Patented Oct. 8, 1946 2,408,853 UNITED STATES PATENT OFFICE I 2,408,853 I GUAYULE RUBBER BY FERMENTATION Samuel R. Hoover, Philadelphia, Pa., Paul J. Al len, Robles del Rio, Calif., and Joseph Naghski, Philadelphia, Pa., assignors to the United States of America as represented by the Sec retary of Agriculture No Drawing. Application July 21, 1944, Serial No. 546,020 4 Claims. (Cl. 195-1) (Granted under the act of March 3, 1883, as amended April 30, 1928; 370 O. G. 757) 1 This application is made under the act of March 3, 1883, as amended by the act of April 30, 1928, and the invention herein described and claimed, if patented, may be manufactured and used by or for the Government of the United States of 2 agar medium which containsthe resin of guayule rubber as an emulsion, along with common bac terial nutrients, if the latter are desired. Resin digestion is shown by the appearance of cleared areas around the colonies as they grow. We have America for governmental purposes without the demonstrated that the microbiological digestion payment to us of any royalty thereon. of the crude resin extracted from the rubber is In the recovery of rubber from guayule by the a selective process, 1. e. only certain constituents conventional mechanical extraction method, the present in the resin are removed by the organ shrub is defoliated, crushed or cut, and milled 10 isms. The data presented hereinafter shows that with pebbles in a slurry containing appropriate marked improvement in physical properties re solids until the rubber is agglomerated into gran sults from incubation of the rubber with these ules, called worms. Upon dilution of the slurry organisms, which leads to the conclusion that with water, the Worms ?oat to the surface and the impurities present in the rubber that are are skimmed off. The product recovered by these 15 removed by fermentation are those which lower operations is boiled, usually under hydrostatic pressure, to waterlog the particles of plant ma terial contaminating it, and then is dried and marketed. the physical properties. A variety of organisms thus selected have been shown to improve the quality of guayule rubber by pure culture fermentation of the worms in Commercial guayule rubber produced by this 20 an appropriate medium under proper conditions process contains appreciable quantities of resinous of aeration and temperature for the particular contaminants and plant debris. Various methods organism. Typical data is given in Table 1, of further purifying the product have been ‘pro below. posed, extraction of the resins with acetone or Examples other solvents having been employed commer 25 100 gms. wet weight of‘ guayule worms were cially from time to time. ' steam sterilized 30 minutes at l5-pound pressure, We have found that the rubber worms pro then fermented in 250-300 cc; of Allison’s min-_ duced by the conventional mechanical extraction eral salts medium 1 in 2-quart jars at 30-32° C. method can be subjected to a process of fermen tation. By this method, only 5 to 20 percent of 30 for 10‘ days. The pH was adjusted and organic the plant mass material is required to be handled amendments were added to the medium as indi and pure culture inoculum may be employed to cated. The worms were washed and ‘air dried at 32“ 0. Physical properties given are those at maintain an essentially pure culture fermenta optimum time of cure, usingthe following formula. tion. We have also developed a method for eval ulating the ability of organisms to grow on and 35 and conditions, referred to as “ERRL Standard”: digest the guayule resins. Guayule rubber worms are particles averaging %" to 1/4" in size which on microscopic exami Rubber ___ Captax __ parts" 100 do___~v 1 ZnO ____ do“; 5 nation of cross sections are shown to be quite inhomogeneous, with numerous voids and oc 40 Stearic acid _____________________ __do____ 1.5 S > do____ 3.5 eluded particles of bagasse. Apparently, the basic visible unit in a worm is the contents of one cell, many of these being pushed or stuck together to form a worm. Mill'I'_______________________________ __°F__ 1.15 Vuld T_____________________________ __°F__ 274 1Standard Allison’s medium was diluted to half Evaluating the ability of organisms to grow on 45 strength. and digest guayule resins comprises inoculation All values are given at optimum time of cure and growth of a pure or mixed culture on an for tensile strength. 2,408,853 Table 1 Analytical results Sample U25W2~1°¥ Organism I‘ ” Organic amend. ' bor , pH _ Benzene Acetone Benzene Tensile Elon- Hug extract extract sol strength gation shot; 7. 1 76. 28 17. 79 5. 93 2, 960 830 32 Micrococcus (unident.) _ .____ 7. 0 76. 65 17. 43 5. 92 2, 650 950 31 Achromabacter lacticum l___-_ N 6. 3 6. 7 7. 4 6. 6 7. 7 7. 6 7. 0 76. 03 77. 49 78.87 78. 30 78. 04 79. 11 76. 34 17. 75 17. 21 16. 74 16. 62 17. 27 16. 25 19. 06 6. 22 5. 30 4. 39 5.08 4. 69 4. 64 6. 60 2. 280 2, 810 2, 860 2, 850 2, 860 2, 810 2, 230 970 880 840 940 910 940 940 27 32 35 33 37 31 27 . _ .. _ . Nutrient broth ...... -. Physical tests _ _ . "do- _ 0.2% N114 citrate ____ _. 6. 8 75. 49 18. 75 5. 79 2, 350 940 32 6. 7 75. 47 18. 80 5. 73 2, 540 950 29 1 Less satisfactory growth took place in this medium. This data is given to typify the negative results obtained with unsatisfactory growth conditions or with organisms which do not produce the desired efiectrin any of the media tried. tions of said resinous contaminants by fermen On the basis of ease of culture and nutrient re-_ quirements Pseudomonas boreopolz's gave the best 0 tation, whereby the quality of the rubber is in creased, thence removing and washing the re results, as indicated by the following example: sulting high quality rubber and drying it. 1000 g. wet weight of worms were incubated with out sterilization at room temperature 2. The process of producing an improved rub (about ‘ ber from guayule rubber worms, comprising sus 86-88° F.) in 5 liters of half strength Allison's solution to which was added 0.1% ammonium 25 pending the worms in water, inoculating the mix ture with Pseudomonas boreopolis organisms, citrate and 0.15% KzIH’Or. 100 cc. of a 2 day conditioning the medium for growth of the or nutrient resin broth culture of Ps. boreopolis was ganisms, allowing the organisms to grow until added at 0 days and 100 cc. more of a 1 day cul the resin content of the worms is substantially de ture was added the third day. Fermentation was carried out in an open crock and aeration was 30 creased, whereby the physical properties of the rubber will be improved, then removing and maintained by compressed air bubbling vigorous washing the improved rubber and drying it. ly from a tube running to the bottom of the crock. Table 2 Analytical composition ERRL standard formula std'?l?gyg’agle'ggggnodi‘ Day Benzene Acetone extract insoluble Tensile Per cent Per cent Per cent P. s. i. 75. 45 77. 77 78. 75 80. 31 80. 99 18. 20 16. 28 14. 61 13. 87 12. 45 6. 5. 95 6. 64 5. 82 6. 66 extract C ontrol .... __ 5 ___________ __ 8 ___________ __ 12 __________ __ 20 __________ __ Benzene - 2, 400 2, 860 3, 020 3, 060 3, 280 Modulus at 600% - Elong' Tens?e Per cent P. s. 1‘. 270 440 500 430 660 940 890 880 900 860 3, 120 3, 380 3, 450 3, 710 3, 790 Modulus at 600% Elong' Per cent 030 1, 070 l, 120 1, 190 1, 080 840 800 800 790 800 The accelerating e?ect of p,p'-diaminodiphenyl methane upon the physical properties of the rub ber was essentially additive upon the improve 3. The process of producing an improved rub ber from guayule rubber worms, comprising sus pending the worms in water, inoculating the mix ment due to the fermentation. A boiling treatment or a heating under hydro static pressure (paila treatment) is convention ture with Aspergillus fumigatus organisms, con ditioning the medium for growth of the organ isms, allowing the organisms to grow until the ally used in the guayule rubber industry to water log the cellulosic debris present in the worms, thus causing it to sink and be separated from the resin content of the worms is substantially de creased, whereby the physical properties of the rubber will be improved, then removing and Washing the improved rubber and drying it. ?oating rubber. The worms coming from this step of the process are at least partially sterilized and in excellent condition for a subsequent fer mentation step. The worms treated by our proc 4. The process of producing an improved rub beer from guayule rubber worms, comprising sus pending the worms in water, inoculating the mix ess can be washed in water by a variety of means, 60 ture with Trz'choderma sp. organisms, condition ing the medium for growth of the organisms, al lowing the organisms to grow until the resin con tent of the worms is substantially .decreasedi, such as on a screen or by successive changes of water, and then dried in the conventional man ner. Having thus described our invention, we claim: 1. The process of extracting high quality rub ber from guayule rubber plants comprising me - whereby the physical properties of the rubber 65 will be improved, then removing and washing the chanically separating rubber worms from the plant bulk, which worms will normally contain resinous contaminants, suspending the worms in water, selectively decomposing substantial por 70 improved rubber and drying it. SAMUEL R. HOOVER. PAUL J. ALLEN. JOSEPH NAGHSKI.