1162
Vol. 19. No. 10
I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y
Production of Lactic Acid by Fermentation of Wood Sugar Remaining after Alcoholic Fermentation' By E. A. Marten,? E. C. Sherrard,3 W. H. Peterson,* and E. B. Fred? U. S. FORESTPRODUCTS LABORATORY, A N D UNIVERSITY OF WISCONSIN, MADISON, Wxs.
The residue from the alcoholic fermentation of woodcessful. As the liquors were N THE commercial prosugar liquor was fermented by bacteria with the formaduction of ethyl alcohol quite acid in reaction and did tion of acetic and lactic acid. An available source of by the fermentation of not contain a quantity of nitrogen and an excess of calcium carbonate must be n i t r o g e n o u s material suffithe sugars that result from added to the liquor in order for the fermentation to cient for the growth of the the acid hydrolysis of wood, take place. organisms, it was realized a difficulty is encountered in The ratio of acetic to lactic acid production is dethat adjustments would have utilizing the sugars of the pendent upon the kind of sugar present in the liquor. t o be made before any results pentose type. These sugars, This varies with the wood that has been used and the could be expected. which normally amount to extent of the previous alcoholic fermentation. The The first successful fermenabout 35 per cent of the total fermentation of liquor obtained from softwood protation resulted only after an sugars produced from softduces a larger proportion of lactic to acetic acid than equal volume of a 10 per cent wood, or about 65 per cent of that obtained from hardwood. yeast-water suspension and those produced from hardIn this fermentation, lactic acid is produced from a an excess of calcium carbonwoods, are not attacked by cheap material which is otherwise wasted. ate were added to the liquors. yeast and are discarded with T h e s e l i q u o r s were-then the still residues. A saving would be realized if it were possible by means of organisms reinoculated and incubated a t 27" C. Fermentation was other than yeast to ferment the remaining pentoses with the noticeable a t the end of 24 hours and after 3 days an appreciable reduction of sugar had occurred. production of a valuable product. Fred, Peterson, and co-workers4 have isolated a large Selection of a Culture number of pentose-fermenting bacteria and have determined the products formed from various sugars by these microThe number of strains of lactic acid organisms is very organisms. The pentoses are fermented to acetic and lactic great. All of them produce lactic and acetic acid. However, acids, and the hexoses almost quantitatively to lactic acid. they vary in their ability to ferment pentoses, and likewise Dilute sirups containing pentose sugars which were obtained the ratios in which the acids are produced is dependent by the hydrolysis of corncobs, peanut shells, and oat hulls, on the particular strain of the organism employed. I n and which were fermented by these bacteria, gave the same order to find a strain which fermented pentoses vigorously products as were obtained from the pure sugars.6 It there- with the production of large amounts of lactic acid, four fore seemed probable that the pentoses which remained cultures were selected to inoculate the liquor. These orafter the fermentation of wood liquor by yeast could be ganisms were isolated from sauerkraut and silage, where fermented by the same bacteria. they play an important role in the production of acids, The method of converting wood into sugar by acid hyTable I-Sugar Yields drolysis and the alcoholic fermentation of this product by yeast are described in detail by Sherrard and his assoCOOK WOOD ciates.6 The general procedure was to hydrolyze the wood by digesting for 15 minutes a t 115 to 120 pounds pressure in the presence of 2 per cent sulfuric acid. Peu cent d r y wood Per cent P e r cent
I
Source of the Liquor
Yeast-fermented liquors obtained from two sources were investigated and the products of fermentation determined. The first sample was obtained from mixed hardwood shavings; the second from a n authentic source of Douglas fir. A portion of the Douglas fir liquor which had not been fermented by yeast was also used. The yields of sugar from these woods, together with the amounts of fermentable and nonfermentable sugar, are given in Table I. Preliminary Bacterial Fermentation
A preliminary attempt a t bacterial fermentation of a 2 per cent solution of the spent liquors directly was not suc1 Presented before a joint session of the Divisions of Biological Chemistry and Organic Chemistry at the 73rd Meeting of the American Chemical Society, Richmond, Va., April 11 t o 16, 1927. 2 University of Wisconsin, Madison, Wis. * U. S. Forest Products Laboratory 4 Peterson, Fred, and Anderson, J . Biol. Chem., 48, 385 (1921); 6% 111 (1922). 6 Fred and Peterson, THISJOURNAL, 18, 211 (1921); 16, 126 (1923). 0 Sherratd and Blanco, Ibid., 16, 611 ('1923).
366 367 370 378 374
Douglas fir Douglas fir Douglas fir Douglas fir Mixed hardwoods
21.52 20.71 21.98 22.19 19.40
65.88 63.48 64.99 73.67 30.1
34.12 36.52 35.01 26.43 69.9
Each of the four strains were used to inoculate samples of spent liquors (Table 11) that had been prepared in the following manner: A quantity of the liquor was clarified by boiling with Norit, neutralized with milk of lime, and filtered. A volume of yeast suspension and an excess of calcium carbonate were added to the clear filtrate. This mixture was tubed in 10-cc. portions and sterilized. Duplicate tubes were inoculated with 4 drops each of a 24-hour-old culture of each organism. The cultures were incubated from 4 to 10 days a t 27" C., and afterward were analyzed for unfermented sugar by the Shaffer-Hartmann method.' During the fermentation the tubes were shaken frequently to neutralize the acids formed. The evolution of carbon dioxide resulting from the reaction of the acids with calcium. carbonate stopped after 10 days. This was taken as a n index of the time required for maximum fermentation, 7
J . B i d . Chem., 16,366 (1920-1921).
.
INDUSTRIAL AND ENGINEERI,VG CHEMISTRY
October, 1927
1163
I n Table IV it is seen that within a range of 2.14 and 4.53 per cent sugar the fermentation proceeds best at the lower concentration, which incidentally is an advantage, for this is the strength of the liquor as it leaves the still after alcoholic distillation. The figures of Table IV show again that malt sprouts may be substituted for yeast as the source of nitrogenous material. The small difference in the amount of sugar converted when yeast or malt sprouts were used is within the limits to be expected in a biological process. Effect of Adding Liquor at Intervals during Fermentation Table 11-Fermentation of Wood Liquor by Various Cultures The method of adding sugar to the cultures a t intervals SUGAR AS GLUCOSE AGE during the fermentation is often used as a means of producing Before After FERSOURCE OF OF ferferMENTED W O O D LIQUOR CULa greater conversion of sugar than can be obtained when menta- mentaTURE the entire amount is present a t the time of inoculation. tion tion -Its successful use in the alcoholic fermentation of woodGrams Grams P e r cent Days sugar liquor suggested its application to this fermentation.
and in all later experiments the cultures were allowed to incubate for a 10-day period. Table I1 shows that all the organisms fermented the sugar to some extent, but culture 24-2 surpassed the rest and was used in all subsequent experiments. I n 4 days about 53 per cent of the sugar in the fermented Douglas fir liquor was destroyed, and 76 per cent in 10 days. The hardwood liquor showed about the same fermentation. Approximately 92 per cent of the sugar in the original unfermented Douglas fir sirup was destroyed.
118- 8 41-11 24- 2 A 24- 2 24- 2 24- 2
Douglas fir, fermented Douglas fir, fermented Douglas fir, fermented Douglas fir, fermented Douglas fir, fermented Douglas fir, unfermented Hardwood sawdust
0.852 0.852 0.852 0.852 2.17 2.32 2.38
4 4 4 4 10 10 10
0.470 0.530 0,401 0.823 0.51 0.18 0.60
44.8 36.6 52.9 3.4 76.4 92.2 74.8
Table IV-Effect of Concentration of Sugar on Fermentation
I
Kind
Effect of Various Nitrogenous Materials on Fermentation
While yeast proved satisfactory as a source of nitrogen an effort was made to find cheaper materials. I n this experiment dried blood, ground malt, malt sprouts, and tankage were used. The coarsely ground blood, malt, etc., were added in varying quantities to 10-cc. portions of the liquor, which were then sterilized. Sterilized calcium carbonate was added and the tubes were inoculated with the selected organism. The cultures were then incubated for 10 days a t 27" C., after which they were analyzed for unfermented sugar. The difference between this figure and that of the unfermented liquor was taken as an index of the extent of fermentation. The results (Table 111) show that nitrogenous substances other than yeast can be used as a nutrient. I n fact, equally good fermentations were obtained with malt sprouts and with yeast in the same amount. I n each instance the higher concentration of nutrient gave the best results. I n later experiments, however, 5 or more per cent of malt sprouts gave no better results than 4 per cent. Considering both cost and nutrient value, malt sprouts and ground blood meal would be preferable for commercial fermentations. Table 111-Effect
Blood meal
iyDir P e r cent 1.00 2.00 4.00
SVGAR AS
P e r cent 2.04 2.04 2.04
hfalt Tankage Malt sprouts Dry yeast Yeast water" a
4.00 1.00 2.00 4.00 1.00 2.50 5.00 2.5c
2.12 2.12 2.06 2.06 2 06 2.25 2.31 2.36
P e r cent 1.33 1.16 0.68 1.33 0.78 0.63 1.28 0.87 0.69 0.95 0.63 0.54 0.57 0.61.
Fermented
P e r cent 29.9 42.3 66.6 37.2 63.2 70.3 33.0 57.7 66.5 53.3 72.7 77.9 73.8 76.4
Original sirup diluted with an equal volume of yeast water extract.
Effect of Concentration of Sugar
The concentration of the sugar in the liquor is an iniportant factor in determining the extent of fermentation.
Fermente'
P e r cent 4.0 4.0 4.0 4.0 4.0 2.5 2.5
P e r cenl 72.2 67.2 69.5 54.5 46.8 73.8 61.4
P e r cent 2.45 3.47 3.86 4.05 4.53 2.14 4.36
P e r cent 0.69 1.12 1.18 1.84 2.41 0.57 1.68
of Adding Liquor at Various Intervals during Fermentation
Table V-Effect
SUGARAS GLUCOSE
SAMPLE
Before fermentation
P e r cent 1.43 1.40
19
21, 3a 4b
(~UCOSE
After fermentation mentation
Malt sprouts Malt sprouts Malt sprouts Malt sprouts Malt sprouts Dry yeast Dry yeast
SUGARAS GLUCOSE
Before After Amount ferfermentation mentation
Four 100-cc. Erlenmeyer flasks, each containing 25 cc. of a 10 per cent yeast-water suspension, were inoculated. Sterile calcium carbonate was added to this, together with varying amounts of the Douglas fir liquor as shown in Table V. The liquor of samples 1 and 2 was first treated with Norit to see if clarification increased the fermentation. Samples 3 and 4 consisted of the untreated liquor. At the end of 5 days all corresponding cultures had received the same amount of liquor, so the effect of periodic additions and the treatment with Norit could be demonstrated b$ comparing the cultures.
of Various Nitrogenous Materials on the Fermentation
I
MATERIAL
1 2 3 4 5 6 7
I
NUTRIENT ADDED
SAMPLE
l
After fermentation
Fermented
P e r cent 0.42 0.57 0.37 0.51
P e r cent 70.6 59.2 71.7 60.7
2 5 cc. of liquor added at time of inoculation. b 5 cc. wood liquor added daily for 5 days. a
Table V shows that the periodic addition of the liquor does not increase the amount of sugar that can be fermented. The maximum fermentation was obtained when all the liquor was added a t the time of inoculation. The data show that the treatment with Norit does not increase the percentage of sugar fermented. Fermentation Products of Pure Sugars and Sugar Mixtures I n all the foregoing experiments the reduction of sugar was taken as a measure of the extent of fermentation. No attempt was made to determine the amount of products formed. I n this experiment the amount of volatile and non-volatile acids in 100 cc. of culture was determined by methods described in previous papers.* In all cases the SFred, Peterson, and Davenport, J. B i d . C h e m . , 39, 347 (1919) Peterson and Fred, I b i d . , 41, 431 (1920).
ISDUSTRIAL AND E,VGISEERIA-G CHEMISTRY
1164
sugars was obtained when glucose was added, than when the liquor was fermented alone. Apparently the glucose aids in fermenting the more resistant sugars of the liquor. Samples 7 and 8 contained a good grade of cane molasses. This produced large amounts of lactic acid, after the manner of glucose. From the ratio of acids formed with mixtures of pure sugars, it appears that each sugar produces acids as if it were alone and is not influenced by the presence of others.
controls were treated in exactly the same manner as the fermented cultures. The amount of acid obtained from these controls was subtracted from that obtained from the fermented samples, and the remainder considered as the products of fermentation. The values thus calculated are shown in Table VI. P r o d u c t s of P u r e Sugars a n d S u g a r Mixtures
T a b l e VI-Fermentation SUGAR
sAMPLE0 KINDO F SUGAR
VOLATILE
pERMENTED
ACID
SONVOLATILE
SUGAR
:zy:;-E
Fermentation Products of Fermented Wood-Sugar Liquor
RECOVERED A J & ~ ~*'ID LACTIC AS PR&%TS VoL*TILE ACID
1 2
Xylose Xylose and glucose Glucose Glucose Glucose and slop Glucose and
3
4 5
6
SlOD
7
Cuban molasses Cuban molasses
8
Grams
Grams
Grams
Gvams
Gvams
1.90
0.698
1.028
1.726
1:1,5
1.88 2.38 2.47
0.323 0,103 0.098
1.645 2.184 2.175
1.968 2.287 2.273
1:j.l 1:21.2 1:22.2
2.01
0.110
1.968
2.078
1:17.8
2.16
0.139
1.916
2.055
1:13.7
2.36
0.151
2.175
2.326
1:14.4
2.34
0 124
2.159
2.283
1:17.3
The fermentations were conducted in 250-cc. Erlenmeyer flasks, using 150 cc. of the liquor. Four per cent of malt sprouts was added in each case, and an excess of calcium carbonate a t the time of inoculation. The fermentation was allowed t o go on for 10 days, after which 100 cc. of the solution were analyzed for unfermented sugar, volatile and non-volatile acids by methods mentioned in earlier publications. It appears from Table VI1 that the liquor contains other sugars than pentoses, perhaps hexoses of a type not fermentable by yeast, such as galactose. This would account for the variation in the ratio of acetic to lactic acid; and also for the variation between the liquors obtained from different woods. I n the case of the liquor obtained from hardwood, the ratio of acetic to lactic acid was about 1:3, while an average of 1r7.1 was obtained from Douglas fir liquor. Hardwood on hydrolysis gives 65 per cent of nonfermentable sugar, most of which is of the pentose type. The comparatively low ratios of lactic to acetic acid from samples 5 , 6, 7 , and 8 may be due to the fact that the original sirup from which this liquor was obtained underwent a vigorous alcoholic fermentation. The percentage
Samples 1 and 2 contained yeast water; 3 to 8 contained 4 per cent malt sprouts.
The lactic acid was determined not only by titration, but the barium salt of the ether-extracted acid was analyzed by the Iron Furth-Charnasg method for lactic acid. The results obtained from this analysis agreed very closely with those obtained by direct titration. The zinc salt of the acid was made and its percentage of water of crystallization determined. This was found to be 18.3 per cent. Inactive zinc lactate crystallizes with three molecules of water of crystallization. T a b l e VII-Fermentation ~~
P r o d u c t s of Wood
1 2
3
4 5 6 7 8 9
10
11 12 13 14 15 16
igar Liquor
~
WOOD-SUGAR LIQUOR SAMPLE
Vol. 19, No. 10
Source of liquor
Douglas fir Douglas fir Douglas fir Douglas fir Douglas fir Douglas fir Douglas fir Douglas fir Douglas fir Douglas fir Douglas fir Douglas fir Hardwood sawdust Hardwood sawdust Hardwood sawdust Hardn ood sawdust
Treatment before lactic fermentation
Fermented Fermented Fermented Fermented Fermented Fermented Fermented Fermented Unfermented Unfermented Unfermented Unfermented Fermented Fermented Unfermented Unfermented
SUGAR Fer-
Before After ferfermentation mentation
mented
Grams
Grams
Grams
Grams
Grams
Grams
2.34 2.34 2.28 2.28 2.26 2.26 2.26 2.26 2.74 2.74 2.78 2.78 2.93 2.93 2.15 2.15
0.58
1.76 1.75 1.78 1.75 1.27 1.19 1.32 1.49 2.42 2.47 2.51 2.49 1.52 1.25 1.64 1.58
0.166 0.179 0.182 0.141 0.238 0.254 0.217 0.240 0.209 0.217 0,209 0.176 0,348 0.339 0.384 0.354
1.487 1.517 1.498 1.537 1.129 1.072 1.260 1.377 2.199 2.280 2.219 2.298 1.053 0,999 1.234 1.053
1.663 1.695 1.680 1.678 1.367 1.326 1.477 1.617 2.408 2.497 2.428 2.474 1.401 1.338 1.618 1.407
and gave a ratio of acetic to lactic acid of 1:1.5. Sample 2 was a mixture of equal parts of xylose and glucose, and gave a ratio of 1:5.1. The theoretical value for such a mixture is 1:4.5. Samples 3 and 4 contained glucose, and samples 5 and 6 an equal mixture of glucose and fermented liquor. The ratio of acids produced from glucose indicated that this sugar is converted almost completely into lactic acid, with the formation of only small amounts of acetic acid. It is noteworthy that a more complete fermentation of the liquor
0.59 0.50 0.53 0.99 1.07 0.94 0.77 0.32 0.27 0.27 0.29 1.41 1.68 0.51 0.57
SUGAR
sOuoRpc&
Douglas fir
~
O sawdust
,
"g'$yG?
By yeast
SIS
Lbs. 436 436
~
388 388
Lbs. 222
~
~
~
116 0
o
Lbs. 96 389 129 291
~
1:8.2
1:lO.S 1:4.7 1:4.2
1:5.8 1:5.6
1:10.5 1:10.5 1:10.6 1:13.1 1:3.0
1:2.9
1:3.2 1:2.9
BY
& :,:::
YEAST Ace- Lactic tic
B y bac-
teria
1:s.s
ACIDSB Y ALCOHOL BAcTERIA
FERMENTED
LIQUOR
1:8.9
Lbs. 48 47
Lbs. 149
143
59
32
97
.. .
68
205
97
.. .
Lbs. Lbs. 12 84 30 335
October, 1927
INDUSTRIAL A N D ENGINEERING CHEMISTRY
this sirup contained all of the glucose formed by the hydrolysis of the wood. Table VI shows that glucose is converted almost quantitatively into lactic acid by these bacteria. Commercial Applications
From the data in this paper it appears that the production
1165
of lactic and acetic acid from spent wood liquors is a feasible process, although many technical details would have t o be worked out before the process could be put on an industrial scale. If these laboratory experiments can be duplicated on a commercial scale, the yields of products per ton of wood as given in Table VI11 might be expected.
Evaluation of Transformer Oils'9' By J. G. Ford WESTINGHOUSE F$LEC!TRIC
& &fANUFACTURING
N EVALUATING oils for transformer use, consideration must be given to flash and fire points, viscosity, specific gravity, pour point, dielectric strength, presence of inorganic acids, alkalies and salts, sulfur content, and finally to the tendency to form deposits, known as sludge. It is a simple matter to choose an oil having the desired physical characteristics, but it is more difficult to select an oil that is desirable from the standpoint of sludge formation. Service tests in the apparatus in which the oils are to be used are the most reliable criterion of this property. Transformer oil has two principal functions-( 1) to act as an insulating medium, and ( 2 ) t o carry the heat generated in the windings and core of the transformer to the cooling surf aces. Sludge is usually itself a good insulator and therefore does not interfere primarily with t'he insulating qualities of the oil. The rate of sludge formation varies with temperature but, not necessarily directly with the temperature. This sludge formation is due t o chemical changes the mechanism of which is not well understood. The precipitation of this sludge in the ventilating ducts and on the cooling surfaces results in increased temperature of the transformers and this increased temperature increases the rate of sludge formation. It is therefore necessary to select an oil that will have a minimum of sludge formed under normal operating conditions and to recondition the oil when the amount of sludge formed becomes sufficient to increase operating temperatures. Recondit'ioning should be necessary only occasionally with a properly designed transformer and a properly selected oil. It is well known that sludging is caused by oxygen being in continual contact with the oil. This oxidation takes place not only at the surface but throughout the body of the oil, since it readily dissolves oxygen. I n fact, new oil before being placed in a transformer cont'ains considerable amounts of oxygen in solution. I t is also known that heat, light, and the mat'erials used in transformer construction accelerate this oxidation. All mineral oils in the presence of oxygen oxidize to a certain extent under operating conditions of a transformer. Some oils, chiefly the water-whit e type. form little or no sludge but produce on oxidation considerable amounts of a large number of organic acids, the lower boiling members of which are mostly vo1at)ilized a t operating temperatures. Mineral oils slowly oxidize, even a t room temperature, in the presence of light, moisture, and oxygen. The steps in the development of oxidation products have already been described by other investigators.3-6
'
Received June 16. 1927. Presented before the Division of Petroleum Chemistry a t the 74th Meeting of the American Chemical Society, Detroit, Mich., September 6 to 10, 1927. Scienlil:c Pa$er 245 from the Westinghouse Electric & Manufacturing Company. Sligh, PYOC. A m . SOL.Testing M'nieviais, 24, 964 (1924). 4 Staeger, THISJ o n R x A L , 17, 1272 (1925). Haslam and Frolich, I b i d . , 19, 292 (1927). 6 Musatti and Pichetto, Ann. chim. agplicala, 15, 238 1:1925).
*
COMPANY,
EASTPITTSBURGH, P A .
There are a t present numerous testing methods employing elevated temperatures and various accelerating means which are supposed to classify oils according t o their value for transformer use. Considerable controversy exists among different investigators concerning the temperature of test, measurement of end products, and the use of catalysts such as copper and iron. I n general, the results of these different tests show little agreement among themselves. An oil which may be indicated excellent when tested by one method may give poor results when tested by another. The author believes that none of these tests can be intelligently criticized until more definite knowledge is obtained concerning the action of oxygen on oils of different chemical constitution, what products of oxidation are det)rimental t o the transformer, and the effect of temperature and catalysts on the rate of oxidation and formation of end products. However, two criticisms of the majority of tests are that the time consumed in making them is disadvantageous and it is difficult to reproduce results. According to Staeger4mineral oils may contain the following groups of hydrocarbons: I-Unsaturated hydrocarbons ( A ) Cycl1c (a) Aromatic hydrocarbons (benzene, naphthalene) ( b ) Alicyclic hydrocarbons (terpenes, polyterpenes, dihydroxy, tetrahydroxy combinations) ( B ) Aliphatic (olefins, polyolefins) 11-Saturated hydrocarbons ( A ) Cyclic (naphthenes, polynaphthenes, condensed naphthenes) ( B ) Aliphatic (paraffins)
Experience has shown that under the same operating condition some oils fail in one or two years while others function properly after ten years. This difference in the behavior of oils is undoubtedly connected with their chemical constitution. Since oils may be composed of any or all of the above groups of hydrocarbons in various proportions and these groups probably act differently towards oxygen, it is not astonishing that oils behave differently in transformers. It seems reasonable to suppose that if we knew the approximate composition of a transformer oil and how the various hydrocarbons therein acted towards oxygen, we could predict just how this oil vould perform and therefore choose the most suitable oil for a given set of operating conditions. To isolate and test the individual hydrocarbons occurring in petroleum would be almost out of the question on account of the difficulty and labor involved in obtaining the pure hydrocarbons. On the other hand, to simplify matters a5 much as possible, some valuable information might be gained by a proximate analysis of a number of oils taken from various geographical locations; determining, if possible, the percentage of the different groups of hydrocarbons present. These oils may then be subjected to oxidation tests in an
