Cellulose, a complex sugar, can be broken down through saccharification by some microorganisms in the soil to yield glucose. After further action by other microorganisms, it may be converted to ethyl alcohol (grain alcohol) for use as a fuel. The discussion that follows deals with some work done at South Dakota State University (SDSU) to ascertain the feasibility of this process in a farm or community-scale fuel alcohol production facility. Presently, the limiting factor in obtaining fuel alcohol from cellulose wastes is the cost of producing the complex of the three enzymes needed to carry out the process of saccharification. To date, improvements in enzyme production have been made only in the research laboratory. An owner-operator system that would convert cellulose to glucose has yet to be economically developed. The fuel alcohol production facility at SDSU is similar in size to what might be used by an individual owner-operator. This study sought to determine how effective it would be to scale up from the laboratory production level to a rudimentary pilot plant

Cass, Carla M.

Gauger, W. Kennedy

English

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South Dakota State UniversityOpen PRAIRIE: Open Public Research Access InstitutionalRepository and Information ExchangeAgricultural Experiment Station Technical Bulletins SDSU Agricultural Experiment Station1985Fuel Alcohol Production : Conversion of Celluloseto GlucoseCarla M. CassW. Kennedy GaugerFollow this and additional works at: http://openprairie.sdstate.edu/agexperimentsta_tbThis Article is brought to you for free and open access by the SDSU Agricultural Experiment Station at Open PRAIRIE: Open Public Research AccessInstitutional Repository and Information Exchange. It has been accepted for inclusion in Agricultural Experiment Station Technical Bulletins by anauthorized administrator of Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. For more information,please contact michael.biondo@sdstate.edu.Recommended CitationCass, Carla M. and Gauger, W. Kennedy, "Fuel Alcohol Production : Conversion of Cellulose to Glucose" (1985). AgriculturalExperiment Station Technical Bulletins. 81.http://openprairie.sdstate.edu/agexperimentsta_tb/81TB 87 Fuel Alcohol Production Conversion of cellulose to glucose -Agricultural Experiment Station South Dakota State University SDSU LIBRARY MAY 15 1985 STATE DOCUMENT :-~:"'."'."""":-~-TA S TY JUNO , 1985 l. y Brookings, SD 57007 FARM-SCALE CIJNERSI(m OF CE:,LUI.OOE 'IO GLUCD>E FOR~ ALCXBJL mcDJcrICE SDSU LIBRARY MAY 15 1985 STATE DOCUMENT Carla M. Cass and W. Kennedy Gauger Microbiology Department, SDSU Cellulose (a cx:>mEX)nent of plant tissue) is the IIK>st abundant organic material on earth (15). Farmers produce, often without subsequent use, IIK>re cellulose than any other group of people. This exists as cx:>rn and wheat stover, beet tops, grain dust, and other forms of crop residues which are of limited economic value except when returned to the soil. Cellulose, which is a cx:>rnplex sugar, can be broken down through sac-charification by some types of microor-ganisms that typically inhabit the soil. Saccharif ication yields glucose. If the glucx:>se is subjected to further action by different microorganisms, it may be converted to ethyl alcohol (grain al-cohol) for use as a fuel (6,8). The discussion that follows deals with some work that we have done at South Dakota State University (SD.5U) to ascertain the feasibility of this process in a farm or cx:>rranunity-scale fuel alcx:>hol production facility. Saccharification is carried out by specialized proteins called enzymes that act as biological catalysts to hasten the breakdown of cellulose to glucx:>se. Actually, this process is carried out by a cx:>mplex of three enzymes, cx:>llectively called cellulase, that act together to achieve the degradation process. This complex of enzymes is IIK>st frequently obtained from strains of the IIK>ld Trichoderrna reesei chosen for their ability to produce large amounts of cel-lulase (6,8,9). L. reesei also holds the greatest promise for use in corrmer-cial fuel alcohol production facilities. Presently, the limiting factor in obtaining fuel alcohol from cellulose wastes is the cost of producing the en-zyme complex. Studies have shown that 50 to 60% of the cost of producing 1 glucose is directly related to the ef-ficiency of cellulase production (2,12). The only viable options a plant manager has are either to buy the enzyme, which is expensive, or to produce his own, which is difficult, as the following discussion reveals. To date, improvements in enzyme production have been made only in the research laboratory. They inv:olve in-corporating additives (5,7) to the production medium, pH cycling and tem-perature profiling, and using continuous culture processes ( 4 ,11) • An owner-operator system that would convert cel-lulose to glucose has yet to be ecx:>nomi-cally developed. The fuel alcohol production facility at SDSU is similar in size to that which might be used by an in-dividual owner-operator. This study sought to determine how effective it would be to scale-up from the laboratory production level to a rudimentary pilot plant. L. reesei cellulase in our studies at SDSU was produced in a stainless-steel dairy culture vessel with a work-ing capacity of 74 gallons (280 liters) and fitted with a stirring paddle, a pH monitor and control device, heating-cooling coils, and a sterile air supply. A EX)lyethylene reservoir provided am-monium hydroxide (2N NH40H) for pH ad-justment. This ferment6r ooubled as a vessel for both enzyrre production and cellulose saccharification. The mold was grown by a subnerged culture technique where it was mixed throughout the culture vessel to achieve maximum production of the saccharifying enzyme. As L. reesei grows, the pH of the culture medium drops to about 3.0, usually within a 24- to 72-hour time span. It is at this pH that enzyme production begins and must be maintained (up to a pH of 3.5) for ~irnum yields. After a period of a week to 10 days, the pH will rise rapidly, indicating the end of the production process (7,12,14). The cellulose substrate employed for saccharification was Solka Floe SW-40 (Brown Co, Berlin, NH). Solka Floe SW-40 is a purified form of alpha-cellulose derived from spruce wood. (It is typically the most difficult to break down; therefore it is used as a standard for comparative studies.) It is ap-proximately 95% pure and is ground to 40 mesh fibers (3,8). Saccharification ex-periments were performed on both the laboratory and pilot plant scales. Enzyme production was monitored by observation of the Fil profile il-lustrated in Figure 1. The pH dropped rapidly after 24 to 72 hours and was maintained at 3.0 with the addition of ammonium hydroxide (13,14). After several days the pH rose. This is at-tributed to the release of arranonium ions and is indicative of the end of enzyme production (12,14). Data observed in both the laboratory and pilot plant facility depict this type of profile (Fig. 1). There was some variance in the amount of time it took for the pH to drop initially for some of the pilot plant experiments, but they still fol-lowed the same basic pattern. These variances of time were probably due to the lack of control over process para-meters. For example, although tempera-ture was constantly monitored, it was necessary to adjust it manually. Consequently, temperature fluctuated over the production period as much as 3 to 4°c. Control of temperature to achieve optimal production is considered critical (10,12). Another variable was inoculum size. 6---------------~----------, 5 :c 4 C. 3 Cellulase production begins Saccharlflcatlon I Initiated 2~-------.--------,,--------~------1 0 50 100 150 200 Time, Hours Figure 1. Typical pH profile of a plant experi.rrent. 2 There is no rapid method for estimating inoculum size. Therefore, even though all experiments were treated alike, there were no adequate means to ensu~e that the inoculum size was rraintained consistently between experiments. This variation affected the initial lag period (when the microorganisms are mul-tiplying). Of all variables, it affec-ted cellulase production Il'K)St seriously; lag time should be kept to a minimum, if possible. Saccharification was not initiated until the pH began to rise on its own ( 6 to 9 days in our studies) • This in-crease in pH indicates the end of enzym: production as well as additional enzyme release from the cell wall on the or-ganism's death (1,7,13,14). Saccharification efficiency of the Solka Floe 5W-40 substrate showed poor results for pilot plant experiments when compared to a typical laboratory experi-ment (Fig. 2). The pilot plant experi-ments showed an increase in sac-charification for the first four trials. This suggested that problems associated with the scale-up and the process con-~ Pilot Plant ~ Laboratory 1 2 3 4 5 BENCH Experimental Run Figure 2. Saccharification* of Solka Floe Sv-40 (5% w/v) by Trichoderma reesei CM 9414 cellulase. *Saccharification period for pilot plant and laboratory experiments were Run 1, 2 days; Run 2, 3.5 days; Run 3, 2.6 days; Run 4, 4.3 days; Run 5, 2 days; Bench, 5.5 days. 3 trol in the pilot plant were diminishing and overall improvement was being achieved. The final pilot plant experi-ment (#5) showed a decrease in sac-charification efficiency, which was roost likely attributed to a small inoculum size. Statistical analyses were performed to determine if characteristics of sac-charification efficiency differed from trial to trial. An analysis of variance (ANJVA) test established that there was a highly significant difference (0.01 level) between plant and laboratory ex-periments. In other words, pilot plant saccharifications were nowhere as suc-cessful in achieving conversion of the cellulose to glucose. As a consequence, not as much glucose for fermentation to fuel alcohol was produced in the scaled-up process as was theoretically p:>ssible. To ascertain how the individual pilot plant trials compared to one another, a least significant difference statistical test was computed. It showed that trials 2 and 5 were the only two plant scale trials which were not significantly different from one another. In other words, although the saccharification efficiency was p:>or when compared to laboratory data, ef-ficiency improved as roore experiments were performed, with the exception of the last one which had a low starting inoculum. Our data confirm the variability of saccharification efficiencies between experiments performed in the enzyme . production facility. The extent to which saccharification occurs is prop:>r-tional to the amount of enzyme produced. The differences in efficiency between individual plant experiments and between plant and laboratory data can be at-tributed to a variety of problems. The major problem was the need for improved process controls including tem-perature, pH, aeration, agitation, and process sterility. Ideally, this production procedure should be control-led by a microprocessor or other com-4 puterized system. This would eliminate error associated with manual operation of parameters and ensure that processes would be adjusted more frequently and probably roore economically. Furthermore, it would release personnel to carry out other n:cessary activities. Our results confirm that process control is critical during the sac-charification grocess. The optimum tem-perature of 50 C was difficult to main-tain manually. This fluctuation can lead to a decline in enzyme activity as well as saccharification efficiency (10,12). Saccharification periods varied from plant experiment to plant experi-ment as depicted (Fig. 2). The time periods listed were the amount of time for which maximum glucose was produced. All plant experiments showed a severe reduction in saccharification efficiency after those times. This decrease was roost likely due to contamination by bac-teria and other microorganisms. The source for contamination could have originated from the substrate, Solka Floe, which was not sterilized prior to its addition. Sterilization of the sub-strate would be energy intensive, and therefore, it would not be an economi-cally viable option. Removal of samples from the production-saccharification vessel for analysis could not be achieved without introducing contamination. Consequently, airborne microorganisms could have gained entry and caused con-tam~nation. Saccharification conditions (50 C, pH= 4.8) are not extreme enough to retard bacterial growth by the majority of organisms. A problem may also have been associated with glucose, the product of conversion. This undoubtedly provided an essential growth substrate for any of the contaminating organisms, including any viable I.... reesei cells. The total production period from enzyme production through saccharifica-tion was generally 11/2 to 2 weeks. Over this amount of time it is not surprising that contamination would oc-cur, especially with such rudimentary facilities. There was no contamination associated with laboratory experiments, ·suggesting that rigorous control over process parameters in the plant is essential. Surmacy We have determined that, with the owner-operator facility at SDSU, enzyme production is not feasible. Updating the facility for optimal enzyme produc- · tion requires a large amount of ItK>ney. Along with ItK>re sophisticated equiµnent, a microprocessor or computer-driven sys-tem would be necessary if the process is to operate smoothly and without constant supervision. Furthermore, enzyme production and substrate saccharifica-tion require personnel with extensive training in microbiology, chemistry, and engineering. Other conversion methods such as acid degradation are currently being compared with enzymatic breakdown at SDSU. At present, the owner-operator system for enzyme production and cel-lulose saccharification appears not to be feasible without a substantial in-vestment in equipnent and technical ex-pertise, which is impractical for a farm or conmunity-scale operation. An alter-native to .in situ production would be to purchase the enzyme, but presently this is not cost effective. LITERATURE CITED 1. Andreotti, R. E., J.E. Medeiros, C. Roche, and M. Mandels. 1980. Effects of strain and substrate on production of cellulases by Trichoderrna reesei mutants, p. 1-12. .In Second international course-cum-symposium on bioconversion and biochemical engineering. New Delhi, India. 2. castanon, M., and C. Wilke. 1980. Adsorption and recovery of cel-lulases during hydrolysis of newspaper. Biotech Bioeng 22:1037-1053. 3. Fan, L. T., Y. Lee, and D. H. Beardmore. 1980. Mechanism of the en-zymatic hydrolysis of cellulose: Effects of major structural features of cellulose on enzymatic hydrolysis. Biotech Bioeng 22:177-199. 4. Ghose, T. K., and V. Sahai. 1979. Production of cellulases by Trichoderrna reesei QM 9414 in fed-batch and CQntinuous-flow culture with cell recycle. Biotech Bioeng 21.:283-296. 5. Gupta, J. K., N. B. Das, and Y. P. Gupta. 1973. Effect of cultural conditions on cellulase formation by Trichoderrna viride. Agr Biol Chem .3.6.: 1961-1967. 6. Herr, D. 1980. Conversion of cellulose to glucose with cellulase of Trichoderrna viride ITTC 1433. Biotech Bioeng 22:1601-1612. 7. Mandels, M., and R. E. Andreotti. 1978. Problems and challenges in the cellulose to cellulase fermentation. Process Biochem ll:6,8-9,11-13. 8. , L. Hontz, and J. Nystrom. 1974. Enzymatic hydrolysis of waste cellulose. Biotech Bioeng 16.:1471-1493. 9. , J.E. Medeiros, R. E. Andreotti, and F. H. Bissett. 1981. Enzymatic hydrolysis of cellulose: Evaluation of cellulase culture filtrates under use conditions. Biotech Bioeng .23.:2009-2026. 5 10. , D. Sternl:erg, and R. E. Andreotti. 1975. Growth and cellulase production by Trichoderma, p. 81-109. ln M. Bailey, T. M. Enari, and M. Linko (Eds.) • Symposium on enzymatic hydrolysis of cellulose. Aulanko, Finland. 11. Mukhopadhyay, s. N., and R. K. Malik. 1980. Increased production of cellulase of Trichoderrna Sli2· by pH cycling and temperature profiling. Biotech Bioeng .22.:2237-2250. 12. Ryu, D. D. Y., and M. Mandels. 1980. Cellulases: Biosynthesis and ap-plications. Enzymes and Microbiol Tech 2.:91-102. 13. Sternl:erg, D. 1976. Trichoderrna viride. A irethod for increasing cellulase production by Biotech Bioeng .JJi:1751-1760. 14. , and S. Dorval. 197 9. Cellulase production and armnonia iretabolisrn in Trichoderrna reesei on high levels of cellulose. Biotech Bioeng 21.:181-191. 15. Tsao, G. T. 1978. Ferrnentable sugars from cel lulosic wastes as a natural source. ln Proceedings of the second annual fuels from biomass symp., piper #30. Rensselaer Polytechnic Insti tute. Troy, New York. Published in accordance with an Act passed m 1881 by the 14th Legislative Assembly, Dakota Temtory, establishing the Dakota Agriculture College and with the Act of re-organization passed +n 1887 bv the 17th Leg1slat1ve Assembly, which established the Agricultural Experiment Station at South Dakota State Universi ty Fi le : 6 . 3-2--2-85 5 4 3 2 1 6 

Fuel Alcohol Production : Conversion of Cellulose to Glucose

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Fuel Alcohol Production : Conversion of Cellulose to Glucose

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