Although petroleum-derived non-biodegradable plastics have been indispensable materials in modern society over the past several decades and have enabled human beings to live in more comfortable circumstances, the increasing plastic wastes have been severe environmental contaminants causing waste disposal problems and even a threat to human. In an effort to prevent the harmful environmental effects of non-biodegradable plastics, the development of environmental-friendly biodegradable plastics has been going on all over the world.
Microorganism-derived biodegradable plastics have a number of advantages over conventional petroleum-derived plastics. They are biodegradable, biocompatible and biologically renewable, making them as useful candidates for replacing conventional synthetic plastics (Steinbuchel, A. and Fuchtenbusch, B., Appl. Microbiol. Biotechnol., 51: 13-21, 1999; Poirier, Y., Curr. Opin. Biotechnol., 10; 181-185, 1999). Target markets for biodegradable plastics include packaging materials, disposable fabrics, hygiene products, consumer goods, agricultural tools, fine chemistry, and other disposables, which are being enlarged with increase of consumption (Gross, R, A, and Kalra, B., Science, 297: 803-807, 2002).
Polyhydroxyalkanoates (PHAs) derived from microorganisms have drawn much attention as candidates for manufacturing natural and biodegradable thermoplastics and elastomers for a wide range of applications, because they are degraded completely to CO2 and H2O under optimal conditions and they possess material properties similar to various petrochemical-based synthetic plastics currently in use (Holmes, P. A., Phyl. Technol., 16: 32-36, 1985; Lee, S Y. Biotechnol. Bioeng., 49: 1-14, 1996). Although a company (Metabolix, USA) sells PHA under the trade name of BIOPOL, the use of PHA is limited because of its high production cost (Choi, J. and Lee, S Y. Appl. Microbiol. Biotechnol., 51: 13-21, 1999).
When PHA is produced in bacterial fermentation systems, the unit cost of production is at least five times higher than that of chemically synthesized polyethylene. Therefore, the use of microorganism-derived PHA is limited to a narrow range just because of high production price. Although the potential for such biodegradable plastics is great, they cannot compete with conventional petroleum-based plastics in production cost, so that the use of biodegradable plastics is limited to special applications (Choi, J. and Lee, S Y., Appl. Microbiol Biotechnol., 51: 13-21, 1999; Lee, S Y., Nature Biotechnol., 15: 17-18, 1997). In order to commercialize PHA, every effort has been made to reduce production price by the development of better host strains, improved fermentation processes and more efficient purification processes (Choi, J. and Lee, S. Y., Appl. Microbiol. Biotechnol., 51: 13-21, 1999).
Polyhydroxybutyrate (PHB) is accumulated as a carbon and energy storage material in various microorganisms and is the best-characterized member of the polyhydroxyalkanoates (PHAs), which have drawn much attention as biodegradable substitutes for conventional nonbiodegradable plastics. It belongs to a class of polyesters of 3-hydroxy acids and has physical properties similar to those of polypropylene (Schubert, P. et al., J. Bacteriol., 170: 5837-5847, 1998; Hai, T. et al., Microbiology, 147: 3047-3060, 2001; Anderson, A. J. and Dawes, E. A., Microbiol. Rev., 54: 450-472, 1990; Lee, S Y., Nature Biotechnol., 15: 17-18, 1997). PHB is derived from acetyl-coenzyme A by a sequence of three consecutive enzymatic reactions (Schubert, P. et al., J. Bacteriol., 170: 5837-5847, 1988; Slater, S. C. et al., J. Bacteriol., 170: 4431-4436, 1988). Polymerization of two molecules of acetyl-CoA is catalyzed by β-ketothiolase to form acetoacetyl-CoA. Then, acetyl-CoA reductase reduces acetoacetyl-CoA to β-hydroxybutyryl-CoA, which is then polymerized by PHB synthase to PHB. Three genes involved in the synthesis of PHB have been cloned from Alcaligenes eutrophus (Schubert, P. et al., J. Bacteriol., 170: 5837-5847, 1988; Peoples, O. P. and Sinskey, A. J., J. Biol. Chem., 264: 15298-15303, 1989; Slater, S. C. et al., J. Bacteriol., 170: 4431-4436, 1988).
Various host strains have been investigated as candidates for the production of PHA. Among them, A. eutrophus, A. latus, methylotroph, Azotobacter vinelandii, and recombinant Escherichia coli are considered to be good PHA producers (Lee, S Y., Biotechnol. Bioeng., 49: 1-14, 1996; Lee, S Y., Trends Biotechnol., 14: 431-438, 1996; Lee, S Y., Nature Biotechnol., 15: 17-18, 1997). In particular, production of PHB by recombinant E. coli having PHA synthesis gene derived from A. eutrophus has been studied by several groups (Fidler, S. and Dennis, D., FEMS Microbiol. Rev., 9: 231-235, 1992; Lee, S Y. et al., Ann. N.Y. Acad. Sci., 721: 43-53, 1994; Lee, S Y. et al., Biotechnol. Bioeng., 44: 1337-1347, 1994). In addition, several groups have developed fermentation processes to increase productivity and these efforts are still largely ongoing. It has been reported that a high concentration of PHB can be produced with high productivity by a fed-batch culture of recombinant E. coli harboring a stable high copy number plasmid which carries phbCAB genes and content greater than 80% of dry cell weight (DCW) (Lee, S Y., Nature Biotechnol., 15: 17-18, 1997; Wang, F. and Lee, S Y., Appl. Environ. Microbiol., 63: 4765-4769, 1997).
A number of PHA recovery/purification methods have been suggested. Although extraction using solvents such as chloroform, methylene chloride, and dichloroethane can result in pure PHA, this method requires large quantities of toxic and volatile solvents, causing additional environmental problems (Ramsay, J. A. et al., Biotech. Tech., 8: 58-9-594, 1994; Choi, J. and Lee, S Y., Bioprocess Eng., 17: 335-342, 1997). The recovery cost is considerably decreased with increasing PHA content (P/X, %). Lower PHA content, though, results in a higher recovery cost mainly due to the use of a larger amount of digesting agents for separating PHA and the increased cost of waste disposal (Lee, S Y. and Choi J., Polymer Degrad. Stabil., 59: 387-393, 1998).
In addition to the factors described above, finding cheaper carbon sources has also been considered to be important because the cost of the carbon source also contributes significantly to the overall production cost of PHA (Yamane, T. et al., Biotechnol. Bioeng., 50: 197-202, 1996; Yamane, T., Biotechnol. Bioeng., 41: 165-170, 1993). In addition, carbon conversion rate is an important factor affecting the overall production cost. The goal is to have the carbon be 100% converted to PHB, resulting in the maximum possible glucose conversion rate (theoretical value of 0.38).
PHB is accumulated as a cell storage material in microorganisms, making it a useful candidate for a biodegradable plastic replacing conventional petroleum-based plastics. However, its use is limited to a narrow range of industrial applications because of high production cost, which makes it not compare favorably with conventional petroleum-based plastics. Various approaches to producing PHB on a commercial scale have been made. Nevertheless, commercial production of PHB remains distant, because production cost of PHB is still 4-5 times higher than that of synthetic plastics currently in use.
After all the researches on production of PHB, the present inventors have confirmed that PHB production could be effectively induced by inhibiting generation of lactate in cells by initial low inoculum. And the present inventors have completed this invention with the development of a method for over-production of PHB on a commercial scale, in which necessary medium components are provided double for high productivity of PHB, production of PHB on an industrial scale is increased by adding high concentration of glucose, the concentration of PHB in a host cell is absolutely increased and extracellular secretion of PHB is induced to simplify PHB recovery/purification process, resulting in the decrease of production cost of PHB.