Uma aula sobre os processos fermentativos no rúmen
(Parte 1 de 5)
Microbial Rumen Fermentation
JAMES B. RUSSELL 1"4 and ROBERT B. HESPELL 2'3
Departments of Animal Science and Dairy Science
University of Illinois Urbana 61801
With the ruminant animal, we have essen- tially two ecosystems, namely, the microbial ecosystem within the rumen and the animal's external environment. In dealing with the mi- crobial ecosystem, we have made significant strides in the last few decades toward accom- plishing 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 approxi- mately one-seventh of the mass of the animal, is maintained at a relatively constant tempera- ture (39°C), is buffered well by salivary secre- tions, 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 Univer- sity, Ithaca, NY 14850.
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 repre- sent 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.
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 manip- ulations 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 culti- vation 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
1981 J Dairy Sci 64:1153-1169 1153
1154 RUSSELL AND HESPELL 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 in- cludes one or more carbohydrates (e.g., cellu- lose, cellobiose, starch, xylan, glucose), ammo- nia 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 stimu- lated 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 carbohydrate- utilizing subgroups (but not to the species level) from ruminal contents (3, 34, 56). Specific details on the nutrition and taxonomy of ruminal bacteria have been discussed else- where (13, 26, 48).
With respect to cultivation and studies with ruminal protozoa, microbiological effort has not been as successful as with bacteria, proba- bly 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 essen- tial 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 parti- cular 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 pre- dominated 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).
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.
Fermentation c Species Animal diets a Functionality b products
Bacteroides succinogenes Many C, A F, A, S, --C Ruminococcus albus Many C, X F, A, E, H, C Ruminococcus flaveJ?tciens Many C, X F, A, S, H, --C Butyrivibrio fibrisolvens Many C, X, PR F, A, L, B, E, H, C Clostridium lockbeadii Coarse hay C, PR F, A, B, E, H, C Streptococcus bovis High grain A, S, S, PR L, A, F (?) Bacteroides amylopbilus High grain A, P, PR F, A, S, --C Bacteroides ruminicola Many A, X, P, PR F, A, P, S, -C Succinimonas amylolytica Forage-grain A, D A, S, -C Selenomonas ruminantium Many; grain A, S, GU, LU, PR A, L, P, H, C Lacbnospira multiparus Legume pasture P, PR, A F, A, E, L, H, C Succinivibrio dextrinosolvens High grain P, D F, A, L, S, -C Metbanobrevibacter ruminantium Many M, H M Metbanosarcina barkeri Many;molasses M, H M, C Spirochete species Many P, S F, A, L, S Megasphaera elsdenii High grain S, LU A, P, B, V, CP, H, C Lactobacillus vitulinus Lush pasture; high grain S L Anaerovibrio lipolytica Forage; high lipids L A, P, S, -C
|Vibrio succinogenes||H S|
Eubacterium ruminantium Forage S F, A, B, C
~z I m >
< o ox ox alt is doubtful any one species is completely absent from any rumen, but given diets indicate where the organism is more numerous.
bc = cellulolytic, X = xylanolytic, A = amylolytic, D = dextrinolytic, P = pectinolytic, PR = proteolytic, L = lipolytic, M = methanogenic, GU = glycerol-utilizing, LU = lactate-utilizing, S = 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.
1156 RUSSELL AND HESPELL POLYNERS ~21 ~
LACT#TE CAPROATE H 2 + C0~" OXALOACETATE
FIG. 1. Generalized scheme for ruminal degrada- tion 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 micro- bial metabolism and species. Under abnormal circumstances, such as either unusual feeding practices or host animal sickness, other prod- ucts 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).
Degradation and fermentation of poly- saccharides essentially can be conceived to occur in three general stages (Figure 1). The initial stage includes attachment of microor- ganisms to plant particles and disassociation of carbohydrate polymers from structural plant cell matrices. Relatively little definitive infor- i POLYPEPTIDES i AMINO ACIDS + SHORT PEPTIDES + NH 3 + CO 2
CETATE ISOBUT RATF "" I"~[ " ~ I MICROBIAL GROWTH I --Im'~IE TH YLBU TYR ATE " ~ I
"-NH 3 + CO 2 ~ AMINO AC[DS
FIG. 2. Generalized scheme for rurninal degrada- tion of proteins.
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 extra- cellular enzymes of which "cellulose com- plexes" are the predominating types. Here again, our knowledge is scanty. Since the studies of King and his colleagues (54), re- search on ruminal cellulases and other hydro- lases 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 estab- lished that the major biochemical pathway employed by ruminal bacteria for hexose degradation is the Embden-Meyerhof-Parnas (53, 1). 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 pectino- lytic, 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 pyru- vate 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 (1), which is decarboxylated to propionate by organisms such as Selenol~lonas ruminan- tium (96).
Another major factor regulating fermenta-
Journal of Dairy Science Vol. 64, No. 6, 1981
RUMEN FERMENTATION -- 75TH ANNIVERSARY ISSUE 1157 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 hydro- gen gas accumulates when methanogens are absent. If methanogens are inhibited in vivo by low pH or chlorinated hydrocarbons, hydrogen accumulates and these more reduced fermenta- tion products are formed. These concepts have been demonstrated amply by Wolin, Bryant, and their colleagues (5, 65,94, 96).
Nitrogen and Protein Metabolism
(Parte 1 de 5)