Cell Immobilization Studies. Cell immobilization can greatly increase the cell density in bioreactors. Immobilized-cell biocatalysts are well suited for energy — efficient bioreactor configurations, such as airlift and fluidized-beds. The suitability of several support matrices for immobilization of Butyribacterium methylotrophicum has been evaluated (34). The mass of cell protein immobilized was measured as a function of time for celite, wood powder, activated carbon, ion exchange resin, molecular sieves, and alumina during batch growth on CO in the presence of the support. The fluidization properties (bed expansion as a function of superficial liquid velocity), rate of product formation, and proportion of two — and four-carbon products were also measured for celite, ion-exchange resin and molecular sieves. All of these latter three supports were deemed satisfactory for immobilized-cell culture of B. methylotrophicum.

Continuous, Cell-Recycle Fermentations. High cell densities can also be achieved in bioreactors using cell recycle. This approach was evaluated during long-term, continuous CO fermentations by B. methylotrophicum, in which a

0. 3 ^im pore size cellulosic membrane was used in a cross-flow mode to achieve total cell recycle (77). Runs were conducted at different pH values, because previous experiments had shown that pH strongly regulates this fermentation (75,79), as evidenced by changes in relative proportions of the products. The experiments were performed with the same dilution rate used during previous continuous runs done without cell recycle (19) to study the effect of cell density on reactor productivity. At pH values of 7.2, 6.8, 6.4, and 6.0, steady-state was achieved. At pH values of 5.75 and 5.5, oscillations in the concentrations of several fermentation products were observed. A viable culture could not be maintained at pH values below 5.5.

Butyribacterium methylotrophicum has perhaps the most versatile metabolic capabilities of known microbes capable of anaerobic CO metabolism. It grows on a wide range of carbon and energy substrates, including 100% CO, H2/CO2, methanol, formate, and glucose (20). When grown on CO, it produces acetate, butyrate, ethanol, and butanol as catabolic products. The direct pathway from CO to butanol is apparently unique to B. methylotrophicum (21).

The reaction stoichiometries for the steady-state runs, balanced for carbon and electrons, are given in Table I. As was evident in similar chemostat experiments done without cell recycle (79), a reduction in pH resulted in the production of less acetate and more butyrate and alcohols. This trend is also evident in the Tables II and III, which show the partitioning of carbon and available electrons in the fermentation products. The fractions of carbon and electrons going into alcohols approximately double between a pH of 6.4 and 6.0. Production of 4- carbon compounds (Le., butyrate and butanol) accounts for over 50% of the total electrons from CO at a fermentation pH of 6.0. Between a pH of 7.2 and 6.0, the partitioning of carbon and electrons to acetate decreases by approximately 35%. However, even at a pH of 6.0, on a molar basis, acetate remained the predominant product. Previously, butyrate was found to be the major product during batch culture with a pH shift from 6.8 to 6.0 at the onset of the stationary phase (75).

At pH values of 5.75 and 5.5, the cultures initially exhibited transient primary butanol production, followed by prolonged oscillations in acetate and butyrate formation. Table IV shows the initial fermentation stoichiometries for these two oscillatory fermentations during the period of primary butanol production. Average product concentrations over the time period of primary butanol production were used to obtain these balances. These initially high butanol production levels, up to 2.7 g/L, were significant in that they demonstrated that B. methylotrophicum is capable of producing butanol as the major product from CO metabolism (21). Butanol accounts for as much as 44% of the total available electrons from the CO feed at a pH of 5.5. The AG0’ of butanol production is sufficiently exergonic to drive ATP synthesis (75). An obvious research challenge is how to control the pathway fluxes so as to sustain high butanol yields. Interpretation of the experimental data with the metabolic model described below can help elucidate the trends in pathway regulations.

Table I. Effect of pH on steady-state fermentation stoichiometries

pH__________________ Fermentation Stoichiometry___________________________

7.2 4CO —> 2.21C02 + O.4IOCH3COOH + O. IO5C3H7COOH + O. OI9C2H5OH + О. ОЗ2С4Н9ОН

+ 0.387 CELLS

6.8 400 —> 2.25C02 + О. ЗЗ4СН3СООН + 0.124C3H7COOH + O. O25C2H5OH + O. O4OC4H9OH

+ 0.377 CELLS

6.4 4CO —> 2.26C02 + О. ЗІ6СН3СООН + O. I52C3H7COOH + O. OI8C2H5OH + О. ОЗ2С4Н9ОН

+ 0.279 CELLS

6.0 4CO -> 2.32C02 + O.26OCH3COOH + O. M2C3H7COOH + O. O5OC2H5OH + O. O55C4H9OH

+ 0.279 CELLS

Table П. Effect of pH on Carbon Partitioning During Steady-State Fermentations

Table HI. Effect of pH on Available-Electron Partitioning During Steady — State Fermentations

Table IV. Fermentation Stoichiometries During the Initial Period of Oscillatory Fermentations

pH______________________ Fermentation Stoichiometry_____________________

5.75 4CO —> 2.36C02 + 0.126CH3COOH + 0.074СЗН7ССЮН + 0.021C2H50H + 0.115C4H90H

+ 0.595 CELLS

5.5 4CO —> 2.40CO2 + 0.112CH3COOH + 0.049C3H7COOH + 0.029C2H50H + 0.149C4H9OH

+ 0.533 CELLS