For practical application of PHAs on commercial scale, consideration has to be given to the origin of carbon source used as well as the reducing the cost of production through the use of economical carbon sources. Carbon source such as 4- hydroxyvaleric acid derived from fossil fuel is often used as raw material for producing PHAs. However, it is known that the consumption of a large amount of fossil fuel results in the increase in CO2 concentration in atmosphere. Waste materials such as lignocellulose is desirable from the viewpoints of utilization of unexplored resources and solution of the green house effect. Xylose is one of main components of hemicellulose contained in wood waste. At present, few types of commercial products have been produced from xylose. Young et. al. reported that Pseudomonas cepacia could produce P(3HB) from xylose and lactose, but the productivity was very low(29). Lactococcus lactis 10-1 isolated in our laboratory is able to produce L-lactic acid and acetic acid at a high production rate from xylose (30,31). L-Lactate also has the potential to be used as raw material in the manufacture of a biodegradable plastic, poly(L-lactate). A. eutrophus cannot utilize xylose but utilize lactate as carbon source. We therefore developed a culture method for the production of PHA from xylose employing these two bacteria. This culture method consisted of an initial fermentative production of L-lactate from xylose employing L. lactis 10-1 and a conversion of L-lactate into PHA by A. eutrophus. Flask culture experiment showed that the growth rate of A. eutrophus decreased according to the increase in L-lactate concentration in the medium and the cells could not grow above 30 g*dnr3 of L-lactate. In pH-controlled batch fermentations, a maximum specific growth rate of 0.6 h_1 was obtained when 5 g»dnr3 of L-lactate was used. The growth of microorganisms is generally inhibited by the presence of lactate, however, the specific growth rate of A. eutrophus when using L-lactate was higher than when other types of carbon source were used. According to our study for A. eutrophus, the maximum specific growth rate with using fructose was about 0.2 h_1 and the maximum specific growth rate in autotrophic condition was 0.42 h_1. Such growth characteristic of A. eutrophus on L-lactate is favorable for production of P(3HB) by the two-stage method. The accumulation of P(3HB) by A. eutrophus was next investigated using the culture supernatant containing L-lactate and acetate converted from xylose by L lactis IO-1. A pH-controlled batch culture of L. lactis 10­1 was first anaerobically carried out using 30 g»dnr3 of xylose as carbon source. When xylose in the culture was completely consumed, the culture broth was aseptically centrifuged and the supernatant was returned to the fermenter. A. eutrophus cells was then inoculated and the second stage cultivation for P(3HB) accumulation was aerobically carried out. The initial L-lactate concentration in the second stage culture was adjusted to 10 g*dnr3. After 24 hours of cultivation, 8.5 g*dm~3 of cells were produced. The final percentage of P(3HB) in the cell reached 55 %(w/w) without nitrogen source on medium being limited (32). The growth of A. eutrophus is inhibited by high lactate concentrations, therefore high cell density cultivation can be achieved by pH-stat batch culture with substrate feeding to control

L-lactate concentration in medium at low level. As the C/N ratio for the consumption of L-lactate and ammonium by the cells was determined to be 10 (mol/mol) by a standard-type batch culture, the feed solution in which the C/N ratio was prepared to 10, was first used in the pH-stat batch culture as feed substrate. However, it was impossible to control L-lactate concentration at a constant level by using this feed solution. It was observed that the microorganism accumulated P(3HB) in the cell even during exponential growth phase and excreted a small amount of an unknown organic acid, then the acid-base equilibrium was not balanced in the culture system. The C/N ratio in the feed solution was, therefore, changed from 10 to 23.3(mol/mol) after 12 h of cultivation and phosphate and other organic nutrients were also supplied. As a result, cell concentration increased to 102 g»dnr3 (Fig. 8). The P(3HB) content in the cells reached about 60 %(w/w) although nitrogen source in culture medium was not limiting. We are now investigating substrate feeding strategy to increase P(3HB) accumulation.

1. Conclusion

The practical cultivation systems for hydrogen-oxidizing bacterium, A. eutrophus to produce a biodegradable plastic, P(3HB) from CO2 and xylose were developed and P(3HB) accumulation was improved by incorporating new strategies. The application of such culture systems should contribute to the solution of the global environmental pollution problems caused by increased CO2 level in atmosphere, disposal of non-degradable plastics and utilization of industrial waste materials. For practical application of biodegradable plastics, obviously considerable technological challenges must be overcome, especially in the reduction in production cost and improvement in extraction and refining process of the product. We are tackling this difficult problem and also investigating the conversion of various types of industrial waste materials to other useful compounds.