Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.

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Microbial cellulose utilization: fundamentals and biotechnology.

A method is proposed for estimating the percentage of dietary protein that is degraded by microbial action in the rumen when protein supplement is added to a specified ration. The potential degradability, p , is measured by incubating the supplement in artificial-fibre bags in the rumen and is related to incubation time, t , by the equation p = a+b (1 – e -ct ). The rate constant k , measuring the passage of the supplement from the rumen to the abomasum, is obtained in a separate experiment in which the supplement is combined with a chromium marker which renders it completely indigestible. The effective percentage degradation, p , of the supplement, allowing for rate of passage, is shown to be p = a +[ bc/(c+k) ] (1- e -(e+k)t ) by time, t , after feeding. As t increases, this tends to the asymptotic value a+bc /( c+k ), which therefore provides an estimate of the degradability of the protein supplement under the specified feeding conditions. The method is illustrated by results obtained with soya-bean meal fed as a supplement to a dried-grass diet for sheep. The incubation measurements showed that 89% of the soya-bean protein disappeared within 24 h and indicated that it was all ultimately degradable with this diet. When the dried grass was given at a restricted level of feeding the allowance for time of retention in the rumen reduced the estimate of final degradability to 71% (69% within 24 h). With ad libitum feeding there was a faster rate of passage and the final degradability was estimated to be 66% (65% within 24 h).

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The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage

The VFA, also known as short-chain fatty acids, are produced in the gastrointestinal tract by microbial fermentation of carbohydrates and endogenous substrates, such as mucus. This can be of great advantage to the animal, since no digestive enzymes exist for breaking down cellulose or other complex carbohydrates. The VFA are produced in the largest amounts in herbivorous animal species and especially in the forestomach of ruminants. The VFA, however, also are produced in the lower digestive tract of humans and all animal species, and intestinal fermentation resembles that occurring in the rumen. The principal VFA in either the rumen or large intestine are acetate, propionate, and butyrate and are produced in a ratio varying from approximately 75:15:10 to 40:40:20. Absorption of VFA at their site of production is rapid, and large quantities are metabolized by the ruminal or large intestinal epithelium before reaching the portal blood. Most of the butyrate is converted to ketone bodies or CO2 by the epithelial cells, and nearly all of the remainder is removed by the liver. Propionate is similarly removed by the liver but is largely converted to glucose. Although species differences exist, acetate is used principally by peripheral tissues, especially fat and muscle. Considerable energy is obtained from VFA in herbivorous species, and far more research has been conducted on ruminants than on other species. Significant VFA, however, are now known to be produced in omnivorous species, such as pigs and humans. Current estimates are that VFA contribute approximately 70% to the caloric requirements of ruminants, such as sheep and cattle, approximately 10% for humans, and approximately 20-30% for several other omnivorous or herbivorous animals. The amount of fiber in the diet undoubtedly affects the amount of VFA produced, and thus the contribution of VFA to the energy needs of the body could become considerably greater as the dietary fiber increases. Pigs and some species of monkey most closely resemble humans, and current research should be directed toward examining the fermentation processes and VFA metabolism in those species. In addition to the energetic or nutritional contributions of VFA to the body, the VFA may indirectly influence cholesterol synthesis and even help regulate insulin or glucagon secretion. In addition, VFA production and absorption have a very significant effect on epithelial cell growth, blood flow, and the normal secretory and absorptive functions of the large intestine, cecum, and rumen. The absorption of VFA and sodium, for example, seem to be interdependent, and release of bicarbonate usually occurs during VFA absorption.(ABSTRACT TRUNCATED AT 400 WORDS)

Energy contributions of volatile fatty acids from the gastrointestinal tract in various species

1. The effect of ammonia concentration on microbial protein production was determined in continuous-culture fermentors charged with ruminal contents obtained from steers fed on either a protein-free purified diet, a maize-based all-concentrate diet, or a forage–concentrate (23:77) diet. Urea was infused into the fermentors to maintain various concentrations of ammonia in the incubating mixtures.2. Under nitrogen-limiting conditions, microbial protein yield measured as tungstic acid-precipitable N (TAPN) increased linearly with supplementary urea until ammonia started to accumulate in the incubating ingesta. Increasing the ammonia concentration beyond 50 mg NH3-N/l had no effect on microbial protein production.3. The molar proportions of volatile acids produced were not affected by the level of urea supplementation. Total acid production was decreased slightly under N-limiting conditions, but not to the same extent as microbial protein production.4. Estimated yield of microbial dry matter/mol ATP produced averaged 15·6 when non-limiting N as urea was provided with the purified diet.5. These results suggest that addition of non-protein N supplements to ruminant rations are warranted only if the prevailing concentration of ruminal ammonia is less than 50 mg NH3-N/l ruminal fluid.

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Effect of ammonia concentration on rumen microbial protein production in vitro

A review is made of the ruminant digestive system in its morphophysiological variations and adaptations relating to foraging behaviour, digestive physiology, to interactions between plants and ruminants and to geographic and climatic diversity of ruminants' ecological niches. Evidence is provided for evolutionary trends from an extreme selectivity mainly for plant cell contents and dependence upon a fractionated fore- and hindgut fermentation, to an unselective intake of bulk roughage subjected to an efficient plant cell wall fermentation, mainly in the forestomachs. The review is based on detailed comparative morphological studies of all portions of the digestive system of 65 ruminant species from four continents. Their results are related to physiological evidence and to the classification of all extant ruminants into a flexible system of three overlapping morphophysiological feeding types: concentrate selectors (40%), grass and roughage eaters (25%) and intermediate, opportunistic, mixed feeders (35%). Several examples are discussed how ruminants of different feeding types are gaining ecological advantage and it is concluded that ruminants have achieved high levels of digestive efficiency at each evolutionary stage, (including well-documented seasonal adaptations of the digestive system) and that ruminant evolution is still going on. Deductions made from the few domesticated ruminant species may have, in the past, biased scientific evaluation of the free-ranging species' ecology. The main threat to a continuous ruminant evolution and diversity appears to be man's neglect for essential ecological interactions between wild ruminants and their specific habitats, which he alters or destroys.

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Evolutionary steps of ecophysiological adaptation and diversification of ruminants: a comparative view of their digestive system*'**

The artificial fibre bag technique was evaluated for assessing the proportions of dietary D. M. and N which disappear in the rumen. The most important factor determining the variability in disappearance from bags incubated together was the sample size in relation to bag size. For incubation of 5 g air dry feed, a bag size of 17 ✗ 9 cm was found to be adequate. There were also variations in substrate disappearance between animals and between days of incubation. It was estimated that three sheep and the measurement of substrate disappearance twice were necessary in order to obtain acceptable repeatability.The technique was found to be satisfactory as a simple and rapid guide for measuring nutrients disappearance in the rumen such as protein degradation and carbohydrate fermentation. It has been used in subsequent work to measure the effect of features of the rumen environment, such as pH and NH3 concentration, on rate of fermentation.

https://www.cambridge.org/core/services/aop-cambridge-core/content/view/S0021859600037321

A study of artificial fibre bag technique for determining the dig estibility of feeds in the rumen

Comparison of in vitro gas production and nylon bag degradability of roughages in predicting feed intake in cattle

Associative effects of mixed feeds. I. effects of type and level of supplementation and the influence of the rumen fluid pH on cellulolysis in vivo and dry matter digestion of various roughages

1. Four sheep were fed from automatic continuous feeders on whole barley fortified with graded levels of a urea solution. This approach was to a large extent successful in maintaining relatively steady states of rumen ammonia concentration. 2. Rates of barley fermentation in the rumen at various rumen NH3 concentrations were assessed by measuring the disappearance of barley dry matter from polyester bags suspended in the rumen of these sheep. 3. The minimal NH3 concentration for maximal rate of fermentation was estimated as 235 mg/l rumen fluid.

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E. R. Ørskov

Papers

The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage

A study of artificial fibre bag technique for determining the dig estibility of feeds in the rumen

Comparison of in vitro gas production and nylon bag degradability of roughages in predicting feed intake in cattle

Associative effects of mixed feeds. I. effects of type and level of supplementation and the influence of the rumen fluid pH on cellulolysis in vivo and dry matter digestion of various roughages

Rates of rumen fermentation in relation to ammonia concentration