Production of volatile fatty acids from bagasse by rumen bacteria

Appl Microbiol Biotechnol (1984) 20:170-175

Production of volatile fatty acids from bagasse by rumen bacteria

C. H. Matei 1 and M. J. Playne 2

1 Department of Microbiology, University of Melbourne, Parkville, Victoria 3052 2 CSIRO Division of Chemical and Wood Technology, Private Bag 10, Clayton, Victoria 3168, Australia

Summary. Conditions are described for converting bagasse lignocellulose to volatile fatty acids (VFA) by anaerobic fermentat ion. Although yields of V F A were as high as 74% by weight of digestible organic mat ter (or 54% of dry bagasse), limitations were imposed by both fermenter design and fibre digestibility. All fermentat ions were substrate-limited up to the max imum initial concentrat ion examined of 50 g bagasse . 1-1 and no product inhibition was evident (up to 260 m M VFA produced). Maximum V F A productivities of 0.25 to 0 . 6 5 g - 1 - 1 . h -1 were obtained in batch fermentat ions and this is greater than those previously repor ted using lignocellulosic substrates. Batch fermentat ions neared complet ion after 66 h.

Unmodif ied bagasse has a digestibility in vitro of about 0.25 g • g - l , has less than 0.15 g • g-1 of water- soluble substances, and has a low concentration of nutrients essential to microbial growth. Consequent- ly, the yield of volatile fatty acids (VFA) f rom bagasse is low unless the material is pre t reated to allow easier access by cellulolytic bacteria to the plant cell components . Addit ional nutrients are also needed for microbial utilisation of bagasse.

This paper reports the conditions required for fermentat ion of bagasse by mixed populations of rumen bacteria. The quantities of lignocellulose degraded by the bacteria, and the concentrations of V F A produced were measured for bagasse substrates which had been subjected to various pretreatments.

Introduction

Ferment ing lignocellulosic material to acetic, propionic and butyric acids is proposed as a means for producing bulk chemicals and liquid fuels (Langwell 1932; Depasse 1945; P6aud-Leno61 1952; Sanderson et al. 1978; Playne 1980; Dat ta 1981). Advantages of this approach are: the substrates are cheap and are not used for human food; chemical hydrolysis of the substrates is not required, although physical and/or chemical p re t rea tment may be often necessary to improve fibre digestibility; mixed cultures of anaerobic bacteria can be used which enable heteroge- nous substrates to be more completely utilised while avoiding the costs of sterilisation; and xylose, derived f rom the hemicellulose fraction, can be fermented.

Bagasse, a waste product of the sugar cane industry, is a suitable lignocellulosic substrate.

Offprint requests to: M. J. Playne

Materials and methods

Preparation of the inoculum

Inocula were prepared from rumen contents of sheep maintained on a uniform diet of grass and legume hay. Fresh rumen content was collected for each fermentation experiment. The rumen liquid was strained through a double layer of cheesecloth, and a volume equal to about 30% (v/v) of the final fermenter culture volume was centrifuged at 10,000g for 20 min at 4 ° C. The pellet was resuspended in a reduced volume (33% of the original volume) of an anaerobic dilution solution (Bryant and Burkey 1953).

Preparation of the substrate

Bagasse was obtained from sugar cane (variety NCO 310) processed at the Racecourse Mill, Mackay, Queensland. It was air dried immediately and stored at 20 ° C. Samples were milled through a 2.25 mm aperture screen (untreated bagasse), or were ammoniated, after milling, in sealed bags for 35 days at 20 ° C using 50g NH 3 (aqueous) per kg dried bagasse (ammoniated bagasse).

C. H. Matei and M. J. Playne: Production of volatile fatty acids 171

Composition of the substrates

Untreated bagasse contained (as g/kg dry matter): neutral detergent soluble organic matter (NDS-OM), 123; hemicellulose, 303; cellulose, 413; lignin, 100; ash, 61; total nitrogen, 3.9; phosphorus, 0.16; calcium, 0.30; and sodium, 0.09. Ammoniated bagasse contained: NDS-OM, 164; hemicellulose, 236; cellulose, 438; lignin, 104; ash, 57; and total nitrogen, 21.0.

matter (DM) weights from consecutive 10 ml samples. The 20 ml suspensions taken at each sampling time were centrifuged at 10,000g for 10 min at 4 ° C. The supernatant was decanted and stored for VFA analysis, and the residue was resuspended in 100 ml of neutral-detergent solution (Goering and Van Soest 1970) for fibre analysis. The second 20 ml suspension was used to determine DM content of the sample, and to provide material for total nitrogen and in vitro digestibility determinations.

Batch fermentations

Apparatus. Batch cultures were made using 21 glass culture vessels (Coming FV2L) fitted with flat flange and multisocket lids (Coming MAF 3/52). Culture temperature was maintained at 39 ° C by placing the vessels in a controlled temperature water bath. The culture was mixed by a magnetic follower (76 x 13 mm) which was turned by a Janke-Kunkel magnetic stirrer (model REO). The pH of the medium was maintained at pH 6.8 using a combined pH electrode (200 x 12 ram) made by Titron Pty Ltd, Melbourne, coupled to an EIL pH controller (model 2838, Kent Instruments Ltd) and a peristaltic pump (Minipump, 10 rpm, Sieper Pty Ltd) which dispensed alkali (2 M NaOH + 2 M KOH in 9 : 1 ratio) as required. An automatic pH switching unit was built to enable one pH meter to monitor and control up to four fermenters. Design details of this unit are available from the authors. A one-way gas release valve was fitted in the fermenter lid, and a gas supply line was fitted so that the fermenter contents could be sparged with an anaerobic gas mixture of 10% (v/v) CO2 and 90% (v/v) N2 (the gas mixture contained less than 10 ppm 02).

Procedure. A weighed amount of milled bagasse (42 or 85 g) was suspended in 1,400 ml of mineral salts solution, and then sterilised in the culture vessel (121 ° C, 20 rain). The mineral salts solution contained (as g .1-1 of final concentration): KH2PO4, 2.0; K2HPO4, 2.0; (NH4)2SO4, 1.0; MgSO 4 - 7 H20, 0.4; CaCI2 • 2 H20 , 0.1; and FeSO4 • 7 H20 , 0.003 g • 1-1. After the solution was cooled to about 50 ° C, the fermenter was assembled. The contents were then gassed with the anaerobic gas mixture for 30 min, and supplemented with 8.5 ml of a 20% (w/v) sterile aqueous solution of yeast extract (Oxoid L21), and 20 ml of a sterile VFA solution consisting of isobutyric (2raM), n-valeric (2 mM), isovaleric (2 raM) and 2-methyl butyric acid (2 raM). Cysteine HC1 • H20 (5 g) was neutralised to pH 7 with NaOH (1 M), made up to 200 ml with water, and sterilised at 121°C for 15 rain. 17ml of this solution was added to the fermentation solution to act as reducing agent. Resazurin (1 g) was made up to 1 1 with water, sterilised, and 6 ml of this solution was added to act as a redox indica- tor.

The fermentation medium was adjusted to 39 ° C, and to pH 6.8 with 2 M NaOH or 2 M HC1 (sterile), and was then inoculated with a 250 ml suspension of rumen micro-organisms prepared as described above. The final working volume in the fermenter was adjusted to 1,750 ml with sterile mineral salts solution. The culture was rapidly stirred to suspend bagasse particles uniformly, and sampled. Between sampling times, stirring was reduced to 80 rpm which was just sufficient to maintain movement of the bagasse particles. The culture was gassed (10 ml • min -1) during the first 17 h of fermentation, but subsequently only for 15 rain during and after sampling.

Sampling. Samples (2 x 20 ml) were taken at 0, 17, 24, 41, 48, 72, 96, and 168 h. Representative sampling of the suspension was achieved by increasing the stirring speed, and then removing samples rapidly with a 10 ml open-ended sterile pipette attached to a Volac 10 ml pipette regulator (J. Poulten Ltd, Essex, UK). This method was found to give less than 1% difference between dry

Chemical analysis

VFA. Individual and total VFA were measured using a Packard Model 427 gas chromatograph fitted with a flame ionisation detector, coupled to a Hewlett-Packard Model 3380A integrator. Samples were prepared as described by Holdemann et al. (1977) and 1 ml of ether extract was injected onto a column of 15% Supelco 1220 - 1% H3PO 4 on 80/100 mesh Chromosorb W-AW (Supelco Inc., Bellefonte, PA). An internal standard of 4-methyl n-pentanoic acid was used. The chromatographic conditions used were the same as those of Britz and Wilkinson (1979).

Fibre. Neutral-detergent fibre (NDF), which contained the cellulose, hemicellulose and lignin fractions of the bagasse, was measured by the method of Goering and Van Soest (1970). When dry samples were used, a weighed amount (0.3 g) was added directly to a 100 ml neutral-detergent solution. Acid-detergent fibre (Goering and Van Soest 1970) was determined on some samples to enable calculation of hemicellulose, cellulose and lignin contents. All residues were ashed (550 ° C, 2 h) to allow all results to be expressed as grams of organic matter (g O.M.). Thus, NDF is expressed on an ash-free basis as neutral-detergent fibre organic matter (NDF-OM).

Digestibility. A modification of the pepsin-cellulase bioassay of McLeod and Minson (1978) was used to determine fibre digestibility (Playne 1984). The cellulase enzyme was Onozuka 3S (Yakult Biochemical Co., Japan). Digestibility was normally only determined on samples before fermentation.

Redoxpotential (Eh). A combined electrode assembly containing a Ag/AgC1 reference electrode in KC1 (3 tool. 1-1) and Pt wire (Metrohm EA 217) was calibrated using a Metrohm pH-mV meter (model E603) and saturated solutions of quinhydrone in pH 4 and pH 7 buffers at 20 ° C. The Eh for these solutions was +258 and +85 mV respectively with an acceptable deviation of +5 inV. The electrode, after calibration, was rinsed with 70% (v/v) ethanol prior to insertion into the fermentation solution. During Eh measurement, stirring was stopped, but gassing of the fermenter headspace with N2-CO~ gas mixture continued throughout the measurement.

Results and discussion

Rumen fluid was used as the inoculum because it was a convenient source of a relatively constant popula- tion of cellulolytic and hemicellulolytic anaerobic bacteria. A simple medium containing mineral salts, yeast extract and branched-chain VFA was adequate to achieve efficient cellulolysis of bagasse by these organisms, provided pH was maintained at pH 6.8 and Eh at -300 mV. Direct enumeration of total, viable, and cellulolytic bacteria were not made

172 C.H. Matei and M. J. Playne: Production of volatile fatty acids

IO0

8O

60

X3 40

O I [a- " 20 Z

0 I I 0 150 180

. ~ 0 ~ ~, ,-0

/ I I I I

30 60 90 120

Time (h)

Fig. 1. P r o p o r t i o n of N D F - O M u s e d v t ime. Subs t ra te : a m m o - n i a t ed bagas se at 25 g • 1-1 (@) and 50 g • 1-1 (©) concen t ra t ions . E a c h p o i n t on the curve is a m e a n of th ree ba t ch f e rmen ta -

t ions

.-..

tJ 0.8

c~ v

0.6

g 0.4

0 I m 0.2 a z "8

0

:/./ I I I I

0 I0 20 30 40

Init ial NDF-OM concB (gL - I}

F i g . 4. R a t e s of N D F - O M d e g r a d e d dur ing first 50 h f e r m e n t a t i o n v in i t ia l c o n c e n t r a t i o n of N D F - O M

250

~ o o I ~...o 200 O~ /

O

£ ,so / z, /0 I/O • =o O/ • 0~ 6 ioo

> 50

0 I I I I I I 0 30 60 90 120 150 180

Time {h)

Fig. 2. V F A p r o d u c e d f rom a m m o n i a t e d bagasse at 25 g • 1-1 ( t ) and 50 g • 1-1 (©)

:50 /.// O0 o E

10 ~ 50

2 e~o o o

m 50 > >

I I I I 5 I0 15 20

NDF-OM digested (g) / / I I I I I I

0 5 I0 15 20 25 30

Total DOM digested (g)

Fig. 5. The relationship between VFA produced and OM degraded at 66 h of f e r m e n t a t i o n

20 B __________i _._.____-B -, i / 34g!_

/ 15 • A

/ / ' 2 4 gL -~

i

z

5 ~l [.I I ~I 8gL- ' II1,, i t l l ~

I I1 ,/ 0 V / / ~ - I I I I I I

0 30 60 90 120 150 180

Time (h)

Fig . 3. A m o u n t s of N D F - O M d e g r a d e d v t ime for va r ious in i t i a l c o n c e n t r a t i o n s of 1 1 v o l u m e of N D F - O M in a f e r m e n t a t i o n

because preliminary work showed that a significant proportion of bacteria were strongly adherent to bagasse particles and resisted removal even when samples were treated with methyl cellulose (Minato and Suto 1978), or with detergents, or were macer- ated (Leedle and Hespell 1980). Instead, we measured microbial activity indirectly by measuring solubilisation of the NDF fraction of bagasse, and by the amounts of VFA formed.

The solubilisation of NDF-OM during the course of batch fermentations of ammoniated bagasse (at 25 and 50 g .1-1 ) is shown in Fig. 1. There was no difference in the proportion of NDF used which indicated that the fermentation was substrate limited. For the 25 and 50 g . 1-1 fermentations, 82.6 and 84.1% of hemicellulose and 54.4 and 63.0% of cellulose were utilised respectively with the production of approximately 130 and 240 mM VFA (Fig. 2). The proportions of individual VFA were quite constant (acetic, 60 - 68; propionic, 18- 25; n-butyric,


Table 1. Digestibility of bagasse pre-treated in various ways before fermentation, and amounts of VFA produced per unit digestible OM. Substrate concentration was 50 g bagasse DM per litre

Substrate and treatment a Initial Pepsin cellulase NDF-OM digested VFA/g DOM b NDS-OM OM digestibility in fermenter by 66 h (as g VFA) (%) (%) (%)

1. Untreated bagasse 12 2. NH3-bagasse 13 3. NH3-bagasse, 3% NaOH 27 4. Untreated bagasse, steam exploded 21 5. Untreated bagasse °, Ca(OH)2, Na2CO3 23

26 43 0.72 45 58 0.73 72 82 0.46 55 73 0.71 66 76 0.74

a For details, see Playne (1984) b Digestible organic matter = NDF-OM digested + 0.9 (initial NDS-OM) c Bagasse (85 g DM) + 1,360 ml slurry containing 6.8 g Ca(OH)2 and 8.6 g Na2CO 3. Treatment 5 was autoclaved 1 h at 121 ° C immediately

(at pH 12) and then neutralised

8 - 1 3 molar %). The redox potentials of these cultures were similar and ranged between - 3 1 4 and - 4 4 8 mV. The most reduced conditions were present immediately after inoculation (mean - 4 0 9 mV), and the least reduced conditions were found after 188 h fermentat ion (mean - 3 3 6 mV). These values are similar to those found by Dat ta (1981) during anaerobic fermentat ion of corn stover.

A fermentat ion containing 50 g • 1-1 ammonia ted bagasse had 108 m g . 1-1 'Captan ' (as 83% active ingredient; Hort ico Ltd) added. It has been claimed that this fungicide increases cellulolytic activity in the rumen (Theuninck et al. 1981). No effect on the amounts of N D F solubilised or of VFA produced was found.

Using ammonia ted bagasse, the effect of substrate concentrations between 7 and 34 g -1-1 of initial N D F - O M on N D F solubilised and V F A produced were examined (see Figs. 3 - 5 ) . Substrate concentrat ion was still the factor limiting fermentation, and no product inhibition by the VFA occurred at the highest substrate concentration (see linearity of Figs. 4, 5). It was not possible to examine higher substrate concentrations of ammonia ted bagasse because the stirring system of the fermenter was not able to mix slurries containing above 60 g . 1-1 of milled bagasse. Adequate mixing was essential if representat ive samples were to be obtained at intervals during the fermentat ion.

The amount of bagasse that could be degraded also depended on the effectiveness of the pretreat- ment of the substrate. Pre t rea tment was designed to solubilise N D F and to make the fibre more accessible to micro-organisms. Playne (1984) examined a range of pre t rea tment methods which were superior to the simple ammonia t ion pre t rea tment described here in terms of improved digestibility. The fermentat ion characteristics of some of these pre t rea tments were examined (for details, see Table 1). Digestibility of N D F - O M up to 82% was achieved with substrate

concentrations of 50 g • 1-1. These digestibilities were markedly higher than the total OM digestibilities predicted f rom pepsin cellulase bioassays. This may have been because the rumen bacterial cellulases were more effective than the fungal cellulase complex used for the bioassays. Production of V F A per unit of digestible OM was quite constant, and the yield was high (up to 74% by weight). Proport ions of individual VFA did not vary between t reatments and were similar to those found previously.

Schwartz and Keller (1982) consider that a V F A productivity of greater than 5 g - 1 - 1 , h -1 and a fermentat ion p H of 4.5 or lower are required for economic viability of their proposed process in the U S A to produce acetic acid by fermentat ion of glucose with a monocul ture of Clostridiurn thermo- aceticum. To achieve this productivity, they believe a dilution rate of 0.1 h -1 and an acid concentration in the fermenter of 50 g • 1-1 (830 mM) are necessary. Torre and G o m a (1981) fermented sucrose at p H 6 with a mixed culture and achieved a productivity of 1.7 g . 1-1. h -1 and a VFA concentration up to 42 g . 1-1. With batch cultures, they only achieved productivities of 0.17 g • 1-1 • h -1. We have obtained productivities of between 0.25 and 0.65 g • 1-1 • h -1 during the first 40 h of our batch fermentat ions of bagasse by mixed rumen cultures.

For experimental convenience, the bagasse was suspended in mineral salts solution in the fermentation vessel and sterilized in an autoclave at 121 ° C for 20 min. This procedure may have enhanced the digestibility of the bagasse by acting as a steam/pres- sure pre t rea tment . Thus, the digestibility of bagasse before and after sterilization was examined. Pep- sin-cellulase digestibility of untreated bagasse was found to be 23.5 + 3.9% prior to autoclaving and 21.7 + 2.9% after autoclaving. Values for ammonia ted bagasse were 43.3 + 2.3% and 44.2 _+ 1.6% respectively. Similar results were obtained in dupli- cate fermentat ions of untreated bagasse at 50 g • 1-1

174 C. H. Matei and M. J. Playne: Production of volatile fatty acids

initial concentrations of substrate. The fermentation patterns of both sterilized and non-sterilized cultures were the same, with the fermentation complete by 80 h with 42% of NDF-OM digested and 210 mM of VFA present in both treatments.

A fed-batch fermentation was conducted over 9 days with a substrate concentration of 25 g • 1-1 of ammoniated bagasse which was maintained by a twice-daily introduction at approximately 12 h intervals of fresh sterile substrate and media (200 ml) into the 1700 ml stirred fermenter, immediately after removing 200 ml from the fermenter. The retention time of the bagasse and of the liquid was 4.25 days. A steady-state degradation of lignocellulose was achieved after 48 h with a mean of 44% of NDF-OM being digested. Production of VFA became stable after 88 h with a concentration of 120 mM VFA in the fermenter.

Conclusions

A linear increase in VFA production occurred with increasing substrate concentrations up to 36 g • 1-1 of digestible OM fermented. Fermenter configurations different from the design used in this study are needed to test higher concentrations of bagasse. Product inhibition by VFA was not evident in this study, however VFA concentrations did not exceed 260 mM (about 18 g • 1-1), which was less than levels likely to cause inhibition at this pH of 6.8 (Playne 1981).

Yields of VFA per g digestible OM were high (72%) and were close to stoichiometric yields of VFA from glucose. Thus, pretreatments which allowed over 75% of NDF-OM to be digested will permit VFA weight yields of over 54% of the weight of bagasse used. These yields were obtained after 66 h fermentation, and are similar to yields obtained by Datta (1981) after 24 days fermentation. Sanderson et al. (1978) also have developed a VFA production process which has a long retention time in the fermenter in excess of 16 days.

The VFA productivities that we have achieved (0.25 to 0.65 g . 1-1 • h -1) are much greater than those previously achieved with a lignocellulosic substrate (e.g., Datta 1981), but they are considerably less than those obtained with sugar substrate, and the 5 g VFA • 1-1 • h -1 productivity considered necessary by Schwartz and Keller (1982) for an economic process. However, pretreated bagasse can be a cheaper substrate than monomeric sugars, and aseptic fermentation conditions are not required for mixed-culture fermentations. Hence, an economically-viable process may be developed at productivities lower

than that suggested by Schwartz and Keller (1982). In any case, it is likely that the most crucial unit cost in a VFA process will be the cost of separation of the acids from the fermentation solution (Smith and Playne 1982). The laboratory batch fermentation studies described here will provide a basis for further studies on the fermentation of bagasse in continuous culture systems, which incorporate 'on-line' product recovery.

Our present work is aimed at increasing the proportion of propionic acid produced by adding ionophores (e.g., 'Monesin') and altering hydrogen balance in order to improve yields of acids and energy recovery; and at carrying out fermentations at a lower pH to reduce costs of separation of acids from the fermentation broth.

Acknowledgements. The National Energy Research, Development and Demonstration Council of the Australian Government is thanked for financial support of this work. The authors are grateful to Dr R.G. Wilkinson of the University of Melbourne for helpful discussions, ideas and facilities; Mr D. Foster, of the Sugar Research Institute, Mackay, Queensland for donating the bagasse; Mr L. Alexander for the design and construction of the pH switching unit; Dr H. Mamers for helping prepare steam-exploded bagasse; and to Miss M. Reilly for fibre analyses.

References

Britz ML, Wilkinson RG (1979) The isolation and properties of metronidazole-resistant mutants of Bacteriodes fragilis, Anti- microb Agents Chemother 16 :19-27

Bryant MP, Burkey LA (1953) Cultural methods and some characteristics of some of the more numerous groups of bacteria in the bovine rumen. J Dairy Sci 36:205-217

Datta R (1981) Acidogenic fermentation of corn stover. Biotech- nol Bioeng 23 : 61-77

Depasse E (1945) Vue d'ensembte d'une production industrielle de c6tones. Bull Assoc Chim Sucr Distill Fr 62:317-339

Goering HK, van Soest PJ (1970) Forage fiber analyses. USDA Agriculture Handbook No 379

Holdeman LV, Cato EP, Moore WEC (1977) Anaerobe laboratory manual, 4th edn. Anaerobe Laboratory, Virginia Poly- technic Inst, State University, Blacksburg, Virginia

Langwell H (1932) Cellulose fermentation. Chem Ind 51 : 988-994

Leedle JAZ, Hespell RB (1980) Differential carbohydrate media and anaerobic replica plating techniques in delineating carbohydrate-utilizing subgroups in rumen bacterial populations. Appl Environ Microbiol 39:709-719

McLeod MN, Minson DJ (1978) The accuracy of the pepsin-cellulase technique for estimating the dry matter digestibility in vivo of grasses and legumes. Anim Feed Sci Technol 3 : 277-287

Minato H, Suto T (1978) Technique for fractionation of bacteria in rumen microbial ecosystem. II. Attachment of bacteria isolated from bovine rumen to cellulose powder in vivo and elution of bacteria attached therefrom. J Gen Appl Microbiol 2 4 : 1 - 1 6

P6aud-Len6el C (1952) La production d'acides gras volatils par fermentation. Ind Agric Aliment 69:211-220


Playne MJ (1980) Microbial conversion of cereal straw and bran into volatile fatty acids - key intermediates in the production of liquid fuels. Food Technol Aust 32:451-456

Playne MJ (1981) Volatile fatty acid production by anaerobic fermentation of ligno-cellulosic substrates. In: Advances in biotechnology, vol 2. Proc 6th Internat Fermentation Symp (London Canada, July 1980). Pergamon Press, New York, pp 85-90

Playne MJ (1984) Increased digestibility of bagasse by pretreat- ment with alkalis and steam explosion. Biotechnol Bioeng 26 : 426- 433

Sanderson JE, Wise DL, Augenstein DC (1978) Organic chemicals and liquid fuels from algal biomass. Biotechnol Bioeng Syrup 8:131-151

Schwartz RD, Keller FA, Jr (1982) Isolation of a strain of Clostridium thermoaceticurn capable of growth and acetic acid production at pH 4.5 Appl Environ Microbiol 43:117-123

Smith BR, Playne MJ (1982) Recovery of volatile fatty acids from fermenter effluents and their conversion to liquid fuels. Proc 1st ASEAN Workshop on Fermentation Technology, Kuala Lumpur (ASEAN Committee on Science and Technology), pp 510-524

Theuninck DH, Goodrich RD, Meiske JC (1981) Influence of Captan on in vitro and in vivo digestibility of forage. J Anim Sci 52 : 377- 381

Torre I de la, Goma G (1981) Characterization of anaerobic microbial culture with high acidogenic activity. Biotechnol Bioeng 23:185-199

Received December 29, 1983

Documents

Production of volatile fatty acids from bagasse by rumen bacteria