Journal of Biotechnology, 30 (1993) 141-148 141 © 1993 Elsevier Science Publishers B.V. All rights reserved 0168-1656/93/$06.00

BIOTEC 00902

Aspects of lignin degradation by rumen microorganisms

P. Susmel and B. S t e f an o n

Dipartimento di Sc&nze della Produzione Animale - Universit?t degli Studi di Udine, Pagnacco (UD), Italy

(Received April 1991; revision accepted 26 January 1992)

Summary

The paper discusses the problems presented by lignin to animal production, particularly in countries with hot climates, where the degree of lignification in plants is extremely variable causing unpredictable reductions in digestibility. The difficulty of chemically defining lignin compounds is reviewed with the conclusion that a combination of traditional and advanced techniques is required if the biological role of the lignin in the plant is to be explained. Experiments in ruminants have often shown incomplete recovery of compounds traditionally referred to as lignin, leading to the suspicion that a limited digestion of lignin could occur under anaerobic conditions, with fungi playing a predominant role in the process. As is well known, the problem is difficult to study in both aerobic and anaerobic conditions. White-rot fungi - P h a n e r o c h a e t e c h r y s o s p o r i u m - has be- come a standard model for in vitro aerobic digestion. The in situ technique appears to be the most suitable for studies under anaerobic conditions, and some results are presented indicating apparent lignin digestibility in forages.

Rumen; Lignin degradation; Microbes

Fiber in animal nutrition

Structural carbohydrates (cellulose and hemicellulose) represent from 50% to 70% of total dry matter in legumes and grasses but, unfortunately, they are not

Correspondence to: B. Stefanon, Dip. Sci. Produzione Animale, Universit?~ degli Studi di Udine, via. S. Mauro 2, 33010 Pagnacco (UD), Italy. Paper supported by EEC, Project No. 8001-CT900022.

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generally digested by animal enzymes. The ability of animals to use structural carbohydrates for nutritive purposes depends on the presence of symbiotic mi- croorganisms, such as bacteria, protozoa and fungi, in the gastro-intestinal tract, which can degrade cellulose and hemicellulose to produce volatile fatty acids and other simple compounds digestible by the animal host; the latter can also digest the microbial cells. Symbiosis between animals and microorganisms is common, even though the digestive sites, in which microorganisms are present, can be different. The most simple form of symbiosis occurs in cockroaches, termites and wasps that eat the microorganisms, mainly constituted of fungi growing in plant and vegetable detritus. This situation is defined as ectosymbiosis, since the insect uses fungi on the outside of its body. In other species of insects a different kind of symbiosis - endosymbiosis - occurs (Prins and Kreulew, 1990); the animal host uses microbes, such as the flagellate protozoa, which live in postgastric sites in lower termites and woodroaches (Criptocercus), or cooperates with cellulolytic bacteria in postgastric sites, as in higher termites, beetles and cockroaches to digest fibrous substrates and utilize the endproducts. The higher termites can also utilize cellulose without the symbiotic bacteria since they have endogenous cellu- lases (Prins and Kreulen, 1990).

Higher animals can be divided into two groups: animals where the symbiotic microorganisms live before the true stomach (ruminants, hamsters, voles) and those in which symbiosis occurs in the large intestine (pony, rabbit, rat, elephant). The absorption of nutrients is high in the small intestine but low in the large intestine and so postgastric decay is generally of low nutritive importance. More- over, the utilization of feeds with high fiber contents (structural carbohydrates) is generally higher in animals with a pregastric digestion site and, of these, ruminants are of particular interest. This is one of the reasons that ruminants are so widespread in the world and why these species are of high interest from a nutritional point.

Rumen microbes are constituted of strictly and faeultative anaerobic bacteria (1-10 × 109 per ml) and protozoa (several million per ml). Orpin (1975) first reported the presence of an anaerobic fungi - Neocall imast ix frontalis - as a normal inhabitant of the rumen ecosystem. This microflora degrades plant tissue - mainly constituted of cellulose and hemicellulose - to satisfy their own require- ments (energy, protein, minerals) and synthesizes new organic matter and protein which are subsequently digested in the host ruminant's abomasum and small intestine. The amount of microbial organic matter synthesized in a lactating cow eating 20 kg d -1 of dry matter can be as high as 2.5 kg d -1, the value varying with diet composition (Susmel and Stefanon, 1987). According to Susmel and Stefanon (1987), the potential bacterial protein synthesis can be predicted from the energy that can be drawn from the degradation of substrates (carbohydrates and protein).

The possibility of increasing fiber digestibility of high fiber content feeds by ruminants to obtain high protein quality products (meat, milk) is of particular interest in developing countries, where these feedstuffs constitute the main or only dietary component for animals, since feeds of higher energy and protein value (cereals, legumes etc.) are reserved for human needs. In this respect, it is

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interesting to note that rumen microflora can produce protein from non organic nitrogen, such as urea, and so the nutritive value of some straws and by-products, low in protein and high in carbohydrate content, can be improved simply by adding an amount of non protein nitrogen. Cellulose and hemiceIlulose are potentially totally digestible in the rumen by microbes, even though it is well known that these polymers are actually incompletely digested. Direct measurements in the rumen, obtained by incubating in the forestomach samples of forages held in polyester bags for different time periods (Susmel et al., 1990), have indicated that the potentially (asymptote) degradable percentage of cellulose and hemicellulose varies between feedstuffs but is always lower than 90%. Pure compounds are, however, likely to be 100% degraded. The incomplete digestion of structural carbohydrates is partly due to the outflow rate of feeds from the rumen. It can thus be assumed that the time required for complete digestion of fiber is longer than the residence time in the rumen.

Lignin is another factor which contributes to reduce fiber digestibility. Several experimental results have shown that high lignin contents are associated with low digestibility values of both organic matter and cell wall constituents. Many regres- sion equations between organic matter digestibility and lignin content have been published (Demarquilly and Jarrige, 1981; Giger, 1985; Mika et al., 1981; Sauvant et al., 1985), with high coefficients of determination and low residual standard errors. In these equations lignin always has a negative regression coefficient (Table 1). The presence of chemical linkages between carbohydrates and phenolic com- pounds have been shown to limit the extent of cell wall attachment by microorgan- isms (Akin, 1988). According to Brice and Morrison (1982), a vast matrix is formed by lignin and hemicellulose, to which cellulose is b~und. Moreover, the inhibitory effects of phenolic monomers (especially p-coumaric acids) on rumen bacterial activity in vitro have been found by many authors (Borneman et al., 1986; Ford and Hartley, 1990; Hartley and Akin, 1989; Op den Camp et al., 1988; Reed, 1987). Akin and Rigsby (1987) have found that some phenolic compounds, such as p-coumaric acid, sinapic acid and vanillin, can cause strong inhibition of in vitro digestibility, with values respectively equal to 78%, 65% and 75%. In animal

TABLE 1

Provisional equations for organic matter digestibility based on chemical composition of feeds

Equation r 2 RSE

Dry matter digestibility Sauvant et al. (1985) 99.0-0.841 * N D F - 0 . 6 1 6 * A D L 0.98 4.60

Organic matter digestibility Demarquilly and Jarrige (1981) 87.8 + 0.172 * CP - 3.767 * ADL (grass) 2.30

73.4 + 0.601 * CP - 2.792 * ADL (legumes) 2.79 Giger (1985) 99.1 - 4.38 * KL (14 forages) 0.93 3.20 Mika et al. (1981) 80.7-3.22 * KL (4 lucerne + 14 maize silage) 0.76 5.30

r 2, coefficient of determination of regression. RSE, residual standard error of regression. ADL, acid detergent lignin; rE, Klason lignin; NDF, neutral detergent fiber; cP, crude protein.

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nutrition research, another group of phenolic compounds is of particular interest, the tannins, which are predominant in legumes, feeds and crops grown in hot climates. Tannins are classified as 'hydrolyzable' (carbohydrate moiety with hy- droxyl groups esterified to gallic acid, m-digallic acid or hexahydroxydiphenic acid) or 'condensed' (oligomers of flavan-3-ols, catechins). One of the effects of these compounds is the inhibition of rumen digestion of the plant carbohydrates from 13°£ to 25% (Reed, 1987; Susmel et al., 1989), but the main effect is found with reduced protein digestibility from 9% to 43% due to the formation of proteo-lignin complexes (Mangan, 1988; Waghorn et al., 1987).

Chemical determination of lignin

From a chemical point of view, lignin is not easily defined and the generic term 'lignin' is used to describe a group of three-dimensional polymers of phenyl- propane structural units, aromatic alcohols, p-coumaril, coniferil and synapil, associated with the cellulose and hemicellulose components of a plant. The deposition of the lignin compounds occurs in a random, non-enzymatic manner, so that it is not possible to define a typical structure. In the plant tissue, native lignin is intimately bound to hemicellulose, so that its isolation or extraction, i.e. by strong oxidation with nitrobenzene (Galletti et al., 1989), can cause some rear- rangement and/or condensations.

From an analytical point of view, scanning electron microscopy, transmission electron microscopy, histochemical techniques, pyrolysis mass spectrometry, nu- clear magnetic resonance, size-exclusion chromatography and near infra-red re- flectance are promising techniques to qualitatively describe the degradative pro- cess (Akin, 1982; Cheng et al., 1983/84; Engels and Brice, 1985; Reid et al., 1988), although, at present, they are likely to be applied only to limited numbers of closely defined samples rather than the necessarily large number of samples required in extension work. An easy routine method to describe carbohydrates and lignin in plant tissue was proposed by Goering and Van Soest (1970); it is based on fiber fractionation in different solvents (Fig. 1): the first, neutral, producing a residue constituted by plant cell walls (NDF); and the second, acid, corresponding to a lignocellulose complex (ADF). Lignin is then measured after strong acid hydrolysis or oxidation with permanganate. In this way it is possible to distinguish between soluble cellular components and cell wall constituents - hemicellulose, cellulose, lignin, cutin and minerals. The above method has the advantage of general application because it is comparatively easy to perform and gives an adequate description of the compounds of nutritional relevance. Some other methods, either gravimetric or based on optical or calorimetric properties, have been proposed (see the review of Giger, 1985), but they have not found a wide application. Although the above method is useful for the characterization of the site of digestion, it gives little information about the extent and nutritive value of the digested fiber. This means that if the biological role of the lignin in the plant is

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SAMPLE

! I~I/TRM. DETERGENT RESIDUE

Neutral detergent extraction

Acid detergent extraction

ACID DETERGENT RESIDUE

~_~_~ S04 Hydrolysis KMn04 Oxidation

LIGNIN CUTIN RESIDUE

I KMn04

CUTIN MINERALS AS RESIDUE -

IAsh at O°C

MINERALS AS RESIDUE

CELLULOSE CUTIN RESIDUE

72% H2S04 Hydrolysis

CUTIN MINERALS AS RESIDUE

Ash at 550°C

MINERALS AS RESIDUE

r- --I CUTIN LIGNIN measured as measured as weight loss weight loss

Fig. 1. Scheme of Van Soest analyses.

SOLUBLE CELLULAR COMPONENTS

HEMICELLULOSE measured as weight loss

LIG measured as wei 'ht loss

I

CELLULOSE measured as weight loss

I

CUTIN measured as wei !ht loss

t CELLULOSE measured as weight loss

to be explained, the characterization of its composition is needed as well as its quantitative determination.

Rumen degradation of iignocellulose

Initially, lignin was considered 'indigestible' in ruminants, and for this reason it has been used as an internal marker to calculate the digestibility of feedstuffs. However, experimental data has shown that lignin, determined either with sul- phuric acid or potassium permanganate, is apparently digested to a variable extent with values ranging from 2% to 53% (Galyean et al., 1979; Susmel, 1979; Thonney et al., 1979). The formation of a soluble lignin-carbohydrate complex in the rumen, due to microbial activity, was first reported by Hartley (1973) and Gaillard and Richards (1973). The complex is not recovered as lignin in the faeces and this can cause the disappearance of a variable proportion of the lignin present, which can account, according to the authors, for up to 50% of the total.

Recent evidence has also shown that lignin disappears from feeds incubated in bags suspended in the rumen. In an experiment conducted by Susmel et al. (1990),

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TABLE 2 Effective degradability (%) of NDF, hemicellulose, cellulose and 1990)

lignin in some forages (Susmel et al,

Feed NDF Hemicellulose Cellulose Liguin

Maize silage 51 AB 51 bc 43 BC 7 Cocksfoot hay 41 B 48 c 49 •c 8 Timothy hay 51 AB 56 b 50 B 9 Fescue hay 62 A 64 a 64 A 9 Lucerne hay 42 B 55 b 42 nc 10 Meadow hay 44 B 53 b 40 C 6

Mean sE 1.0 1.2 1.2 0.5

A, B, C on the same column denote significant differences (P < 0.01). ~. b, ~ on the same column denote significant differences (P < 0.05).

the in situ degradability of hemicellulose, cellulose and permanganate lignin of six forages was measured (Table 2). The results seem to indicate that lignin can in some way be solubilized or transformed during its residence time in the rumen but, if the biochemical pathway of degradation is unknown, it is not possible to state if lignin was used as an energy substrate for the microbes.

In aerobic conditions, degradation has been shown to occur in the white rot fungi basidiomycetes, a process which involves extracellular peroxidases, oxy- genated compounds (Prince and Stiefel, 1987) and requires the presence of readily available substrate (Ander and Eriksson, 1976, 1978). In anaerobic conditions, the degradative process is much more complicated and some studies have used monoaromatic lignin derivatives (Colberg, 1988). In this case, it has been supposed that the cleavage of the aromatic ring is subsequent to its reduction; in anaerobic photometabolism, the reduction involves a reductive enzyme coupled to a light induced transport system (i.e. ferredoxin). In microbial consortia, such as that found in the rumen, a similar metabolic pathway could be proposed, in which aromatic compounds serve as carbon sources and HCO~ as the electron acceptor. Animals in intensive grazing systems are likely to consume considerable quantities of NO 3 , a potentially powerful electron acceptor which has been demonstrated capable of reducing aromatic compounds (Taylor, 1983). Degradation of lignin-de- rived monomers can occur in mixed cultures in the presence of methanogenic bacteria, which can use hydrogen as an electron donor in the CO 2 reduction process (Wuhrmann, 1982). The hydrogenation of polyunsaturated fatty acids, naturally present in vegetable oils, could be an additional sink for hydrogen. Anaerobic degradation of lignin fragments has also been reported, caused by bacteria and fungi, with the production of soluble compounds (Colberg, 1988).

Future prospects

The potential degradation of lignocellulose by ruminants has been recently investigated by microbiologists and nutritionists, as the biochemical utilization of

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these compounds and their subsequent transformation into animal products would be an alternative way to the oxidation generally achieved by aerobic degradation, burning or other industrial processes.

However, some topics are not yet fully understood and require further efforts by researchers, such as the chemical identification of lignin monomers, their rear- rangements in polymeric compounds and the anaerobic pathway(s) of biochemical degradation.

In this context, the isolation and identification of microorganisms able to anaerobically convert lignin into simpler compounds would represent a very important step in the improvement of the degradative ability of the rumen ecosystem by means of propagation of the enzymatic ability, if needed, to other microbial species using genetic engineering.

This last aspect is very encouraging, as the potential utilizatior, of lignocellulose represents an important target for underdeveloped countries.

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