Abstract:
In vivo and in vitro cellulose fermentation by cellulose-digesting microorganisms is increased by conducting the fermentation in the presence of a minor amount of a 2-(chloromethyldithio)acetic acid.

Description:
BACKGROUND OF THE INVENTION 
     The effect of chemical additives in microorganism fermentations has been extensively studied. For example, P. P. Williams et al, App. Microbiology, 11, 517 (1963) describe rumen bacteria and protozoal responses to insecticide substrate; J. J. O&#39;Connor et al, J. Animal Sci., 33, 662 (1971) describe the in vivo effect of chemical additives on production of volatile fatty acids by rumen microorganisms; and L. W. Varner et al, J. Animal Sci., 33, 1110 (1971), describe the influence of ammonium salts upon rumen fermentation by steers; and T. W. Dowe et al., J. Animal Sci., 16, 93 (1957) describe the effect of corn treated with fungicides (N-trichloromethylthio-delta 4  -tetrahydrophthalimide) on the performance of fattening steers. 
     PRIOR ART STATEMENT 
     U.S. Pat. No. 2,553,778 of Hawley discloses parasiticidal perchloromethylacetic acid esters. U.S. Pat. Nos. 3,442,941, 3,595,915, 3,629,313 and 3,718,687 of Emerson et al disclose pesticidal polyhaloethyl alkanoic acids. Although the compounds of these references are structurally related to the compounds of the invention, the compounds of the references have been found to be ineffective for increasing the rate of cellulose digestion by microorganisms. 
     DESCRIPTION OF THE INVENTION 
     The cellulose-fermentation-accelerating compounds of the invention are 2-(chloromethyldithio) acetic acid, 2-(dichloromethyldithio)acetic acid and 2-(trichloromethyldithio)acetic acid. The preferred compound is 2-(trichloromethyldithio)acetic acid. 
     The amount of compound employed in the process of invention depends in part upon the type of cellulosic material and the particular microorganism(s) employed. Generally, weight ratios of compounds to cellulosic matter in the range of about 1:10 to 1:1,000,000 are effective, although weight ratios in the range of about 1:100 to 1:10,000 are preferred. 
     In in vitro cellulose fermentation processes, the compound is generally added directly to the fermentation process. In in vivo cellulose digestion, the compound may be orally administered to the aminal along with the cellulosic feedstuff. Alternatively, the cellulosic feedstuff may be pretreated with an effective amount of the compound prior to feeding the animal. 
     The process of the invention is generally applicable to in vivo or in vitro cellulose fermentation by microorganisms. Examples of in vitro cellulose fermentation by microorganisms are the aerobic and/or anerobic destruction of cellulosic wastes in sewage plants; conversion of cellulose to sugar by microorganisms such as Trichoderma viride; conversion of cellulose to singlecell proteins by microorganisms such as Bacteroidaceae, Cellulomonas and Alcaliginis; and the biodegradation of lignincellulosic plant material. Examples of in vivo fermentation by microorganisms are cellulosic digestion by rumen microorganisms of ruminant animals, cecum microorganisms of animal intestines, and other cellulolytic organisms in the alimentary tracts of herbivores. 
     The process of the invention is suitably employed for all types of cellulosic material such as paper, municipal waste and plant products, e.g., wood, cotton, straw, bagasse, rice hulls, etc. 
    
    
     EXAMPLES 
     Example 1 -- Cotton digestion by Bacteroides succinogenes 
     The organism, Bacteroides succinogenes, was obtained from the American Type Culture Collection, No. 19169. 
     
         ______________________________________        Bacto-fluid ThioglycollateNutrient Source:        (29 g formulation/liter of H.sub.2 O)______________________________________      Bacto-Casitone                    15.0      g      Bacto-Yeast Extract                    5.0       g      Bacto-Dextrose                    5.0       g      NaCl          2.5       g      1-Cystine, Difco                    0.5       g      Thioglycolic Acid                    0.3       ml      Bacto-Agar Resazurin,      Certified     0.001     g______________________________________ 
    
     The rate of cotton digestion in the presence of several test compounds in the above nutrient broth with Bacteroides succinogenes was determined by the following procedure: 
     Cotton (100 mg) was placed in screw-cap tubes. To these the test compound (1 microgram) and the nutrient source (20 ml) were added to completely fill the tube. 
     The tubes were then sterilized, cooled and inoculated with the microbe (1 loop of inoculation needle), their caps tightened, and incubated in a water bath at about 40° C. 
     The tubes were stirred throughout incubation and the caps loosened every 2 hours for the first 18 hours and every 6 hours thereafter to release gases produced by the fermentation. After 70 hours of incubation, most of the fermentation processes had subsided, as noted by cessation of gas accumulation. 
     After various periods of incubation, the tubes were emptied on previously weighed filter paper. The filter paper was washed several times and dried to a constant weight. The weight of the undigested cotton was determined by difference. 
     The cellulose digestion results are tabulated in Table I. The results are based on the average of 48 runs and standard deviation analysis showed the results to be significant 1% level. 
     
                       TABLE I______________________________________              CottonTest Compound      Digestion %                         Acceleration______________________________________Control            38.6%      --2-(trichloromethyldithio)-              43.6%      13%acetic acid2-(1,1,2,2-tetrachloroethyldithio)-              38.0%      0acetic acid______________________________________ 
    
     Example 2 -- Cotton digestion by Bacteroides succinogenes 
     The rate of cotton digestion with Bacteroides succinogenes in the presence of several test compounds was determined by a procedure identical to that of Example 1. The test compounds and the results are tabulated in Table II. 
     
                       TABLE II______________________________________              CottonTest Compound      Digestion %                         Acceleration______________________________________Control            37.1%      --Methyl 2-(trichloromethyldithio)-              36.2%      0acetateMethyl 2-(1,1,2,2-tetrachloroethyl-              36.7%      0dithio)acetate______________________________________ 
    
     Example 3 -- Plant Cellulose Digestion by Bacteroides succinogenes 
     Lignin-cellulosic matter of herbaceous plant forage was digested by Bacteroides succinogenes in a purified medium in the presence of 2-(trichloromethyldithio)acetic acid at a concentration of 10 micrograms/ml by a procedure identical to that of Example 1. After 70 hours incubation, the percent cotton digestion was 54.3%. In an untreated control run, the percent cotton digestion was 41.1%. 
     This example exemplifies the in vitro separation of cellulose from lignin-cellulosic matter by biodegradation of the cellulose. 
     EXAMPLE 4 -- Cotton Digestion by Ruminococcus albus 
     Ruminococcus albus was obtained from the America Type Culture Collection. It was cultured on Pseudomonas medium broth which contained the following (per liter of distilled H 2  O): 
     
         ______________________________________Nitrilotriacetic acid  1.91   gK.sub.2 HPO.sub.4      8.71   gNa.sub.2 SO.sub.4      0.57   gMgSO.sub.4             0.25   gFeSO.sub.4             0.5    mgCa(NO.sub.3).sub.2     0.5    mgAgar                   1      g______________________________________ 
    
     About 20 ml of the medium and 0.1 g cotton were added to each of 48 screw-cap tubes and sterilized. The tubes were then inoculated with 1 loopful of the microbe. To half of the tubes was added sufficient 2-(trichloromethyldithio)acetic acid to give a concentration of 10 micrograms per ml. The tubes were then sealed and incubated in a water bath for 70 hours at 40° C. At the end of the incubation period, the weight of undigested cotton was determined. 
     The treated tubes (average of 24) gave 29.8% cotton digestion. The control tubes (average of 24 gave 23.2% control. 
     Example 5 -- Cotton Digestion by Bacteroides succinogenes in rumen fluid 
     The rate of cotton digestion in the presence of 2-(trichloromethyldithio)acetic acid in sterilized rumen fluid with Bacteroides succinogenes was determined by a procedure identical to that of Example 1. After 70 hours incubation, the percent cotton digestion was 46.6%. In an untreated control run, the percent cotton digestion was 39.8%. 
     Example 6 -- Solka Floc digestion in rumen fluid 
     Digestion of Solka Floc was determined using a modification of the two-stage digestion procedure (Tilley and Terry, J. Brit. Glassl. Sc. 18:104-111, 1963). Substrate (in triplicate) was treated with either 0, 40, 60, 80, 100 or 150 ppm of the test compound and incubated with buffered rumen fluid for 24 hours followed by 24 hours pepsin digestion. In vitro digestibility was measured at end of incubation with buffered rumen fluid (ruminal digestion) and at end of pepsin digestion (total digestion). 
     Rumen fluid was obtained from donor animal maintained on alfalfa hay - corn grain mineral supplement ration at maintenance plus level of intake. The test compound was added directly to Solka Floc on dry matter basis. 
     
                       TABLE III______________________________________Level of     Percent Ruminal                       Percent TotalTest Compound.sup.1        Digestibility.sup.2                       Digestibility.sup.2______________________________________ 0 ppm       10.34          26.5040 ppm       13.96 (35.01)  29.53 (11.43)60 ppm       12.73 (23.11)  30.00 (13.21)80 ppm       13.18 (27.47)  29.93 (12.94)100 ppm      12.91 (24.85)  29.16 (10.04)150 ppm      11.96 (15.67)  28.26  (6.64)______________________________________ .sup.1 Values in parentheses represent percent increase. .sup.2 Test Compound added on substrate dry matter basis.