Susan L. Fox

Document Type

Thesis - University Access Only

Award Date

1997

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

Abstract

Calcium magnesium acetate (CMA), an environmentally friendly, non-corrosive road deicer, has been investigated for use as a possible replacement for NaCl or CaCl, due to their corrosive and environmentally damaging nature. The main obstacle preventing widespread use of CMA is its high cost, 15-20 times as much as NaCl. The most expensive ingredient in the production of CMA is acetic acid, which accounts for 72% of the cost. To reduce acetic acid (and thereby CMA) production costs, I evaluated the biological production of acetic acid by an acetate-tolerant mutant strain of Clostridium thermoaceticum (ATCC 49707). The main advantage of C. thermoaceticum is that it can quantitatively convert a wide range of substrates into acetic acid. Unfortunately, this organism has extensive and diverse nutrient requirements and most investigations have used a chemically defined medium (modified Ljungdahl's medium [MLM]) which is prohibitively expensive for use on an industrial scale. Therefore, the first objective of this project was to acclimate this strain to grow and produce acetate on a less expensive medium previously developed in this laboratory. Another key limitation of C. thermoaceticum is its sensitivity to acetic acid, resulting in relatively low acetate productivities and final concentrations. While mutagenesis has improved the acetate productivity (0. 49 g/1/h) and tolerance (43 g/1) of ATCC 49707 on MLM, still greater acetate production is needed prior to commercialization -- and this must be achieved on an industrial production medium. Consequently, the second objective of this project was to enhance acetate production by the acclimated ATCC 49707 strain via further chemical mutagenesis. In the first phase of this study, C. thermoaceticum strain ATCC 49707 was acclimated to grow on condensed corn solubles (CCS) medium in place of the standard laboratory medium (MLM) . This involved progressively transferring the strain into hybrid media consisting of gradually increasing levels of CCS and decreasing levels of MLM. After five months the strain was able to grow and produce acetate on the CCS medium and it was therefore designated strain 49707A. Strain 49707A was then characterized with respect to its tolerance to initial levels of formate, pyruvate and acetate in the ccs medium. Strain 49707A on ccs medium was more tolerant of formate (50 mM at pH 6. 5, 80 mM at pH 5. 5) and pyruvate (80 mM at pH 6. 5, 20 mM at pH 5. 5) than the unacclimated ATCC 49707 strain on MLM (50 mM formate at pH 6. 5 and 5. 5; 50 mM pyruvate at pH 6. 5, 80 mM pyruvate at pH 5.5). However, acetate tolerance of 49707A in ccs medium (25 g/1) was significantly lower than the unacclimated strain (ATCC 49707) in MLM (43 g/1). To determine if the above dissimilarities were due to differences between strains ATCC 49707 and 49707A or changes in medium composition, fermentation comparisons were made between strain ATCC 49707 in MLM, strain 49707A in MLM and strain 49707A in ccs medium. Similar acetate productivity, tolerance and yield data were obtained when either strain was grown on MLM, suggesting that acclimation to ccs medium did not significantly alter the metabolism of the microbe. On the other hand, the acclimated strain exhibited increased acetate production on MLM compared to the CCS medium, suggesting that ccs medium may lack certain critical nutrients which allow C. thermoaceticum to achieve its maximum metabolic rate. While adding back these critical nutrients may restore improved acetate production to strain 49707A in ccs medium, it would also increase medium costs. An alternative approach to improving acetate production would be further mutagenesis of strain 49707A on ccs medium to select for mutants with reduced nutritional requirements. To this end, chemical mutagenesis with nitrous acid was employed, with surviving cells challenged under various selective pressures (i. e. , either a range of higher acetate concentrations, lower pH, etc. ) to enrich for mutants with improved acetate productivity and/or tolerance on the CCS medium. Six mutants were isolated and characterized in this process, with two showing the greatest potential (A4 and A6). Strain ATCC 49707 and strain 49707A on MLM medium could tolerate 45 g/1 acetate as could isolate A4. In contrast, strain 49707A and isolates A4 and A6 on ccs medium could only tolerate 25 g/1 acetate.

Library of Congress Subject Headings

Clostridium

Acetic acid

Calcium magnesium acetate

Deicing chemicals

Roads -- Snow and ice control

Corn products industry -- By-products

Format

application/pdf

Number of Pages

86

Publisher

South Dakota State University

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