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INDUSTRIAL AND ENGINEERING CHEMISTRY
(10) McRorie, R. A , Mosley, P. hl., and Williams, 1 %'. L., Arch. Biochem., 2 7 , 4 7 1 (1950). (11) Nelson, H. A., Calhoun, K.-M., and Colingsworth, D. R., Abstracts, 118th Meeting ACS, p. 16A, 1950. (12) Novak, A. F., U. S. Patent 2,447,814 (Aug. 24, 1948).
(13) Pfeiffer, V. E., Tanner, F. W., Jr., Vojnovich, C., and Traufler, D. H., IND. ENG.CHEM.,42, 1776-81 (1950). (14) Rasmussen, R. A., Smiley, K. L., Anderson, J. G., Van Lanen, J. M., Williams, W.L., and Snell, E. E., Proc. SOC.Expt. Biol. )Wed., 73, 658-60 (1950). (15) Schaible, P. J., ChemuigicDigest, 9 , No. 4, 6-9 (1950). Wright, L. D., and Bosshardt, D. K., (16) Skeggs, H. R., Huff, J. W., J . Bid. Chem., 176, 1459-60 (1948).
(17)
Vol. 43, No. 6
Snell, E. E., Brown, G. M., Peters, V.J., Craig, J. A., Wittle, E. L., Moore, J. A., McGlahon, V. M., and Bird, 0. D., J . Am.
Chem. SOC.,72, 5349 (1950). (18) Stiles, H. R., U. S. Patent 2,483,855 (Oct. 4, 1949). (19) Stokstad, E. L. R., and Jukes, T. H., Proc. Soc. Expt. B i d Med., 73, 523-8 (1950). (20)
Wickerham, C. J., Flickinger, M .H., and Johnson, R. PI.,Arch.
(21)
Wlliams, W.L., Hoff-Jorgensen, E., and Snell, E. E., J . Biol.
Biochem.,9 , 9 5 - 8 (1946). Chem., 177,933-40 (1949). RECEIVED October 27, 1950. Presented before the Division of Agricultural and Food Chemistry a t the 118th Meeting of the AMERICANCHEMICAL SOCIETY,Chicago, Ill.
Thermophilic Fermentation of Cellulosic and Lignocellulosic Materials GEORGE J. HAJNY, C. H. GARDNER, AND GEO. J. RITTER Forest Products Laboratory, U . S . Department of Agriculture, Madison, Wis. Research was undertaken on thermophilic cellulose bacteria i n order to find new methods for the utilization of waste wood. A thermophilic culture of cellulose bacteria isolated from garden soil was found to ferment a wide range of carbohydrates, comprising the simple sugars, hemicellulose, holocellulose, cellulose, and part of the cellulose in untreated wood. The main products of the fermentation were acetic, butyric, and lactic acids in yields approximating 5070 of the carbohydrates consumed. Carbohydrate material in the untreated wood was not fully utilized. The yield of acids calculated as acetic acid from untreated sweet gum sawdust w-as 15.970, based on the weight of the dry wood. In sweet gum wood previously given a mild acid hydrolysis, the yield of organic acids calculated as acetic acid was 26.4% of the wood. Pretreatment of delignified cellulose with dilute acid at high temperatures decreases the rate of fermentation of the residual cellulose. Studies on the viscosity and alpha-cellulose content of the cellulose residue at various stages of the fermentation strongly suggest a gradual shortening of the cellulose chain rather than a random splitting of the cellulose molecule. Contrary to much of the reported work in this field, it has been shown that wood can be attacked by microorganisms to produce useful products.
M
ACFSYDEN and Blaxall(6) were the first investigators to repoSt on the thermophilic fermentation of cellulose ; since that time the subject has received considerable attention. Until recently it had been generally accepted that highly lignified material such as wood could not be fermented by thermophilic bacteria. Thus Langwell ( 4 ) states that typical ligno- and cutocelluloses are not fermentable and only become fermentable in proportion as the cellulose is liberated from combination. Olson, Peterson, and Sherrard (6) found that small amounts of lignin in a wood pulp inhibit fermentation and to obtain a readily fermentable pulp the lignin content had to be less than 1%. Acharya ( I ) , using mesophilic organisms on rice straw and other materials, found that resistance to fermentation varied with the degree of lignification of the material. Fontaine ( 2 ) , too, found t h a t finely ground wood saxdust was not fermentable, whereas holocellulose, the total carbohydrate portion of wood, was readily fermentable. On the other hand, Virtanen and coworkers (16-16) have stated that finely ground sawdust can be fermented by their cultures of thermophiles. In fermentation periods of 3
t o 4 weeks, as much as 68% of the cellulose and 87% of the pentosans in the wood were consumed. Research was undertaken a t the U. S. Forest Products Laboratory to determine the feasibility of fermenting cellulose and wood by means of thermophilic bacteria. A thermophilic culture supplied by A. I. Virtanen was used in the experimental work reported on in the present paper. This preliminary report comprises only the more pertinent results that have been collected up to the present time in small scale laboratory thermophilic fermentations. The authors are unprepared a t this stage, to undertake trial fermentations of a promiscuous variety of cellulose material. Fundamental research that is essential before commercial applications can be considered is being carried on a t the Forest Products Laboratory. MATERIALS AND METHODS
The culture comprised a mixture of organisms of differing morphology; three types of bacteria were present. One type was a slender, filamentous rod often bent or curved ; the second was a shorter, thin rod bearing a terminal spore; and the third a bacillus-like organism with a subterminal t o terminal oval spore. Under anaeorbic cultural conditions the bacillus-like form disappeared. Since the general activity of the culture for cellulose fermentation was maintained, the two forms remaining were considered the important types to study. The long, thin, often-bent rod type of bacteria occasionally forms long chains. I t has a diameter of about 0.3 micron and a length varying from 1 to 10.4 microns. The results of the Gram test are variable; the younger cells generally being Gram-positive, whereas in older cultures they are Gram-negative. Spore formation has not been observed, but is presumed to be present since the organisms survive boiling temperatures and remain viable over extended periods in soil suspensions. The thin, straight, rod type of bacteria is Gram-positive with a terminal spheroidal spore. It has a diameter of 0.4 micron and a length of 1.4 to 8.1 microns. The diameter of spores in sporangia is from 0.6 to 1.5 microns. Spores are readily formed in the culture medium. Their formation is induced by allowing double the time necessary for the completion of visible fermentation. The cultures are then removed from the incubator and placed on a suitable medium. Sterile soil to which a little calcium carbonate was added proved satisfactory. Stocks prepared in this way have remained fully viable for more than a year.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
The culture is anaerobic and thermophilic. It grows well in deep culture and in the complete absence of air a t temperatures from 55 to 63' C. Above 65 O C. the fermentation weakens and a vigorous culture cannot be maintained. At 50" C. no fermentation could be detected in a period of 3 weeks. In the work reported here, all cultures were incubated a t a temperature of 60" C. The basic medium used in this work was that of Viljoen, Fred, and Peterson as modified by Tetrault (11). Its composition is as foll0.u.s: *
I
Two grams of sodium ammonium phosphate, 1 gram of potassium dihydrogen phosphate, 0.3 gram of magnesium sulfate, 0.1 gram of calcium chloride, 5.0 grams of peptone, 30 grams of cellulose or other carbohydrate material, 1000 ml. of t a p water, and 15 to 30 grams of calcium carbonate. This medium was used in all work unless otherwise stated, The pH of the medium a t the start of fermentation was 7.8. At the completion of fermentation the fermented liquor had a p H of 6.9. The cellulose used to carry on the vegetative culture was added as ground filter pa er prepared by passage through a Wiley mill. When wood sawfist was used as a substrate, it, too, was ground in a Wiley mill. Aspen and maple hemicellulose, which were used in several fermentations, were prepared by the method of Rogers, Mitchell, and Ritter (8). Fermentation was allowed to proceed until no further evidence of gassing was seen. This usually required from 5 to 7 days with cellulose as the substrate. Inoculums were prepared by adding a small amount of the spore stock to 20 ml. of the basic cellulose medium. Heat shocking was usually not necessary. When the cellulose was being actively fermented, usually in 48 to 72 hours, 5 ml. of the fermenting liquor were transferred to 100 ml. of fresh medium. Fermentations were carried out, in general, in 500-ml. Erlenmeyer flasks containing 300 ml. of medium, except when larger scale fermentations were made, as will be discussed later. Routine analysis was made of the fermented liquors for volatile and nonvolatile acids. The volatile acids were removed from the liquor by steam distillation and determined by a modification of the method of Virtanen and Pulkki (16). Nonvolatile acid in the residue from steam distillation was extracted with ether, titrated with standard sodium hydroxide solution and calculated as lactic acid. It was identified as lactic acid by the preparation and identification of the paraphenylphenacyl ester.
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TABLE I. PRODUCTION OF ORGANIC ACIDSBY ACTIONOF THERMOPHILIC
BACTERIA ON SUGARS AND HIGH MOLECULAR WEIGHT CARBOHYDRATES AT 60' C. Concn. of Sugar
,:!,
Acetic- Yield of Acid Produced, G./100 M1. ~ ~ ~ t~ G./lOO Total Acid Acids, Substrate M1. Acetic Butyric Lactic Acids Ratio %a 56.3 2.98 0.480 0.225 0.973 1.678 2.13 Glucose 52.5 2.98 0.577 0.268 0.717 1.562 2.15 52.4 2.72 0.656 0,288 0,484 1.428 2.28 52.3 2.72 1.046 0.215 0.160 1,421 4.86 49.1 3.05 0.373 0.583 0.544 1.500 0.64 Galactose 49.6 3.05 0.411 0 , 6 0 3 0,500 1,514 0.68 2.52 0.302 0.154 1,060 1.516 1.96 60.0 Mannose 76.8 2.52 1.85 0.360 0.195 1.380 1.935 51.9 1.01 0.382 0.379 0.880 1.641 3.16 Xylose 1.00 46.4 0.356 0.355 0.754 1.465 3.16 48.3 0.71 0.325 0,455 0.668 1.448 3.00 Arabinose 48.5 1.02 0.379 0,370 0.706 1.455 3.00 58.3 0.298 1.138 0.315 1.751 0 . 9 5 3.00 Sucrose 52.3 0.671 0.241 0.656 1.568 2.78 3.00 47.3 0.314 0.326 0.778 1.418 0.97 3.00 Starch 45.2 0.555 0.567 0.232 1.354 0.98 3.00 32.3 0,400 0.328 0.187 0.915 2.83 1.20 Commercial 47.8 0.506 0.736 0.114 1,356 0.69 hemicellulose 2.83 44.0 0.460 0 , 6 9 8 0.106 1 , 2 4 4 0.66 2.83 (pretreated by bisulfite) 0.550 0.629 0.023 1.202 0 . 8 8 43.2 Maple hemicel- 2.78 0.443 0,640 0.051 1.134 0.69 40.8 lulose 2.78 51.2 As; )en hemicel- 2.73 0.286 0.811 0.302 1.399 0.35 0.405 0.750 0.125 1.280 0.54 46.9 11ulose 2.73 0.359 1.033 0.049 1.441 0.35 48.0 )ha cellulose 3.00 Alr: 51.7 0.399 1.090 0 , 0 6 3 1,552 0.37 3.00 0.635 0.720 0,060 1.415 0.88 47.2 3.00 a Yield of organic acids based on weight of carbohydrates in the medium
value decreases from 0.484 to 0.160 gram. Gain in the acetic acid is equivalent to 0.390 gram and the combined loss of butyric and lactic acid is approximately equivalent t o 0.397 gram. The results suggest that, after the sugar had been converted to acids, the bacteria began converting butyric and lactic acid to acetic acid,
RESULTS AND DISCUSSION
4
Since the object of this research was to ferment cellulose and the cellulose in wood by means of thermophilic bacteria, it seemed desirable to secure information on the fermentability of the individual sugars that are formed from the polysaccharides in wood on hydrolysis-namely, glucosq, mannose, xylose, galactose, and arabinose. As recorded in Table I, all these sugars were found to be fermented by the thermophilic bacteria. The yields of organic acids obtained were approximately 50% of the original sugar, with the exception of mannose, from which the yield of acid ranged between 60 and 77%. Sugars. Data in Table I show variability in the amouQt of organic acids produced during the fermentation of the sugar. In the first two fermentations of glucose, the two glucose substrates in the medium weighed 2.98 grams. During the fermentation of the two substrates the bacteria produced acetic, butyric, and lactic acid. When the fermentations were halted the acetic and butyric acid values were 0.480 and 0.225 gram, respectively, in the first fermentation as compared with 0.577 and 0.268 gram, respectively, in the second fermentation. I n contrast, the lactic acid values show a decrease from 0.973 to 0.717 gram from fermentations 1 to 2. Also, a glucose substrate of 2.72 grams was used for each of the third and fourth fermentations. The third fermentation was halted when gassing ceased after a total fermentation time of 7 days, but the fourth was allowed to stand in the incubator for a total of 11 days, although gassing had stopped a t the same time as it had in the third fermentation. An acetic acid value of 0.656 gram for the third fermentation compared with 1.046 grams in the fourth fermentation shows a substantial increase. In contrast, the butyric acid value shows a decrease from 0.288 to 0.215 gram between fermentations 3 and 4, and likewise the lactic acid
FEUMENTATION
TIME DAYS)
Figure 1. Relation of Reducing Sugar Conoentration and Organic Acids to Fermentation Time for Aspen Wood Sugar
On the other hand, it appears from other data that lactic acid can be converted by bacterial action t o both acetic and butyric acid. As shown in Figure 1, the peak of lactic acid production from aspen mood sugar occurred a t 7 days, a t the time the sugar residue had almost reached its minimum level. From then on, the gain in acetic and butyric acids approximated the loss in lactic acid, and the sum of the three acids remained constant. For example, the seight of the three acids recorded on the vertical right-hand ordinate is 1.70, 1.72, 1.69, and 1.66 grams after 7, 8, 10, and 13 days fermentation, respectively. In fermentation 4 of glucbse (Table I),the substantial increase of acetic acid a t the expense of butyric and lactic acid resulted in a high acetic-butyric acid ratio of 4.86 as compared with approximately 2.2 for the corresponding ratio in fermentations 3, 2, and 1 for glucose. The acetic-butyric acid ratio from mannose approxi-
t~
~
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INDUSTRIAL A N D E N G I N E E R I N G CHEMISTRY
TABLE 11. EFFBCT OF FERMENTATION TIMEON AMOUNTSOF ACIDS PRODUCED FROM COMMERCIAL HEMICELLULOSES BY ACTIONOF THEILVOPHILIC BACTERIA AT 60" C. (Hemicellulose substrate equal to 2.83 grama per 100 ml. of medium) Fermenta$ion ~ i Acid ~ Produoed, ~ , G./100 M1. % in Total Acids yie;d, % Days Acetic Butyrio Lactic Total Acetic Butyric Lactic 4 0.362 0.334 0.237 0.933 38.8 35.8 25.4 32.9 5 0.362 0.378 0.239 0.979 37.0 38.6 24.4 34.5 7 0.399 0.470 0.228 1.097 36.4 42.9 20.8 38.7 8 0.407 0.491 0.189 1.087 37.4 45.2 17.4 38.4 11.6 41.5 12 0.475 0.566 0.136 1.177 40.4 48.1 0.487 0.570 0.120 1.177 41.4 48.5 10.2 41.5 13 18 9.5 42.8 0.518 0.378 0.115 1.211 42.7 47.6 0.540 0.624 0.098 1.262 42.7 49.4 21 7.8 44.6 0.546 0.635 0.089 1.270 43.0 50.0 25 7.0 44.0 8.0 47.5 0.585 0.653 0.108 1.346 43.5 48.5 28 a
Percentage yields based on weight of oven-dry hemicelluloses in rnediuni.
mates 1.9, whereas those from galactose, xylose, and arabiuosct appear to approximate 1.0 or less. Cellulosic Materials. Theoretically, the cellulosic materials listed in Table I may be considered as intermediates between sugars and wood, which are lignocellulosic materials. The starch, the maple and aspen hemicelluloses, and the alpha cellulose were prepared in the laboratory and were lignin-free. The four materials were fermented readily by the thermophilic bacteria. I n contrast, the commercial hemicelldose, which was a by-product of a wood-conversion operation, contained considerable noncarbohydrate material, some of which appeared toxic to the thermophilic bacteria. Two methods were developed to overcome the toxic property of the commercial hemicellulose, after which the material fermented readily: The first method involvcd a steam distillation of a water fiolution of the commercial hemicellulose a t a constant volume. The second was simpler than the first; a quantity of sodium bisulfite equivalent to 0.3% of the hemicelluloses was added t o the aqueous solution and the mixture brought t o a boil. No doubt the two methods can be simplified further. The five carbohydrate materials are polymers of anhydrosugars and must undergo more complicated reactions than the sugars. The reactions seem to involve initial bacterial hydrolysis into sugars and then bacterial conversion of the sugars into acetic, butyric, and lactic acid. Results of the conversion of the starch, the hemicelluloses, and the cellulose are given in Table I. The data indicate that the thermophilic bacteria, in general, produce a higher proportion of butyric acid and a lower proportion of lactic acid from the cellulosic materials than from sugars. The ratio of acetic acid to butyric acid, in general, is lower from cellulosic materials than from sugars. Yields of the total acids produced in the best fermentations of the cellulosic materials approximate 50.0'%. The values of lactic acid are erratic from the apparent duphGate fermentations of starch, commercial hemicellulose, and aspen hemicellulose. With no data to the contrary, these variations in lactic acid production were assumed t o result from differences in the duration of the fermentations. It is not a simple matter to confirm or disprove thip assumption. The term "fermentation duration" involves more than actual time by the clock; it comprises such factors as lag time, vigorous fermentation time, and the total incubation period. The question inevitably arises, whether actual or only approximate duplicate fermentations can be performed. Relation of Time of Fermentation to Lactic Acid Yield. Yo far in this research experiments have been especially designed and conducted t o determine the effect of the duration of the fermentation on the lactic acid yield. The effect of other factors has not yet been determined. The effects on the lactic acid yield of the total time of fermentation are shown in Table 11. These data were obtained by setting up fermentations in which 3 liters of medium were used, in order to have ample
Vol. 43, No. 6
supplies for the periodic withdrawal of samples on which the amounts o€ the acids were determined. The results show definitely that the yield of lactic acid decreases with increase in time of fermentation, whereas that of acetic and butyric acid increases. The data offer nothing definite on the fate of the lactic acid that disappeared during prolonged fermentation. Some data were obtained by experiments in which calcium lactate was inoculated with the thermophilic bacterial culture and allowed t o ferment. Analysis showed t h a t the fermented medium contained only acetic acid in yields of 63.0% of the lactic acid in the original calcium lactate. The increase of butyric acid was thus not accounted for. Variation in Concentration of Substrate. Information on the relation between the cellulose substrate concentration in the medium and the maximum acid production by the bacteria is highly desirable for economic reasons. Available time, however, permitted only scout experiments on this phase of the work. Concentrations of 1.0 to 8.0% of the cellulose substrate were employed in the medium. The data obtained are given in Table ITI.
T IBLE 111. EFFECT O F SUBSTRATE CONCENTHATION ON PRODTiCTION O F A C I D S
(Action of thermophilio bacteria on cellulose at 60" C.) Conon. of Cellulose Aoids Produced, *./lo0 hI1. yiey, in Medium. % =tic Butyrio Lactic Total % 0.136 0.311 0.036 0.483 48.3 0,026 0.772 0.665 0.181 38.8 0.359 1.033 1.441 48.0 0.049 1.305 0.068 1.878 47.0 0.605 2.141 1.470 0.178 42.8 0.493 0.800 0.101 1.254 20.9 0.333 0,912 18.1 0.126 1.449 0.411 Yield of acids based on weight of oven-dry cellulose in medium.
As in Table I, the acetic acid values are low compared to those of the butyric acid. Also, like the values for lactic acid from cellulose, the values for lactic acid are extremely low. A high yield of butyric acid is desirable, as demand and price for the acid are good. The trend in yield of total acids appears to be a t a maximum of 47.0 to 48.0y0 from 1 t o 4% concentration of the cellulose substrate and then decreases. Although the 42.8% yield of acids from the 5.0% substrate concentration is lower than the 47.0% yield from the 4.0% substrate concent,ration, nevertheless the 2.141 grams of the acids produced from the 5.0% substrate concentration is greater than the 1.878 grams produced from the 4.0% substrate and should receive serious consideration in production operations. Source of Nitrogen. Some source of nitrogen is necessary for conducting fermentations. In t,he medium used for t,his research, the nitrogen was supplied in t.he form of peptone and sodium ammonium phosphate. Virtanen (13) used only ainmonium chloride as a source of nitrogen and reported good results. By using Virtanen's medium, however, the authors were unable to initiate growth from spore stock. If vegetative inoculum were used, fermentation could be accomplished using Virt'anen's medium, but results were somewhat erratic. These results led to a series of experiments in which it was attempted to demonstrate the nitrogen requirements of the bacteria. Mediums were prepared each of which used only one of the following sources of nitrogen: Peptone, sodium ammonium acid phosphate, urea, ammonium chloride, ammonium sulfate, and sodium nitrate. The amount of nitrogen in the mediums waa held a t the same level as in the basic medium. Only the mediums containing peptone, ammonium chloride, or ammonium sulfate showed any evidence of fermentation. In the two mediums which contained mineral nitrogen and showed fermentation, longer lag periods were encountered before vigorous fermentation begari than in the medium that contained peptone. Another series of fermentations was run in which mineral nitrogen was substituted for part of the peptone nitrogen, with the total nitrogen in the medium remaining constant. Table IV shows that mineral nitrogen does not substitute quant,it:rtivelg
June 1951
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INDUSTRIAL AND ENGINEERING CHEMISTRY
for or anic nitrogen in so far as yields of organic acids are concerned As the pro ortion of mineral nitrogen was increased the yield of organic aci& decreased. These experiments indicated that peptone will stimulate fermentation. To check whether the stimulation was due t o organic nitrogen or growth factors, in which pe tone is quite rich, an organic nitrogen source in the form of h y g o l zed vitamin-free casein was used in several fermentations. ?yhis medium produced results as good a s the one in which peptone was used. It was accordingly concluded that growth factors are not essential for vigorous fermentation. Corn steep liquor also proved to be an excellent and economical substitute for peptone in that it furnished some nitrogen and initiated vigorous fermentation; i t is also lower in price and was used in subsequent research.
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TABLE IV. EFFECT OF REPLACING ORGANICNITROGENWITH MINERALNITROGEN ON PRODUCTION OF OSLGANIC ACIDS (Action of thermophilic bacteria on cellulose at 60° C . )
Grama of Nitrogen per Liter yield of From Organic
Medium
No.
From peptone
mineral aource
1" 2 3
.0.706
0.804
0.134
4
0.497
0.060
0,244 0.268 0.424
0.848
0.908
Acids,
%
64.2 45.8 41.5
46.5
Mineral Nitrogen Source NaNI4HP04.4HaO NHIHZPOI NaNH4HP04.4H20 (NHc)aHPOd (NHi)nHPO4
82.5 Composition of bmic medium used in most of this work. 6
a
0.683
Total 0.938 0,960 0.961 0.921
Size of Inoculum. Very small inoculums were found t o be unsuccessful in initiating fermentations. I n order t o determine the optimum size of inoculum, studies were made on the rate of cellulose fermentation in relation to the quantity of inoculum. In the fermentations the quantity of cellulose used was such that when the inoculum had been added the cellulose waa a t a 3% level. Inoculums of 1, 2, 5, 10, and 20% by volume were used. Two such runs were made and a third run, using only 10 and 20% inoculums, waB made in which the quantity of noncarbohydrate nutrients WM doubled. During the fermentations aliquots were periodically withdrawn, acidified with hydrochlorio acid to free them of carbonate, washed, dried, and weighed, and the percentage of residual cellulose was calculated. The results were judged by the time necessary to complete the fermentation and yield of acids. On this basis an inoculum of 2.5 to 5% was apparently the most satisfactory. FERMENTATION TIME [DAYS)
Figure 3. Effect of Acid Hydrolysis on Rate of Fermentation of High Alpha Cellulose Wood Pulp PERCENT ACID ACOTTON nrmoirzm
Figure 2.
d
w m
0.8
Effect of Acid Hydrolysis on Rate of Fermentation of Cotton
Acid-Treated Cellulose. Rate-of-fermentation studies were made on cellulose treated by a mild acid hydrolysis. The results show that the previous history of the cellulose has a marked effect on the rate of decomposition of the cellulose. Two types of cellulose were used in the studies: One was a cotton linter and the other a high alpha cellulose from wood pulp. The celluloses were hydrolyzed with 2.25 times their weight of either 0.4 or 0.8y0sulfuric acid a t 180"C. for 15 minutes. After the hydrolysis, they were washed with distilled water until acidfree and then with ethyl alcohol until no more color was extracted. Cotton linters hydrolyzed with 0.4% sulfuric acid gave a yield of hydrolyzed cellulose of 83% and when treated with 0.8% acid gave a yield of 77%. The yields from the alpha cellulose pulp were 83 and 67% from the 0.4 and 0.8% acid hydrolysis, respectively. These celluloses were then made up in the basic medium and their fermentation rates determined. The results are shown in Figures 2 and 3. In each case, the untreated materials required only about one half the time required by the treated material to be decompoBed completely. The reason for this behavior is ob-
scure. Acid hydrolysis should give a random splitting of the cellulose chains. The material of very low molecular weight, which became water soluble, was, of course, washed out. The residue used in the fermentation, however, should have been smaller than the original in average chain length and hence should have been more easily utilized by the bacteria, since some of the hydrolysis had already been performed. The results, of course, make this view untenable. It is now generally accepted that cellulose is made up of crystalline and amorphous portions. The amorphous portion is more susceptible t o chemical attack than is the crystallme so that on acid hydrolysis a more resistant residue is obtained. This residue might also be more resistant t o enzymatic hydrolysis than the amorphous portion of the cellulose. If this view is valid, some portion of the rate curve for the untreated material should have the same slope as a portion of the curve for the treated material, since, presumably, there is resistant or crystalline cellulose in both materials. This is not the case, since the slopes of the curves for the treated and untreated materials differ through the entire period of active fermentation. The fact t h a t the slopes of the curves for the material treated with the 0.4 and 0.8% acid are almost identical might be explained by assuming that both concentrations of acid remove all the amorphous cellulose from the samples. The removal of the amorphous cellulose by the acid hydrolysis may have an effect on the ultimate bacterial population of the cellulose fermenters. If the remaining cellulose is crystalline in nature and is more resistant to enzymatic hydrolysis than amorphous cellulose, it would be available to the bacteria only with difficulty and in inadequate amounts, so that a low population of cellulose fermenters would result. A low bacterial population would in turn be expected to have a low rate of fermentation. The fact t h a t the lag period in both the untreated materials is much shorter than in the acid-treated materials lends support to the idea of an easily hydrolyzed portion of cellulose. The behavior of the acid-hydrolyzed celluloce during fermenta-
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sawdust was subjected in either the untreated condition or after mild acid hydrolysis to thermophilic bacterial fermentation. The proportion of the three organic acids obtained from wood was similar to that obtained from delignified wood cellulose. In subsequent work, instead of determining the individual acids, all acid in the mixture was extracted from the acidified fermentation liquors and titrated as acetic acid. Acetic acid has a lower molecular weight than either butyric or lactic acid. The weight of the total acids, therefore, is greater than the minimum value obtained by titrating the total acid in the mixture as acetic acid. UNTREATED WOOD. As shown in Table V, triplicate fermentations were conducted on untreated woods and allowed to proceed for 14 days to reach equilibrium. The average yield of acids was 15.9% from sweet gum, 13.8% from aspen, and 19.4% from yellow brich. From western larch heartwood and sapwood and from hard maple these yields nere 6.4, 3.1, and 8.370, respectively. The low yield of acids from larch is typical of those obtained from most woods in the early Tvork on this project. Although the larch wood contained 8.0% of galactan, it had been shown previously that this crude water-soluble extractive, before being hydrolyzed v,ith acid, resists thermophilic bacterial conversion t o organic acids. ilfter it had been partially acid hydrolyzed to galactose, however, it produced on bacterial fermentation 43.0% of organic acids, which is somewhat lower than that shown for refined galactose (Table I).
TIME (HOURS) ~~
Figure 4. Relation of Fermentation Time to Residual Cellulose, Viscosity, Alpha Cellulose, and Potential Sugars of Residue
The rate-of-fermentation curve is normal. -4fter an initial lag period of approximately 20 hours, the rate of cellulose utilization becomes uniform and is so maintained almost to the end of the fermentation. The viscosity of the residual cellulose falls slowly throughout the fermentation. With the exception of the last point, all the data fall on a straight line. The average degree of polymerization of the residual cellulose was calculated on the basis of a relationship with viscosity developed by Staudinger and Reinecke (IO). The original cellulose had a degree of polymerization of 975 and when the fermentation was 80% complete, the degree of polymerization of the residual cellulose was 750. The percentage of alpha cellulose in the residual cellulose decreased slowly during the first part of the fermentation and then more rapidly during the latter half. The theoretical yield of potential reducing sugars from cellulose is 111.1%. During the more vigorous part of the fermentation, the potential sugar values were almost a t the level of the theoretical yield. When the activity slowed down, debris accumulated on the residual cellulose, with the result that a t the completion of fermentation the yield of potential sugars on the residual cellulose was only 12%. Thus 99% of the original cellulose was utilized, rather than the 92% as shown on the rate-of-fermentation curve. The plotted alpha cellulose and viscosity values for the fermentation, too, are minimum values, since no correction mas made for the bacterial debris in the residual cellulose. The results of these studies on the alpha cellulose and the viscosity of the residual cellulose suggest t h a t the bacteria attack the cellulose on the ends of the cellulose chain, rather than a t random. Random splitting of the chains should give a large initial decrease in alpha cellulose, viscosity, and degree of polymerization. Fermentation of Wood. Results so far obtained indicate that wood ferments less readily than does delignified cellulose. Lignified cellulose, such as that in wood, offers resistance t o the attack of thermophilic bacteria. Apparently, the close association between the lignin and the cellulose must be disturbed physically or chemically in order t o increase the susceptibility of the cellulose in the wood t o the bacterial action. The source of wood was coarse sawdust passed through a Wiley mill. The
TABLE.'1 P R O D U C T I O N O F ORGAKIC ACIDS BY ACTION THERMOPHILIC BaCTERIA OK UNTREATED '17'OOD Yield of Acids, %" Species Run 1 Run 2 Run 3 18.2 14.6 Sweet gum 14.9 12.6 15.0 13.8 As en 20.4 18.9 19.0 Yeylow birch 8.1 8.2 8.7 Hard maple 3.7 3.0 2.5 Western larch sapwood 4.6 8,5 Western larch heartwood 6.1 a Yields based on weight of oven-dry wood.
OF
Average 15.9 13.8 19.4 8.3 3.1 6.4
HYDROLYZED WOOD. Because delignified cellulose was found to ferment more readily than the cellulose in wood, a mild chemical treatment was given the wood t o disturb the close association between the cellulose and the lignin. Wood sawdust was subjected t o 4 parts of 1.0%sulfuric acid a t 15.0 pounds steam pressure for 1 hour and neutralized 15-ith calcium carbonate. Sawdust so prepared from sweet gum, yellow birch, hard maple, and n-estern larch was fermented; the resulting acids obtained are given in Table VI.
TABLEVI. PRODUCTION OF ORGAXICACIDS B Y ACTION OF THERMOPHILIC BACTERIA O N WOOD (Wood treated with 1% sulfuric acid for 1 hour at 15 pounds steam pressure) Yields of Acid (Based on Weight of OvenDry Wood), 70 Species Run 1 Run 2 Run 3 Average Sweet gum 23.4 22.7 26.5 24.2 26.6 26.5 27.2 26.4 27.2 25.8 27.0 26.6 22.2 23.8 22.5 21.6 Yellow birch 20.9 20.0 21.0 22.2 Hard maple Western larch sapwood 23.0 23.6 21.5 22.7 21.2 21.9 Western larch heartwood 21.6 21.6
The yield of acid from three sets of triplicate runs on sweet gum ranged from 22.7 to 27.2%. Average yields for the three sets of triplicates were 24.2, 26.4, and 26.6%, respectively. -4s the acids produced were approximately 50% of the cellulose consumed and as sweet gum contains 74% of holocellulose, which is equivalent t o 37.0y0 of acid, the yield of acid from the last sweet gum triplicate indicated t h a t a t least 72Y0 of the celluloae in the wood was utilized. Average yields from western larch sapwood and heartwood were 22.7 and 21.6%, respectively; average yield from
June 1951
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INDUSTRIAL AND ENGINEERING CHEMISTRY
yellow birch was 22.5% and from hard maple, 21.0%. These values are all substantial increases over those from the untreated woods (Table V). Because the values are reported as acetic acid, they are lower than the actual yields. The actual yields can be estimated by calculating the percentage distribution of the three acids in the acid mixture. A basis for doing so is given in the distribution of the acids, if calculated as acetic acid, from the fermentation of the second sample of commercial hemicellulose in Table I. This calculated distribution is 48% each for acetic and butyric acid, and 4% for lactic acid. Thus, the yield of 26.6% acetic acid from sweet gum (Table VI) would approximate 33.0%, if calculated on the basis of the three individual acids. LITERATURE CITED
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(1) Acharya, C. N., Biochem. J., 24, 1459 (1935). (2) Fontaine, F. E., Ph.D. thesis, Univ. of Wisconsin (1941). (3) IND. ENG.CHEM.,ANAL.ED., 1, 52 (1929). (4) Langwell, H., .J, Soc. Chem. I n d . ( L o n d o n ) , 51,988 (1932). (5) MacFayden, A., and Blaxall, F. R., Trans. Jenner, I n s t . Preu. M e d . , Ser. 11, 162 (1899).
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(6) Olson, F. R., Peterson, W. H., and Sherrard, E. C., IND.ENG. CHEW,29, 1026 (1937). (7) Paper Trade J . , 94, 38-9 (1932) (corrected Dee. 15, 1937). (8) Rogers, S. C., Mitchell, R. L., and Ritter, G. J., A n a l . Chem., 19, 1029-32 (1947). (9) Saeman, J. F., Bubl, J. L., and Harris, E. E., IND.ENG.CHEM., ANAL.ED.,17, 35 (1945). (10) Staudinger, H., and Reinecke, F., Papier-Fabr., 36, 489 (1938). (11) Tetrault, P. A., Zentr. Bakt., Parasitenk., Abt. 11, 81, 28 (1930). (12) Virtanen, A. I., and Hukki, J., S u o m e n Kemistilehti, 19B, 4 (1946). (13) Virtanen, A. I., and Koistinen, 0. A., Svensk K e m . Tid., 56, 391 (1944). (14) Virtanen, A. I., Koistinen, 0. A., and Kiuru, V., S u o m e n Kemistilehti, 11B, 30 (1938). (15) Virtanen, A. I., and Nikkila, 0. E.. Ibid., IQB, 3 (1946). (16) Virtanen, A. I., and Pulkki, L., J . Am. Chem. Soc., 50, 3138 (1928). RECEIVEDJune 30, 1950. Presented before the Division of Cellulose Chemistry a t the 118th Meeting of the AMSRICANCHEMICAL SOCIETY, Chicago, Ill. The Forest Products Laboratory, U. S. Departmemt of Agriculture is maiutained a t Madison, Wis., in cooperation ivith the University of Wisconsin.
Aqueous Dispersion of Carbon
Blacks E. M. DANNENBERG AND K. P. SELTZER Godfrey L. Cabot, Inc., Boston, Mass.
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T h e development of carbon black-synthetic rubber masterbatch is one of the most progressive advances made by the synthetic rubber industry in recent years. In the preparation of black masterbatch an aqueous dispersion of carbon black is mixed with latex, and the mixture is coprecipitated, washed, dried, and baled. The wetting and slurrying behavior of carbon blacks is of great importance i n this process, since it has been observed that carbon blacks of the same grade or type may show marked variability i n these properties. This paper presents information on the factors responsible for such differences. The rate of wetting of carbon black is influenced by traces of benzene extractable material and to the bulk density of the dry black. The quantity of dispersing agent required to give a stable and fluid slurry is related to bulk density, surface area, water-soluble inorganic contaminants, and surface oxidation. An understanding of the factors influencing the slurrying behavior of carbon blacks is necessary in order that carbon black and masterbatch manufacturers, may modify their operations to mutual advantage.
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NE of the major achievements of the government synthetic
rubber program is the large scale production of carbon black masterbatches. Developed during World War I1 under the pressures of increased production, lack of processing equipment, and labor shortages, black masterbatch has found wide postwar acceptance by many sectors of the rubber industry. The production of black masterbatch was about 70,000 long tons in 1949. Some of the advantages of incorporating carbon black into synthetic rubber latex to form a masterbatch are inherent cleanliness, simplicity of handling, and a reduction of mixing time and power requirements. Mixing is done a t a lower temperature. A more uniform mix is obtained with less trouble and no loose carbon black is lost to the dust collecting systems.
Carbon black masterbatches can contain as much as 100 parts of black per 100 parts of synthetic rubber with most mixtures having about 50 parts of black. They are available in a number of different combinations of grades of black and types of synthetic rubber. Their use by rubber product manufacturers requires mastication and mixing in Banbury mixers and roll mills in order completely to disperse the carbon black and t o incorporate the curing, softening, and other compounding ingredients. The development and production of black masterbatch have been described in publications by McMahon and Kemp ( 6 ) ,O’Conner and Sweitzer (9),Rongone, Frost, and Swart (IO),and Madigan and Adams ( 7 ) . The sequence of operations in making masterbatch is carefully controlled mixing of an aqueous dispersion of carbon black and synthetic rubber latex, followed by creaming, coagulation, washing, drying, and baling, similar to the techniques of normal synthetic rubber manufacture. In order to incorporate carbon black satisfactorily into the synthetic latex, the pigment must first be dispersed in water. Knowledge about the preparation and colloidal properties of aqueous carbon black dispersions is therefore of importance to the masterbatch producer. This subject is also of obvious interest to the carbon black manufacturer, since it is desirable to predict and control the slurrying performance of carbon blacks used for latex incorporation. The purpose of this investigation was to determine the dispersion characteristics of various carbon blacks in water and the fundamental properties of a black that influence its dispersion characteristics. The reinforcing ability of carbon black has long been attributed to its small particle size. I n order to develop high reinforcement, carbon black must be dispersed in rubber to a degree commensurate with its particle size. In ordinary dry mixing the dispersion of carbon black in rubber is accomplished mainly during roll milling, where high shearing and elastic forces disintegrate the carbon aggregates and bring the newly exposed carbon surfaces into immediate contact with rubber. In contrast to rubber
