Acetate Production Characteristics of Clostridium Thermoaceticum Strains Grown on Various Media

Calcium magnesium acetate (CMA) is an environmentally friendly, non-corrosive road deicer which has been identified by the Federal Highway Administration as the preferred alternative to rock salt. Its use is limited, however, by its prohibitively expensive cost currently 30-40 times as much as sodium chloride (NaCl). Acetic acid, the major component of CMA, accounts for 72% of its production costs. To make CMA economically feasible for deicing applications, the cost of producing acetic acid must be significantly reduced. This study examined the biological production of acetic acid via fermentation of inexpensive, condensed corn solubles (CCS)-based media by Clostridium thermoaceticum. Traditionally, this acetogen has been cultivated in modified Ljungdahl\u27s medium (MLM), an expensive chemically defined medium. However, two strains of C. thermoaceticum were previously acclimated in our laboratory to grow in media consisting of only tap water, ccs, sodium bicarbonate, and glucose, thereby reducing medium costs substantially. C. thermoaceticum strain DSM 521 had been acclimated to grow in wet mill CCS media. Since many commercial ethanol plants employ a dry milling process, strain DSM 521 was acclimated to dry mill ccs media via transfers in shake flasks with successively higher ratios of dry mill CCS. Unfortunately, this medium did not support acceptable acetate production. Consequently, all batch and fed-batch fermentation trials were conducted using wet mill CCS media. Batch fermentation trials were conducted to determine the optimum initial glucose concentrations needed in order to maximize acetate concentrations, yields, and maximum and overall productivities. C. thermoaceticum strain DSM 521 was examined in a series of benchtop trials using initial glucose additions of O, 5, 10, 15, and 20 g/L. The 10 g/L addition level resulted in the best overall performance –average maximum acetate levels of 24.1 g/L, yields of 100%, maximum acetate productivities of 0.23 g/L/h, and overall productivities of 0.19 g/L/h -- and was thus used in all subsequent trials. C. thermoaceticum strain ATCC 49707 and a mutant strain (A6), isolated in our laboratory, were studied in identical 10 g/L initial glucose addition batch trials to compare results to those of strain DSM 521. Strain ATCC 49707 produced a maximum acetate concentration of 23.7 g/L, yield of 89.8%, maximum acetate productivity of 0.32 g/L/h, and an overall productivity of 0.22 g/L/h. The mutant A6 strain produced a maximum acetate concentration of 23.3 g/L, yield of 81.8%, maximum acetate productivity of 0.22 g/L/h, and an overall productivity of 0.19 g/L/h. Although yields of strains ATCC 49707 and the A6 mutant were 10-20% lower than those of strain DSM 521, maximum acetate concentrations were nearly equal and overall acetate productivities were slightly higher. Therefore, fed-batch fermentation trials were conducted with all three strains to compare their performance. Fed-batch trials were conducted using initial glucose additions of 10 g/L to explore the possibility of improving acetate production by adding media at selected intervals during fermentation. Two types of additions were examined: raw, unfiltered CCS vs. diluted, filtered, and condensed ccs. The raw CCS additions caused growth inhibition of C. thermoaceticum and therefore acetate production was substantially lower: strain DSM 521 produced average maximum concentrations of 19.9 g/L, yields of 81%, maximum productivities of 0.16 g/L/h, and overall productivities of 0.15 g/L/h. Strain ATCC 49707 and the mutant A6 strain were not examined with this method. The filtered/condensed CCS medium did not cause notable C. thermoaceticurn growth inhibition in fed-batch trials. Strain DSM 521 produced average maximum acetate concentrations of 30.4 g/L, yields of 98.8%, maximum acetate productivities of 0.23 g/L/h, and overall productivities of 0.20 g/L/h. Strain ATCC 49707 produced a maximum acetate concentration of 24.1 g/L, yield of 82.6%, maximum acetate productivity of 0.19 g/L/h, and overall productivity of 0.17 g/L/h. The mutant A6 strain produced a maximum acetate concentration of 22.9 g/L, yield of 91.4%, maximum productivity of 0.22 g/L/h, and overall productivity of 0.21 g/L/h. In fed-batch trials, strains DSM 521 and the A6 mutant outperformed strain ATCC 49707 in yield, maximum productivity, and overall productivity. Strain ATCC 49707 generated a slightly higher maximum acetate concentration than the A6 mutant strain, but both were lower than those produced by strain DSM 521. The improved acetate productivity and yield exhibited by the mutant A6 strain relative to its parent strain (ATCC 49707) suggest that further efforts to develop mutants may ultimately generate a strain capable of producing acetic acid on a commercial scale