t-,/rrr',r47c

t-,/rrr',r47c

PROTEIN ENRICHMENT OF RICE 

POLNIIINGS THROUGH METABIOSIS AND 

ITS BIOLOGICAL EVALUATION 

BY 

MUHAMMAD IKRAM-UL-HAQ 

M.Sc. (Hons) U.A.F 

A'THESIS SUBMITTED IN PARTIAL FULFILMENT 

OF THE REQUIREMENT FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 

IN 

ANIMAL NUTRITION 

DEPARTMENT OF ANIMAL NUTRITION 

LINIVERSITY OF AGRICULTURE 

FAISALABAD 

2000

To 

The Controller of Examlnations, 

Unilerslty of AgriculrurGt 

faisslabad. 

'We, the Supc.visory Conmitrce, cerrify lhar rhe cotrtenrs and 

forn of tltcsis subnitted by Mr. Muhamnad Ikrrm-ut-haq, 82-a8-E60, 

have bcetr found satisfacto.y ind recommeod rhat ir be proc€ssed for 

ev.luation, by the Exrernal Examinc(s) fo. rhc awa.d of degree". 

SupGrvlsory Commitae€ 

3. 

_d4+? 

CHAIRMAN 

(Dr. Altaf-ur-R Barque) 

2. MEMBER 

MEMBER 

(Dr. Sh id Rasool) 

\t 

it;. fi ;;;i;;d-Sh;ihl----

DEDICATED TO 

-.MY DECEASED BROTHER 

DR.MUHAMMAD ZIA-UL-HAQ 

AND 

DR.ABU SAEED HASHMI 

AND 

DR.ABRAR IIUSSAIN GILANI

ACKNOWLEDCEMONTS 

. ll nnie5 lor Allih. who 8u,Jes us ,n d.rknBr an,l h.rtt n, d,|]j!,,trier 

drd,'rr 'e.r(t( h' l|n poph.l AzfuAt MU AMM D (nrry pr,a b. u|rr litrr) 

oriislrurin{ or con*ienc. wirh fic.6senc. or tailh in A[ah. "ou"etging tris ti,,,tu*s 

I t*tsE.r pt.uuc !o.rpEs! my hdr.tr sErirudc and linc.r.sr rhanks 

r' u' ^rr0r u' Rchnah uarqu.. prctersr ond ChDnmn, D€nlnnFnr ot Arrul 

N nrnr lor lk tind su'da ce coniist.nl .n@uds.nEnr, h;atfiy (iri.n drl 

udainr! kndtresr thrcugh lhe c!!M otfiis studv, 

,. _ , I rirl nrcu'i urtFins rtE nud.nl;rt,r.Abu S..cd .st,n rnJ t)r.Ah,al 

rrrqn.rir, r, rrc \oy \nrd xnJ.lrccrioh!i. r.a.he,r, Inos[,s rtE nrit.sroncs in ltN 

.., ........,,,.:lll'1"1'..11*,ru.,!DrshahidR6mr.Asnr"h,rror.io,.Dena,rherl 

:'iTni:? ',J D, vurdnnnr,i Ahru . A$isrsnr prore{!u,. r,.i*,i,""i "i 

i,j;;,;J}"filii$illl;Jlli:, suidena.,n.crionok,n,rd. !d !.,uabr su*s.liotr1 

-,,'..,,,','". ",1:i.ll'"1l:::"'"'" 

quore r' DeE ot Dr'Rana riirdos sho r'|erted De s 

r,r.d nkm,tr. rn ntn(. .n r.cord n,y ohtig.rion. ro ,tr, L,en,k 

11 lli'llil'ii"lllti,i*"llt'"1,1il*;",;tiiT*::*',,;'qb"' s",;' 

^;;;;;;: 

., , -, ..]l': i!tr,$teitaDenrr wi L imornpt.r€ u,rtDur reteaDL. dr Na/i, 

^hmHr \rur'iit slltr. Nazr *rnor. M!n,&r. 

qr 

Khatid. Javed. 

!u,fsaler Cr,"r.., 

$.rtr 

Sr,"r,,,a ,,r,., 

De o(h more dutrtre whote Firod otmy Ese,crr ""a 

, wnn,a dRp (trk ot ipnr.(,arion. 

\LT,nrj,.rd I otT.r nry 

IJ a, 

sihc.,.\l rhsnt\ 

u+us lo nr\ 

d V k.t, t.tDrc qllr(ia 

Irlj , O,.,r.t"" 

rt,tr. rr". t)rEcror 

U, 

vckrinrry 

v.,r,,,,_,; 

lf 

tt€$srch Innir;rc. t r,. 

.,1;ll, i,.;;"l;,tl;1, :' J:ffi ;',]fit:,:itri:,ffi ,::y,)liiil:,",tlllj"lll 

I ufter xry hea ielr ftanlrs |o hy ,ft ctioture poenls, 

ror u{tr broth.u and sislel 

tE}cn d.t noatsppon,nd my eiic dd my 

Mtr^,ln,wrD t u..}Eps N@r ut udda and 

Iny 

'enaimJ rann.B du,,ns 

astro\reds. ., .*rol.wi,r, 

nry r!d'e{}hw. 

au. ,".*i l 

my bbrhr6{n:t!w. 

nccr{d my sist.".i"-r"., 

Mo.F,.,tr+w. a,r,.r 

"i,a ",, 

.ft*i, b.sr *i"h"" rd pdyos:,obl.d n" . *r^" l,"l],.1i 

(Muh.mm.d tkrdn uth.q)

CONTENTS 

LISTOTTABLF^S ." 

LISTOFFICUERS 

LIST OF PHOTOGRAPHS 

r NTRODUCTION 

2 REVIEW OF LITERATURE 

F.rmetrtlalon Parrm.teru 

Soblanae tr.rrn€nt 

D€fatting 

Acid treatmcnt 

Md.s.rclu.d. Soppl.D.nr.tlor 

Con Step Liqoor (CSL) Slpptd.trr.atoD 

Crrbor to Nltrose. R.tio 

Ircubrtior Pe od 

Multl{rg!ft n F.rn.!t tion 

s!ppl.d.n|.tio. of y.$t stud!. 

Growth Kln.ris 

Biologiel Prnn.ten 

3 MATERILAS AND METHODS 

D.fritiry 

ct.mlc Composiaion 

Pnprntiotr of ltrsuhD for Arnhaiot6 spei.f 

Optlmlz{tlo. of Ferh.nrrrior C.diatons 

E recl of Typ€ of Rice polishinss 

ElTet of Acid THtrndlr of Subsrrar. 

*.w 

^ltf \l:tr'rtEUta 

,.l"nr.j Atrt,|l,|t. 

-x!u'1wt' 

lor Aroch n lotut spe.l.t 

Additio. of Diffe@l La€k of Mots$€s rt 486 Hour of Fcrmenbtion 

Setting Carbon to Nilrog€n Rario 

SednA lihe for Additio otMdllg 

Addition of Com Step Liquor (CSL) r! 48 trous 

sertine Tim. for Addilion of csr. 

HaNastirg Time 

Pig€ 

vii 

I 

4 

1 

4 

5 

9 

l6 

l9 

22 

3l 

38 

4l 

43 

51 

57 

57 

57 

5E 

5E 

59 

59 

59 

59 

60 

60 

60 

60 

6l 

6l 

6l 

6l 

62

PreDrmtio. oflooculum for Bt@iba.tetiadflaran 6, 

llocdrn 62 

Clltoe Cnllir.iioo 66 

Hrdesting 66 

Opinlation ofrcrnenlrtiotr Condilions for A,Jooran 66 

Autoclavod vs Umut@laved Firsi Step Fementation 6l 

Addirion of Dillerenr Levek of l,lolNes ar 48'i Hour olFcrm.hLd'on 67 

Addition ofDilT€rcnt GIU@se Levcls at Dir€rent Tim€ P.riodi 61 

Co'nparison ofclucose md Cuffh 67 

Ysl Sludge Suppleinenlatiol 68 

He*1in8 Time for 2d Phde 68 

Growtb Kitr.tic! 6E 

Ldrge Scrle Productior of Biomrss 59 

Biologic.l Evrlulion ?0 

Elp.rlnental Rr$ons 10 

Etp.rioeolal Bird! 7l 

Mrnrgcmeot 72 

D.tr Collecliotr 72 

Prnm.l€6 71 

Slrrhricrl Analysis 73 

4 RESULTS AND DISCUSSION 71 

A TO OPIIMIZE THE GROWTH CONDITIONS noRAtochniotb 75 

sp..irs USrNG RICE POLISHINGS AS SUBSTRATE FoR THE 

PRODUCTIOIN OT BIOMASS PROTEIN 

Rarlls.od Dtuctrssiob 

Effcct ofTr?e ofRice PolishinSs 

Ercct ofAcid T!€atrnol ofSubsrmte 

D€temimtion of Optimm Level of Motdss al48 lrou$ 

Ef.c( of Different Carbon ro Nitog€D Ratios 

Dctennioation ofoplinu Tnnc for Addilion of l% Molsscs 

Deldnination of Optinm lcvcl of Com Steep Liquor ar 4E Hou6 

D.tcDinarion of oprimum Timc fo. Addiiio' of 0.5% con 

Liquor 

Del€rmination of Oprimrm Harvdting f ime 

Oveniev for A-Prt of Cbrpter-4 

B TO OTTIMIZE THE GROWTH CONDITIONS TOR 

B,.vlba.t.tiun Jlaeun ON \HE BIOMASS PRODUCED ITURING 

FIRST PHASE OF TERMENTATION 

R.sulb rd Dbcu$ion 

75 

75 

76 

79 

EO 

82 

E3 

9l 

92 

92 

92

Brcvibdctein llatun C rue Cnttiyarion 

EITecr ofAddition ofDiffsetrr L.veh ofMotssq 

Erer of Dirsenr L€vels of ctu6e ar Diflercnr Timc periods on 

crcwth of R rc v i ba. E I i M Jl av un 

ConDarison berween 2% Ctucose and 2zocunh 

Ydr rludge Supplemenrsrion 

Detemimtion of He€srinS l-ill|le fo. Brevibact.ri@ flarM 

Growt[ Kin.tic 

Ov€nlew for B-Pfft ofChiprer-4 

C NUTRITIONAL EVALUATION OF BIOMASS THROUGH 

AMNIO ACID COMPOSITION AND CHICK ASSAY 

objcctiva 

R€sulh ud Dtu.u$ion 

Atuino Acid CoDlosition 

Biologlcrl Evrtq.aion 

F€.d CoNumption 

WeightCain 

Feed Convdsion Ratio (FcR) 

Nel Prcrcin Uliljarion (NpU) 

prcrein Imcien€y turio (pER) 

Oveni€w for C-Prrt or ChrDr.r_4 

SUMMARV 

ACHIEVEMENTS 

Rf,COMENDATIONS 

BIDLIOGRAPHY 

APPENDIXCES 

I Ch.mical composition of rhc subsFare us.d duing he prfts of 

92 

93 

ihe 95 

91 

9l 

9E 

l0l 

104 

105 

r05 

105 

105 

t0E 

108 

t08 

109 

ll0 l 

ll4 

llE 

ll8 

119 

r!6 

t36 

I Percenl chemicdt composition ofbromass t3l 

lll Amino ac'd prcfileofbiomass_A snd biomass-B rDMd,o) D8 

rv chemr€t composition ofingredienrs us€d in rhe raxons {pscenu tl9 

r ( ompo$tjon ot vilaDin and minersl premix used in I he Eriols i qO 

', 1'lllf'. o, \d,ance or da'a shosing influence or def! ,ns of rice iai 

,,., po|snrngs on b,ooEs prorein yietd 

fdata showing inftu.nce oreid reunenr ofrice t42 

.. por6hh$ on bromass pror€in yield 

viir Amlysis of v&ianc. ot dara shosing e{Ier ot addilion of di0cr.nr l4l 

teveh otmolases on biomGs Drorein;ieH 

,^ 111{: -glJ-'I*- "f dar! ;howins rh. ,i.rd of bioms3 psrcin ar r44 

otrhar corbon lo nirrogs drios 

x Analysis ofvariance of data showing prcdrcrion ofbiomass prorein as t45

effecled by addition of I % nolasses al vadous tirne periods 

Xl Analysis ofvtrime ofdata on thc eff€ct ofvarious lev.ls ofcom slcep 146 

liquo. supplenenbtion on biomsss protein yicld 

Xll A.alysh of varianc. of dala showing erecl of supplcmenfing 0.5% 14? 

com stecp liquor at various nme inlerals of f€rm€nialion 

XII Analysis of vari.ncc of data on lrue !rct.in yield of the biomas l4E 

hdsted !t diflercn( time pcriods 

xlv Analysis of va.idrcc ofdala showing effcct ol aul@lavine rhc growlh 149 

ot Anch ot6 species on nu. ptot in tield of B.lNun 

150 

xv Analysis of variancc of ddta shoping effcct of supplorentaiion of 

dir@t l.vels of molasq at 48 hou6 of h. grcwlh otAlM 

XVI Aralysis of vdiece showing erect of addirion of dircrdt lev.ls of l5l 

Slucose st ditfercnl lime peiods 

XVll Analysis ofvdidce ofdata showing effcct of2% cwh on dE biomrs 152 

XVIII Amlysis of variance of data showing cffecl of y.ast sludSe 153 

supplementation on biomass pro€in yield 

xlx Analysis of variancc of data showing innuence of hrwesring riDe on 

tue prcr.in yield by Enribrct timJlew 

XX Analysis of varianc€ of dara on fced consumprion 

XXI ADalysis of varianc€ of dsta on weighl gain of chicks 

XXll Analysis of vdime of data o! f..d @nve6ion mtio 

XXlll Analysis of vdioe otdara ner prct.in uritiation 

XXIV Analysis of vdiec. of dala on prorein efnci€ncy ralio 

Prcinrt. Anrlysi! 

Ether Extract (E.E) 

crudeFibrc(c.F) 

crudePmrein(c.P) 

Nitros.D Frce E(racr (N.F.E) 

Solubl€ Crrbobydnt.t 

E linrtior ofcirbon 

ardo.tio! ofTru. Protetu |I.P) 

Amim Acid Analysi! 

Ribose Nu.leic Acid (RNA) E fltn,rion 

F,stia.tioo of C.lcirn lnd phorphorus 

Estimation ofcalciurh 

Esti@tion ofPhosphotus 

154 

155 

156 

151 

158 

159 

160 

r60 

160 

160 

160 

i6l 

t6l 

t62 

r62 

163 

165 

165 

166 

l6E 

l6E 

168

LIST OF TABLES 

Trbl. Ttate 

I CotrrpNitiotr ofag& ncdiun (B/L) 

2 Coop6ition of in@lumm.dirm f6 traclniotg sp.lejtL, 

3 Compcition of itr@ufu n dltn t6 Bftibactqim ltavun 

4 Composilion ofrle exp.rimentat rarios 

5 M.s I SE tue prcrein producrion sr affe4t€d by using 

Atuchniotw sp. 

6 Mcm i SE lne proreh production a! aff@red by cing 

tl 

l2 

ll 

Rraiba.Eri@ ll@@ 

58 

59 

@tL) 62 

.11 

? M@ prorein yieldardiflrcnr t.vckotStu@sedd dilTcrcnt rime a7 

E V.rious Grcs,$ Kirclica par&ncr.rs of femenrative or8anisms 

9 Amino acid compGition (c/l6CN) ed Chemical Saor€ of ric€ 

- polishinss, bionss-A and biomass-B 

l0 Average values of Weidr Cain, Fced ConsmDtion. F.cd 

Convclsion Ratio, Prolein Eflicicncy Rdio and Ner pforcin 

Utiliation of rhe exDc.im.nral Erions 

Eslination of Solublc Calbohydnrd 

CalibDtion ofstandard curvc for Ribo!. slimarion 

Eslimalion ofPh6phoru! 

77 

94 

t02 

lo? 

lt2 

162 

167 

169

LIST OF FIGUERS 

Flturc Tltl. Prr. 

I Trend ofCroslh During Borl Phase ofF.rm.Nurion t03 

? Showing lrsd of Suh6lntc utilialio[ Cell ||e fomtrio, od t03 

3 

5 

Prcdel (T.D fo.natim 

SllDdlId Curvc fq Eeination of Solublc clrbol|l/d|.r.s 

Slardrrd Clivc for Dstination ofcsrbon 

St ndad Curvc for RNA Brinution 

t64 

164 

t67

LIST OF PHOTOGRAPHS 

_ photo8rrph! p.8. 

rnowmg resn cufrur. Janr ot A.&hniorut q.ciet 6l 

ShNirg fr€sh crdtur! rlrtrt of B,etlbocEritat'Iwts 64 

Showirg rotrry.h*d pLcd ir ovc4 pFp.rios inadoln mdium gj 

Showins Inbor.tory sc.lc f€rn€oilr (qp.city 5 Lit in op€nrion 86 

Showing fdrnqtition rt irs!€{k 89 

ErperimerDrl p.m showinS serara{c anlngcmm(! of fc.din8. ea|tring 90 

8nd droppings collccrjon for @ch exDcrimcnrlt unil 

Showins arrng.mcnb made in thc,p.[t3 for dtoppinss co .crion b€tow 100 

|n. net 

Srtn*ing cxpdirD.ntil clicrs killcd !|ld opencd fd &ying in ovor ofi lod I 13

Chapter - I 

INTRODUCTION 

Tle grcwing f..d industry of Patdsre ha always fac.d lhe challense 

oi shodage ofqrality ingredients bolh of vegerabl€ ad eimatorigin. (Anonymous, 

l9E8). The .xpesion rnd incEase in production @paciry of this indur.y ha! 

lurther brcadened th. gap beiw€en thc dcmand and supply and rhe prices of lh.se 

coonodilics s€lated. Althougi rhc cxpdding agriculture induiry hs atw.ys 

srppon.d the needs of feed indusrry but lhe equalion neve. bec.ne b.lanc.d. Th. 

requirements for en€r8y of rh€ slions are met onb way or the othq by a vdiery of 

cer€l Shins but lhe requircnenrs of rh. qualiry pror.ins arc n.vq mel wilh b€c|use 

of the non availabiliry of siandtrd prorein sou.ces and also of then limited suppty. 

This situation nccesitated att atong lhe se@h for novcl prcrein sources. The 

dcv.lopmenl ofsienritic methods to .nhance th. nutririvc value of exkring non 

coDverlional leed rsou.ces eirhq by nechoicat, chemical, physi@hemical o. 

bioreclnolosical mehods hat dwaF ben fie priorjty. 

lementalion rechnology tad been practiccd long sgo for the 

production of food ftom a vari.ry of matc.ials_ This is $€ p.@!, thrclgh wt,ich 

the carbohydare rich products, b}!@ducts .nd wasre producG ee f.menred with 

differenl kif,ds of microorgaxis'ns like bacrqia, y€lsr and fungi. Each typ€ has ib 

oM pot€ntial to urilize subshte and synlhesia the produ.rs like Drotejn. fr€e 

anino acids, enzymes, vit rnins and many other usefii lroducts (Aab .nd Amnir. 

1994 i Bonna.det er dt t 995 ; S@narh er al l99j). A nunb.r or exp.rihenrs hav. 

bcen conducted iD Pokistan to iftprove rbe outrjtive value of a8rc_induslriol by- 

producls throuah fcootarion 6ing mic@rg.nisms for then udliuiion in

livestock/poulq l€eds. Di €rcnt kiDds 01 lungi, yeast dd bacl.ria have been 

grown or a vdi€ty of fibrcus wastc matedah (Bajwa er a1. J99l ; tlathni €t a/. 

1989: Kiani e, a/' 1989: AkBm, 1992 i Ar|l'at et al. 1995 r SaiDa, 1996) ro 

i'rcrc.sc rhc prolein quantity along *ith impbving the qualily d sell a lh. 

digslibility. 

The incrc66 i! lsricultuG produclivity in the @unilt duritr8 rcc.nt 

yeaB ha ault d i! a l&ge mounr ofagricultural p.oducts md byprcducts. Ri@ h 

one of th€ major cqel crop.r of Palistan and its production is incrcasing cvery 

year. Acco.dirg lo a Repo.t ofNational Commission on Agriculrure (Anonymouq 

1 988) 325 5(000) M.T oi rice was prcduc€d d rins the year 198?-88 and $€ target! 

set ior lhe y€af 19992000 are 5320(000) M.T. About 106.4(000) M.l of ricc 

polishinss (2% orrice) will th€reforc be available du.ins the yd 1999-2000. Rice 

polish'ngs conlain 12-14% oil ed l2-139lo poor qlaliry prorein. High {ibre conteirs 

( | 2- l3%) limits ils ue in broiler ntions. By impoving i1s digestibilny ald lurritivc 

vallle it c€n be ued upto l5% in all tinds of lslions. 

In th. pst cfforts wer. nadc ro imprcve the nutiirive value of dcc 

polishinss firough fementation witl sinSle orgeism Atrch"iotw sp.cies ot 

Canlida stilis in tle presence of y€ast sludSe, a wasre producr of distitlary. 

Atdchriotus species wer€ initially used as an artagonklic fungi agaiDsr planr 

palhosens (Akltar, 1982). Its potenrial for synlhesis of sin8le cell pforein (SCp) 

vas, however, rccognied larer (Ha!hni, 1987; Bajwa er ol. l99l ; Sarrraz, 1990). 

This organisn w6 rcported to havc sumcicnt clllulolyric ability ed has pot€nlial 

lo enrich pmlein of Ihe fementliion medium by ulilizing the soluble su8a6. 

Kecping in view lhc cunent situarion of fed industry and fie dcmand 

tor ch@! qMlity p.otein $uces, a proj€cr ws @nducr€d ro imprcve rhe nukirivc 

value oflhe .ice polishings, d aboundantly available @nodiry in rhc counlry. This 

was pr@Ned otrough femcnrarion fitst wirh ,4fa.,riord rpeci4 followed by

BrcvibrctetiM llavM. Bretibacteriw low hst b..n rcW.ted to Foduce lyrine 

in th€ ferm€ntation mediurn alotrg wi$ ofier arnino &ids (Cher e, dl. l9E7 ; 

Frantirek ?r al l9E8). The mrt8nr strain ofthis organhm is reportcd to produce 558 

lysnlc p.r litc. of femeorarion mcdium s ia hydrochtoiic rcid satl (hamu cr at 

1964). bdB&ial byprcducts of sug.r industry likc mohss.n corn st rp liquor dd 

y61 sludge of suga industry werc also uritized during $. proccss of fermc orio, 

to .c[iev. th€ following objecrives. 

Objectives: 

r. To optimia fie g.o*,$ condirions of Atuchnio,w speciq nd oI 

Brcvibackntn lavrn fq msimum yield of bioma!! p.orcin. 

2. To produc. rhe biomass on targe $ate foltowing the opimizcd condirions 

dd to assss ib rulritional porenrid by ch.micrt 0nat),sis. 

3. To dcrcmirc rhc nut.itionlt vrhe of lhe biomass prodoc.d ihrough cc 

culte ush8 broitd .iick.

Chapter- 2 

REVIEW OF LITERATURE 

Recycling of w6t€ hal.rials ud indarlial bypiodlcis noi only h.lps 

in educing the pollution but also l.ads io th. p.oduction ofmby useful prcducts. 

The recycled wasle materials @ usefully conve.r.ble io edibl€ prcducrs like mitk. 

and €ggs lhiouSh livestocl atrd poljtry. This pmces has lso fold advantag., 

'neal 

it meets the nutrienrs .equiremenls for livestocupoullry on one hdd and hetps in 

bridSing th€ prct€in shonaSe gap for humans on the orher hand. A 8@d deal of 

ro.k ba bem done on th. bioconv€rsion of rice polishings into high gradc nutrienr 

ihrolgh tenenhrion by diffcrcnr kin& ofmicroorganisds. The avaitabte lh.nturc 

otr dirqenl dpels of femstltion as wcll s bioloSidl aatuation of diff.rcnl 

f.nnctrtalion products hd ben @iewed in rhc following pdgEphs 

A. FERMENTATION 

I. SUBSTRATE TREATMf,NTS 

lJ D€frtting 

Kimi er o/. (1989) wo*ed on protein e.richnent of defated rice 

polishings rhrough its fement^rion virh Candi.la Uilij. They found thal yeasl coutd 

synllt€size 22.125% lru€ polein whei 9yo sljbsl.al€ was fermenr€d uder oDrinlrn 

conditions for a pe.iod of?2 hon$. ]'hc biomass pfot€in was, howevd, d€ficienlin 

aDirto acid, valine. Kho (1990) convedcd dcfarlcd ri@ potishings to prorein 

conc.nlrale dd biolosi@lly evalnared il in broile. chicks. The fementrrjon ws 

.atied onr nith Ttichodema hanihuh. He s$c...d.n h inoeasing the infterenr 

prot.h of ri@ ralishings ftom 14 to 24.80%. n. ftngat bioms was f.d ro b.oil.r 

chicks by rcplacinS veg€rnbl€ protcin sour.es at ldcls of25 and 50yo of thc Etion.

Hc repon€d non signifi@l difcr.nc.s in wcidt gain ofthe chick. Mal|'nood./ 

dl (1990) obrain.d l7.ll% prctcin funSal biomlss on fcmcnhtion ol defaltcd 

'n 

rice polishinSs vith ,{rddlrrrbr6 pe.ier at pll 4.0 md 30 "C t.mpentur. to. a 

pcliod of ?2 houls und.r lcmishalc .rndition. Ih. ionic conentnlion of thc 

ncdium was adjust€d rt 0.00lyo CiCl:,0.001% MsSO..7H1q and 0.075% 

Kf IrPO.. Thc pEs.ncc of mymtoxins w|s l6lcd Uairst F6oriu dysryM by 

spo!€ germination est. Maximum production of my@toxins wa rcportcd in fi. 

filra|€ all€! 6 drys ofincubatid. 

1,2 Acid Trerhn€n. 

Acid tlattnc t has b.en caFicn ot by $v.61 wdtcB with limitcd succcss. 

DilTcEnt l.vcls mgirg from | !o 6010 of HzSOr or HCI or c.mbin0tion of both hlvc 

been appli.d toge .r *ith hc.r lnd pBurc rr6trndi!. Exccrs of acid in thc 

rftrted md.rial has b.qr n.lrrllized wirh alk li likc NsoH. Tr€{rnor wi0r highly 

concenlllcd acids padicularly HrSO. produc.d rcducins su86. lt h.t b€en no ced 

lhat acid lEllm.nl followcd by ncutEliation with .lksli incrcrs.d thc nin€l.I 

matcl of thc ucatcd subslmlc. Funllcr. &id tcdncnl incrciscd th€ cosl of rMrcd 

subslralc. 'lle Litemlur. otr dilT.rcnt typ.s oflignoc.llulocic mnerills in rhis r.gffd 

ha ben Bicw.d rs undcr. 

Ri.ch. er ./. r 1966) wd*cd on prodrction of a h'gh qualiry prcr.in f.cd 

frcm pot tocs. Thc potalo€s werc pulpcd ed hldrolyz.d by l% .cid and h.!r. Thc 

mtrtur. so obroin.d wa fcmcnt d eirh Candido utilb for 4 ro 5 d.,s lRcr 

adjutint it5 pH ed tddirS mnoniun phdrph.L lnd 0mmoniM 3ulph.t. in th. 

mcdium a nioogd sourc.s. Yield of y6t dry m.ttq wa 44 to 468 pcr l00t 

rcducibl. sugar. Thc r.sulta fed luficodaincd 36% c$dc pml.in. 

He .r dr (1971) studi.d tlte .ffcct of subsrtarc pErMtrncnr on producrion 

or si'Blc ccll prorcin (SCP). Th.y reporred rhar rhc soblrrare te3|m. with rthti d 

acid or ofid oxidinng lgcnt d.lignified lhc lign@llulosic substrar., allowi.S

norc ulilizlion of cellulose which uhitrsG9 improved the @ll gtlwth and SCl, 

production. In thlir study they wd cellulase producing b6.tetia C.ltulohotu' 6s 

Han md Callihm (1974) cofrpared ih. erech of chemiqt, physic.t and 

enz.ymic l.ealmenls on rice stBw ard suSd c6ne bagdse to inprcve th€ mioobi.l 

dig$Libiliry of ellulosc- lhcy rcportcd rllu| trornc ei|l| 4% Naolt tb. t5 

oinutes at 100 'C incrersed $c diS6ribiliry of crllulose ftclrn 29.4 ro 1l%. 

Treahent wilh 5.2% NHr @uld incnase dig€stibility ro 5?.0% md rr.rhcnts wilh 

sulfuric acid ed crude cellulse prcpsElions solubalizd c€llulose but did no1 

in(.N. th. digestibilily. crinding or hi8h prcsure cooking of the subslErc had 

little eff€c1 on incrcdinS the dig€stibilily of cellulosic subtrares by the 

C'yabyab et al. (1971t gt.s Cadida trcpicari on acid hydrolyzcd .ie 

slraw dd '@vqed 46.E lsent y€.et biom.ss contlirirg 5l% pror.h lt wls 

rcport d rhat rhe eltrciocy of conv.Bid of 3ugr i o y4t bims! wB mximud 

d pH 5,5,lcmp.Flurc 29oC &d sugar con@nt ion L6%. Th€ ye{sl bioms had 

aDibo acid composirion compmble eilh thc FAO refe.en@ prct€in. 

Moo-Young d, al. (1977) conrpared $e cellule activity Md single cell 

pror€in production belween t*o cellulolyric funSi Cr,ebui@ cellulollti.un and 

Trichodemd tiri.le. mes. organisms were gfown on 1wo substratcs Solka,floc (a 

purilicd, predominanlly amorphorcN fom of cellnlose) and padially deli$ifi€d 

sawdlst (consistitrg of a mixrw of hardeoo&) wirh alkali, pe.sccric .cid ud 

HrOr. Thcy obsded lhat T.vtide p..drc.d morc ellul8e md d.sradcd morc 

slbstrate b the SCP produclion wrs higlEr in crsc of CC.n/./oUriM. 

Quierzy rr zr (1979) rraicd rhc ground pear wirt acid dd dlncls qere 

os.d ,s fm€nration n€dia fo. lhe production of SCP 6ins Candida ttilir. A.id 

Iydrclysis of grcund pa1 (40-60 m.sh) wu achicv.d in d allochve op.Er.d

under a sel of condilions: acid strenglh, 0.3-1.5 % v/v H,SO. holdin8 time, 14 

hou$ t€mp€rature, 100'l65oc and weighl ralio of dry pent to solurion, 3.1-16.? 

e/1009. Lo* concenFalion i.e., 4.1 I dry pe.Vloog elution rsulred in high yield 

of ca.bohydnre. Tle bs1 yield ws obtain€d for & holding lime of I hour ar a 

leDp..alure ot | 52qC. Lower yiclG wcn foud with inc@in8 p@t conc.nhrions 

upro I6.79 dD p.aL/ l00g solurion. 

volfova €r 4r (1979) treGd st6w w'1h .cid ehich sollbalized stBw and 

Cah.lida ttupicalis fennentation of stEw resultcd in 8-9% proGin in fte producr. 

The f.molalion wa canied out at l0oc ed al pH 4.6 for 72 hol|6. Thc biomass 

prolein was rich in all $senlial amino scids ex@pt melhionine. 

Balarubrarnanla md Bhaiawd€kd (1981) assc$cd rhe reasibiliry ot 

femenraton of co(on-sralks, rur sralks and groundnut shells for lheir enrichmenr of 

prctein with Penci iM fwi.ul6w and Can li.la srt16. Th. slbsrars scrc 

lrualcd wiL[ watcr and 0.5N lIrSO4 heated at l2l'C for ]0 minurcs ar a ptt adjusred 

belwcen 5.0-5.5 before incubstion- Enhdcdenr ofdude prcGin 9s achi.ved in 

6ll the cars wilh both the cultuGs. 

Dlvcy &d Brue (r9E3) srudi.d the .rccr of mild H:so. hydrctysis on 

cellulosic compon nts of pigerry slurry. Acid lreahetrl bclween | .rd l0% (w/v) al 

diff€rent Icmpcmtur€s was studi.d witi dE rim to maximize rclelse of solubt. 

cebolydmtes. They obtained highesryield ofl9.8 g/t ofDicrobiatbiomass havinS 

22.? pcrcenl protein by hydrolysis with 5olo sultunc acid ar 90oc for 3 hoN wirh 

Sporctic hM ptlverc uh ntn. 

Chahal (1984) in d inlegrated pl@ss for food/fed and tuel producrion 

used eheat staw after grinding to 20 m.sh &d lralin8 wilh 0.5 N IITSO4 in rh. 

mlio of l:10 at l2l'C for halfd hour. The trcatn.nr solublisied ov.r Eoyo of 

hemi@llulse. Dodng rreatncnl some roxic compound! (turtuEl, hydioxlme$yl 

furfuBl and theif prccursoB dd phenolic odpounds) w.rc prcdlced by

d€hydotion of p.ntoses and h466 which suppr€s!.d lhe oicrcbial &livity. 

Dificrent levels ofpH varying from 4.5 lo 7.0 wqe d$ studied in different 6€dia 

for fte growth ot lrt roL.a4 'eerei 10 overcome thc cff@t of toxic compounds 

Thc inhibilotr .ff@is of toxic compounds wcrc ov.rcotn€ by increlsing the pH of 

rhe fcmenrarion mediun lo 5.0. The inocula of oth€r fuisi, C. c.lltlalrti.uh a d 

Pr4,r-.a/, could .lso bc prcdued on tlis ftution. Thc surplls hcmicclhlGcs 

would be convcd.d to SCP by th* fungi. All of tbcs. funSi wer. c.Fblc of 'rsint 

the sugd found in the hemicellulose fraclion at pH 6.0. lt {as repoded thal the 

SCl, produced by thee fungi conlained 4047% crud. protoin on dry weigh basis, 

qlricl can be used u an animalfeed. 

Ch^h^l .t al. (19E7) found tllat toxic cobpounds lil€ furlitral, 

bydroxymelhyl furfml dd lheir prc.!$m and phcnolic compounds rvcrc 

produced du.ing acid lrdtment of lignoccllulcs. Toxic compoun& identified 

vere furfuml, sydngaldeyd. and syringic acid CnCr acids mong the fany acids 

found in hydm!.zate ll wB noticed lhst th. inhibilory eff@l of such compound 

lras olercome by Srowing thc fuSi in mcdim naintain€d at pH betwen 6 and 7. 

Hdlhni (1987) report.d rh.r 5olo HrSO. tF.tnenr of the rice polishinS, 

rcsulled h inc@led myelill prctein yicld rs conp.rcd to unlr.rt d substraG ancr 

fenenr^ti,on with TtichodeMo h@zionun tot 3 daw inder oplimum conditiotrs of 

pH. tcmpemlure and ionic conmtration. 

v.zgla et ol. (lv)l-ll) tezted Pinus pindt . bark s!d6 in a rwo st.p 

process. In 1i6t st.p it was lrcated with NIOH acelic 6cid and HrO, to incr.asc lh€ 

polysacchdidc conlenl of thc solid sidu€ In se.ond slep etzymic hydrolysis was 

achieved ro yi.ld susN \rhich w@ us€d as ferme talion medium for singlc ccll 

protein production. 

Gupt md Dath (1997) studied lhe ploduction of cellulosic enzr'r.s on 

basals. under solid srate flmdration by @olrt . of .ltpetsilla e ipticw tnd

Asperyila Iufligarts.l\e @culture system slowed imprcved hydrolylic and tcla 

Bluc6id6e rclivitis a comlaed io rh. occasion when th€y weE us€d scpararely. 

Various pflreal])ol melhods were ued 10 Dake celtutose acccssible to enzymatic 

a ack. llest reulrs were obr|inej rhmugh prerslrnenl *ith 2% (Wv) catcium 

2. MOLASSES/GLUCOSE SUPPLEMtrNTATION 

SupplemenIation of 8ro*lh mcdiurn wi$ sugds for de supply ot 

enc!5/ b@ones e$ential when ir is depteted ar a ce.lain poinr of rime durinS 

fementatjon. Diflerc sources ofsugars hav. b.cn tried as enerSy suppt€m€nr for 

lltis puQose. Each rype of sugar has its own benelir and uailiry. Some are more 

soluble hence provide readily availabte enerSy. whilc oncrs conrarn some growlh 

racrols alonS wirh rhe avaitabl. en€.$,. Il€ benefit achieved by providing different 

types of sugars fo. $e .nhancemcnr of microbial 8roqah has been reviewcd as 

Yarez er at. \t972) dctcfi ned the chemitlt composlron and biotoSicat 

quolity of rhe p.otcin biomass of Can tida tnitis. Motasses tiom sugar-b€€l 

pMesing w6 used.s a substBte. /\mino acid of|wo sampte of Cnl,s showed a 

high correnl of tysin. (6.9 ud 6_5 g/t68 N) and rnrconr. (a.5 tud 5.lg /l6gN). 

Howeve. Ihe biomas contrined tow conrerrs of S,conraining amino acid 

(ne0'ioninc i.e., l.4 rl6s N). 

Dovhych (19?5) us€d small doses (0.01 ro 0.05yo) of suse beet sri asc. 

mnize exrncf and yea$ sludge to increa* ttr Uiomus ot the yeut Cahdida 

topicalii K-41 g.own on oit paia{in. He .€poded rhar $e addirive of molass€s, 

maie extmct .nd yeasr sludSe rcsutled in 8 to I I % increse m biomass. 

Fod^ et al. (t9'16) used fie stop (vinsse) liquo., a maJor byproducr of 

ahohol ffln€nlation industries as growth mediutn for plod n rton ot Cwdida ttilh.

l0 

They reporled $at supplementation of fie slop wirh 0.2% (NHrzSOr dd l-5 yo 

c.ne molas*s improved rhe yeasr @llyi€ld, significandy. 

Yoso ?r 4l (19?5) comparcd difte€nr yqt @Is tor prcducrion of sinSte 

cell protein on m€dium containing Stucose, glacrose or f.uclose, glyc€rol. MeOIt. 

EIOH. HOAC. They .eponed $ar whcn Cadidd MOy,657 ws culrwcd wilh 

shaking l0'C for 48 houB on a m.diurn at pH 6.0, it produced yeasr mass 

conlainine 55.5% c.ude prolein, whi.h was 313% jn rems ofM.olt. 

ElAshwah., a/. (1978) delehiD.d rhe cfiecr of motGscycom slep tiquor 

on yield aod piotein concenb.iioD of thc fungi. They repofted thar medium 

.o.taining only molasses could nol suppon adequatc tungal 8rofth, bu( rddido, of 

com sreep liqDor rsultod in a narked iDdease in tnycelial yietd and protein 

concenuation. It was filtttx.r r(ted tttrit Spicorio cbaaN, Ctado4oi$ sp. and 

Clad$potiw .kdosporiodes produced crude prolcin 34.06, 26.69 a^d 2:J.4s Vo, 

Pa@nml e/ dl (1978) ferncnlaled tyi alkatiprerre.&d hadwood sawd$l 

lq 48 hou6 sr an initat pH of7.O{Enrr5.0sd.r 37,C tcmp€mrue ed obseded 

12.5olo 6rudc prolein in rhe biomss. Thcy atso rcponed thar lhe addition ofgtucosc 

@ 0.5% naRedly incrcased rh.prctcin yietd. 

LaNford d! dl (1979) used cane ,nolases s source of 8row$ limitinS 

carbon in rh€ fermenrarion media ior rhe prodncrion of sinste e prorin using 

Cdhdido trtilit NRI(L y,900. Ar one percenl level of reducing sugr wi$ $e 

addition of zinc powder, 6utt€d in an increse in biom6$ producrivir, ircm L? ro 

2.6 g/t/h wifr rhe elo*,th yield of 0.559 dry biorrB /g rcducrng srge utilizcd at $e 

naximum dilution rale. t he ye!$ biomass contained 5G55 o/o prolein_ 

Rosl*ova (1979) compared fE biomrss yicld of two y€,st slrains viz 

Catdida ttain 2672 ed 2671M on acerst. and m.rhanot medim lnder o imun 

condnions. Maximum yield ofbiomass for srrain 2672 was 56.5% dried yeast after

20h ofculturins md 44.3 % rne.4Eh for strain 2673M. Th. crud. prcrein @nicnls 

w.e 51.9% md 4E.7% fo.3taiN 2672 ud 25?3, cpectiv.ly- rvh.n glucos. w4 

addcd dlotrg *itb .ceLate drd melhanol in th. mcdium for lwo straif,s, litll. 

difltrcnc. in composition oflheirbiodss wd obsedcd. 

Dowson and Sleinh q (1980) sludied lh€ cell cyclc of Cdndida uilis i, 

rlmc nlcdia of .elntcd conrposition. Medidn(a) was thosPhorus liniled ond 

connined 30g tlucose, 2.58 NlIi)rSO. md 0.18 KHrPOy'L Medium(b) wa 

nirrosen limited and conlain€d 308 slucose, L08 o\lHrrsor and 2 58 KHfoa p€. 

liIer. Mcdium(c) w6 carbon limited md contained 108 slucos€, 2.5g (Ntta)lso{ 

and 2.5g KTHPO4 per lite.- Each medium al.o @ntained 0-58 M8SO.THrO, 0.059 

Coclrsd l0 nrl winzlcr miqo nullienr s.lt solution ln @bon limitcd mcdium ell 

division occured a1 4.5 to 5 houB. while in N and P limiled mcdia it occwed 

betweon 5.5 to 6 hou6. ln carbd limited Dedilm bud cvcle fom.tion sbrred s@n 

aRer the stan ol cell cycl€, wh@ s in N md P limit d mcdia bud did not fom 

utrlilafter 1.5 to2 hour. 

El-Ashwah e, al. (1980) Drcduced nDsal biomas bv cultivarinS different 

fungal $lairls on subsltatc comprising molass.s, swcet Pot toes od com stcep 

liquor. The resullrnt biomess (24.2Isll) €odai'ed 34 38 percent Prctein' l-7.8 tL 

to(al crud€ prolciq 14.33 g/L crude fat, 50'lo N.F.E and 400 mg/ 1008 NPN. 

Chen e? al. (1987) emined the efTecls of cullurc mcdium on lvsine 

fomation duinS fermcntation with Br.tibocteriuh fuw strain FM 84415 The 

effccrs of salls, urea, bran, pH, sugar and com powder werc studied. High€sl lvsine 

prcduction (64.39 lysirc HCyL) was obsewed with a medium containing l2-ll% 

slucose, l0% molasrar, l"/o clm powdcr, 0.5% hair hvdtolver€' 4016 (NHr)$O. 

0.02010 KrHPo1.3HrO, 0-02% (HrPOr,0 06% MgSOa TH1O and 4% cacor at pH 

7.2 in a fmentation pcriod of ?2-E0 houB.

t2 

Diaz Ricci .r al (l9E?) fepo ed lhat whcn c!/i/ir was continNusly 

cultured in sugar qne snllhSe wirhoul any supplmotllion, the y*r ulilized the 

stillnge rilrog€n only panially. s a result the ccll biomass producdon was low and 

fic rcsidual COD, as woll N the totul Ditrcgen @nte of the efluenl werc high. 

The addition of24gll mmo M phosphare and smlt amounb offe, . Cu,' and 

Znr' Ddkedly enldced both the ovenll nirrcgen uprake and cetl biomass 

production, decreaed the COD dd higber cdtical diturion r.te w6 auained_ No 

subsldlial differ€ne werc found wh.n pH was kept ar 5.0 or allowcd ro vary 

freely. The optimu,n lemporature range was found between 30 and 150C. 

Atsushi and Isamu (196E) dedved mur.nts from d./?dl@ No.l5-8 si$ 

cirmle synlhase activiry and feed back resists1. Ih€se mutants prcduced above 40 

g/L oflysine d ilJ Ilcl salr in rhe medium containing t0% 8luc6e. Strain No.6647 

with normal activiry and completety feed back resistanr, produc€d 45 g/L of lysine. 

Chavez €/ al (t988-I) produced microbial biomass usins celtulotyric fungus 

with glucose o! molas$s subsrrare 6 min csrbon sourcq. The chemical 

composhion and Dulrilile values of rhe bionass were compmd Nilb othe. 

microbial protein i... Totuto !41, brcw6 ycr5t and dricd mushrooms. Tlr 

.erlulolyric fungi C.ce ulollti.@ MAp products coniained about 45% crude 

prolein on dry mattcr bsis. lheir ajnino acid p.otile, aner hydrolysis was conpar€d 

well with rhar of dried skim mitk. Torat amino acids accounted for onty 75_6 and 

62.5% ofthe crude prcrin ofMBP ofglucose and molNes substrate, rospectively. 

ljad.i-SNi ?, al. (1988) srDdied rhe influence of initiat concemrarions of 

grucose from 60 to 223 8/L on the prcducrion of L-lysine by Corytub@teim sp. tl 

wa studicd fisl in batch culturc. The maximm convcBion rare into L_tysine was 

oblaired .t 165 g/L.nd thc best specific prcduclior de for LJ)sin. was obsen.d 

at 65 g/L ofglucose. In fed balch feflrenralion bcfler and specific produclio, dies 

rve.e obtained. Maintiining of a high gtu@sc cotr@l.arion h the f€d batch

ll 

tcchnique allowed r 54% incrcase of L-lysine prcduclion @mpaEd lo batch culturc 

i.e.2l vs 50 s/L L-lysine HCl. 

Gomez (1989) culturcd Cddidd ,7 i NRRLY 660 on substrate containing 

cane f&lory mixed juicc ed molNs, diluted juic. ed eith€! mdNs, ckined 

jrice or vina$e.In each casc th€ constiluenb rere used in s@h proproiions fial fte 

mlios of tolll tducing s!8& wcrc 0:100, 70:10, 80:20. 90:10 ed l0O:0. lt wc 

concluded that mol6s wa ! good slpplmcnt to 3ubsrat6 bacd on filmre o. 

vin.sse, bur rh. prot.in yi.ld and produciivity d.crcdcd with molsses of abnomal 

@trposition or with t@ low proponion ofmolases, 

Hir.o.t dl. (1989) clltuftd CtlsrznriM rtrnin in nediuo bdcd on cde 

n]olasss, inilially conraininS 3% susar (a, slucos€), f€d wnh a nedinm containing 

12,20 or 28% suga$.'i]]ley obsedcd fEl L-\isine output was slD6l constsnl 

durins 50 hours. Tle maximum L-lysine concenlration @ched was 105 g[ and it 

d€cr€ded with increasing dilution rete in the rarg€ 0.03-0.07/h. Productiviry 

ircMsed wi(h .gitatio! ntc in the rdg. 450-?00 rpm, it inc@l.d who oxygd 

cnriched.ir was us€d. Thc nnximw yield wG 5.6 g/tfh,2.5 limes rhar in fed 

Pf&jy ud Ulb.n (19E9) isol.t.d C.sl anitu 9166-AEq tdosedrc 

dependcnl, aillant to S-(2-minoethyl)-L-Cysteinc ed sensilive to fluoropyruvde 

and culturcd on a medium corbining 2540 % molases dd 18-24 % Sround nut 

meal hydrclyzt.. It produced 40.4-49.58 lysine{- rhtr was hiSher rhe orh.r srnins 

tested. Yield was mdimum wirh 250lo molasse, 24% hydrotyzrr€, l% com steep 

liquor and | .6 o/o CNItr)rSO.. 

Abdel-Hameed (1991) usen sugar cue mol.sses as a cr6on sourcc for 

growth and single ceu protein (SCP) produclio. by Sadcrarohyce, cenisiae lal 

cdrlsbereehsit. He obtair.d suffici@t single ell prcrein yi.td ar t0lo sugd 

cotrccnldtion, while 50,6 wos c@plcd wilh gmd grcwrh dd minimat chotqtol

@nlcnl. Fudher impmlenent of biomass and single c.ll prolein prodlction vas 

achi€ved on 0-3% KHTPO. and L50,6 M8SO..THrO addilio to lhe mdass 

NL'diuD. Thc coDparative @lysis of ycst c.lls grown on synthetic "Phaf' 

medium wilh thal ofoplimal molass€s m.dium rev€led an increa5€ of4l5% SCP. 

52?0/0 peplides, l0l8% arnino acids, 2010/0 ammonia, 394% lotal soluble nitrosen. 

394% lolaltrilrogen and 522% in dry weight eain for laler.It was ako obseNed that 

lhc cholestrol cont€nt decr€ased by 40% on moldsesmediu'r. 

Bajwa er dr (1991) oblained a biomass co.taining 15.17% protein whet 

0.5vo alkali lreared dce slraw was fenn€nt€d wi$ Auchniot8 spe.ies. They 

ferndled the sDbslrate in the prcscnc. of Caclr 0.0l%, no|ldg 1.0% wilh C:N 

mrio of 25: I at pH 4.0 od 30 oC temp.mturc after 6 days of incubalion. 

Abo-Ahmad (1993) hydrolyscd com sFaw with 0.5 N H,So. dd srew 

Sacchotohlres ceflisioe on ils diff€rent mncenk tions. The high€st concenlralion 

of clm sFdw hydrolyz.tc (CSH) !t which thc maxirnum cellular prorein (SCP) was 

obhined wa! that mnlaining 15% sugors. H€ added dilTercnr conenlralions ol cane 

molasses (CM) to lhis n€diun ed obtaincd $e muimum biomai, containing the 

highest cdcenlnlion of SCP wi$ a m€dilm conrainingCM ar a ocotmtion of2 

e,I' 

Alhd er al. (1995) used bcer pulp for bieonvcrsion inlo nicrobial biomN 

iD fed batch cultu€ syscm. Tley suce€ded in improving the qumlity of mioobial 

bionds produced at first st€p i.e. 5.9 mgml to 7.78 mg/ml by supplemetrling the 

femmtation medium with 1.5% molass€s and 0.75% coln ste€p liquor at 36 hou$ 

of fementation. I het r€ported that the ,.J4rvrm inoculalion with 40lo glucose added 

in Cltlir medium enhanced prolein f.on ?.92 mg/ml to 10.74 mg/ml and lysine 

contents from L29 to 3.08 p€rcent. 

nerc6 er al. (1995) producri biomass having hieher prctein conlenrs frcm 

sugu cane juie with a liltle proportion ol molasss. They obhined a grcatcr

l5 

nunber of yeast c.ll in 10-14 h dd found nccessary to ae frcsh su8e @e juic. 

qhoce rH $s 4.5-5.2. ar incubdrion remptrsture or 12 t. Tle oprimum 

concentntion of rcducing sugar at initial nage wc 70 8/L. 

Elsabacny and Hlus (1996) femmted d.i.d vhey *ith ltutobtuiUa 

delbub uec*ii sub sp.cies dg@r'tu 369 and cpo.(ed the hi8h6t producrion of 

lactic acid l.7l 8,{-{r by using medium enriched wi$ whey (8% q/v} at pH 5.5. 

Spccific activily oflactic acid dehyd.oSenrse was 1.03 U/mg prolein and single cell 

prclein produced was 32.8%.In balch culture, the addirioD of20 tL elucose plus 

0.759 nilrcSen/L a (NHaLSO. reruX€d in highst prcducliviry. By contrar when 

xylose ed cellubios. rw 6.d d a eure of fm.ntlbl. clrbohydrar€s all 

vad.bl6 serc reD.escd. 

Han.t 41. (1997) isolared a'nicrcorg.nism from th€ provincial soil of Soulh 

Korea. Tlt orgarism was idotilt d ts Candida sp. Dt t6. 't h€ oigrnism had ability 

lo grcw in minimal poultry fa€B extEct. Supplddtation ofglucose to poulry 

fa€c€s exhcl medium helped in compl.t upirte of soluble protein by lle 

micrcorgatrism. Uric acid in lhe poultry famq cxctEct mcdiun could b. 

con,plerely dcsodcd du.iDg ,nicrobial Srowth, but this deSadation wa faler in 

taecs exkact thar in lhe gllcde oomplement medium, They obtlined maximun 

cellsro*lh (l.8xl0'CUF/ml) at 36h of incubation. Addilion of4% neopetlore ro 

th€ oinimal poltllry faecs edEct medium promoted Inaxilnal growth ofcells. 

Lo sr o/. ( I 997) studied the cffecl of th. gl$coser'yeast cxtEcr 6tio (c/YE) in 

$e medium on c€ll srowth and xmthan production in various op€.aling models 

includios barch, two stages batch atrd fed barch fermentarions. They {o!nd thar 

incred€d C/YE in the m€dium r.sultld in incrcdcd x.ntlan Drduction bur 

d@l%ed c.ll yicld Md speific growth ra1c, lt ws @nclud.d that two slag6 balch 

fmatation wirh C/YE shin frcm (2.5% 8lu@ser'o.3% y.!st .)dtnct ro (5.9% 

glucose/0.3% y6si €itdc9 at th. €nd of.xponential gowth ph&c€ ws prcf.nblc

l6 

for xanlhan produclion md fast cell growth. In contr6( fed-balch fementalion with 

idrnniLrcnt oddilions of glucose to rh. fcrbentltion medium durine th. srationary 

pha3e wss not tavoumble for mlhan prcducrio. b@!se of reladvely low O/YE 

rcsultinS in low xutho production rat.andyi.ld. 

3. CORNSTEEP LIQUOR (CSL) SUPPLEMENTATION 

Com step liquor is . by prcduct of @m itrdurry snd is produccd 

durint the com wshing. ll is t rich eurce of orgdic nitrogcn, vitMins md 

minedls. lt is d$ rich in sotuble su8$s snd th@f@ @ be sed bo|h as .n.r$/ 

$ppl.,nerl s well $ source of nilrogen Equicd fo. li. microbial gtoslh. ft ha 

been us€d ei$er 3 exclusive growlh m€dium o. 6 a supplement in lh. grost' 

mdiuD for enhancedent of microbi.l g.ov"4h. Lit€dture hd becn rcvicwcd on 

corn sl€cp liquor s supplement in the 8ro*1h m.diu md k sho{n as follows. 

Foda .r al. (1973) u5ed com st..p liquo. conlainins procitr 5.2, tolal solids 

I L4 lnd cdbohydotes 0.2% fot sowr|i of Candida utilis. Tney obldted oqimum 

microbill yiclds of prcteio ild dry matler a 38 to 55% ed 15 lo 29 8/L, 

rcspectivcly ancr p@ipilation of liquor bctw@n PH mg€ of 6.0 lo ?.0- The 

femenl.lion ws cMied orl at 30'C for 72 hou6. Shellan (19?5) ptoduced tunsal 

bio'na$ by fennedting l0 g/L corn sc.p liquor havins C;N ss 6rl with 

n i.hod.nM viri.le td 48 hot6. 

Smirh d, a/. (1975) delermined lh€ anino ecid composition and prolein 

nutilivc values in rais and p'gs f€ed'nS trials. Six diffd€nt funSal my@lia gro$n 

on diff.renl subslEtq wee l6led. Th€ protein qulity of fungal biomas of 

Rhizops dthbu M 261 sFtin 8rctn on @m slep liqror rcv€lcd NPU, Prclein 

ditestibility, biologi@l value and chemical s@re 3s aa,69,64 and 60 rcspecliv.ly. 

lle bio'noss ws repoftd lo b. deficient in sulphcr @ntaioing amino,c'd!. Trte 

RNA conrenr oflhe biomN wd 1.2 %.

t1 

Yogo er al. ( 1976) krmentql Cattlida Moy-657 wirh shakir g at 30 'c for 48 

hours on a medim (pH 6) onrairing NttaNor 4.0, KrHPo. 4.0, Mgsor 0.5, 

t:eSO. 0.02. Caclz 0.01, yelst exnact 1.0 ed com st.ep liquor 18. Miror amounts 

ofMnSOi, ZnSOi,lhiamin€ and biolin, wiih lh€ addition of l0g methanol/L were 

udded. Tle yield of ye6!r conrain€d 55.5 % crud€ pror.h rnd w4 31.3 % in rer6 

Svrki .t at. (1977) qfta.n Cardida l@alr,?a 

on a medim conuinilt r- 

alkanes 2.0, (NHrrHPO4 0.4, KCl0.l. MgSOa.?HrO 0.1 ed com steep liquor 0.1 

pcrcent plus lrace amount ofNacl, CaCl! FcSOa and y€lst extract, at ptl4.0and 

ls"C lemp€nlDie. Sp€cific growlh Erc wd noled s 0.3?6, yield baled on n- 

alkanes wd 103.8 pcrccnt dd @d6 prct.in @ntdr of 0|. c.ll w3 59.1%. 

El-Ashwah ./ a/. (l97El) rcported thal ir fermentation medium having 

nolssses, sweet polaloes and com sGep liq or the produclion of biomas wss 24.2 1 

e/L containiry 34.3E percst protein. They .le oh6.rv€d d oprimum rsle of 

slalin8 ror 6 isobt.s of fiDsi Impcrf.cri. A nte of 250 rpm w4 found suirabl. for 

all culturcs ex@pt Clddospo.,i@ claosopqioides (150 ry'i). 

rrantisek er r/. (1982) precultured Corywb@teliuh slutamicun in a 

mcdium conbining ACONa , slcrs€, com st€ep liquor and watcr. thcn flrlh€r 

cullured fo. 96 h !1 29-30'C i! medium @ linin8 addni@l pcanll nql 

hydrclzte and mineBl salls with feeding fron 5th 10 24th hour ad a mixturc of 

ACOtl, ACONHa, lactic ac;d md sucrose and an cqual volume of air. l he yield or 

33-40 g L-lysine[ medium was oblain€d. 

GaB and N.clalelln (19E2) fem.nted I pcrc.nl alkli trealed bagas 

alon8 with €ttle uring urea, com st.cp liquo., (Nt!)rSO., NH.NOr, ltNH.SOt, 

Ntlacl a NaNOr (fumishing 250-600 g NlL, wi|Jl. Asperyi s tefteus.'rhey 

r*o!e.ed 32,8 pscent crude prc&in 6rd stlt€d that all sddili!6 lsed 6! nitogo

l3 

s,urces prcv€d bcrl. Com sle.p liquor wos the besl ofall, whcrc s licNtlrso. wos 

fdrnd as lhe poorcst rourc€ ofnil.ogen. 

El-Ma$y d, al. {1987) uscd DreLreaied rice straw for s(udyins choico ofhost 

suitable nitrosen source ro increase th. prcduction of fun$l biomds 6 w.ll as 

prcteinby Tri.hodem hani@@. They r€poned that u@ wls gMtly dsimilal.d 

than other nirrcgcn sources and addilion of us in conjuction *ifi ammonium 

sulfate in ! ralio of I I I gsve a tunsal yield of 8.7 g/L, with 30.4% crude prolein md 

2.6 g/L rotal prot.iD. Com steep liquor used !t a level of20% (.quivalenl to 400 mg 

N/L) gready improvcd the tungal yicld to ll.4 e/L and protei! cont€nt to 5l.l 0/0. 

TIE mino acid profile ofbioN showed the pr*nce ot lE amino acids- Tb.y 

obsered rhat thc composilion of T.hoBionun ary@cned |Jrat ofsoyb@ m@l md 

sug8sted thal it can be used a! a supplement to plant prolcin in mimal feed. 

Mahmood ul lldsan (1992) produced a biomai, having maximum of 280lo 

true prclein aller fennenlalion of 3% beet pulp wi$ ,4rp?r8ilr' ,e/'e6 undcr 

optimi&d condirions. Tle ionic concenlration of lhe medium wa! adjust d al 

0.0075% Caclr, 0.01% I{gSOi.7H,O, 0.125 % KHIPOr and supplemenlcd wid) 

1.2% corn sle.p ljquor. 

Athar er ai. (1995) reporl€d thc bi@onveBion of b..t pulp into microbial 

bion6 h.f.d ht.h cultuc sysr€m. Th.y succeded ir att.inirs 5.98 ms/ml of 

mic.obial biomsss prct.in aI inirirl 3t.p by f.mdtltion wi,h Candida ttilis wn rc 

d fol batch of 1.5 % ndaq and 0.75 % 6m ste.p liquor ai 36 hou6 of 

fcarcnlation causcd tunlE ris€ of micrcbial prolein to 7.78 m8/ml. 

cm€lilrs €l al. (1996) worked on . new liquof com sre€p mmDfactured by 

Roque(e Freres, which rcpB.ded a source of.nitroge4 tace elements and sev€ral 

vilmiN to cvalulte ir for industrial prcduclion oflactic bacl.ria. They found thst 

So/ltr l-3b 2218 advantaseoullr coorp4ed Iactobacilla planrd'!' for biomals 

production, at miDiDal y6t extacl addition. Cultur€ m€dium containing Sotutr

l9 

dd yeasi exkact in con@nrntions of8-10 g/L and t-2 gr_ rcspecdvety with 20 g[ 

ofD-dexhse led 10 similar biomN..lcs rhan.ulture mcdium wnh onty 5 gil_ of 

4. CARBON TO NITROGEN RATIO 

Shellan (1975) used com st@p liquor for rhc prcduclion of fungal prccin. 

tle obtained 48% cNde funSal protein by fermcnt'ng l0 g/L ofcom steep liquor 

havingC:N as 6ll vitll Tti.hoderna vii.le tor 48ta@. 

Osmm and Voino ( 1976) studicd rhe effect ofCN mtio on the prcducrion of 

ye6l procin. They ob$wed thal drc protein conlenls deoreased from 52.30lo lo 

42.0y0 by de.reasing C:N nlio white r,he biomas yietd incr€s€d. In orhcr 

experment rt wa5 obseflcd that aner keeping the suSar t€vel constanl and by 

dsresing the C:N rario f.om t? to 8 rhe prcreil| conient ofyessr ircrcas€d from 

2E.lto5l % and th€ biomass yield likcwise droppcd f.om l8 to t2 g/L. 

Prmment er dl (19?8-s) cordrcred $lid slale femenradon of alkali 

prerred.d haplc s.wdut jn rmy and ronbt. fem.nld usinA Chaeroniuh 

ce outicth as fenenrative ofganism. Th€y reporred rhat crude pror.in contenl of 

rhe solids roso f.om 0.9 to I tyo i! r.ay t mcnr.r and s% in rhc tumbte femencr. 

Tle pH of llle medim was set al 5.0, cebon ro nirroge, mtio ar l:10 and 

tempenturc al 37oC for 20 days. 

Abdullah e/ 4l (1985) srudied ih. .ffer of C:N nrio on rhe produclion of 

tuneal biomss proreir f.om wh€1 srrav wirh Chaebhiun cellulolrticud. .Ih. 

fdnenltlior ws cMied oot uder sotid sbd eidr mmonia fMa cxprcid 

followcd by srem telted whcat stoq (100 8 at 80?o ww mostu.e conrenr) and 

C:N ratio of l0:t at 30oC rcmpemrure in rhe prcscrce of KHrpO., ZnSO. ard 

Feclr. They obt|ired mdimum bionas, pmbeh yietd (27.2%) ane.3 days. 

Camal €r d/. (1985) pmpagaLd h. Candido utitis tn .ontintes cutrue usint 

bagasc hydoly?rle medium suppli.d ar diffe.crr diturion d6 .atrting from

20 

0.0122 to 0.02E p.r hour. The highest productivity w rcco.d.d d ! diludon 6re of 

0.022 pe. hour. 11 was obs€ryed lhal Yeast dcnsity, C/N conten( (4.46) and nilrcgen 

consurnplion rate reached their maximum. Wahing out of crllurc occuir€d at a 

dilulion mle of 0.028 pq hou- The moul of @sumed ca6otr rcqnired for 

produclion ot lg carbon biomN wd found to range fiom 2.21 lo 3.05. The amount 

of .oDsund CN bioorN werc 9.37 \nn 4.24 ^10.022 p.r ho!. dilulion rale. The 

prolein conront, carbon conlent md total asb of Cad,ida urilis CBS 62 I *ere 42. I , 

93.4 ad 6.5 p.rc.nt, respectively. 

H6hmi (1987) prcdlctd biomsss protein aner femcnlation of nce straw 

eitlTticho.Lrtu hMiuw. Mrximm prctcin of 13.3 perc.nt was ob(qined when 

fementation wa! clded out und6 c:N latio of l5:l for 120 hou$ al pH 4.0 and 

l0'C le,np.ratur€ on 5% slbslrate. 

Hashmi er al (1989) culued Alachhiota sp. on slkali n.ded rice strdw lor 

lhe produclion of micrcbial biol)N. Th€ fcnnentatron medium .ontained subslrale 

Mler ratio 5% w/v od wbon lo nirogetr latio of25:1. Th. biomass prcduced in 

srill md submerg€d cullure showed almost lh€ sme di8€stibilily valu€ ftr dry 

Dauer, crudc fibre and crDde p.olein uDd€r optimum colditions. From I ks ofalkali 

keated rice stmw ?30g biomass w!! produced. 

Kiei .? al. (1989) studi€d production ofbiomds prct€ii fion detatted rie 

polisllings by f.nncntirg tr wirh Caftlida lrilir. They obtlin€d lhe muimum 

biolsss prctein of 27.E% udc. C:N ntio of 12.5:l for 72 hom at 35'C 

Bashir (1990) produced lhe biom8s proein ftom whcd bnn ihrcug} ib 

tenent ion with Cahdidd ,tilis. He obbin€d lhe mdimum biohass Prcl€in by 

fmotation under CrN otio 12.5:l in th. pr€sence ofCaCh 0.005%, MgSoi.THro 

0.0050/0, KHTPOT 0.001% al 35'C temp€Elue ud pH 6 using whet bm 6 

g/l00ml.

2l 

Mahmood ?/ at (1990) sludicd lhe innu.nce ofdiffercnr C:N ralios on the 

pulNrior of furS. bionms ptotcit bt /lro.hniot6 sp- usin8 ricc tolishangs d 

subslral€. l hey obsined lhe maximum crude prolein under C:N ratio of l 5 r l dd in 

fie prcsence of CaCl, 0.001%, MgSOa 0.00 l% ud KHTPO. 0.075% ar ptt 4_0 and 

30'C tempcrature for ?2 hou6. 

Bajwr et ar (1991) obraincd $e maximum crude prorin of 15.17 p€rcent 

Birh 0.5% NaOH kearcd .i@ srraw (5yo) femsrcd wjth Atetniotw Sp. 'fhe 

opttum conditions used were cacl,0.0l%, MgSOr 0.01%, KH,PO4 0.2%, 

molasses (cane) 1.0 % *ith C:N Btio of25:l at pH 4.0 and 30.C rcmperature qirh 

Chanem er a/. (1991) damined rhe eflcct ofsubstote ro wsler and carbon to 

nitroeen ratios by !si!g (l) lxcd dounl of nutri.nr medium md incr€asing h€.r 

puh levels (2) usirg conslart b€€r pulp level ed incr€aling mcdium votlnes. 

Subsrate 2 g/l0ont proved the oprinat Cn! ratio for prctein production and high 

convesion elficicncy of 23%. TIE resulrs atso indicared rhat ls (NHrlSOy'L was 

opnnum for maximnm biomass yietd. Higher coocentrations of nirrogen sfiowed 

inhibirory effects on c.llulasc production makin8 tec, slbon avaitable for 

Ak.am (1992) prcduced fun8al biomss using whear bra, os subslrare and 

Asper{llu tercLt I fcmenrative ol8eism. He obrained fte maximum tungal 

biomass production having 32.88 0/o crude protein with 8% subsrEre (s/v), 

0.0075o/o CaCb.0.l% MgSOi.THrO, 0.25% KHrpOa and 7:l carbon to nitroS€n 

Mahnood ul Hassan (1992) oplimied conditions for p.oduction of fungat 

bioDass. He us€d 3% b€et lulp ( v) under the ionic conce,rratio, of 0.00?50lo 

CaCl2, 0-01% M8SO+?H!O, 0.125% KHrpO..?HrO and carbon to nitrcgcn mrio of 

5:l vith fr suppl€me.tarion of 1.2% com s1€ep liquor. He obtain.d rhe maximum

22 

bioffi prctein of28% by f.mdtlrion ||ilh Asperyill6 terreu. H. obsewed th.t 

the biom6s w6 @mpler.ly lackiog in cscnrial dino lcids, llsirc, his dine dd 

Arhar er al. (t995) rcponed lhe bioconveBion of beel pulp inlo hicrobial 

biomN in fed barch culrue sysrm, Followirg 3% sutEtEtc to \rarq Eiiq 

0-00?5% C.Ch, 0.125% MgSOrTHrO, 0.30% KHTPO. 0.000?5% F.SO..1H!O 

and l5:l carbon 10 nitrog€n ratio drcy sll.ined 5.98 mg/ml of niorobial biomass 

prorein at initial sbge by fermorrrion vit\ Condida utilis. Fed batch of 1.570 

molasses and 0.75% CSL ar 36 hou6 of fermenlation caused funhe. rise of 

microbial prcrein to 7.78 mg/ml. Tle baclerial inocul.rion with 404 glucose added 

lo the abov. fermenied medium ohanccd prclein upro 10.74 ng/ml od lysine 

content fioo L29 lo 3.08Y0- 

5. INCUBATION PERIOD 

This is onc of rt. very imporrer palama.r for 8cning ndimum 

yield Aom m orguism od is also iDpo.ret to loow tbolr ib p.liod of mdimum 

8ors'1h. Th. incDbalion pe.iod of any org&ism dcpcnds on ih€ avaitability of 

optimun conditions. Once he 8ro*th conditions arc optmized it is necessary to 

tnow abou( the inobation p€riod of thar orgmism for Setiin8 majdmum yield. 

Some o.gdbms gmw in shtrt period of ine while orh6 take mor. rime. Srudi€s 

have beer conducled 10 know about th€ inqibarion Friod ofdifferenl ogranisms. 

Follosing arc a few rcterencs which gives info.tnarion aboul rhe incubalion p€riod 

ofeme o.ganisms and its .r.ct oo final prcducr yietd. 

Rieche er a/. (1966) fem€nrcd t% acid lre3t€d hydrolyed potaroes with 

C.Utl,r sl.. adj61in8 pH ed adding .|moniuD ph6ph.t ed mnorium 

sulphac as oirlogen sources. They obrained rr4 ro 469 yea$ dry mane/100g 

rcduciabl. sugd md 36% crude prolein wilh incubarior period of4-5 days.

2J 

t'oda d al. ( | 971) Iirmented supemaranrs of corn sbep liquor obtained atter 

precipiiaron of protein *i1h C,dda d ir for goducrion of fodder yeast. Ihey 

$rcceeded ii prcducinS yean biomass containing 3E lo 55% prorein aft$ 72 hou6 

of f€.ncnlalion at lo'C. 

I le { | 975) obtai,rcd lungal biomrss having 18.6% c.ude prorcin atier 3 days 

of fernentation of alkali prctre3red rjce srraw wiln mixed cullurc of Cellutohow 

sp. Md Alcalieenet f.ecatis. He reponed that c.llular biomas was rich in all 

essenlial amino acjds ard vilsmins nnd had 4t.2 lo 55% in- viro proiei! 

dieeslibility. 

Pcn6.n (1975) optimiz€d rhe conditioB for two srr.ins of @futotyric funsi 

Tri.hodetM vitide. Tlc conditons w.re blsed on max'oum yietd of biomds 

prol€in and cellulase enzym. prcd&rion, He found thoi l_2 % ofalkati rcared 

barley srmw when fennenled for 3 days at pH 3.5 ro 4.5 yielded maximum .rude 

p.otein (2t-26y'\ whercas t0 days of fernedalion yi.tded maximum ce utsse 

R€nde and Cregory (19?5) $rou8h fennentalion of cssav a vjrh Atperaitrus 

lutrigota obtd,rcd microbial biom0ss after 20 hours of femenlarion. The final 

yi.ld lvas 249 dry product conraining 36.9% crud€ p.otein. The detcrmin€d prorein 

on amino acids b6is ws 2?.1%. 

AroM md Fitzpatrick 0976) obLin.d mdimum prcducron ot e ure 

.nzya. wirh Trichodema ei,id.. Th.y trr.d m.dium amp.bing dihydrogen 

phosphale, dimmonium sulph.r€, CaCl, atrd other lrac€ elen.nts with an 

incubation period of ?0 hous. 

Yogo., zt. (1976) culrwd yejsr Card,Za MOy-657 in a m.diun having 

l0g MeOH ed lg com sreep tiquortiter at 30.C for 48 houB. tt war observed lhal 

lh€ crud€ prolein in rhe yesl cells w6 55.5 percent, which was 3t.3% in tems of 

M.OH.

24 

Fuska md Kallarova (19?7) used mixcd cllrwe of Mw steritiM and 

cibbetettalrjitwoi on sav dusr hydrolyale for a pqiod of52 hou6 and obtained 

1.28-1,45 a dry uDucr/100 mt othcdiuh. the dty matter ofDyelilm @nldned 

33.3'35.7% amino acids ot which |6.I-16.6% wcrc escnri.l amino acids. 

Kag€aki d, a/. ( 1977-a) cutrurcd Toltopsis nahatutophags 6 ^,i.dinnl 

ror 30 houB and obtained 3?s/L ofdrj.d ce s. Kas.aki sr d/. (t9??-b) in second 

stut\ ,crobic.rry cuhurcd //a Nehuta trcthrlororu 91 on ^ tncdiuh for 30 houB and 

obrained cell yi.ld oflsg/L. Kagc*i et al. (t977-c]l i^ another nudy obraincd 20.1 

stL ..ll prcdwr u'ing Hmicota net,ytovorc 97 . The naubation period in lhis caje 

PeileBen (1977) oblained n]dnnum of24 tL mycelial prolein by culturin8 

Irichodena vnid. witt, 4-t I g/L of subsrnlc. Hc used (NlL)rSO4 t.S. KHrpOl 

2.0, !rea0.4, Cactr 0.3, MgSOa.?HrO 0.9S, ZnCIr o.Et, CoCl, L0 (gl0g ccthtose) 

md peplores (t8/kg cellulose) in thc mcdium hd obseryed an hcubaiuion pedod 

Patmenr er dl. (1978) srudied rhe fementarion of atklti rrelted sw dost 

with Ch@toni@ c.UutotltiM ed obhiocd 12.5 p€rccnr prorein iD 48 houB. Thc 

subskate when f.rnored in solid srar. for 20 d.ys wirh Cra etonitun ce utot ti.w 

produced onty lt.0 and 8.0 percont celt mass for tray fermenl€r and rumbtc 

feflnenler, resp.ctively indicating much slowq rate rnan wben substrare was 

feflne ed in shate nask. 

Silamm ?/ al (1978) srudied the abitiry of some tunsat cutrures to produce 

microbial prolein on 3 difterent !rclr$ted c€llutosic subslrates (rice slraw, bagr$e 

ed groundnut stElk). pe,.tidr M chtysoeenW go\ed s ve bd or8rnism fo. rhis 

purpce. It produed 85 mg prorcin p€r g of alkati rentcd rice straw (ATS) all€r 62 

h of incubalion wlEn fte iniriatpH wd kepr in fte nnge ol 3_5.6.0 at t% Vv of

'neal 

25 

Ba.jrach.rya and Mudse( (1979) repoded (hat prolein r€covery from atfatfa 

residue increed from 47.270 tor conrrol sanples upto 64.5% fo. temenred 

sampres an r irs fmcntation wilh different fuSal stnift e.A. Aspergilt6 sp. eM 

9994, AsFErgilb hiqet QM a77 a d Rhitopt$ "igica6 eM 387 for 5 daF. Kamel 

(1979) obse ed lhal yeasl vcry ellicicntly convedcd 4-5 perce dates soluble 

solids to 4.E6 percen( sinSle cettprotein ater I2 hours incubation. 

L.. et al. (r97g) studi€d rhe chmctdisrics or 6 isotatG of @ltulosic 

bacleria. All werc idenrified .s mcmbeB of rhe gens C€itutaroer. Diff.ml 

condirions w@ optimiad fo. hqimun yietd of biomass. rn rwo separ.i. rrials 

one at l% and orhd at 6% sobsrEtc tevet recove.ed IZ ed 25 g e msyl lnd 

48 houls of f€mcnradon havinS 4 t .55y0 and 25.6% proem, resFcrrrely. 

Quie.zy ar ar (t9?9) obrained sphagnum peal exhcb as hydrotyzates aod 

osed that as culrurc mediun fo. rh. prcduction of Cdrd,i/a lrilir biomass. Shake 

fldks werc used for f@Enlarion. fte ,naximum bioma.r, conenraton (?.5 8r-) 

rvs obtqined after 60 hou$ incubarjon ar jo.C and 200 rpm. 

Roshkova (1979) in a srudy cuxivaled rwo srrains of Caddd viz 2672 and 

2673 M on difleEnl nedia unde. oprimun @ndiriol!. The biomN yictds w.rc 

56.5% and 44.3./, 6 dtied y6r for stEirs 2672 .nd 20 hou6 Md for 267j M atter 

volfova er al (t979) aner $e fermedarion ofscid rrerted srmw hydrol)zrle 

vith Candido topi@tis for 12 hou6 at pH 4.6 and l0.C remp@turc succ.cdcd i! 

ar|aininS 8-9 % prolein in fitraly fcmcnlej biomN. Atberro ed D.sou (t9E0) 

fef,neDt r€d ellulcic hydrotlzrle wjrl marine yesr cutrur€ tor 2 days .nd 

obrained 5-6 g/L of dry yeasr m€ss. 

Hirchne. and L€{therw@d (19S0) fermeded celulos€ wilh 

derepresscd nurant slEin ofceldouoro rp. for 96 hou6 and producrd 

protein havinS 75% crude prcrein and 68% rrue protein

26 

aaraquio ?/ a/. (1981) prodrce/ singlc cell prolein ftom shey lsine 

Cur.lkld tls.udoh oticolis nnd Sacchdrunyces ,agi/jl. Ti € nrotcin yield q.s l l .8 

pcrdcnr b rlt sliokc ll'$k culturc usils dcp.otcini4d whcy lbf batch and 21.4-3?.7 

pcrcent lbr olher merhoG Gemiconrinlow and supplem€nted batch collures Ning 

unlrcated whey). Il. hislEt c.ll mas yi.ld ws 12.5 g/L and 12 hou6 ot 

cullivarion wirh Candida pvttlolropicalb 6ing whey supplooled with 

(NHr,so. od KHrPoi. 

cds dd Ne€lakantatr (1981) produced single c€ll protein from bagasse. 

-l-hey su@eeded in obtainine 20.1% biomals pmtein after fem€ntation of I%alkali 

rreared bagasse wirh ,4rpfal6 ,ere6 ancr 7 days of incubation- 

Muindi and Hsnssd (l9El) suc.lded in improvinS $e crude prolcin 

conl€nls of.assava rool meal ftom 2.40lo to 3E% after sub'n€r8ed cultivation wilh 

Trichodem hdBiMun at 3O'C lempeatwe, 4.0-4.2 pH for a period of 60 hours. 

Ca|e and N€elakanrd (1982) fcm.nred l% alkali tGat.d bagdse alonS 

with €de urine. !re!, c@ st + liqlor, (NTLLSO6 NHaNOj, FeNtlrsoa, NH4CI 

and NaNOI @lf€ctively by /spetgillw tene6 for 3 days.. They produced 16.4 

percenl p.otein/1009 bagase- Davey and Bruce (1983) femenGd ll0 9,4, of 

Iiggerf dury solid ,netrer wlth Sporctti.htm pulterulqtrz at pH 5.0 od yi€lded 

22.? percent biomass p.olein aflq 168 hours. 

Eriqrcz and Rodriquez (19E3) f€me ed alkali k atcd bagasse at leo 

levels 25 and 40 gil with Ce ulononas sp. llbc fot 4E hours in a shalc medium at 

pH 6.5 ard lemperature 35'C and r€covered bacterial biomass containing 49 pe.cent 

Mill$.nd Srinivasan (I9E3) f.nncnred ellulose conrai.ing synlhelic 

neditm w'nn AspetSill6 tenw at pU 4.0 in sdni continuous batch. They obuined 

23-28 pe@nrliomas prclein aller 36lou6 of fementarion,

27 

Sandhu 4d woraich ( 1983) sludi€d a numbcr of yeast spe.is for production 

of single cell protein uing chcese whey s rh. subslrale. The ch.es. whey was 

suppl€m€nled wilh minorah ed yeasr exlract. They reporred that of lJ)e lliaeen 

speciq srudied df,4rdralrr, C.c@ata, P.polyiqpha dd W.robe sii ^lained 

'naximum bioD6s aner 72 hous of incubation, Tt. prorein conlerrs ofrhe skains 

varied lion l0 to 60%. 

Laukevics.r a!. (1984) subjected steam treated wheat sr6w (wheat sraw 

907c, whcat bm l07t 1o solid stac femenratlon with Tlichoderno ,eesei d a 

Dixed cultue of tf€6et and E"dohttopsis frbutiget ar 29.C. Ttcy obrained 13 

perco1 prclein aner 168 hou6 of incubrrion. The besr prcrein pirducrion ws 

obtaincd if, stalionary layer fermsters. lt was conctuded that SSF had towd ovedll 

elficiency but higher pfoduct concenkarion per rcacljon votume than other 

Molina.r u/. (1984) studied the production ofsingt€ cell prorein frcm lolo 

NaOH lreded ud unfrcared bagase pith wirh a mixcd culrt rc of Ce ohohos sp. 

and Aacillu rdrilrc. The shond incubarion period rcquired for fie producrion of 

mqmM prot€in wa 24 houB 31 25oC temp€aturc. 

Abdullah e, o/. (t985) Opiinized the gro$th corditions tor crderariM 

ce dol,ti.un tt producrion of bionas gotein from whet srmw The b6r 

lreatnent was IoLlnd 1o be ammonia fieeze exllosion. Tl€ feme .tjon was canied 

oul ar 3/C usirS 2ol, ineutum, substrar€ hiakness of I to 2cn for ?2 houls. 

Maximtn of 27.2t/r biomas p.or€in w.s obrained under optimum condirios. 

Mixed culluing of Carddo //,ir fuflhe. inqeesed biomrss producrion by 20%. 

Tley concluded lhat the besr mode of femslltion was scmi conrinuors f€d barch 

fcnrenration wherc hslf of the formenred mat..iat was rcmoved a1 ihree days 

nrtewal dd rcpl&.d by frsh subs1raL.

28 

Maciel-d.-Mecilha er dt (1985) uscd cacao pod husk as a sole cafton 

source wnh @ncenoations of3.0, 5.0 and ?.59/L fo. sitrgl. cell prorein producdon 

tttotcb CellutMo@ flsise@ 8d Alcalieeres faecoti'. They obrained a 

mdiDm of 63.79% proteh lt subctrat€ @@tlirion of 3.0s/L !ft.r E0 hoE of 

remenrariotr. Th. valft of dry mdrq oblained vei€d fiom L93g& ar sobsrare 

conc€ntElion of 3.09/L aner E5 lrculs of femenrarion and 7.040g/L a1 ?_58 

substnter'L afi$ 65 holl6 of fmentarion. 

Pdaiotov e, al (19E5) used browr supmarrnt msinint and rhc 

@guhtion of alfalfs prclein as grcwlh mcdium fo. miqobial f.mcnr.tid. Duing 

72 hou6 of f.mentation Caadi.h topicalit, Car.tida utitis sid percitih 

rcntuculosun con\.ned 50, 6l and ?l% of rotal suga6 ed accumutated 2 I ,26 md 

3 I g solidyl0oml containing 32, 35 6rd 3 I yo prcrein, respecnv.ry. 

SmaAnna (1985) grew Crrilir using m inr€mirl@t supply of concenlnGd 

spenl sulfite liquor (black liquor) h.ving ll.9% lolal reducir8,ugs or psnial 

substBtc subsdlutim nediw. He ohain.d 54.95% yi.td afte. t?2 hous of 

AIam ( 1986) opnmird rhe fennentdion condiriom for maximum produclion 

of biomass proreiD. She f.rmented rice polishings with Arcehriotus sp. in sttrlke 

n6k usinS 5 pcrccnr (Vv) subsrale ar pH 4.0 ;d 30.C remp..atur€ for ?2 houB 

dd obtaiDed maximum bimass p.oteil|. 

chen d dl (1987) eMDined ih. .lT.cls of culrurc m.dium on tysine 

fofrarion dudng fcmenrarion wi|}. Brcvibactqiunlawn FM E4415. The effecrs 

of salts, ur€a, brm, suge. com powd€r and pH of lhe medium w€rc srudied. HiShesr 

lysine p.oductiotr (64.3s Iysir,€-HCl/L) was obtained aner ?2-80 hours of 

fenrdlalion undcr optimum @nditions.

29 

Ilashini (1987) srudied the produclion of biom.is pmtein fmn rice 3traw, 

tsfig Trichadema hdtziahttu as ferl,eniative orgsism. He achieved maximum of 

I 3.3 percenr pslein aRe. 120 hou6 of fennentltion. 

Ftrrisek er dl (lgEE) u.d chrolnoEqic m'rrats of Brcvibactel'M sp. lol 

,r. srudied the bicytrlhqis of L-lysin.. Undd optimum fmenrarion condirions, 

L-l)sine production of 43-49 gil in 96 houls with a @Ne6ion of 45-49% wrs 

noric.d. Kiani eI a/. (1989) producal yeall biomass ftom defatled rice polishings 

lsinA Candida !116 d f.m.nlalive orgmism. TIE mdimum cnde protcin 

oblained was27.8l percenl for an incubatior periodof?2 hou6. 

Moltrnood "r a1. rlcao' oprimized f.ment rion penod for maximum 

production of biomds protoin by Arcchkiota 5p. lsingri@ polishinSs as subs|ml.. 

A maximum protoin percenl of 17.23 ws produ@d anc. 72 hours of f€rment tion 

!l{ 4.0 and 30"C temperaturc. Bajwa et dl. (1991) obtained the maximum crude 

prolein of 15.17 percent when 0.5 p€cent NaOH t@ted dc. st"q (5yo) wes 

f.nnenr.d wirh ,{racr,tor6 lP. for 6 days. 

Yans (1993) obtahcd 32.4y0 crudc pro&in rner 4 days of femenrdion of 

sweet potatoes residue al 30oC with I I I cGculture of Mylol)lic mycelial fun8i. 

Hcn.B €r a1. (1995) conducted a study 10 get maximum numb€r of yenst cells by 

10-14 hours fermentalion. ftey uled sugar cane.iuice havinS liule proportion of 

mola$es thrcugh a rcNl€rilized process. Tley suggesl€d thal juice should be flerh 

havirspH 4.5 to 5.2 and be incubated .l 32'C. 

El-Nawi er at (1996) fcrmenred alkali rcated bagasse al conc. of 1.5 

o/dw/v) under various culor. condilions by ,1vel8il6 teneu' fot prcre;n 

produclion. The optimum cooditions l|cle, pH 4.5, tenperature lsoc, culturc broth 

l:5 (v/v), inoculm 4% (v/v) and cortinuous shakirs fo.7 days. They obtained 

biomss hrying prcIein conrenis 2l-2870 (dw). The protoin re@vercd wls ll, 

14.5g p€l l00irba8asse.

l0 

El,sab..ny ard HNd ( t996) arcmptcd rhe producrion of taclic acid, tacric 

acjd d€hydrogeE€ (LDH) and singlc cell protein (SCP) fiom dried whey by 

fem.ntarion with bcbba.iUw .lelv@ckii, subspei€s ,,taartu6,369 on ! 

ftdium €nriched with dri€d whey (8% v). Tl.y rcDoncd rh.t ferb.nralion cdied 

out ar pH J.5 for 24 hou6 gavc hqimM of32.Eyo SCp. 

Cao e/ al. (199?) dev€loped a snnple and €fI€crive melhod for rrca$nenr of 

fig"Mflulo6ic nile.ill for tlE prcducfion of 2,3-buranediol by Ktebsie o ottroca 

AICC 8724 frcm comcob f€rmenralion. TIey prcpared subskaie comprized 90% 

cellulose. They werc able 10 produce but nediol @nc€nlsrjon of25 g/L and an 

ethanol concentalion of ?g/L by Kotttoca ftor'|.E' gL ol comcob celtutose wirh s 

@llulae dosage of 8.5 IFPU/g @llulose of coh@b atler ?2 houB of solid slarc 

Dahol ar al (1997) conparj $c anounr of singlc cel protein production 

trom rice h$k rreared wirh two differ.nr atkati solutions !r variow concenrrarions 

(0. | 5, 0.3, 0.45 ald 0.6 N) ed femotcd with p.tui in .xrv&M. Th. 

fcmentstion was canied our bstch whe in shake fl6*!. They r.ported m.rimum 

rungal biom&ss p.orcin (2.63%) ar t92 hom on 0.3 N sodilo hydroxidc Uered 

nc€ husk rs comparcd to l.3l% on 268 houls of 0.45 N ammonium hydroxide 

Gupt. and Darl! (1997) fen€nr.d bagassc undd solid statc condirion with 

*ctllrve of Aspergll8 ellwics and Aspergi us luni4atus for producrion of 

cellulol'lic enzymes. They foutrd thar rhe pcrrcatrnent of baeass. wilh 2% (Vv) 

calcium hydronde .nd lh€n E da)s of fermentqrioD savc maximum Foduclion of p- 

HM et al. (t997\ isotated Candida rp€.riar ftom rhe soit atrd found rhar lhe 

organism was abl. io d€gradc uric acid of poutry f&ces pl.s.nt in rh. groqi 

medium. Msximurn cell srotrth (3.8x10v CUF/ml) wa! obraincd at 16 hou6 of

3l 

in.lbalion- ]l'ey obseNed that volalil€ fi(y acid!, known as major compounds of 

poulq f..c.s odor w.E .lmost rcmorrd ftom lhc mirinal louliry fa€ces drr&r 

Lec .r a/. (199?) cullivated blue gr€en algae CNIES 19,19,46) for production 

of singlc ccll protein. ]tey rcponcd rhar after ? &ys of cultivarion mdd 5000 

Wm2 lieht inleffity, Iiml cell conc.nhlion srs 2.8 (g/L) fld chlorcphyl,a w.s 

4.9 ms/L. rwirh inilial concenrrarion of NlHcoj, l.7olo and NsNor, ws 0.250lo, 

ndimm ccll @ncentrarion vu obtti,n d. Spnulitu ploreNn NIES 39 show.d 

faste. growrh nre lhd NIES 39. 

Sdndhu and Jorhi (1997) studied fep factoB for producrion of ethanol and 

protein th.ough enher solid s1a1€ feanenlation (SSF) or natu.ll f€rmenration of 

apple ponuce Ning dwc diffcEnt y61 sr-aiB. The fesults showcd rhar produclion 

of ethanol, crude and solubl€ prclein rhrough natural fcmentarion was atmost hatf 

rhd thal of fmnenration witr added inoculm of Saccn@ony..t cenisiae. The 

oplimum timc for ethmol orcdlction in SSF ws 96 houB. 

6. MULTI-ORGANISM Ff, RMf, NTATIOIN 

Vadely of ways have been adopt€d 10 fe neot diffefent types of subslrat€s 

for fie productim of ei$er sinsl€ cell prot€in or other kjnd of usctut prcducrs. 

B6ide uing methods of b3tch, f€d bakh, solid srrr. fmenrarior or subne.g.d 

fernentation, the biotechnologists have kied the use of more than one orgdism for 

$e enhancemenl of fermenraiiotr products. Sone workers had adoDled $€ co- 

cullure renncr ation whil. olii6 hav. used fmarlriae orgatrtum one ancr rhc 

olher. An available lircorure on multi-culrue fem.nirdon has b€€r rcviewed as 

Han ?t a/. (1971) used leo orSdisms for ferm€nration, one C./t lo'ora 

(cellulas. lroducinS bicf.ria) .nd oth.t Alcatieenet faecatis (ccllobiose urilizing 

bactefia). Tb€y succeedcd in obrainin8 6.248 of dry c€ll per lii€r i, symbiotic

,2 

fenncntation as compared ro I -?g of dry c.ll p€r litrc wirh prre cultu. t-€mrenHion 

orC. ulohon's. They .mmqled rhar lhe SCP prcdrclion $.ou8h f€mentarjon of 

cellulGic substr.l€ wilh a singlc o.sanbm b nor a emcienr s in c6€ of 

fermentation wjth nore ftm one organhn. Synergistic erect oltwoorgdnisns wa 

suggested for lhe enhoc€m.nt of the atnoun! of dry @lls production, 

Hm (1975) f'motcd rice straw wirdt Cdlulono@ sp. sd ltcaliEeaes 

Jae.alis. H. rcported rhar'l5yt ol tlE ricc struw slbslrale wc digetcd .nd I E,6% of 

tolal substEtc wcidl lhat disapp.€red eas recovercd d micrcbial prorein. FrntE 

lhe micrcbial cell fFction wd 3?% prorcin ud 5% crude fib€!, Gido. wa! 12% 

prorcin and 45% crude fibd. In virro digetibilny ofthe microbiat piolein was 41.2 

ro 55% ard that of cellulose was 52%. 

Fuska and Kollarovs (1977) fermenr€d 5-7 peI@r sawdust hydrotyzate with 

a mixed culrorc of Macor rte riliun atd cibberclh l\jikuoi and yietded I .28- l .45g 

dry !na&r/lo0,nl of the nedium- The dry marler of$e mycetium was obseded to 

be rich in essential mino acids ofwhich m.rhionin ed cysreire @ntcn|s w.rc 5.4 

and 7.0%, Gsp€ctively. 

Banaquio er a/. (1981) srudi.d rhe producrion ed nutrilive vatue ofsinglc 

cell proLin tro'n whey Ning Cd4lida ps.unouopicolis ad Sauhotok!.es rasitis. 

'liey obsened the prot€in yield of ll.E perc.nr in the shajc fldk culrure and 2l.4- 

17.7 perccn( fo. tle other melhods. Thcy obhined fte highcsl cell mass yield .s 

12.5 g/L all.r cuftivatiotr with Cah.lida pseudonopi.alis tsilg whcy supptcmenled 

with (NH.ISOa md KHrPO.. The av.rog. pror.in mrenl ofccttnass eas jtyq 

and bolh the y6ts prodoced all f'e ess.ntial amiro &ids. 

Sek r dd Prablu (1982) isotalcd and cheacreri&d 1wo bacr.ri0l slrains 

liu rumen and liquid wasle (bio-liquid) from a bio,eas ptanr urilizing basasse, 

capablc of growing on alkali lreated baeasse. Borh isolats were id€ntified .s 

Ce onon6 sp. Nd verc.nlturcd on alkali rreted bagass€ at thc rate of l%

ll 

substrate in balrl n.dium ar pH 7-7_5. Ttc orgeisos showed 50-55% 

decomposition of substr.t. in 5 days sh.ting cutrurc. A mixed cutture of 

ce 'no|onos sp. nnl Ttichoddma sp. showed 95% dccomposilior of thc subsrrare 

in a day culrurc. The prolein contenr of Celtulonow IAM l2l0E, lhe 

Ce ulonuas sp. rrcm b. rume and lhar fron (h. bio-tiquid wcrc 62, 52 dd 57 

klkevics e/ al. (1984) Ned solid sra& femcnr.rion of sr€M hcarcd whqr 

smw (wheat srra- 90% md whear bM l0%). Ttichoderda re.sei or ^ mixed 

culturc of lreerei nd Edoht@ptit libtlig.t was us€d fof fermeDblion and 

dcoveEd 13 pcrc.nt prorcin. Th. best proi€in pmducrivity Ms obraired in 

stationary layer feim€nters. Tley concluded lljat SSF had low€r ovemlt ef|ciency 

bui hidq prcdlct conc.ntnriq Fr @rion votum. rhln othe. conv.Bion 

schcmes. I wls aho conclud.d rhar teo orgeiss fcmenrarion prov€d besl rhd 

single organisn fcrmentalion. 

Molina a al. (1984) incubarcd slkati hydrotyzcd and unr..aGd bagass pilh 

with mixed cuftlrre of Cethtowtus sp. îd Baci 6 rrrrtii Th.y reported 

'naximum myceli3l biomals p.orein prodocrion by co-qjtrue. 

Maciel-dc-Manciltal er al. (t985) used @cao pod husk 6 a sote carbon 

source wnh concenkarions of3.0, 5.0 and 7.5 g,,I_ for singte cel protein production 

thto\t$ Cellulonow ll@ieetu s d ,ltcatigd.s hecatis. A ndinum ,i.t.l of 

63.79% to total prctein wss obiained !r q€o pod huk conccno-ation ofl.0 e/L 

aner 60 hous of fermentation. 

Pqoni al|d Os@ (t9E?) used slk.li rre{ed ni ed comcobs of differcnr 

gnnulometric fncrions d caftohydnt. euce for fcmenrario, wir,'r nixc.t culrurc 

of Cellutodotus tp. Nd Rocir8 s,rrilir. They found no difcrcnce berwcen rh€ 

bacldill prcrcin production from rh. tinc fiacrion (_25m6h), th. cerse fmction 

(+25 mesh) and rhe clmcob mitted *ithout fracrionaring by gmnulomcrry. They

14 

reported th.t rhe li.!l composition of th. fcrmcnl.tion Prcducts changed according 

|o rhe @m cob conce ration us.d in the cont.nt (65.070), and lov fibd @ntent 

(0.6?vo) suitable as protcin supplemenl fo. fowl forag€s. Iley staled that by 

increE'ng tle inirial com cob concenlralion upto 609 litrc ', it was possible to 

produce a final biomass with higl liber co .nl (l7.E7o) and 35.70lo of prot€in. It 

was suSAested that srch biomass could b. us.d as rumin.nl fccd. 

Manilal er al. (1991) inoculat€d two cullurcs of Csrlis and ,lr!/tge' or 

Car,7,ir ond ;uas'6i simulbn.oNly into lhc l00ml sl.ril€ emuenl of cassava 

starch for the production of biomals. They mixed th. .mu€nl wirh (NH.):SO. 2.8g' 

KllrPo. 0.7& CaClr.2r|O 1.0& MgSOa.THro 0.2& NsCl0.lg and y.an exhcl 

0.01 8/L. 'Ih€y obtai.ed a biom.ss contsining 22% (*/w) prolein, which rcmained 

unchuBed during f.rm.ntation. 

Rodiiquez .nd OsU.rdo (1991) proddd siDsl€ c.ll prct i! usins rwo 

orsanistu viz. C. ulontu sp..nd Ps.udodo@ sp. usinA b!8tss€ pith. Th. 

nnge of prcponion of tnq.lwo orgsnism! \{rs found to b€ 4:l to l00rl 0nd lhc 

b.sr pH vas ?.0. A nuturl symbiodc relarionship was found b€tw€en rhese two 

orearisms in such a way thal tl'c C. ulononas sqpljin hc cubon (Slucose 

produced from blaaisc dcgradation) 1o thc PreudoafirJ .nd thc lattcr producing 

lhe virrmin supplcocnb nc..ssdy for minimal m€dium, wihout addnion of any 

grcwfi factor. They concluded lhal thc co-culturc syslcm was succ€ssful for F.d- 

b.rch clllivarion givinS ri$ ro hi8h biomN Ptoduction (19.a g/L) and utilliziioD 

of lisnoccllulosic subslrat . 

Vst$trez et al. (l93ll) culth€led a mix(w of orsrnisms, 3 yasts (l 

Car.li.lo uttlis ,nd 2 Sacchoronlees .enbi@, 6d a fungus (C.ofidi@ 

.drdtd@) od leo slcp Geld Pinus pi.litcr buk sd!p16. Thc substntc w6 fiEt 

r.eated wnh akali and al sccond step wift ac.tic .cid HrOr solution. Tt.y w.rc 

succcssful in 8€dng incrcsscd biomass yi€ld ard singlc cell prolein

)5 

Jin €r al. (1994) succeed€d in trmsfonning com residue inb lrigh qualir, 

mixed prcrcin f.ed using solid 3kre femedtation wirh a miaed culrurc of a y.sr 

Sa.charon'tNes ther|'antiton@ and ^ tu.Ci Pletot6 oslreztu on com rcsidue. 

lhe Iennenkrion proJud ubrained I'ad l7-20s. prolein 'irh iomptere srnino sciJ 

prolile, rre€ ofpoisonoN compounds and rajLfut. 

Kallef-Mhni €/ al (1994) used nixed cuttuc of Sa.cralhrc.s .eflisiE 

CAS 8066 and Kluleercdycer r'.fa8ir:r for continuous p.oduction of bionass on 

nEdia conlaining whcy pe.mear. and glucce (WG) or whey permearcj malrose and 

Slucose (wMC). clucose snd maltose w@ lrovided ftom hyd.otyate srffch. Tte 

yield varied frcm 0.26 to 0.48 g/9. Ttrey suSSested that the rcsutls obrained werc 

duc to rhe difl€rcnt kinerics of glucose rmspori in Ktrr\eroarca l.agitis nd 

So c cha rc ntN. s c e n is i a e. 

Athd dr al. {1995) used beet lutp for convenion irro rnicrobial bio,nass in 

fed batch culrure sysr.m. They obrlined 5.98 rne/ml ofnicrcbiat biomN prctein at 

iridal scp by f.menbrion with Cdn lida ,litit wh.rc $ fen balch of L5% molalg 

and 0.75% CSL at 36 hou6 offemeniorion caused turlher rose of microbial protein 

1o 7.76 mg/ml. Tley rcpon€d :flr EreibaaeriD ,Idun io@ulared wilh 4% 

Slulle addsl lo the n€dim enheced proreiD upto 10.74 mg/ml and tysine conrent 

from 1.29 b 3.08%. 

Saima ( 1996) obrained an increased crude proaein frcm t4.29 ro 3O.Soh by 

fementing *heat brsn fiIsr wirh Candida ilis snd lh€n $iri Brcvibacte,iu 

layuu. Sh€ repoded 2.5 rimes increase in lysine contenrs. 

Desai and Tanvi (t99?) 3clected six isolares of lacric acid bacreia on rie 

bNis of their homo .nd hctro femenlation chander, ssh rolemce od nt6 of acid 

product'on. UsiDs these six isolates in combinarion. TtEy cdied out con0olled 

r€m.nl.1ion of b.ined be.! ffo! cabbaee, cucunber, €uliflower. singq, 8rM 

chilli, onion, rldish dd rmip ws cmi.d o!r. Th.y rcporred thar rlE fm.nt€d

36 

brined vegetabl.s axained lhe desh€d acidity wi6in 4 days at 28-30.C by adding 

0.1% sorbic acid to $e brine. 

Guple and Datla (1997) atcmtlcd fie productior ofc€llulolr,lic enzymes oD 

b.grse undq sofid slale femen(qtion by @cullu€ of,llp.rgill6 euiptic6 ad 

Atpersittu fwigtw.ItEy rcpoded th.r the cGculrurc had tEuer hydrcb,ric ed 

0-elNo6idasc activitie, a5 compeed to rhe @ions whcn they were us€d 

Gutienez-Coffe3 and Tengedy (1997) ,tudied elfect of @culture 

cultivation of Trichodema ,e.sei LM-UC4 ed its mulanl LM-UC4E with 

Aspe.gila pho.nicit onbngass€ duing its solid s1are fmenbtion process. Mutual 

syn€rghD rlas obsened between Trichodenna nonmutant s]fain and AspetAillut 

resulting in enl€nced biomasr production and conesponding increase in c€llula!€, 

endoslucanase and p-sluGidde activitj.s. They could not obsede such synergism 

betweo mutet ftichoder@ sn in 8d AtperyiU8. They wcre of the view rhal 

mutarion 16l thc coopcmtive intdction lbility of tr,i.lod.tua. 

lkesamiand Yasuhno (1997) investisaled the cret ofcarbon marerials on 

lhe g.owth of ycast du.ing a co-crllure f€rn€ntation ofpotato sloch with skains of 

Sdccharcry,cet sp. 8d Asperyillu otrza€. They reporled that by acBrion of rhe 

liquid cullurc thc mycelia formcd pcllcis. The y6t could groe pEdominmlly ar 

30'C dd yelsl tr€€ c€ll nunb€. hcr.ascd wi$ the saarch cont.nl. By the addirion 

of Cebon marcrials, aclivat€d-C or charcoal, ye3sl Srowrh was accelemred wif) 

rcg&d lo the sizc of chtrcoal, smaller was more effeclivc for y€aslsrcMh. Th.y 

suggesled lhat the carbon mterials apparcnlly acted as a carder for immobilirarion 

Tsiguchi dr o/. ( loa?.a) deleloped a sysrem of cecullue femenrltion for 

the production of ethdol from glul:N md xylose thrcugh f.rm€ntlrion wilh Piclio 

nipitis nnd Inrls[tl of Stu harcnycer c.nrlrrbe in a batch cultrr. process. It was

J7 

nported lllat lhc f€mentation widr Prttplir was stronSly dcpcnd€nt on o(ygen 

lra sfer mle (OTR). On $e basis ofotR values the relationship betwen optimum 

spcrific oxysen lptakc ruLc (qot and m!:(imun specific cthanol production (q!- 

md) w6 evaluated. Tlte oplimum qo, for ctheol prcduclion was 14.3 and 66.7 mg 

G ccllr,A'r) when xyloce md glu@se *rs us.d $ t'e qfton souM 6psriv.ly. 

Whe! a oixturc ofaluc@ ud x/@ wa usld d lhe clrbon su@ in a culturc of 

erriptir alone, fi. yicld 8nd prcduclivity of eltool wse significetly enhdced ai 

qo: adjBt€d to an optinm level dep.ndcnt on the typ. ofsugaf @nsmed. Tley 

obseN.d that thc co{ulturc oflhese lwo organisms r€sulted in mar-yield ed ftc 

highert Foductivily of ethaDol, from a mixture of xylo6. and 8luco6€, because qoz 

for xylose fem.ntation by Prr;Dtit ws$ suGsfiily mlinbincd due lo the loq 

oxygen consmption by th€ mulart y€ast stnin. 

Taniguchi dl di. (1997-b) develop.d a novel co,culture system consisling of 

lwo fementeB and two mimfi[eEtion moduls for effici.nt etharcl p.oducrion 

from a mixturc of 8l'|me and xylosc by c{)o[DE of Pi.hia stipits a d 

Socchuodrces .enbia.. Thay h@ulat d th. two dgdisru in a singl€ f.mcntcr 

whcre i1 was imposible io naintain the specific oxygen uptak. (qor) for elhanol 

prcduction from xylos. by Crtipirlr due to O, conlumption by AcdryiJrb.. Th. 

.lhanol produclion by conventiodl co.cul(w slstm ws lowd lhd a singlc 

culturc ofPJtlr,?ir .lon. in which qo, w3! ldjusled to e oplimum ldel depcndinS 

on tbo kind of suSu @nsumcd. Thc proposed cr-culture systcrh !l'o*€d re$lalion 

of dissolved oxyg.r concetrlralion at a level suitable for an individual y€61 in esch 

fcrmenler as well as successful exchang. of culture media b.lwc€n two fermenters. 

'Irrcy obbincd higher yields ud prcdocrivity of elhmol from a mixture of glu(rF 

dd xyl@ in a ncw cGcult@ s,,rt€m the h singl€ cultw ot P.ttipitis al6.-

7. SUPPLEMtrNTATTON OF YEAST Cf,LL MASS. 

l8 

llar (19?5) uscd ! growrh mcdium havins o.5e y@r cxlEcr pc. lir€r atoDs 

Fnh 10,50e ot atkali p.erreated cellulcic subslrar€ of ricc sl.ap ad snall 

quantitis of orher salts fo. irs fermcnrarion wili Celllonoadr sp. and Atcatigehes 

/ae.all'. lle rcporred that rbe microbiat c. mas kotaled aft€. femenrarion had 

37% nrotein and 5%.rude fibcr. 

YoBo et at. (1976) Femenled difleren( yeasr slmins on cutlure mediun 

containjng MeOH (6?-56-l), EIOH, glycefot, HOAC, glucos€, galaclose or fiuclose. 

Candida MOy-657 (IERM,P 3601) wss culrured wirh shating at 30"C for 4E 

hou.s. The m€diun had pH 6.0 snd conbined NH.Nor 4. K,Hpor 4, MsSOa 0.5, 

FcSOi 0-02. CaCl:0.01, y@t qtlact 1.0 and com sreep xquor lg plus minor 

amoDnb of Mnso4, znso4, thimin and biorin with l0g MeoH per Lire. They 

obtainedayield otyeast conbining 55.5% crudc prolein, which w6 3l_j% in lenns 

ofMoOH- 

Moo-You!8 "r al. (1977) in a srudy compared fie ce|utase and sjtr8le cell 

protein pfoduction by lvtride and C.@ utolyticM. Th. basrc media formulared for 

study lrd 0.1% ycasl exracr along wirl, urea and orlc. satrs. The subsrnlcs used for 

fenc arion were Solka-noc S.W.40 and paniarty dcriSrified sawdun and fie 

fcnnenhlion wss carried our ar pH j.0,30.C and 37.C lempemture for lritrile and 

C.cellulolrticun, rcs!€.tively for 22 hours. The resuls showed Dorc celtrlase 

prcdrcrion and substmre degmdation hy T.viride. Morc SCp p.odudion by 

C.CeUulolytic"h i\ rhe presence ofyeasr extiact ws noticed, 

Suzuki et at. (1971) fermened shight chain hydro.dbons wirh Catr.rila 

parul|iai.a. Tne microo.ganism was ae.obically cuhured at J5"C on m€dium 

having pH 4.0 containing n-atklns 2, (NHa)rHpOa 0.4, KCt 0.1, M8SO|.7H?O 0.1 

ano com s&€p riquor 0.t% plus race amounts of NaCt, FeSq ed yea$ exrracr. 

Tle) obEined a produ(r conraining 59.190 crude prolein.

l9 

Burgets (1994) dcveloped the mutsrt st6in of y@t Sa..hdrotht\es 

.endh". {hich wcrc dcficienl in prol4e enzym.. 'Ilt stains e€e used for 

preparation of yeasr €xtrdcts Siving maxnnM yield of nuclear prol€ins. They also 

developed anothe. mcthod for avoiding prclolysis during erlraction process llal h 

blendils c.lh with 8lN b.tds. 

Kalld-Mhiri dr aI. (1994) in a @culture fem€nlation prc.es studied lhe 

bioniass production on nedia eilhd ortlinin8 whey pemot dd glucose (WC) 

or whey permeatc. elucose and maltose (WGM). The orgmisms used for 

f€nneniation w* Sd..rarou)r.r ..nnide CBS 8066 .nd &utaetuht!.es Iragilis. 

Fo. eoch medium thc efist of y.st exlracr concotralios (0.1 and 0.5 e/L) was 

in!€stigated, Tley rcported lh.l the y€asi exlracr @ncentntion Ddledly elfect d 

the equilibriun of the co-cullue and c.ll yield, shich wd also influ€nced by the 

dil(ion.ak. Theyield varied from 0.261o 0.48 g/9. 

Niwa and Hirct(o (1994) prcparcd a cullurc medi! @ntaining yeasl extract. 

glucos. ed srdch havin8 pH 3-5. Th.y rcpon.d that lhe cultua d€dium wos 

useful for sel€ctive growth ofacidoplilic thermophilic bacteria toud in acidic fiuit 

juice at 40-60"C t mp@ture. 

Tabata €r d/. (1995) obse.v€d lb€ feding valu€ of th. yerst €itrEct- T1€ 

e\trlct cotrlsitred 5 'neGlcolide, frce amino acid ad mannon and was lscful for 

enhancement of feeding efrrciency dd imono stimulalion. Tte extdc! 6 

prepar€d by autolysk ol ycasl celk grown in sulfile pulp spenl liquor healing 1o 

inacrivate lhe enzymic activilics and incubation of c.ll lysale wift 5 - 

ph$phodiestcBe ard 5 -adenylic acid deaminas.. The yeatt seleclcd were rortla 

yeast and sacc haronryes. 

El-S.baeny and Halsan (1996) in a study ob6€rv.d lhc influencc of mediom 

composition on producdon of laclic acid, lactic acid dehydrogenate (LDll) and 

single cell prolein. The] reportcd thal dried \fhey endched mediu'n (870 w/v)

40 

having pll 5.5 whcD femented wilt, LacbbacillB btlgdt !JJ69 fo.24h result€d 

in Dqi,rum produclion of lactic acid. Addition of y6r extEct plus MnCl, gave 

the highBl production of ladic acid (1.?l g/L/h), sp6ific acriviry of LDH (1.03 

U/'r8 prolein) and ScP (32.8%). 

K.rthikeyan d dr (1996) oprimized rhc nutienr medium compcirion 

(conla'n'ng sucrose, yeasl exkact and K:HPO,, lempc.alure and inilial ptl 

conditions for batch dexrr.n producrion in shate flask fementalions using a srain 

of LelconNtoc nesenteliodes NRRL B 512 (F). T}'ey rcpon€d rhe optimal valu€s 

6 sucrose 3009/1, y6t extact l0g/L, KTHPO4 308/L, rempemture 23 oC ed iniliat 

pH 8.1 wirh ipredicred dexhnyietd ot t54 gtL. 

Okada and Shingo (1996) compared rwo media fof s€lectiv€ gr0{rl) and 

diagnosis of Ba.ir6 .er€rr. They found ttal $e yeasr cxtract subsriurion wirh 

mear exkact in O€ cultue medium havjng p€ptonc, NaCl, Stycine. meat exrnct, 

laclose, phenol red, polymyxia B suthre, agd a8d .nd ess yotk lsulred in stabt€ 

dd sup€rior media t@d for $lective gron1h dd diagnosis of A cef.rr 

Lo et al. (1997) srudied the synergelic effecrs of ca$on (Slucose) aM 

nilrogcD 06t qrract substr.les otr cclt growrh &d brhs producrion. Tle eff€cl 

of Slucosdyedl €xrract ratio (C/yE) was studied in veious opemting models 

includiog batch, 1*o srage barch 8d fcd brrch f.nnenLtiols. They obs€ryed that 

both xdrhe yield .nd specific produdion nte inc@sed wirh ind€asin8 C,fyE in 

rhc m€diuD. Bur thc cell yield and spccific grcw$ deoea!€d 6 c,ry[ shitl fron 

ard inili.l low lcvcl (2.5% glucose/0.3% yea{ exrncl) ro a tigh tdel (5.0% 

sluaosc/0.30l, yeast extocr) ar rhe end of exponcnrial Browth phlse which was 

prcfeEble for xanthm pmduction. lr wss also found rhar a ,noderatcly high yeast 

extacr concentrar'on in (ne mcdium rcsutred in high cell dcnsily beforc the cutturc 

ent€red in slalionary phase.

Okmolo (1997) rcported that L-liprophan (I) @ b€ nanufacrucd by 

cultuinS l-prcducins micrcors hh such $ d.r/l6 in a m.dium conraininS Et 

l@t y@t .xt .ct Md Try?tophe dd th! melllod did not €quirc substdr.. 

8. GROWTII KINf,TICS 

Rieche er o/. ( I 966) piepsred high quality pmrein f€€d fron potalB through 

rhejr fe.tndrrarion wift C4rddo /r/ii.&. Thcy repo.r.d 44 ro 46g ofy.an dry m.rrcr 

yield per I00g reducible sugar. Penels€n (19?5) studied rhc producrion ofprctein 

srd enzyme @llulasc from alklti prcr.€{ed barlcy sraw rirough fc.menrarion wirt 

Ttichoderha ytide.Ihe yield constant (y) were catcutared ro be 0.40_0.56, of rhe 

sane order ,s lhN, which m be obtain.d by growing rhe fungu on gtucN. 

Yogo €r ar. ( f976) cultured y.$t Ca"dida MOy-657 (FERM_p 3601) wirh 

shrting on a medium uder oprimiud condinons. yi€ld of y..sr containing 55.5% 

crude prorein $a3ll.l9o in l€ms ofM€OH. suzutr d/ at. rt977r in ! srud] repon.d 

that \ahen Candida parujinia e6 cutturcd on suligt chrin hydrcarbons, fic 

specilic Bfowth rar. (p)was 0.376 md yield coefficjent (y) basen on,-atksnes was 

l0l.E%. 

Pamm€nl er al (19?8) reponed average sp€cific groslh nte (I) value for 

shdle flask fcxDenradotr of l% akatipretrearcd hdd wood Medusl as 0.057hr 

dudng thc pcriod ofactive grc*t. On th. ofier hand rhe f.mentarion ofprcF€ared 

sawdust liquor followif,g same condnions hsd specific S.owth 6tes of0.t96h.r at 

pH ?.0 and o.l5lh'r ar pH 6.0 @mparcd to 0.22h., fo! &e orgeism srcwn on 

glucose al pH 5.0. 

Panmqt €r a/. (197E-s) @nduccd femcnhrion of d!3ti pct e3red mtpte 

savdust in rwo conditions first solid slaG, second slurry srate Bing Chdetoniuh 

cellllotitic@. Th. ale61e sp.cific 8ms1t| nr6 duing slid soe feme lrion 

were 0.01h_r dd 0.0092hr for tumbl€ femenrer and rEy femenrer rcspeciively

42 

alte. 9 days of fmenLalion, wheres for 20 days of fmenrarion rhc vatue w6 

0.0046h_r. -the avcng. spccilic growlh dtes du.in8 sluFy-srae felnenration wcrc 

o.o329hr and 0.0204hr for tol, subsrralc an.r 3 ard 5 days of fem.irado. 

r€spectivety, whercas 0.0l9Ehr fo.5% subsrrG and 5 days offementarion. The 

prcGin production ot./iniri.l culruro vollm. (c/l-41) w6 0.0197, 0.0t95 and 

0.0114 for solid srate fermcntation in lumbt. and 0-ay f.mcnie. aner 9,9 ud 20 

days of fernenHion rcspectivety. For sturry starc fernenhtion lie valucs werc 

0.00E42,0.00554 md 0.0196 fo. l% and 5% subshale aner 3,5 and 5 days of 

incnbalion rcspectively. 

Larvford €/ dr (1979) used cane molasses as gow{r timiting carbon in the 

Iafln€ntalior medium lo produc€ singte ce prorein $rough fementation wi$ 

Cakdida utilis.Ihey obtained bionass yietd @ 2.6 g/LA wilh gro{,lh yietd of0.559 

dry biorEsvg reducing sugu utilized at th€ maimum drtution rate. 

yolfo!^ et at. (1919) Ereq Caadida trcpicalb on acid ryd.olyad straw 

alotrg wilh o. without lkaw solids wilho $e addilion of virmins or mineEh_ The 

yield coellicieot (YZo) aad specific gro$ri arc U, on non sepanled skaw 

nydrolyzte $erc r.poned as,gyo and 0.34 respccdvct,. 

Abdullah er ul (t985) oplimizcd rhc grorvrfi condirions for SSF of wheal 

stmw with Chaetoniun c. ulot j.ru. Thc r€chnical paramele$ or rhc oprimized 

condilions were, dcgrec ofsubsrrdre uritizarion 27_2%,prorei, yieldsubstrare 0.099, 

bro'nass yield/bioconverted subslrab 0.408, deSfee of bioconveBion of toral 

availabl€ sugars in rhe subsr.are 60.5%, specific clficiency of bioconversion 70.80/0. 

olerall emciency of bioconveBion fron subsrale 42.? 4. 

Kallel-Mhirier at (1994) studi€d lhc €ffccr ofy6rex&acr supptmenlation 

al two lelvcls (0.1 and 0.5 g/L) duinS rho co,culbre cuttivarion ofa medium eirher 

conlarnin8 wncy permeare and glucose or wh€y p€mea(e, 8lu@se ard matrose with 

Saccharonws @isio. CBS 8066 ard Ktt eercnyc! fagitis. The y€asr extrrcl

4i 

markedly inll €nced fic cGcuhurc €quilibrium as well as cell yield, whicl $as 

high at higl€r yeost extract level. The yield varied from 0.26 to 0.48 g/9. 

Alhar e/ al. (1995) reporred thc specilic grcwln 6lc 0r) 0.180hr, s ofcell 

mN fmed ps t of substmre udliT.ed {Y*) 0.49 ed I of prcduci (prolein) fomed 

p$ 8 ofcell mas (Yed 0-59, during fenncnration ofbe€t plllp in fed barch culturc 

sysrem, first witlf ca"dlda ,rtli' t']Jen wln B.Jlaw. 

shojaoradali and Chaali (1996) hol.led twenty colonies of Dethylotophic 

b.cEia fioo soil sanples of oil lield dq ed wte yaler ol oil disrilldies for 

singl. ceU pmtein ploduction. The conlinuous culrre t@hnique und.r chemctat 

qd Bed 10 optimi?e the growrh conditions ofbacrerial culturc CA 16,1. Opimll 

param€res such as pH (6.8), l€nperalur€ (3?'C), aemrioD rare (lvol/vollr), 

asiralion speed (a0orpm) and dilution rale (0.15h') werc mainrained. The 

manmum biomass yield coeflicienl (Y$) oblain€d uder optinizcd condilions was 

0.4lgg'- 

Vevinit ?r al. (1996) 6ed barch dd conlinuolr cultivarions to invesdgsr. 

the single cell prcGin (SCP) prcdn rion by CepJel^potiW ei.hhorni@ 152 in d 

air lin rcactor. fie culture medium consisl€d of (sava ss a sole ca$on source, 

(Nr!)rSOa 65 a nilroso souc md KHlPOr !,3 a buffcring ag.nt. The pH of rh. 

medim w4 adjulted to 3,E. ln batch f.m.niadon l% c,gva &v.1he hiSh.sr 

yi.ld of 0.4. The optinum coodition fo. ontinuou fm.ntaion wls I % 6s&va in 

feed m€dilrn ar a dihtion 6G of0.4hi. 

B. BIOLOGICAL EYALUATION 

The cheDical analysis givcs us only infomarion abour rhe presence of 

nulrients but it does not lell us about rheh digesiibiliry ud .vailabitiry for thc 

biological system. rtis @ only bc jld8ed tlnousb biological rriah. Sisniticanr 

available litcElw on the biologiql cvllution of products ohqined s a rcsutr of 

femmt lion har been.wiqed wi$ special refeftn@ ro fed conslnltion, wcighr

sdn. prcrcin digcstibilily, biologi€l vatue oer prorei! utiliario4 f..d efilci.nc1 

Erio and prclein efliciency ratio. 

Gitl.reral. (1958) rcplaed ftc fish malwnh Toruta yest .l 25, 50, 75 and 

100 perccnt lqeh in rhe basal dier of lats. They concluded $at 25% subslitulion 

did rot cffect the grcw1h but subsritDiion of50, Z5 md I00% decrea.d the groMh 

rate by I 8, 29 aqd 4E peunt respsliv.ty .s conpsed to contrcl. Thc p€llaing of 

Torula yeasl improved ib perfomance and 50% r.placmenr tevel was comparable 

with that of control. But subsritution ar ?5 or 100% of rh. fish m$t rcsutr€d in ! 

reduced Srosth by I ? md 35% r€spectively. 

Bo.k .t al. (1968) d.l@ined rh. nuritiv. vshe ofscp prep@d ftom thrc. 

diff.renr year stmins yiL, C.tdida ttilx, Candi.t ttoDi.alb and Condida 

Iipo,tica in a ftr fea ing nial. C.ttilit ai C.lropicatis werc rich in lysine $at is 

nore tlan 6.5% oftotal prcbin and in .ll l:as the S-mino acids wft limiring thar 

is less thon 2% of the prcl€in. Tl€y obseNed lhat biotogicat value of Ct,rotr.ca is 

bclow 50 but the addirion of melhioninc inro tyo sept€s of C.tropicatis incrcas.d 

biological value from 55 to fron 8910 93. 

Breesani (1968) obsen€d the prorein digesribitiry, proreiD efliciency l.rio 

and biologisl vafuc of the SCP prcp r.d ft@ Candida dittu s 88%, r .4 and 48yo 

resp@liv€ly. Tho ndhionine suppl€mctrralion of the sct, at 0.5% level was 

obswed to improve rhe valo.s ro 9070,2.1 ud EE %, respecrivety. 

Calloway and Kumar (1969) hsesled the cells ot Hrdroeehonon8 

4!r.oprd fron senimtinuous autrophic cdtu !. Th. ells conraincd |3% nitroS.:n 

on dry wciShr basis. Th. biotogi@t vatue $6! found to be iTvo borh for cc ut.r 

protein ard @ein whefeas diSestibility wd 93% and 99% ofcellutar pror€in and 

cdein prolctn, repcctively. Tle amino acid @mposirion ofrhe badc.ia as in rhe 

dse ofcas€in indicaled methionine a! dc fiBl limiting anino acid.

45 

Dods ed Kuns (f969) had5ted th. eel]]s of Hr&ognohon8 eutopla 

from semi continuous auircphic culrurc and yashed f@ ofsrbsrrate. The ccls w€re 

tunlyed fo. ch.mical @nposition ed nuLilionat vstue. Tho @lh wcrc obs€rv.d to 

onlain l37o nillogen on dry sotids basis. Thc biotogicat valuc del€min€d tttrough 

rar f€edin8 was 77o/o \]ll,ifofllttf for casein md bact.rial prepamtiotrs. Cas.in 

nitrogen was 99% dig*tibte, \rhile borh whole boilcd and sonicalty ruprurcd 

baclc.ial cclls had 93% digcstibiliry. The nel prclcin uritiarion of bacrerial Drclein 

w.r 72% !s cep.red to ?6% of c4ein. The Miro &id compGidoa delted (h.r 

sulfer cortaining smino acid ws thc fisrlimiring. 

Han et at. (\971) cvaluarcd the sirgle ce protein prodrc.d through 

femenbtor of ellutosic aubsrite wi$ C./idoho@ dd ,lbarigw faecalis. 

The c€lluld m6s isotated had *solial dnim eid, qc€pr sutphd conraining 

dino acid (methiorin€) th. qusnliry ofwhich was towd lJ)an nandrd DroreiD of 

milk ed e88, bul wa! at par with thar of FAo referencc prcrein. The w€igh1 8ain, 

nel pforein urili?rtion and pror.in digcrribihy of lh€ g.oups fed diers conraininc 

micrcbial prorein were markedty tow€r $an 6e co,npa.abte latues ro rtre eroup tcd 

r l0% casein protein dier. How€ver the m€thionine supptemenl.tion of micrcbial 

prcleitr mhmced irs ncr p.olcin utilization. There ws a nnear relarionship berw€co 

fte dieraly oicrobiat t.rct .nd the facc.t nirroilen coni.nr of rh. r.st hrs. Ttey 

suSSested thar rhe high fa@t trir.osen was prcbabty d@ lo tne Esisrance of rhe cetl 

*all of lricoolgdisms to dig€stion. Howeve. fie cett tysis prior lo t.din& 

improved thc eflici.ncy ofprorein untizarion. 

Kihlb€rg (1972) Bcd 30% dd 40% of y4t 6 the sot. source of nnrosd 

in Oc 6t di€t ed rcporled that . f.cding pdiod dceeding one yee dcvctoD€d scv.. 

livef l€siors. The lonS lerm feeding foi sev€rat gcnenrions caused stos Srowth, 

which was due io deficiency ofslltfer conraining amino acids. ln anorher srudy a

slignlly lower cgg prcducrion md fenilty ws noled for tay.6 fcd 20% y@t 

Shacklady and Carlhet (r9?2) suppl.mcncd rh. y6( prct.in wnh 0j% 

derbionine end ien tsled that in ra!s. Th.y reporred rhe di8etibility and 

biolosical vslue s 96% and 9l yo, rcsp€ctively. 

Yan z et dl. (t972) der€rmin€d rh. chcmical composirion a|ld biotogicat 

qu.lily oftwo sanples ofyeasb of C'.!r'lrs prol€in viz., St and 52. Tte 52 was heat 

lrcaled and slored ar room temp€rature tor ore year and then analyad and lhc olher 

*d analyzed as such. fhe biologicalquati(y of.p.otein was medured by lrEtt in 

2l-dats old nrs. They reponed (har rhe Sl had pER value ofl.8o, where u of52, 

l.l5 as comparcd io cdein cortrcl, whjch h6d pER vatue of 2.54. I.hey ako 

obscrved thal nethionine supptementation of Sr @ 0.30lo irrproved ils pER vatlle 

Bello e, al. (1923) studi.d |he pror.in qlarity of biomds ot caadidd ,tith 

SrosD on waie tiquo. trcm olivc pro.$sing, When y€.!1ws! 8iv€n to.atr ,s 26.4 

or 31.7% of the dier 6 solc prorein source supplying 8.0 ,nd l0.6yo prorejn. it did 

nor supporl Srowrlt. Ttey gave a dicl wiLh 31.7% prolein 10 hypo proreinaenic nrs, 

il could not prcducc a no.mat reave.y whcn compued to orc bascd on cascin. A 

.lr€r conraining 62_2% yc6l provoked signs ofsrrss md dqlh. 

cmwfo.d ed Etizbc$ (t973) pmdrccd microbist biomsss prorein d.ou8h 

fenrcrration of pulp fines wi t1 Themonospon f6.a. the biomsjs so obrqined had 

30% lrorein comprising alt rhc mino &ids. The bionN ws red io baby chicks 

and dey showed simitar g.otr'ln as conpred to lhe conrcr grorp. 

Woodham ard Deans (1973).vatu.r.d rhe o,omrss produced throuSh 

fennertatio! of I p.@cnr betey with Asperyittus orrz@. Ttey epo(cd lhar th€ 

boma$ pror€in had alr ftc amino acids .xca1 sulphq conurnmS amino aci&,

Ljpiosky and Litchfield (1974) reponed that tsyo addition of SCp in diet of 

chickens show.d cquivale f.cd @rvcnion efliciency to tiore obhined with 

conlrcl gNp. Hoeever in pctlcrcd mdons fecding dtov. rhis tcv.t 6rtr€d in 

excesive accumolation offa@6 on flmr scrccns in batlery b.ood.B. 

Reade and cregoiy (t9?5) ferme ed cassava non as€prica y wirh 

,4speryiUus luhigat6 and prcduced dried mass..Whetr the biomas was aalyzed 

tb. its mino acid profile, it wss fourd deficie h merhionin€. Tlc r.r fedinS t ial 

indicaled tial supplernotation of biomssr wirh m€thionine lesulred in compahbte 

nel prolein atio and feed efliciency vatuc wifi 6ein dier ehen fie pdein valne of 

biorDrs conlainitr8 diet wa! c.lculared on mino acid profil.. 

Smilh e/ dl. (1975) dere.mined th. feeding valu. of six direrent tunsal 

biomass obhin.d after fermentation of dillerent substates. ficy observed sna 

diiIererces in lh€ contents of essenrial amino acids berween diff*nr or8disrns 

uder sludy. ]I€ rals fe€ding rrial Save ner p.orein utilizarion vahes dging 

berweeD 34 ro ,g p€I@1 dd true dig€sribitirig ftom 69 ro Ej pcrc. F@ding r.ial 

also .qqlcd S-mino acid s the fiBr limiting mino acid dd turrher 

supplemcnlation with methionine showed marked inc.esse in ncl prot.in utitizion. 

TeEa (1976) delemined 6€ nuldlionat vatue ofrhree dicrs all$ a rat feeding 

lrjal. fte fced effciency values obtained by feeding ca3ein, biomass and biomas 

plus whey prct€in, wcrc 0.32t4, 0.2086 and 0.254t and prorcin clficiencv ratio a 

3.3309,2.4403 ard2.6?49,Bpecrively. 

C^yabyab .t at. (1977) obtained ycasr bionass ane, fem€nration of acid 

hydrolyzed dce st6w vitl Cahdida hopicatis. me biomass had 5t.0 Dercent 

prolein. The amino acid composirion ot the biolrrN was coDpaiabt€ with thal of 

FAO refennce piotein. Comp.mrivc srudies showed Crolicatu had gMtd lysine 

conrents rhn c.tuilis v3 rher@ohitia.

48 

Fuslo and Kallqova (1977) obtain.d l.2E-1.59 dry biomds aner 

fmenlalior of sawdusr with Mlcor stetiliffi 6d Gibb.tella ftjikwoi. The 

biomass conraincd 5.4 dd 7.0 perccnt mcihionine ed cyslcinq repslively. 

Disetibility t i.l of$e biom.ss r.vcalcd that E0-90 pd@t of th. totll protein wa 

Moo-Youn8 "r al. (1977) produccd single c.ll p.olcin (SCP) from lhc 

tennentation of Solka{oc .rd pa ally delisnified s.wdust with Crhaerouiuu 

celtulolytiM. rt]€y fowd .ompaEble arnino .cid composil'on ot lhe biodals witl 

lhos. ofthe FAO rcf.H.e prot€in, alfilfa snd sya nenl. Thc feeding tials on.ds 

showed no adveBe effecrs oflhe SCP prcduc€d. 

Singh .r dr. ( l9?8) femetrted spent gain wi$ Mt|othe.iuh eeftucatia ed 

prcduced 24.7 perc€nt myc€lial protein. Tl€ fermentation caus€d 8.8 fold increase 

in the substrate prclein. The amino acid profile rwealcd lhc nrcsence of.ll the 

csential arnim acids except S{ntainin8 mino acids- 

Roshkova (19?9) detminerl lhc crude pmlein ontcnls dd biolosicil wllc 

of two sr.ai$ of y!6t Ca'.tAa 2672 and 2671M cullival.d and hwsted f.om 

acerate and methdol nedim respecrively. They rcpon d 5l.9.nd 48.7 percent 

crude prc€ins and 68.4 and 69.12 percenl biologicalvalue, respectiv€ly. 

El-Ashwah er dl. (1980) 8rd diffeFnt fiugll cultures on m€dia bsed on 

molasses, swect potalo€s dd com slcep. The my@lial protcin rhus obtain.d 

differd ma*cdly in composilion. tloeever, tle maximun funSal biomass obtained 

(24.21 g^) had 34.3E p€rceni p.orcin. Tle 0mino acid patt€m show€d hisler valu€s 

of lysine, histidin€, dginine, tryptophan. methionine, phcnylalanine and ryrosin€ 

thrn in cosein. wlE.eas leucine. isoleucine and valine wcrc lowd than casein. The 

threonine wd found 10 be delicienl in all cultwes. 

Hitchrd ed tstheBood (1980) @ver€d ellul* bioma*q fmm 

f@dlalio. m.diom of ellulo3e f.mcntcd wirh @llule dcEDrcssent mutanr

srain of Cellulonore specie!. They rcported rh.r bioos had crudc prorein v!tu. 

or 75% a.d had good anino acid prcfir€ boing .ich in tysine contenrs i.e., 6.5%. 

They fed methionine supplcmerbd biomals lo wentitl8 rars ed obsened 50.4% 

NPU value as compded ro cdein dier (NpU, 58.1%). Th. wejght 8ain on bolh rhe 

dicts \os almos( snme. 

Kellqnr./ al (198t) proess€d rh. SCp by certdfuSa on or by addition of 

polyacrtlmide polymef bcfore beinS pul thfough a b.l1 process and dri€d eith 

sotric dchydnre!. They obwed rhlr rhe scp rs high in merhionine miro acid 

than collonseed meal (CSM) bul had a sibitar lysine levet. The peFin digstibiliry 

of the prctein so prcpeed w6 found ro be 16.2 to 36.E yo_ Th. digesnbitiry y6 

repon€d to increase upro 6.1 to I1.3 percenr whcn SCp woe incubated in buflered 

rumen liquor for 24 hous. 

Muindi and Hdss€r (1981-a) studied $e nurririve value of cassava r@r 

meal (CRM) endched by cultivalion wilh the fungu, t /tclo/e,n a harziahln.They 

observ.d ME value of erriched CRM as 9.t MJ kg_l dry maue. and crude prc|ein, 

37.6% of which 30% was NpN. The bioma!., amino acid profile rev€aled deficiency 

of ,rcthionin.. The m@ appddr digestibiliry lftflicisl of rh. c.p ws 66% 

whercas thal ofA.A w.s El%. 

Zafat er at. (198t) evatuared ile fsg.t biomrss obtdned ihrcush 

femenrarion of ri@ sriaw wirh ptetotw sajdcaju nd t.poded rhar rhe crudc fibrc 

and cellulose decreded from 32.6 ed45_0 ro 6.3.rd 17.8 p€@nl respecrivcly ed 

in-vivo djgasribility ofthc biom.ss incre€sed by j4.l pe.cenr. 

Cudoso and Nicoli (1982) ferm€nred \in{flse \|tth phaaeroch@te 

chlrospotiw and obtztnd ttq bionsss havinS pER and pD valucs .s 1.9410.20 

and 64.2612.08 coDp&ed to 2.46i0.26and 90.0 812.29 r€specrively torcasein. 

hShn ed ScU (1982) deiannin.d lhc limirimg anino .cid of y61 singte 

c.ll protein. Thcy obsed€d that yoast prorein wls limiring in m.lhionine, a€inine

50 

.nd leucine as fi6t, second and thnd limiting Mino acids, r.sp€clively compsred 

wirl NRC valucs. The soya prolein dict sened s onlrol 6nd th€ othe. YSCP as 

basal. Seven addnional diels we.e prcped wirh 0.55,0.34 strd 0.3?% methionine, 

esinine dd leucine, Fp€cliv€ly alonc sd in all pNibl. combinstions. Tlcy 

rerort€d simild fced inrake, weigbt gain and feed conv.rsion eoiciscy d 

compded {i1h di.t conraining YSCP in broiler chicks qhcn compared wilh soya 

prolein di€r whcn f€d in equiliz€d form. They conclDded that n€rhionine was 

limilinS mino acid ir yeast prolein and arginift did not give rcsponse in the 

absence of fi61 limiting eino acid. 

Gu@l od Vogr (1982) cull!rcd !.tst Sa..h@ontc.t .eniria. in nulti.rr 

solution ofwh.y. The biomass obLir.d had crude protein of 46.40/r, ctude fat7 _6o 

and phosphorus 0.99%. The biomass $as fcd 1o day old brcil€r ohicks upto 6 we€kj 

ot a8e by rcplacing the soybean oilmellal0, 5, 10, 15. 20 aId 2570. They fosd 

rhar upto 25% y€lst had no dotrimenlal cllecb on weishl gain and iL€d @nve6iot 

Lee and Yan8 (1982) obseN.d non sisnilicrnt diff.rcnc€ in weight gain, 

feed convc|sion elnci€ncy ed nulrient compcition of carcajs of broiler chicks 

when they weE f€d @lions havinS soybean m€al rcplaced wirh yeast prclein 

obtain€d ftrcug) femenlalio! of mcthanol ^r 20 nd 40oh levels in the basal 

ralions. Howcver the crude prorcin in musclcs was signific$rly gr€arer ;tr rhc 

@trt ol (24.48%) tlan 40% y6t in diet (23.58%). 

Rab€cha €r al. (1982) produced fungal biomass havinS 26% prote'n aner 

fennenfalion of glucose soltrrion w;ti Asp.rgi a siget.The protein was found ro 

be deficient in sulpher conlaining amino scids as well as lysine, isoleuc€in, 

ph€nylala nc dd vrlin. and lfiat it5 protcin efficiency Erio wat found ro bc 46 

pcrcent of that of6cin.

5l 

Enriquez and Rodriquez (1983) conduccd a biologi@t lrial on rals ro loow 

llN prorci,r qualiry ot blcreial prorcin. Thc digstibjliry and biological value of the 

baoterial biomAs wos ibund lo be 90 and 62dl0 r€spectivety, *hich h good index of 

protein qDalityardirs utilizarion. T16 NpU vatue Ms reporEd !o be 56%. 

fou ora e/ dl (1983) cotrdoctcd a fecdiDg rrial wilh ycasr mass fcd to 

broiler chicks ar staner age using rwo t€v€h viz., l0% aDd 20%..l.te r€sutrs 

reveal€d non significanr direrencc in weighl gain and feed consump$on by chicks 

fed l0% yeast mass when comparcd wirh thc 20% suppremenred grou!, 1504, I 539 

g/bird ard 3499,3501 g/bi.d r€spectively. Th€y obswed thar 20% Gpt.cemcnr 

si8nifi cantly educ€d FCE, 

soros dd come (198t) f€d fungat pror.ir diel ro 6r! for r p€.iod of 28 

drys. The 6rs lhow.d p@. w.ighr gain s coDprca rc cs.,n .@1rct di.r, 

consurned tess feed atrd gained lcss weigh. p€r unir of feed consumed. Th.y 

obsedcd lhar fic nefrionine supptemenrarion improved rhe qually of prorein, and 

Bls gained sinilar ycighl ro thar ofcas.in diet. 

Leeson dr al. (1984) produced sinSte @ proteir ttrougi ferncnrado, of 

Dair, sbver and putp sludge wl|lt Chaetohirn cellutot ti.ra. Tiey reponed oon 

signilicanl difference in ften chemicjt composilion exceF proten cpntents which 

were higher in case of pulp sludSe SCp. Tie comparison of net proFn uriliziion 

value of maize srover fungat bionds prol€in procesed by two wsls, ov.n d.ied 

md lj€eze dri€d wi r rhai of soybco mcal (4E% Cp) wa rdund b b. 25.0% and 

33. 19i, 60.3% and 61.3% respectivcty. 

Prokop ?r 4l (1984) yi€tded 3?-56 pcrcenr true pror€,n ot t|,e biomoss by 

conli.Dous fcrm€nbtion ofinerhanot yith a.robic ihemophilic nell.oot uilizirS 

bacl€jial cuhure (NBR). ft€ nur Lional €vatuarion of the produ.l proved irs 

sul,e o ty over both casein and soybean with rcspecl ro ner prote,n tiliation and

52 

pror€ir efliciency mrio. Thc biom8 was rich in sllph€r @ntaining amino acids. 

which w€r. abov. FAO stddsr*, 

Su.dhiiska €r 4t (t984) fcd nix.d feds wirh o. wirhoui blcleriat prcr.in 

(cultrtred on mcthanol) 4. E and 12% reptacing soybean oil meat ro day otd broit.r 

clickcns lill J6 doys. h w.s found thal mean final w€ighi *6 l9lj, t9t2, 1745 and 

17218 resperivcty ed t.d iltake 2.39,2.42,2.57 ud 2.60 l(8/kg f.in 

Ba(yuzhevskil e/ d/. (1985) obtained biomass after fenentatioD oflpirit and 

liquor indusrry by-products with s.lectcd srains of microo.ganisms. Th. biomN 

had 50 to 7trlo crude prctcin and was low in me$ionine. In a fesding l.iat whcn 

100% rcllacemcnl of feed yeasl yith biomass protein wss Irade, tEre wa! found 

ro difference in weiSht gain belween cortol ed experimental eroups of p€kin 

du.ks, white leghom hens and tukey hens. 

Petukhova er ?i. (t985) in firsr dpedmena rcptaced mep.in (a midobiat 

prot€in) ir dier Sivq 1o tcns 57 ro 390 days ar lone o. 10, 20, 25 dd 30% of 

normalprotein requiremenr Respective avemge daily feed intake was 104.3. 108.4, 

I I0.3, 109.9 dd I08.8g. In th€ s€cond exp€dment replacement pu €15 from 70 lo 

310 days old were givcn a diet wirh meprin added ro rcFace none or 5, 10, 15, 20 

and 25% of the protcin. Avcragc daity int tc of f€sd wss 91.J, 9t.5, 83.6, 85.1, 

88.8 and 98.88, reslecrively. The bd resulrs werc obta,ned wnh mcprin al l0 of 

200 . 

Richrd er aa (19E5) f€d shndard feed mixrurc to qSed whir. lcg hom tayer 

as aloio (control) o. ar lhe cxpeD$ ofsoybenn oil mcat, fish mcal ed sutfire liquor 

y€ai wnh 5, 7.5 o. t5% femosin yc61. tn 8€neration 2 snd 3 the f€n6iD 

'nixlurcs wcre supptem€nted with Dl-mcrhionine. Wirh femosin rl l5ol0 level, fecd 

inrakc ad fed .ffc cncy ofnost layirg p€rfomancc exclu.ting .gg guatity and

5l 

body weight gain werc rcduced ro smc exl.nl, Tley @nclud.d lhat lhe lolerance of 

thc biomN wd @nsid@d 8ood, provid.d baeic nutie .equireBents werc m.t- 

Halda$/l-Valte. ?r zl (19E6) includ.d singl. @U protein havi.e 69% crudc 

prcrcin i! Ihe dicts of brcileB at 5yo l.vel- They obsdved d in€r€e in thc bodt 

wcieltt al Oo €nd of 42 dlys @mpared *iO thlt of brcilcF siven diets conbining 

nsh meal dd helt m@l (1481 Vs 1340d. FCR wN inprcved wirh mioobial 

prorein (2. I Vs 2.26). 

Lyurskanov and Sokolov (1986) comparcd the biomass produced $rough 

rwo direrenl yeasl st aiN viz., Condido ttopicalis and Cardida r.o(i grown on 

wood hyd.olyaie ed sDgd cane viDas€ m€dia respectively- They found thar th€ 

medium did nol caus€ significel change in crude, true ed diSeslible prolein, lhe 

values of which were 50-2-61-30/0, 43.1-44.80/o ar'd 40-5-41.770, rcspecrively. Thc 

biological value ofboth slrains was hid r,'8irg 69.4-71.1 perctnt. 

Viklorov et zl (19E7) inv.stiS .d d. p.orein quality ofnep.in (midobial 

prcducr) rhroud a r.eding tri.l in which m.prin wB rcptacd wirh fish met in rhc 

ralion of brcilq chicls at 0, 25, 50, 75 lrd 100 o/o l.vels. It wa found rhar thc 

.ve68e daily weidt Sain w.s high in sll thc Broups f.d rnepritr. Ir was cdctuded 

llEr ire tneprin had no advcBe erects on mctabolism ud hetlh ofchicks. 

clavez e/ ar (1988-l) produced rwo kitrd or fungal biomass protein, one 

irom glucose slbsrrate (lW) and othcr f.om molasses substrale (2w) as the main 

carbon sources. 'fte bioloSical trial conducted on wsnling rats rev@ted rhar lhe 

yeasl prolein had highest PER but signi,icantly lower lhan calein @nlrol d'er, ils 

value was sigificurly grealer thsn lW wi(h simil& pmrein inrake. Torul. yeasr 

showed a signilicotly hiSher PER value lha, lW, althougb prorein inLke wa3 

low€r. The NPR EIus beinS gr.arly influlnced by inrate lev.ts, prcvcd to b. 

nuch lss Etul m6ue of !rcr€in qu.lity. Thc lW md 2W di.L rere infqic to 

rh€ Torul. y6t od tftweas y8t di.ls in dry m.rrd diAqribitily, Diseribitiry of

54 

crude protein was high€st for the ca!€in die! avernainS cto6e ro 9l% for 20d and 

40\ week meaurcmenrs, followed by fo'uta y6t of 82%. The lW aveag. 

digestibility of?E.7% wa simite to &.r of brc*cr's y6r (?7.1%) ed $Derior ro 

rhat of 2w ed dried mushroom diets. 

chsva,r at (l988ll) prcrcd rhe micrcbiat bim4 por.itr (MBp) lsing 

rtneus ChaetoniM ce ulolt idu dd d.i.d in olcn st 6O.C (oD) or frc€z dri.d 

ar -25oC (FD). P!.ified dicis cltcutarcd ro enoin l0% crud! prclcin s6 fed io 

w@ling mts for two week. A clnrrol diet conraininS cascin as rle onty prcrcin 

Bource and other having isolal€d soyb€a, prcrein (tsp) * $€ sole sourcc ofp.otein 

vere also prcprEd. ln two o$q dier! on€ hatfof rhc crudc pblei! ws provid.d by 

IsP md odEr half by MBP-OD or MBP,FD. Thc diets conlaining OD mpt€s 

yielded poor growth, feed corsumplion and feed uritiation efnciocy than $e diels 

conlaining lSP or ISP plus FD smptes. Alt tltr@ ofrfiesc dicts showed sig ficanr 

i6ponse to sutptementation wilh 0.4% melhiori.€ dd slight bur non sisnilicanr 

irhpmvm.nl wifr funher 0.t50lc Ltysine. Thc doubte supplmenr.d MBp dicrs, 

howev€r showed a gMter weidt gain lhm rhar of the sinitarly suppl€menred ISp 

or c.$in conlrol di€r. The r!l! eeiving MBp bsal die( ,howed dte low6r f..d 

cotrmption wh.rca with Mim aqid suppl.m.nradon rh.y showcd hisn$t 

consunption. Relarively !oo. fed/gah sno of rhe rars tcd MBP_OD alone or 

$ppl.mencd indicdcd low prorei. uritiation emciency than conirot or tullv 

supplem.nted MBP-FD 8roup. 

Ch^yez et al. (l988lrr) ltudied fte nurririve vatue of Ch@tmiuh 

Micrcbial bionass proGitr (l,lBp) obrained ai.r srcwio8 on slucose (lw) or 

molases (2W) in combination wirh isolated soybean prcrein (rsp) in lhe diets of 

chicls. SiSnificad dill@nc6 w.E rcflered in th. body wciShr gain of fic chicts. 

Fecd emciency da1! $ve non sisnificant difte!€nces. Thc contrct ISp di€r ws 

bercr than the b.ewe. s yejlr diet bul rhe besr f@d cmciency was obsened with ltre

55 

co'nnercial diet. Feed consumprion of 3eni puified diet was much lower than thal 

of lhe comrercial chick srarrer Thc MBp septes in conbinar'on with ISp 

appeai€d ro have good acccprabiliry and consurnprion by $. chick. Consunpfo, 

of rh€ ISP diel incfeased wi$ l})€ addirion of fuigat MBp. The values ofapparent 

proteh digestibility ofisolared eyb€ln probin (tsp), tsp pts MBp and lw' lsp 

pl6 MBP dd 2w ad ISP plv Tottlt y.s\ w.rc 6|yo, 59yc, 6t /o ant 56%, 

Chrlal (1969) culriv.ted P/.wottq tajq-@jl in sfi ncr8cd condirion on 

J./o (w/v) of coh slovd in Mandels atrd Wcb.r m.dium wirhour lry orher grcwll 

iactoa addql and produced hyc.tiat bioma$ @ntainine 40-490/0 crude prorcin. 

The mino acid prcfile ofrhe prorein wss as good as edible funSus exceDl rhat ir 

was deficienr in sulphcr cont ining amiro acids. Th. prctinioary feding rri.k on 

rals indicated thal aboul 500/0 ofprctein in $e di€r can b€ r€Dlac€d witb p.raDr_ 

caJ! biomass wiIlloul oy delerqiols eff.cl'. 

Maxte and Hamld (t994) fed bro'ter chick a modemre {22%l o. a hitl 

prctein (300/0) well balanccd diet conraining yesr ,taccl@roarrcs ..tutuiae (20/r) 

artd soya protein isolate (Soxzii) or a pu.ified anino acid mixrurc. .lte viramin- 

86 statu of thc chicks ea! rss€ssed and ir w.s foud rhal the pr.scnce ofy.asr did 

nol augment metabolic r.quirem€nts for pyridoxinc. Th. removal of yeasr frcm ih. 

dicl provoked sever sympronstic d€ficicncy. It qe found rhal a low level of0.6mg 

viranir BrtS ofdier wrs adequare ro mai dn normat srowlh and ro aaoid ncusl 

symproms. The nomal r€quireme for pyridoxinc hr! been r€poned ro be 3 m8&g 

Arhar €/ aa (1995) fed rhe biomds ro day old broilcr chicks prcpared 

thrcugh f€rm€nration of bler pulp in fed barch cullure sysren first wnh Cadrira 

utilis rhenwirh Brqibetetunlav"nl. Tle biomals was f€d for a period of I 0 days 

by rcplacinS wirh lsh mcat in th. bas.t mrion al0, 25, 50, 25 and 100% lcv.h.

56 

They concluded $at 25% replacenenr grve bst results. They tound significet 

dirercnce in w€i8hr gaio and f@d .onsumprion berwc.n conrml and rhat of 

rcpl4.'nst wilh micmbial bio'nols prcrein (Mttp), rhe btcr showcd lower vatu6. 

A gmdual deprc$ion in feed convedion ralio (FCR) u0, obseNed by incrcasidg 

ieplacemol level of fishmql wilh rhe biomass. Thcy rcported the Drorein 

(iigcslibihy s 74.1%. Th. biolo8idl vsluc (Bv) *6 50.2% at 25% subsliturion 

with nsh meal. The ne1 p.ot.in utilizarion (NpU) vatue obrained al 25% 

r€placemenl w6 39.35% ss comparcd ro rhrr of fish m€d which was 40.74%. 

Simil.rly Inc value of prcrcin crtrcia.y 6rio (pER) i125% rcptllmert was bet 

ofall h&ving value of2.lE3 s compded ro @nrrol vatue of2.3jJ. Thc !ni!o lcid 

prolile reve.l€d koleucinc as lh. firsr timiting amino acid. 

Kdleczn! et ot. (1996) cvaluared prorein pEparrtions of dilTcml kinds 

wirh felevance to m€al processins. The hydrophilic_lipophitic batarce (HLB) 

numb€r PA used as c.irc.ion. pamfiio oit(HLB=I1.0) and soybean ojl (HLB=|5.0) 

1vfrc sclecred ro detemine rhe HLB vatues of non m€at proteiB (btood Dlasma. 

'nilk proteins, ptanr proleirs, bacteriat biomsss)_ Emuts'on stabitity curves a! a 

function of tlLB indi€red $.t burlennitk prorein and bacre.i.l bioma$ prorein 

were lhe per.sr cmulsili.rs. Th. reminiry prol.in had Elrriv.ty tligh mutsion 

stsbility (>80% emulsion phsse) 

Vulchevdal. (1996) obs.Ned thst thc ca!.in_s singte celtprorein corraired 

75.7y1 Votein (57.4oA Wpsin digcalibt€). Th. prcrcin cdrtined 6.56./, Irsi\., 2.6% 

methionine, 3.51% m.thioninc+cystine, lAyo ryptophu dd 6.9y0 vatin€. clrrin 

conlaincd all .ssedial ma$o ard micrc eteocnrs. Tle enc.S/ conrdt or the fed 

va 55 14 Kc.L/ke.

Chapter - 3 

5l 

MATERIALS AND METHODS 

'IIre rcs@ch was oricnted lowdds rhe bi@nv€rsion of dce 

polishings inlo piorcin biomss rtrough ils fermenbton wilh rwo micr@rSanhms 

i..-, ^ ttt9i (Ara.haiot6 speci.r) ad s ba.ren^ (BrceibactetiuM jtat6). Th. 

details of prccmment of subslrate and thc lwo femcntariv. microorsrninB, 

detauinS of ll€ substrare, adapralion snd cultivarion of thc fernenlalive orSanisms 

on thc gmMh medium, 8roMt conditions oprimization of both organisms for 

mdimum yield of biomars prc&in, grcyth kircncs of bofr orgsnisms, tar8e sslc 

production of biomass, biologicat elatuation and chemicat eatylis of rhe biomss 

and statisdcal merhods uscd for anatysh ofdata havc been discussed hercsepamlely 

m a sysr.naUc rnoner. 

l.Substrate 

l l Procurement 

,,1-t\ ) 

V o,r 

inh 

Tle fresh third step fine ac6 polishinSs was pro.urcd 

niot, Distrid &ang and ya used 

ol ah oven a170oC lemperalure. 

1.2 Deffating 

as subshle, Ir *6 dricd ro a 

Five hundred gmms .ice potishins was laten i, one lilrc ertenneyer 

{lask and one litrE perrol (super) wN added ro ii. tle flark was stoppered with a 

cork and rhe conieDrs were srhr€d for 15 minures on o.biral shale.. lt was then 

allowed to sraod for 5 minures. Tle slpcmat nr p.lrol us poured ofand prcs.ned 

for rcuse. The residue w6 washcd rwice *ilh p€hol. Thc defalbd rice polishings 

was tansfened to sn oper iron tray (15..x 8..x l_5..) lined with paper and ws 

d.ied to a consler weight in an oven ar 70oC. The d€fatred @ polishings so 

obtain.d eas plac.d in sealed poblhen bags for fidher e.

l-3 Chemical Composition 

58 

fte dried detaned ri@ potishinss was flagzd for crude ,rclein, 

ether extracl, crudc libre, sI ed nitog.n free.xracL lblowing AOAC (tgBO) 

nethods. True protein corlent was esrimared by rh. m€lhod of MuDro and Fleck 

(1966). Aoino acid prcfile ws esrimared by rhe merhod of Moore ard Srein 

(1954). Crlcium ard phosphorus conlenrs w.re ako cnimared (Anonymous, t954). 

-llle cebon conrenl3 ofrhe ri@ polishings ed rhar of oth.r subsr,rnces uscd d!in8 

lh. cxpe.imen were Brinared by colorimetiic m.fiods dBcribc by (clnhms. 

I 948). The compocition of 0rc suhc|mcs is gircn itr Appendix L 

2.Organisms 

The certified pur€ cuttuH of fe.mmbrive 

tpe.i.r .ad Brcvibocteriu lI@M rerc obr.in.d ftum 

Palholo$/, Univ.Eiry of AgricultuE Fabatsb.d. ,.d 

Culture Collecrion of Micrcorganisms, Instiille or 

Univ€rsity of Tokyo, Japan, respecrively. 

orgsnisms,,,{'daruiors 

|ne d.pattnenr of PIor 

Jap.n@ Fcdcralion of 

Appli€d Microbiolory, 

'l he cultues w€re majnrained on Czap€k,s a8ar stan6 (Beneke, I 95j) 

flnd Modified Nude Asar (NCIMB, 1990). The coDtosrtron or,utienl ,sar 

medrum and 

'uodifi€d agarsta m€dium h shown itr Tables l. 

NaCl 

DefaGd .ice polishinSs _ 50 

._ 1.t25 

M8SO..?HrO : l?: 

KH,PO4 

Aaar ,n ' 

5 

5 

,14.0 tot Atdch iorzs v andat1.ofo,;t1;;;"'""' r"'r h trr'dim *a adrBLcd

3. Preparation of in oc|uhm for Arachniotus sn. 

3.1 lnoculum 

59 

In@uluD w6 pEp&e

60 

gram ofdn€d biomass was tat€n for estinatior ofru€ prot€in by rcating wilh 30 

ml of5% Trichloroaceric acid (TCA) in warer bati ar 90'C rempemrure. 

4. Optimization of Fermentation Conditiotrs for 

Arschniotus sp. 

4.1 Elfect ofType of Rice polishings. 

h lhis expcrimenr codpsrison was madc b.rwed undefafi.d rice 

polishings and defatled .i@ polishings for ihe producrion of biomals protejn. 

Triplicate flasks of€ach type of rice polishings containing 50 mt grcMi medium 

were prcpared md incubarcd for 3 days udcr prcdctemined conditioN viz., .ice 

polishius 5.00lo, urca 0.3125yo, Caclr 0.0025%. MgSO..THrO 0.0025%, KHrpO. 

0.2%. pll4.0 add l€npe.ature lo"C (Mahnood, 1990). The biomass so preparcd 

was dried and esrimared for rrue lrolein. nle amount aod B?e of subsrnte used in 

Ois qperiment w8 adopted in thc suts.quenr exp€rimenls. 

4.2 Effect ofAcid Treatment ofsubstrate 

An experiment wa! @nducled usiDg defalM rice polishinSs and fte 

ellect ofrrcarme of sqbsrnre wirh 5% Hrsoa @ I mlrg wrs srldied. on the basis 

ol reull of rhis cxperimcn! r.€rcd subsrmre w6 used ,n tnc subs.qu.rr 

4,3 Addition of Different Levels of Morasses at 4E hours of 

F€rmentation. 

The effecr of addition of motasses at 48,i hour of Srowth ol 

Atachhiot6 tp. was studi€d in this cxperim€ni For lhis purpose 5 leveh of 

molassd werc resred viz., l, 2, 3, 4, and 5 p€rc€nl tor lne maxrmuD producrion of 

b,omss protein and hawestcd after 72 hous of fcmenration. The opii,rum tcvel of 

molass€s was used in rhe subsequ. experin€nts.

4.4 Setting CarboD to Nit.ogen Ratio 

An€. dcrenninalior ofop num lev.lof molscs !o b. added ar 4E( 

hours of ferne.rarion, anorher €xp.lim.nr was conducled for dcrerninin8 the 

opliDnn carbon to niroSen iatio to decide how mLlch nilrogcn is to be added along 

with the addjtior of motarses. For fis purpose five differenl C ratios viz.,27:1, 

20:1, 15:1, l0:l and 5jl werc tcst€d for addiiion to rhc Brofin nedium al48 ho!6. 

The opdminm cs6on lo nitroSe! rario obffed in rhrs expdimenl was used in 

subsequenr €xperiments fo. ,naximum producrion of b,omass prcrem. 

4.5 Setting time for Addition of Molasses 

FollowinA $e optimu,h l.vel of molsses ro b. add.d ard .ftd s€ninc 

C/N 6rio, uorh.r cxpcrimcnr war @nducred lo deide rhe rime ot incubarion ; 

vhich this con€nkarioo of motrscs wd to b. add€d. For this purpose rhrcc fim6 

ofincubation were setecred viz.,48,60. and 72 houB. Alt the fl.sks were harlscd 

alter 96 houls of f.rn.nhrion. Ttlc olrimum incubal 

experim€'r fo, addition of mo'",,* -", r"r"-"r," Jil::,":ffil:* -" 

4.6 Addition of Corn Steep Liquor (CSL) at 4g Hours 

An qpenmed was conducred ro rest thc tevel of CSL to be added in 

the femenblion medium ar 48 hou6 of incubation for moxmum yjetd of biojrsss 

proreir. Fu this purpce six lev€ls ofcom saeep tiquo. werc resred viz_, 0.5. t.0, 

1.5, 2,0, 2.5 and 3.0 perceni anq selting CN ratjo $ del.min€d pr€viouly. t}le 

optunum levet obs€ned durin8 this q\perimcnr was used in $bs€quenr 

4.7 Setting Time for Addition ofCSL 

Ir wd required to determin€ fie opdmun rime for supptementation of 

ophnized lwel of CSL. for riis purpose lh@ rin.s of hcrlbation wcre rcsrcd viz., 

4E. 60 ed 72 hou6. The ft€sk ofalt rlE tine fE.iods we.c narvesrcd at 96 houB of

62 

fennsralion. Th. optimum lime for CSL suppl.menration obscded dudng this 

experimenl was followed in rhc subsequent €xpqimdts. 

4.8 Harvesting Time 

Under optinjad condilions for fi6t phssc of fcrmenrarion, final 

exp€irmenl was conduct€d to find out the oprimum harvestinS time for maximum 

yield of biomals protein. For $ir p rposc $e fcrmcntsrion tr?s r.rminared sr tlree 

di|fcrcrt time periods viz., 12,84 an 96 hours. T1€ oprimu,n hanesting timc 

observed during this experimenr wa! tollowed in the subsequenr experim€nb for 

obraining rhe highsl yield ofproEin. 

5. Preparation of Inocrtllum Ior B.Jlavum 

5.1 Inoculurn 

Fo. second phasc of fcmenlation, the oryrnisn Brevibacteriun 

lavM was used. The inoculun ofs.,/la,M wls prepnred from the pure culrurc of 

rhis orsanisn mised on agar slantj mcdium. Cc[s of E.jtovun vqe oanstercd 

aseptically from agar slants to rlree auloclaved 250ml erlenmeyer flasks each 

containing 50ml of iroculum medium. Tl|e composilion of f,e mcdium is shown in 

Table 3. Thc pH ofmedium prior to steritialion ws adjusred ar 7.0{1.2 wilh tN 

NaOH. Flasks wse $o incubared on oftiral shak* running at a rale of 100_120 

rpm to. 48 lou.s at J7.C. 

Tabfe J. Composixon of inocutun n ditm fot Brcvbackriw narw 

t0 

5 

NaCI 

5 

l0 

Dc'ilkd wa'.. addcd ro nEke ;;tu;;u!61 ljtt.. pH of the nediun m

I'hrr L Sl,oni'r I lr'csll culfo.c shnt of,l ntuthtuiotus :

l'lrr(:. !!lorr ins lrc\h !tr1(urc shDt

65 

Alter 48 houls it€ nrmb€r ofcclls ofrlav@ werc counGd,n rhe inoculum 

mcdium following Brced.s Smear Method (Collin d 4/., l9E9). .Ih. iollowing sreps 

]vcrc rollorved. 

l) Loop of 4 didmeter (one toopfull=0.0tmt) 

'tun 

w6 prepared and dipped in 

2) Tte loop fu culrure waj sp€ad on mioos@pjc alide in a.e of tcm by first 

nrakinS rht d€s on nide rhm metinS it *irh mdker 

3) Slide wd air dried (n.n fixcd on flame. 

4) Staining was ptrfomed using Cnam.s st in, $e lta,mng ruft of which was as 

a) Crysralviolet 3 minul€s 

b) C.ah.s iodine 3 minur€s 

c) Acid alcohal washing 

d) Saf.ainine 5_r0 seconds 

5) The sljde so lrepeed w6 examined under l00X oil te!s. 

6) The microbial counr was pe.formed in five microscoprc lietds, to catculale rh€ 

average mic.obiat count per field. 

?) Calcularions werc done as follows 

whe.e, 

Tobtnumber oforgeisnll,/mt - Ave.ae. No. ofo.aaaisms r M.f x pilurion 

o-ol 

M f= Micccopic fadorô or micrcscopic fi.los per cn.. which w.s 

bY 

1,,{.F "tt- ' 

A =l0ru (mearx Icrn? ale, is equal to l0rmicrons) 

a = AEa, which is carculared by t',

t = 2217 and r = Radius of micG@pic ti.td which is mcasrrcd with 

the help of micronelg. ln one midomerer one dilision is eq@l ro t0 

'nic.ons(p). 

r= lEo !r /2 = 90 

As rhcE w€rc l8 d'vhion3so it becahc 180 p. 

So arca of micrcsmpic fietd came oul b be. 

^= 

22n a9O1 

Tle trmber of elh of Aldla per mt we.e mintdned at 4xt0t by 

dilutin8 the inoculum wi$ srerilized dislilled water 10 ger a honogenous suspension 

of thc o.g&ism. The inocutm thus prepe€d ws lsed in the grcwlh mediun @ 

5my50ml lo invcsligale various conditions dudng second phare of femeDlaton. 

5.2 Culture Cultivation 

Th. r.covcrcd tungal biomds du.ing fiBl phas. of f.Dcnration was 

usr !s grcwih Dedium duing rhe s@nd pndc of fcmenlation. A rcqui.ed 

number of llasks were prepded accordihg ro the condition undd ltudy and 6eir pH 

*as ad.iusrcd ar 7.0 wnh lN NaOH aseplica y. Tt€se flask were ften inocutared 

$ill 5ml of inoculum wirh Oe hetp of stcriti&d ptperre. lh. n6ks eer. rhen 

incubated foi 72 holrs on o.biral shakcr workirg ar I 00,120 rpm ar 3 ?.C. 

5.3 Harvesting 

Trilli€re flsks of €ch treonen! werc heested by 5 minurcs 

sle.inirS. fte bionss ftw recovered ws dried in chiDa dish.s sepaErely attd 

putting then in oven at ?ooc remperarure for 24 houE. The died biomas was 

grcund md malyzcd for uuc prorcin. 

6. Optimization 

B.Jlavum. 

Expedmenb werc condu.red for opdmizing 

naxinln producrion of bion)N prolein 

of Fermentatiol Conditions for 

Following conditions werc optimied.

6,1 Autoclaved Vs UnNutoclaved First Step Fermentation 

61 

Ar expquneni w6 conducled ro decide whelhq first and second 

organisms should be growD in synerSism or one aner $e olher_ For this pufpose six 

flasks haNeued aner fi6l phase of fcmemadon eere divided in lwo groups. Out of 

these, ttuee flatls wcre adjusled for their pH ar 7.0 rnd then aur@lavcd whe.eas rhc 

.emainin8 were adjusred ar pH 7_0 wirh srsitized tN NaoH d.plicalty wilhour 

autocraving. AII the fldks werc inoculated wirh inoculum ot B.FlMn asepti.a|y 

and $en incubated ^t tl"C tot 12 hours. Atter 72 hours all rhe flasks were 

hanesled md lhcir tiue prot€in contenb w.re d€brmined. Tte opdnum condnion 

so achieved ws followed in subsequmt expcrimenls. 

6.2 Addition of Different Levels of Molasses at 48rh Hour of 

F et mentation vriah B.llavum 

for improvjng lhe prcduction of bionss prclei! during s€cond phase 

of fennenhdon. ir ws deided to supplenenr lhc 8rcw|n medium wilh notas€s. 

For Ois purpo*, sir levck ofmolases were 1sr€d viz, l. 2. 3,4, 5, and 6 D.rccnr 

and add.d at 48'r hour of g.owth of B.Jtdwn and hanesr€d aner 72 houu of 

rermentatjon. The tevet of mola$es giving maximum prolein was used in the 

subsequenl expedmenb. 

6,3 Addition.of Different Levels of clucose at Different 

Time Periods 

For rhis expsjment rwo lerels ofglucce ,2., Z ed 4 p€rcenl veE 

rested. Tlse tevets werc resred fo. rhree rime periods vz., 0, 24 ed 48 ho!6 of 

incub.[on. The oprimum l.vel of ootarss and rime or ternentdtion giving 

maxnnum producrion of bion.ss p.olein was us.d in subsequerl experihenrs. 

6.4 Comparison ofGlucose and Gurrh 

To d@ide if gurh @n rcplacc gtucose ar s,mitd 

w.s conducred in whicn gtucose sid gurh w.rc supplemenrcd

6E 

nEdium ir sepamte flsk at 24 hou! of s@nd phsse of f.menrarion. On the basis 

of higher biomass protein producrion .iiher of th. rwo w.s usd in rhe subcqnenr 

6,5 Yeast Sludge Supplemertation 

To srudy the €ff@t of strpplemcnt.tion of y.ast studge on rhe 

cnri.hhcnr df fcnnenlation 'nedilm for .nhancement oferowrh of BJIarun.lning 

secotrd phAc of femen(ation, the arr@taved yosr srtd9 of Sac.huonyces 

ceNtrr?e was added to th€ medim al0.5, 1.0, 1.5, 2.0,4.0, 6.0, 8.0 ad l0 percenl 

levcls. The supplemenrarion was done !t 0 nou$ of s@d phase of fem|cnlation. 

Th. optidun level so achieved wa 6ed unde. the prcviously opridized 

coDdilioB. 

6.6 Harvesting Time for 2nd Phase 

To lchiev€ lhe mosl suirable hawesting lime for maximum yield of 

biomas lrotci4 direren( hs€sring rim€s w€.. r€sred during seond phe of 

tbnnenlarion. Tte hadesling of biom.s wEs done sr 24, 4s ed ?2 houB of 

feanenta on. lle he€lting tibe Siving oplhruD biomss prorei, for sccond 

phe of fennentation *as foltowed during sqlc up fermmralion. 

7, Growth Kinetics 

crca1h are of Atdchniotu sp. afi Brdibacterim JI@M, lt td of 

products and sp€cific subshle uptake 6G dDring borh phas€s of f€mcnlar'on werc 

olculated using the following fonnulas. 

F = _l_ x 

Yp/s = 

Yp/x - 

hr n: 

x,- x, 

S:-Sr 

Pl-Pl 

Sr_Sr 

lo& XL 

XI

69 

X:-Xr 

gP = Fx YPlx 

q, = p xYx/s, whcre, 

& = Final Producl 

Xr = InilialProduct 

la& = lng naltrnl 

P = SPecilic growth rate 

Y'i" = Gm3 ol microbial ccll produc.d/g sublrate utilized 

Ypis = cItm of pmduct flm€dg of subslrat€ ulilizcd 

Yp* = Gram! ofproducr rormed/s of c€ll forn€d 

Qp 

qs 

S) - s' = 

X: - Xr = 

P: - Pr = 

SPecific product quotienl i e I 

Specific subsrrare uptake rate 

Diflcrctre in sub$rakJh' 

Difleren(e;n c.llprcducliotr/h ' 

Diffcrence in Product/h ' 

br - br = Difl.r€nce in Time 

producug @ll/h 

as = volumekic rale ofsubstmle utilization Gnrh) 

a, = volum.rric iat€orcellma formation (tt4r) 

a" 

Vol@.tric mle ofproduc. fomrtion (8/Uh) 

8. Large Scale Production ofBiomass 

Large sol. femcntalion wd (sied out in Iwe litr. @Pacit) glass 

fementer. Th. go$'th medium eas prcparcd in fernent r, it wd then autoclaved al 

l2l"C for 45 ninutes. Anq auto.lavinS $e pH of the mediun was adiusted al4 0 

with lhc help of lN H1SO. added drcp wise silh the help of peruslaltic pumP Thc 

leDperalur€ of $e fef,ncnler wr! adjuslcd al 306C, by circulatinS water in the oucr 

jacket of the fmenter lftei ehich the 8ro*1h medim sas indulaled *ith frcsh 

inocn'r,m of lreluiota tp..s.plically @ 5%. Tle femente. rls tun at 300 rpm 

speed !lo!s with contituou supply of frcsh filtd€d air. Tte pH of the mcdium was 

checked md adjusted at 4.0. After 56 hours of fcrm€nlation lhe f€rmenler was onc. 

again auloclav.d at 12 I 'C for 45 ninulcs dd before 3tad of autoclave y6( sludSe 

was add€d 1o the nedium @ l%. Ancl autoclaving tl|c PH of lh€ mcdium was

70 

adjrKed at 7.0 wirh lN NaoH md temperaturc of thc fermenrer ws adjuned at 3? 

'C. l lre fermenter was then in@ulal€d with fresh jnocnlu it of B.Iavuh @ 5% ot 

the gorv(h medium. Again fernenrer \yas run at sp€ed of 100 lpm atong *ith 

contntuous supply of fresh nltqed ah. The pll of lhe mcdiun wN checked and 

adjstcd al ?.0. tq 24lou6 offtc addition ofsecond inocullm o. E0 hou6 of 

toral lbrmedtation pe.iod the biomds was rccovercd aner 15 minlles stcamirg.-the 

biomGs wa then dried in llal tra)s in lbrced air oven at65oC. 

Following eoc pbclice th€ f@.rls was run five rimes sd rh€ 

biomass rccovered wa dried and g.ound. It was aratyzed for crud. protein, crude 

fibre, erher extracr, .sh md NFE following AOAC (1990) DiomN was 

'nethods. 

aho analyzed rof lrue protein (Munro and Fteck, 1966), amino acid profil€ (Moore 

and SGi\ 1954), Calciu'n sd Phosphorus (Anony'nous, 1954) dd RNA conrenls 

(Muro.nd Fleck. 1966).ltese ealysis ee shown in Appendixlr ort llr. 

9. Biological Evaluation 

Fo! biological cvahadon rhe biomass wd fcd ro 7-days old broil€r 

chicks for a period of 9 days. The triat ws @lducted.t Raja Mrham,nad Alcarr 

AniDal Nur.iton Rcs@h Cenre., Univ€Firy of^sricultur.. Fais,labad. Eighrccn 

sepdalc pens werc construclcd and birds we.€ placed on {ire net fo. rhe collecrion 

olrhen droppings on rhe plaslic sheets plac€d on $e fl@r. h this tial fish meat of 

the b.sal €tiotr was replaccd with thc biomass at 0,25,50, ?5 and t00 percenl 

lelels oD protein equiv.lenl basis. 

9.1 Experimental Rations 

Six cxperimenlal ralions viz., A, B. C, D, E and F wcrc prepared. 

Ration l: was prolcin free lo derermin€ endogenous |lilrcgo loss6. Ration A had no 

bioDass ond sewed as conrrol.In ralions, B. C, D and E the quarlityoffish mealot 

conrrol was replaccd yith biomss prorein on prorein equiv.tcnr bajs a.d rhese 

conlaincd 2.5, 5.0. ?-5 @d 10.0 percent bioh.ss, r.specrively, nE nnrient t€vels in

1l 

rll $. rariom werc.djuied accordinS to NRC (1984) nandards. lhc choNical 

conrposiritr ol rhe experimenrat rarions is shown in Tabte 4. t.hc ch€nical 

.otr'psilh' ofingrdicnk used irr Dtions is Sivcn in rucndixrv. 

'l.blc 4. Conioosilion olrhc expernnurl{t rariops 

oit 

ME Kc /ts 

' 

54.74 

18.80 

7,00 

5.00 

3.00 

t00 

0.41 

,i 

0.25 

0_25 

24,54 

3211 

1.54 

0.95 

0.58 

|.228 

0_547 

0.t 8l 

0.10 

B 

(25% 

50.09 

20.98 

7.00 

6.00 

2.50 

0.92 

5.25 

0.64 

010 

0.25 

":' 

24.51 

3219 

3.77 

0.87 

0.57 

t.219 

0.542 

0.187 

0.1t7 

c 

6e/, 

5l.ll 

1J.17 

8.00 

.00 

500 

0.96 

4.15 

0.91 

0.ll 

0.2t 

0.04 

o:n 

24.60 

1242 

t.92 

0.82 

0.57 

0.t43 

0.186 

o.J29 

CoDtposilion ofPremi; isshown in Appetrdix V. 

9.2 ExDerimental Birds 

D 

05rA 

49,t7 

15.50 

8.00 

'I 50 

0.91 

4.82 

Ll6 

0.15 

0.25 

0.05 

o:' 

24.51 

120 | 

0 ti6 

0.55 

| 215 

0.518 

0.180 

0.125 

E 

(twr 

46.96 

17.20 

8,00 

t0.00 

087 

l.l8 

0.40 

0.25 

0_06 

o.:r 

24.54 

320t 

4.15 

0.51 

|.224 

o.5J2 

0.tE3 

0.116 

1.50 

100 

300 

L00 

80.01 

I L00 

3.91 

J202 

I l(i 

o:, 

0.131 

0.2E2 

sevenly day-old brciler clicks werc procnred lroD markel. Atlerone 

$cck llty-lbur bnds were sel€cted otr wcighr uniformity basir. These birds w€re

72 

'l h€se birds were dividcd into €ightcen €:(pe.imenbl units conlaining hree birds in 

cach unil. Each exp€rioen(sl nnir ws kepl in sepuarc p€n_ Th* bnds Ner€ 

rihdomly nlloled ro six expcriDent.l ralions in such a way rhar there were lhree 

cxperimental mir! on @ch mrion_ 'llte Erions were offe,€d to the birds ad libirum 

tor nine days. Iresh warer was svaitabte lo the birds all the rime. Weigh ot each 

bird was @rded prior to rhe exp.rimenr ed rherean€r a! the ed of rhe 

9.3 Manaeement 

The day-old chicks were reded under br@der for one Ne€k and 

drirs lris period lhey wcrc v!@inaGd agaiBr Newcdrt. diseasc. red and warer 

wrs suppli€d ad libilum. Feed and wat€r was supplied sepanlely in each pen and 

elcctric bulbs wefe fired in p€ns for mainraininS rhe lempe.ature. The droppinSs of 

lhe chicks were collecled on ptstic shets ak ady spr€ad on floor 

'n 

each pen. 

DrcppiDgs or dch pen qere *eiSbed daily, mixed woll ed r o% of qch was placed 

in bcakers having Ilrsoa. 

9.4 Data Collection 

Following data were recoded during the expeiment. 

'lhc trilrcg.n conrenti of eacl ralion w6 estimared 6.ough Kjctdahl s 

Weight of chicls in @ch expe.imenlal unir w6 re@rd€d a( the srart of 

qperinenl ard thereanq daily. 

Wcighl of drick in cach cxpc.jftnral unit was rccordcd at Lhe end of 

Feed oflered to each unit al srort 

Feed rcfiscd by ech unit al rhe e|ld 

Total weiShr of droppings voided by each uri1.

1J 

opetrirr8 rheir abdomisal cavilies. Afler dryinS birds werc wcighed and grornd and 

ll'e,r their body nirrogcn was esrimarcd by Kield.ht.s crhod. fhc d,opping 

collccrcd nr berke6 \!.rc wcighed. lhorolghly nrixed {nd irs niriogcn con(cnrs $.crc 

9.5 Parameteis 

'lhe fotlowing pahmelers w€le wo.ked out usin8 rhe fofinut.e of 

PelleleaDd YouDS ( 1980) 

a) IeedcotrveEion RaddFCR) = red consrncd / Cain in eeislrr 

bl Net l,rclein utilialion (Npu) = u_(Bk,lk)/t x t00. whqe 

B = .rod body nitrogen on rsr dict 

r.ik = .tolltbody nirrcgcn on l,n,tcir ftccdicl 

| = Intakeofnihsen on rest dier 

tk = lDrake ot nirrcgcn on prol.in tree dicr 

c) ltociD Bliciency Rado(pER) - Cain in wer8hr / r,rol.in imale 

10. Statistical Analysis 

]-he experimcnbt dara weE subjccred 1o shrisrjcat analysis usins 

CoDplcrcly Rando'nized Desien_ Fo. rhis puQose the mean varucs ot the each 

h.nn.nt rcplicate werc c.tcut.red. Ttresc repticalc rcrns wcrc lhcn !.cd for 

staristicdl analysh o. (r conrpurer rd anatysis ofrrnnncc r{orcs \verc ltup rcd 

for each experimenl. [inally the healmenl means \verc compared r,! t)uncan.s 

Mrlliple Range test(Sreetand lbdje, |980).

Chapter-4 

RESULTS AND DISCUSSION 

The nudy wa unde&kcn to enrich rhc Drorein conrents of rice 

polishirSs $rcugh lbmentaion in a inutri cutrure system tid wnh cellulolyric 

frngi. Arachniotw rp..ier md rhen wi$ Iysine synrhsizing bacteria. 

Rrevibacteri@ l@u. Thc prctein biorEss so produced ws thcn evatu.r.d 

throuSh chick feeding rial and throuSh chemicat dalysh. For rhi! pDrpose the 

research w$ conducred in thre phas€s. Tte rcsutts obbjned during rh* $le 

phases have ben described herc sepamtely. Fof rlis purpose rhe chaprer ..Resuhs 

and discussion" has been divided inro lhK subohapteB for elaborsring the resutts 

ofeach mder separate headinss, which arc as follows. 

A. To optinize rhe grorvrb .ondiiiors for,4tbhrtot6 spe.ies $rng nce 

polillirys rs slb6rr.le for th. prodrcliotr of bion.ss p.oa.ii. 

B, To oprimi* rh€ giowlh condiriotrs fo. Ar.riracr., iah ltarun usial tttst 

pb.s. prcdu..d bioD.$ rs subsrnte. 

c. 

Nutriliolal.evdmlion of biomo$ lhrooSb rmlno rcid compd ion rtrd 

For each sutchapter lhe objecliver, resutb and discussior and 

overview h.s be€n given sepamtely.

'15 

A. TO OPTIMIZE THE GROWTH CONDITIONS 

FOR Arachniotus Species USING RICE 

POLISHINGS AS SUBSTRATE FOR THE 

PRODUCTION OF BIOMASS PROTEIN 

Thc grcwrh cond'tions were optinjzed tot ,ittuhniotus species virn 

rhc ra.8et io prcdue more biomN prct€in ke€pir8 in yiew $e fo owing 

Objectives: 

i) 

:vlE$s d.farkn de polishinss @ b. used .s subsrrsre ror proErn 

orom6syrcld, 

ii) Wlether prctr$tm€nt of the sLlbsr-dre wi$ acid is nccessary to yictd 

morc biomass p.orein. 

iit To see if lhe adding of molasses ed com steep liquor al differcnt 

levets and dme inrrats ro rfi" fen"*,!d- mcdi; j* n";;i;; 

improving rhe prcrein biomas yictd. 

ir) lplErher rhere-was a ne€d ro chan8e rh€ carDon ro n,trogen ralro wirh 

u. aqoruonot molassev@m sl€€p tiquorro fermenkrion m€drun. 

vr io,estabt|sh rh€ oprirnun haNesrin8 rime tor rhe mdimun 

prooucuon ot bromas Drcteir. 

Results and Discussion: 

The Hutrs obLNined on optimized fementation condirions arc 

discused in $e lishr of available tit{arue s !rdq. lhe aveBSe vatua for rrue 

p.ot.in p.oduction as affecred by dillemt femmt.tion condiliotrs sE shown in 

Tlble5. 

l. f,Ifect ofType ofRice polishings 

Rice pothhings conbins 12-13% oil ard is uualy dcfalted b avoid 

rancidity lrd its spoitage duinS stohge. Thc oit has a good ma.kct vatu€ md rfic 

extraction rnales the product a cheap one. An all€nDL wd made to use rice 

polishings !s a substrare for rhc femenring orgeisns |o oDra,n morc prcrcin yietd. 

To deide wiether defallcd rice polkhines could be uscd ro oblain highe. yietd, 6w

76 

dod dcfafied dce polishings wde compred.s flrbst ar4 for femanbrjon by 

Ar@hniots tp.cies- Tfu tv prctei! contors of ric. DotishirSs rft.r f.moralion 

nay be seen i! Table 5. Tl. analysis of variance ofrhe dala h shown in Apperdix 

vl. ltre .ornparison revealed non signiticdr diffem@ b€twM ihe biomass 

proteins oftwo tnes ofrice polishings. 

Th. result! were encouraging. Cheap d€falted fice polishings reptac€d 

ccily undeflned .ie polishings. A @mp@bte rue prclcin yietd of biomss with 

dcfatted and undefauetd ric. polishings (17.20 vs t?.33%) was obtained. Direrenl 

wo*ers have slso used defaned potishinSs a! subsrrare and obrained biomass 

prot€in yiefds of Z2.l3oh \|ith Andida ulilit (Kini, l9E9), 24.80% with 

Trichodema hdziaaM (Khe, 1990) ed tj.33o/o with ,lrrchaiotu, ,pe.i.s 

(Molr'nood er 41., t990). The biohoss prcrein yi.ld by fem.nriry d€fa(ed ricc 

polishings by Artuhniotts speciq is in confimity wifi rh. prcvioB work. Ir was 

lhe.eforc decided to use def.tr€d ricepolishings in furtncr cxpea,nen6. 

2. Effect ofAcid Trcatmena ofSubstrate 

Th€ subslral€ should have maximum f€ffen1a1ion in rte D€diun. Fo. 

this purpose lhe substnte ar€ trsred in diffmnr ways, viz. physicrt or chemicll. 

Physical treahenrs degrade rhe subsral. at ce utar level while chenicat rrearn€nG 

help in deending fte chemical bonds. Exkemes ot pH play majo. role in this 

Espccr. The d.faned rie polishings wss lr@ted wirh 5% HrSOl in a 6rio of t:l 

(Vv, subsrale : HrSO4 ) and auroctav€d .l l2loc for 30 minules. The lrealed s 

w.ff .s untreaLd substratcs were ferm€oted with Arehiiot6 specif \nd.r 

previously optimired condirions. Th. dara on uuc proteiD conrent ot hrrysrcd 

bionus, (Table 5)was subj€cted ro stalkrical snalFis (Appendix VII). t.he analysis 

of varianc€ of thc data ftvcded Don significanr diff.i€nc€ in biomN prorein yi.ld 

oI the acid lrearcd substrale as compared wirh unrr.ared.

ablc 5. Mo t SE lde p.or€in production s affect€d by u stng Atu h,iotu sp. 

UMcfan.d ri€ Dolishinss 5t.t5 

t7.tl 

ltiircilo|s!b!|d|. 

2 

11 

56,75 

57_0J 

s5,t96 

t6.870 

r4 o87ro.,2rd 23.E81 

l3.9tl 

1.125 

25:- r.58 

20: I 

t.08 

t2,76 

, rE or rcotrpn or por.s.r (HB) 

uYss d eonbn or (sL (y, 

5r 31,19 

17.197i0.s1. I 57.31 

53.19 

I5 tt_t8710:t" 13.95 

2.0 

l7.21 

2.5 

3.0 

24.70 

,0'o0 

---l.r-r 56.72 

53.00 

17.05 5599 

72 r 7.09 i 56.16 

- - 

t?Jt- --r 54.t7 

17.22 

t1.21 J8.00 

sDp.Bci,p! otr @inr h . .otum *hhtn .mh ondiG;;;w sisntf6a(p

7E 

Results iodicared rhai rh. lcid k ated subsL.t did nor yietd beter 

tue pro(eitr llan uDtroated ri@ polishings in the tinally hawesred biomas. 

Sulphuric acid lreatment solublizes hemicclluloscwirbolt in(.rsins irs micrcbial 

digestibility (H!n and Calliha[ l9?4). Moo Young e, a/. (t97?) obsded pa.lill 

delignifi@tion ofew dust wirh lakali, pa setic Ecid or H1SO. and s. reulr ih@ 

was morc @llullac producli@ by Ttbhoderw yitide. C.celtutolttiM ti.td.,l 

highe. single c€ll protein, indic.tirS the prefe.enc. of microoreanism for 

substrares. Acid hydrolysis of $€ substnte somelimes results in the production of 

toxic faclors such as furfuBl, hydroxy ncthyl finfinal, rhc prccursoE md ph€notic 

compouds {hich supprcrs the microbial acrivity. The inhibirory eff@r of srch 

compounds could be minimiad by growing the fungi in a mcdium mainrained at 

pH around 6.0 or 7.0 (Chalal, 1984 and Chahal er al. 1989). Low pH (4.0) oflh€ 

medium maintained in th. prgent study could be alributed ro poor biomals yi€ld as 

cotnpded to pr.viou studies. Ricch. .r dl. (t966) rcporr.d y.a$ biomass 

conrlirirg 36% prcrcin on l% rcid or hot hydrclyzed porlrG subskarc f@ent.d 

wilh c'lrltr. simildly Qui€zy er al. (t979) dporr.d hi8h.r singl€ e[ protcio 

production fiom fermerlatio! ofacid k..ted g.oud pear with Crrilir. In borh ih€ 

above ref€red sludies rhe pH was mainllined al 6.0 or abov. which mighl hsd 

am€lionted the ellcr of loxic prcducrs from acid Lertmenr. Vollova ?r ul (t979) 

oblained 6low as 8-9 o/o prolein biomass yieb aner feme arion ofacid lMl.d 

subslrate wirh a y6t Cardtd, ttopicali. ar low (4.6) pH. T his vuialion could b. 

due to low pH snd lhe type ofy€ast used. Pre lrealm€nts of subsrrale can also havc 

a signiiicanl effect on th€ hemicullolose acc€ssibility to enzymic atlck. cupta and 

Datta (199?) achiev€d b€tte. Esulrs rhrouSh prei@tmenr of blsssc with 2% (Vv) 

calciun hydroxid.. SinG no improvem.nt i! prolein yield w6 nored wifi acid 

prctrc.t rot of ricc polishings, this t(c.rmcnr ws trot u.d i. funh.r GxDerimenrs.

3. DetermiDation of Optimum L€yel of Mohsses at 48 Hours 

To .nhan@ rhe growlh of fcrnentltivc orSanism, du ng rhe ta8 

phase of fementltion, molasses wrs add€d a! surce oflolubt€ sugars. lt is a che.p 

source of rcadily availsble €ner$, and ws supplem. al 48rr hour of tie srowrh of 

Atachniotu sp.ciet. Five differe levcls of mdtNs wer. lesr€4 alons wirh a 

conlrol (without mola$es). Table 5 shows thc averag. true prorein vatues oblained 

fiom biomats al v&ious lev€ls of motrss€s. Thc entysis of vdisnc. of rhc dal! 

(Appendix vlll) revslcd a signifi(jnt (p

EO 

lhe biomN containing bighgl Frcenage ofsingt€ elt prorein hs been obt ined 

alier fmenting com staw hydrolyale (Abo-Ahmad, t993) snd suge @e juie 

(H€rca .r dt 1995) yith liLlle prcportio. ottnotajsa added ro rhe Dedium. 

Dovhych (1975) obtained E to I l% incr€se in biomals yietd du€ ro 

addilion of moldss !o gro\4,th nedium but ir sss ar small doses of 0.0 I to 0.05%. 

An increse in the rrue protejn vslue ofbiomNs from j.4r4ro t5.t7% (Bajva d 

al. l99l) dd fro,n 5.9 mrml 10 7.78 mg/ml has be. repon.d in conpeison ro 

$ar or the subsr.al€ (A{iar er at 1995). Maximum yields q.rc obrai.cd ar I ro 

1.5% levels of molasses. Thes. differ€nc.s werc duc ro the variation in rime of 

supplemenradon compd€d ro rhe pres.nt sludy. ln tie above r.fercd studi.s rhc 

supplemenration wa! done und.r oprimum conditos. 

4. Effect of Dilferent Carbon to Nitrogen Ratios 

tn th€ previou r.ial lhe suppty of nitrogen $zs nol €noogh 

cor$sponding to incrcdiDg t€veh of nolasses. 11 was fiercfore decided lo scr 

ca6on lo nfuogen dio fo. minimat level of molasses in order to enhdce the 

biomas p.otcin yield. For lhis purpos€ four wbon ro nuogen mrrcs were lcsred. 

Ttc conrrcl without .xrmd .ddilion of nitrogcn had jnh.Ml cebon lo nitroscn 

ntio of25:1. Because I pcrc.nt notss.s eos supplenedcd |o lhe 8rcwrh medim 

al48$ hoors, sterilized ue was added atong wi0r rhe molasses for mainraining rhc 

Equircd @bon to Dibogen dio. Thc avemgc r.uc prcrein v.lu6 for @ch CrN dc 

show! in T.ble 5. Thc drts on biomas prct.in yietd wcrc subjer.d ro $aiistiel 

analysis (Appadix IX). The rerults reloted significrnt (p

EI 

I1*as obs€Ned tat !t l% levcl of molasss, biomess prorein yield 

increaed from 16.037010 17.E60lo aft€r setting carbon to ritrogen ralio. However 

lhis yield wd not sigiificandy higher than thal of contfol, t7.5% (wilhout 

'nol8cs). This ag.in indicated rhat rhe subsrnre (rie polishirgs) @nlained 

suflicient soluble c€rbohyd6t6 and pmbably suppleme ing $. growth medium 

wilh nolasea, is no( nccded. An incrss€ in prclcin contenr of yeest from 2E.l ro 

51.0% wa5 rcporled by Osman and Voino (1976) when the sugar level was kepr 

constant and CrN ratio decr$sed frcm 17 to 8. Howev€r, lie bionass prorein yi€td 

likqis. dropped frcm 18 to 12 glilrc. Cebon !o nitrog€n ario of tort ws found 

10 b€ the optmum for diffcrent ki.ds of subsrare undq differcnl fcrmentation 

coditions (ldnmenl ?r al., I9?8-a fld Abduttah e, dl 1985)_ the vsriations in 

oprimum ca$on to nirrogen ratios could be due b $e kind of subsr.ares fern€nled 

(Kiani .r a/. 1989 md B6hir. 1990) s the subsrare wa! defarled dce polishjngs in 

fi61case and whcat bran in second casc. Thc oprimum carbon to nikoSen mrio was 

Iotnd to be 12.5:l wirh Can lad dilir. 

Lower crrbon ro nitroscn mri6 12.1, 4.46tt, 7:l and 5:t) werc 

feporrcd lor opimum biomoss protein production (She arr, t9j5 i Crmat et al., 

1985 i Alram, 1992 sd Malmood-ut,Hassd, 1992) whcr€js Hajhmj (1987), 

Hashmi .r d/. (t989), {jhanem et at. (1991) and Athar €/ a/. (t995) reDon€d 

.cspectively higficr C:N dios of tJ:1, 25:1, 2j:l ed l5rl for oDrimum biomass 

protein yield. Thc va.idions in rll€s Etios e.E due 0o eirhcr dilTcr€ncc in kind of 

inicerganism used fo. fenhenraftn or diffcMc. in kind of substtarc femdted. 

These lindings suppon $e fact that diffcr€nr kinds of microorganisms hav. 

direrent requireDents fo. niliogcn for dsiDitaling €$on for prorein synthesh. 

Howev.r rcpod! cisls whcrc different C:N ratio (15:l) for the 3ame or8&nism nd 

$ne subscale (Mrl'm@d d al, 1990),nd @bon 10 nirrogo mrio of25:t fo. 

s4ne o€oism bur fo. differe subsrlle (Bajwa €l dr, 199 | ) werc obseded.

82 

5, Determination of Optimum Time for Addition of lyo 

Molasses 

Atlcr delerminirg rh€ optimal levet of oolases for maximum 

bronrds produclion, a rial w6 conducr€d io d€r€rmine rhe time for addilion of 

Dolasq to Ure termenlation medium, For this purpose ooe pefent motasses eas 

added lo lie m.dium a1 486, 60'h md 72d hours oI fennentlrion. A conrot wiliour 

supplemcntalaon of molasses wd rsed for compsrison. t.h€ fcrnenrarion was 

teminared an.r 96 houls. The avenge biomds prorein vatus obrained ee shoM 

in Table 5. The data w.rc subject.d to slarkricat datysh. The anat).sk ofvadance 

of rhc dard hd be.r shown in Appodix X. t1c rcsuls revcaled significsnt 

(P

83 

periods during fmenlatioo fo! prorein yietd. Such addition rhercfore was not 

adopied in tu(her €xperimetrts. 

6. Deferminalion of Optimum level of Corn Stcep Liquor 

(CSL| at 48 Hours 

Coh sr.ep liquor (csl) is a w6r. !rcducr or com sb&h indNlry ed 

ir.railabh in obundsnr qtrexry ar ch.ap @re. Beides it,, rich in viramms, 

rnDerdls and rilrogcn. An .ficry)r ws m.de to supple,rent rh. fermcniatio, 

Dedfun to deterftirc lhe opftnum levet for hjSher biomass prcreitr yietd. The 

suppreDEDlalton ofstcritizd csl ws done an.r &ljLrslinS ca6on lo nirrcSeo hrio 

at l0:l and Oe harvcsiinS ws donc ar 72 hou6..I.tE avenge true prorein vdws 

obtaincd as elTecied by supplenentalion of diffsent lerek ofCSL arc pr*enred in 

Table 5. The dsta werc subjected to st lhtical afttysts and rhe anatysis of veiarce 

ol rhe data has been shown in App.ndix XL Thc Esrrrs .cvealed significant 

differcnces in rruc p.or€in valu.s ofthc biomass amorg diIarc l€veh ofCSL. A 

gadual decrcasing rend i, biomass protein yietd was oDsefred by incieasing rhe 

levcl ofCSL. Tl€ prorein valuc obtained from conror wd comparabte wjth rhar 

oblained when 0.5% CSL was supplemente4 the difference betwen thc two bcing 

nolsiSnificant. TIe pdein vatue signific€ntty drcpp.d beyond rhis levet dl 1.0% 

CSL. The reujrs indicarcd thar CSL supptenmi€d ro ft€ ferDenrarion medium at 

0.5% l€vel wa3 good for its uritialion for b€rer bjomass yretd. However. the exacl 

ln€ for;tr supplemenhtion was requh€d to b€ del.rmined. 

Berause CSL conhins . numb.r of mincsrs, the quanriry of any of 

lhe mineral, lniehr had bsome hiSh by incre4ing ib levet. fth probabty had 

seprcscd grcwrh of th€ fenenr6rive organi$n and ulrimalety rh. biomass proiein 

yield was lowered d highef tevet. However, its supptementadon at lower l.vel 

proved 8ood. Yogo e/ al (1976) and Suaki er al (1977), aho .eported b.!er 

resulrs of towq l.vel (o.tyd) of suppl.menrafon. CSL n4 becn shown as good 

source of nitogen for supptem€nblion ro thc femc arion mcdium (Care and

84 

Ne€lakantan, 1982 dd El-Masry.r al. 198?). Mrlmood-ul-hasw, (t992) and 

rh.r ., d/., (1995) while oprinizin8 rhc 8r@th condnions or orSoisds found 

tusp.crively, 1.2% od 0,75% of co step liquor 6 opthu for gcrrina 

maximum yield of biomals and bionass protein. Tl€s€ veiarions mighr b€ du. ro 

lhc veiation in typ€ of femenbtive orgrnkn, which mighr had diff@t toleMc. 

for lhe minerals in lho gowth mediutn. CSL has also b€en used rs temcnrarivc 

medium yielding snlficient mounr ofprotein (Foda er a4 1973 ; Shetlar! 1975 and 

Smith €t al. 1975). 

7. Determination of Optimum Time for Addition of0.5% 

Corn Steep Liquor 

A posilive effec! of supplcmentinS CSL to rhe fermenration medium 

was noliced in the previous expcdncnt and 0.5% lev.l of CSL was found as 

oprinum. In the presenr experiment oprimum time for rhe addilion of 0.5y0 CSL 

*s d.r.mined. The b'ontlls wa hd.srcd at 96 hom of f€menhrion. Th€ 

averaSe lrue protein valus of biomas, obtain.d d a 6ull of.ddirion of CSL ar 

diffennt tine irtcflals arc prcs.nt d in Tablc 5. Thc srarisricat mtysis of rh. d{a 

were pdlomed atrd rhe mal)ris of variecc of dara is shoM in Appendix X . The 

results revenled non siSnilicanl difcrcnccs in biomds protein yield anong 

differcnt tin€ interyals i.e., 48, 60, and 72 ho!6 of f€mentaiion. The oprimum 

rim€ fo. the addition of 0j% CSL ro th€ f€m€ntarion nedium was. theretore. 

considered as 4E houB. 

Minor effect was obseryei by supplementing 0.5% com sreep liquor ar 48 hours bur 

$e yield dropped at 60 and 72 hours, ahloud riis change was non significanr. 

Almosl simild lrend was obsdcd in cile of molsses supptem€ntarion, lhat an 

@ly supplemenlalion suppo.!.d $c growlh whilc er(l| lare supplm€norion ir 

Emained uutilized, rh. diff.rcnc€ bcint thrt rhc supplmdtltim of molarscs 

bcyond 4sd hou dDpped prct in yietd siSrili@rty white rh. drcp h prot€in yi€ld 

wirh CSL wd non signili@r.Ahar .r al. ( 1995) fm.nred b€er putp with a y€asr

E5 

Phac lI1. Showib rotrrv lhrker phced h ov€tr 

llqqulqm medi[m

86 

Plrte lV. Showing lrborrtorv scde fermctrtcr (crprciav 5 Lit) in 

gpgllgS!

87 

candido utilis and ewn dqty linc (l6ho!6) 3 opti'num for supplc'ncnt rion 

ol0 75%oo(r st co liduor. 

8. Determination of Optimum Harvesting Time 

In rhe prese cxpsiment an attempt w8s mlde to ass lhc ol,rimum 

h vcstirg li,ne. Tlrc alcrutc lruc protcio values oflhe bioma$ harvcslcd al three 

dii&.€nl ttu€ periods i.e., 72, 84 and 96 hou6 are pr.s.nted in Tabl€ 5. I1t data 

werc stalistically anallz€d and the an.lysis of vari.nc€ of the data h$ be€n shown 

in Appendix XIII. The rcsulls rcvaled non siSnificanl diffd€nce in thc mean rrue 

protein values of lhe biomss harvested .l diflerenl line pe ods The hiehesl 

biomass prctein yield was obsened al 72 houB. Tlh lime period w.s thecfore 

considercd &s the optimlm fo. tungal8rc*1h. 

Similarly hwesdtrg ti'rc fo. mdimun biomas prct.in yi€ld hs 

been reporred fo. olher sp€li€s of'€ast and tunei (Foda.r al., l9?3 ; Reade and 

crcsory, t9?5 ! PciteBen, l9?5 ; volfova er 4l, l9?9 i Gar8 ed N€elaletan, 

l9E2 i Sandhu ud Waraich, 1983 and Abdullah €/ al, 1985). UsinS ,{/oc}'ior"r 

v..ier as f€rmentative organism Mahmood et al (1990) also repod€d 72 hours a5 

rhe op(imum harvesling lime. The period of f€rmentation vades with r€spect to kind 

of org.nism, numbs of organisns, kind of substratc and fmentatiot conditions. 

Le e/ al (19?9) obtaincd maximum c€ll mss yield nner 48 hours ot fementation 

using Crlllonotras (c€llulosic bacteria), whereas YoSo er,/. (1976) and Alb€no 

sd D Soua (19E0) obraincd mdimum biomss yi.ld throu8h fm.trlaion u5in8 

rwo dillcEnt y6t specis. As re8.rds ih. kind ofslbstrtt HM.t al. (1997)wh.n 

fement.d poullry fa€6 wilh . ye$t obtsined m.ximum @ll growth sRer 36 

houB. Rcade md Gresory (1975) forhd 20 hou6 a! n.ximum linc for conv.Fion 

of cassava Mls (a starchy lropical rcot crop) into sinSle ell protcin. The 

synsgis$c etl€ct belween two organisms promote thc Ere of femcntalion ad 

hence rcduce lim€ of maximun yield. Fuska dd Kallarova (1977) obsencd 52

EE 

hom of f.rn ntitioi c 'n dmun for utiliz.lion of lrw d||d lry|}otyzd. usine 

mbed cultulc of lrEor rr.rt 16 ud Ctbb.r.lla rdhrl'ttc !{dirnm hrv.6rine 

lim. havc b..n r€pqbd for.tt|tDl ed dryL..ll Eo!.ln produ.ltd !! 95 ho6 

(sddhu md Jo!N, t 99?), lougb , 60r! !.llul$ic dc. !n.w .! 120 ho!$ (rhlhrni, 

l9E7), ric. ltraw a! 144 hours (Bajwa., at, l99l) lDd rtc. hurk.! 192 ho|ll! 

(Dahot ,r al,. 1997).

89 

Plat€ V. Showins fermcntalion !t its peek

Pl3te Vl Erpe mental pens showihs se arrte rrranqements of 

feedinp. weterine and droooin s collectiotr for e.ch 

experimental unit 

90

L 

2. 

l. 

4. 

5. 

9l 

Overview for A- art ofCha 

'fte defancd rice polishings gave comparabte prorein yield wili undefar€d. 

Ior oprimizing filnler femctrr4ion conditio$ defalred ricc polishinSs was 

Tle pretreahenr of substrate wirh acid did nor cause significanl 

imp.ovemcnr in prot€in yield, so h wd not uscd ir tunh.r rrials. 

The addilion ofmola3s.s at lyo lev.l ar 48d hour of fmenhion did nol 

Sive any signiticanl ifrprovement in protein yietd sd ws nolsupptemenGd 

ro the glowrh medium du.ing n.xt rriats. 

The addilion of com steep ljquof (CSL) ar 0.5% l€vel at 48,i hou of 

fementation also did not give significa improvemenr in prot€in yield. 

Howeler, the improvemenr in this case was bet|er as 6mpe@ ro mojsses 

supplem€nhtion- It wa! fierefore adopred during $e nexl datr. 

Carbon to nit oSen mrio of l0 : I gave significrnt impruvm.n ,n pole tr 

yield and was adopted durin8 rhc next tdals. 

Delaying of hNeslirs ft)e above 72 holrs did not give sjgnitior 

imprcvem.nt in prolein yield. The hadering rime of 72 hou6 was thercforc 

odoprcd becaus€ funhcr delay was unc@nomical.

92 

B. TO OPTIMIZE THE GROWTH CONDITIONS 

FOR Brevibacterium lavzz USING FIRST 

PHASE PRODUCE BIOMASS AS 

SUBSTRATE. 

The biomass prQared thrcu8h femenlation of rice pothhinSs *ilh 

Arachniotu species udet $eviously oprimized condirions was funh.r femenred 

wih A./zy@ with th. t.lset ro achicvc improyed pror.in yietd p.diculdly irs 

enriclmlct with lyside. 

Objectives: 

i) \llh.thct R$@M q g.ow in presence of,tfa.rziona spe.r.r or not for 

funis prciein.trrichmdt. 

ii) To se€ if the addilion of molass€s, glucN or 8un, com sl.cp liquor and 

yeast sludge a1 diflercnl levels and tine intervals ro rhe fermenrarion 

medium de ne.€ssary for improving rte protein biomN yield. 

iii) To eslablish the oplimum harystinS time for mdimum producrion of 

bionrass lrctein. 


The results obtained regarding the optimiafon of conditions for 

fef,ncnraiion ee discusred c under. Thc biomss prorein coorsls obkined lndd 

difiererl conditions acsbown iD Tablc6. 

l. Btevlbqclerium ldtaft Culture Cultivrtion 

Rteyibrctetiu IM is known d a tysin. synrhsizing bacraia dd 

was cultivated on lh€ prc-fennenred m.dium aner adjuring i.s pH ar 7.0. Thc 

fennenlation was cani€d ou1 fo.72 houls. Inirially the f€fln€ntarion was canied out 

lo see if fie lwo organbm! can grow in syne€isn. 11'€ biomass thus obrained war 

hades(.d and analyz€d for ils truc protein clnrenrs (Table 6). Th€ data wer€ 

subjected to slatistiel analysk ed the analysis ofvadance ofthc dara is shoM in 

Appendix XIV. Significant differenc€ (P

t] 

biomass harvested unde. two conditions was obseded. Under unauteldvcd 

coMiriotr r incrcsc in nrolcin *.s 52.28% ov€r the inri.l vatue, but oulmlavins 

read 1o an increase of 7026% in prolein bionN yi€ld. This indicated the 

nnpoftnce ofaul@laving the nedium dudng co-culturc form€nrarion. 

Tl'e rcsulls showed $at th. b.cteria (r.,/rzw) @ nor prcbably 

grcv in symbiosis with the fuga (Arcch"iotw tpecia) s rh@ w.s p@. product 

yicld. Tle po$ible realon for poor yield could be due ro the prcscncc of 

mycoroxins produc€d by th€ fiu8ttt. Mohnood et al. (t990) discuss.d an inhibired 

gtoitrh of B.fM and assisn€d it to toxins. Tle rsults tudd showcd tiar 

autoclaving prcbably detoxified thc toxic irhibnory facloE produc.d by rhe furgi 

(.4tuchniotrs speci6). The findines oiAthar e, al. (1995) and Saima 0996) also 

support the fact l]],at B.ll@M .an not grcw in symbiosis willj yelst of tungi. 

lDprcvcd prcduct yield have.lso becn achiaed thrcugh symbiolic fermenktion of 

dillsot srrais of bactdia (go. l97l i Han, 1975 dd Rod.iqu€z aId Calledo. 

1993). Some wdke6 have rcloncd d improvcmcnt in the yicld rhrough symbioric 

f€rmentation by ccculture cullivalion ofy.asr 0nd tuogi (Vazquez et at., t993 i lin 

.r al, 1994 and lkegami md Yarhiio, 1997). Usins two shins of fungi ed rwo 

slrsins of y€ast in symbiosis, Fuska and Kallafova (1977), and Buaquio ?r a/. 

(1981) got nnproved prodDcl yi€ld lhrough fermentaiion. The possible reason Iit 

symbiosis among two kinds of fun8i of ye{st and between two kinds oi fungi and 

yeast is rhal they do not produce []c bxic compou& hmful fo. th. olhcr or one 

deioxify the loxic ompomds pmduced by thc oth.. 

2. Effect ofAddition of Dilferent kvels ofMolasses. 

For enhmeme of growth of A./ar!r ad tunhq enrichm€nl of protcin, 

lhe srcwth medinm was supplcmcnt€d with various levels of molasses ancr 48 

hours of fermenlalion. Five levels of molasscs were tesl€d along with a conlrol 

(withoul mobsses). The avemge lru€ prolein values obtained in the bionass,

Table 6. MerD t SE Luc protei,r producrion a! atf@red by diller€nr femcn(ation 

Ewibackrirn JlMn culrw clttwrion on 

UMUI*lav.d ,4facr,r',rr sp cuhur€ l7lolo.35b t8.28 

Auroclav.d A@hniotu sp. .ultue 18,6110.11' 70.26 

L€v.ls ofaddirion of nolM l%J 

l8.Jl7!0.4d 67.58 

17.281r0.2f 5E.12 

t0.42010.55' 4.66 

-12.25 

8.750r0,00' -t9.94 

7.9E310..]61 -2696 

Compdden of 20,6 clu@sr nd 2% cuEn 

Ctucoe tE.u' 1l_3t 

16,18?' 48.09 

!.=r-Ir4&+#!"!tu 

70.35 

0.5 ?8 16 

1.0 t9.63t0.2{ 79.89 

L5 t6.223!0.2J" 48.4) 

2.0 1t.81010..t6d 44.41 

4.0 t5.467tO,l{ 41.51 

lr.l0l10.rl 19.88 

8.0 ll.0l010,l6r t9.21 

t0.0 lt 02lio.l6r 0.85 

24 19.86 81.52 

72 

sup.EcriDrr d.norc aisnifi 6nr dnr@-m:ali 

t9.7E 

19.84 

40.91 

8t,70

95 

haN*ted aner 72 houB of ferm€ntlrion for ditfercnt lcvels of nota$$ are shown 

in l-able 6. Tle dara wde malyzd sbtisricauy Md rhe analysh of variMce h6 

becD slown in Appendix Xv_-the.esulrs show.d sigrifiant (p

96 

Sine molasses supplemcnrariotr to rfie Srowrh nedium of A.r4@2, 

showed depressanl effecr duc po$ibty ro high minerol contenrs, the €ffect of 

dificrcnt l€v.ls of purc glucos. supplcmentarion to the growlh medium was 

detonnined on microbial bionass prorein yield. Two t€v€ts ofSllcose wer€ lested 

al lwo different time inedals of rhe growfi of A.,{ara along with control. Th. 

biomass w6 hapested ar 72 hous. Tabte 7 shoss the averase true protein values 

or lhe biomds obrained al diflqe levels of glucosc added ar differe time 

periods. fte analysis ofvariance ofrhe dala is shown in Appendix XVl. The r€suth 

revealed siSnificant (P

O.,4dvrm in srnerghn wirh the yeasr, 

table ?. Mean protein yi€lds ar dillerent 

(HB) 

0 

24 

48 

pt*r ipr ;n corunnr ma rc*s acrcc i6iiiii?iftE-D.5il 

ct 

0 2 

which might have improved fte gtucos€ 

lelels of8lucos€ ar difl€rent time periods. 

18.56 13.597 ltls3 

18.56 18.727 t2.go3 

1E.56 16.073 t0.367 

--t &i6"--C-- r l.IiF- 

4. Comparison between 29lo Glucose and 2yo Gurrh 

cunh is a tocal lweerene. prepff€d frotr, lugar caneJu,cc ar rhe fsrm 

level.Ir conrsins Bood quurity ofsuea ud is avaitable at cheap rales. A trial was 

planned ro subsiirute Z% costty glucose wi$ 2% cheap.r hydrolyzrd gurh. Tabte 6 

shows the avdage true prot€in values of ttc bionss obtahed s eflccrcd bv 

supplemcnrarion or2qo etucosc and 29, cunh. nre tmtysis orvaimce tanmnOix 

XvlI) of $e dat! rev.den si8nific{t (p

98 

molas€s inchding unfernented sueas, .nd mircral siducs 6f s!lr5 add.d during 

rh€ @u6e of f.mdtltion sd somc of dr virsmins. K@ping in vi.w $c 

impodance of y6t extract s s conmon ingrediert of differeni gro*1h mcdia for 

.nhancing lhe grcwth ot dir@dr org&isms! it wB deidcd 10 use y41 rludge as . 

supprcDrnr to rhe g.owth 'nedin6 ot B.Iow. The avqage rtue pdein vatu€s of 

rl'c bioNass hlrvcscd s i hcnced by supplcnpnrarion ofdillcrcnt lcvcts ofye.sl 

sludge ar€ lresenled in l able 6, and Ihe ltatistical ealysis of Lhe dara is Drcsentcd 

in Appendir XVIU. The rcsults..vealed signifi@1 diffcrcnc€s among truc proi.it 

values ofthe biomas obtained as a eult of supplmentllion ofdirercnt levels of 

yc.sl sludgc. ll ws obscned tlut rhc supplemcntador ol yed( sludSe lplo L0% 

Ievel rcsulkd nt muitnum incr€3le in bioma$ pfot€in yield. Tlis incr€ale was 

sigoincan y hi8her at 0.50lo and 1.0% lev.ls d comparcd with the contrcl and orher 

lcvels, wher€ as non significint diffss@ re fosd b.lw6 truc protcin !?lu€s 

obtained at 0.5 and Lo%o levels. 

Ycasl cell mals or y4t €xlract has ben .€poned to enhance the 

nulriliv€ value of lhe growth medium (Yogo e, a/., 1976 i El-Sabaeny and Hdsan, 

1996 md t{ .r a/.. 1997). In all rh€se c!s6 ll|c }61 

extract was supplem€nted ro 

the medium within rhe rang€ of0.l to 1.0 %. Simild findinss werc observed in the 

present study wilh sludge, which contained sufficicnt Inin€lal conl€nts slong with 

the y€ast bionass. A neSalive eff€cl was observ€d when sludge was used above I 0/o 

in presenr study. This @uld b€ duc lo thc pso@ of high minqal conrcnis in rhc 

sludee. Addilion of yoa3t sludgc at 1.0% level gave the maximum biomass proGin 

yield and wa! $crefore adopted. 

6. Determination of Ilirv€sting Time for 8.jfI4r4m 

For se.ting maximum dd €conomical yield of biomass prctein $€ 

femotllion was cuied od follosing all the dclennined groMh conditioG on 

fonnulated medium. The fermentation was l€rminated at $ree titne Deriods. Th.

99 

averae. true protein valu4 of the biomas hsd.sled at thesc rime periods de 

Fsenr.d in Tablc 6. The odysis of aridcc of &ra (App.ndix XIX) rcvated 

non siSrili@l diffcr€nce bctwa rhe tue prolein values of biomass han€sred ar 

24. 48 and 72 houls. This indicalcd rhat mdimum fernentarion r@k plac. llpro 24 

houF rlg which il rehained in statidary phe till 72 hou6. 

Tle incubalion period oflhc o.ganism deDcnds on i1s kiid. Bacl€ria 

ulually rqke short p..iod for mnplelior of rheh fem. ltion_ The incubaliot 

period fou d ollimum for fermentdion of ala",l (bacreria) in pEseni study was 

24 hours. Molina rl a/. (1984) and Elsabaeny and H6se (1996) also reponed 24 

hom incubation p.riod d th. m.ximum for producrion of fcrmeotlrion producrs 

for diffcrenl shains of bacleri! in the shak€ @ndition. Using same organism as in 

case ofMolina.r di. (1984), Enriqu€z and Rodriqucz (1983) however, obsd€d 48 

hoG Lmentation as optimum fd maximllln yield of bactc.ial biona$ of atkati 

l.eated bag6s. with C.//!/ohonar lP, This vaiation .d be aitibutcd to rhc kind 

of $bsll:te ferm.nted. The rBulls iodicaled 24 hours itrcubario pdiod d rh. 

optinum and was lhercfore us.d in funh€r studies.

t00 

Plate VII Showinp rrrsnaenent msde in the pens for 

droppids collection below the net

0l 

Growth Kinetics 

For l}te explan.liotr of trend of g.oMh and cfficiency of subsrrare 

ulilialion for the synti€sis of pmlcin by borh thc organisDs, the crc{1h Kinetics 

was worked out. Vdjo$s pa.an€tcs w.rc catcularcd for both Aractuiots ipecies 

^nd Btivebacteriutu favun, sepaatety. Th€ vatues for Crowlh Kineiics are 

presenled in Table E. The rfend ofgrowlh durir8 borh the phases of femenrarion is 

tresen&d in Fig-l ed rh. hends ol subsiral€ urilialion, clll mss fomation ed 

product (T.P) fomarion arc presented in lig,2. 

The Srars of product formarion p€r gram of substrate utitized fo. 

Iilrgi is usually repoaed as 0.5 + 0.05 (Ri€che.r at. t966., peireBen. 1975., 

lAwtod et al. t979 al.d Arhar et at. t99j). A $imitar vatue was obtained in lhe 

pGent study. Yo8o .r dl (1976) however, showed dir€rent varue b€cause of rhe 

E6on that rhe substtnre rhey w.d corraircd t€ss q@rily of cdbon requicd for 

Sp@ific 8rcr'lh 6rc (!) obrlined for )trrchniots spcci* (0.035 h-1.) 

h rhe p.$cnt srody us in line wift rhe nndings of pmoenr., at. (197E_a) qho 

Eported ! as 0.0l2ohr duing slurry srlr€ femcnlation. The specific ercsdr mre 

for any fementation p.ocess depcnds lpon $e kind of substrat€ femenred. This 

varue is reponed b be hiS}d (Suaki .,,l t97?) in cas. of easity fem€nrabte 

subsrmie Gt@'8ht chaiD hydrc.5rbont. Thc sp€cific growth n1e also depends upon 

the state of fermentation. Pammcnt er.r1. (1978) r€porred lower values for p (0.01h. 

I and 0.0092h') because $ey us€d sotid stale of flenerralion. Tle findinss of 

Alhar et al (1995) ,re not in line wilh $e pr€s€nl srudy although they used shske 

process of fmentation. 

The rate of prcducr fomation (yrJ for bacr..ia is cpo.red 10 be 

hisher (0.60 + 0.05). This val!€ for pr$cnt sludy wa! 0.614 ggr. which is close ro 

the value reporled by Atha., al (1995). Thc prcdrcr (T.p) c@flidor (y*)

t02 

obseoed durios ile present work (0.432 gg') is also in line wirh rhe r$ult! of 

Vanvinit .r at ( 1996) who rcpod€d ypt vetuc as 0.4g&r. 

'l-abl. 

E. Vldous Crcwrh Kinclics psnmcte.s of fenft {ive orSanishs 

Kind ofMic@llmism 

a 

a, 

a, 

0.5 

0.25 

0.5 

0.0175 gproducug ell/h 0.0098 g producrg etuh 

0.07 g c.lug subottltdh 

0.275 guh 

0.t175 glltl 

0.0681g,{Jh 

0.614 

0.432 

0.7033 

0.4228 g.ell/A 

0.115 SlLlh 

0.1l5 gilrth 

0.08 Snth

I 

; 

60 

50 

40 

30 

20 

10 

0 

3 

2 

1 

0 

-1 

t0.] 

fls-1: Tmd or corrh Dudn! Borh ph-n .t 

Fmmn on 

Fis-2: Shdhc t $d ot sut k . uthnbn,c.I mu. 

fiqn.lon .nd pFdsl (T.p) rom.lon 

50 100 150 

+Sub gI 

+Ce .M g/ 

T.P gn

l 

2. 

3, 

Overview for B-oart ofChapter-4 

'I}te se@d orseism (r.,fzv@) imprcvcd tte protein conr@ls ot lh€ biomss 

significmtfy when it wd grcwo in absen@ ot Arutniot8 spe.ies_ tn rtg 

subsequent tials the inoculation of rhe sccond qguim in rhe fe@ellation 

m€dimwd done an.. autoclaving. 

Supplemenlation of growlh D.dium with motarss reduced lhe protein conlenrs 

or the bioI]rN at all $. leveh, mohsses ws the.etore nor used dudng n€xl 

lriah. OD the otler hand rhe glucos€ supplemented ro th€ groMh medium 

significblly €nhmced $e protein conrenrs, whereas gunh depressed il. So 2% 

glucosesupplemented at24'"hourof $e grcwfr ofBl@u,ws aoopreo. 

The y6t sludge supplcmerted !t l0lo levct sigrifi@nrly impioved rhe proGin 

conlents ofthe biorn !s, so it \ra adopted. 

The delayinS of fednentatiotr lbove 24 hou6 did nol imprcve (he prctein 

@ntenlJ, e it '6 adopled d msximum rime for fmolari on of B.fow.

105 

C. NUTRITIONAL EVALUATION OF BIOMASS 

TIIROUGH AMINO ACID COMPOSITION 

AND CHICK trSSAY 

'the qualily of aay fememcd prcduct is c$aycd by rwo merhods. one 

is chemielanalysis dd olhcr is biologial elalualion in oy biotogiclsynem. t.he 

chetrial mlysis leth orty aboul rhe pisence of nulrienls ad oth€r non 

rurrirional factos present in the product but ir does nol rell about drc actual 

utilization of nutricn$ and effet of anlinulritioDal factors preserl in rhe producr_ 

Tlte bio,nass pretar€d lhrough ${o nep f€menlalion during pres€nt sludy was 

evaluated wilh rhe following objecrives. 

Objectives: 

t. 

2. 

'lb asess lhe chemicat scorc and essentat odno acid index of rhe produced 

To ds€s,rhe prcLeh qutrry oi rhr biomass rhrough a brotogr{al rriat In 

:lii,1 .*"q rcadm8- rhcr w.isl| cain, f.cd consumprion, fced 

converson ratro, protrin emc€ncy ratrodd nerprol€rn DlrtialioD. 


The resulrs obrained reg&ding (he n .itionat cvaluarion of 

biomN ee discussed as undei The &rino acid prcfite atrd anino acid indq 

shown in l able 9 and biological €vatuarion of the bioos is showtr i, Table I0. 

l. Amino Acid Composition 

Tle amino acid composirion of ric! poljshinss and har of biomas-A 

obrained thougl ferinenrarion of rice polishings wifr the froaus (ArochniotE 

rp?.ier) dd biomssA obbined through femenraiion of bjomds.A with the 

bactei^(Ilreeibacteriwfovr,r) is show, in Tabte 9. 

There was a resonable incrcase in amino acid contents of rice 

polishings aner femenr.fion wirh fung$ and bacreria. A pan,cular increase w6

t06 

seen lb| lysinc, lir€onine, arginine,leucine, valin€. phenylalanine, slycinc, alalinc, 

alpallic acid and Sluiamic acid, at firsr ncp of fenrentation white an incredc was 

scen tor lysinc, leucirc. valino, pheaylaleine, atanire, dparlic acid and gluramic 

acid ul seco.d step of fement rion. tnoease in tysin€ @nlenrs was of panicular 

interest because higb lysine biorrms w6 inrended tor replacine fish m@tin poutlry 

rations. In ricc polishiDgs the tysine increded from inherenr 0.68 ro l.j8 ttre 

ircrease being more fts doubte the qudrity in lhe finafiy had.sled biomass. 

llotlever. the lysine content of fisb meat war 3.30g/l6gN b€ing 2.5 rime! mofe 

lhan the biomas. Arhar ?r a/. (1995) obtained 129% lysine in yeast bioous snd ir 

was r.oE% in finally haw.sted biomass with BrcvibKteriunl,t vrn al second slep. 

'llie direrence my b. audbuted ro b€ller adapbion of B/e vibdctetid lldun on 

b€er pulp subsrnte a! comparcd ro ricc polishings used in oursrudy. Tte 1o|atmino 

acid contenk as percentage oflolat prolein for biomss,A and bron6$B were 7l 

to 74 percenr, respectiv.ty. .mis h a gmd indication of rh€ bjomas protcjn qualiry 

as rhese had low NPN conrenrs. 

To dcremine tte qualiry of biomass prcr.m ne contenB of ils 

essentiat amino acids weE compa€d with lhal of Egg p.otcin and FAO Reference 

ProLciD. I|E chemi@t scorc wcre also calcuhted for Dromass_A and biomass-B. 

Esseorial anino acid indices (EMI) of bionass_A dd biomass-B we.e atso 

Otr rte basis of chemical scorer, ir was observed $ar melhionine wa! 

lhe fi.$ limiti.g mino acid bolh for biomass_A and biomass_B. fiis is one ofrhe 

soeral lirniador of most of rle fernenred biohass p.oreiB rhar $ey arc {teficie 

in sulpher cotrrdninS amino acids parriculely mcrhionirc (Ca oway amt Kudur, 

1969iH et.t.,197) i Sinsh., ar, l97B ad Deghir and Sell. l9S2). However 

Alhd dr a!. (1995) repofted tsolcucine s fte f6r tih(ng .mDo acid in bioDass 

prolein. The data on chemial scorc turtier show€d lsoleucine s rhe secodd

lnniring diDo acid es wa rcported by Et-Ashwah e, at ( 1980) snd Rabccha e/ at 

(1982). Lysinews thc third liniritrg anino acid borh for bioh.ss-A bd biom.ss_B 

(Ilabecha e, at t9E2). 

11 may be seen that the quali9 of p.orein funher 

secood phdc of f.mcnbtior wirh ep€cr ro @nGms of csorial 

is indicaled by the higher vatue ofEAA| ofbiom6s$8. 

Tablc 9. Amino acid compGirion (c/t6cNJ and Chm,@l Score. 

lishinss, bioma$ A and bi 

0,31 

0.13 

0.68 

0.5? 

0.42 

0.45 

1.08 

r.06 

-24.291 

+t2.t4 

B 

0.45 

0.40 

l,l8 

|.20 

-4t_J1t 

+2t.14 

1.0? 

LU 

0.16 

0_39 

0.61 

0,80 

0.67 

1.59 

1.30 

t.l5 

0.60 

l.l.l 

t.33 

Ll5 

+ t0.62 

5.24 

+19.64 

1.54 

L:! 

1,50 

q$ 

t.24 

L1! 

+16.E7 

+t _42 

+25.71 

|.t1 

0.1t I.l4 t.2l 

0.89 1.56 

!12 

r.l5 2.25 

2.49 

t.9l L19 J.46 

0.51 0.97 -lt.4l: l.l0 -25,48. 

t).68 ,21.08 22.a3 

t2.95 29.78 30.97 

97.92 't0.19 

13.72 

rro 

-cr,..i.ar sc*: r*."o*cao--r,iiiiiii'r6iiirr,c -fiGiiiB 

l:1.:1f l-'j:^!tq Tq og'icurure oB;i'dion 0.5312 0 1468 

or d. u red Ndion; 

RequiEftnts. FAo Ndiomt Srdi.s. No. t6. p.j2r. 

s=SaondLimrtine, T=ThirdLinirins. 

lsMs o, hrno.cid, Gr 100 m ctrd. 

Lr!n. (.20).Th6tuG(2.E0). v.tincr4loiMdl,;;;Al6r, 

rsreu( in? I 1.20,, r.uc,n.!L!0Lt!4!rhE4iqLLE0t

2. Biological Evaluation 

108 

'lltc biorrass produced on pilol scatc was biolosicaty evaluared usina 

.lrichs.-lhc resutls obtairred durins rhc rriat are given herc under. Thc aveE8e 

values on vaious pamnerels used fo. biotogiql evalualion ofbiomas are gjven in 

ldble 10. 

2.1 Feed Consumption 

Tltc dara on reed consumption of fie bids on experimenral .ations 

$cre subjccted to statistical anatysh. l.tre analysh ofvariarc€ or.rhe dala is slo\u 

i Appcndix XX. Non{ignificanr dirTerences were obs€rved in feed consuDntion 

li) lhe chick led difcrent arions_ lecd consumplioD values ranged f.om 322 

-the cellular prctein (biomass) gave cooparabt€ feed consumption by 

lhe chicks as Nirh conrot, as the ditTerences in fecd consumprion vatues were non 

siSDificnl. Thk is an iodiqrion of good quatily of tte bjomds. Foundom s/ al 

(1983) olso found nor sisnilirant differcnce for feed consumprion by rhe chicks 

al$ough they rycre fed ye.sr biomass upro 20"/o in fte mr|on wnerc as in the pres€nl 

Nork rl'c bionrass was fcd to a maximun of tO% in ue ratron. Almosr simila. 

rcsults rvcrc obtained by petukhova ?r d/. (1985). Tr,ey rcptaced upro l0% protein 

olthe rario,r \vith lhe nricnlbiatproleii. 

2.2 Weight Cain 

lhc dala on rveiSht ganr (Tablc t0) of lhe birds fed diflerenr 

cNperiDental rarions were subjecred to sratislical anotysis ,nd the analysis ol 

v.flanceofllE d.r! is shown in Appendix XxI. AppaMlty lhe highd weight gain 

\vas aMincd bt the birds tedRation-E, in which fish mealwas coDptetety replaced 

try lhe biomas. lle Neight gains EnSed frcn 22E gEms ro 27t gms .nd rhe 

dill-ererces a,ron8 rations were non si8nificanl.

t09 

Similar ro fed consumpriotr the weidr gaio of $c chicks did nor 

direr significanrly due lo nlions. Thh is anorhe. indication of good qlality of 

nridobial biomass prepared during rhe erpedmcnt. Crawford and Eliab.th (t973) 

also could not fild ey significdt diffcrcnce berw..n wcjghi gain ofbaby chicks 

f.d biona$ proei! and corrrcl. This was prcbabty du. to rh. facr fi.r lh. 

dclicidcy of Lysine od Merhionin. wss met thrcugh soppl.n@lalion ofsynrhclic 

amino acids. Daghir and Sell (1982) alro foud non significant difer€nce in wcigll 

gain oI broiler chicks fed di€ls, cont.ining soya protein or yealt pror.in 

supllemsled wi{r deficienr amino acids. Similar rsults were rcpoded by Foundra 

d 4r (19E3), viktorcv "r al. (198?) and vulcrev e, aa (1996). However, poor 

weighl gair was shown by rhe expsim.rtat .nimats wirh $e inc6e in fie levels 

ol-micrcbial b'omass in the mrions (Citter er o/., 1958 ; cunz€l dd Vogr. 1982 ; 

Surdzltiiska ?r al., I 984 ; Cha,hal, 1989 and Alhd .l 41. 1995). This could b€ du€ to 

the deficied.y ofsonc €sntialmino acids i! rhe microbial biomN. 

2.3 Feed Conversiotr Rstio (FCR) 

The data on F€€d Conv.$ion Rado (FCR) wer€ subiecl.d to 

sl.lislicsl malysis ed the analysis of variance ws shown in Appendix xxtt. The 

r€sults showed significant (P

ll0 

quality offungal biomas wa5 rorally ameliorated by meerinS the dcficiency ofi|3 

cssenrial .mino acids. Sioilar findinSs wcE also rcpod€d by Daghir and Sell. 

(1982) dd Chavezer at. (t988lI). 

One fact is noticeabte in prcsenr work is rhar the trend of decrease in 

FCR of th€ qperi'nenr.t otions wd nor gndult by insqing thc bio@s t.vet in 

ihc ration *hich mighl b. due 10 sode.xpdmenbt crc., 

2.4 Net Protein Utilization (NpU) 

Tte darr on Ner protein Ulitialion values for differe0t expedmenral 

ralions w€rq subjccled ro stathricdl anatysis and anatysis of variance k show, in 

Appendix XXll. Thc rcsulrs showed siSnitimr (p

2.5 Protein Elliciency Ratio (PER) 

l 

The dai. on PrcieiD Elficiocy Ratios (pER)of diff€Ml expe.i,nerlal 

ralioDs were subject€d to sr.risrical matysis ed the s.lysis ofvariance afthe dara 

c shown in Appendix XXIV. Tte rcsulrs show€d signiti@r (p

2 

lu m be said rhar rhe biomN p.epdrcd dudng rhe pnsenr srudy 

allhngh \ens not parlcl in quolily wnl fish m€al, hs vatue was quil€ saristacroiy 

\rl'cnaddcd h driols upro t2%. 

Tablc l0 Avemge vatue! ofreed Consumprion, weigh cnir. F€€d Conrerio,l 

Kotro. Net t.rolejn ulitizluon and prolein EIkie,rc' Ralo ot ltre 

experDenral mrrons, 

ltercnick(s) 

chick(s) 

&rio (fccdlsain) 

\4jo 

lMarion (%) 

(8ain /rrotein) 

B 

(25o/r 

n(hi *p.M"prs o"ftml;;;;;;;;6fi;ii,n-c* 

c 

(50yo 

D 

(75o/, 

E 

(to0% 

32t.66 403.22 361.77 180.00 194_22 

242_44 261,66 22E.77 27 t.t l 

l,J2r 1.58' 

63_42' 56.14! 49.61d 47.5I', 

-lE 

5t.12& 

3.37', 2.69' 2.91J

I tl 

l'hte Vlll Showi'rg cxperincnral ctrici{s ki €d rnd ooered for 

dryins in oven on 10" dry ofcxperim.nt

Overview for C- art ofCha 

L Thei€ wa m inclle in rhe qurndty ofdl lfic mino acids duinS lhe 

l14 

phase of fermenration. However the increase in lysine wm nore 

compmd lo olher .mino scids during second phsc. 

l Methioaine, lsol€ucine and Lysine were found firsr, second 5nd rbird 

2. 

3. 

4. 

5. 

6. 

limiting amino acids Br'ectively in fte rdulring biomass. 

The esential amino acid index of lJ)e resulling biomars wa! tbund 10 be 

0.5466 

Nor sisnilicdt dittd€nces jn fe€d consumption and weighl gain of $e 

chicks fed different expe.imentat |aliom were noriced. 

R rion A hrving fish meal only t|ad hiahest FcR_vatuc .s @mpaEd to atl 

Simildly the ner prolei! ulitialion (NpU) value of nlion_A having fish 

meal only ws siSnific&lty hiSher 6 @mpeed 10 all other ations. 

The sMe tr.nd was found for prclein cfficiency rario (pER) as fo. FC& 

and NPu-vslue. Here as.in rarion-A h.d highest pror.in eficicncy nlio al 

compred toall orhe.nrions. 

fi6t

||5 

SUMMARY 

Rice polishings is a by-product of ricc industry ed is availabl€ in 

lbuDdant qu.ntity in Palhlan. Althoueh it is quit€ ich in encrgy bur its high fiber 

cuncDls limirs irs usc i! pooltry raions.It m bc lscd in poulrry drions at 5lo 8% 

level. lhe surplu quan y oftbis agriculLure byproducl is wasrcd.s tUet lbr brick 

nakjng. It wa so pldned lo upg8de its nulritional valuc $.oud fermentalion 

with ref.rcnce lo its prcl€in conrents and also d.gEde ils {ib€r conretrls to make ir 

more digeslible. The ain of the sludy under report w6 to use rice polishings as 

subslrate for the prodrclion of mic.obial biomass protein rh.ouSh fennentalion firsl 

wilh,4ruchhiot6 specie6 (ftngus) ed rhen wi$ Bfeviba.r"/ iun lawn (ba.tedaJ. 

Il was also envisiged $al lhe biomass eilt be evatuacd tmugh 

chemical ana!/s'! as well as $rough chick assay ro know lhe nurritionat porentiah. 

The pmtein yield was enhanc.d thiouSh stuEing rhc eff@l of defauins and acid 

reamenr of ii. subshte eitt. Arachriotw speci.s. Supplcmentation of growth 

Dedium wilh molass€s ud @m st6p liquor was also anenpted. Optimum carbon 

lo niliogen mtio and hdestinS rime wcre €srablished_ For funher enhanc.ment of 

prolein esp€cially with respect 10 lysine cont€nls, ll)e growlh condilions for 

B-JlavM ||rc opnn.irld. Eff@ts of supplemahlion of molass6, Btucosdgunh 

and y6t sludge werc delermined and rhe optimum hsesting tine for bacrerial 

tbmentadon was esiablhl.d. followinS all rhc oplimiz€d condiliom biomN was 

prcparcd at ldge scale. Th. bioh6 so prcpared was subjecred lo chmic.l analysis 

and biological trial broilerchick. 

'n 

Thc rcsrlb of rhe tnng.l fcmsralion shoeed |har def.(ing of ric. 

polishings Bavo compalable biomais prctein yield wi$ undefa$€d one and was 

adopred becaue ot behg cheap. Supple'nenralior of mdr$s lo lhe growth 

nedium duing fisr phe of fcmenralion result.d in depNd grofih above t%

level. The preiein yietd ai t% tevel of supptemohtion had .on sigrificanr 

difercncc wirh conlrot (wirhotri motasss). .Il,e besr carbotr to nitrcgen ralio 

esrablished was I I I 0. foflowin8 this .d(io when I % molasse wa! supplem€nled ro 

the 8rowrh mdlium ar direcnr rime inleNars,46,r hour supDtemcnhrion gave 

ldcir yicld having nor sianincatrl dircmccc wjrh €ontrol (wnhoul ,notasscr. 

llislEr lcvch stDecd siarificlDtly dcpesed prcGin bjooas yietd. Corn stcep 

lr{tuor ai 0j% levcl supptcmenhdotr b rhe grcu,th oedium at 48ft hour proved th€ 

bcst ofatt th. tevels rcsred, howeve. irs addirion ar 60d or 72d hod6 did nor give 

funh.. inprovenor in biomrs yietd. Unde. lhe oprmEed condirions when 

IaFcsrinS lime wa derermined, ?2 hou6 of fcrmenhtion gave marimum pro&in 

Yield of l1 .3lo/r. 

An inocularion $i$ &.erl.ra. teriM ltMn wa, Eiy.n io th. stetitized 

nLdtua ot,lra.hhiot6 ,tEcier. Durin8 ba.lqiat fcmenrauon supplcmenrarion of 

molasss ro lhe medium dcprcssed the gro*ln ar alt the tevels .vm al t% t€vet. 

Stpplemcnta on of ghce a12% Ievel ro rhe grcwr medilm a124o ho!. g.v. 

besr resulrs as omparcd lo 4% tevct and ofter rime periods ofaddilion. tlow.v.r 

the diffemcs wefe non signifierl when compared wih rh. contot. 

Suppledenrarion of gwh lo fte mcdium at 2% Ievct dcpqsed ft.8roqh while 

yeasl sludge supptencDblion al l% lcvel improved rhe prcGin yjcld significan y 

(19.66370) s comped to @nkot (18.62%) md orhq levels. 1.he besl hNsting 

lime for femenbton witl A,{rvrn war found ro be 24 nouls. 

Tle specitic growrh 6le during fi|sr phsse was 0_0j5h_r whci@ for 

sccord pl6e it w$ 0.0t4h.r. C@n of ell bN produccd rE.8rm pr subnEte 

ulilizld wd 0_5 ed 0.6t4 fo. fi6r ed for second phase of fermcntarion 

respcclvery. Specitic substrate uprake mre was 0.07 g/&/h for frtst aN O.O22S S/gh 

for second phase of femenrarion_ The gran of producr (r.p) fomed pcr gram cetl

|7 

Dds lbdned was 0.5 ed 0.7013 for tirst ed s€cond phasc of fenn€nrarion 

The chemicar anarysis ot prolein biomrss show€d rhar during 

lc(ncntation sufli.icht iNrcas in.nrino acid contenG t@! ptae,-t.he tysinc 

coDrcnrs ilcru.s.d tior 0.68% jr {N subsL.utc ro t.08% trfic. lirsr phasc and lo 

l.lE% ali€r secod phEs€ of leme alion. Merhionine, tsotacucine and Llsinc 

weR lbund ro be fiE! second.nd $id li|niting anino acidr, resp€crivety in Umlty 

rc$vcred biom.ss when compared wirh rhe Egg prorein add FAO Refer€nce 

Dudng biotoSical iiat non sigrifiqnl diflerocs in fccd 

connnnption and weiSht giin among diffcr.nt expe.imenr.Nt dtions wde obseped. 

Significant differcnces werc found aDong FCR values, I .32 (.onkot) !s I .54, L5E, 

l.4J r'nd 1.45 fo. iarions , B, C, D and E respecrively. The nel prct€itr urilization 

v0luL oI ration B (56.14) ws significanrly poo. lhan mlion-A (63.42) but rh. 

diltfenc.s weE nor siSnifica amonS .alions C and E (49_6t md 5l_12). 

SiErificant difTereDc€s sec found amorgprcteh emciency raliosofa the nrions, 

r tktrlA laving thc lighcsr (3.3?). Ralions D, D, E and C lad pEtt 3.04,2.98, 2.93 

€nd 2.69, respecrively.

|lE 

ACHIEVEMENTS 

Dudng th. p'!gt strdy rh€ following achievcmcnts werc nad.. 

L The ricc polhhings was enriched with rcspet of irs protein contenrs. Tte fioalty 

prcparcd bioeas had 37% proleiD as compse ro rhe subst ale which had t2% 

2. Tlc lysine conient of .i@ polishings also inFoved frcm 0.59dlo (subsrrarc) to 

L38 % biomass 0uough two steps fermcntation. 

3. The aim !o uie by-products ofother industries i.e., com steep liquor and yeasl 

sludS€ duing lhe proccss of femmration was also lchiev€d. 

4. Tle prolein biomass proved good protcin supplene although some whar poor 

as compared to fish,r€al. 

RECOMENDATIONS 

l. h is rccotunended th8t l)sine conrenB of biomAs should funher bc incrded 

by us. of mutdlstmitrof Af etibaderimJl@u. 

2. Il is also recomnended liat the methionine deficiency in fedenred biomas, 

should ale be imprcved trouSh furthn lmcntation with mebionine 

3. l1 is suS8esl€d thar lh€ polenrial ofteast sludge a! an additive 1o lhe medium for 

the produclion of biom.ss protein and iE other us may be cxploited. 

4. The €{.nomic fssibility for @ycling ofa8ric$lbrc waste thrcush largc scale 

prcducliotr of bionass and its us€ in lhe broiler chioks (p€rformance rdal) 

should be allempled.

lt9 

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rrcm peat extracts. Biolechnot.and BioenS.2lU). 75_|l90. 

Rabcchr,M., C-Billoud. dd J.Adrid. t982. Food efficiency of tu Asperigiu8 

,,A./.uxurc. [STA. l4(3): cl5l. 

Read.3.L . 

K F.Crc8roy. t975. HiSh rcmpemlurc p.oductron ot Drckin 

and 

cmfteq le.d ^ rrcm casslva by fun8i. Applied Mi!rcbioto$/. 1016) : 8o?_ 

Riehe,A" W.Nordhcim, A.Martini. and c.Mull... 1966. Tobl proc.ssins of 

porarocs infr'odkrio of h,gh quatir, ked s\h Cahdtda uilis. Z:*h; 

neminelt.2t : 144_151.(Nuu. eU". *a n*.

.l2 

Richter,C., H.Bcntz, H.Richler. and A.Heming. 19E5. A multigenemrion rdal wirh 

he.s Siven Fe'nosin i! feed- L.ying pdiod. Arch'v fur Tiq.oak tu8. 

36(6): 391-399.(Pouk.Abst. l2(9) :2093). 

Rodriquez,H. ed P-Callddo. 1993. Siogle @ll prot€in prcduction fom bag8c 

pith by o mix.d bacreri.l culrlre. Acia Biolechnol. ll(2) : l4l-149. 

(chen.Absr. 120(?) : 132415r. 

Roshliova,Z. 1979. Dynamics ofyc.st pro!,agation and sludy of biomasca donnS 

ycst cultivalion on aelltc md n.thdol. Vkusovs Prdrut., Plovdiv. 26(l) 

: 20?-216. (chem.Abst.95(10) : l5709ld). 

Saima. 1996. Bi@onv.$ion of uh@1 bru 1o biomass prcr,ein, its biologicsl 

evaluation 6d dctoxiltcation of aflatoxb in brcilq chicls. M.Sc. 

Thesis.Univ€Bitl of Ae.icultnre. f.isalabad, Pakistan. 

Sd(hu,D.K. od V.KJoshi. 1997. Solid state fennenbrion of apple pomace for 

concomitant production of €tDnol ud animal feed. J.Sci.lnd.Res. 56C) : 

86-90. (Chem-Abst. 126(23) | 3049429). 

S.Ddhu,D.K. and M.K.Wraich. 1983. Conve6ion of ches€ wh.y lo singte c.lt 

protein. Biorechnol.Bioeng. 25( l -3) : ?97-806. 

Sdr'Ann4E.S. 1985. Cahdida ilis 8:.o*n in sp€nt suttile liqror. Lxperim.nt in 

fen€nto!. Rcvisra-dcMicrcbioloSia. 16(4) | 305-t0E. (CAB ; 7L 

Biodelcrioration Abst. 003-03215). 

Santos.J. dd C.Gomez. 19E3. Fungal p.olein produced on cNava for srowing rats 

dd pi8r. J.A!in.sci. 56(2) : 264-2?0. 

Sdf.az,A.R. 1990. Biomass productjon from wer feed by,{ractniots species and 

its biological evaluation in broiler chicks. M.Sc. Ihesis. Univelsirv of 

Asdcullurc, Faisalabad, Pakisrd. 

Sdkar,C. dd K.A.Pmbhu. 1982. Urilizalion of bagasse by bacteria. 

,.Fement.T@hnol. 60(4) ; 297-303. 

Shacklady,c.A. and G.Catunel. 1972. The nutritional value of yelst Srown or 

alktua. In ProEin fiom Hydroc!.boi!" €d. counellc de pontanet,H., p 2?. 

Academic Pr*s, New York. 

Sh€lla(r.A. 1975. A nodel study otr prcrcin Aom com wstc efituents. H.Veenme 

nnd Zon.r B.V, Wageningc, p I I

ShojaoBdati.S-A. .nd M.N.chazlj. 1996. Single @ll prorein (SCP) prcduciio, 

from mellleol urilizinS bacreria in l.R.lmn. Amirkabn. 8(30) : ll-4t. 

(Chem.Absl. 126{13) : 170464s). 

Sibbald.l.R. 1979. A bi():Ny of available mino acids and rrue melabotizrbte 

ener$/ in feedirg_sluffs. poull.sci. 5Ed-3) : 66E_673. 

Sin8h/A., c.S.Dhillon, Lp.cupla 

lroduction from spenr gnin 

(Biol.Abst. 68(10) | 60t01). 

ed M.S.Kalm. 197E. Singtc cell prorein 

wale. Indian J.Erp.Biot. 16(12) : l]17-1316. 

Sitarm,N., A-A.M.Konhi, B.R.ceethad.vi. ed T.N_panschddra R.o_ t9?8. 

lTlerloD or funeal prorein frcm @[utos'c plani materials. rrdia; 

LMicrobiol. l8(2) :90-92. (Biol.Absr. 67(8) :47a0s). 

shirh,R.H., R.Palmei ed A.E.Rsde. 1975. A chemi@l dd bioto8icat sssmcnt 

o! ,,lsperyillw oryze atd orh€r filmentous fuigi a5 p.ole-in sources for 

simple stomached eimals. J.Sci.Fd. and A8ri. 26 : ?85_7a5. 

Srcenarb,H.K., P.c.Cmdalt. Brkq ard A.Roben. 1995. 

produ(b ed ea

134 

Tani8uchi,M., T.Tohma, T.Itaya. ,Id M.F!jii. l991_b. Ethanol productio, from ! 

mdl@ o! al_u6. .nd ryt6. by cccutL@ ot pr.r@ rrprir dd a 

rcrpinlory defi(ienr mutur ot Su&orony.es .er.visiae. J.t.crm.tli6nB. 

El(4) :164170 

Tena,N.N. 1976. whey 6! a subsra!. for edibtc y6t. Retrisr. d. Fsmacia e 

trroqum'ca de UnivcBidade d. Sao pauto. t4{1,:55.0E. (NukAbsr and 

Rev. S€.is.A- 4E(4) : 25ll). 

Vtudini!-S, P.Srilhongkum, C.De-Ehankul, A.Assvrnig. 6nd K.Charcensiri. 

1996. Prcdncron of singte cc prorein fiom cdsala sLarch in 0r tin 

t.frent

135 

V"[OS$ !?3.. Pldir dt br of nrtc !d.ro 1! i.rE $h co-cllh.. of 

eryotyuc tuui Dy totid rt i. ftn&otirion. Btobclbot.Adv, ll(3) : {95_ 

s05. (Cb.o.And.l2o(l l) : l90l6tc). 

Yogo,rr" Y ir*, cd r,f,Tluh lf6. yd c.[r. JF-Kdd 7t 03, j80, 

_ l3pp. (Clbcd. Abr. 8E2l) : l506l9q). 

Z!fir,SI, TXtllS... ed pfl.sbd_ l9Sl. Blod.g.ddin ofc. o.. c@ohl 

d rb ed b! phrors ,qjo, "e,. F"u. r6,.bt. t(jj , tg+:ii__

t!5 

APPENDD(CES 

Ar?6dir-r: c|*".|l .o@o.td..r oa t!. ,|t.tg u 

&8Dauioo 

&n..Erhc 

N.F.E 

Sodim 

CbloiE 

dui.t |[. F!... of 

IUc. Folnils Ccre.Gg Sdlhrcshd!. 

liql[(.sd 

10.0 

13.t2 

t0.q| 

9.0 

ll33 

t0.0 

56J5 

0.05 

44! 

0.02 

0.ll 

17.13 

5.0 

3.0 

t2.o 

830 

0.E 

oo 

al.0 

ot5 

1t3l 

50.0 

23r 

10.0 

c,.0 

t7n 

ol 

0.4 

73.0 

29.06 

t7.91 

t.(tr 

29.0 

4.U 

3.05 

t.075 

I,IE 

t.E2

NuEicn 

crudc Fibr. 

EIbE;d 

N.F.E 

C.lolun 

XNACcht|' 

Sodi!!r 

Chhh. 

137 

12.00 

37.14 

2523 

10.00 

lt.dl 

2lt7 

0t0 

lt)9 

2JE 

0t47 

ot'7

Ili!tidi!. 

m.tjrldoi!. 

Olt to 

l3t 

0I2 

oa5 

o.n 

lI|8 

t.6 

1.59 

09 

l.l0 

l-t5 

0.50 

l.tl 

lJ3 

t.l5 

t.t4 

1.56 

225 

!.t9 

Idl (vl6d,llt{r) 21.0E 

1.95 

0.45 

O,l0 

ot5 

138 

IA 

r.54 

l.l0 

l.9l 

150 

0J9 

t.u 

1.40 

r-t7 

t2l 

t.72 

249 

3.rt5 

nt3 

L1l 

Totd 23,t2 2523

oil 

!5.00 

":oo 

lz0o 

3300 

2200 

2500 

$m 

139 

2.46 

7,O3 0.20 

lt.9l 0,t5 

- 35,00 

0.25 

o.N 

0J0 

0.20 

,.., 

F..d Sbtr Cdn ocrid T|bt., FlbtEh.d b, Do.rl@l 

^grirurift, Prisbh.! 

F@d stoth, M.y t4, t990 (t!b!.lt,c.H,, t990) 

: 

2.@ 

t,t5 

'lt 

0.t8 

0.65 

0.55 

1.78 

t,36 

":' 

0.02 005 

0.05 0.22 

0-3! I.3l 

'l' ol'

Biolrn 

CholiE Chlod.lc 

Zirc 

Burylared hydbxy lolue@ 

t40 

ComDosition of vitrDir lld niBil 

t2,000,000 tu 

2,000,000 tu 

40,000 ng 

1,500 ma 

6,000 m8 

5,000 n8 

10,000 hg 

30,000 ng 

1,000 n8 

5,000 nS 

1,0{D ng 

60,000 n8 

30,000 nt 

1,000 b8 

3,000 ng 

35,000 m8 

125,000 rns

A!?.dir-vl ADlti. of n l@.fin turtla bftreofdqffiBof de 

loli*ilgoU6-FLh',L& 

sdBd D.sFof Edo( lL- F.t*. l|!h. 

ntidid t!..16 .it!t!. rFuu. 

Ine.d I 0.006 0M 0.0lt 

Brr!. .( 1359 O?lO 

. No dgi6.d 

l{l

t12 

4fqdir-vlt Ar.h6i. ofnd|F ofdar &dvbr i!6rrc ofdi !..tu ofdc. 

ffi 

lmit* ft.dod .qrr! ql!! 

I 0lt0t o.flI 0txlr. 

. 1.615 0.121 

. Nc.tr-6fd

. sbdfqd (Pd.ot) 

IrlS 

Ald)rir of v.ri.aa ofd.L &o$i!S .&d o{ddl&a ofdft..ot 

l4! or m|.!sr 00 btd|u orD!.io vt td. 

Hotr gL=-rcE-EC 

?3t76 

\n1 

11tr95 

0.1/||

t4 

AEadr{C rrbi.otwlo.oad.lb*|brLddUdpb d 

dlli|tlr...nd b ftrr.! rda 

8o@of D.g6of lrbof M.r!squ{! LV.Io. Pl!b. 

a..iabn n!.dom r[e 

Trffi :1 lo6.t{4 265t6 t'a3/4. orE} 

Etu l0 2:05 UA 

. sir'ifolt d (P4.ol)

t45 

AFadix-X: Ad'tndv'.ic.ofd-dwirrF!&dk dbir-Fdr,i.ld_ 

_ __ @g| bl-ddidqof t%mtlsdt/ddd4.Ei.d!' ' 

sdrc ot DryBof srnor--8ry!--Tfi; 

I sisalfc&t .r (P{.01) 

l5.l7t) 

l.l3a 

5tFr 

0.t42

la6 

ApFdk-)c: Adylit of Eira of d- o t ! .&.r of v.doa bvd! of c6rD *.p 

LIc.@ld.d..e htEdrn+u -'_-sow.or 

D,.scaa .'nA---6;i;;---FEE 

. 8i6nor d (P

147 

Alp.ldix-Xll ADdrrir of!{drn . of&. &r! rtorbs .ft.. ofrlpC.a.!ri!8 0J% 

0.05 

ora 

00166 

0.1(5 

ott!.

' 

No! Si!!if@t 

ta8 

Arlrdr dwiE of tb dd d ru F.dr ridd oftt bdns 

tfqi.d .r dlftEt tn 

D.4Fof Suof [r..!t4rnE lrr.rr 

ftdd 

2 001 o.Q5 

6 

o52 

0,0c66

App6dix-XIV: Aidyrk ofwidc. oflh. dsr. showin8 cfer of EuioMtving rh. 

- - goN|1hot_Ar@t-jota lxcE onkEorotliLlrqBot B.furw. 

rr.Ec or s@ot I'iatr{uE F,VdE preb 

. Sis,ifiturar C

Ito 

A{Fdr.Xv: Arbi ofwlc of ddn qi!r.&ca ofqddim ofdieGd 

lsvd! ofml.g d rU holn ofrh! 

D.tF.of Suof Mo.Sr! F.V.tr 

.5 

t2 

. sitlincd d (P{.or) 

3mJt5 

1.500 

6tf57 

ol25 

/19!rt8l. 0.0(x)

ADFft-XU: A!.Bd. ortrlF turhr.&.r ofddt of dm.t. bra& of 

-'='=,,=" SE9dftP9"-t9* =,., 

So||Eof D{reot 8uoa r,rdiri- nEG 

o|e(o) 2 

HorB(H) 2 

OiE a 

Ellor 18 

.8itiif..!a-(FO.ol) 

lD.AB 

2053l 

t 2r3 

LE50 

t5l 

r0(949 

t0Jti6 

7gt 

ol03 

r@0.9065' 

9W. 

7tIr3l. 

0.0m 

0.000 

o.qp

I52 

Ap@in-Xvtr: Ardrd. o€vdoa ofih. drr lhorrll8.&.a of2tt crd d tb 

&reof D.rrEd sDof Mo.C'i Fi-E 

Frllr 

. Sigrn .n d(P{.01) 

9577 

o27 

9.8n 

oI(n 

o000?

Apt odit-xvle Arttn of whE ofrbdr-oriry6dofyd ds. 

. 8i8!i6.dd(F4.0t) 

2z,:tt 

0r39 

153 

n.w 

oM7

154 

ApFodix.XX Andyi! ofEiec. of&ir rlDwbg il8ue4 ofLlv€dle de! od llle 

vr&tfub!&}tu't'tuu. 

So!rc.of D.gFof &nof lr,Lrqe! l. VdE 

ff!.d6or 

Tdtrat 2 0Ol o.qE 11625. 

E|lor 5 0.06 0.m8 

. Nm dgnfcd

I5J 

Ap'codtx.lo( A.d}n ofyrirE ofdd oo S.d.oarE0dd 

Dcs.c of Sleof Maisrc F. vde 

. Non Sirdnc.nt 

l0 

xyBrfi7 

zltw.67 

6'A1L7A 

zt?a.81 

lJt9. OIN13

. Ncsitdficd 

3t3n257 

t(,!,567 

156 

ua.8l 

rtrgt.67

t57 

Ar!.odir-)Ofil ofrrttt4. oftb ddr o! &.d odnd6 rdio 

thof Ma!!qa! l-V.lG 

1 

to 

. siiEcrlt .t C{.01) 

0355 

0.(xll 

0.61 

(Iu) 

t209.900. 0.0(I)

Etrtr 

. sig.itrqd- (p-ii) 

t2,;t919 

139.0t0 

t5t 

323230 

r3.901

. 3i9i6cd1(P4.0t) 

z 

0.ml 

159 

0.631 

olm

160 

Analytical Methods 

l. Proximate Antlysis 

AO C (1990) nethods. 

1.1 Moisture 

Th. prorimate nnalysis of subsrrate ud biomsss wa! done fo owins 

For noisture esrimalion sampts w.€ w.iShed .rd rhcn dricd 

oveD al ?0"C dll consbnl wcishr. Weisht of dried samptes ws rccoded and 

the direrencc in initial dd final weighl lhe moisrurc conrcnrs we.c esrrmared 

following fomula 

Moisturc'/o = 

1.2 Ether Extract (E.E) 

Inidal Wei !:Einal wei 

Inilial WeiShl 

!x 100 

A known weiShi of $e s.nple es d(en in fiimbl. ard then 

cxhcled wirh dierhyl elher in Soxnlefs appaBtus. Aft€r 5_6 exrmc ons the clh€r 

ext"ct wd tat.n in p€rri dishes rhen pul in oven for drying fi conjtant wei8ht ar 

?0"C t€mperanrr.. The p€rccnr .tft.r exrract ws calcutared by using following 

1.3 Crude Fibr€ (C.F) 

Eth.r Exkact o/o 

A klo*n quartiry ofdried and far tile sarnpl. wss rakell snd refluxed 

li6l *ith L25 per@n( sutfuric acid fo. half sn hour, &ercalt€r it was fijrecd md 

$orouslly warhed wili disritt€d wale. and rhcn rcnuxed again now wnh 1.25 

percot sodium hydroxid€ for hatfan hour. .nE suspcnsio, was fitrercd and Siven 

lhorcu8h washing wirb dki €d water. The r$idue was dncd in oven ar 70.C 

lenpedtu.e rill coNtant weight Tte weighr of dried r€sidue was rcco.ded and rhcn 

using 

Weilht of Etb Extracl X 100

I6l 

isnir.d in lhc numc tumac€ a( 550 oC. Thc wciShr of s5h wa! rcc4rdcd and from 

lh. difrcrenc€ in wcidt thc crudc fibrc pcrenrlg. ws @tcutd€d 

= Wci.ht ofR.sidu. - r .iqht oftuh X t00 

wcidtofseple 

1.4 Crude Protein (C.P) 

Thc crud. prorein was dcr.mind following Kjctdlht.s m€dnd. A 

lnow|l qu.entily of srinplc rrs digestcd wilh conclnltGd sun ric acid in rl,. 

pier€ncc ofdigestion mixrutr m aining KrSO{ CuSOa and F.SOi innrioof100: 

l0 : 5. The digcsrcd sampl. yes diht€d upro 250rnl votumc wirh disri cd *iter. 

Tcn ml of thh digc,sr.d s$pte plus l0 ml of 40% N.OH was transtdr€d b 

MicroKj.ldhat dislillalion applrrtus. Thc mi)(furc was distill.d wili srcEm and rhc 

disrilhlc wa! collccrcd in a conicrl flrsk (l00ml) having l0 ml of 2yo boric acid 

solution and mcrhyl nd indic{or. Thc conr.r s of r}e ll.sk lr€r. titrar.d aSainsr 

standafd N/10 HISO. ro lighr pir* color md poinr. The oirogcn and ,rorcin 

conlcors w.Ic catculdcd using lhe fo owins formula. 

1.5 tuh 

Nitrog€n % = 

Protcin pcrc.naaSe = 

vol. ofN/l0 

Vol. of digcsld sffnplc 

lrl(.n for disriuarioo 

% NitroSq X 6.25 

Wcielt ofsanplc 

X talen 

A rno$n quantity ofsrhptc was r.sten in cruciblc,nd rlcn igniGd on 

oxidizing flsm. of bumcr, till tro srnok. was Sivcn our rhcn ir wrs ignir.d in a 

Mumc tumac. at 600oC rcmpcErurc rill .ll rh. o.glnic mrng wa! oxidiz.d. Thc 

weight ofash w8s rccordd and rh.n ash per@nl.a8c w!! cltcutarcd uling fotto\|linS

1.6 Nitrogen Fr€c Extr.ct (NFE) 

162 

ueigllelAL X 100 

NFE wa olculatd by ditT.Ene usins $c followirg fomulr 

NFEo?6 r 100-(voC.P+o?6E.E+96c.F+o/6Ash) 

2. SolubleCarbohydrates 

The solublc erbohydrat€s io lhc biolt'ris filtra(c $.!5 estinrr.d by d|c 

m€thod of Dubois er al (1955).'ne @nseurivc votllmcs of0.l p€rccnr gtucos. 

solution w.E trlt|sferrcd ro rhrc. rcat rub6 (I!btc-l l), wh@!s rhc 4rh rcsr rubc 

e.v!d a bldk. All rh. rdr tubc qs trclr.d with lmt @h of 0.25N H2SO. plB 

lml of 80% aquous phenol. Finally 5ml c-onccntrared H2SO. yas rdd.d ro .lch Gsr 

tubc and mix€d. 'nlc ab6orb6rcc of orangc y.llow colour devclopcd, was mcjsurd 

on spc.lropfioaom€lcr at 490 nm. A gnph wss conslrucr.d by ploning rhe 

rbsobanc! lgainir conc€ntalion of gtuc{sc, Fig-j. 

TrblFl l: Estimnior ofSolubl. Cartlotvdtlre 

0. l',t Gluco6. 

0.25%H,SO. 

0.E5%Phcnol 

Distill€d V.tcr 

conc. H?SO. 

0.1 

t.0 

t.0 

0.9 

5.0 

Dilutions (mL) 

3 4(Bla ) 

o.z 

t.0 

I.0 

0.6 

5.0 

Onc nL cich ofbiona$ fitrllrc (€st nlltcrial) wrs diturcd ro t00 mL 

usinS dislillcd wrt€r Ocn L0 mI ofthe ditu. dnwn was ttercd wirh rhc r.o8.nrs 

.s u!€d in thc clse of sbndlrd cud.. Thcn f.om tlc aranddd curv. rt. ab6o6cncc 

of thc r.action mixrurc wa! r6d .nd cx&.polar.d fo. warcr sotubtc clrbohy.tBr.s. 

0.3 

1.0 

t.0 

0.7 

5.0 

t.0 

1.0 

t.0 

5.0

3, Eslidlr|iotr ofcrrbotr 

l6! 

'nc flto co6 ns! .titn t d by colqin ric n rtod d..c.ibcd 

R (h@. (lgaE). o.tn sE of d.6rd, drt d od gqod (40 m.rt) ric. 

poftbils! w.t rat d wtr 8dl of .'c..ljd ruttuic rcld (969i p,r!) |!d 5nL 

of 2N K,Ci:O, h ! lm rL b.&.r, A b|er wl! Fw,t ir tb lmc yry. Atl 

&. r|lDpls w.rc tld h 0! ov.a fo. G .bd r hrlf off,oor .. I l(rc Il. ,upL. 

w!.! coolcd nd dild.d !o ii06t ,i6 irdiflld r,a.r. Ih. or6c.l dolig w!| 

Ir .lulld od lpcctophotmdd st 590 on rtc. drutioS z€rc .ltu(tac. *i6 

bh.l" Thc d.ab.occ \r|' th.a ctu Ild et6lb tubd fivc FEIttd fdm 

gluco!., Fig.l.

154 

Ff3: St.||iLrd Cu .ftrEffii of 

Sof$b C.ftolqtrh.. 

I o.e 

t- 

EEo.z 

E$ o.' 

I or5 

! E ot 

f fi o.os 

! 

0 0.'l 0,2 0.3 0.,t 0.5 

coft.|' dc! of O|Uooa (mg) 

Flg{: SfrndrdcurcJ 1 

E|{m.lbn of Cartorl 

0510 15 

Co.|of|ffio.r otof.a (|rtl

165 

4. Estimation ofTrue Protein (T.P) 

'l-he ruc protein $G €srimlred brough (Munrc and flcck, t966) 

mclhod. One gm of dri€d and finely groDrd sampte war treared with jomt of 5% 

T.ichloroac€tic acid (TCA), mixed welt and lhen hqted h eare. ba$ at 90.C for 

20 minutq. Smple was shaken occasionally during healing. Alter heal tr€ar,renr 

the mixtre was filrercd rhnu8h filr* paper and Siven 3_4 wdhio8s with disriled 

water. The reidue along -ith filter papq ws dig$red in coicenlr.ied H:SO4 along 

wilh digestiod mixrur€ ofKrSO., CUSO..5H1O al|d FeSOa in rhc rario of 100 : l0 : 

5 (Hiller sr 4/. 1948). A bldk ws atso run. AS.r dig.stion n was dilurcd nDlo 

250m1 volumc $ith dislilled water. Ten mj of diSesred ssmple plus l0mt of40% 

NaOH @ raken in rcceivs o I micr oKj€ldalt.s disnltarion appdatus. The mixrurc 

was ($'ned and the disrillat. wa, collected in a b&k€r (100 ml) having lomt 2% 

boric acid solution along wilb methyt red s indicalor. The conl€nrs of6. flask 

were tirated against slsnded N/10 HrSOr tilt lighr pink color end Doinr. The rrue 

proten {"s etimated using lbc fomrla, 

vol. ofN/10 

T.P % = x 0.0014 x 

vol. ofdisesred sample 

tlken for dhtillddon X sample 

The rqulranr fiace ws Bultipli.d wilh 6.2j fo. conv.rrirS rhe 

nilrogen inio protein. The sme above procedur. was ako fo ow€d for th. bl@k 

dd iis @ding wa subhcled frcm rh. smplc @ding for rhe qtim.rion of % rrue 

5. Amino Acid Analysis 

The amino acid contenis of the bionass was d€r€rmined by lhe 

merhod of Moo.e and Srein (1954) usi,8 auroneric amino acid analyzer. .nte 

sample containing l.5t0.l mg of protein w$ w.igh€d inlo l6xl50 mm pyrex resl 

tube md 0.5m1of Mg.nt gBdc con@nkared HCt *rs added follow.d by 0.5mt of

distill.d wlter. Tle conlcnts wc frozen with dry ice dd tet tube w6 evacuated 

with ihe helt ofa slclion punp. Thc tube wd Bealed with pin poinl oxy8en suigas 

flame and placed in uprigil posilion in an ovcn reeulaled ar I looc for 22 houc. 

'fhe hydrolyate so folred ws evapoEred unde. vaculn 10 d.ynes ro reDove 

Ilcl. TIE d.ied nln of hydrolrate was susF ded witb 2rnl buifer (rrH 2.2). Tle 

solublc maicrial was ccnhiluged to get rid of visiblc sedihen$ sd samplc wd 

loaded on ion exchdS€ column of aubmatic miro acid analy&r for g€ring 

compl.le profile of mino acids lr€sent ir the smple. Standard solulion of rhino 

acids qaalsorun in lhe aminoacid analyzer under simila. conditions. 

6, Ribos€ Nucleic Acid (RNA) Estimation 

Tlt€ co.centralion of RNA5 in th€ fermenr€d biomass was €stim.led 

by rh€ method ofCerioui(1955) as Dodifi€d by Munro and Fleck (1966). For fiis 

purpose a stand{d cupe Nas conlrrucred by rakin8 different votumes ofsranddd 

dbose solution (0.01% in concentrared HCt) in six rest tubes. whereas the 7th lube 

ser.d as blank (Table- l2). '[re direrenr dbose corccntarions as well as th. blank 

werc lrealed *ith lhe rcagenls as illustmcd in rhe rabtc. 

TIle test rubes shaken rhorouShty were immersed nr boiling watef bath 

for 40 minutes and tlen cool€d undef tap water. Tte cotou| was rhen exracled {i1h 

5mL anyl alcohal by mixing md c€nlifu8ing ar 4000 rpm for five minutes. Tle 

extracted blue colour€d solution was @nlrituged again ed iB optiql densily was 

Md on spectrophoromeler ar 675 nn againsl rhe blank. The stmdard cune so 

prepared is presenr€d in Fi8,5. 

finely ground and seiv€d lhroud 40 mesh sMpl€ of fermenred 

biomais (0.1 g) was exkacred rwic€ with chillcd absolure alcrhol (5ml each iime) 

lhen centrifuged for five minules al 4000 rpm. The residue was once exracred wilh 

chilled l0 percent Ticholoro-ac.ric acid (TCA) by cenkifugation and rhe 

supemalrnl wd discard€d. l.lle r€sidue was rhen fina y treal€d with 5 peoent TCA

t67 

(5 mL) m'C for 15 ninurq and caLifuged. Supem.rarl (0.05 mL) w keed 

in th€ smc mamd s d$ib.d for sr.rndad @re fo. rhe $rim.tion of ribos.. 

RNA concnts of tlE sdpl. {.s qtcutrt d by Ntriplyin8 rib$. concoralion 

witb 3.77 (C..iotti,1955). 

Table-l2: Calibratioo ofst ndard cud. for RibGe eslination 

T$l Tub. Volumcs (mL) 

|23456. 

Sr,ndard 0.01% 0.r 0.t 0J O4 -j--- 0j , 

Ribs€ Solurion 

conc.HCl 

(sp.sr. I.l9) 

2.4 2.3 2.2 2.t 2.0 Lg 2.' 

OrcimlR.ag.nt. 2.5 2.5 2,5 2.5 2.5 2.5 2.5 

2oo Ds or oEin.t Bdis@i. 

g'enhr.d,H€t Gp4r. t.t9) E !dd.d !o il ed tn. rotuo. eB m.d. upro loo;i *i.n

6E 

7. Estim.tion ofCalcium and Phosphorus 

These estimations were nad€ followinS the Anonymous (1954) 

herhods. 0.58 of smples wde wct disest.d in l00ml- conic.l oasks. To esch flask 

w.s add.d l0 mL conc.nrracd Nnri. Acid. SmDte w.rc het€d n tow 

lc,rp.ralnrc @1il solid !{licls had nedly di!!pp.{.d. Then 5hL of p.rchloric 

Acid (72%) ws add.n in esch fl.sk. It {6 fiNt hsred g.nrty rhd aigorouly mril 

2-l ml clear solution was obtljned. Th. di8.sred samptes sere transfen€d ro 

l00ml volM.lric fllsk! and volumes werc madc uplo mark using disrilled war.. 

7.1 &timrtion of Cslcium 

Five nillilirer digesred sotution was taken in china dish. To it 20mL 

of dktilled water was addci alone wi0r l0 drops of 4N NaOH and 50 m8 

moniun puryu.alc (0.5g ammoniuo puQrnrc plB l00g K6O.). h ws rirated 

lsainst0.0lN Erhyl€n.-Diamine-Tera Ac.rare (EDTA) solulion 10 prptecolorend 

poinl. P.l@ll8e of calciM (Ca") in rhc sapl. wd @lcut0red frcm the volume 

of 0.0 IN EDTA ued asainsl 5 t L of solulion. From the volum.s ot th€ soturions 

used, nodaliry of th. sample soltrion wirh rcspecr to Ca| wa catculat..t ed 

therefrom % age ofca-' in th. sdpl€ was calculated. 

7.2 Estim6lion of Phosphorus 

For phosphods estimation fi61 of all srock solulion ws pr.parcd 

sing 0-3519 dried Mono Pot.ssium Phosphab. Ir ws dissolved in 200mL of 

dislilled wacr atrd resfmed quanribnvcly to a l000ml volumelric fl6k. l0 mL 

of loN HrSO. was added Dd volumc wa! madc upto rhc mdk wirh dhrilled water 

and mix€d. The solution contaircd 0.4m9 phosphoru per 5mL. A stand{d cuwe 

va prcped uiing diffffit dilurions (0.25 tol.sml)- To cach dilurion lml 

AmbDium Molyb&te, 0,5u!L Amino N.lhthol Sdphonic Acid soturios werc

169 

added and lhe volurnes werc mad€ upro lomL with distill.d w.rer (Tabl.-33). 

Opricaldensily ofthese solulionswea Deasured at 700nm on speckophotometer. 

Tle tesr tubes werc shaken thoroughly and allow€d lo sland for 5 

minutes. Optical density was lhen m€asured on spe€lrohpotometer al ?00nm 

wavelengih aner sefling zdo .bsorbaDc€ with blank. 

Table l 3: &!!83!!e!_etlbe!phea! 

Tsr Tubcs Volum€s (mL) 

t234561 

0.15 % KHTPO. 0_25 0.50 0.7s r.00 r.25 l_50 

Amnonium Molybdal€r 1.00 1.00 1.00 1.00 1.00 1.00 1.00 

12.s%) 

Aminonephthol 0.50 0.50 0.50 0.50 0.50 0.50 0.50 

Distiued Water 8.25 8-00 7.75 ?.50 7.25 7.00 8.50 

r Dksol\rd 25cin 200 tr,Ldisrilled warq in lL vollmelric fl.sk, plaoed,l00mL loN H,SO., 

,dd.d ro molyfi.t elution.nd dilur.d ro |L silh dhtill.d s.t.r 

.r l,ls.d l9tnn of 15% sodiun bisulphit€ solution in a slass sloppecd cylindq !dd.

t-,/rrr',r47c

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