동시당화발효를 이용한 초산 생산

윤현희, 이윤영*

경원대학교 화학공학과, *Auburn University 화학공학과

Simultaneous Saccharification and Fermentation of Cellulose to Acetic Acid

H. H. Yoon and Y. Y. Lee*

Department of Chemical Engineering, Kyungwon University

*Department of Chemical Engineering, Auburn University

Introduction

Acetic acid is one of the most important organic acids and has a wide range of food related and industrial applications. Recently, application of acetic acid for producing Calcium-Magnesium-Acetate (CMA), a possible replacement for chloride salts which are used for de-icing road in winter, has created renewed interest in producing acetate as a bulk chemical commodity.[1] Much work has been done on producing acetic acid from glucose-based feedstocks. Product yields higher than 90% have been reported in anaerobic fermentation.[2] It has, however, been limited to a corn derived glucose as the primary feedstock. The high raw material cost is one of the major obstacles for large-scale commercialization of the fermentation process. The alternative feedstock we are referring to is cellulosic biomass. Cellulosic substances are abundantly available resources and could be utilized as a feedstock for producing fermentation products. Possible cellulosic substances include agricultural forest residues, municipal solid waste, and pulp and paper mill wastes. Converting these cellulosic substances requires the polysaccharides to be enzymatically broken down into simple sugars and the sugars to be microbially fermented to the final product.

The microbial and enzymatic processes can be combined into a one process termed as Simultaneous Saccharification and Fermentation (SSF).[3] The benefit of the SSF is that it enhances the enzyme efficiency and reduces the equipment cost since it uses one bioreactor instead of two. It is a process technology proven to work well in the conversion of cellulosic biomass into ethanol. This reactor configuration has not been investigated for acetic acid production. The applicability of the SSF to acetic acid production from cellulose was studied. The optimum conditions and associated process data (productivity, yield, and terminal concentration and conversion of substrate) for the SSF were determined.

Experimental

Clostridium Thermoaceticum (ATCC 49707) was cultivated in an incubator at 55℃ using Difco Reinforced Clostridial Media. A complex media containing 5g/L Yeast Extract, 9g/L KHCO3, 1.4 g/L K2HPO4, 1.1g/L KH2PO4, 1 g/L (NH4)2SO4, 0.25 g/L MgSO4·7H2O, 0.04 g/L Fe(NH4)2(SO4)2·6H2O, 0.03 g/L CoSO4·7.5H2O, 0.0033 g/L Na2WO4·2H2O, 0.0024 g/L Na2MoO4·2H2O, 0.00024 g/L NiCl2·6H2O, 0.00029 g/L ZnSO4·7H2O, 0.000017 g/L Na2SeO3, 0.25 g/L Cysteine·HCl·H20, and □-cellulose(or glucose) as needed was used in all fermentations.

The cellulase enzyme, Rocksoft NCE-L-600 was obtained from CPN International Inc., USA. The optimum temperature and pH of the enzyme are 55-57℃ and 6.5, repectively.

Pure nutrients were autoclaved, added to a glass fermenter vessel, and mixed with water to make a total volume of 400 mL. The vessel was then inoculated with a three-four day old cultivation of C. Thermoaceticum. Inoculation was taken as time zero, and samples taken accordingly.

Sample analysis was accomplished using a Biorad Organic Acid Analysis Column (HPX-87H Ion Exclusion Column).

RESULTS AND DISCUSSION

Acetic Acid Fermentation

A batch fermentation with 20 g/L initial glucose concentration gives a yield of 73% of theoretical, as shown in Fig. 1. It is believed that considerable side product formation takes place after inoculation until acetic acid is being produced. Considerable improvement is shown from batch to fed batch mode. Shown in Figs. 2 and 3 are the productivity profiles over time. At pH of 6.9, (Fig. 2), the maximal productivity is 0.42 g L-1 hr-1. Productivity at pH of 6.7 is expectedly lower (0.237 g L-1 hr-1) than at a pH of 6.9. The decreased productivity is due to the stunted growth rate at the lower pH. When applied to SSF, it is believed that pH-limited growth is favorable over nutrient-limited growth. Yields for the fed batch operation show slight improvement. At the higher pH (6.9), the overall yield based on glucose consumed is 69%, while at the lower pH the overall yield is 70%. The fact that lowering the pH does not greatly affect yield is positive for SSF, which will need to be run at a pH lower than the organism's optimum.

Enzymatic Hydrolysis

To effectively apply the SSF method to produce acetic acid from cellulosic substance, the microorganisms which produce acetic acid should be compatible with  enzymatic hydrolysis conditions. The cellulase enzyme(Rocksoft NCE-L-600) used in this study is a commercial grade neutral cellulase produced for the purpose of fabric softening. Therefore, no IFPU data was available on the enzyme. It is a CX enzyme, showing considerable formation of low MW glucose oligomers as well as monomer glucose. The enzyme's optimum temperature and pH (55-57℃, 6.5) coincides with that of the bacteria (60℃, 6.9).

Initial studies indicated that the Rocksoft enzyme encountered product inhibition even at low glucose concentrations. In Fig. 3, it is shown that even the highest enzyme loading (10%v/v) is almost completely inhibited at a glucose concentration of only 2.4 g/L. Because SSF is intended to eliminate glucose inhibition, initial glucose productivity is the critical parameter for SSF. Table 1 shows initial productivity of Rocksoft on 1% wt/wt cellulose at differing enzyme loadings. Higher enzyme concentrations (10% v/v) resulted in higher initial productivity (0.381 g/L hr). Also, because there will be effectively zero glucose concentration in the fermentation media during SSF, this will be the rate of glucose production.

Table 1. Maximum productivity, glucose concentration, and yield for various

enzyme loadings.  Total liquor volume in all cases  400mL.

Enzyme Volume [%v/v]	Max. Prod. [g/L hr]	Max. [Glucose] [g/L]	Max Yield (%)
1.25	0.0104	0.46	4.6
2.5	0.0207	0.75	7.5
5.0	0.0638	1.06	10.6
7.5	0.0951	1.61	16.1
10	0.381	2.4	24.0

Simultaneous Saccharification and Fermentation

SSF runs were made with varying pH (6.5 and 6.9). These are depicted in Figs. 2 and 3. The temperature and enzyme loading in each case is 57 C and 7.5% v/v. A lower pH produced better cellulose conversions than a pH of 6.9 due to more favorable saccharification conditions. The SSF indeed proceeded well giving acceptable yields of acetic acid, indicating that glucose inhibition on the enzyme was eliminated and the efficiency of the enzyme raised during the SSF.

In all SSF operations, the production of monomer glucose is the rate-limiting step. Therefore, any increase in acid productivity is a direct result of an increase in saccharification rate. In this instance, it is vital to match as closely as possible the rate of glucose uptake and glucose production. This is due to the behavior of C. Thermoaceticum under glucose starvation conditions. Specifically, previous literature (Parekh, S. Cheryan, M., Advances in Applied Microbiology, 1997) indicates side product formation of fructose under starvation. Also, the SSF data shows that considerable amount of lactic acid is produced as a byproduct. It is not known whether this is a result of starvation or some other constraint imparted by SSF.

ACKNOWLEDGEMENT

This work was supported by the Ministry of Construction and Transportation and by the Kyunggido Regional Research Center at Kyungwon University.

REFERENCES

[1] Marynowski, C. W., Jones, J. I., Tuse, D., and Boughton, R. L., I&EC Prod. Dev., 24, 257 (1985)

[2] Parekh, S. R. and Cheryan, M., Enzyme Micro. Technol., 16, 104 (1994)

[3] Takagi, M., Abe, S., Suzuki, S., Emert, G. H., and Yata, N., in Proceedings of the Bioconversion Symposium, IIT, Delhi, Ghose, T. K., ed., 551 (1977)

Fig.1. Time course of batch fermentation    Fig. 2. Time course of fed-batch

of C. thermoaceticum at pH 7 & 57℃.    fermentation at pH 6.9 and 59℃.

Fig. 3. Time course of fed-batch          Fig. 4.  Time course of saccharification

fermentation at pH 6.7 and 59℃.          at varying enzyme loadings.

Fig. 5.  Time course of SSF at pH 6.5,    Fig. 6. Time course of SSF at pH 6.9,

57℃, and 7.5% v/v enzyme loading.       57℃, and 7.5% enzyme loading.

Cellulose loading = 25 g/L.               Cellulose loading = 10 g/L.