APPnr MICROBIOLOGY, Apr. 1975, p. 510-514 Vol. 29, No.

4
Copyright 0 1975 American Society for Microbiology Printed in U.S.A.

Microbial Fermentation of Rice Straw: Nutritive Composition

and In Vitro Digestibility of the Fermentation Products
YOUN W. HAN I
Western Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture,
Berkeley, California 94710
Received for publication 18 November 1974

Rice straw was fermented with Cellulomonas sp. and Alcaligenes faecalis.
Microbial cells and undigested residue, as well as chemically treated (NaOH or
NH4OH) and untreated straws, were analyzed for nutrient composition and in
vitro digestibility. In a typical fermentation, 75% of the rice straw substrate was
digested, and 18.6% of the total substrate weight that disappeared was recovered
as microbial protein. The microbial cell fraction was 37% protein and 5% crude
fiber; the residue was 12% protein and 45% crude fiber. The microbial protein
amino acid profile was similar to alfalfa, except for less cysteine. The microbial
cells had more thiamine and less niacin than Torula yeast. In vitro digestibility
of the microbial protein was 41.2 to 55%; that of cellulose was 52%.
The major limitations of straw as an animal 7-mm screen, was used as a substrate. Part of the
feed are low digestibility and low protein con- chopped straw was treated with 4% NaOH solution
tent. Efforts have been made to increase the (10:1, water-straw) at 100 C for 15 min, the excess
feed value of cereal straws by chemical and caustic liquid was expressed, and the treated straw
was oven-dried (60 C). The other part of the straw
physical treatments, as well as nutrient sup- was sprayed with aqueous ammonia (5 N NH4OH) in
plementation. The digestibility of various crop the amount of 5.2% of the substrate, and kept at am-
straws can be increased by treating with NaOH bient temperature for 30 days before being subjected
or NH4OH, but the low protein still requires to microbial fermentation (20).
nitrogen supplementation. Additions of urea, Fermentation. Fermentations were carried out at
molasses, branched-chain fatty acids, sulfur, 35 C in 40-liter Humfeld fermentors (13). The fermen-
and other minerals have met with varying tation mixture was agitated (140 rpm) and aerated
success (2, 4, 5, 6, 12, 17). (0.5 vol of air/vol of medium per min) with sterilized
Recently efforts have been made to produce air. The medium (20 liters) contained 3 to 5% (dry
weight) of rice straw. Separately grown active cultures
high-protein animal feed by microbial fermen- of Cellulomonas sp. and A. faecalis were inoculated.
tation of cellulosic substrates (9, 10). We now

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Fermentation continued for 3 to 5 days.
report the nutritive composition and in vitro di- In vitro digestibility. In vitro cellulose digestibil-
gestibility of the products of microbial fermen- ity was determined by measuring disappearance of
tation of rice straw. substrate insoluble dry matter upon treatment with
MATERIALS AND METHODS cellulase ("Onozuka" SS, Kanematsu-Gosho, Ltd,
New York) and protease ("Pronase" B grade, Calbio-
Microorganisms, media, and growth. A species chem, Los Angeles, Calif.), and was expressed as total
of Celluomonas and Alcaligenes faecalis, isolated by solubles after enzymes. In vitro protein digestibility
the author, were used. Their cultural characteristics was defined as the decrease in substrate nitrogen after
and conditions for growth have been reported else- treatment with fungal protease (Streptomyces gri-
where (9, 10). The growth medium contained seus) and chick pancreas acetone powder (18).
(NH) S04 (6.0 g); KHPO4 (1.0 g); KHPO4 (1.0 g); Rumen metabolism. Rumen microbes were ob-
MgSO4 (0.1 g); CaCl2 (0.1 g); yeast extract (0.5 g); tained from a fistulated sheep that was maintained on
FeCl, .6H20 (16.7 mg); ZnSO4.7HO (0.18 mg); alfalfa hay with free access to dried range grass.
CuSO4.5H20 (0.16 mg); CaCl2 (0.18 mg); ethylene- Susceptibility of substrates to rumen microbial attack
diaminetetraacetic acid (20.1 mg); and 10 to 50 g of was determined by measuring total gas and volatile
cellulosic substrate per liter of distilled water. fatty acid (VFA) production. VFA samples acidified
Substrate. California rice straw (Oryza sativa Lin- with meta-phosphoric acid were analyzed on an
naeus), field-dried and chopped to pass through a Aerograph 204 gas liquid chromatograph. The rate of
IPresent address: USDA, ARS, Western Region, Depart- ruminal fermentation was measured by anaerobic
ment of Microbiology, Oregon State University, Corvallis, Warburg procedures and described by Oh et al. (17).
Ore. 97331. Vitamin, amino acids, and chemical analysis.
510
VOL. 29. 1975 MICROBIAL FERMENTATION OF RICE STRAW 511
All vitamin aasays were microbiological: thiamine crobial cell precipitate had been produced. The
and niacin by the methods of Gyorgy and Pearson (8), residue fraction (mainly undigested straw plus
folic acid by the method of Jukes (14), and pyridoxine some microbial cells) was 12% protein and the
by the Association of Official Analytical Chemists (1). microbial cell fraction (mainly microbial cells, a
Amino acids were analyzed by ion-exchange chroma- small amount of fine fibers, and precipitated
tography after 10 mg of protein was hydrolyzed with
10 ml of 6 N HC1 under vacuum at 110 C for 24 h. minerals) was 37% protein. Thus, 84 g of protein
Cystine plus cysteine was determined after pretreat- (18 g in the residue and 66 g in the cells) was
ment with performic acid. Lignin was determined obtained by digesting 450 g of the initial 600 g of
according to Van Soest (19). Protein was calculated rice straw, a net protein yield of 18.6%.
by multiplying the difference between total nitrogen Table 1 lists the chemical composition of the
and NH8-nitrogen by 6.25. All other chemical analy- rice straw substrates and products of their
ses, unless otherwise specified, were carried out ac- microbial fermentation. The untreated rice
cording to the Association of Official Analytical straw contained 0.67% nitrogen, 29.8% crude
Chemists methods (1). fiber, and 15.8% silica that accounted for most
of the 18.6% ash. NaOH treatment removed
RESULTS AND DISCUSSION about half of the silica, probably by solubiliza-
When a mixed culture of Cellulomonas sp. tion of the amorphous silica. It also removed the
and A. faecalis was grown on rice straw, sub- reducing sugars. The ammonia treatment in-
strate utilized depended on the initial substrate creased both total and NH3-nitrogen, possibly
level, inoculum size, fermentation time, and by formation of ammonium salts and amides
substrate pretreatment. Figure 1 shows mate- of cellulose. Ammonia treatment, however, re-
rial balance of a typical fermentation of NaOH- moved silica less than NaOH.
treated straw. After 3 days, 75% of the initial The undigested fermentation residue was
substrate had been digested, 150 g of undigested about 12% protein and 45% crude fiber. This
fermentation residue remained, and 178 g of mi- level of protein was equivalent to that obtained

2L Cultu re Medium
19. 5L Mineral soln.
6OOg NaOH-treated rice straw
rCellulomonas sp. & A. faecalis

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days, 35 C
Itration a

150 Residue 18L FiltrateJ

18g protein 66g protein
Centrifugation b
.-

178g precipitate |18L Effluent|

132g cells c
46g silica, fiber, etc.
FIG. 1. Schematic diagram of rice straw fermentation. Weights expressed on dry matter bases. (a)
Filtration by passing through two layers of cheese cloth; (b) centrifugation by Sharples centrifuge at 50,000
rpm; (c) cell mass determined on the basis of 50% protein in the cell.
512 HAN APPL. MICROBIOL.
TABLE 1. Chemical composition of the fermentation products (percentage of dry weight)
Rice straw Rice straw Udgse irba
Component Rice straw
(untreated) (NaOH
treated)
(NH4OH
treated)
Undigested
reiu Microbial
cla

Total N 0.67 0.52 1.59 2.30 6.65

NH,-N 0.03 0.03 0.43 0.24 0.51
Ether extract 1.24 0.81 1.03 1.48 0.93
Crude fiber 29.8 32.5 34.5 45.0 5.71
Lignin 3.72 4.34 4.96 9.24
Reducing sugar 0.55 0 0.23 0.10 1.63
Ash 18.58 21.84 15.95 20.69 25.09
Silica 15.8 9.9 13.4 16.9 19.9
Ca 0.12 0.19 0.19 0.21 1.37
P 0.10 0.10 0.07 0.41 0.98
S 0.14 0.08 0.08 1.02 0.43
'From fermentation of NaOH-treated straw.
by urea (2.5%) supplementation of straw (6). TABLE 2. Amino acid composition of microbial
The increased crude fiber in the residue may protein (g/100 g of protein)
have been from preferential microbial utiliza-
tion of readily digestible carbohydrates, leaving Amino acid Cellulo-
mornsa ligenesa Mixed
Alca- culture Alfalfac
cellulose and lignin. Lignin in the residue in-
creased from 4.3% to 9.2%. Thus, 53% of the ini- Lysine 8.00 9.92 6.62 6.70
tial lignin was recovered in the residue, and the Histidine 2.96 2.53 2.13 2.53
rest was solubilized in the effluent. The digesti- Arginine 6.18 4.85 6.82 5.54
bility lowering effect of lignin may be caused by Aspartic acid 8.30 9.15 10.67 12.54
Threonine 4.73 4.46 5.28 5.12
its chemical complex formed with cellulose. Serine 4.11 3.44 3.75 5.25
Therefore, the lignin in the residue, which may Glutamic acid 18.49 17.08 14.47 11.30
be partially dissociated from the plant cell walls Proline 7.51 4.46 4.71 5.10
by mechanical and microbiological action dur- Glycine 4.17 5.16 6.51 5.73
ing the fermentation, should not be as detri- Alanine 8.12 8.94 11.07 6.33
mental as the native lignin in the straw. Valine 6.79 7.01 7.15 6.70
Ca, P, and S in the undigested residue were 2, Methionine 1.69 2.70 1.45 1.96
4, and 7 times, respectively, those in the un- Isoleucine 4.12 5.40 4.11 5.54
treated rice straw. These minerals are especially Leucine 8.66 7.64 8.70 8.43
low in rice straw, and their increase could be an Tyrosine 2.41 3.06 2.98 3.72
Phenylalanine 3.69 4.11 3.76 5.75

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additional benefit from fermentation. Cystine/cysteine 0.41 0.47 0.54 1.40
The microbial cell fraction was 38% crude
protein, 5.7% crude fiber, 25% ash, and 0.9% a Cultures of Cellulomonas sp. and A. faecalis were
crude fat. The high ash may be due to silica and grown on Trypticase soy broth.
salt accumulation during neutralization of alk- b Mixed culture of Cellulomonas sp. and A. faecalis
ali-treated substrates. Membrane filtration grown on rice straw.
only reduced ash to 23.4% and silica to 15%. As cData from Livingston et al. (15) was converted to
a comparison, the same microbial species grown
g/100 of g protein base.
on Trypticase soy broth (BBL) contained 11.9%
ash and 1% silica. Although silica is nutrition- TABLE 3. Vitamin content of microbial cells
ally inert, its abrasiveness may damage the (/g/g of cell)
digestive tract of animals. More than 50% of the Vitamin Microbial cells" Torula yeastb
silica in the filtrate of fermentation mixture
could be separated from cells by allowing the Folic acid 20.0 21.5
mixture to stand for a few hours at room Thiamine 36.0 5.3
temperature. Niacin 222.0 417.3
Table 2 compares the amino acid composition Pyridoxin 35.4 33.4
of microbial and alfalfa protein. The essential aMixed culture of Cellulomonas sp. and A. faecalis
amino acid profiles of Cellulomonas sp. and A. grown on rice straw.
faecalis were similar, except for more methio- I
Bressani (3).
VOL. 29, 1975 MICROBIAL FERMENTATION OF RICE STRAW 513
TABLE 4. In vitro digestibility of the fermentation products
Protein Crude fiber
Product Contenta Digestibilityb Contenta Digestibilityb
(%) (%) (%)(%)
Rice straw 4.2 29.8 30.3 0.50
NaOH-treated straw 3.1 32.5 73.2 ± 0.27
NH4OH-treated straw 7.2 34.5 57.4 X 0.41
Fermentation residue' 12.8 55.5 :1 0.69 45.0 51.7 0.44
Microbial cells 38.2 41.6 0.25 5.7
a Percentage of dry matter.
b Means and standard deviation.
cFrom fermentation of NaOH-treated straw.

nine in the latter. Organisms grown on Trypti- TABLE 5. Ruminal metabolism of the products
case soy broth contained a higher essential
amino acid content than those grown on the Gas
production Net VFA
straw-mineral solution. The amino acid profile Product (mmol/8 h/ production
of the mixed culture grown on rice straw was 10 g of rumen (mg/100 ml)
similar to that of alfalfa, except for less cys- content)
teine. Eighty percent of total nitrogen in mi- Rice straw 0.799 207
crobial cells was recovered as protein nitrogen; NaOH-treated straw 0.977 378
the rest was the nucleic acids and other nitroge- NH4OH-treated straw 0.885 345
nous compounds. Fermentation residuea 0.579 312
Table 3 compares the vitamins in microbial Control (alfalfa) 1.307 409
cells grown on straw with Torula yeast (Candida
utilis). The microbial cells contained more thia- aFrom fermentation of NaOH-treated straw.
mine and less niacin than yeast. Folic acid and
pyridoxin in the microbial cells were equivalent products from straw fermentation produced as
to Torula yeast. Thus, microbial cells from much gas or VFA as alfalfa.
straw fermentation may provide vitamins as Thus, the laboratory data indicate that the
well as protein. microbial cell fraction, with its high protein
The in vitro digestibility of protein in the content, may be used as a protein source for the
fermentation products ranged from 41% to 55% nonruminants, and the fermentation residue for
(Table 4). This relatively low protein digestibil- the ruminants.
ity may be attributed to microbial cell walls,

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since Yang (21) has shown that sonication of ACKNOWLEDGMENTS
microbial cells improved their protein utiliza- I acknowledge the assistance of J. H. Oh of the Uni-
tion by animals. Crude fiber digestibility (total versity of California Hopland Field Station, who performed
solubles after enzymes) was 30% for raw rice the rumen metabolism study; A. C. Waiss, who supplied
NHr-treated straw; and the members of Field Crops and
straw, 73% for NaOH-treated straw, and 57% for Chemical Physics Laboratories of WRRL, ARS, who per-
NH4OH-treated straw. Some of the digestible formed the chemical analyses.
matter in straw was apparently used by micro- LITERATURE CITED
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