Glucose와 lactose 그리고 whey로부터

Acaligenes eutrophus에 의한 PHB 생산

박기용, 박준성, 김철경．, 김남기

성균관대학교 공과대학 화학공학과

．신흥대학 환경관리과

Production of PHB by Acaligenes eutrophus

from glucose, lactose and whey

Ki Yong Park, Joon Seong Park, Chul Kyung Kim．, Nam Ki Kim

Dept. of Chem. Eng., Sungkyunkwan University

．Dept. of Environ. Mag., Shin Heung College

1. Introduction

Current and growing concern about the environmental fate of plastics and polymeric materials has generated much interest in the development of biodegradable and biocompatible materials. Thus, various investigations have focused on the biosyntheses of a class of polyesters, known as poly -hydroxybutrate(PHB). PHB is naturally occurring, optically active polyesters that are produced metabolically from the bioconversion of alkanes and alkanoic acids by a number of bacterial strains.

A. eutrophus has been studied most extensively due to its ability to accumulate large amount of PHB, ca. 80%(w/w) of dry cell mass, from simple carbon source(ICI, now ZENECA Bio Products, UK).

Initially A. eutrophus is grown in a glucose-salts medium containing only the calculated amount of phosphate to support a desired amount of cell growth.

Cells encounter phosphate limitation after about 60hrs, and accumulate PHB during the next 40 to 60hrs from supplied glucose.

This paper describes strategies for the high cell density cultivation of A. eutrophus for the production of PHB in glucose, lactose, and whey based medium.

PHB could be produced to a high concentration when glucose, lactose, and whey were depleted.

2. Materials and Methods

Bacterial strain, medium, and growth condition

A. eutrophus NCIMB 11599 was used in all experiments.

Fermentation A was carried out in a 4.5L jar fermenter(Korea Fermenter Company, Incheon, Korea). The initial medium was used as 3L containing 50g/L glucose; In fermentation B, the initial medium was used as 3L containing 50g/L lactose, and in C, the intial medium was used 3L  containing 80g/L whey(whey consists of minimum 11% protein and approximatively. 65% lactose). In fermentation C, whey solution was pretreated to remove precipitates.

The initial medium consisted of(L-1) MgSO4·7H2O 0.36g, KH2PO4 3.4g, (NH4)2HPO4·2H2O 2.67g, citric acid 0.43g, NaClO 10mL, and trace element solution 2.5mL(The constituents in the trace element are listed in Table1.).

Cells were cultured at 30℃, 250rpm and 10□/□g, and initial pH was adjusted at 6.5-7.0 by the addition of 2N NaOH.

Initial inoculation was carried out as 10% of initial medium.

Table1. Constituents in the trace elements solution

constituent	concentration(per liter)
HCl	5g
FeSO4□7H2O	10g
CaCl2□2H2O	2g
ZnSO4□7H2O	2.2g
MnSO4□4H2O	0.5g
CuSO4□5H2O	1g
(NH4)6Mo7O24□4H2O	0.1g
Na2B4O7□10H2O	0.02g

Analytical procedures

Cell growth was monitored by measuring the absorbance at 660nm. Cell concentration was defined as cell dry weight per liter of culture broth. PHB concentration was determined by gas chromatography(Carbowax 20M, Hewlett-Packed, USA) using benzoic acid as an internal standard. The residual cell concentration was defined as the cell concentration minus PHB concentration. The PHB content was defined as the ratio of PHB concentration to cell concentration given in as a percentage. The concentration of glucose was determined by enzymatic method. The concentration of lactose was determined by liquid chromatography(□ Porasil, 3.9□×300□, Waters, USA) using acetnitrile as mobile phase.

3. Result and discussion

Figure 1., Figure 2., and Figure 3. show the time profiles of cell concentration, PHB concentration, residual cell concentration, glucose concentration and pH during fermentation shown above. At the end of fermentation A(48hrs), a cellular dry weight of 5g/L, PHB concentration of 2.75g/L, residual cell concentration of 2.25g/L, PHB conversion of 55%(Max. 60%), glucose concentration of 6.42g/L and pH of 6.25 were shown. At the end of fermentation B(48hrs), a cellular dry weight of 3.0g/L, PHB concentration of 1.24g/L, residual cell concentration of 1.76g/L, PHB conversion of 41%(Max. 48%), lotose concentration of 9.9g/L and pH of 7.38 were shown. At the end of fermentation C(48hrs), a cellular dry weight of 10.3g/L, PHB concentration of 3.1g/L, residual cell concentration of 7.2g/L, PHB conversion of 30%(Max 40%), lactose concentration of 9.84g/L and pH of 8.34 were shown.

This study demonstrated that PHB could be produced efficiently when carbon sources were fed by A. eutrophus completely. Glucose could produce PHB more than lactose and whey in three kinds of carbon sources.

If initial inoculation concentration is high, and precipitates is removed by pretretment, the production of PHB will be increased by use of whey.

In addition, the same result can be taken through the proper adjustment of pH.

4. Reference

1. Eggink G, P de Waard and G Huijberts, Formation of novel poly(hydroxyalkanoates) from long-chain fatty acids, Can J Microbiol      41:14-21, 1995

2. Stelenbuchel A and U Pieper, Production of a copolyester of 3- hydroxy -butuyric acid and 3-hydroxyvaleric acid from single unreated carbon sources by a mutant of Alcaligenes, Microbiol Biotechnol 37:1-6, 1992

3. Wang F, Lee SY, Production of poly(3-hydroxybutyrate) by fed-batch culture of filamentation-supressed recombinant Escherichia coli,  Appl Environ Microbiol 63: 4765-4769, 1997

4. Yang ST H, Li Y, Hong G, Contiuous propionate production from      whey permeat using a novel fibrous bed bioreacter, Biotechnol Bioeng      43;1124-1130, 1994