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Department of Dairy and Animal Science, Pennsylvania State University, University Park, PA 16802
ABSTRACT
INTRODUCTION
FIBROLYTIC MICROORGANISMS
MICROBIAL ADHESION AND HYDROLYSIS
GENETIC MANIPULATION OF
MICROBIAL SPECIES
ANIMAL FACTORS AFFECTING MICROBIAL FIBER DIGESTION
ENHANCEMENT OF FIBER DIGESTION: OPPORTUNITIES
FOOTNOTES
LITERATURE CITED
ABSTRACT 
The ruminal microbial populations attack, degrade and ferment structural carbohydrates in forage cell walls and thereby provide volatile fatty acids and protein to the host animal. Microbial colonization of fiber is quite rapid; however, the rate and extent to which fiber is degraded is determined to a considerable degree by factors such as microbial accessibility to substrate, physical and chemical nature of the forage and kinetics of ruminal digestion. The physical and chemical nature of forages can present a barrier to their complete digestion in the rumen, especially the association of lignin with polysaccharide constituents. Adhesin proteins allow bacteria with cell-bound enzymes to come into intimate contact with their substrates, ensuring that the degradation products are preferentially available. Research on various fibrolytic enzymes and cellulose binding domains may allow for the transfer of novel genetic material to bacteria for enhancing the hydrolysis of plant cell walls. Fungi may also play an important synergistic role in the ruminal digestion of forages by physically disrupting the lignified stem tissue. This allows the ruminal microbes greater access to the plant stem and the digestible portions of the plant. New developments in fiber utilization by ruminants are currently under investigation and include genetic manipulation of ruminal bacteria, chemical and biological treatments of forages, and manipulation of dietary inputs and feeding management.
KEY WORDS: ruminants · fiber · digestionINTRODUCTION
Ruminants have adapted to a variety of ecological niches because of diverse ruminal microbial populations, which consist primarily of bacteria, protozoa and fungi. Ruminant animals have the ability to convert low quality feeds into high quality protein and to utilize feeds from land not suitable to grow crops for human consumption. This is made possible by the ruminal microorganisms that synthesize and secrete the 
1-4 cellulase enzyme complex, thereby allowing hydrolysis of plant cell walls. However, the actual conversion of feeds, especially fibrous forages, to meat and milk is not very efficient. Only 10-35% of energy intake is captured as net energy because 20-70% of cellulose may not be digested by the animal. If a greater percentage of the total dietary energy from forages was available to ruminants, lower cost diets could be formulated and environmental resources would be used more efficiently.
Four major factors regulate ruminant fiber digestion: 1) plant structure and composition, which regulate bacterial access to nutrients; 2) nature of the population densities of the predominant fiber-digesting microorganisms; 3) microbial factors that control adhesion and hydrolysis by complexes of hydrolytic enzymes of the adherent microbial populations; and 4) animal factors that increase the availability of nutrients through mastication, salivation and digesta kinetics (Cheng et al. 1991
). The effect of plant structure has been discussed by Buxton and Redfearn (1997) in this symposium. This review will focus on limitations of the fibrolytic microorganisms in converting fiber to usable end products and the effect of animal and dietary factors on digestion of fiber.
FIBROLYTIC MICROORGANISMS
). Butyrivibrio fibrisolvens also produces a cellulase, but it is probably more important in the hydrolysis of hemicellulose. Fibrolytic bacteria tend to degrade the more readily digestible structures such as the mesophyll cells, although F. succinogenes digests parenchyma bundle sheaths, epidermal cell walls and leaf sclerenchyma (Akin 1989). F. succinogenes interacts synergistically with non-cellulolytic bacteria during forage digestion. Examples of these synergistic actions include a twofold increase in the utilization of orchard grass hemicellulose and pectin by co-culture with the hemicellulolytic bacterium Prevotella ruminocola over that by F. succinogenes alone (Osborne and Dehority 1989).
Fungi.
Fungi account for approximately 8% of the microbial biomass in the rumen (Orpin and Joblin 1988). The fungi seem to have an important role in fiber digestion because they are able to penetrate both cuticle and cell wall of lignified tissues (Akin 1986). This suggests that fungi have cutinase activity (Kolattukudy 1984). In addition, fungi can degrade more recalcitrant cell wall materials, including the sclerenchyma and vascular tissue (Akin 1989). Fungi degraded 37-50% of barley straw, whereas rumen bacteria digested only 14-25% (Joblin et al. 1989). The fibrolytic activity of the fungi, which includes both cellulase and hemicellulase activities, is enhanced by hydrogen-utilizing methanogens (Joblin et al. 1989), which decrease the repressive effect of hydrogen (Orpin and Joblin 1988). The fact that fungi do not predominate in the rumen is primarily a function of their slower generation time in comparison with bacteria (6-9 vs. 0.5-3.5 h, respectively). In addition, fungal growth is repressed by culture with some bacteria, such as Ruminococcus spp. Research to elucidate the interactions of the bacteria and the fungi may result in the development of methods to circumvent this growth repression.
Protozoa.
In vitro studies have suggested that 19-28% of total cellulase activity can be attributed to protozoa (Gijzen et al. 1988). However, digestion seems to be limited to very susceptible tissue such as mesophyll cells (Akin 1989). Studies have demonstrated that defaunation reduces fiber digestion (Bonhomme 1990, Yang and Varga 1993). However, in the absence of protozoa there is an increased requirement for non-protein nitrogen because of an increase in the bacterial population. A shortage of nitrogen may therefore account, at least in part, for this reduction in fiber digestion (Ushida and Jouany 1990). Because the difficulty of culturing protozoa free of bacteria, this microbial group has been ascribed a minor role in the digestion of fiber.
MICROBIAL ADHESION AND HYDROLYSIS
). Bacteria usually gain access to readily digestible inner tissues through stomata, lenticels or damaged areas, and digestion essentially proceeds from the inside out. Ruminal fungi also degrade the more vulnerable areas of plant tissue, but in addition have the ability to penetrate the plant cuticle. This aids in reducing the tensile strength of the tissue and provides additional sites for bacteria to access and attach (Akin 1986). Ho et al. (1988) described the production of appresorium-like structures where rumen fungal rhizoids come into contact with undamaged rigid cell walls. The appresorium can produce a fine penetration peg at the site of cell wall contact, which penetrates and grows inside the plant cell, thereby forming normal rhizoids.
). Several researchers have demonstrated that attachment of ruminal microorganisms to their substrate is a prerequisite for the digestion of forage particles in the rumen. The degree of colonization and mode of attack are specific to each microbial species (Kudo et al. 1990). Cellulolytic bacteria adhere to substrates by means of an extracellular glycocalyx coat and possibly by protuberances called cellulosomes. Of considerable interest is the fact that a large fraction (10-50%) of cells within a tested population for each strain of cellulolytic bacteria do not adhere to feed particles, even upon prolonged incubation (Weimer 1993). This may be due to differences in the ages of individual cells or to some subtle genotypic variations within an apparently homogenous microbial population. Information regarding the activity and composition of the cellulase complex of these nonadherent populations could be useful for genetic selection and transfer into other species.
, White et al. 1993). The anaerobic fungi also possess a broad range of fibrolytic enzymes, including cellulases and xylanases. Neocallimastix frontalis has the highest cellulolytic activity of any organism ever reported in the literature. Borneman et al. (1990) demonstrated the presence of both ferulic and coumaric acid esterase activities in two monocentric and three polycentric fungi. The protozoa also possess cellulases, xylanases and a broad range of glycosidases, although none have been purified (Cheng et al. 1991).
Microbial interactions.
Although cells of F. succinogenes alone are unable to clear cellulose agar, these organisms can digest, in combination with Treponema and Butyrivibrio species, large areas of agar (Kudo et al. 1986). Fondevila and Dehority (1994) demonstrated through sequential addition studies that hemicellulose utilization resulted from initial solubilization of the hemicellulose from the forage by non-hemicellulose utilizers (F. succinogenes A3c and R. flavefaciens B34b) and subsequent fermentation of the soluble polysaccharide by the utilizing, but non-degrading organism (P. ruminocola H2b).
GENETIC MANIPULATION OF
MICROBIAL SPECIES
, Teather 1985) remains largely unrealized. Strategies proposed have included the following: 1) increasing the competitiveness of cellulolytic organisms (F. succinogenes, Ruminococcus) by conferring the ability to utilize xylose and pectins, thereby allowing earlier colonization of particulate matter; 2) inserting the cellulase gene into numerically predominant species (B. ruminicola); 3) increasing the competitiveness of cellulolytic species present in the rumen in low numbers (C. polysaccharolyticum) by according the ability to adhere to feed particles; 4) inserting an acid-tolerant cellulase gene into acid-tolerant bacteria (Lactobacillus) to allow fiber fermentation at a ruminal pH less than 6; 5) developing a cutinase activity in predominant bacteria; and 6) allowing predominant species to degrade arabinose side chains, thereby overcoming the rate-limiting effect of lignin. There have been at least 50 scientific reports on the cloning of genes coding for fiber-degrading enzymes (Wallace 1994).
ANIMAL FACTORS AFFECTING MICROBIAL FIBER DIGESTION
). Because the rate of degradation of structural carbohydrate is of the same order as passage rate, at high levels of intake the depression in digestibility of structural carbohydrate can be two to three times greater than that of the faster degrading, nonstructural carbohydrate. Although a high level of intake may depress ruminal fiber digestion, compensation occurs through increases in gross energy intake and hindgut digestion (Bourquin et al. 1990).
Composition of dietary fiber.
Rumen available energy normally limits growth of bacteria, and any additional organic matter fermented in the rumen as a result of changing the forage:concentrate ratio will probably increase microbial protein synthesis by providing more energy. Sniffen and Robinson (1987) suggested that the yield of bacteria was maximized with a forage content of 70% in the diet dry matter. Because structural carbohydrate-fermenting microbes are usually limited by a ruminal pH less than 6 (Hoover 1986), the depression in fiber digestibility at higher inclusion rates of concentrate can most likely be explained by the rapid degradation of nonstructural carbohydrate. It is likely that fiber digestion will not be maximized at a single forage:concentrate ratio; rather, it will depend on the various rates of digestion of structural and nonstructural carbohydrate supplied by the forage and the concentrate. This may be shown indirectly by the studies of Tamminga (1981), who reported no relationship between forage:concentrate and bacterial yield. This study used by-product ingredients with a high fiber content, in contrast to the traditional cereal grains.
Particle size and chemical and biological treatments.
Although physical processing of forages by grinding and pelleting does provide a greater surface area for attack by enzymes, utilization of structural carbohydrate is not increased; rather, improvements in animal performance arise primarily from an increased digestible energy intake (Bourquin et al. 1990). In fact, fiber digestibility is reduced by 3.3% as a result of reduced residency time in the rumen. Chemical treatments such as sodium hydroxide, potassium hydroxide and ammonia will partially solubilize hemicellulose and lignin, as well as hydrolyze acetic, phenolic and uronic acid esters. Oxidative treatment of forage with sulfur dioxide or peroxide results in the degradation of lignin and extensive solubilization of structural carbohydrate (Fahey et al. 1993). Potential improvements in fiber digestion could result from the use of alkaline hydrogen peroxide, which is a combined hydrolytic and oxidative process. The use of white-rot fungi for converting lignocellulosic materials to more digestible feedstuffs for ruminants has also been intensively investigated. In vitro dry matter digestibility was increased 30% and 13% for rice straw leaf and stem, respectively (Karunanandaa et al. 1995). Fungal treatment enhanced digestion of the mesophyll tissue and improved access for ruminal microorganisms by collapsing the vascular bundles.
Effective fiber.
Recent use of the term effective fiber (eNDF) acknowledges the different functionality of dietary fiber. Milk fat, chewing rate, and particle size have all been used as an index of effective fiber. Currently, the Cornell Net Carbohydrate and Protein System (CNCPS) uses eNDF to adjust ruminal pH and passage rate (Sniffen et al. 1992). Factors other than particle size that influence eNDF include the degree of lignification of the fiber, degree of hydration, and bulk density. The importance of eNDF can be seen in the reduced growth rate of structural carbohydrate-fermenting microorganisms and the reduction in total microbial yield when ruminal pH is lower than 6.2 (this being related to a dietary eNDF of 20%). Research to further quantify the value of eNDF for a range of feeds is much needed.
Feeding strategies.
Robinson (1989) indicated that fiber digestion may be limited by the order and frequency of substrate presentation to the rumen. A total mixed diet provides an optimal balance of nutrients to the microorganisms, thereby stabilizing fermentation. The potential to modify the ruminal environment is perhaps greater when separate, twice-daily feeding of forage and concentrate is practiced. Feeding diets, especially those that are highly fermentable, more frequently than twice a day is generally thought to stabilize the ruminal environment. This reduction in diurnal variation of fermentation end products, in conjunction with improved coupling of protein and energy release in the rumen, can increase the rate of fiber digestion (Robinson 1989).
Additives.
The addition of buffers and alkalinizing products (sodium bicarbonate, sodium sesquicarbonate, magnesium oxide, sodium bentonite) to the diet of lactating dairy cows can improve fiber digestion by reducing the period of time during the day that ruminal pH is less than 6. A buffer may overcome limitations to fiber digestion in diets that have a high proportion of low pH silages, fermented feeds with a moisture content greater than 50%, an acid detergent fiber <19%, finely chopped haylage, a high proportion of concentrate, irregular feeding of high levels of concentrate, or finely ground concentrate (Hutjens 1992).
). Ruminal fill and rate of passage are also influenced. Other ionophores reported to produce similar results include lasalocid, salinomycin, lysocellin, narasin and tetronasin (Wallace 1994). Further research is required to determine if these function as an antibacterial or an antifungal agent in the rumen and if the efficiency responses in the animal are a result of ruminal or post-ruminal effects.
). The further development of strains of probiotics to stimulate the growth of specific types of ruminal bacteria may result in diet-specific additives. Recently, the use of extracellular enzymes that have been protected from the digestive process has been proposed as a method to improve fiber digestion (Wallace 1994).
ENHANCEMENT OF FIBER DIGESTION: OPPORTUNITIES
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Table 1. Research areas that could lead to the enhancement of fiber digestion |
FOOTNOTES
LITERATURE CITED
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