Fermentation of Douglas Fir Hydrolyzate by S. cerevisiae Elwin E. Harris, George J. Hajny, Martha Hannan, and Sedgwick C. Rogers ti.
S. Forest Products I a b o r n t o r y , Madison 5, Wis.
wood sugar solutions produced by the hydrolysis of Douglas fir sawmill waste contain inhibiting substances that make them difficult to ferment by the procedures usually used in fermentation industries. Previous reports show-ed that it was helpful to remove furfural produced by the decomposition of pentose sugars. The useof large quantities of yeast was also helpful. This report shows that neutralized wood hydrolyzates ferment more readily if they are pretreated with aluminum chloride, or if they are produced under conditions of rapid sugar remobal to prevent decomposition. Pilot plant fermentations of Douglas fir hydrolyzates used Saccharomyces cereuisiae in batches from 7 liters to 300 gallons. Yeast was trankferred without
intermediate treatment from a completed fermentation to fresh 5 % sugar as many as fifty-nine times without lose of alcohol yield. When 1% by volume of yeast was used, fernlentations were complete in 10 to 14 hours. If larger quantities (6 to 8 % by \ o h m e ) of yeast were used, complete fermentations were accomplished in 5.5 hours. 4Icohol yields from 5% wood hydrolyzates were 39.2 to of the total reducing nubstance, or a yield of 2.64 gallons of 95% alcohol from 100 gallons of hydrolyzate. The fermented hydrolyzate produced about 0.3 gallon of heads cnnbisting principally of aldehydes and methanol, and about 0.3 gallon of a fusel oil fraction for each 100 gallons of 95% alcohol.
P
range, flashed to atmospheric pressure, cooled to 30" C., anti filtered). They were then treated with sodium carbonate until the hydrolyzate had a pH betreen 5.6 and 5.8. Total yeast counts were made by a procedure using a moldcounting chamber. Wet yeast volume was determined in a graduated 15-nil. centrifuge tube by centrifuging 10 ml. of B represent,ative sample of yeast suspension in the fermentation liquor. The wet volume was read directly from the tube; 0.1 ml. of wet yeast is equivalent t o approximately 1% wet yeast volume. The dry weight of the yeast was determined by the method described by Peterson, Snell, and Frazier (6). Reducing sugar, fermentable sugar, and alcohol were determined hy methods desrribed by Saeman, Harris, and Kline ( 7 ) .
REVIOUS work a t this laboratory deruoiistrat,ed that the sugars produced by the saccharification of wood could be used to propagate yeast and produce ethyl alcohol by fermentation, provided that treatments were given, or conditions used, which overcame the effect of some of the inhibiting materials i n the wood sugar hydrolyzates. Leonard and Hajny ( 6 ) demonstrated that addition of sodium bisulfite, a heat treatment a t neutrality, or the use of large quantities of inoculum aided in overcoming the effect of the inhibiting materials. Peterson and eo-workers (6) found that the use of a rich inoculum was beneficial to the growing of yeast on wood hydrolyzates. Grondal an? Berger ( 2 ) found that removal of lignin and furfural, plus the addition of autolyzed yeast, aided in the butanol fermentation of waste sulfite liquor. Sjolander, Langlykke, and Peterson (9) found that clarification of the solutions with activated carboil produced solutions that, were easily fermented. Many other notable contributions have been made by others, especially in connection with the Scholler process for wood saccharification in Germany (8). Many improvements which affect the ease of fermentation have been made in the wood saccharification process developed a t this laboratory ( I , 3). Hydrolyaates prepared by these proresses were used for study in these experiments. The need for the removal of furfural from liquors to be fermented was pointed out by Leonard and Hajny (6) and by Heuser (4). I n recent work a t this laboratory a method of flashing the hydrolyzate after neutralization has been used. This procedure removes all but traces of furfural and, a t the same time, permits removal of calcium sulfate a t elevated temperatures. This report describes the effect of these changes on the ease of t'ermentat,ion, and reports a study of the problems connected with pilot plant scale fermentations, such as yeast requirements, conditions for growth of yeast, methods for removing yeast from t'he fermented liquors, nutrient requirements for growing yeast and for fermentation, ebc. The yeast used was a strain of Brewer's yeast S. ccrcvisiue, S o . 49, from the University of Wisconsin collection, selected because of its high alcohol-yielding capacity. Wood hydrolyzates were prepared according to procedures described in earlier reports (neutralized with lime a t 130" to 135" C. t o a pH of 4.7 to 5.0, filtered within that temperature 896
INOCULUM PRODUCTIOh
111 the production of alcohol by large voluuia oi preformtd inoculum, it is desirable to keep the cost of yeast production a t B minimum. Since alcohol is the desired end product, there is no loss if alcohol is produced while the inoculum is propagated. I t is possible, therefore, to obtain higher utilization of air and to decrease the cost of yeast production by employing a ratio of air tcr medium lower than that of the usual yeast production. Thtt use of less air during the growth of yeast on wood hydrolyzrttes 3lso reduces the amount of organic material which precipitates. For the growing of yeast (Table I), inoculum which had been tleveloped from a slant on a glucose and malt-sprout medium nah propagated on 2.6 gallons of a sterilized 2% sugar solution. The hugar solution was obtained by diluting Louisiana second-crop ~nolasses,2 grams of ammonium sulfate, 2 grams of disodium phosphate, 8 grams of urea, and the aqueous extract from 200 grams of malt sprouts. This solution was aerated through a cloth sparger for 16 hours, and then added to 4.2 gallons of B sterilized solution containing 8% sugar from Louisiana secondcrop molasses, 12.7 grams of disodium phosphate, 12.7 grams of animonium sulfate, and 50 grams of urea. This inoculum (6.8 gallons) was aerated as described for 8 hours and then placed in B 75gallon fermentation tank. While the inoculum was aerstetl a t a rate of 4 cubic feet of air per minute, neutralized wood hydrolyzate was added a t a rate of 2 to 3 gallons per hour for It; hours. The inoculum thus produced was placed in a 500-gallon fermentor and, while it was aerating a t a rate of 0.2 volume of
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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
September, 1946
pvr volume of liquid per minute, bvood hydrolyzate \vas added a, rate of 0.75 gallon per minute for 7 to 8 hours. In general, the more concentrated solutions were acted upon :nore slowly. Fermentation P5 (Table I) did not appear to follow this general observation because the rate of feed of the wood wgar inoculum was greater, and therefore the amount of yeast xdded to the large fermentor was greater. The sugar in fermentat ioiis P4 and P5 was utilized in the time allowed, whereas sugar in [lie higher concentrations of fermentations P6 and P7 was used riiore slowly. The experiment was iiot continued, but it is believed that a longer period would have given high yeast yields iii fcsrmentations P6 and P7. .1series of fermentations (P8 to P18, Table 11) were carried out 1.0 study the effect of both increase in time and variations in the ratio of air to medium on the yields of yeast and alcohol froni inore concentrated sugar solutions. For this series, yeast was qtarted on 5 gallons of 3 % sugar from diluted Louisiana serontlrrop molasses; this solution contained 20 grams of ammonium -illfate, 20 grams of monosodium phosphate, .40 grams of urea, arid the extract from 570 grams of malt sprouts. This niediurn :vas sterilized for 30 minutes at 15 pounds per squarr inch stearii ~ i r e s s u r e ,adjusted to pH 4.8, and cooled to 30" C. The yitast \vas added, and the mixture was aerated through a 3.5-inch cloth -parger for 16 hours, a t a n air-liquid ratio of approximatrl~ 2 . 5 . This molasses inoculum was placed in a 75-gallon fermentor. .und wood sugar was added while aerating and stirring took plarts. In fermentations P8 through P13 the sugar was fed in slon-ly, thtd d i t i o n requiring 6 to 8 hours. Samples were taken at the end of 9 hours; aeration was then continued to the end of 24 hours. when samples were again taken. An increase in aeration rate decreased the rate of sugar utilization in 9 1iour.q but increased the total amount of yeast produced. The total yeast produced was limited by the amount of nutrient present--namely, the amount required to produce a 20% growth of yeast. In feriiientations P14 through P18 the sugar was added as a batch and aerat,ed. Fermentation P14 differed from P16 in rate of agitation. I n fermentation P14 the agitation was half the rate used in series P15 through P18. Yields of yeast in 9 houra in ~ e r i c sP14-Pl8 were higher than those from the series that was l'od slowly, because the full amount of sugar was present the enrive time and a greater utilization of air resulted. The yield in 24 hours, however, was about the same. I n t'he series P8-Pl8 tht' nlcohol produced in 9 hours was, in most ferrncntations, al:tir
st
1'1 1 1'8 1'9 1'12
0.2 0.17 0.20 0.19
1'13
0.17
' Cnlr,ulated on total sugar.
3 49 3 39 3.4I 3 42 :j 39
Slow feed Slow feed Slow feed
Slow feed Slow feed and
n
5:i r1,5:1 I 0:I 2 0:l 0 .i: 1
most equal to that produced in 24 hours; this indicated that only yeast was produced after the first 9 hours, or that alcohol was (.ither used in the gron.ing of yeast or lost by evaporation. PILOT PLANT SCALE FERMENTATIONS
I n fermentations shown in Table 111, yeast was first grown on 5 gallons of diluted mola transferred to wood Sugar in a 75gallon fermentor, and ac I fur 16 hours for further develop ment of yeast. It \vas then transferred to a 350-gal1on batch of wood sugar for anawobic fermentation. I n the scriw P20, P21, ant1 P22, the ye:rtst, ivhich had increased from about 0.35 to 1 pound (dry basis), was added t o about 350 gallons of approximatcsly 59* wood sugar solution. The inoculum in t,hese fermentations did not contain enough yeast to carry the ferment,ation to rompltation in 2-1 hours. Fermentation P22 was allowed t o continue: for 40 hours. The yield of alcohol was normal for the amount of sugar subjected to alcoholic fermentation. Except for tht, time required, fermentation P22 was satisfactory. St-ries P21,P25, P26, and P29 comprised a study of fermentation rates, using varying amounts of inoculum. I n fermentations P2-1 and P25 a large amount of inoculum was used, 1.8 t o 2.0 ~iomidson a dry hasis in 76 gallons. Fermentation \vas compltste in 5.5 to 6.0 htrurs. Half that amount of inoculum, as in lenientation P29, rcquirrd 14 hours. Fermentation P26 was not criinpleto in 7.5 hours hut would havo hcttn in 0.5 hours. The sugar suhjrctcd to alcoholic fermentation gave gootl yic:ltlb, but because the amount used to grow the ycast was Ixrgc', the over-all yield of alcohol was Ion-. Growth fartors in the form of rnola s and extract. of n d t sprouts were added to fernicntxtions P and P28, but thr rate of fermentation did not appear to be affected. Growth factors which may have been carried over in the solution from the molasses inoculum were removed in fermentations P30 through P33 hy centrifuging and washing the yeast before transfer t o a wood sugar solution. The effect of adding various amounts of growth factors was determined by adding molasses to the fermentation. I n fcrmentation P30 molasses was added for the growing of inocula on wood sugars and also for the fermentation. I n P31 and P32 molasses \vas used in the production of the yeast only, whereas in P33 the only sources of groivth factors wvcre those in the wntrifuged and washtd yeast. Fermentations w r e slower tts CP was decreased, but all were complete in 24 hours, x i t h good convrrsion of sugar to alcohol. Ovc~-all
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898
INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 58, No 9
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FE€i.\llSNTATION O F (:L.iHIFIKL) WOOD HYDROLYZATE
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Alcohol Yield. %, f r o r n : Total Fermentable dugar mgar 35.2 46 J 84 3 4 4 . .j 33.8 41.3 35.2 44.0 35 5 13.: 35.0 13.r 35.6 31.5 35.0
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INDUSTRIAL AND ENGINEERING CHEMISTRY
September, 1946
899
Alcohol Produced, Based on: Total Fermentmi reduciiig reducing sugar iugar 35.2 44.6 33.8 44.4 38.5 16,E 39.2 .17. 0 37.9 4 6 . li 39,4 47.0 38.3 46.0 39.6 47.4 34 1 44.0 34.0 44.0 19. R 39.7 40.3 49.8 87.0 1 6 .l i 39.3 47.2 37.0 46.6
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~0.003~1, i , i , i i ~ ~ ~ ~ ~ i tio~ p~* ;n ~i i ti ri ,:t , 20yo ~ ~ ~ iiicm at i o i i i v a , ~varritd through fol hoiirs. Tht. r t w i l b arc sllo\v11 in Tab1 It appears that an iiicrtxast. i n t h i ~uti cvrivcwion into alcohol result through y(':wt, There was no dific.rilt y in maintaining yeast concentra.tions. ,411 frrrnrnt at icins o n clarified solutions were complete iii 6 to 12 hours. but the yeart vas left i n the beer to the end of 20 hours to determine any loss ill vitality that may have resulted I'roni standing. Tht, 1-t:as.t \%-asvrr>-active and seldom more than half an hour itlapstvl htJfort, vigorous fermentation started; in all fermentors thc yet~st in(wase(1 beyond the amount required for rnaintenanctb of satisiactiiry fermentation rate in 20 hours. ATIOS R Y YEASTTRASSFER. Fermentatiow o f wood hydrolyzates using solutions clarified with aluminuni sulfate were also studird in 77-gallon hatches. I n the first of t h e wries (Table 5'1) P3R as madcs on the clarified hydrolyzatrs, whereas P42 was madc 011 liquor that had been clarified and t,ht,ri aerated for 21 hours prior t o i t $ iist' for the growing of p a s t and for fprnientation. ieriiiciit
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i)i,oxiiiiattbl>. a.oocl >ugar ~ l J ~ l l 1 i i i ~ ~iurther yeast groi5 1 1 , . 'Tlii. niisturt: was stirred anti at,r.ated s l o ~ - l yfor 16 hours \vitlr :ihout 0.7 volume of air per volunir of liquid per minute. Tlit~ii 23 gallons of this inoculum w r + ' placed in a fermentor and i i i n d ~ , ilp t o 77 gallons with wood sugar solution. At the end 01' 20 hours the yeast \\-as reniovrd by crntrifuging; about 8 gallons I ) i y t m t cream were obtained. This p a s t cream was added to t 111, next ft,rmentor and made up to 77 gallons with 5% wood sugar solution. The ratw for fi~rnicniatiorisP3R and P42 are shown i l l Figure 1. The over-all yield of alcoliol based on the total sugar used f o r ytviat production and alcohol for frrmentation P39 was 38.2%, alierc~~ that s for fermentation P42 was 37.3%. The aeration 01' i't~rnientationP42 produced a precipitate which made the yt'asi rnuch darker and which may h a w h e m responsible for t he lo\r t h r alcohol yield. The clarified wood sugar solution was used for two other scritsr 01' fermentations, C a n D, involving yeast re-use by transfer of t lit, yeast froni a conipleted fermentation to fresh sugar solution+. Series C was carried out in 55-gallon batches t o which 54 nil. o f Louisiana second-crop molasses had been added to supj)I> growth fac*tors. Fermentations were usually complete in 12 t l i I4 hoiir
