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some of the rats and produced acute physiological symptoms in the balance killed for examination.
Selenium i n Gluten Fraction Most of the analyses given in Table I are based on the analysis of the gluten fraction. Some preliminary work indicated that nearly all the selenium present in wheat could be recovered from the gluten. The gluten of wheat sample B14.l.98showed on analysis slightly more selenium than could be recovered from the quantity of wheat from which it was extracted, presumably because of losses in igniting the larger masses of wheat and errors involved in the determinations of such small quantities. Eighty-five per cent of the total selenium in sample B14824 was recovered from the gluten fraction. The toxic wheat from South Dakota showed 80 per cent recovery of the selenium in the gluten. Most of the figures given in Table I are probably somewhat too low, since it appears that as much as 20 per cent of the selenium may be lost in the process of concentrating it in the gluten fraction. No work has been done on the relation between the selenium in the wheat and the flour milled from it. Some idea of the distribution of selenium in the wheat may be obtained from the following: The toxic wheat from South Dakota containing 26 p. p. m. selenium was separated into bran, starch, gluten, and soluble fractions by working 1 kg. of the ground wheat with about 17 liters of water. Eighty-eight grams of Hurd-Karrer’s wheat were treated in a like manner with 2.5 liters of water. All that settled out overnight was considered starch. This starch probably contained some fine particles of bran. The table which follows shows the selenium content of the various fractions in p. p. m.
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Toxic Field-Grown Wheat Hurd-Karrer’s Wheat mihole wheat 26 90 Gluten 121 340 Bran 22 94 Starch 6 7 Sol. and suspended matter lBa 180b a 8.9% of wheat. b 7 . 2 + % of wheat (some lost).
Since the gluten of the wheat carries most of the selenium, the examination of glutens prepared for special diets for selenium becomes of interest, since presumably the dry-land wheats would be used for gluten extractions. Several prepared glutens were examined for selenium. Of two samples of gluten obtained from M. J. Horn of this bureau, one contained 0.8 p. p. m. selenium and the other 16 p. p. m. A gluten purchased in New York contained 12 p. p. m. selenium. The gluten separated from the wheat raised by Hurd-Karrer on artificially selenized soils contained 340 p. p. m. selenium. A gluten of this selenium content and the gluten separated from the toxic field-grown wheat would be dangerous to use, and one containing 16 p. p. m. could hardly be considered wholesome.
Literature Cited (1) Byers, H. G., U. S. Dept. Agr., Tech. Bull. 482 (1935). (2) Byers, H.G., unpublished data. (3) Hurd-Karrer, A. M., J . Agr. Research, 50, 413-27 (1934). (4) Munsell, H.E.,De Vaney, G . M., and Kennedy, M. H., to be published by U. S. Dept. Agr. (5) Robinson, W.O.,Dudley, H. C., Williams, K. T., and Byers, H. G . ,IND.ENG.CHEV..Anal. Ed., 6, 274-6 (1934). (6) Strock, L. W., Am. J . Phnrm., 107,144-57 (1935). (7) Williams, K. T., and Lakin, H. W., IND.ENQ.CHEM.,Anal. Ed., 7, 409-10 (1935). RRJCEIVED February 4, 1936.
SULFITE WASTE LIQUOR Laboratory Study of Its Anaerobic Decomposition When Discharged into Water Bodies ULFITE waste liquor (8. w. 1.) is the liquid waste resulting from the manufacture of cellulose pulp from wood by the acid sulfite process. It contains in solution about 10 to 12 per cent of organic matter, which consists of lignin in the form of “calcium ligno-sulfonates,” sugars formed by hydrolysis from hemi-celluloses of wood, and small quantities of acetic and formic acids, methyl and ethyl alcohols, furfural, and acetone. Polyhydroxy acids, such as mannonic, xylonic, and d-gluconic formed by oxidation of sugars, are also often present (3). Although s. w. 1. can be disposed of in a number of ways, such as by evaporation and combustion, as a binder for road building, for alcohol production, in the manufacture of yeast, and for preparation of tannic substances from lignin, most of the liquor is still disposed of by discharging it into water courses as an industrial waste. It is generally agreed that the water pollution effect of s. w. 1. is not due to the presence of any poisonous substances in the liquor but arises from the fermentable organic substances it contains. Bacteria and other microorganisms present in water attack these organic
H. K. BENSON A N D A. M. PARTANSKY University of Washington, Seattle,Wash.
substances of the liquor, converting them into carbon dioxide and water a t the expense of the dissolved oxygen. Thus s. w. 1. changes the gaseous balance in the water; oxygen concentration is decreased and carbon dioxide concentration is materially increased (4). It has been found, however, that properly aerated liquor in concentrations as high as 1 to 50 has no injurious effect on fish (6). The question of what happens to the liquor itself after it is discharged into a water course-that is, how rapidly and to what extent it is decomposed-except for the work done in this laboratory ( I , I, 6, 8), has been very little investigated. The present paper summarizes the findings and conclusions of two and a half years of work on fermentation of sulfite waste liquor.
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Methods of Study
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The pollution effect of sulfite waste liquor is due chiefly to the sugars. The latter, together with the minor constituents of s. w. l., can be destroyed by gasification i n anasrobic methane-type fermentation, which is, however, relatively slow and as yet cannot be controlled. The sugars can also be changed into butyric acid by a pure culture fermentation and the acid recovered. I n both cases the residue contains only lignin, and its IO-day B. 0. D. is only about one-fifth that of the original liquor. Therefore, when lignin is first removed from s. w. I., the methods of disposal are greatly simplified and similar to those used i n the disposal of sugar wastes. All organic matter of s. w. l., including lignin, when sufficiently diluted is oxidized biochemically to carbon dioxide and water in both fresh and sea water. This oxidation should be complete i n about 5 months. Evidence shows that the fear of pollution by s. w. 1. has been greatly exaggerated, and that the problem of s. w. 1. disposal is similar t o that of sebage disposal. Sufficient dilution accomplished by discharging into large bodies of water, where the liquor is destroyed biochemically, should solve the problem.
I t was found that if the fermentation were to proceed t h r o u g h o u t a t the maximum rate attained in the fermentation, it would be complete in 3 to 4 weeks. It was believed, therefore, that by using selected cultures and maintaining optimum conditions it should be possible to reduce the f e r m e n t a t i o n time to a matter of days, in which case it would be feasible to stabilize the s. w. 1. before its discharge into the waterways, thus a v o i d i n g the pollution and a t the same time utilizing the heating value of the fermentation gases. S u b s e q u e n t work (discussed in the following paragraphs) showed, however, that the methane ferm e n t a t i o n could not be speeded up, and the 2-3 month period required for its completion is too long to be used commercially.
When free o x y g e n i s available in the process of biochemical destruction of organic material, the ultimate end products of decomposition are carbon dioxide and water; under a n a e r o b i c conditions the end p r o d u c t s are carbon dioxide and methane, water serving to supply the necessary hydrogen and oxygen. For this reason-especially in the case of anaerobic feron the m e n t a ti on-data kind, the rate, and the total amount of the gases evolved depict accurately the course of f e r m e n t a t i o n . For instance, when the initial carbon content of the fermenting substances is known, from the volume of carbon dioxide and methane evolved the percentage of the m a t e r i a l gasified is easily c o m p u t e d by the method used in sea water mud fermentation studies. In studies with fresh water mud, on the other hand, in addition to gas determinations the disappearance of the individual organic substances was f o l l o w e d by c h e m i c a l analysis of the fermenting s. w. 1. solutions, using analytical methods described in another publication (7). The relative amounts of carbon dioxide and methane evolved a t any time is also important, for the ratio of the two gases is fixed for any compound fermented ( l o ) ,and any variation from that ratio shows formation of intermediate products. For instance, when the composition of the substance undergoing anaerobic fermentation can be represented by a formula CnHPnOn, the volumes of carbon dioxide and methane evolved should be equal. If carbon dioxide predominates, as is usual a t the beginning of fermentation, it is a sign of accumulation of intermediate products, such as butyric acid, which are richer in hydrogen and poorer in oxygen than the original substance.
Anazrobic Decomposition by Bacteria of Sea Bottom Mud The influence of temperature and dilution by sea water on the rate and extent of decomposition and stabilization of neutralized s. w. 1. by the bacteria of sea bottom mud under strictly anaerobic conditions was studied; the experimental set-up and other details are described in an earlier publication ( 2 ) . The temperature of incubation and the dilution affected only the rate of fermentation but had no effect on the extent of gasification or the final end products. The gases evolved consisted of carbon dioxide, methane, and hydrogen sulfide, and their heating value was equivalent to over 3,000,000 B. t. u. per ton of pulp manufactured. The average extent of gasification was 25 per cent of the carbon input. The residue was a biochemically stable liquid. The gas data are summarized in Table I.
Fermentation Studies with Fresh Water Muds for Inoculation
New experiments were undertaken (6,8)to study in greater detail than in the first work anaerobic ferm e n t a t i o n of s. w. 1. under the mixed culture conditions, to isolate active s. w. 1. fermenting bacteria in pure culture, and to study the optimum conditions for their fermentation of s. w. 1. Fresh water muds which were found to contain more active s. w. 1. fermenting bacteria than the sea bottom mud were used for inoculum, s. w. 1. was used in 30 per cent concentration, and the cultures were incubated a t 36" C. for 340 days. These experiments are described in detail elsewhere (6, 8). With all of the twelve muds used, a methane type of fermentation took place which resulted in a complete gasification of all organic matter of s. w. 1. except its lignin. That is, all sugars, both pentoses and hexoses, organic acids, alcohols, furfural, and other volatile organic compounds were destroyed; and the lignin lost nearly half of its methoxyl content (8). The gases evolved were carbon dioxide, methane,
TABLEI. ANAEROBIC GASIFICATION OF WASTELIQUOR IN SEA WATER 0rigin a 1 Dilution Carbon Total CaOt Carbon Incubation of Content and CH4 Evolved Carbon Temp. S. W. L. of S o h . Evolved as Gas G-asified C. V'ot. % Brams/liter Cc./liter Grams/Ziter 76 25.1 342 0.183 1.4 0.73 26.0 1376 0.738 21 5.3 2.84 8.77 4186 2.246 26.2 16.4 3.250 23.5 26.4 14.13 6072 26.7 1418 0.758 5.3 2.84 2.661 30.4 8.77 6009 . 36 16.4 3.450 25.1 26.4 6437 14.13
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and some hydrogen; the latter was formed only at the beginning of fermentation. No hydrogen sulfide was formed in this series of cultures because the s. w. 1. was neutralized with calcium carbonate a t boiling temperature, the precipitated calcium sulfite was allowed to settle out, and only the decanted liquor used for fermentation. The sulfonic group attached to lignin is not attacked by anaerobic bacteria (a, 6). It was found as in the previous study that the fermentation was all but complete in the first 3 months; thus in a typical culture 73 per cent of the total gas produced was evolved in the first 40 days of fermentation and 92 per cent in 70 days (8). The 10-day biochemical oxygen demand of fermented waste liquor was equal to that of the lignin solution containing the same amount of solids and was only 18 per cent of the original unfermented liquor. The B. 0. D. of the fermented and unfermented waste liquor is given in Table I1 (averages of three determinations).
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the final end products. For this reason we call methane fermentation desirable, and butyric undesirable. The experimental evidence points to the conclusion either that several species of microorganisms are required to produce methane ferment'ation or that the sludge has to be first brought to and then maintained a t a certain state of anaerobiosis by other microorganisms before the methane group can develop (6). The problem of methane fermentation in pure culture has not yet been solved. TABLE111. COMPARISON OF PERMANGANATE OXYGENCONSUMED WITH IO-DAYBIOCHEMICAL OXYQENDEMAND Substance Glucose Lignin Acetic acid Butyric acid .4v. fermented waste liquor Av. unlermented waste liquor
Mg. of 0 2 per Mg. of Substance Permanganate IO-day B. 0. D. 0 650 0.600 0.820 0.100 0.004 0.530 0.016 0,900 0 803 0.087 1' 045 0.226
TABLE11. BIOCHEMICAL OXYQENDEMAND OF WASTELIQUOR Mg. 0 s per Mg. Organic Matter Substance 3-day B. 0. D. 10-day B. 0. D. 0.600 Glucose 0.325 0 100 Lienin - from 8. w. 1. 0 . 0 6 3 Fermented liquor from cultures: 0,077 No. 6 0.046 0,090 No. 7 0.068 0.062 0.095 No. 15 Unfermented liquor: No. 1 0.177 No.4 0.170
0.234 0.218
Mg. Oa per Cc. Original Liquor 10-day B. 0. D.
4.61 4.59 5.22 Av. 4 . 7 9 25.1 26.1 Av. 2 5 . 6
These B. 0. D. results are significant, for they show that by allowing s. w. 1. to undergo anaerobic digestion prior to its discharge into a water course, the pollution value of the liquor, as measured by the B. 0. D. test, is reduced to less than one-fifth of the original. The second significant point is that lignin in the dilution used for the B. 0. D. determinations is oxidized biochemically as shown by the progressive consumption of oxygen with time. Both fresh and sea water were used in the B. 0. D. studies, and the results agreed. It has been calculated that if the rate of oxygen consumption continued to be the same as in the first 20 days (20-day B. 0. D. for lignin was 0.190 mg. of oxygen per mg. of lignin), the lignin of s. w. 1. would be completely oxidized to carbon dioxide and water in 140 days. To extend the incubation time in B. 0. D. tests beyond t;he 20-day period and verify this figure experimentally did not appear to be a fair test; in nature the water is mixed and aerated continuously and the microorganisms are kept vigorous, whereas in a closed bottle with lignin as the only food material we would expect them to become attenuated before the digestion is complete.
Controlled Fermentation Processes When fermentation in the cultures described was a t its peak, a large number of bacteria were isolated in pure culture; those which were capable of fermenting s. w, 1. with evolution of gas were studied in detail and are described fully elsewhere (9). All these bacteria were strict anaerobes and fermented only s. w. 1. sugars, converting them into butyric and acetic acids, carbon dioxide, and hydrogen. Of the two usual types of anaerobic fermentation (butyric and methane types), only the methane type results in complete gasification of fermentable material into carbon dioxide and methane, although volatile acids-the intermediate products-frequently accumulate at the beginning. In the butyric type of fermentation, however, acids as well as gas are
The newly isolated organisms mentioned, especially Clostridium polyfermenticum, can be made under proper conditions of temperature, liquor dilution, and nitrogenous matter content to convert s. w. 1. sugars into butyric and acetic acids in the course of 2 days. This change reduces the permanganate-oxygen-consumed value for the liquor, owing to resistance of the acids to oxidation by permanganate, but not the B. 0. D. value (the true test of pollution) for biochemically the acids are oxidized just as readily as the original sugars, so that there is no gain from such a change from the pollution standpoint. Permanganate oxygen consumed and 10-day B. 0. D. values are compared in Table 111. However, if all sugars of s. w. 1. are converted into butyric acid and the latter is removed from solution by distillation or extraction with a solvent, the residue will contain practically only lignin and be much more stable biochemically, as has already been shown in Table 11.
Acknowledgment Acknowledgment is made to a group of Puget Sound pulp mills for a research fellowship by which this study was made possible.
Literature Cited (1) Benson, H. K., IND. ENQ.CHEM.,24,1302(1932). (2) Benson, H.K., and Partansky, A. M., Proc. Natl. Acad. Sci., 20, 542 (1934). (3) HRgglund, E.,Ahlbom, J., and Johnson, T., Ber., 62B, 437 (1929). (4) Heiduschka, A.,and Munds, E., 2.angew. Chent., 42, 11 (1929). ( 5 ) Marsh, M. C., U. S. Ceol. Survey, Water s u p p l y Irrigat. Paper 192,337 (1907). (6) Partansky, A. M., doctorate thesis, University of Washington, 1935. (7) Partansky, A. M., and Benson, H . K., Paper Trade J., 52,No. 7, 29 (1936). (8) . . Partansky, A.M., and Benson, H. K., Proc. Natl. Acad. Sci., 22, 153 (1936). (9) Partansky, A. M., and Henry, B. S., J . Bact., 30,559 (1935). (10) Symons, G. E., and Buswell, A. M., J . Am. Chem. SOC.,55,2020 (1933). RECEIVED January 28, 1936.
