Utilization of Agricultural Wastes 11. Influence of‘ Nitrogenous Substrate on Production of Butyl and Isopropyl Alcohols by Clostridium butylicuml 0. L. OSBURN AND C. H. WERKMAN
Agricultural By-products Laboratory, Bureau of Chemistry and Soils, U. S. Department of Agriculture, and Engineering Experiment Station, Iowa State College, Ames, Iowa
.+*
w* N CONNECTION with g e n e r a l inu
vestigations of the utilization of agricultural wastes and of byp r o d u c t s of industries using agricultural raw m a t e r i a l s , a study has been made of the utilization of waste glucose, gluten, and other by-products of the corn sugar refining industry in the production of butyl and isopropyl alcohols. This present paper deals with the products and fermentation characteristics connected with the attack of these industrial wastes by Clostridium butylicurn.
The production of butyl and isopropyl alcohols by the fermentation of by-product glucose sirup with Clostridium butylicum (Beijerinck) Donker occurs in the presence of various proteins. The growth-stimulating factor described by Tatum, Peterson, and Fred may be considered beneficial but not essential to the fermentation of glucose. The yield of butyl alcohol from corn mash increases from about 3.0 to 14 per cent when the stimulating factor is added. The proteolytic power of the organism is weak. With hydrolyzed proteins the rates of the fermentations are slower than when peptone is present, but the percentage yields of alcohols compare favorably with the yields obtained with peptone. With mixtures of corn steep water and malt sprouts or corn gluten as sources of nitrogen, glucose may be fermented in 4 per cent solution writh the production of 20 to 25 per cent of butyl alcohol and from 4 to 9 per cent of isopropyl alcohol.
of its possibilities in the industrial u t i l i z a t i o n of “hydrol,” a by-product glucose sirup resulting from the manufacture of c o r n s u g a r as well as other substandard sirups and sugars r e s u l t i n g f r o m t h e refining process.
Culture Methods and Inoculations The organisms were cultured for 3 days in 4 per cent corn mash containing 1 per cent of yeast extract or of potato extract. The cultures were then immersed in boiling water for 1 m i n u t e a n d cooled rapidly, and l a . portions were used to inoculate new tubes of corn mash. Four or five sets of transferswere made in this manner. F r o m t h e l a s t series a large number of culture tubes was inoculated and allowed to sporulate. These c u l t u r e s were stored a t room temperature and the spores used to inoculate all flasks in the experiments to be described. Ino c u l a t i o n was made as follows: One tube was immersed in boiling water for 1 minute, and 1 cc. of the suspension of spores was inoculated into a sterile flask containing Lgrams of peptone, I gram of yeasit extract, and sufficient corn sugar*to represent 6 grams of glucose in 300 cc. of tap water. After a 4-day incubation nt 37’ C., the mixture of spores and organisms had formed a heavy sediment on the bottom of the flask. Most of the clear supwnatant liquid was decanted, and the smalI quantity of remaining liquid with spores was shaken up and transferred aseptically to sterile test tubes. The spores were centrifugalized and resuspended in sterile water. One cubic centimeter of the suspension was used to inoculate each flask.
Historical Review Beijerinck (1) first isolated C1o s t r i d i u m butylicum (Beijerinck) D o n k e r in 1893 and d e s c r i b e d it as Granulobncter butylicum, an organism capable of vigorously fermenting malt sugar with the production of butyl alcohol. The same organism was again i s o l a t e d f r o m malt in 1920 by Folpmers (d), who showed that, in addition to butyl alcohol, isopropyl alcohol was produced along with small quantities of n-propyl and isobutyl alcohols, and butyric, isobutyric, and acetic acids. In 1928 van der Lek (ij), working in Kluyver’s laboratory in Delft, Holland, found a n old spore culture which had been stored in the Delft laboratory in 1893 by Beijerinck. Peptone-glucose broth and yeast-glucose broth were inoculated with the spores, and a vigorous fermentation of the sugar resulted. After a few transfers the organisms readily fermented 2 per cent glucose with the formation of 28 per cent of butyl alcohol and 9 per cent of isopropyl alcohol (by weight of sugar fermented) with small amounts of ethyl alcohol, and butyric and acetic acids. Carbon dioxide and hydrogen were also produced. Kluyver kindly sent to the authors a transfer of this culture used by van der Lek. The high yield of alcohols produced by this organism led to the present investigation
It soon became evident that the nitrogenous substrate exerts an important influence so far as industrial application of the organism is concerned. It is known that the organisms are not capable of hydrolyzing proteins to any appreciable extent. I n all previous work on Cl.butylicum, the proteins used were partly hydrolyzed, van der Lek using yeast extract and peptone, and both Beijerinck and Folpmers using malt sprouts. The corn augar uaed in these experiments contained 92 5 per cent glucose. Crystalline corn sugar was uaed inatead of sirupy hydrol in these experimenta, because of convenience.
A previous article in this series sppeared in February, 1935, pages 195 to 200. 1
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Tatum, Peterson, and Fred (8) found that some strains of butyric organisms apparently require the presence of some widely distributed growth-stimulating factor for the production of butyl alcohol from corn mash. In the present study of the production of butyl and isopropyl alcohols, the two factors of growth stimulation and nitrogenous source are intimately associated with the proteolytic power of the organism, and some doubt exists as to whether certain proteins are unsuited as nitrogen sources because they are not easily hydrolyzed by the organism, or because of the absence of the growth-stimulating factor. Potato extract, corn steep water, and yeast extract3 were used as stimulants. The potato extract was prepared by grinding the potatoes, adding two or three volumes of distilled water, and pressing out the juice. The juice was allowed to stand for 5 hours, decanted, and boiled down to small volume. Three volumes of alcohol were then added. The remaining starch settled out, and most of the protein was precipitated. The mixture was filtered and the alcohol evaporated on the steam bath. The resulting aqueous solution was made up to such volume that 1 cc. represented 1 gram of potato. The solution was starch-free and contained only small amounts of protein. In the manufacture of corn sugar, before the corn is ground it is steeped in water containing sulfur dioxide. The steep liquor is then concentrated to about 12" BB., the concentrate being known in the trade as "corn steep." The material used in this work contained 2.896 per cent of nitrogen, 34.0 per cent of which precipitated out (glutelin) when the solution was neutralized. Thirty-two per cent of the total nitrogen was in the amino and amide form. It is assumed that the remaining 34.0 per cent consisted largely of partly hydrolyzed protein and albumins. Tatum, Peterson, and Fred (8) have shown that corn steep is relatively rich in the growth-stimulating factor. Yeast extract is a good source of nitrogen for the butyl organisms and is also rich in the growth-stimulatirlg factor. AS KITROGEN SOCIRCE, WITH POTATO AND TABLEI. PEPTONE YE.4ST EXTRACTS AS GROWTH-PROMOTING FACTORS
(Glucose in 2 per cent solutions) Ratio G1ucose Alcohols ProFermented ducedb as Per Glucose Fer- to Cent of Glucose ~l~~~~~ Nitro en mented presFermented Use$, Stimu- Used, Gram lanta Grams Grams Per cent ent Butyl Isopropyl Peutone 0.016 P.E. 6.0 1.4 23.3 87.5 .. 0.048 P. E. 6.0 1.5 25.0 31.3 ... 0.080 P. E. 6.0 2.0 33.3 25.0 25:5 4.3 0.128 P.E. 6.0 3.8 63.3 29.7 ... 0,240 P.E. 6.0 4.1 68.3 17.1 24:5 4.5 0.321 P.E. 6.0 5.8 96.7 18.1 26.5 4.2 0,321 P . E. 6.0 5.8 96.7 18.1 26.0 4.0 26.2 4.3 96.7 12.1 5.8 P . E. 6.0 0.480 98.3 18.4 26.5 4.2 6.0 5.9 0.321 None 18.4 27.5 4.1 98.3 6.0 5.9 0,321 iXone 0.321 Y.E. 9.0 8.9 98.9 27.7 28.5 4.6 0.321 Y.E. 9.0 8.8 97.8 27.4 26.0 4.5 Hydrolyzed PeDtone . . 0.037 P.E. 6.0 2.1 35.0 56.8 ... 24:5 4.2 66.6 55.5 4.0 P.E. 6.0 0.072 58.7 24.7 4.1 80.0 8.0 6.4 0,109 P.E. 25.0 4.5 83.7 46.5 8.0 6.7 0,144 P.E. yeast extract Y. E . a Potato extract = P. E: b Ethyl alcohol percentagAs omitted, pending further study.
zp-
-.
...
-
The primary objective of this investigation has been the study of the effect of various nitrogenous materials on the yields of butyl and isopropyl alcohols. Observations on the effect of the stimulating factor have been made only in so far as they have been of assistance in the interpretation of the nitrogen relationships. As will be seen later, the results 8 All yeast extract used in this study was the Bacto yeast extract prepared by the Difco Laboratories.
417
of such observations have been more or less indefinite except when corn mash was fermented. Four per cent corn mash was not readily fermented by C!. butylicum, only about 3 to 4 per cent of butyl alcohol being formed. However, when yeast extract (1 gram) or corn steep water ( 5 cc.) was added t o 300 cc. of 4 per cent corn mash, a complete and vigorous fermentation of the starch resulted with the production of 12 t o 14 per cent butyl alcohol, based on raw corn.
Sources of Nitrogen PEPTONE.The results of several fermentations in which peptone in various concentrations was used as the nitrogen source are given in Table I. In some cases growth stimulants, potato extract or yeast, were present as indicated. The object of these experiments was to find the optimum concentration of peptone, or preferably the ratio of glucose fermented to peptone nitrogen present a t the optimum concentration of peptone, and to compare other nitrogen sources with peptone. TABLE 11. MALTSPROUTS AS NITROGEN SOURCE WITH POTATO EXTRACT AS STIMULANT Alcohole Produced Total aa Per Cent of Total Sugar per 100 Sugar Fermented m%"t',;l to Sugar Fermented Nitrogen Sugar Present, U s e d 0 Cc., Per Nitrogen IsoGrama Gram's Grams Grama cent Preaent Butyl propyl 0.048 5.4 2.0 2.0 37.0 41.7 0.064 5.5 2.0 2.9 52.7 45.3 20:O 410 2.0 4.0 71.4 35.7 ... 0.112 5.6 0.144 5.8 2.0 5.0 86.2 34.7 23:O 4.2 2.0 5.5 93.2 31.2 25.5 4.5 0.176 5.9 27.4 2.0 5.6 93.3 0,204 6.0 2.0 5.7 93.4 25.4 25:O i:3 0,224 6.1 2.0 6.0 96.8 22.1 26.0 4.2 0.272 6.2 6.0 36.0 60.0 51.0 18.0 4.1 0,705 60.0 1 . 4 1 0 100.0 5.0 54.0 54.0 38.3 15.3 4.5 6.0 36.0 60.0 51.0 18.0 4.0 0.705 60.0 Including starch and maltose.
In order t o estimate the commerical value of a nitrogen source, it is necessary to consider among other factors: (1) the ratio of sugar fermented t o nitrogen required; (2) the percentage of the total sugar fermented; and (3) the yield of products of fermentation expressed as percentage of the sugar fermented. In addition, the rate and vigor of fermentation are of importance. The data in Table I indicate that a t the optimum concentration of peptone the approximate ratio of glucose fermented to nitrogen present was 18 to 1. For 2 per cent sugar solutions the average yields were about 26 per cent of butyl and 4.2 per cent of isopropyl alcohol. In addition, 2 to 3 per cent of ethyl alcohol was usually found, and from 1 to 4 per cent of volatile acids was formed. The alcohols were determined by the method of Stahly, Osburn, and Werkman (7). In fermentations to which neither yeast nor potato extract was added, the percentage yields of alcohols and the sugar utilization were substantially the same as in the experiments in which the stimulants were present. However, the rate of fermentation was slower, requiring 4 to 5 days for completion; whereas in the experiments with added stimulants, especially those with yeast, fermentation was usually completed within 3 days. Therefore, while the growth-stimulating factor is undoubtedly beneficial, it cannot be considered essential, In the last four fermentations shown in Table I, the peptone used was completely hydrolyzed with 20 per cent sulfuric acid. The data show that more sugar was fermented a t the lower concentrations of peptone. About 23 per cent of the total nitrogen of peptone is in the amino nitrogen stage, whereas 85 per cent of the nitrogen in the hydrolyzed peptone was amino nitrogen.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
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MALTSPROUTS. The malt sprouts used as a nitrogen source contained 3.52 per cent of nitrogen, of which 35 per cent was soluble in hot water and 30 per cent of the total nitrogen was amino nitrogen. They also contained 10 per cent of maltose and 18 per cent of starch and dextrins. Schjerning (6) has shown that marked proteolysis occurs during the malting process and that the protein in the malted barley is broken down to various stages.
readily utilized by the organism, the ratio of sugar fermented to protein present is high, but the percentage yield of butyl alcohol is low. The yield of isopropyl alcohol usually remains unchanged. I n experiments 10 to 14, inclusive (Table 111),corn steep water and malt sprouts were mixed as shown. The quantity of glucose fermented was large, but the percentage yield of butyl alcohol was rather low. I n these experiments, however, the percentage yield of isopropyl alcohol was nearly 4 u doubled. I n experiments 15 to 18, inclusive, steep water was mixed FIQURE 1. PERCEIVTwith corn gluten which contained about 45.0 per cent proAGE HYDROLYSIS OF tein equivalent to 7.2 per cent nitrogen and 35.0 per cent PROTEINS TO WHICE starch. “Corn gluten” is the trade name given to the protein STEEPWATERWAS residue of corn after the starch is floated off in the manuADDED $ 1 I facture of corn starch and other corn products. The proj 0’ 20 40 60 80 100 teins consist largely of zein and glutelin. PERCENTAGE HYDRO1 YSIS Again the data in Table I11 show a fairly high ratio of The percentage yields of alcohols (Table 11)were calculated sugar fermented to nitrogen present with a low percentage on total sugar fermented, including starch and maltose. yield of butyl alcohol. Corn gluten alone, except that to In column 1 the grams of nitrogen shown are calculated which steep water had been added a t the factory, does not support fermentation. Casein, gelatin, zein, and other unfrom the grams of malt sprouts added. These data show that slightly more sugar is fermented hydrolyzed proteins also fail to support growth of the organper gram of malt nitrogen present than per gram of peptone isms. Analyses of the residues in experiments 15 to 18 show that about 20 per cent of the protein in the gluten had nitrogen, but the percentage yield of alcohols, especially of butyl alcohol, is lower and more variable. With increasing become soluble. concentration of sugar, the percentage yield of butyl alcohol HYDROLYZED PROTEINS.Such proteins as those in malted is markedly lower. In fermentations such as the last three barley, corn steep water, and peptone will support sufficient in Table 11, the broth becomes quite viscous and foams growth to bring about vigorous fermentations, but the native badly, and the percentage yield of alcohols lags considerably proteins from which such substances are derived are apbehind the disappearance of the sugar. This phenomenon parently not utilized, owing to the inability of the bacteria is now receiving further study. to hydrolyze them. The proteins indicated in Table Tv CORN STEEP WATER. The corn steep water contained were hydrolyzed by boiling with 25 per cent sulfuric acid for 2.896 per cent of nitrogen, of which 32.0 per cent was in the 24 hours. The acid was removed with calcium carbonate. amino form. The results with corn steep water alone and The neutral filtrate was decolorized with norite. The tyrosine with steep water mixed either with corn gluten or malt was allowed to crystallize out of the zein and casein hysprouts are shown in Table 111. drolyzates. Volumes of the hydrolyzates, sufficient to represent the quantity of protein (calculated from total nitrogen) The quantity of nitrogen in each of the first nine experiments in which steep water alone was used is represented indicated, were added. ’r%
.’
TABLE111. CORNSTEEPWATERALOXE,AND WITH CORNGLUTEN OR MALTSPROUTS AS NITROQEN SOVRCE (4 PERCENTGLUCOSE)
Expt. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1s
-Source Steep water, cc. 2 3
4 5 “ 6 8 10 12 14 2 4 6 8 10
5 5 5 3
of NitrogenMalt Corn sprouts, gluten, grams grams 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.0 5.0 5.0 5.0 5.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.0 5.0 5.0 5.0
Total Nitrogen, Grams
Total Glucose, Grams
0.067 0.097 0.135 0.168 0.202 0.270 0.336 0.405 0.470 0.243 0.272 0.378 0.446 0.512 0.528 0.528 0.528 0.457
24.0 24.0 24.0 24.. 0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 24.0 20.0 20.0 20.0 20.0
by the volume of steep water expressed as cubic centimeters in column 2: The results show that a t the lower concentrations of nitrogen \he ratio of glucose fermented to nitrogen present was very high, but a maximum effective concentration was soon reached above which no more sugar was fermented. This maximum concentration gave percentage yields of alcohols uniformly higher than the yields secured at the lower concentrations of nitrogen. A hint for further investigation arises from these observations. Whenever a growth stimulant is present with either a low concentration of nitrogen or a protein which cannot be
Glucose Fermented Grams Per oent 4.0 6.0 11.5 11.8 12.5 14.1 15.1 14.9 15.0 17.6 12.5 18.7 18.0 24.0 19.0 18.9 13.0 10.5
16.6 25.0 47.9 49.2 52.0 58.7 62.5 62.0 62.5 73.3 52.0 77.9 75.0 100.0 95.0 94.5 65.0 52.6
Ratio ~l~~~~~ Fermented to Total Nitrogen 59.6 62.0 85.2 70.2 61.9 52.2 44.9 36.8 31.9 72.4 45 :9 49.5 40.3 46.9 36.0 35.6 24.8 23.0
Alcohols Produced as Per Cent of Glucose Fermented ISO-
Butyl
propyl
20.0 27.0 22.0 28.5 19.3
5.0 4.2 4.3 3.6 6.0
29:5 25.6 28.4 21.7 20.5 22.5 19.5 20.3 20.7 19.8 25.0 23.6
3.6 5.0 4.2 9.1 9.5 8.8 10.0 9.5 11.0 7.3 5.7 5.0
...
All these hydrolyzed proteins gave rise to a fairly vigorous fermentation, with normal yields of alcohols. When n o potato extract was added, the rate of fermentation was somewhat slower, and in some cases fermentation failed to start. Glycine, lysine, and tryptophan (except for traces) are lacking in zein; gelatin contains only traces of tryptophan, valine, or tyrosine, yet these incomplete proteins supported the fermentation better than did the casein. The monoamino acids were removed from casein by the Dakin method (2) and used in the last three experiments reported in Table IV. Only a trace of tyrosine was present.
APRIL, 1935
INDUSTRIAL AND ENGINEERING CHEMISTRY
Fermentation occurred a t substantially the same rate as in the case of the hydrolyzed casein. The yields of butyl alcohol were low. The optimum concentrations of nitrogen were ZEIN, GELATIN,CASEIN,AND AMINO TABL IV.~ HYDROLYZED ACIDSAS NITROGEN SOURCES Hydrolyzed . protems, StirnuGrams lant
Alcohols Produced a8 Total Per Cent of Glucose ~ lGlucose~Fermented ~ ~Fermented ~ ~ Grams Grams Per cent Butyl Isopropyl Zeina
2.0
2.0
1.5 1.6
0.9
0.9 0.9 0.9
a
None None P.E.
P.E.
2.0 2.0
P.E. P. E. P.E.
2.0 2.0 2.0
P.E. P.E.
2.0
6.0
P.E. P.E. P.E. None
P.E. Hydrolyzate.
6.0 6.0 6.0
5.9
5.9
5.8 5.9 Gelatin' 5.9
98.3 98.3 96.7 98.3
98.3 70.0 90.0 5.4 5.7 95.0 Casein0 6.0 5.9 98.3 6.0 5.5 91.7 8.3 55.3 15.0 Monoamino Acide from Casein 78.3 6.0 4.7 66.7 6.0 4.0 54.1 12.0 6.5
6.0 6.0 6.0 6.0
4.2
28.5
27.5 29.0
26.5
4.1 4.5
4.6
4.1
26.3
4.0
26.6
4.5 5.3
20.0 27.6 29.7 28.0 21.1
20.5 21.5 25.5
4.1 5.0
4.6 4.0 4.2
4.0 4.1
not determined. Further studies of the amino acid requirements of the organisms are being made. The foregoing experiments indicate that the organisms can utilize various proteins which have been partly hy-
419
drolyzed. Casein, zein, gelatin, and corn gluten (the crude commerical product) were hydrolyzed to varying extents by subjecting 2-gram samples in 25 cc. of water with increasing amounts (0.5 to 3.0 cc.) of concentrated sulfuric acid to 20 pounds per square inch (1.4 kg. per sq. cm.) pressure in the autoclave (265' F. or 130' C.) for 2 hours. After removal of the acid, the residues and extracts were recombined and the percentages of hydrolysis determined by the van Slyke , method. Three cubic centimeters of steep water were added to each flask. In Figure 1 the curves show a gradual increase of sugar fermented with the increase in hydrolysis of the proteins, more than 100 per cent increase being shown with zein. The difference is less pronounced with casein than with the other proteins.
Literature Cited (1) Beijerinck, M. W., Verhandel. Akad. Wetenschappen Amsterdam, &de. Sectie 11, No. 10 (1893). (2) Dakin, H.D., Biochem. J., 12, 290 (1918). (3) Donker, H.J. L., Thesis, Delft, 1926. (4) Folpmers, T., Tijdschr. uergelijk. Geneeskunde, Nos. 5-7 (1920-
22). ( 5 ) Lek, van der, Thesis, Delft, 1928. (6) Schjerning, H., Compt. rend. du lab. CarZsbad, 11, 45-105 (1914). (7) Stahly, G.H., Osburn, 0. L., and Werkman, C. H., Analyst, 59,
319-25 (1934). (8) Tatum, E.L.,Peterson, W. H., and Fred, E. B., J.Bad., 27, 207 (1934). R E C ~ I V EOctober D 5, 1934.
Haddock Meal Effect of Manufacturing Process upon Nutritive Values H. S. WILGUS, JR., L. C. NORRIS, AND G. F. HEUSER Cornell University, Ithaca, N. Y.
C
OMPARATIVE studies of fish meals manufactured by methods in common use have been made in several laboratories. Ingvaldsen (4) reported that fish meals dried a t 195' C. for 15 minutes showed a diminution in arginine and cystine and (6)that putrefaction of the raw material lowered the tyrosine, tryptophan, and cystine contents of these meals. Daniel and McCollum ( 2 ) , Maynard, Bender, and McCay (6),and Maynard and Tunison (7)found that vacuum-dried or steamdried cod or menhaden meals were superior to flame-dried meals for growth of the rat. The latter authors and Schneider ( I I ) , using pigs and rats, obtained results indicating that these differences were due to the detrimental effect of the heat treatment on both the digestibility and the biological value of the proteins of the flame-dried meals studied. While this work has demonstrated the effect of the various common methods of manufacture on the nutritive value of fish meals as measured by chemical means and by biological studies with rats and pigs, no attempt has yet been reported t o compare the effects of vet- and dry-rendering and of
In a study of the effect of the manufacturing process upon the nutritive value of haddock meals, the relative protein efficiency and the relative growth-promoting vitamin G content, as determined by White Leghorn chicks, were used as criteria. Dry-rendered meals were found to possess a greater protein value and 50 per cent more vitamin G than those wetrendered by similar processes of drying. A flame-dried meal was of inferior value. The use of vacuum with steam-drying materially preserved the vitamin G content but did not improve the relative protein efficiency over that obtained without the use of vacuum. Doubling the size of charge had a slight beneficial effect. By not pregrinding the raw scrap, the vitamin G content was best preserved. variations in the temperature and length of application. The present study was undertaken, therefore, in order to investigate the effect of these factors on the nutritive value of haddock meal. This investigation was conducted on the theory that the growth-promoting properties of this class of feedstuffs are due chiefly to the quality of their proteins and to the quantity of the growth-promoting phase of the vitamin G complex present. Results (IS) have been presented in support of this theory. The necessity for an adequate source of pro-
