November 1947
INDUSTRIAL AND ENGINEERING CHEMISTRY ACKSOWLEDGMENT
T h e authors wish to thank J. K.Polsky, n-ho conducted the majority of the laboratory tests, and also express appreciation to W. H. &- L. D . Betz for permission to present these data. LITERATURE CITED
(1) Baker, Combustion, 12, 31-4 (1940). (2) Betz, Handbook of Industrial Water Conditioning, Chap. 1E. p . 547 (1945). (3) Flickinger. P o m r , 85, 118-20 (1941).
1443
Heiskell, I bid., 90, 747-9 (1946). ( 5 ) Hundesshagen, 2. ofeentl. Chem., 24, 159-67, 175-86 (191s) (6) Leick, T'om F u s s e r , 7 , 197-205 (1933). (7) Naguire and Tomlinson, Combustion, 11, 26-39 (1939). (8) Mehring, Cliem. &. -Vet. Eng., 21, 629-32 (1919). (9) Paris. Chaleur &. industrie, 5, 277-9 (1924). (10, Paris, Chimie 6 industrie, 4, 722-30 (1920). (11) Itodman, Chem. 6 X e t . E ~ Q 35, . , 221-3 (1928). (12) Ypcrry. Combustion, 10, 27-33 (1939). (4)
RECEIVED September 6, lQ47. Presented beiore t h e Division of Water, Sexyage, and Sanitation Cherni>try a t the 112th Meeting of the .\VEP.ICAN C R E M I C ASOCIETY, L S e x S o r k , S . I'.
Butanol-Acetone Fermentation of Wood Sugar REID H. LEONARD AND W. H. PETERSOY
GEORGE J. RITTER
Cniversity of FVisconsin, .Madison, W i s .
Cnited States Forest Products Laboratory, Madison, Wis.
Wood h3droljzates were fermented with Clostridium b u t y l i c u m No. 39 to butanol and acetone. The wood species and method of h?droljsis affect the fermentabilitj of the liquors. I er) mild or i e r ) \igorous conditions of hjdrol?sis do not produce an e a s i l ~fermentable solution. Complete utilization of sugar could be obtained up to 3Yo concentratioiis. Solient jields ranged from 21 to 38Y' of the sugar fermented.
by the JIadison wood sugar process as described by Harris and Beglinger (4:. These samples xere neutralized to pH 4.2 with lime at 138" C. (4, 7 ) . The oak sample represented the first 2 5 5 of the hydrolyzate ~eceivedfrom the digester, and the Douglas fir liquor was from a normal run.
T
HE fermentation of vvood hj-drol>-zatesby butanol-acetone
bacteria is difficult. Sjolander, Langlykke, and Peterson (10) conducted butanol-acetone fermentations on hydrolyzates prepared by a method similar to the Scholler process arid obtained a fermentable medium aftcr precipitation of metals a t pH 10, neutralization, and clarification with Sorite decolorizing carbon. I n the present paper other types of hydrolyzates have been studied, and attempts have been made to simplify the pretreatments for fermentation. Two cultures had previously been selected for wood sugar fermentations: Cl. felsineum Carbone S o . 41 by Sjolander et al. (IO) for hydrolyzates and Cl. butylicum (Fitz strain) No. 39 b y Wiley et al. (11) for sulfite waste liquor fermentation. These two cultures, as well as Cl. butylicum S o . 37, Cl. beijerinckii No. 67, and C1. butylicun KO.69, were compared on wood hydrolyzate, and No. 39 was selected as the most suitable organism. Fermentations were conducted with cultures transferred three t o five times from the spore stock. Iiutrients were supplied by 1%malt sprouts and 0.1% (NH4):HPOd. A trace of reduced iron was added t o the media before autoclaving. From 0.1 to 0.3% calcium carbonate was added to the media after inoculation. Inoculum was produced on glucose-malt sprouts medium and used after 12 t o 20 hours a t 870 of the fermentation volume. Determination of reducing sugars was made b y the method of Shaffer and Somogyi (9); furfural by a colorimetric method ( 1 ) ; ethanol and butanol by Johnson's procedure (6); and acetone by Goodwin's method (3). Volatile acids were determined by titration of 11 volumes of distillate from 1volume of sample. Hydrolyzates of maple and spruce were prepared in a rotary digester similar to t h a t described by Plow et al. (8). RIaple sawdust was hydrolyzed by 3% sulfuric acid with a n acid-wood ratio of 1 : l a t 181' C. for 30 minutes. A milder hydrolysis of maple and of spruce was made with 1.8% acid, ratio 1 : l at 173" C. for 5 minutes. Oak and Douglas fir were hydrolyzed
FACTORS AFFECTIKG FERMESTATIOX
I t was first desirable to repeat the results of Sjolander et al. In their work the amount of decolorizing carbon was not stated, arid it was found that n-ith maple hydrolyzate, folloving their procedure, 10 to 20 grams of Sorite decolorizing carbon per 100 nil. n-cre required to duplicate their fermentation results. Fermentation of high temperature maple hydrolyzate prepared horved that 92% of the sugar in a -1.04 grams per 100 nil. solution was fermented in 5 days. The quantity of decolorizing carbon was found to be important; the use of 1 gram per 100 nil. resulted in the fermentation of 2 5 5 of the sugar, 5 grams gave 6-15, 10 grams gave 72y0,.and 20 grams gave 937,. .iftcr the furfural vas removed from the sample, the quantity of carbon required was decreased to less than 5 grams per 100 ml. The pH was found to be important, since 1 gram of carbon a t p H 2.0 gave 17% fermentation, while 1 gram at p H 6.8 gave 51 7,. The difficulty found with maple wood hydrolyzates was also found with spruce, Douglas fir, and oak. The fermentations were characterized by a long induction period and a slow sugar utilization. I n some samples much of the inhibition could be accounted for by the presence of furfural-for example, the maple hydrolyzate made a t 181" C. contained from 0.5 to 0.8 gram of furfural per 100 ml. On synthetic medium 0.1% furfural decreased the fermentation b y 15%. Hydrolyzates containing more than 0.1% furfural stopped the development of the bacteria completely. Furfural was removed easily by distillation or by passing the liquor through a steam stripping column. When furfural was added back to the stripped liquor, t,he fermentations were not inhibited t o the same extent as initially. This indicated t h a t substances other than furfural were removed by the distillations. When the concentration of the inhibitory substances was decreased b)- dilution of the liquor, the extent of fermentation was improved. Complete fermentation of sugars in wood hydrolyzates v a s usually obtained a t about 3% concentration. With glucose-malt sprouts medium 5% concentration was about, the maximum quantity which could be completely fermented with
INDUSTRIAL AND ENGINEERING CHEMISTRY
1444
TABLE I. E F F ET~OF DILUTIOS o s FERMESTATIOS Fermentation Period, Hr.
Source of Sugar (;lucose
90
Xylose Glucose a n d xylose, 1: 1 Spruce. 173' C Maple, l i 3 ' C. Maple, 181'
f'.
90 65 65 65 120 120 120 120
90
90 65 65 65
Oak
Initial Sugar, G./100 M I . 6.50 4.95 4.00 4.00 4.00 6.49 4.85 4.65 3.90
4.65 3.45 5.45 4.16 3.12
Amounc Fermentedu,
R 81 99 98 49 95 3ib 62b 44c 83C 71 86 dY 86 91
65 3.9: 40 Media co1:tained less t h a n 0 02% furfural except for cases noted h e h a .) Media contained 0.03% furfural. e Media contained 0.09C; fiiriural.
I h u g l a s fir 'I
the organism used. Xylose did not ferment so well as glucobt! and usually gave acids rather than neutral products. \\700tl sugars were more difficult t o ferment, and frequently a quaiitit!. of sugar corresponding to the hexose fraction, as determined by yeast fermentation, was utilized, and the remainder \\-as l(3f1 untouched. D a t a are shown in Table I. Since furfural was a minor constituent, of these liquors, most oi' the inhibition results from the action of other substances. The d a t a are too diverse to permit definite conclusions. The soitwoods could contain wood oils, b u t spruce was found to ferment more easily than maple or oak, and Douglas fir was more difficult than either oak or maple. -1partial explanation may be fount1 in the fact that, under comparable conditions of hydrolj maple produces more pentose, furfural, and furfural degradation products than does spruce. I n the vertical hydrolyzer the suga1.k are subjected t o somewhat more vigorous conditions than in thc. single stage hydrolysis. Alkaline precipitation \vas firat applied as a means of removiiiy metals (10). It may also produce reducing substances from sugars ( 7 ) . If reduction were a prime factor for induction of the butanol fermentation, then the addition of a reducing agent other than reduced iron would be favorable. Some results are show1 in Table 11. T h e reduced iron can be replaced with sodium sulfite. Alkaline precipitation usually improved fernientations containing reduced iron. Metals were not present a t inhibitory concentrations in the liquors used. The action of the alkaline precipitation in improving the fermentation is difficult to explain; it ma>result from the removal of some type of solid indicator acid coming from degradation products of furfural as dencribcd 1)y Dunlop, Stout, and Swadesh ( 2 ) .
ASD Itcur-crsc, TABLE 11. EFFECTOF XLK.ILISE PRECIPITATIOS
AGESTSos FERMESTATI~S
.J.:noullt
Treatmenta
Source of Liquor
Kone Alkaline p p t n . None Alkaline p p t n . Alkaline pptn., iron omitted Kone Added 0.1% SaPSOs Alkaline p p t n .
Maple, 173' C.
Initial Sugar, G./100 111.
I,'erinented,
Vol. 39, No. 1 1
Various other methods have been tried for iinpruvemeiit of the liquors for fermentation purposes. Precipitation tvith zinc, lead, barium, and iron salts were of no use. Clarification procedures employing carbons, lignin, sawdust, and proteins were uscful only when the agents were applied at 1 t o 2OC; concentrations. The inhibitory substances are not present in maple n-ood txfore hydrolysis, since u p t o 10 grams of sawduet per 100 nil. did not harm the fermentations. The inhibitory materials originate during hydrolysis and, since they are not metals, must come from the wood. In nearly every case yeast fermented the hydrolyzates more easily than did bacteria. For example, yeast fermented 63("C of the sugars in maple liquor and S o . 39 only 23%. it is nccessary to employ large amounts of yeast for in the alcohol fermentation; this is not a feasible procedure in a tiacterial fcrmrntation. EFFECT OF HYDROLYSIS
In order to obtain information on vihy the hydrolyzates were so difficult to ferment, the effect of the method of hydrolysis was 5tudied for a simple case. Previous demonstrations of the effect of hydrolysis on fermentation have not been clear1 Russian workers, Zubkova, Bochukova, and Zats (1 summarized the difficulties of relating the fermentation data with the method of hydrolysis and suggest t h a t the optimum fermentation conditions should be determined for each type of liydrolysis procedure. Harris et al. (5) published fermentation d;tta for various hydrolysis procedures. However, the drastic conditions required to hydrolyze wood produce so much inhibitory material in the liquors t h a t any hydrolysis variables t h a t might affect the fermentation are not evident. For the most part the only knoivn fermentation differences are dependent upon the total yield in quantity of sugar and the relative composition of fermentable hexose and pentose.
TABLE 111. EFFECTOF PERIOD OF HYDROLYSIS o s EXTENTO F FERJIESTATIOX Period oi hydrolysis, Inin. 0 Products, ' 2 of d r y ivood'l I n w l re-idue 71.5 Redurine sugar as glucose 15.3 Furfural 0.0 Yolatile acid as aretic 3.6 Other so1 -ub*rance, difference 9.2
.\foistlire i i i pre+ ciike, ; Hydrolyzate I i q ~ i i nIh. , C o m p n . of hydrolyzate, g. 100 nil. Reducing sugar a - glacose I'uririrnl Yolatile acid as ttcetir
41.5 14.0 10.9 0 02 2.3
10
30
90
66.1 18.8 2.5 4.5 8.1
58 0
64 6 11 5
47.4 21.6 8.T 0.28 1.7
15.0 4.4 6.0 17.6 49 0 21 2
4 0 4 9 15 0 50.4 23.6
7.1 0.42 1.8
4.3 0.35 1.4
Extent uf fer:nentatio:i :w ' ; of iiiear 94
75
95 95
64 57 50 45 30
92 81 72 63 25
96 96 85 79 70 63 28
96 9e 86
76 67
..
l i e l d of neutrnl solvents. ' of Ier28 inented s u g a r l l a p l e sawdu>t, 3'; arid o n wood, 1 : l water-wood ratio, 181" C.
5~~
Spruce, 173' C
Douglas ti,
0.16 6.16 5.04 5.04 5.04 3 95 3 95 3 95
~~
7
33
:: 6%
40 59 87
Sone Oak 5.45 58 Kone 5,PD 78 5.4: 60 Added 0 . 1 % TazSOa Added 0.1% SazSOa, 5.45 61 iron omitted Alkaline p p t n . 5.4,j 52 0 Furfural-free medium containing reduced iron unless otherwise indicated.
.Sftcr preliminary ~ o r kfour hydrolyses m r e carried out on bark-free air-dry maple sawdust. Hydrolysis of 10 pounds of sa\Tdust \vas accomplished with 3 ' 3 sulfuric acid (on weight of wood), a ratio of acid solution to wood of 1: 1 a t 181 C. The hydrolysis period was varied from 0 to BO minutes; the 0-minute period v a s obtained by heating the charge to 181 and then bloxing immediately. With a preheated digester about 1 minute was needea to reach the required temperature with occasional venting of gases during this period. T h e temperature \ m s maintained by manual control. The digester was relieved through a condenser capable of permitting a pressure drop to atmospheric
November 1947
,
INDUSTRIAL AND ENGINEERING CHEMISTRY
in about 2 minutes without loss of vapors other than noncondensable gases. T h e contents were removed quantitatively. When the pressure a-ithin the digester was lowered rapidly, many of the wood particles were ruptured by expansion of steam inside the wood, and considerable mechanical disintegration a n d mixing occurred. The wet residue was pressed in a lever-action screw press until all possible liquor was removed. Comparison of free-draining hydrolyzate with the press liquor shoived t h a t , with rapid blowdown of the digester, the two liquors were nearly identical. Analytical values per 100 ml. of drainings and press liquor were, respectively, 7.08 and 6.93 grams of sugar, 1.05 and 1.05 grams of sulfuric acid, 1.46 and 1.46 grams of volatile acid as acetic, and 0.56 and 0.36 gram of furfural. Since agreement was good, the t x o liquors were mixed together. I t was assumed, therefore, t h a t the residual water in the press cake contained the same concentrations of sugar and other substances that n-ere found in the press liquor. I t n-as possible t o calculate from the data the percentage composition of the hydrolysis products formed from the wood. Results are shown in Table 111. T h e hydrolyzates were steam-stripped to less than 0.05% furfural, neutralized with lime to pH 6.8 and filtered. Dilutions to various initial sugar concentrations Tvere made, and then the samples were fermented for 5 days. The amount of sugar fermented in duplicates was espressed as per cent of the initial sugar content. The results arc shown in Table 111. The four hydrolyzates all fermented to the same extent with 1% initial sugar concentration. S o appreciable quantity of polysacchai,ides could have been present. 3Iaximum yield of sugar and fermentability were obtained with the 10-minute period. Hydrolysis for periods longer than 10 minutes decreased the fermentability, the yield of sugar, and the insoluble residue. As the period of hydrolysis n-as iucrea.sed, the nioisture content of the press cake, the per cent of furfural, and the per cent of unidentified soluble products increased. Explanation of the poor fermentability of the 0-minute cook is based on the degradation products froni the xvood. Since the furfural formation was small, the substance causing this early inhibition must have been derived from degradation of noncarbohydrate material. I t vias found that the residue after hydrolysis had n o detrimental effect when added t o the fermentation medium; in fact, in some cases the addition improved the fermentation. The improved fermentability a t 10 minutes would indicate that these first-formed substances rvere destroyed with extended hydrolysis. As the hydrolysis period vias lengthened further, the carbohydrate degradation t o furfural proceeds, and the furfural is apparently decomposed gradually with the formation of a second class of inhibitory materials.
TABLEIv.
PRODUCTS CfiLCULATED PER TON OF WOOD
Utilization of niaple wood by fermentation of single-stage hydrolyzates n-ould require, per ton of dry wood, 60 pounds of sulfuric acid, 100 pounds of hydrated lime, 1 0 pounds of malt sprouts, 4 pounds of dibasic ammonium phosphate, and 12 pounds of calcium carbonate. The yield of neutral solvents varied be-
SOLVESTS
FROM \f*OOU
8l.ti.4R
FERMENTATIOKS
Sugar Initial FerAcid a8 Source of Sugar, mented, Se'tra' Solrents, G . l L . 1-ieldh, -\cetic, G./L. Butanol Ethanol Acetone % G./L. Sugar' N a p l e , 181' C. 5 1 . 0 85 9.5 0.3 4.3 33 .. . XIaple, 181O C. 4 3 . 5 68 6.07 0.05 3.45 32 ,, Maple, 173' C . 3 6 . 7 88 9.0 0.1 3.4 38 ,, Spruce, 173' C. 5 3 . 8 04 .. .. 35 .., 0.15 3.41 25 0 6 Oak 52.0 79 6.78 00 6 . 1 9 0 . 3 9 Oak 54.5 2.92 2q 0 9 91 4.04 0.22 Oak 31.2 1.17 19 3.7 Douglas fir 39.2 40 3.21 0.22 1 39 32 0 5 Douglas fir 31.6 83 4.34 0.28 1.86 23 2.6 4 Liquor stripped free of furfural (less t h a n 0.03C,:, neutralized with lime t o pH 6.5. b As per cent of sugar fermented.
.
..
.
trveen 75 and 100 pounds pel' ton. I n addition about 50 pounds of furfural and 120 pounds of calcium acetate are present, in the neutralized hydrolyzates from which the furfural must be renioved before fermentation. Ctilization of Douglas fir hydrolyzate from the Madison process ( 4 ) yielded, on the basis of 50% conversion of ivoocl t o sugar, about 200 pounds o f neutral solvents per ton of wood. The method of hyilrolysis is a most important factor in determining the final j-ieltl of solrents from wood sugars. SU\IMARY
Wood hydrolyzates have been fvrinented to butanol arid acetone JTith CZ. butyl2'eoni S o . 30. Several suitable methods havr bcen used t o prepare the hj-drolyzates for fermentation; t h r simplest, was to remove the furfural by distillation and neutralize the liquors to pH 6.5 ITith limr. The efftxt of hydrolysis on fermentation has been denionstrated for the temperature variable in a single-stage hydrolysis procedure. . It1% concentrations of sugar all samples fermented to the same extent. The most poorly fermented hydrolyzate was obtained at 0-minute hydrolysis. hbove 3 q sugar the rstent of fermentation decreased with increasing period of hydrolysis. Yields of neutral solvents ranging from 21 t o 38% of the sugar fermented have been obtained. ACKNOWLEDGMENT
The authors are indebted to Elizabeth McCoy of the Department of AlgriculturalBacteriology for t,ransfers of the cultures. Part of the maple hydrolyzates were kindly supplied by C. 0. Guss and the oak and Douglas fir samples by E. E. Harris of the Cnited States Forest Products Laboratory.
SOLVEST YIELD
The yield of solvents from fermentation of n-ood hydrolyzates m-as low, since it {vas difficult to obtain complete fermentation of reasonably high sugar concentrations. Frequently the fermentations become acid and stop. Some typical results are shon-n in Table IV for fermentations of furfural-free liquor neutralized t o pII 6.8. S e u t r a l solvents varied from 24.5 to 38.57, of the sugar fermented, or from 5.6 to 14.1 granis per liter. Apparently it vias more difficult t o obtain a high yield of solvents from the hydrolyzates niade a t higher temperatures or acid concentrations than from those made under milder conditions of hydrolysis.
T I E L D OF
1445
LITERATURE CITED
Duncan, I. J., ISD.ESG.CHEX.,Asar.. ED.,15, 162 (1913). Dunlop. -1.P., Stout, P. R., Swadesh, S.,ISD. ENG.CHELi., 38, 705 (1946). Goodwin. L. F., J . -4m. Chem. SOC.,42, 39 (1920).
Harris, E. E., and Beglinger, E., ISD.ENG. CHEN.,38, 890 (1946).
Harris, E. E., Beglinger. E., Hajny, G . J., and Sherinrii. E. C., I b i d . , 37, 12 (1045). Johnson, 11.J.. ISD.ESG.CHBY., . ~ s . L L . ED.,4, 20 (19:IL'r. Leonard, R. H., and Hajny, G . J., ISD. ESG. CHEAr.. 37, 390 (1915). Plow, I t . H., T u r n e r , H. D . , Saemsn. J. F., and Slierrntd. E. C . , I b i d . , 37, 36 (1945). Shaffer,P. A., and Somogyi, AI., J . B i d . Chem.. 100, 6% (1933). Sjolsnder, S . 0.. Langlykke, -1.F., and Petersoti, Ti-. I I . , ISD. ESG.C H E Y . , 3 0 , 1251 (1938). Kiley, A. J., Johnson, XI. J., lIcCoy, E., and Peterson, TI-. II., Ibid.. 33, 606 (19411. Zubkoi-a. S. R., Kochukova, N. R., and Z s t s , 11.R., Biobhimi y a , 1, 49 (1836). RECEIVED S o v e m b e r 4, 1946.
Published with the approval of the Director
of the Wisconsin Experiment Station.
