TABLE 1. Ruminat bacteria and their fermentative properties.

c

~a

Species

Animal diets a

Functionality b

Fermentation c products

Bacteroides succinogenes Ruminococcus albus Ruminococcus flaveJ?tciens Butyrivibrio fibrisolvens Clostridium lockbeadii Streptococcus bovis Bacteroides amylopbilus Bacteroides ruminicola Succinimonas amylolytica Selenomonas ruminantium Lacbnospira multiparus Succinivibrio dextrinosolvens Metbanobrevibacter ruminantium Metbanosarcina barkeri Spirochete species Megasphaera elsdenii Lactobacillus vitulinus Anaerovibrio lipolytica Eubacterium ruminantium Vibrio succinogenes

Many Many Many Many Coarse hay High grain High grain Many Forage-grain Many; grain Legume pasture High grain Many Many;molasses Many High grain Lush pasture; high grain Forage; high lipids Forage ...

C, A C, X C, X C, X, PR C, PR A, S, SS, PR A, P, PR A, X, P, PR A, D A, SS, GU, LU, PR P, PR, A P, D M, H M, H P, SS SS, LU SS L SS H

F, A, S, --C F, A, E, H, C F, A, S, H, --C F, A, L, B, E, H, C F, A, B, E, H, C L, A, F (?) F, A, S, --C F, A, P, S, - C A, S, - C A, L, P, H, C F, A, E, L, H, C F, A, L, S, - C M M, C F, A, L, S A, P, B, V, CP, H, C L A, P, S, - C F, A, B, C S

,<

alt is doubtful any one species is completely absent from any rumen, but given diets indicate where the organism is more numerous. < o ox

z

0

ox ",O

b c = cellulolytic, X = xylanolytic, A = amylolytic, D = dextrinolytic, P = pectinolytic, PR = proteolytic, L = lipolytic, M = methanogenic, GU = glycerol-utilizing, LU = lactate-utilizing, SS = major soluble sugar fermentor, H = hydrogen utilizer. CF = formate, A = acetate, E = ethanol, P = propionate, L = lactate, B = butyrate, S = succinate, V = valerate, CP = caproate, H = hydrogen, C = carbon dioxide.

Z

Z ,q > ~q ~z I

m > Z z < >

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RUSSELL AND HESPELL CARBOHYDRATE

POLYNERS

(CELLULOSE, ST RCH PECT N . . . . . . . . . . . . . ~. . . . .

I

~21 ~

S~;ALL SACCH~RIDES

(CELLOBIOSE,

LACT#TE H2 + C0~"

MALTOSE,

SUCROSE,

XYLOBIOSE, HE×OSES, PENTOSES)

~

BUTYRATE* CAPROATE

OXALOACETATE

CH 4 •

SUCCINATE

~

PROPIONATE"

FIG. 1. Generalized scheme for ruminal degradation and fermentation of carbohydrates. The products marked by an asterisk are those which represent terminal products and accumulate in the rumen.

butyrate, carbon dioxide, and methane. Ratios of these products vary with diet and frequency of feeding and are caused by changes in microbial metabolism and species. Under abnormal circumstances, such as either unusual feeding practices or host animal sickness, other products like formate, lactate, or ethanol may appear in the rumen. Proteins also are degraded in the rumen, and ammonia, carbon dioxide, and either short straight, branched-chain, or aromatic fatty acids are formed (Figure 2). Carbohydrate Metabolism

Degradation and fermentation of polysaccharides essentially can be conceived to occur in three general stages (Figure 1). The initial stage includes attachment of microorganisms to plant particles and disassociation of carbohydrate polymers from structural plant cell matrices. Relatively little definitive infor-

DIETARY AND OTHER PROTEINS

i i

POLYPEPTIDES

AMINO ACIDS +

I "CETATE ~[ "

SHORTPEPTIDES +

ISOBUT RATF ~ I ""

NH3 +

I--Im'~IETH YLBU TYR ATE "

~

I

I

]

~

CO2

MICROBIAL GROWTH

~--~--ISOVALERATE "-NH 3 + CO2

mation is known about this important process, but some recent studies indicate both bacteria and protozoa are involved (4, 24). The second stage, hydrolysis of released polymers to small saccharides, is catalyzed by numerous extracellular enzymes of which "cellulose complexes" are the predominating types. Here again, our knowledge is scanty. Since the studies of King and his colleagues (54), research on ruminal cellulases and other hydrolases has been relatively dormant. Part of this stagnation can be attributed to the complex number of organisms (Table 1) and enzymes (see 37, 78). The final stage, the intracellular fermentations of small saccharides, is relatively well understood, primarily because of the use of pure cultures in studies over the last two decades. Pure and mixed culture studies have established that the major biochemical pathway employed by ruminal bacteria for hexose degradation is the Embden-Meyerhof-Parnas (53, 111). For pentoses and deoxyhexoses, there "is less information available, but the most likely pathway is probably a combination of a pentose cycle plus glycolysis (106). Pectins, which are abundant in lush clovers and alfalfas, are degraded rapidly and fermented in the rumen. Several bacteria/ species are pectinolytic, and numerous other species can ferment the breakdown products (103). Although the extracellular degrading enzymes have been characterized partially in recent years (103, 117), the specific intracellular enzymes in fermentation of these uronic acids are not known. The major intracellular products formed from hexose or pentose degradation are pyruvate and phosphoenolpyruvate. The compounds are converted to an array of fermentation products (Table 1) by various pathways; however, some of these products (ethanol, succinate, and lactate) rarely accumulate in the rumen. The lack of some of these products in rumen fluid can be explained partially by the fact that the terminal fermentation product of one species may be catabolized further by other species. For example, in the rumen most of the propionate is derived from succinate (11), which is decarboxylated to propionate by organisms such as Selenol~lonas ruminantium (96). Another major factor regulating fermenta-

AMINO AC[DS

FIG. 2. Generalized scheme for rurninal degradation of proteins. Journal of Dairy Science Vol. 64, No. 6, 1981

RUMEN FERMENTATION -- 75TH ANNIVERSARY ISSUE tion products produced in vivo is interspecies hydrogen transfer. Within the rumen the partial pressure of hydrogen is low, but the turnover of hydrogen through this pool is high mainly because of its rapid utilization by methane bacteria (48). Because the partial pressure of hydrogen is extremely low, the formation of hydrogen gas from reduced pyridine nucleotides by non-methanogenic species is thermodynamically feasible (116). Thus, reduced pyridine nucleotides can be oxidized directly with production of hydrogen gas rather than by alternate means of oxidation - such as formation of lactate, propionate, succinate, or ethanol. These latter products are produced by pure cultures, because hydrogen gas accumulates when methanogens are absent. If methanogens are inhibited in vivo by low pH or chlorinated hydrocarbons, hydrogen accumulates and these more reduced fermentation products are formed. These concepts have been demonstrated amply by Wolin, Bryant, and their colleagues (55, 65,94, 96). Nitrogen and Protein Metabolism

Proteins can be degraded extensively and fermented in the rumen as shown by pathways in Figure 2. The extent of ruminal protein degradation can vary greatly, but studies directed toward definitive information on factors controlling proteolysis have not been performed yet. Most studies have dealt with preventing ruminal degradation and increasing the flow of dietary proteins to the omasum by chemical or physical treatments of dietary proteins and alteration of rumen functions (22, 23). However, considerable knowledge has accumulated in the last two decades concerning nitrogen sources that can be utilized for bacterial growth. Considerably less in known about nitrogen metabolism in ruminal protozoa; but since engulfed bacteria (and particulate protein?) can serve as a major nitrogen source for many species (29, 30, and references therein), proteolysis is an important aspect of their ecology. Pure culture studies on nutritional requirements of ruminal bacteria have shown ammonia to be a major nitrogen source for growth (20), and in vivo studies using lS N have confirmed these observations (63, 79). The ammonia is derived either from bacterial urease action on

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urea (dietary and transferred blood urea) or from microbial catabolism (Figure 2) of amino acids and peptides (1, 80, 81). This latter catabolism does not appear to produce sufficient energy for growth in the absence of carbohydrates of the various bacterial species having these catabolic abilities. The amino acid catabolizing enzymes in various species of ruminal bacteria have not been characterized biochemically. Although ammonia is a major source of nitrogen for bacterial growth, peptides and amino acids are also important, particularly with low quality diets (high fiber, low protein) where up to 40% of the bacterial nitrogen does not come from ammonia (72). As discussed recently (40) insufficiency of these compounds at certain times after feeding may be a major factor causing energetic uncoupling, resulting in continued production of fermentation products without concomitant bacterial growth. Direct evidence that this can take place in vivo was shown over 10 yr ago by studies of Hume et al. (44), which indicated that microbial cell yields in the rumen are proportional to dietary nitrogen. Finally, peptides and amino acids are needed as precursors to produce the branched-chain fatty acids that are growth factors for a number of bacterial species, particularly the cellulolytics (2). However, definitive studies have not been done to explore whether dietary supplementation of branched-chain fatty acids are efficacious in promoting ruminal digestion beyond what can be achieved by urea additions to poor quality diets (e.g., high forage, low protein) as would be encountered in range grazing or possibly used in future years when grains may not be as amply available. Ruminal Protozoa

Much of the foregoing discussion on biochemical aspects has centered on ruminal bacteria because of the preponderance of specific information that is available for them. Furthermore, for any one biochemical process in the rumen, such as production of a volatile fatty acid, it is difficult to partition quantitatively contributions by protozoa and bacteria. However, protozoa are less metabolically active on a cell mass basis than bacteria simply because of their larger cell volumes. Rumen Journal of Dairy Science Vol. 64, No. 6, 1981

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protozoa are fermentative anaerobes, and their fermentation products include acetate, butyrate, lactate, carbon dioxide, and hydrogen. Besides contributing to volatile fatty acid production, protozoa aid in sequestering carbohydrates from rapid bacterial attack by engulfment of starch grains and other particulate carbohydrates. Without this, a significant portion of the carbohydrates would be fermented rapidly to lactate, and a lower ruminal pH would result, both aspects of which are detrimental to overall rumen functions. A homologous situation may occur with particulate proteins, whereby engulfment would allow for extended proteolysis, slower release of products, and less catabolism of amino acids/ peptides to volatile fatty acids. However, evidence for this latter role is scanty at this time. Various interrelationships between the protozoa, diets, and the host animal are discussed more fully in a recent article by Coleman (29). D E T E R M I N A N T S OF R U M E N E C O L O G Y

Since many microbial species inhabit the rumen and have evolved together over millions of years, one would expect numerous interrelationships among them. Within microbial environments several types of ecological interrelationships are possible (67). If two species have no effect on one another, one can say that a state of "neutralism" exists. When the growth of one species is promoted by the presence of a second species, but the growth of the second is unaffected by the presence of the first, the relationship can be termed "commensat". In a "mutualistic" relationship the degree of interaction is even stronger, and the growth of both species is enhanced by the presence of the other. If two species are dependent on the same limiting nutrient, a state of " c o m p e t i t i o n " exists. When toxic products are produced, "amensal" relationships are possible, and if one species can consume another, "predation" can occur. Within the rumen ecosystem, there are examples of each of these types of relationships. A particular microbial species may be involved in several types of interactions at a given time. Realistic estimates of total microbial growth as well as relative numbers of individual species within the rumen is largely dependent on a Journal of Dairy Science Vol. 64, No. 6, 1981

quantitative understanding of microbial interrelationships. These interrelationships are determined ultimately by the nutrition, biochemistry, and physiology of individual rumen microbial species, and studies of their properties have given us further insight into the dynamics of rumen ecology. As will become evident in the discussion below, this insight is still qualitative when one considers the true in vivo situation. Maximum Growth Rates

When soluble nutrients are plentiful, an important determinant of relative microbial success is maximum growth rate. At such times, an organism with a higher maximum growth rate is able to grow faster than an organism with a lower maximum growth rate. When maximum growth rates were compared among several rumen bacteria, differences were large, and maximum growth rates were dependent on both energy source (85) and pH of incubation medium (91). Because bacteria grow exponentially, it is impossible for them to maintain high rates of growth for extended time. Streptococcus boris is able to achieve a doubling time of 14 min (86). At such a growth rate, one S. boris cell with a volume of approximately 1.2 times 10-13 cm 3 would be able to fill completely a 60-liter rumen in less than 14 h and would equal the mass of the earth in approximately 34 h! Thus, other factors also must limit bacterial growth in the rumen. Substrate Affinities and Preferences

During much of the feeding cycle soluble substrate concentrations are low in the rumen (48, 97). At low concentrations of substrate, increments of substrate will cause microbial growth rate to increase, and this pattern follows saturation kinetics typical of enzyme systems (69). The affinity constant, K s , is defined as the substrate concentration that will yield one-half maximum growth rate. Since affinity constants of most bacteria are low, affinity usually is determined with chemostat cultures. Recent studies have compared substrate affinities of rumen bacteria and showed that affinity for the same substrate can differ greatly among species and that a species can have higher affinities for some substrates than others (86). These

RUMEN FERMENTATION - 75TH ANNIVERSARY ISSUE data are consistent with some in vivo observations. F o r example, a strain of Butyrivibrio fibrisolvens had high substrate affinities for a variety of soluble carbohydrates, and this species has been observed to proliferate in the rumen on poor forage diets (13, 48). In studies with nonrumen bacteria, organisms can utilize preferentially some substrates to exclusion of others (59, 77, 83, 112). These preferencing mechanisms allow bacteria to select substrates that yield the highest growth rates and to minimize the synthesis of degradatire enzymes. When pure cultures of rumen bacteria were grown in batch culture with a mixture of fermentable carbohydrates, some substrates were not utilized until others were depleted, and the "more preferred" substrates inhibited utilization of "less preferred" substrates (85). Comparisons of organisms indicated that they had different patterns of substrate preference. Differences in substrate affinities and preference patterns suggest 1) rumen bacteria have evolved different strategies of growth and 2) these physiological factors may affect competition among rumen bacteria. Cell Yields and Maintenance

The early work of Bauchop and Elsden (8) indicated that yield of bacterial cells in grams per mole of ATP produced (YATP) was 10.5. However, theoretical calculations by Stouthamer (100, 101) indicated that YATP could be as high as 32.5 for growth of an organism in complex m e d i u m . The differences between theoretical and observed yields are presumably due to an organism's relative efficiency of ATP utilization for growth. Experimental determination of maximum YATP with several rumen bacteria have indicated that these organisms were efficient utilizers of ATP for growth. Indeed, Selenornonas rurninantiurn exhibited a maximum Y#.TP of approximately 28 (87), and as discussed recently (40), the rumen microbial population as a whole has a potential YATP of 22 to 26. With the advent of continuous culture techniques in the 1950's (70, 73), it became apparent that microbial cell yields were lower at slower growth rates (39). These observations suggested that energy was used for both growth-related functions and functions not

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directly related to growth (ion balance. protein turnover, etc.). Nongrowth functions have been termed maintenance energy expenditures and are analogous to maintenance energy of animals. The underlying principle of maintenance is that growth only can occur after this requirement is met. When growth rate is high, maintenance makes up a relatively small proportion of total energy utilization. However, when growth rates decrease, maintenance becomes more significant, and all of the energy is used for maintenance when growth ceases. Within the rumen, bacterial numbers are high, and average bacterial growth rate is low. Under such conditions one would expect cell yields to be lower than the maximum obtainable because of the increased involvement of maintenance in total energy utilization, and this concept is supported by data of Isaacson e t al. (51). These maintenance expenditures are reflected partly in heat of fermentation and represent a loss of microbial protein (but not necessarily a loss of volatile fatty acids) for the animal. Although the maintenance energy of the total rumen population is low as compared to other bacteria (51), maintenance for individual rumen bacteria can vary greatly (87). Butyrivibrio fibrisolvens and Sefenornonas ruminantiurn had low maintenance expenditures compared to other species. B. fibrisolvens and S. rurninantium often are found in high numbers in the rumens of animals fed poor quality diets (13, 48) where microbial growth rates would be expected to be slow. These results are consistent with the idea that organisms with low maintenance energies would be able to grow faster and dominate the population when substrate availability and growth rates are low in the rumen. Tolerance to Low pH

Although the rumen is buffered by bicarbonate, phosphate, and proteins, production of fermentation acids sometimes can exceed the buffering capacity, and pH can decline to the point where rumen function and, hence, animal p e r f o r m a n c e is decreased. Low rumen pH usually is associated with an accumulation of lactic acid in the rumen, and during this time Streptococcus boris and lactobacilli increase in numbers (50, 58). One of the factors responsible for this shift in the populaJournal of Dairy Science Vol. 64, No. 6, 1981

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tion appears to be tolerance of these organisms to low pH. When pure cultures of rumen bacteria were grown in chemostats, S. boris and Lactobacillus vitulinus were more tolerant to low pH than cellulolytic organisms or the lactate utilizer M. elsdenii (90). Furthermore, S. boris produces more lactic acid at high growth rates or at low pH values (89). Thus, the combined effect of inhibition of growth by low pH on many rumen bacteria along with continued lowering of rumen pH from lactate production by the acid-tolerant S. boris are major factors influencing onset of rumen acidosis (89, 91).

tion when they were cocultured with cellulolytic species (33). A simultaneous crossfeeding of carbon and nitrogen also can occur. For example, when B. ruminicola and R. albus were cocuhured on a medium containing cellulose and casein, the proteolytic B. ruminicola was able to provide ammonia and branched chain fatty acids essential to the growth of R. albus. In turn, cellulolytic R. albus provided the hexoses required by the noncellulolytic B ruminicola. Many rumen bacteria have specific vitamin requirements (13, 14, 16), and crossfeeding by cell lysis or excretion probably occurs to a large extent.

Cell Lysis

Most studies with rumen bacteria have dealt with growth of organisms, but considerable cell death and lysis occur in the rumen as evidenced by studies of nitrogen turnover (63, 72). Cell death in most cases results from lack of nutrient during part of the feeding cycle and causes a decrease in potential growth capacity when nutrients are available at other times. Pure culture studies with rumen bacteria generally have indicated that they are particularly susceptible to cell death and lysis. Rumen bacteria differ from each other in their abilities to maintain viability during times of nutrient starvation (56, 66). Since ruminal bacteria have limited survival abilities, death and lysis from nutrient starvation are factors controlling the total as well as species numbers of microorganisms in the rumen. Cross'feeding

In addition to crossfeeding of succinate and hydrogen, which was discussed earlier, other types of crossfeeding between microbes may occur in the rumen. When S. rurninanium was cocultured with B. succinogenes on a cellulose-containing medium, S. rurninantium was able to grow even though it was unable to degrade cellulose (96). Such findings suggest that there could be a considerable crossfeeding of cellulose degradation products in the rumen. Hemicellulose is degraded primarily by cellulolyric bacteria, but many noncellulolytic strains are abld to utilize the xylans that are released. Experiments by Dehority showed that B. ruminicola and L. rnultiparus were able to utilize the products of hemicellulose degradaJournal of Dairy Science Vol. 64, No. 6, 1981

Predation by Protozoa

Under most natural conditions, the rumen is inhabited by protozoa, and part of the bacterial population is eaten by protozoa. Most of our knowledge regarding protozoal engulfment of bacteria has been derived in the last 15 yr from studies by G. S. Coleman and coworkers (28, 29, 30, and references therein) using in vitro incubations with in vitro grown protozoa. In summary, and realising differences between protozoal species, rates of engulfment range from 130 to 21200 bacteria!~protozoan per hour at bacterial densities of 10 cells/ml. Intracellular digestion rates of bacteria range from 345 to 1200 bacteria/protozoan per hour. Ingestion of or availability of carbohydrates to protozoa may influence digestion, but other influencing factors have not been examined. Depending on engulfment rate and for a high protozoal concentration (106 cells/ml), anywhere from 2.4 to 45 g bacteria could be digested protozoally per day in a sheep's rumen. Recent studies using in vivo grown protozoal species have indicated that Entodinium and larger species are selective in their engulfment of bacteria, preferring mixed ruminal bacterial suspensions (30). Entodina species engulfed cellulolytic bacteria more rapidly than other ruminal or nonruminal bacterial species. Optimal engulfment occurred at pH 6, with drastic declines in rates above or below this. As pointed out by the authors, these recent findings may have important significances in vivo. Namely, previous in vitro estimates of bacterial engulfment and digestion may have been

RUMEN FERMENTATION -- 75TH ANNIVERSARY ISSUE too high. Additionally, selective engulfment of bacterial species in vivo could lead to altered ruminal fermentation patterns. Properties of the Feed

Bacteria possess transport systems capable of taking up low molecular weight soluble nutrients, but most feeds are composed of large, relatively insoluble, complex polymers (cellulose, hemicellulose, starch, pectin, proteins, etc.). Thus, the" chemical, physical, and structural properties of feedstuffs must be important factors affecting rumen degradation. Feedstuff polymers first must be degraded to lower molecular weight compounds by extracellular enzymes before they can be utilized by rumen bacteria. As extracellular enzymes of rumen bacteria must act on the surface of feed particles, and as smaller particles have a greater surface to mass ratio, particle size is an important affector of fermentation rate (48). Within the rumen, particle size can be determined by feed treatment (pelleting, grinding, chopping, cubing, flaking, etc.), rumination, and digestion itself. Although reduction of particle size would be expected to increase fermentation rate, small particles can pass unfermented from the tureen. If this passage rate exceeds the increase in fermentation rate, the overall extent of rumen fermentation can be reduced (110), and total animal digestion is then more dependent upon the degrees of postruminal enzymatic and fermentative digestion. Because intracellular enzymes come in contact with feedstuffs through interactions of water with feed, one would expect polymer solubility to affect rumen fermentation rates. Starch granules of most plants are inherently insoluble and as such are resistant to the hydrolases of bacteria (37). This insolubility is partially responsible for the rather slow fermentation rates and high passage rates of many native starches (76, 105, 109). Heat and chemical treatments have increased solubility and likewise increased both rate and magnitude of starch digestion in the rumen (75). Proteins also differ in solubility, and protein solubility is determined largely by the distribution of hydrophobic and hydrophilic amino acids on the periphery of the protein molecule. All proteins whose tertiary structures are known have many hydrophilic residues at the surface, and hydrophobic residues usually are

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buried in the interior of the tertiary structure (113). This arrangement increases solubility, and more soluble proteins in some cases have been degraded at a faster rate by rumen bacteria. Denaturation by heat causes unfolding of tertiary structure, greater exposure of hydrophobic amino acids to the surface, and decrease in solubility. Heat treatment of feedstuffs can cause a reduction of protein fermentation in vivo (25). However, ovalbumin is a soluble protein; y e t it is resistant to proteinases (61). In this case, resistance appears to be from the cyclical structure of the protein. Recent work by Mahadevan et al. also has indicated that disulfide bridges within a protein render it more resistant to hydrolysis by rumen bacterial proteinases (60). Soluble and insoluble soybean proteins were degraded at nearly equal rates, and such results suggest that factors other than solubility must influence fermentation of proteins within the rumen. Cellulose and hemicellulose fractions of feeds are relatively insoluble and are degraded slowly within the rumen (33, 107). However, structural factors also influence their fermentation rates. For example, within forage fibers, lignin is in close association with cellulosic materials, and it appears to present a physical barrier to rumen bacterial cellulases. Delignification of fiber fractions by various chemical treatments has increased the extent of cellulose digestion in vitro and in vivo (108). Crystallinity also appears to affect rate and types of rumen bacteria digesting cellulose fibers. Cotton fibers have a highly crystalline structure and are more resistant to certain rumen bacterial cellulases, t3. succinogenes is able to degrade cotton fibers at a rapid rate, but the rate of cotton fiber digestion by R. albus and R. flavefaciens is much slower (14). The significance of crystallinity to in vivo forage digestion has not been elucidated (107), but more crystalline types of cellulose may select for

B. succinogenes. Hemicellulose (xylan) is a complex polymer, and its chemical and physical properties have been studied in less detail. However, the amount of hemicetlulose that could be digested by R. flavefaciens and R. albus differed markedly with the type of plant from which it was isolated (33, 107). From these results it is reasonable to assume that the arrangement of Journal of Dairy Science Vol. 64, No. 6, 1981

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various constituents within hemicellulose can affect its rate of digestion. As mentioned earlier, hemicellulose degradation involves a symbiotic relationship between cellulolytic species that are able to degrade the molecule and noncellulolytic bacteria that utilize the breakdown products. Thus, greater hemicellulose degradation can result from combined growth of cellulolytic and noncellulolytic strains than growth of cellulolytic strains alone (33). The actual degradation of intact feedstuffs by rumen microorganisms in vivo is complex, but experiments suggest that chemical and physical properties of feeds are not only important determinants of rate of fermentation but also types of microorganisms. Yet, a relatively clear understanding of how any one plant polymer is degraded and by which organisms is simply not known. The dynamics of rumen ecology will not be understood until chemical and physical properties of feedstuffs are elaborated in more detail. Rumen Dilution Rates

Because feed usually enters the rumen at regular intervals and because digesta and microorganisms are washed out of the rumen to the lower gut, the rumen in many ways operates as a continuous culture device. However, there are major differences between kinetics of a laboratory chemostat and the rumen. These differences sometimes have been ignored by ruminant nutritionists. Dilution rates in most laboratory chemostats are controlled by addition of nutrient medium. These nutrients are usually in a soluble, readily utilized form, and a single nutrient limits growth across all dilutions. In this case, if dilution rate is less than the maximum growth rate of the organisms, the growth rate of the culture is proportional to dilution rate, because increasing dilution rates are associated with an increased delivery of the limiting nutrient. Since soluble nutrients are provided, the system operates under homogeneous, steady state conditions, and all of the organisms in the culture vessel are subjected to the same chances of growth and washout. In the rumen by contrast, most nutrients are fed in an insoluble form, nutrient addition is discontinuous (meals) rather than continuous, and various nutrients can limit growth Journal of Dairy Science Vol. 64, No. 6, 1981

during different phases of the feeding cycle. Because there are both insoluble (feed particles) and soluble fractions, the rumen does not operate as a homogeneous system, and there are at least two major dilution rates - that of the solids and that of the liquids. Feed particles turn over at a slower rate (two to four times) than the liquid fraction, and rumen microorganisms can be associated with both the feed particles and the liquid (48). As such, rumen microbes can be subjected to different conditions of growth and washout depending on their position in the rumen. Thus, measurements of solid or liquid dilution rates alone cannot quantitate microbial cell yields. Moreover, even if both rates are determined, this gives a net microbial growth rate less than the true growth rate because of factors such as cell lysis. Inclusion of mineral salts in ruminant diets will increase rumen liquid dilution rate, and increased dilution rates have altered fermentation products (84 and references therein). The most marked change in this fermentation shift appears to be reduction in the molar quantity of propionic acid. These fermentation shifts also suggest that different microorganisms (both bacteria and protozoa) may be selected at different dilution rates within the rumen. Microscopic examination of animals supplemented with mineral salts have shown that higher liquid dilution rates were associated with increased numbers of cocci (104). Since bacterial growth yields are greater at higher growth rates (maintenance energy makes up a smaller proportion of total energy utilization), there has been an interest in increasing delivery of microbial protein to the lower gut by increasing tureen dilution rates. In some cases increased rumen dilution rates have been associated with an increased availability of microbial protein to the animal (27, 38). However, it is difficult to say whether true microbial growth rates actually were increased. Numerous other possibilities exist, such as the increased microbial protein was from washing microorganisms out of the rumen before cell death and lysis or a reduction in protozoal predation of the bacteria. SOME FUTURE DIRECTIONS

The bulk of our current knowledge on the microbial ruminal fermentation has been gain-

RUMEN FERMENTATION -- 75TH ANNIVERSARY ISSUE ed within the last 25 yr. Although we have made great achievements, our overall ecological picture is still cloudy. Elucidation and integration of the myriad interactions and their specifics between bacteria, protozoa, dietary materials, and the host animal will give a focused, detailed picture of the dynamic ruminal fermentation and ecosystem, but this has not been accomplished yet. It is in this context that the goals of future research must be set to gain the knowledge needed to predict accurately the dynamic changes in ruminal fermentations under various dietary conditions. This will allow for fruitful manipulations of the entire process. Based on our current status, some possible directions for future research are offered below. Sampling/Enumeration

One aspect of many nutritional and microbiological studies that limits interpretation of data has been the lack of good sampling techniques or adequate descriptions of the sampling methods used. The rumen ecosystem is dynamic, but it is not completely homogenous as exemplified by recent studies on ruminal ammonia (115) that show distinct differences in concentration with tureen location, time, diet, and method of analysis. As suggested by the classical studies of Smith et al. (97) similar differences probably exist for many, if not all, other biological components of the rumen ecosystem. Future nutritional and microbiological studies must begin to give considerably more attention to these differences. Further documentation that different diets yield different degrees of ruminal digestion, volatile acid production, or microbial cell yields is of relatively small value. This is particularly true if each aspect is determined in separate studies with different animals and dietary materials. The important point is to determine precisely why these differences occur, and this can be approached only if a more comprehensive picture is obtained by measurement of numerous parameters of rumen fermentation under the same given set of dietary conditions. In microbiological studies dealing with mixed ruminal populations, times of animal feeding and sampling can be and should be controlled. The ruminal site of sampling must be defined and the entry means (stomach tube,

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fistula, cannual, e t c . ) i n d i c a t e d . Future studies must be directed towards a comprehensive microbial analysis that should include as many of the following aspects as possible (or as applicable). First, delineation of not only the bacteria but also the protozoa and their enumeration by speciation is necessary. The microbial population that is obtained should be separated with respect to whether it is "free in the liquid" or "attached" to digesta particles. Each of these subpopulations then could be separated further by appropriate plating techniques. Other measurements of the digesta sample that should be made include pH, types and amounts of fermentation acids, soluble nutrient content (e.g., ammonia, soluble sugars, peptides, and amino acids, etc.), and complete (e.g., Van Soest-type) analysis of digesta solids. Finally, but importantly, rumen digesta samples should be taken over numerous times to obtain diurnal changes or patterns. Presently, the technical knowledge needed to make many of the foregoing quantitative microbial measurements is limited. However, a few initial steps have been made. Some media and techniques have been developed for separating mixed ruminal populations into carbohydrateutilizing subgroups (3, 34, 57), and some fractionation of attached and free bacteria has been accomplished (e.g., see 68). Although much has to be developed, the goals are not unsurmountable. Culture Techniques

In vitro incubations of rumen microorganisms offer means of examining experimental variables under more precisely controlled and assayable conditions than are possible in vivo. However, one must be careful in interpreting results of such in vitro experiments. When rumen inocula are used, extrapolation of the data to in vivo situations only can be made with confidence if both the resulting population of microorganisms and fermentation products approximate those of the rumen. Simple batch culture techniques with high concentrations of soluble carbohydrate can select for a lactate fermentation in as little as 3 h (88) and is caused by the high maximum growth rate in S. boris in comparison to other rumen microorganisms. Selections of other species also may occur if other substrates are used. Thus, it seems that such simple batch Journal of Dairy Science Vol. 64, No. 6, 1981

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incubations are likely to examine the effect of experimental variables on selected organisms rather than the total rumen microflora. However, less selection may occur with short term limited-fed-batch systems that appear to produce more normal fermentation products and maintain the diversity of the rumen microb i a l population (88). In these systems small doses of carbohydrate can be fed throughout the incubation period, and microbial growth rates can be controlled. Since soluble carbohydrates and microbial growth rates are kept low, there is less selection of microbial types during the incubation, and it seems that limited-fed-batch incubations are more apt to mimic ruminal conditions. Conventional chemostats offer an ideal method of examining interactions between a limited number of microbial species. Coculture incubations in chemostats, as discussed earlier, already have been used to demonstrate interspecies transfer of hydrogen and several other examples of crossfeeding. Cocontinuous cultures also have demonstrated that relative bacterial numbers might be predicted from the physiological characteristics of organisms, and it seems likely that future chemostat experiments will allow rumen microbiologists to build a more accurate model of relative microbial growth in the rumen. Although conventional chemostat methods are helpful in elaborating the nutrition, biochemistry, and physiology of individual species, they are usually not suitable for incubations to simulate the total mixed ruminal population. Since one nutrient (usually in a soluble form) must limit growth in chemostats, organisms that are best suited (high affinity, lower maintenance energy, etc.) to the utilization of this nutrient will be selected. Modified continuous culture devices employing ground feed with liquid and solid phases have been able to maintain a diversity of microbial types (32, 43), but considerably more work has to be done with them to validate this point. However, these latter chemostats may offer the means to understand microbial degradation of insoluble dietary substrates. Nitrogen-Ammonia Assimilation

Ammonia is a major source of nitrogen for ruminal microbial growth, yet little definitive Journal of Dairy Science Vol. 64, No. 6, 1981

information is available on interactions between growth rate, cell yields, ammonia assimilating enzymes, and environmental ammonia and other nitrogenous compounds. Affinities of ruminal bacteria] species for ammonia range from 5 to 45 /~molar (93) suggesting ammonia uptake cannot occur by passive diffusion, and it seems likely that specific transport mechanisms are involved. However, nothing is known about ammonia transport mechanism in any ruminal bacterial or protozoal species. Research is needed to ascertain whether these mechanisms require energy and what factors regulate their activity. Once within the cell ammonia can be assimilated (or fixed) by either the non-ATP requiring glutamate dehydrogenase or by the ATP-requiring glutamine-synthetase pathway, and both pathways were found in Selemonas ruminantiurn (98). Whether similar or other pathways exist in other ruminal bacteria and which pathways are used in vivo is totally unclear. Expression of glutamine synthetase is needed for expression of urease activity (99). Thus, in the rumen (and most likely in the cecum and large intestine) an interplay of ammonia assimilation pathways probably takes place at various times after feeding and with various dietary changes, but the nature of these is not known. Protein Degradation

Although it is well documented (42) that a large fraction of dietary protein is degraded by rumen microorganisms to ammonia and excessive ammonia accumulation in the rumen can cause a decrease in the nitrogen retention of the animal (92), mechanisms of rumen proteolysis have not been examined in detail. Early work demonstrated that a variety of rumen bacteria were able to degrade casein (10, 18), but few actively proteolytic bacteria have been identified (9, 10). These findings generally have been interpreted as meaning that proteolysis in the rumen is accomplished by the combined action of many species, but the possibility exists that shifts in the types of rumen microorganisms also could influence the rate of proteolysis in vivo. Studies with nonrumen bacteria have shown that extracellular proteinases are subject to induction (12), repression (35, 64), and catabolite repression (114). None of these mechanisms has been demonstrated in

RUMEN FERMENTATION -- 75TH ANNIVERSARY ISSUE rumen bacteria, but their existence might allow proteolysis to he manipulated by energy status or chemical means. The process of amino acid deamination also has received little attention. Some studies have noted amino acid utilization by several species of rumen bacteria (95), but it is not clear whether this utilization reflected actual deamination or a simple uptake for protein synthesis. When various species of ruminal bacteria were grown in glucose-limited chemostats with ammonia and high Trypticase, the only species that caused a net increase in the steady state of ammonia was M. elsdenii (89). These results suggest that growth conditions can influence amino acid deamination. Maintenance Energy

Many studies with bacteria have indicated that maintenance is not a constant and varies with growth conditions (71, 101, 102). For example, when Klebsiella aerogenes was grown in a chemostat at .1 h -1 under carbon, nitrogen, sulfate, and phosphate limitations, the relative yields were 1.00, .40, .33, and .25, respectively (71). When growth is limited by nutrients other than carbon, bacterial cells will uncouple growth and continue to metabolize carbon in the absence of growth. The uncoupling of growth and energy utilization in rumen bacteria has not been studied (40). Considering that many rumen bacteria have specific requirements (branch chain volatile fatty acids, vitamins, amino acids, micro-minerals, etc.), bacterial growth in the rumen could be limited by nutrients other than carbon during a portion of the feeding cycle. If rumen bacteria uncouple growth like other bacteria, such nutrient limitations could have marked effects on the efficiency of synthesis of microbial protein in vivo. Computer Techniques

Fermentation of feedstuffs within the rumen involves numerous complex interactions of the nutritional, biochemical, and physiological characteristics of the organisms, of the chemical and physical properties of the feed, and of host animal factors such as rumination and dilution rates. Simple descriptions of the system under one set of feeding conditions are not extrapolated readily to other situations. This complexity has confounded prediction of animal performance from diets fed. One approach to

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understanding the system is to feed experimentally all combinations of feedstuffs under different levels of production. However, this approach is impractical because there are infinite numbers of these combinations. Another approach is to develop a quantitative understanding of the basic variables that affect rumen fermentations, and this latter approach seems more promising and approachable. The development of computer technology over the last two decades had made it possible to quantitate and integrate simultaneously a large number of variables, and this technology has been adapted readily in the chemical and physical sciences. Baldwin and his colleagues have applied this technology to digestion in the rumen and have constructed several dynamic models of the processes (6, 7, and references therein). Further development of such computer models offers the possibility of predicting animal performance from feedstuffs as well as expanding our understanding of the rumen system in its entirety. Chemical Agents

As discussed recently by Chalupa (23), rumen fermentations can be modified by chemical additives. Amichloral, monensin, and diphenyliodonium chloride are the chemical agents that have been examined most thoroughly, but we are far from having a clear understanding of their effect on specific ruminal microorganisms or on the true dynamics of rumen fermentation. With most of the chemicals the accumulated data is not sufficient to provide an accurate explanation of the observed effect on animal performance. Future research must be shifted from the current descriptive approach to one of an analytical nature. Such a shift would result in new, more logical approaches to predictable modification of the rumen fermentation. Interactions of Scientists

Regardless of the specified biological process being examined, individual scientists must take on an increasingly more holistic outlook in their research. This stems directly from the tremendous increase of scientific knowledge over the last few decades that has brought into focus how little we really know and how complex biological processes are. However, a holistic approach will require individual scientists to Journal of Dairy Science Vol. 64, No. 6, 1981

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develop a greater interaction, understanding, and even expertise in one or more additional disciplines. For the rumen, there must be high degrees of interactions between nutritionists and microbiologists, and both scientists must have some relative competence in each other's field. Both also must begin to view ruminant digestion from broader standpoints - namely, from a comparative viewpoint encompassing other species (horse, human, swine, etc.) and, more importantly, the entire ruminant gastrointestinal tract. There are large indigenous microbial populations in the cecum and large intestine and numerically significant populations in the small intestine, yet our knowledge of these fermentations in ruminants is virtually nonexistent. The potentials of these ecosystems must be examined, particularly if concepts such as ruminal bypass of dietary materials are to be exploited fully.

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1 Allison, M. J. 1970. Nitrogen metabolism in r u m e n microorganisms. In Physiology of digestion and metabolism in the ruminant. A. T. Phillipson, ed. Oriel, Newcastle upon Tyne, England. 2 Allison, M. J., M. P. Bryant, and R. N. Doetsch. 1958. A volatile fatty acid growth requirement for cellulolytic cocci of the bovine rumen. Science 128:474. 3 Allison, M. J., I. M. Robinson, J. A. Bucklin, and G. D. Booth. 1979. Comparison of bacterial populations of the pig cecum and colon based upon enumeration with specific energy sources. Appl. Environ. Microbiol. 37:1142. 4 Amos, H. E., and D. E. Akin. 1979. R u m e n protozoal degradation of structurally intact forage tissues. Appl. Environ. Microbiol. 36:513. 5 Balch, E. E., G. E. Fox, L. J. Magrum, C. R. Woese, and R. S. Wolfe. 1979. Methanogens: Reevaluation of a unique biological group. Microbiol. Rev. 43:260. 6 Baldwin, R. L., and L. J. Koong. 1977. A dynamic model of r u m i n a n t digestion for evaluation of factors affecting nutritive value. Agric. Syst. 2: 255. 7 Baldwin, R. L., L. J. Koong, and M. J. Ulyatt. 1977. The formation and utilization of fermentation end-products: mathematical models. In Microbial ecology of the gut R.T.J. Clark and T. Bauchop, ed. Academic Press, New York, NY. 8 Bauchop, T., and S. R. Elsden. 1960. The growth of microorganisms in relation to their energy supply. J. Gen. Microbiol. 23:457. 9 Blackburn, T. H. 1968. Protease production by Bacteroides amylopbilus strain H18. J. Gen. Microbiol. 53:27. 10 Blackburn, T. H., and P. N. Hobson. 1962.

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Journal of Dairy Science Vol. 64, No. 6, 1981