Fructose is an expensive carbon source in the fermentation industry, hence we next investigated the application of other economical carbon sources for the heterotrophic culture in the two stage method. We used acetic acid as the carbon source and developed a pH-stat continuos substrate-feeding method for the culture. Flask culture experiments showed the optimum concentration of acetic acid for cell growth was very low (ca.1.0 g-dnr3) and the growth was seriously inhibited by a slight increase in acetic acid concentration. It was, therefore, necessary to control the acetate concentration around this level in high cell density cultivation. The ratio of consumption of acetic acid to that of ammonium by A. eutrophus cells was determined by a standard-type batch culture experiment to be about 10 (mol-acetic acid/mol — ammonium). It was therefore expected that the acid-base equilibrium in culture system would be balanced by feeding the substrate solution in which the C/N ratio was 10 (mol/mol) so as to maintain the culture pH at a constant level, in order for acetate concentration in the fermentor to be also controlled at low level. However, in batch culture with such a feeding, acetate concentration of the culture liquid increased after cell concentration reached approximately 5 g*dnr3. The increase in acetate concentration was thought to be due to the depletion of mineral nutrients. Hence, the mineral concentrations in the medium was increased 5 times as that of the basal medium (this was referred as 5-fold medium). As a result, acetate concentration was controlled around 1 gednr3 and cell concentration reached about 25 g*dnr3 after 18 h. Acetate concentration increased after that due to the depletion of phosphate. A pH- stat batch culture was hence carried out by feeding a solution in which the C/P ratio was 118.4 (mol-C/mol-P). Acetate concentration was maintained around 1 gednr3, and cell and protein concentrations increased to 48.6 g*dm-3 and 35.0 gedm~3, respectively after 21 h of cultivation (Fig.4). Autotrophic cultivation for P(3HB) accumulation was then performed using the protein-rich cell mass obtained from a pH-stat batch culture into which the modified substrate solution was fed. When the cell concentration reached to about 5 g*dnrr3, feeding of the substrate solution was stopped and autotrophic cultivation was performed by feeding a substrate gas mixture into the fermentor. P(3HB) accumulated in the cells up to about 60 % by dry cell weight. This feeding method can therefore be used in fermentation process where the cell growth or P(3HB) accumulation is inhibited by high concentrations of the substrate such as propionate, formate, or lactate, etc.

Time course of autotrophic culture of A. entrophtts in bench-plant scale culture system.

Table I. List of autotrophic cultivations of hydrogen-oxidizing bacteria and fermentaive production of P(3HB) using various microorgansims and substrates

Strains Substrate Culture Cultivation method time (h) Cell concentration (g-dm3) Cell productivity (g-dm J-h‘) P(3HB) P(3HB) concentration productivity (g-dm3) (g-dm3-h‘) Ref. Alcaligenes H2/02/C02 Batch 25 25.0 1.00 — — 9 eulrophus Alcaligenes H2/02/C02 Batch 40 91.3 2.28 61.9 1.55 5 Pseudomonas H2/02/CO2 Batch 48 24.0 0.50 — — 10 hydrogenovora Alcaligenes H2/02/CO2 Continuous 0.40 — — 11 eutrophus Alcaligenes H2/02/C02 Batch 70 18.0 0.26 16.0 0.23 12 eulrophus H16 Alcaligenes H2/02/C02 Continuous 0.33 — — 13 hydrogenophilus Pseudomonas H2/02/C02 Continuous 3.00 — — 14 hydrogenothermophila Alcaligenes H2/O2/CO2 Batch 60 60.0 1.00 36.0 0.60 3 CUltUfjrtUA Recombinant Glucose Fed-batch 42 117.0 2.79 89.0 2.11 15 E. coli Protomonas Methanol Fed-batch 121 223.0 1.84 136.0 1.12 16 елігициепь Alcaligenes Glucose Fed-batch 50 164.0 3.28 121.0 2.42 17 eutrophus Alcaligenes Sucrose Fed-batch1) 18 142.0 7.89 68.4 4.0 18 latus Methylo- Methanol Fed-batch 70 250.0 5.57 130 1.85 19 bacterium organophilum Alcaligenes Glucose* Fed-batch 50 — — 90.42) 1.80 20 eutrophus valerate 1) The inoculum cell size was 13.7 g*dm 2)The product was poly(hydroxybutyrate-co-hydroxyvalerate).