APPLIED MICROBIOLOGY, Jan. 1974, p. 159-165Copyright 0 1974 American Society forf Microbiology

Vol. 27, No. 1Printed in U.S.A.

Cellulose Fermentation: Effect of Substrate Pretreatment on

Microbial GrowthY. W. HAN' AND C. D. CALLIHAN

Western Regional Research Laboratory, Agricultural Research Service, Berkeley, California 94710, andDepartment of Chemical Engineering, Louisiana State University, Baton Rouge, Louisiana 70803

Received for publication 12 September 1973

The effects of chemical, physical, and enzymatic treatments of rice straw andsugarcane bagasse on the microbial digestibility of cellulose have been investi-gated. Treatment with 4% NaOH for 15 min at 100 C increased the digestibilityof cellulose from 29.4 to 73%. Treatment with 5.2% NH3 could increasedigestibility to 57.0%. Treatments with sulfuric acid and crude cellulasepreparation solubilized cellulose but did not increase the digestibility. Grindingor high-pressure cooking of the substrate had little effect on increasing thedigestibility of cellulosic substrates by the Cellulomonas species.

Recently there has been much effort to de-velop a source of protein independent of agricul-tural land use. One outcome of these efforts isthe production of microbial protein (single cellprotein; SCP) from various substrates (9, 16, 22,24). Of these substrates, cellulose not only is themost abundant raw material but also is themajor constituent of municipal and agriculturalwaste. Thus, cellulose is considered to be anideal substrate for SCP production from thestandpoint of availability of the substrate andthe abatement of pollution. In spite of itsdesirability as a substrate, cellulose has notbeen as widely used as have hydrocarbons orother substrates for SCP production. The maindisadvantage in using cellulose is the difficultyof growing microorganisms on it.Even though the degradation of cellulose in

nature is extensive, the microbial degradationof cellulose in the laboratory is difficult andslow. In the laboratory, the growth of microorga-nisms on a cellulosic substrate depends largelyon the nature of the substrate. Whereas growthof microorganisms on native cellulose is sparse,growth on denatured or pretreated cellulose isabundant. Therefore, proper treatment of sub-strate before microbial fermentation is impor-tant for the success of the SCP production fromcellulosic substrates. Although various forms ofpretreatment of cellulosic materials have beenproposed (2-4, 6, 25), their effectiveness varies,depending on the substrate. Thus, optimalpretreatment must be established for each sub-strate.

' Present address: Department of Microbiology, OregonState University, Corvallis, Ore. 97331.

This paper reports the effectiveness of variouschemical, physical, and enzymatic pretreat-ments of rice straw and sugarcane bagasse onthe growth of cellulolytic microorganisms.

MATERIALS AND METHODSOrganisms, media, and growth. The cellulose-

digesting microorganisms used in this study wereCellulomonas and Alcaligenes species, and their char-acteristics have been reported elsewhere (10, 11). Thecomposition of growth medium was: (NH4),SO4 (6.0g); KHP04 (1.0 g); KHPO4 (1.0 g); MgSO4 (0.1 g);CaCl2 (0.1 g); yeast extract (0.5 g); FeCl3.6H20 (16.7mg); ZnSO4.7H20 (0.18 mg); CuSO4.5H30 (0.16mg); CoC12 (0.18 mg); ethylenediaminetetraaceticacid (20.1 mg); and 10 to 50 g of cellulosic substrateper liter of distilled water. The cells were grown inErlenmeyer flasks on a rotary shaker at 35 C. After theundigested substrate was removed, the cell concentra-tion was determined by turbidity measured with aKlett colorimeter and by the protein content deter-mined by a modified Lowry method (13).

Substrates. Rice straw and sugarcane bagasse wereused as test substrates in most of the experiments. Insome experiments, purified wood cellulose, filter pa-per, computer print-out paper, and wheat bran werealso used.

Samples of ground cellulosic substrates passedthrough a 20-mesh screen were mixed with variousconcentrations of alkali, acid, and other chemicalsolutions and stored with frequent agitation atvarious temperatures. A part of the slurry was with-drawn at frequent intervals and washed, with orwithout neutralization, to remove the reagents. Theexcess liquid was removed by squeezing the slurrythrough two layers of cheesecloth, and 1 to 5% dryweight of the recovered, treated substrates was incor-porated into the basal medium.X-ray diffraction patterns. X-ray diffraction pat-

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terns were determined by a diffractometer (GeneralElectric X-ray unit) fitted with a scintillation counterconnected to a scaler and a synchronized recorder.The X-ray beam (35 kV peak and 15 mA) from thecopper target was filtered through a nickel filter. Theground samples were pressed onto the sample holder,and the diffraction patterns were obtained within a5 to 500 range. An X-ray crystallinity index was ob-tained from the height-width ratio of the main peakat 20 = 220, as described by Baker et al. (1), or by theheight ratio of the peaks at 20 = 22° and 20 = 180, asdescribed by Segal et al. (21).

Digestibility. The digestibility of the cellulosicsubstrates was determined by measuring the disap-pearance of insoluble dry matter when the substrateswere treated with cellulase (Onozuka SS, Kane-matsu-Gosho, Ltd., N.Y.) and with protease (Pro-nase B grade, Calbiochem, Los Angeles, Calif.), andwas expressed as "total solubles after enzymes"(TSAE), according to the formula of Guggolz et al.(5). In some cases the digestibility of the substrate wasdetermined by measuring the amount of cell growthand disappearance of substrate after the fermentation.To express digestibility as a single index, the values ofTSAE, cell yield, substrate loss, and production ofsugars were averaged to give a digestibility index.Each method of measurement represented a differentkind of unit, so the weight of the values of eachmethod was standardized by subtracting the meth-od's means and dividing its standard deviation foreach different measurement. The crystallinity indexcorrelated poorly with other measurements, so it wasexcluded from the calculation.

Chemical analysis. The amount of total sugar wasdetermined by the phenol-sulfuric acid method (18),and the amount of reducing sugar was determined bythe dinitrosalicylic acid method (19). Lignin contentwas determined by the method used by Van Soest(27).

RESULTS AND DISCUSSIONAlkali treatment. Figure 1 shows the growth

of Cellulomonas sp. in alkali-treated sugarcanebagasse. The alkali treatment increased thegrowth rate and the maximal cell density of theorganisms. When sugarcane bagasse wastreated for 5 min with 0.5 to 10% NaOH, boththe microbial growth rate and the maximal celldensity increased in proportion to the increasedalkali concentration. When the alkali treatmentwas extended to 2 h, the growth rate did notincrease further. Therefore, there appears to bea limit to the increase in digestibility throughthe NaOH treatment.The NaOH treatment was also the best

method for improving the use of rice straw(Table 1). Rice straw treated with 4% NaOH for15 min at 100 C increased TSAE from 29.4 to73.0%. NaOH treatment also increased the cellyield of the mixed culture of Cellulomonas andAlcaligenes spp. by a factor of three.NH, treatment also improved the digestibil-

C

d.-

0

-0

-

MU

0

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3000

2500 - 5%

2000 - 1%

1500 - I / S A 0.5%

1000-

500 /°Soo0 2I I 0I I I00 20 40 60 80 100

TIME (Hours)

FIG. 1. Growth of Cellulomonas on alkali-treatedsugarcane bagasse. Substrate was treated with differ-ent concentrations of NaOH solution for 5 min atroom temperature.

ity of rice straw as indicated by the increase inTSAE, cell yield, and substrate loss (Table 1).The digestibility index of the NH,-treated sam-ple was 0.15; that of the NaOH-treated samplewas 1.5. There was no difference in digestibilityof NH,-treated rice straw, whether kept under0, or N,, indicating that the increase in thedigestibility was not due to oxidative hydroly-sis.The growth-promoting effect of the alkali

treatment could have been due to the produc-tion of soluble carbohydrates that are readilydigested by the organisms. However, little car-bohydrate was solubilized by the alkali treat-ment, and the soluble carbohydrate, if producedby the treatment, was drained off with thesupernatant (Table 2). Therefore, the growth-promoting effect of the alkali treatment mayhave been due to changes in the structure of thecellulose fiber. The alkali-treated and thor-oughly washed bagasse also supported growth ofthe organism to the same extent as did thealkali-treated but unwashed bagasse.Acid treatment. Various methods of acid

hydrolysis reported (8, 12, 14) fall into one of thefollowing categories: (i) a dilute acid hydrolysiswithout separation of the products as they areformed; (ii) a percolation process that continu-ously removes the products as they are formed;and (iii) a concentrated-acid process followedby a dilute-acid hydrolysis. We applied the lastprocess in treating the sugarcane bagasse firstwith 50% HSO4 and then with dilute (0.5, 1, 2,and 10%) HSO4. Sugar production was maxi-mal when the bagasse was treated for 15 minwith 50% H2SO4 at 121 C, followed by dilutionof acid to 1% and heating for 15 min at 121 C.

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TABLE 1. Effect of chemical treatment on the relative digestibility of rice straw

TSAE Cell yielda Substrateb Reducingc Crystal- Digesti-Treatment % loss (% of sugar (OD linity bility

C C + A initial) at 640) index index

Control (untreated) 29.4 0.44 0.60 27.5 0.14 5.3 - 1.365Steam, 160 C, 4 h 61.5 0.86 0.76 37.0 0.18 6.7 0.003Propylene glycol, 160 C, 4 h 69.0 0.86 0.90 38.5 0.17 8.35 0.1993% NaOCI, 130 C, 4 h 54.2 0.42 0.51 39.0 0.16 3.68 -0.7823% NaCI, 160 C, 4 h 65.5 0.76 0.48 31.0 0.15 4.36 -0.4453% Urea, 160 C, 4 h 64.1 0.70 1.10 0.17 7.05 0.0435% Na2B404, 160 C, 4 h 78.6 0.32 0.30 22.0 0.18 7.5 -0.3423% HCl, 130 C, 4 h 59.3 0.30 0.30 28.5 0.05 5.3 - 1.4383% H2SO4, 130 C, 4 h 53.5 0.30 0.30 26.0 0.07 5.4 - 1.4943% HNOS, 130 C, 4 h 59.9 0.50 0.30 0.09 6.5 -0.9373% CHSCOOH, 130 C, 4 h 43.3 0.60 0.65 26.0 4.85 -0.9493% NH3, 130 C, 4 h 60.0 0.90 0.18 0.1075.2% NH3, RTd, 30 days 57.0 0.98 1.11 45.5 0.17 6.2 0.1465.2% NH3, under N2, RT, 30 days 60.7 1.00 1.11 45.5 0.15 6.68 0.1585.2% NH9, under 0°, RT, 30 days 61.3 1.02 1.10 49.5 0.14 5.0 0.1412% NaOH, 160 C, 10 min 54.4 0.92 0.92 40.0 0.20 6.47 0.0694% NaOH, 100 C, 15 min 73.0 1.02 1.76 53.5 0.28 5.94 1.5004% NaOH, 100 C, 60 min 78.0 1.10 1.62 55.5 0.27 6.56 1.4894% NaOH, 150 C, 50 min 76.0 1.28 1.38 0.24 5.94 1.299

aAmount of protein (milligrams per milliliter)(Alcaligenes faecalis).

produced in the culture of C (Cellulomonas sp.) and A

' Amount of substrate loss after a 5-day cultivation of a mixed culture of the test organisms.e Amount of reducing sugar produced by treating 0.2 g of substrate with 150 mg of commercial cellulase

(Onozuka). OD, optical density.d RT, room temperature.

TABLE 2. Sugar production and digestibility of acid- and alkali-treated sugarcane bagasse

Sugar produc-Treatmenta tion (mg/g Substrate" L. in Digesti-

of substrate) recovery treatedi bilityc(% of substrate(%) (% of

l I~~~~~~~~~~Reducinitial) |Sbtaeliniti~al)Primary Secondary Total ing

0% H2SO4, 15 min, 121 C 1% HIS04, 15 min, 121 C 34 16 80.5 9.410% HIS04, 15 min, 121 C 1% H2S04, 15 min, 121 C 91 16 55.6 20.550% H2SO4, 15 min, 121 C 0.5% H2SO4, 15 min, 121 C 176 120 51.6 36.7 3050% H2SO4, 15 min, 121 C 1% H2SO4, 15 min, 121 C 232 168 53.4 33.5 3050% H2SO4, 15 min, 121 C 1% H2SO4, 1 h, 121 C 230 140 50.0 46.8 2050% H2SO4, 15 min, 121 C 1% H2SO4, 2 h, 121 C 212 192 53.0 38.6 2050% H2SO4, 15 min, 121 C 2% H2SO4, 1 h, 121 C 146 86 49.9 39.7 3050% H2SO4, 2 h, RTd 10% H2SO4, 1.5 h, 121 C 143 167 68.6 16.2 300% NaOH, 10 min, RT 22 7 86.3 7.7 400.5% NaOH, 10 min, RT 22 8 83.2 10.1 551% NaOH, 10 minm RT 25 9 76.2 9.7 605% NaOH, 10 min, RT 32 13 72.5 9.7 5510% NaOH, 10 min, RT 36 16 64.3 8.1 60

aAt the end of primary treatment, the concentration of acid in the reaction mixture was diluted andsubjected to a secondary treatment of 121 C for an additional time period.

"Reaction mixture was expressed with two layers of cheesecloth, and the residue was dried overnight at105 C.

c Amount of substrate loss after a 3-dAy cultivation of mixed culture of the test organisms.d RT, room temperature.

By this treatment, a sugar yield of 23% was the sugar yield. Increasing the acid concentra-obtained (Table 2). Prolonged heating (up to 2 tion in the secondary treatment decreased theh) in the secondary treatment did not increase amount of both total and reducing sugars. This

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loss was probably due to further degradation ofhydrolyzed sugars, such as the formation offurfurals and levulinic acid.The lignin content was considerably higher in

acid-treated than in alkali-treated bagasse.This was probably due to the fact that the acidhydrolyzed the cellulose and the hemicelluloseand left the lignin intact; the alkali treatment,which was milder than the acid treatment, didnot hydrolyze either the cellulose or the lignin,as indicated by the higher rate of substraterecovery. Because the lignin content is nega-tively correlated with digestibility of cellulose(26), the low digestibility of the acid-treatedsample may be attributed to the high lignincontent in the residue. Conceivably, the acidtreatment solubilized the easily digestible part(possibly amorphous region), leaving the highlyundigestible part (possibly crystalline region) inthe residue.

If the acid treatment could improve thedigestibility of the residual cellulose, it wouldbe more economically advantageous than theNaOH treatment because an extra addition of anitrogen source to the growth medium would nolonger be necessary. Ammonium salts producedby neutralization of the acid by ammonia wouldserve as a source of nitrogen. On the other hand,sodium salts produced by neutralization withan acid of NaOH-treated straw are of littlenutritive value, if not detrimental, for thegrowth of microorganisms. The acid treatmentmay also be advantageous by producing solublecarbohydrates and partly hydrolyzed cellulosethat are readily usable not only by the cel-lulolytic organisms but also by many noncel-lulolytic organisms, including yeast. Thus, theacid treatment provides a wide variety of choicein selecting organisms, either as a single pureculture or as a symbiotic pair, for the produc-tion of SCP from cellulosic substrate.The effectiveness of the acid treatment in

terms of increasing digestibility (TSAE), cellyield, substrate loss, and production of reducingsugars was less than that of other chemicaltreatments as expressed by the digestibilityindex in Table 1. The digestibility indexes ofseveral acid treatments were in the range of-0.9 to -1.5, whereas the index of the un-treated control sample was - 1.365. Therefore,acid treatments do not appear feasible forincreasing the digestibility of rice straw orsugarcane bagasse.Treatment with oxidizing agents. Because

of its polyhydric alcohol structure, cellulose issensitive to oxidizing agents and may undergostructural modification. With most oxidants,

the reaction is confined to the amorphous re-gions and the surfaces of the crystals. Someoxidants such as periodates and nitrogen diox-ide, however, are reported to penetrate andreact with the crystalline as well as the amor-phous parts of cellulose without causing mea-surable degradation (17).

In an attempt to alter the structure of thecellulose and to increase its digestibility, anumber of oxidizing agents were applied, andtheir effects on crystallinity and cell growth onthe treated substrate were observed. Table 3shows the relationship between crystallinityand cell growth. Even though alkali-treatedbagasse generally supported heavier cellgrowth, there was no quantitative relationshipbetween crystallinity and the level of cellgrowth. Variations in the crystallinity indexcaused by the treatment with oxidizing agentswere more pronounced in alkali-treated bagassethan in plain bagasse. The presence of thesewider variations may indicate the vulnerabilityin the structure of alkali-treated cellulose,which shows higher digestibility.Table 4 shows the correlation of the effective-

ness of the seven methods used to determinedigestibility of rice straw. The digestibility,measured by cell yield of Cellulomonas sp. andby the amount of reducing sugar produced bythe treatment with commercial cellulase, cor-related (P < 0.05) with all other measurements

TABLE 3. Effect of oxidizing agents on thecrystallinity and digestibility of sugarcane bagasse

Crystal- Digesti-Treatmenta linity ~~bilitybTreatmenta u~nity (Klettidx unit)

NaClO, on bagasse ............ 3.5 410KBrO, on bagasse ............. 2.8 410KlO, on bagasse ............... 3.8 460KMnO4 on bagasse ............ 3.7 450KS,08 on bagasse ............. 3.7 420KCIO4 on bagasse ............. 3.0 410NaOCl on bagasse ............. 4.2 420NaClO, on alkali bagasse 696KBrO, on alkali bagasse 685KIO, on alkali bagasse ......... 1.9 630KMnO4 on alkali bagasse 750KSO. on alkali bagasse ....... 4.2 667KCl04 on alkali bagasse ........ 1.9 660NaOCl on alkali bagasse ....... 3.8 570Control, bagasse ............... 2.6 420Control, alkali bagasse ......... 4.5 680

aSubstrates were treated with 0.01 1

agents for 17 h at 50 C.'Measured by the level of cell growth.

M oxidizing

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TABLE 4. Correlation indexes between seven methods to determine digestibility of rice straw

No. ofMethoda A B C D E F G significant

values

A 1.0000k 3B 0.4348c 1.0OOOb 4C 0.3734 0.6158b 1.0000" 3D 0.0558 0.0456 0.0134 1.0000b 0E 0.4588c 0.01081 0.0915 0.2773 1.0000b 1F 0.5446k 0.6486" 0.6288b 0.0420 0.2341 1.0000b 4G 0.4108 0.8633b 0.9165b 0.2645 0.1371 0.66481 1.0000b 3

a Letters refer to methods as follows: (A) digestibility (TSAE) measured by the method of Guggolz et al. (7);(B) cell yield of pure culture of Cellulomonas sp.; (C) cell yield of mixed culture of Cellulomonas sp. andAlcaligenes faecalis; (D) crystallinity index, measured by the method of Segal et al. (21); (E) crystallinityindex, measured by the method of Baker et al. (1); (F) reducing sugar produced by commercial cellulase(Onozuka); (G) substrate loss after a 5-day fermentation by mixed culture of Cellulomonas sp. and Alcaligenesfaecalis.

b Significant value in 1%.c Significant value in 5%.

except the crystallinity index. Other measure-

ments of digestibility (TSAE, cell yield ofmixed culture of Cellulomonas and Alcaligenes,and substrate loss) also correlated with eachother except with the crystallinity index mea-

sured by two methods. Thus, the crystallinityindex may not be a good index for predicting thedigestibility of lignocellulosic material, even

though Baker et al. report a direct relationshipbetween crystallinity and digestibility of woodcellulose (1).Enzymatic treatment. Table 5 shows the

effect of cellulase on seven cellulosic substrates.A considerable amount of reducing sugars was

liberated by the enzyme treatment. However,the digestibility measured by cell growth andTSAE did not increase. In many instances thedigestibility actually decreased after enzymetreatment. This decrease may have been due tothe removal of potentially usable sugars fromthe substrate by C. component in the enzymesolution (enzyme solution contained both Cand C. activity).The production of sugars was especially pro-

nounced in samples containing modified cellu-lose, such as wood cellulose, computer paper,

and filter paper, whereas the production ofsugars was lower in samples containing nativecellulose, such as sugarcane bagasse, rice straw,and wheat bran. Alkali treatment of sugarcane

bagasse markedly improved the microbial cellgrowth but did not increase the production ofsugars by the cellulase.The test organism Cellulomonas sp. probably

has the C. enzyme but not the C, enzyme;therefore, some form of pretreatment of the

TABLE 5. Effect of enzymatic (cellulase) treatment ofcellulosic substrates

Reducing ~~CellTreatmen Reducing TSAE growth

Treatmenta su(mg/ml) ( (Klett(mg/ml) ~~Unit)

Wood celluloseControl ........... 0Treated ........... 5.5 1,550

Sugarcane bagasseControl ........... 0.8 1,150Treated ........... 1.8 840

Alkali bagasseControl ........... 0.36 1,500Treated ........... 0.36 1,240

Computer paperControl ........... 0.36 1,500Treated ......... 9.80 790

Filter paperControl ........... 0 1,500Treated ........... 12.20 1,730

Alkali rice strawControl .... .. 0.32 73.4Treated.0.42 70.1

Wheat branControl ........... 0.42 41.1Treated ........... 0.89 30.2

a Substrates were treated with a culture filtrate ofTrichoderma viride for 4 days at 50 C. The enzymesolution contained 1.13 units of filter paper activity,1.00 unit of C. activity, and 0.57 unit of C1 activity asmeasured by the method described by Mandels et al.(15).

substrate is necessary in order to use cellulosicsubstrate effectively by the culture of Cel-lulomonas sp. (23). The effect of the alkali

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TABLE 6. Effect of particle size on the growth ofmicroorganisms

Sample Cell growth Substrate(Klettunit) initial)

Computer paper> 10 mesh ............ 1,100 2510-16 mesh ........... 1,250 2016-28 mesh ........... . 1,370 28<28 mesh ............ 1,100 15

Sugarcane bagasse>28 mesh ............ 850 2728-60 mesh ........... 850 28<60 mesh ............ 1,080 35

TABLE 7. Combined effect ofNaOH andhigh-pressure heat treatment of substrate on the

growth of microorganisms

Treatment" Cell yieldSubstrate (mgof

NaOH Heat p i)

Rice straw 0% Control (not treated) 0.440% 400psigb, 30 s 0.300% 400 psig, 50 s 0.250% 400 psig, 90 s 0.42

Sugarcane 0% Control (not treated) 0.54bagasse 0% 400 psig, 60 s 0.46

0% 500 psig, 60 s 0.350% 600 psig, 60 s 0.551% Control (not treated) 2.441% 60psig, 10min 2.161% 80psig, 10min 2.321% 220 psig, 5 min 2.001% 240 psig, 5 min 2.641% 250 psig, 10 min 2.523% Control (not treated) 4.683% 50 psig, 10 min 4.563% 65 psig, 10 min 5.243% 230 psig, 5 min 6.483% 260 psig, 5 min 7.20

aHigh-pressure steam was introduced into a reac-tion chamber, and the reaction mixture was main-tained under specified conditions. After the reactiontime, the pressure was released to atmospheric pres-sure to extrude the substrate.

b psig, Pounds per square inch (gauge).

treatment is, in a sense, similar to that of C1activity in facilitating the degradation of cellu-lose by the C. enzyme. For this reason, variouscellulosic substrates were treated with a culturefiltrate of Trichoderma viride (Table 5) which isknown to produce strong C1 activity (15); itseffect on production of reducing sugars, TSAE,and cell growth was determined. The resultsindicate that the effect of the cellulase treat-

ment was somewhat similar to that of the acidtreatment with regard to the production ofsugars and the decrease in digestibility oftreated substrate.Reduction of particle size. Table 6 shows the

effect of particle size on the growth of microor-ganisms. When the mixed cultures of Cel-lulomonas sp. and Alcaligenes faecalis weregrown on computer print-out paper and sugar-cane bagasse having various particle sizes, littledifference in cell yield or substrate loss wasobserved. When sugarcane bagasse was reducedto below 60 mesh, however, a slight increase incell yield and substrate loss was observed.For a cellulolytic reaction to take place, direct

physical contact between cellulase and cellulosemust occur to produce an enzyme-substratecomplex, which then can break down into theproducts of the reaction. We can, therefore,expect that the rate of reaction in the celluloseshould be a function of the surface area, whichis accessible to the enzyme. Reducing the parti-cle size will certainly expose more surface to themicrooganisms. However, grinding the sub-strate below 60 mesh on a production scale toachieve such a small increase in cell yield iseconomically impractical.

Effect of pressure cooking. Table 7 showsthe growth of the mixed culture of Cellulomonassp. and Alcaligenes faecalis in rice straw andsugarcane bagasse treated by various combina-tions of NaOH and high-pressure heat treat-ments. The cell yield, expressed as proteincontent, increased in proportion to increasingNaOH concentration. The effect of heat treat-ment, however, was not apparent in most of thecases, except when the substrate was treatedwith 3% NaOH and over 200 lb (about 91 kg) ofsteam for 5 min; this combination increased thecell yield. When the substrate was not previ-ously treated with alkali, there was even atendency toward a decrease in the cell yield asthe severity of the heat treatment increased.

Moist-heat expansion (extrusion) and dry-heat expansion (popping) have been used toincrease feed efficiency of grains in animalfeedlots (28, 29). Walker et al. reported thathighly organized starch granules in the endo-sperm were disrupted by these processes so thatthey were easily digested. They also reportedthat rate of digestion and overall digestibilitydepend on the degree of expansion of the grain.These processes, however, do not appear effec-tive in increasing the digestibility of ligno-cel-lulosic substrates.

In the present experiment, an effort was madeto determine a suitable means of improving the

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digestibility of ligno-cellulosic substrates,thereby increasing the cell yield in the course ofproduction of single-cell protein from cellulosicwastes. Of the various treatments applied, al-kali treatment in the form of NaOH or NH,improved the digestibility of the substrate,whereas other treatments, such as acid, oxidiz-ing agents, cellulase, grinding, and high-pres-sure cooking, had little or no effect on microbialcell yield.

ACKNOWLEDGMENTSWe acknowledge the technical assistance of T. L. Huang of

Louisiana State University and members of the Field Corpsand Engineering and Development Laboratories of WesternRegional Research Laboratory, Agricultural Research Ser-vice, for supplying the chemically treated samples.

This study was supported in part by Public Health Servicegrant EC 00 328-02 from the Environmental Control Admin-istration.

LITERATURE CITED1. Baker, T. B., G. V. Quicke, 0. G. Bentley, R. R. Johnson,

and A. L. Moxon. 1959. The influence of certainphysical properties of purified celluloses and foragecelluloses on their digestibility by rumen microorga-nisms in vitro. J. Anim. Sci. 18:655.

2. Cowling, E., and W. Brown. 1969. Structural features ofcellulosic materials in relation to enzymatic hydrolysis.Advan. Chem. Ser. 95:152-187.

3. Doneffer, E., I. 0. A. Adelefe, and T. A. 0. C. Jones.1969. Effect of urea supplementation of the nutritivevalue of NaOH-treated oat straw. Advan. Chem. Ser.95:328-342.

4. Feist, W. C., A. Baker, and H. Tarkow. 1970. Alkalirequirements for improving digestibility of hardwoodsby rumen microorganisms. J. Anim. Sci. 30:832-835.

5. Guggolz, J. R., R. M. Saunders, G. 0. Kohler, and T.Klopfenstein. 1971. Enzymatic evaluation of processfor improving agricultural wastes for ruminants feeds.J. Anim. Sci. 33:167-170.

6. Guggolz, J. R., G. 0. Kohler, and T. Klopfenstein. 1971.Composition and improvement of grass straw for rumi-nant nutrition. J. Anim. Sci. 33:151-156.

7. Guggolz, J. R., G. M. McDonald, H. G. Walker, Jr., A. H.Brown, W. N. Garrett, and G. 0. Kohler. 1971. Chemi-cal treatment of agricultural wastes to improve di-gestibilities as livestock feed. Proc. West. Sect. Amer.Soc. Anim. Sci. 22:71-76.

8. Hall, J. A., J. F. Saeman, and J. F. Harris. 1956. Woodsaccharification. Unasylva 10:7-16.

9. Han, Y. W., C. E. Dunlap, and C. D. Callihan. 1971.Single cell protein from cellulosic wastes. Food Tech-nol. 25:32-35, 56.

10. Han, Y. W., and V. R. Srinivasan. 1968. Isolation andcharacterization of a cellulose-utilizing bacterium.Appl. Microbiol. 16:1140-1145.

11. Han, Y. W., and V. R. Srinivasan. 1969. Purification andcharacterization of 0-glucosidase of Alcaligenesfaecalis. J. Bacteriol. 100:1355-1363.

12. Harris, E. E., G. J. Hajny, M. Hannan, and S. Rogers.1959. Fermentation of Douglas fir hydrolysate bySaccharomyces cerevisiae, p. 1-8. U.S. Dep. Agr.Forest Serv. Rep. 1618, Washington, D.C.

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