Submerged Culture of Fungal Amvlase J S. L. ADAAIS, B. BALANKURA, A. A. ANDREASEN,
AND
W.13. STARK
Joseph E. Seagram & Sons, I n c . , Louisville, K y .
Improt ements are described for the production and culturing of submerged mold for commercial use as a contersion agent of grain mashes. Distillers' dried solubles were found superior to stillage as a substrate for submerged mold amylase production; calcium carbonate was used to replace sodium hydroxide for adjusting the pH of the mold media. Bj-products from mold-coniertetl grain mashes are comparable to those from maltconterted mashes as a substrate for mold growth. In the culturing procedure, O.S$?C mycelial transfer is sufficient for inoculation; the mold culture may be transferred seriallj through several 24-hour submerged stages prior to the final propagation stage. Submerged mold cultures prepared by a method incorporating the impro\ements described may be used to replace barley malt completel) as a conterting agent and results in an alcohol ?ield increase of 0.2 to 0.3 proof gallon per bushel of grain.
T
HE literature contains many references t o the use of fungal
amylases in supplementing or replacing malt for the conversion of grain starches for alcoholic fermentation. Takamine (10) was the first in the United States t o suggest the commercial possibility of such a process. Cnderkofler and associates have published numerous papers describing laboratory and commercial scale production and use of fungal amylase in the form of mold bran (4,11, lb, 13). Hao and Jump ( 5 ) and Roberts et al. ( 7 ) have also reported successful application of the mold bran process t o the conversion of starchy grains. Recently, these laboratories ( 1 ) and others have reported on methods for the preparation of fungal amylase in submerged culture employing a niediuni consisting of stillage from grain mash fermentations. During these st,udies it became apparent that stillage as a mold medium presents a problem that may vary with plant operating conditions. Erb and Hildebrandt (e), working with species of R h i z o p u s , reported a toxic substance in stillage which could be removed by carbon treatment or by the addition of t'races of aluminum powder. Van Lanen and Le llense (14) successfully cultured dspergilllts niger in stillage and reported no indication of any toxicity. I n t h e authors' laboratories it was found that gron-th and amylase production by a strain of A . niger in plant stillage (produced in a low temperature distillation unit) were definitely inferior t o those in stillage from laboratory-prepared mash. Charcoal treatment and the addition of aluminum poivder rvere ineffective in improving the groivth-promoting quality of plant st,illage. The present paper deals with t h e selection of a inore uniform subst,rate for submerged mold amylase production and iniprovements in culturing procedure designed t o facilitate commercial application of the process. The specific problems t o be considered are:
-1. The re-use of stillage from mold-convcrted niashcs ac mold mcdia and as backset. (Backset i. defined as stillage added t o conveited mash ab a nutrient prior t o fermc,ntation.) 5 , Procedures for culturing mold in the intermediate stages betiwen the laboratory culture and the final plant propagator. PREPARATION AND EVALUATION O F MATERIALS
-1 strain of A . nioer obtained from the Sorthern Regional Research Laboratory (S.R. R. L. S o . 337) was employed in all the studies discussed in this paper. Submerged mold cultures were gro1Vr-n in stillage or distillers' dried solubles at a solids concentration of 5 grams per 180 ml. fort,ified with 1 gram per 100 ml. of corn meal. The p H was adjusted and 1 liter of the medium was sterilized in a 2-liter coneshaped flask and inoculated with 2.07, by volume of a surfacegrown mold culture. The culture was then placed in a 30' C. water bath and aerated (by means of a n Aloxite sparger) for 48 hours a t a rate of 0.2 cubic foot per minute per liter. Variations from this procedure are discussed below. Enzyme activities of the mold amylase preparations were measured by three methods. Alpha-amylase det'erminations T5-ei-e run on a portion of the samples by the Sandstedt, Kneen, and Blish 30" C. method (8) and results reported as alpha destrinizing units per 100 ml. of mold filtrate. All cultures were analyzed for saccharifying power by a n unpublished method of Hao (9, revised in 19-16). I n this determination the saccharification value is defined as the number of grams of starch saccharified by 100 ml. of mold filtrate in one hour at 40" C. This method lacks the value of wide acceptance hut has the advantage of speed and is particularly valuable when large numbers of analyses are to be made. All mold amylase preparations were evaluated finally on the basis of alcohol yields from laborat,ory test fermentations by a n adaptation of the method of St,ark el ai. (9). Corn was cooked a t atmospheric pressure for one hour, autoclaved at. 22 pounds' pressure for one hour, and cooled to conversion teniperature (130" F.), and a n amount, of the submerged mold culture equivalent to lo", of the final mash volume was added. The mash was agitated for 5 minutes and cooled t o setting temperature. Ten per cent by volume of stillage was then added as backset, t,he p H adjusted t o 4.8, and the total volume of t,he mash adjusted t o a concentration of 38 gallons per bushel of grain. Control fermentations ivere run with each espei,iment using 1% premalt, i G conversion malt, and 145' F. conversion temperatures. Twenty per cent by volume of stillage was added to the control mashes as backset. The yields of the malt controls are based OIL total grain and in all reported experiments the control yields are at a level considered optimal. Throughout this paper the quality of each mold amylase preparation is evaluated i n terms of the yield of alcohol from test fermentations employing the mold preparation as the sole conversion agent. EXPERI>lE.VTAL F O R K
1. Aicomparison of distillery by-products as media for mold propagation. 2 . The effects of variation of the conversion temperature on eonversion of grain mash x i t h mold amylase. 3. Procedure? for the neutralization of mold media.
The standard malt used throughout, these studies had a starch content of 4i,1yo; Lintncr, 166"; 0-amylase, 46.8 units; Pamylase, 14 units; and sacchsri'fication value, 6.3 S. I-, units 1615
IN D U S T R I A L A ND EN G I N E E
1616
TABLEI.
DISTILLERY BY-PRODCCFY
AS
lIEDI.4
FOR l 1 O L D
PROP.4G.4TIOS
s.
Mold Medium
17.u
Alcohol l-ipld, Proof Gal./,Au., D r y Basis
Laboratory stillage Evaporator sirup 63:s Distillers’ dried solubles 95.0 Control (malt converted)
Plantb Efficiency, ’3’~
6.11
6,l5 6.10 5.99
91.6 92.2 91 4 90.:
a Saccharification value is defined as t h e number of grams of starch saccharified b y 100 ml. of mold fdtrate i n one hour a4 40” C. b I’lant efficiency is defined as per cent of theoretical alcohol yield based on starch in grain used.
T.4BLE
11. DISTILLERS’DRIED SOLUBLEs
DIFFERFXT CO\lPSSIES Source of Solubles for X o l d Media Company Company Company Company Company
1
2 3 4 5
PRODTCED B Y 4 s 3IEDI.I FOR I I O L D PROPIC.lTIOh
Alcohol r i e l d , Plnnt n-.Aniylase, Proof Gal.,/,Bu., Efficiency. Unirs,’100 111. D r y Basis 70 6.06 2783 90.8 6.00 90,s 2390 BO 2 6 02 2351 2941 6.05 90.7 90.1 80 2606 6.01 5.93 Contrul (malt converted) 91.3
S. 7‘. 80 80 80 95
per gram. \Beta-am>-lasecontent of the malt v a s detcrniined by th5 method of Iineen and Sandstedt (67.1 Two st,andard lots of Corn having starch contents of 67.7 and 69.3% were used. SELECT103 O F l I E D I G l 1 FOR l\lOLD P R O P A G A T I O S .
I11 C O l l l -
mercial distillery opemtions, stillage from the distillation column is screened t o remove most of the suspended solids, evaporated t o a solids concentration of approximately 30y0,and finally dried on drum dryers. The end product is known as distillers’ dried solublcs. When the inferiority of plant stillage t,o laboratory stillage n-as cstablished, both evaporat,or sirup and distillers’ dried solubles Xvere investigated a s media for mold propagation (Table I). The yields of alcohol from fermentations converted n-ith mold amylase produced in both evaporator sirup and solubles \wre comparable t o t h e yields from laboratory stillage media. The yield in all cases v a s over 0.1 proof gallon per bushel higher than \\ ith the malt,-converted control. (Proof gallon per bushel is a term used t o denote gallons of 100’ proof alcohol yield from 56 pounds of grain ns received, unless stated as “dry basis.”) I n this and succeeding t,ables each yield figure represents an average of at least three fermentat’ions. Since evaporator sirup has sho\vn some tendency t o vary in quality from time t o time, attention !vas concentrated on solubles a s the medium for further refinement of the mold amylase process. Distillers’ dried solubles throughout numerous esperiments have proved remarkably consistent in quality 8s reflected in mold growth and amylase production. I n order t o test this material further, hon-ever, solubles w r e obtained from five separate companies representing a wide divergence in grain bills and processing equipment. Thcse samples were compared a s media for mold propagation (Table 11). The saccharogenic values of t h e mold cultures groLm in these solubles and the alcohol \-ields from fermentations converted with t,hese ~ n o l dcultures n-;re practically identical. There was a 11-ider variation i n the a-amylase analyses, though not enough l o be reflected in plant efficiency a t t,he concentrations used. (The term “plant efficiency” is used to denote t,he per cent of the theoretical alcohol yield based on the starch in the grain used.) The submerged culture of :I. COSVERSIOXTEAIPERATCRE. ,tiger was normally added to the cooked grain a t 130” F. This conversion temperature has been generally accepted as optimum for fungal amylase action, although the high mash viscosity a t 130’ F. poses a problem in plant operation. Since the existing cooling equipment in most distilleries is designed for conversion at 145’ F., it was thought advisable to test the use of mold amylase a t this higher temperature. The
R I14 G C H EM I S T R Y
Vol. 39, No. 12
effect of conversion temperatures on alcohol yield is shown in Table 111. The higher conversion temperature substantially decreased t h e viscosity of the mash and in addition resulted in higher alcohol yields. .ilthough t h e increase from 6.07 t o 6.15 proof gallons per bushel would not be considered significant on t,he basis of one test, numerous experiments have proved that such an increase is obtained. As a result of these tests 145’ F. was adopted as the standard conversion temperature and all the following experiments were conducted a t the higher temperatures. I n all subsequent data the difference betlveen the alcohol yield from malt- and mold-converted cooks is appreciably greater than in previously discussed experiments. ADJUSTMEKT OF pH OF MOLD~IEDIUAI. Adjustment, of the pH of the mold medium !vas also investigated from the standpoirit of application t o plant processing problems. Prior t o this tirile nicdia for laboratory scale mold propagation were adjusted with sodium hydroxide and 0.5% calcium carbonate added as a buffer. The results of several variations in this procedure are presented in Table IV. I n the first instance the solubles medium was adjusted to p H 6.0 Jvith sodium hydroxide and 0.57, calcium carbonate \vas added. I n the second case, sufficient calcium carbonate was added t o adjust the p H t o 5.0 and 0 . 5 5 , escess \vas added. I n the third case the p H was adjusted t o 5.0 with calcium carbonate but no excess was used. Mold groFvth and amylase values \yere ident,ical with each of the above procedures. The alcohol yields from fermentations converted with these amylase cultures give further evidence t h a t calcium carhonatc is s a t i s f d m - y in replacing sodium hydroxide. I n all cases the alcohol yield of the experiqental fermenters was 0.25 roof gallon per bushel higher than the m$t-converted control. There is a definite advantage in the use of calcium carbona,tc in plitnt operation. The danger of overadjustment due t o operational crror is niinimized and the time required for pFI adjustment is reduced. ItE-L-SE O F S T I L I A G E FROM ITOLD CONVERTED l I A s H E S AS
~ I O LlIl)~ ~ 1 . 4I n the commercial product,ion of mold amylase the distillery by-products used as mold media and as baclisrt n-ould obviously be produced from mold-converted mashes. In all the esperiments previously described, the submerged mold cultures have h e n grown in by-product,s from malt-converted fcmicntations. It was deemed necessary 60 determine whether stillage from mold- and malt-converted mashes differ in regard to suitability as substratcxs for mold amylase production. Experiments \yere run in which laboratory stillage from mold-convtrted mashes \vas compared ivith malt stillage a s mold media and as backset (Table Vj. Tu-o controls w r e employed. The first n-as the nialt-converted control and the second, the mold control in xhich 1Oc&, by volume of a culture grown in stillage from a malt-convert,ed inash \vas used its the conversion a.ycnt. Ten per cent by volume of malt stillage \\-asadded as backset. I n one set of fermentations malt stillage was again used as t h e submerged culture medium T . i i r ~ n111. EFrcc.r
O F COSVERSIOX TEMPERAXRE os ~ L C O I I O LYIELD
Temperature, Cuiirersion .\gent 3 I a l t control l l u l d culture N o l d culture
Neutralizing Agent
KaOH CsCOt CaCOr
F.
s. v.
145 1x0 I45
6i.j
633
.%lcohol Yield Proof Gal.:!3u., D r y Basis 8.99 6.07 6.15
Alcohol Yield, Excess CaCOj, Proof Gal./Uu.. G./100 1\11, 9 . 7’. Dry Basis 0.5 95 6.26 0.5 95 6.26 95 6.24 Sone Control ( m a l t converted) 6 . 0 1
Plant Efficiency,
so
91.5 91.0 92.2
Plant Efficiency,
70
93.8 98.8 93.6
93.2
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
December 1947
T.iBLE
v.
STILLAGE FROM 1IOLD- .4SD .\[.\StIES .4S 1IOI.D 11EDI.i ASD AS
Conversion Agent M a l t (contrnl) l l o l d culture (contrul) Mold culture .\loid culture Rlold cnlture
T y p e of Stillage Mold medium Backset
3 . V.
IIALT-COSVERTED
BACKSET Alcohol Yield, Proof Plant Gal./Bu., Cficienry. D r y Basis 70
...
Ualt
..
6.13
94.0
Alnlt Alnlt, Ilold
Malt Rlold XIalt Mold
80 80 80 80
G 32 6 38 6.27 6.38
94.7 Y5.6 94.0
Aldd
g:.s
TABLE VI. EFFEC'T O F \'ARYISG . i \ r O U S T s O F 2 4 - H o r ~~ I Y C E LI.iL TR.C,-.SFEIE .is ISOCTLA FOR XOLD .I\IYLA~E PRODL-CTIOS Alcohol Propagariiip Proredure u-.iniylase, Yield, Proof Plant of i,-nits/l00 Gnl./Bu , Efficiency, Type.of .kinourit inoculum inoculum, 7 S. V , MI. Dry Basis yo 90 3 Sporulated 2 0 9 ;1 2180 G 10 90.4 Mycelial 2.0 80 2020 G 11 80 2042 6 11 90 4 Mycelial 0.5 Cuntrol (molt converted) 5 . 9 4 90.0
Conversion l g e n t N a l t coritrul 1Iold culture h l o l d culture Mold culture a
s. v. 63:5 80.0 80.0
.ilcohol Tield, Proof GaI./Bn , D r y Basis
Plant Efficiency,
5.95
91.3 94.0 92.9 92.3
6.27 6.20 6.16
mold growth had developed suffiririitly f o r use as inoculum; 0.5% by volume of such a traiisfer is comparable t'o a heavily sporulated surface culture.
TYPICAL FERMEXTATIOS DATArSIiVG IMPROVED CCLTURE METHODS. Table VI1 contains typical d:ita from laboratory fermentations in JThich the submerged mold amylase cultures used for conversion vere prepared by a procedure incorporating all the developments iv-hich have been described. The mcdium consisted of 5 grams per 100 ml. of distillers' dried solublcs and 1 gram per 100 nil. of corn meal, neutralized with calcium carbonate. The culture was transferred serially through three st,:iges n-ith 0.57, by volume inoculum. The fiist tn-rJ stages were aerated for 24 hours and thc last st,age for 48 1ioui.s. A n amount of this material equivalent t o 1056 of the filial mash vvlume was used as the sole conversion agent in tcbst frrriientations. Conversion was conducted a t 145" F. T\-ith this procedure the alcohol yields varied from 6.16 to 6.27 pmof gallons pcr bushel :is compared t o the malt-converted control yield of 5.05 proof gallons per bushel. Plant efficicncies (based in the starvli analysis of the grain) with mold conversion arc' equal t o or higher than the efficiencies with malt conversion. C O S C LUSIONS
%
Incorporating all improi-ements described in this paper.
but s illage from a mold-converted mash !vas used as backsct. Mold stillage n-as also used as the mold medium F i t h malt and mold stillage backset. I t was found that the amylase produetion as measured b y the saccharification values was identical in all cases. T h e alcohol yitlds indicate t h a t the elimination of malt n-ill not affect the by-prcducts in regard to re-use for mold propagation. The average alcohol yield n-ith thf mold amylasc preparations n-as approsimately 0.3 proof gallon per bushel higher than the malt-converted control. SERIAL SUBlfERGED CULTURE TRASSFER. .~nOtheI' probleln n-hich has been investigated from the standpoint of industrial application is the procedure for culturing mold in the intermediate stages betn-een the laboratory a n d the final plant,-scale mold propagator. Inoculations of laboratory submerged cultures have been made from heavily sporulated surface cultures. On a commercial scale i t is necessary t o use intermediate submerged culture stages and the size of these vessels and the length of the iricubation period a t each stage are of course important from the economic standpoint. -1group of experiments n-as run in n-hich varying aniounts cf inocula from serially transferred submerged cultures were coinpared n i t h 2.0% by volume of sporulated surface cultures a3 inocula for the final submerged mold amylase preparation (Table VI). Serial transfer of submerged cultures is termed mycelial iransfer, since sporulaf ion is negligible in submerged growth a t the incubation periods studied. The first propagating procedure listed is the staiidsrd control niethod in Tyhich the medium \vas inoculated with M s;iorulated surface culture, aerated for 45 hours, and used for conversion of a test mash. I n the second propagating procedure one flask was inoculated as above but transfer 11-as made t o a second subnierged culture which was aerated for 24 hours. Two per cent of this culture was used t o irioculate the third and final stage vhich \vas aerated for 48 hours. The last pro9agating p r ~ redure IWS varied from the above in that only 0.jCL inoculum w s employed. The amylase values a n d alcohol yields with the various inoculation procedures were practically identical. Although amylase production in submerged culture was low a t 24 hours, the
1617
-1s a result of the a-ide variation of stillage in regard t o mold growth-promoting quality, distillers' dried solubles which consistently support mold growth and amylase production are recommended. Improvements have been made in submerged culturing niet,hods which will be reflccted in simplification of operating procedure and in a reduction of operating cost. These improvements may be summarized as follows:
1. Adjustment of the pH of the mold medium with calcium cnrbonate, instead of sodium hydroside, and the elimination of esccss calcium carbonate. 2. The use of 24-hour submerged cultures as intermediate stagrs between the laboratory culture and the final plant mold . propagator. 3. The use of 0.5yomycelial transfer for inoeulrition of suhmerged cultures. 4. Elevation of the conversion temperature from 130" to
l G C F.
K i t h submerged mold amylase prepared by this procedure as the sole conversion agent for grain mashes, a consistent alcohol yield increase cf 0.2 to 0.3 proof gallon per bushel can be obtained over malt-converted fermentations. LITERATURE CITED
(I) Balankura, B.. Stewart, F. D., Scslf, R. E., and Smith, L. A,, J . Bact., 51, 594 (1946,, abstract. ( 3 ) Erb, S . >I., and Hildebrandt, F. AI., 1.1.~.E s n . ('HEX., 38, 792-4 (1946). ( 3 ) Hao, L.C., unpublished inetliod. ( 4 ) IIno, L. C.. Fulmer, E. I., arid Cnderkofler, I.. A , , ISD, ESG.
CHEM.,35, 814-18 (19.231. (5) Hao, L. C., and J u m p , J. A . , I b i d . , 37, 52 1-5 (1945:. ( G ) Kneen, E., and Sandstedt, 11. A I . , Cereal Chem., 18, 237-52 (1941).
( 7 ) Roberts, XI., Laufer, S.,Stewart, E. D., and Salatan, L. T., I X D . ENG.CHEM., 36, 811-12 (1944). ( S ) Sandstedt, K. M.,Kneen, E., and Blidh, 11. J., Cereal Chem., 16, 712-23 (1939). (9) Stark, TV. H., Adams, S. L., Scalf, R. E., and Kolachov, Paul. I N D . E X G . CHEX., > I N A L . ED., 15, 443-6 (19.13). (10) Takamine, J., J. ISD. ESG. CHEM.,6 , 824--8 (1911). (11) Underkofler, L. A , Fulmer, E. I., and Schoene, L., Ibid., 31, 734-5 (1939). (12) Gnderkofler, L. A . , Severson, G. M., and Goering, K. J., Ibid., 38, 980-85 (1946). (13) Underkofler, L. A , , Severson, G. hf., Goering, K . J., and Christensen, L. M., Cereal Chem., 24, 1--22 (1947;. (14) Tan Lanen, J. >I., and Le Mense, E. H., J . Bact., 51, 595 (1946),
abstract. RECEIVEDMay 9, 1947.
Presented before the Fernientation Section, Dirision of Agricultural a n d Food Chemistry, at t h e 111th Meeting of the AMERICAX CHEJIICAL SOCIETY,Atlantic City, S. J.
