Influence of heat sterilization and ammoniation on straw composition and degradation by pure cultures of cellulolytic rumen bacteria

Animal Feed Science and Technology, 12 (1985) 195--203

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Elsevier Science Publishers B.V., Amsterdam - - P r i n t e d in The Netherlands

INFLUENCE OF HEAT STERILIZATION AND AMMONIATION ON STRAW COMPOSITION AND DEGRADATION BY PURE CULTURES OF CELLULOLYTIC RUMEN BACTERIA

HADDEN GRAHAM 1, PER ~MAN 1 and OLOF THEANDER 2

1Department of Animal Nutrition and Management and 2Department of Chemistry and Molecular Biology, Swedish University of Agricultural Sciences, S 750 07 Uppsala (Sweden) NAZIF KOLANKAYA* and COLIN S. STEWART

Department of Microbial Biochemistry, Rowett Research Institute, Aberdeen AB2 9SB (Gt. Britain) (Received 12 March 1984; accepted for publication 11 December 1984) ABSTRACT Graham, H., ~ m a n , P., Theander, O., Kolankaya, N. and Stewart, C.S., 1985. Influence of heat sterilization and ammoniation on straw composition and degradation by pure cultures of cellulolytic rumen bacteria. Anita. Feed Sci. Technol., 12: 195--203. Untreated and ammoniated straw was incorporated into culture medium, sterilized by autoclaving, and incubated with pure cultures of the predominant cellulolytic rumen bacteria: Bacteroides succinogenes, Ruminococcus flavefaciens and Ruminococcus albus or mixed rumen liquor micro-organisms for up to 8 days. The residues from the incubations and the water-insoluble fraction of the original straws were analysed for neutral sugar residues and Klason lignin. The relative disappearance of dry matter, cellulose and arabinoxylans was compared with the pattern of straw digestion in vitro. Sterilization by autoclaving solubilized 17 and 8% of the arabinoxylans and 9 and 4% of the glucans in the untreated and ammonia-treated straw, respectively, About 13% of the Klason lignin in the untreated straW and 8% in the ammoniated straw was also solubilized. Ammoniation did not alter either the composition or the relative degradation of insoluble straw polysaccharides, but increased the potential degradability by about 20%. All the bacterial preparations solubilized substantial fractions of both straw cellulose and arabinoxylans. However, mixed rumen liquor micro-organisms and B. succinogenes solubilized the two carbohydrate constituents equally, whereas the ruminococci preferentially solubilized the arabinoxylans.

INTRODUCTION The three bacterial species which p r e d o m i n a t e in the digestion of p l a n t cell w a l l s i n t h e r u m e n - - B a c t e r o i d e s succinogenes, R u m i n o c o c c u s albus a n d R. flavefaciens -- are c a p a b l e o f s o l u b i l i z i n g b o t h c e l l u l o s e a n d h e m i c e l l u *Present address: Department of General Biology, Beytepe Campus, Hacettepe University, Ankara, Turkey. 0377-8401/85/$03.30

© 1985 Elsevier Science Publishers B.V.

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loses in intact forages (Dehority and Scott, 1967; Morris and van Gylswyk, 1980; Stewart, 1985). The t w o ruminococci are, in general, capable of utilizing both hexoses and pentoses, whereas most strains of B. succinogenes are unable to metabolise the pentose fraction of the hemicelluloses they degrade (Coen and Dehority, 1970; Morris and van Gylswyk, 1980). The relative capacities of these bacteria to solubilize cellulose and hemicelluloses depend on the composition and structural organization of their substrate (Dehority and Scott, 1967; Koch and Kistner, 1969; Morris and van Gylswyk, 1980). Celluloses and hemicelluloses are closely associated in the cell wall matrix of cereal straws and are degraded at an equal rate by rumen liquor. Ammonia treatment, whether for a long period at ambient temperature or short period at high temperature, increases straw digestibility, but does not alter the relative degradation of these carbohydrates (Graham and Aman, 1984; Lindberg et al., 1984). Thus straw is a suitable substrate for the study of the relative ability of monocultures of rumen bacteria to solubilize cellulose and hemicelluloses in the secondary cell walls of m o n o c o t y l e d o n s . The thermal delignification of plant materials such as w o o d and straw by steaming is employed for improving the quality of these products as animal feeds (Puls and Dietrichs, 1980). Microbial studies on the ability of pure cultures to degrade plant cell walls normally involve sterilization of the substrate by autoclaving (Ifkovits et al., 1965; Dehority and Scott, 1967; Koch and Kistner, 1969; Morris and van Gylswyk, 1980), although the effect of this treatment on the susceptibility of plant material to degradation has seldom been considered. In the present study the pattern of cellulose and hemicellulose solubilization in cereal straw by pure cultures of B. succinogenes, R. albus and R. flavefaciens, and the influence of heat sterilization and ammoniation on straw composition and degradation was investigated. MATERIALS AND METHODS General methods Barley straw (var. 'Sonja' -- winter variety) was harvested in England in 1981 and a 1-t sample was ammoniated (30 kg anhydrous NH3) at 90°C for 16 h (Theander et al., 1984). The untreated and the ammoniated straw were ground on a Wiley mill to pass a 2-mm screen, then separated into fractions of different particle size using test sieves (Endecotts Ltd., London, Gt. Britain). Straw particles retained by sieves between 422 and 853 p in diameter were employed in the tests on microbial digestibility. Samples were further milled to pass a 0.5-mm screen before chemical analysis, and all analyses were carried o u t in duplicate. Bacterial isolates Ruminococcus albus SY3 was isolated and maintained as described by

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Wood et al. (1982). Ruminococcus flavefaciens 007 was isolated and maintained as described by Stewart et al. (1981). Bacteroides succinogenes $85 was a gift from M.P. Bryant, Univ. Illinois, Urbana, IL 61801, U.S.A.

Culture methods Straw (2 g F.W.) was incorporated into 400 ml of anaerobic culture medium (medium HSM w i t h o u t added carbohydrates, Stewart et al., 1981) contained in 1-1 Erlenmayer flasks. The medium was prepared and maintained under strictly anaerobic conditions by the m e t h o d of Bryant (1972) using O2-free CO2 to displace air. Autoclaving was for 15 min at 121°C. The flasks were inoculated with 50-ml cultures grown for 15 to 18 h in the same medium, with cellobiose replacing straw as the carbon source. Incubations were carried out in duplicate at 39 + 1°C for the stated interval. Differences in dry matter losses between duplicate incubations were on average less than one percentage unit and never exceeded two percentage units.

Rumen liquor R u m e n liquor was withdrawn from the rumen of a cannulated cow fed on a roughage diet supplemented with starchy concentrate, and filtered through 4 layers of surgical gauze before inoculation (50 ml) of the flasks containing straw.

Chemical analysis The water-insoluble fraction was prepared by extracting the straw (2 g F.W.) with water (200 ml) for 6 h at 39°C and freeze-drying the residue. The degraded and water-insoluble samples were treated with 72% sulphuric acid and hydrolysed with dilute sulphuric acid. The unhydrolysed residue -Klason lignin -- was collected by filtration, dried and weighed. Part of the hydrolysate was neutralised and the neutral sugars were analysed as alditol acetates by GLC on an OV-225 capillary column (Theander and /~man, 1979).

Calculations Cellulose was calculated from glucose residues and arabinoxylans from the sum of arabinose and xylose residues -- which constitute more t h a n 85% of the hemicelluloses in this straw (Graham and Aman, 1984). The disappearance of cell wall constituents was calculated relative to the content in the water-insoluble residue, whereas straw degradation was calculated on a total dry matter basis relative to unextracted material. The zero time points in the figures were taken from the control experiment of straw incubated for 8 days w i t h o u t bacteria.

198 TABLE I The i n f l u e n c e o f h e a t s t e r i l i z a t i o n o n t h e c o m p o s i t i o n o f u n t r e a t e d and a m m o n i a - t r e a t e d straw Component

C o n t e n t in unsterilized straw 1

C o n t e n t in sterilized straw 2

Solubilization due to sterilization (%)

U n t r e a t e d straw K l a s o n lignin cellulose arabinoxylans cellulose : a r a b i n o x y l a n s

17.2 36.2 19.2 1.89

14.9 33.0 16.0 2.06

13.1 8.7 16.6

Ammonia-treated straw K l a s o n lignin cellulose arabinoxylans cellulose : a r a b i n o x y l a n s

17.4 37.6 19.1 1.96

15.9 36.0 17.7 2.04

8.4 4.3 7.6

1Calculated f r o m t h e w a t e r - i n s o l u b l e residue, as a p e r c e n t a g e o f d r y m a t t e r in original straw. 2Calculated f r o m t h e insoluble residue following sterilization and i n c u b a t i o n in c u l t u r e m e d i u m for 8 days, as a p e r c e n t a g e o f d r y m a t t e r in original straw.

RESULTS

Water extracted 9.4 and 11.0% of dry matter from the untreated and ammoniated straw, respectively, whereas the corresponding extractives following autoclaving and incubation in the culture medium for 8 days were 17.0 and 14.3%. Ammoniation caused no significant change in the polysaccharide and lignin content o f the straw (Table I). Sterilization solubilized about twice as m u c h arabinoxylans as cellulose with losses of carbohydrates being greater in the untreated straw, while Klason lignin content decreased by 13% in the untreated straw and 8% in the ammonia-treated straw (Table I). Incubation in r u m e n liquor for 8 days solubilized 38 and 50% of the dry m a t t e r of the untreated and ammoniated straw, respectively (Table II). PerTABLE II Solubilization of straw components by rumen Hquor or R. flavefaciens during 8 days of incubation Micxo-organisrn Straw

Solubilization (%) D r y matter

Cellulose: arabino xylans

Cellulose

Arabinoxylans

in residue

R u m e n liquor

Untreated 38 A m m o n i a - t r e a t e d 50

33 50

41 58

2.1 2.4

R. flavefaciens

Untreated 32 Ammonia-treated 41

25 36

41 56

2.4 2.9

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centage cellulose disappearance was similar to that of dry matter, while arabinoxylans solubilization was a b o u t eight percentage units greater. Ruminococcus flavefaciens monocultures degraded less dry matter -- 32% from the untreated straw and 41% from the ammonia-treated straw (Table II). This exceeded cellulose disappearance by a b o u t six percentage units, b u t was approximately 12 percentage units less than that of arabinoxylans. Ruminococcus albus degraded 40 and 47% of the untreated and ammoniated straw, respectively, during 8 days incubation, with most of this digestion occurring in the first 2 days (Fig. 1). Arabinoxylan solubilization was almost double that of cellulose, and exceeded cellulose disappearance by a b o u t 16 percentage units in the untreated straw and 22 percentage units in the ammonia-treated straw. The respective cellulose: arabinoxylans ratios at the sampling times (2, 4 and 8 days) were 2.4, 2.5 and 2.5 for the untreated straw and 2.8, 3.2 and 3.4 for the ammoniated straw. Bacteroides succinogenes degraded 36 and 43% of the untreated and ammania-treated straw, respectively, during 8 days incubation, but digestion was probably incomplete after this period (Fig. 2). Arabinoxylan disappearance exceeded that of cellulose by a b o u t four percentage units for both straw samples. The cellulose:arabinoxylans ratios during incubation with B. succinogenes were between 2.0 and 2.1 at all sampling times for both untreated and ammonia-treated straw.

Untreated

Ammoniated

60

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o

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Fig. 2. The disappearance of dry matter (x), cellulose (o) and arabinoxylans (z~) from untreated and ammoniated straw during incubation with B. succinogenes.

Arabinose residues were slightly more susceptible than xylose residues to solubilization by all bacterial cultures, and the number of xylose to arabinose units increased from 6--7:1 prior to digestion to a b o u t 9:1 when a b o u t 40% of the dry matter had been degraded. DISCUSSION

The solubilization of cell wall c o m p o n e n t s during autoclaving of the straw in the present study is consistent with the known influences of heat and pressure on lignocellulosic materials, but disagrees with Morris and van Gylsw y k (1980) who concluded that autoclaving did not significantly alter hay cell wall digestibility pattern relative to 70% ethanol sterilized samples. Thermal delignification (200°C, 1200 kPa, 20 min in water-saturated steam) of cereal straw solubilizes up to 75% of xylose residues, but little cellulose and lignin, and increases in vitro digestibility b y a b o u t 30 percentage units (Puls and Dietrichs, 1980). This is attributed to the hydrolysis of bonds within, and between, cell wall constituents by acetic and other organic acids released from straw during treatment. The loss of cellulose during sterilization of the straw in this study could result from the solubilization of the more amorphous areas of this c o m p o n e n t or of other glucose containing polysaccharides present as minor constituents in the straw. The untreated straw had an acetyl content of 1.7% and a b o u n d ferulic and p-coumaric acid c o n t e n t of 1.2% (Graham and Aman, 1984), and thus the added ammonia

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(3% of dry weight) could neutralise any acids released from the straw. The neutralisation by ammonia of acids released during autoclaving probably resulted in the decreased hydrolysis and therefore reduced solubilization of cell wall components in the ammonia-treated straw relative to the untreated straw. Ammoniation enhanced straw degradation in all incubations and the average increase in dry matter disappearance of about 20% is similar to that reported for this and other ammonia treatment regimes (Sundst¢l et al., 1978; Graham and )kman, 1984; Lindberg et al., 1984). The absence of any effect of ammoniation on the composition and relative degradation of insoluble straw carbohydrates is consistent with the known action of weak alkali on secondary cell walls (see Graham and Aman, 1984; Lindberg et al., 1984). The observed differences between cellulose and arabinoxylan disappearance in rumen liquor for both straws were due to the greater solubilization of the latter fraction during autoclaving. Thus, as previously reported for straw samples not heat sterilized (Graham and •man, 1984; Lindberg et al., 1984), arabinoxylans and cellulose were degraded at the same rate by rumen liquor. After 8 days incubation the two Ruminococcus species had solubilized around twenty percentage units more of the arabinoxylans than of cellulose, whereas B. succinogenes solubilized about four percentage units more of the arabinoxylans. As autoclaving resulted in the loss of about six percentage units more of the arabinoxylans relative to cellulose, R. albus and R. flavefaciens preferentially degraded the arabinoxylans whereas B. succinogenes digested both components equally. Previous studies employing strain $85 of B. succinogenes have shown that glucose solubilization (88%) in teff grass cell walls exceeds that of xylose (80%, Morris and van Gylswyk, 1980), whereas a substantial fraction of the cellulose, but little or no hemicelluloses, are degraded in bromegrass and alfalfa (Dehority and Scott, 1967). In both those studies, several strains of R. albus and R. flavefaciens exhibited more cellulolytic than hemicellulolytic activity. The greater solubilization of arabinoxylans by R. albus and R. flavefaciens in the present study is consistent with the ability of these species to utilize pentoses (Coen and Dehority, 1970; Morris and van Gylswyk, 1980). The greater susceptibility of arabinose relative to xylose to solubilization in this study has previously been observed during the degradation of various forages by rumen liquor and bacterial monocultures (Morris and Bacon, 1977; Morris and van Gylswyk, 1980). The relative abilities of the micro-organisms to degrade the straw were: rumen liquor = R. albus > B. succinogenes > R. flavefaciens. However, digestion by B. succinogenes, and possibly R. flavefaciens, may have continued after 8-day incubation period. A lag phase in degradation may have occurred, particularly with B. succinogenes (see Fig. 2), and the pattern of dry matter disappearance (Figs. 1 and 2), with R. albus having almost reached completion after 2 days while digestion by B. succinogenes continued up to 8 days, could reflect the growth pattern of the bacteria (Kolankaya et al., 1983). It is known that the growth rate of some strains of B. succinogenes declines

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during maintenance of cultures in the laboratory (Stewart et al., 1981). The relatively slow rate of attack of straw by strain $85 may, therefore, not be typical of this organism in its natural habitat. Many factors, including the composition of the incubation medium, substrate morphology, relative enzyme production and the inhibitory qualities of, for example, lignin, could bring about the preferential digestion of one carbohydrate component. More detailed knowledge of the effects of pure cultures of bacteria on plant cell walls will clearly depend on the development of methods of sterilization less damaging than the treatment used in studies to date. ACKNOWLEDGEMENTS

Drs. Graham and Kolankaya were recipients of OECD fellowships. This work was in part financially supported by a grant from the Swedish Council for Forestry and Agricultural Research.

REFERENCES Bryant, M.P., 1972. Commentary on the Hungate technique for culture of anaerobic bacteria. Am. J. Clin. Nutr., 25: 1324--1328. Coen, J.A. and Dehority, B.A., 1970. Degradation and utilization of hemicellulose from intact forages by pure cultures of rumen bacteria. Appl. Microbiol., 20: 362--368. Dehority, B.A. and Scott, H.W., 1967. Extent of cellulose and hemicellulose digestion in various forages by pure cultures of rumen bacteria. J. Dairy Sci., 50: 1 1 3 6 - - 1 1 4 1 . Graham, H. and Aman, P., 1984. A comparison between degradation in vitro and in sacco of constituents of untreated and ammonia-treated barley straw. Anim. Feed. Sci. Technol., 10: 199--211. Ifkovits, R.W., Ragheb, H.S., Barnes, R.F. and Packett, L.V., 1965. A pure culture inoculum method for evaluation of forage cellulose digestibility. J. Anita. Sci., 24: 1092--1099. Koch, S.G. and Kistner, A., 1969. Extent of solubilization of s-cellulose and hemicellulose of low-protein teff hay by pure cultures of cellulolytic rumen bacteria. J. Gen. Microbiol., 55: 459--462. Kolankaya, N., Stewart, C.S. and Costerton, J.W., 1983. Effect of ammonia treatment on straw digestion by rumen bacteria. Proc. 17th Conf. Rumen Function. Chicago, IL, USDA/ARS. Lindberg, J.E., Ternrud, I.E. and Theander, O., 1984. Degradation rate and chemical composition of different types of alkali-treated straws during rumen digestion. J. Sci. Food Agric., 35: 500--506. Morris, E,J. and Bacon, J.S.D., 1977. The fate of acetyl groups and sugar components during the digestion of grass cell walls in sheep. J. Agric. Sci., 89: 327--340. Morris, E.J. and van Gylswyk, N.O., 1980. Comparison of the action of rumen bacteria on cell walls from Eragrostis tel. J. Agric. Sci., 95: 313--323. Puls, J. and Dietrichs. H.H., 1980. Separation of lignocelluloses into highly accessible fibre materials and into hemicellulose fractions by the steaming--extraction process. In: OECD Co-operative research project on food production and preservation. Workshop 2: Conversion of lignocellulose material to simple carbohydrates. Amersfoort, The Netherlands, pp. 52--68.

203 Stewart, C.S., 1985. R u m e n function with special reference to fibre digestion. In: E. Barnes and G.C. Mead (Editors), Anaerobic Bacteria in Habitats other than Man, Blackwell Scientific, Oxford, in press. Stewart, C.S., Paniagua, C., Dinsdale, D., Cheng, K.-J. and Garrow, S.H., 1981. Selective isolation and characteristics of Bacteroides succinogenes from the tureen of a cow. Appl. Environ. Microbiol., 40 : 504--510. Sundst~l, F., Coxworth, E. and Mowat, D.N., 1978. Improving the nutritive value of straw and other low-quality roughages by treatment with ammonia. World Anita. Rev., 26: 13--21. Theander, O. and ~ m a n , P., 1979. Studies on dietary fibre. I. Analysis and chemical characterization of water-soluble and water-insoluble dietary fibres. Swed. J. Agric. Res., 9: 97--106. Theander, O., Hartley, R.D. and Stewart, C.S., 1984. A note on the OECD collaborative experiment on ammonia-treated straw. Anim. Feed Sci. Technol., 10" 89--91. Wood, T.M., Wilson, C.A. and Stewart, C.S., 1982. Preparation of the cellulase from the cellulolytic anaerobic rumen bacterium Ruminococcus albus and its release from the bacterial cell wall. Biochem. J., 205: 129--138.