Mold as an Adjunct to Malt in Grain Fermentation I r
N. nl. ERB AW I;. hl. HILDEBRANDT . S. Industrial Chernirrils. l r i t - . . B n l t i m o r e , \ I d .
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I his paper describes the direc,t u-t. of
in)celiunl of amylolytic rtlold for saccharifjing starch in the produc*tion of eth?l alrohol from granular wheat flour mashes. Pure culture technique w a s employed in growing the mold i n the distillery. The niediiim conEisted of grain stillage, nutrient salt., a trace of metallic aluminum. and some grain mash. When a solume of mold mycelium equitalent to 6-12% of the fermenter \olurne was added to the mash, i t was possible to eliminate four fifths of the malt pretiouslj used and thuq tn obtain a fipnificant inI'i-e as an aid t o or a substitute for malt. This has bcen piopo-Ctl by a number of investigators. I n most cases the uv' of moltl\ aran is contemplated. Takamine, one of the first t o develop tlic mmmercial use of mold amylase in the United States, took out riumerous patents on amylase production from mold (13, I,$), including some on processes for fermentation alcohol i study ot these and other proposed processes shoas thnt the quantity ( i f diastatic material needed for a n industrial alcohol plant, 1% heit amylase-containing material (such as mold bran) is used a5 a malt Jiibstitute, calls for a manufacturing operation of considerahl+* size and complexity t o supply enough enzyme for the convcrsiori process. If it were possible to provide mold amylase as a c~ompletc~ malt substitute or as a n adjunct t o small amounts of malt nitliout this added manufacturing process, the use of mold nould tie more readily undertaken in distilleries. Underkofler and co-Forkers published a number of papera 011 rlie use of mold amylase as a saccharifying agent for starch) grain mashes (3, 8, 15-19). They conclude that Aspergzliui oryzae grown on bran under suitable conditions is superior t o malt amylase for use in the conversion step in the production of almhol from grain. Underkofler (16) and Christensen ( 4 ) patented methods and apparatus for the production of mold bran. R o b wtq (12) and Hao and .Jump (8) likewiiv report favorahl? OIL fungal amylase. A M Y L 0 PROCESS
this procedure, established in France in 1895, mold :iniyl;ihi. for all the malt in t h grniii fvrmrritntion. Thf. molt1 I S ~ N J W I I submerged in the grain mash itwli n i t h high aeratioii i l l the fermentation vessel; a t first sight t h i h procedurv would i(v'ni mtiPlac,tory for the use of mold amylase iir plncix of m:ili. \(,111
I S substituted
792
1 ~ a l l y ,t tie prwess has had limited applicability because ab Wig1riail.v proposed, it requires rigorous pure culture conditions. The method was developed by Calmetter who isolated from ('hinese yeast (a crude mixture of many typm of molds and other organimid several species of mold which had good starch convcrting Iihility. _. l h e mold finally sc.ltacd as the best :starch converter $\:is one ( 1 1 t l i t b Rhizopus group. Spores of this organkin developed on 1:1~01ccdrice were used t u inoculate starch mashes previously soluliilized by cooking with a small amount of mineral acid, prefr~i~:tblyhydrochloric. I.-iider these conditions the Rhizopu,s r;ipitlly converts the jtarch t u sugar and, more slo~vly,t o alcohol. FIIIplnctical use the alcoholic fermentation should be carried out lly :I ywcial strain uf yeast. 'The process has been utilized suci ~ d u l l yiri the, Cermentnticin of rice, corn, manioc, durra, millet,, :!rid pot:ltoes. Ikwriptionr of tlit. :mylo niet hod are giveii Iry Grow ( 7 ) , Poth iG), Boulard (21,Dc4cmiir ( 5 ) ,and Owen (11). It is evident that a pure culture. prowdure is a principal requirement for the wccessful utilizat,ion (IC the amylo process. Deviation from itrictly pure culture prowdures is >aid t o tie permissible in the fSou1:irtl modification of the process, in that open ftlrmeniation 01s have been used. Sterilization of the grain, howevcr, is recjuirr,tl, and yields are said to he lower in this process. S e u t)auvr ( l O j and Boidin ( I ) discuss relative costs of producing alcohol by t h r malt arid ltniylo processes. The necessity of pure i.ulture procedure, sprcial equipment, laboratories, and trained laboratory and plant personnel has doubtless stood in the way of general adoption of the classical amylo process. Thcrefore, the produotion and use of mold amylase for the average alrohol plant -itill rt,mains R desirable goal for fermentation prndurtinn of grain :i lrnhol,
PRELI\IINARY fi:XPEKIClE'rTS
_I hi$ .
paper dearrikien experimental run6 in xvhich ;i nii)difit.~I aniylo process was used in large scale opera1 ion. The runs \vert. rriadr without establishing pure culture conditions in the alcohol plant. The necessary mold n'a.5 grown on a ,solution congisting iri part oi liiatillery slop; this procdure decreased production eo-1 111 1 o represent a s t i l i ' of the mold. These experimental run3 t o w x i l c rlir practical use of mold amylase. A consideratilc~ :imoutit of preliminrtry laboratory and pilot plant work, arid ic~nielarge' swlc f(lr.mc,ntati o i i * , p r e r w l t 4 thr. plant riins dwrrilicvl in thi-. p a p w
August,
1946
INDUSTRIAL AND ENGINEERING CHEMISTRY
793
mold growth K-ithout further treattileiit; it has not beexi powible to give a reason for this behavior. T h e metal was first thought to counteract some toxicity due t o t,raccs of othcr metals picked up during mashing or distilling; but' this is not, the compkte explanation, since stillage produced in glass is similarly toxic for the mold until treated with aluminum. On the basis of obstwations of t,he effect of the metal, a small amount of aluminum powder was used in the mold-growing solution.. I n tjhe first experiments, undiluted stillage t o which aluminum powder had been added proved adequate as a growing medium. However, since the stillage contained antiseptics used in the mash, it also had to be diluted and supplemented wit'h a small amount of granular flour mash to which no antiseptics had been added. A solution of this make-up, strrilized and cooled, gave an abundant growth of mold. FERMESTATIOS STAGES.The actual operation in the plaiit involved growing mold through three stages. The first was a laboratory stage; the next two were in plant vessels of 75- and 2500-gallon working capacity, respectively. The mold from the last vessel was added with 49% of yeast' to the infusion mash of granular wheat flour in 20,000-gallon fermenters. JL~BORATORYSTAGE. Spores of Rhizopus boulard or Rhizopus delemar were developed on a pad of granular flour paste in a 100-cc. Erlenmeyer flask (25 grams flour, 500 cc. water, 0.1 gram ammonium sulfate), sterilized one hour a t 250' F. Thew flasks CURRENT EXPERIMENTS u-ere inoculated from malt agar slants and allowed t o inculnte 1 hiring t,he recent war the Government desired to use surplus until the mold was \vel1 sporulated. The spores from one pad nxw .?heat for alcohol production; consequently, efforts were again suwended in 3000 cc,. of sterile riatrr. This was the inocvilum made by this company t o utilize mold amylase. Use of this mold for the first plant stage. cvaa desirable because malt is high-priced and scarce, and also FIRSTPLANT STAGE.This was carried out in a jacketed, rtc1.1 i~ccausrhigher alcohol yidds are expected from a conversion using pressure vessel, designed for pure culture opcration and of 75both malt and mold am e rather than malt alone, The probgallon working capacity. The mash was made up as follows: lem \vas to fit the nioltl procedure into the current distillery operaground malt, 20 pounds; zinc sulfate, 5 grams; phosphoric arid, 'ion without modifying equipment or using specially trained per100 cc.; ammonium sulfate, 200 grams; water 1,o make 75 g d l o n ~ . .;onnel. The plant selectrti for trial of this process was operating The mash was st,erilized one hour a t 2#50"F., and its pH w:I~- 3.8 , + I I an infusion method of mashing granular xheat flour and was t o 4.0. rising antiseptics to inhihit tmctcrial infection. The cooked mash vias cooled t o 90' F . :]rid inoculated n.ittl To make certain that the mold process \vas adaptahle to \\-heat 3000 cc. of mold spore suspension. Pure culture procedurcs \vcre i'ermentations, it was shown f i h t in the laboratory that this grain followed in preparation and inoculation of this stage. 'I'ht, ~ ~ o u lhe t l fermented with amylo mold and yeast. .4 fevi pilot plant inociilntcd mash n-as aerated undcr about 6 pounds prcwun.. amylo runs, which confirmrd laboratory findings, were made .I heavy development of mold mycclium required a h i t 20 hours. tinder pure culture conditions. They were on a small scalp, apII .hi. W I ' Ruscd ~ to inoculate the next plant stxpt:. proximately 200 gallons. C'oritinuous $team jet cooking with ' SIKOSDPLANT ST.AGE. A closed, steel, pressure vessel \vith addition of 1% sulfuric arid on the weight of the grain was em\vorking capacity of 2500 gallons was used; it was equipped with ployed to thin the grain and sulubilize the starch. Tields averrwiline coils. Composition of the mold-grotying mash \vnh: aged 10.0 proof gallons per 100 pounds of dry \Thole v-heat and 500 p i l l ins granular flour and malt from plant mashing system, 12.0 proof gallons per 100 pounds of dry granular flour. Control 300 gallonr stillage, 0.5 pound aluminum powder, 20 grams zinc. of temperature and flow n-as difficult in this size of continuous +uli:iteqA pounds ammonium sulfate, 1000 cc. sulfuric acid, and cooking apparatus, but sufficient information was obtained to water t o give about 2500 gnllons (includinR condensate from +how that the orthodox amplo process conld be used for the fer.twm u * c d i o sterilize); the pH of the mash \vas 3.8 t o 4.2. mentation of wheat. Thy -oilition was sterilized by direct steam for one hour at 2-50' F. a n d cooled t o 95' F. It-was inoculated by blowing tht, DISTILLERY PROCEDURE mold culture developed in the first plant stage into the second by .\ practical way of utilizing the mold in the distillery was t o mcm- of' air. The inoculated second stage was then arratctf grow it in a portion of the yeasting system. The Sewark, S. J., undt.r :rhout 5 pounds air pressurr for 24 hours. Very heavy plant at which the trials lvere made had surplus yeasting equipt i ~ t > l ( lpro\vth developed in 24 hours. ment designed for operation under pure culture conditions; its F't;R\IESTER STAGE. Following the preparation of the second size was such that. a mold culture equivalent to 6-12% of the 1)I:int stage, pure culture conditions \wre no Longer maintainotl: rh:it i., the final stage was mashed under exactly the same cnntotal volume of fermenter mash could be prepared. This proportion of mold was found sufficient for good conversion when it was (Litioiii as ohtained for the infusion niashes of regular plant operaemployed n-ith a very small amount of malt (about 2y0 of the t i o n . I n this regular procedure granular flour mash containing total weight of the grain). Its use in this way is analogous to the 190 pounds flour and 20 pounds malt per 100 gallons was ronme of green malt in the usual malting procedure. vc.i,ttd in mash tulis equipped with rnechanical agitators. ('onALUMIXUM POWDER. Early in the lvork the utilizatioii oi \rcr.ion was carried out a t 145' F. for one hour. Ahtiseptic,3tillage for mold development \vas conuidcred desirable. It wx.: ' 0.7.; pouncl ammonium bifluoride and 0.7 pound Dowicidc G 1ii.r foiind, however, that some substance in stillage interfered wit l i LOO0 g:rllona of mash) n-ere added trr the mash. Since stillnpc \v:ic !he submerged growth of the mold. This toxicity could lie ri'i i o t nwtj for fectl, these quantitier: of antiscptiw \\-ere prrniis~i\)l~*. duced by dilution, or rcmovrd by carbon treatment. -4tldition (Ji U I I:iniouiits . could br suh.t:inti:illj- rcduccd or antisvptir- r i b n i siliiminnm poivder 10 the stillage in all c ;)li>ti.ly i~limin:itcdif mashinE w r i irritd ou' I)? thil iibii:il 1)n-
1x1 1933, when there nab a possibility that alcohol producers would be required to use corn or other grains t o alleviate economic nwntlitions on farms, this laboratory undertook a cooperative itudy of the amylo process. The investigation included laboratory experiments, 3000-gallon pilot plant runs, and finally thirteen 50,000-gallon full scale fermentations on degerminated corn ~nval, I n the laboratory and pilot plant runs it was found that, when contamination with bacteria occurred, development of the mold and starch conversion were retarded; lowered yields or Pomplete failure resulted, depending on the magnitude of the Infection. Full scale fermentations were successful, however, because they could be carried out in equipment such that the mash could be completely sterilized and bacterial infection exduded. Corn fermented by the classical pure culture amylo proc~ s gave b normal yields, A mold (Rhizopus delernar) and a yeast (Saccharomyces anamrnszs) were obtained from the Culture Bureau a t Delft, Holland. Two heed stages \+ere employed. The fermentation time averaged 55 hours and the average efficiency of fermentation was 01.6%, based on the sugar equivalent of starch in the corn. ('ontinuation of these evperiments became unnecessary when vononiic conditions improved, so that the method was not used 4s H innniifacturing procedure for an extended period.
INDUSTRIAL AND ENGINEERING CHEMISTRY
794
sure cook met,liod. At the end of the conversion period t h e m a s h wa.: pumped tlirough cwolcrs t o :i ferm t ~ n t t ~4cL r , oiycast \v:1s added, antl tli? i'c~rnicnt:ition \\-:I:ill 6 81 Har). L. C . . Fuliiier, E. I., and Uiiderkoflcr, L. i n t l i e ii(s:ivy (-'HEX., 35, 514-18 (1943). linl W o ~ v t h'Ii 4)Hao, L. C., a n d J ~ r i i pJ.A., , Ibid.,37, 521-5 (1945;. rhe sccolld ])lant 10) S e u l i a ~ e rSI., . I t ~ i d .25, , 712-13 (1933). 11) Owen, TI-. L.. Ibid., 25, 87-9 (1933). t u supplt,mellt 12) I{oherth, SI.,Laufer, S., Stewart, E. D . . and Palatail, I.. 'I.., the reduwtl m:ilr I L i d . . 36, 511-12 (1944). emp1I)yccl. 13' Takamine, J., U. d. Patent> 625,519, 5'25,822-4, aiirl 525,97! r . (1894); 562,103 (1S96): 991,5GO (1911); 1.054,:324 aiiu I hc: rc:sult> 01' 1,054,626(1913); 1,148,938 (1915); 1,263,817 (1918); J. I s I ) . fermentations art' I:VG. CHEM.. ilY14). . 6.824-8 . shown i n TnLle I. I 4 ' r a k a n h e , J., and Takamine, J.. J r . . U . d. Patent$ 1,:391,2I!l Fermenters receiv(1921) and 1,460,736 (195.3). I,?) Uiiderkofler, L. A , , Breuer'.i Digest, 17, T o . 12, 2Y-Y5 (1942). ing 12yomold (Sos. 16) Uuderkofler, L. .L, U. S,Patents 2,219,665 (1841) ant1 2,29l.W!1 9, 3, 2, 13) fininhed (1942). in 40 hours. The 17! Underkofler. L. A . , and Fulnier, I:. I.,f ' h w n i c a Botan., 7, 4 X - - 2 remaining fer(1943). 18) Underkofler, L. A , , Fulmer. I:. I., and Schoene, L., I N D .F,;SL,. menters, receiVirln CHEM., 31,734-8 (1939). 6% mold, had >' (1% Underkofler, L. X., Goering, I(.J., and Bu(,kaloo, G . IY..I ' f o c . fermentation timi, Iou:a Acad. Sci.. 48, 225-31 (1941).
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