목질계 당화액을 이용한 Mannheimia succiniciproducens MBEL55E의 숙신산 생산

Theories and Applications of Chem. Eng., 2003 Vol. 9 No. 1

김도윤, 임성춘, 장호남, 이상엽

한국과학기술원 생명화학공학과

Fermentative production of succinic acid

from wood hydrolysate by Mannheimia succiniciproducens MBEL55E

Do Yun Kim, Seong Chun Yim, Ho Nam Chang and Sang Yup Lee

Department of Chemical and Biomolecular Engineering,

Korea Advanced Institute of Science and Technology

화학공학의 이론과 응용 제9권 제1호 2003년

Introduction

Succinic acid is a dicarboxylic acid (Gottschalk 1986) and has been received a great deal of attention as a green feed stock for the manufacture of synthetic resins, biodegradable polymers and chemical intermediates (Lynd et al., 1999). Among the nutrient sources available, cellulosic and hemicellulosic materials such as wood and municipal solid wastes are renewable and abundant feedstock and enzymatic hydrolysate of wood has been regarded as one of the most inexpensive carbon source for the fermentative production of biochemicals (Lynd et al., 1999, Lee et al., 2003).

In this studies, it is evaluated that economical production of succinic acid from wood hydrolysate by Mannheimia succiniciproducens MBEL55E. We carried out continuous fermentation  in the wood hydrolysate based media to investigate the time profile of growth and end product formation.

Materials and Methods

Preparation of wood hydrolysate

Chips (2 × 4 mm) of oak wood were prepared by a chipper designed and built at the Korea Institute of Energy Research. The composition of oak wood was found to be as follows: cellulose (49.3%); hemicellulose (25.9%); Klasson lignin (21.7%). Steam explosion of the oak wood chips was conducted at 215℃ for 6 minutes in an 8-L exploder. Residues obtained after explosion were washed and enzymatically hydrolyzed with Celluclast (Novo Co., Denmark) and Novozyme (Novo Co.) in a reactor having 500 L working volume at 50℃ for 3 days. The concentration of glucose and xylose in the wood hydrolysate was found to be 75 g/L and 20 g/L after these treatments. The hydrolysate medium was treated with NaOH before sterilization to reduce the formation of inhibitory compound.

Microorganism and growth conditions

Mannheimia succiniciproducens MBEL55E (KCTC 0769BP) is maintained at Department of Chemical and Biomolecular Engineering, KAIST (Taejon, South Korea). Cells were anaerobically grown in sealed anaerobic bottles (100 mL) under CO2 atmosphere. The medium for seed culture contained per liter: 5 g glucose, 2.5 g polypeptone, 2.5 g yeast extract, 3 g K2HPO4, 1 g NaCl, 1 g (NH4)2SO4, 0.2 g CaCl2․2H2O, 0.2 g MgCl2․6H2O, and 3 g MgCO3. The medium was heat sterilized (15 min at 121℃) in the anaerobic bottles under a nitrogen atmosphere. Concentrated H2SO4 was then added to the sterile medium to adjust the pH to 6.5. The nitrogen headspace was replaced by CO2, and Na2S․9H2O was added to a final concentration of 1 mg/L to ensure anaerobic conditions. After 15 min, the reduced medium was inoculated with 1.5 mL of glycerol stock culture and incubated at 39℃ for 12 h.

화학공학의 이론과 응용 제9권 제1호 2003년

Continuous cultures were carried out with working volume of 500 mL in a 2.5 L jar fermentor. The feeding solution contained 17 g/L of glucose, 8 g/L of xylose or treated wood hydrolysate. 5 g/L of yeast extract and 7 g/L of Na2CO3 for a high level of CO2 were used. The feeding solutions for high level of CO2 culture was purged with oxygen-free CO2 gas for 24 h in order to establish anaerobic conditions before use. Other culture conditions were the same as those described for batch cultures. After 10-h of batch operation, the feeding solution was added at various dilution rates while an equal volume of spent medium was removed from the fermentor. The new steady states were confirmed by the constant concentrations of glucose and organic acids in the medium for three-five consecutive samples taken at 3-8 hour intervals after at least 5 turnovers.

Analytical methods

The concentrations of glucose, succinic acid and acetic acid were measured by high-performance liquid chromatography (Hitachi L-3300 RI monitor, L-4200 UV-VIS detector, D2500 chromato-integrator, Tokyo, Japan) equipped with an ion exchange column (Aminex HPX-87H, 300 mm x 7.8 mm, Hercules, CA) using 0.012 N H2SO4 as a mobile phase. Cell growth was monitored by measuring the absorbance at 660 nm (OD660) using a spectrophotometer (Ultrospec3000, Pharmacia Biotech, Sweden). Succinic acid yield was defined as the amount of succinic acid produced from one gram glucose, and was expressed as a percentage.

Results and discussion

The effect of dilution rates on the cell growth and succinic acid production during the continuous cultures. Anaerobic continuous culture was carried out at various dilution using glucose and xylose as carbon sources. When the feeding solution containing 17.54 g/L of glucose, 7.80 g/L of xylose and 5 g/L yeast extract was used, the steady state cell concentration was maintained an OD660 of 3.5-4.0 until a dilution rate reached 0.7 h-1. Glucose was completely consumed when a dilution rate was lower than 0.1 h-1. With increasing a dilution rate (> 0.1 h-1), glucose started to accumulate in the medium and its concentration reached 12.7 g/L at the dilution rate of 0.7 h-1. The steady state concentration of succinic acid decreased with increasing a dilution rate. The steady state concentration of lactic acid reached a peak at a dilution rate 0.4 h-1. After that, the lactic acid concentration decreased with increasing a dilution rate. Succinic acid yield decreased with increasing a dilution rate  (55-33%) and lactic acid yield increased (5-24%). Because succinic acid was a growth-associated product, volumetric productivity of succinic acid increased with increasing a dilution rate. The maximum volumetric productivity of succinic acid of 3.29 g/L/h was obtained at a dilution rate of 0.4 h-1.

Continuous culture using pre-treated wood hydrolysate. Anaerobic continuous culture was carried out at various dilution using wood hydrolysate and yeast extract. When the wood hydrolysate based media containing 18.06 g/L of glucose and 7.16 g/L of xylose was used, the steady state cell concentration decreased with increasing a dilution rate. With increasing a dilution rate (> 0.2 h-1), glucose started to accumulate in the medium and its concentration reached 6.0 g/L at the dilution rate of 0.5 h-1. The steady state concentration of succinic acid and lactic acid decreased with increasing a dilution rate. Succinic acid and lactic acid yield was maintained with increasing a dilution rate (55-61%, 39-44%, respectively). Because succinic acid was a growth-associated product, volumetric productivity of succinic acid increased with increasing a dilution rate. The maximum volumetric productivity of succinic acid of 3.19 g/L/h (Fig. 1) was obtained at a dilution rate of 0.4 h-1.

Acknowledgements

This work was supported by Ministry of Science & Technology

Reference

Gottschalk, G. 1986. Bacterial Metabolism, 2nd ed., Springer-Verlag, NY, p.242-249

Lee, P.C., Lee, S.Y., Hong, S.H., and Chang, H.N. 2003, Biological conversion of wood hydrolysate to succinic acid by Anaerobiospirillum succiniciproducens, Biotechnol. Lett., 25: 111-114.

Lynd, L. R., C. E. Wyman, and T. U. Gerngross. 1999. Biocommodity engineering. Biotechnol. Prog. 15: 777-793

Fig. . Continuous fermentation using pre-treated wood hydrolysate supplemented yeast extract. Symbols are productivity of succinic acid (●), yield of succinic acid (○), yield of lactic acid (◇), cell concentration (□), glucose (◆), xylose (△), succinic acid (■), lactic acid (▲)

화학공학의 이론과 응용 제8권 제2호 2002년

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 2

Theories and Applications of Chem. Eng., 2003, Vol. 9, No. 1

화학공학의 이론과 응용 제9권 제1호 2003년