Document Type

Thesis - University Access Only

Award Date

2000

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

Abstract

Sphingomonas paucimobilis has the ability to convert six carbon mono- and disaccharides into gellan, an exopolysaccharide with characteristics making it suitable as a thickener and stabilizer in various applications. Unfortunately, S. paucimobilis has extensive and diverse nutritional requirements for gellan production. The defined medium typically used in laboratory studies is prohibitively expensive for use on the industrial scale. Therefore, the overall objective of this study was to develop an inexpensive medium which would support growth of and gellan production from S. paucimobilis. To accomplish this objective, I evaluated condensed com solubles (CCS) as a low-cost alternative medium. CCS is a byproduct of the com ethanol industry, and is considered a negative to low-value byproduct. It does however, contain a good balance of carbohydrates, proteins, vitamins and minerals making it a likely candidate for use as a base for microbial growth media. Through a series of shake-flask fermentations, spanning six weeks, I was able to successfully acclimate S. paucimobilis to grow on a medium composed of 240 g/L dry mill CCS (fresh weight, equivalent to 58.56 g dry basis) and 30 g/L glucose. In these trials, S. paucimobilis was sub-cultured in a blended medium composed of progressively lower levels of the defined medium and progressively higher levels of the CCS medium. Initially, S. paucimobilis was only able to produce 5.8 g/L gellan on the CCS based medium, however gellan production rose to 12 g/L after the acclimation. Glucose is typically provided in growth media to increase gellan yields. Prior studies have noted, however, that fermented broth often contains up to 10 g/L residual glucose. Consequently, the second objective of this study was to determine the optimal level of glucose with respect to gellan production and fermentation efficiency. I evaluated initial glucose levels ranging from 0 g/L to 300 g/L and found that 30 g/L glucose resulted in the highest gellan yields with acceptable levels of productivity and fermentation efficiency. Due to the high broth viscosity resulting from gellan production, it is difficult to separate cells from gellan. Therefore, the third objective of this study was to evaluate methods to quantify cell mass vs. gellan levels in fermentation broth. Dilution and centrifugation of fermented broth were not effective in partitioning cell mass from gellan. Therefore, I developed a standard curve relating dry cell weights to viable cell counts in a medium not supporting gellan production. By this means, dry cell weight could be estimated (from viable counts) in gellan production trials, and subsequently subtracted from the precipitate gellan - cell pellet to determine gellan concentration. There are various methods to recover gellan from fermentation broth. In general, most methods first utilize a dilution/centrifugation step to remove the cell pellet from gellan in the supernatant. (As noted above I found this step ineffectual). The supernatant is then heated (90-100°C) for 10-15 minutes, followed by the addition of two volumes of either methanol, isopropanol, or ethanol to precipitate gellan which is recovered by centrifugation. To simplify the recovery process I evaluated various methods for gellan recovery and settled on a process that involved boiling the broth for 15-20 minutes (best accomplished by autoclaving), adding two volumes of cold ethanol, shaking vigorously, and centrifuging for 20 minutes at 10,000 rpm and 30°C. Based on the results of this study, the medium costs for gellan production were lowered from $4.75-5.42 /kg gellan on the defined medium to $2.58 /kg gellan on the CCS-based medium. The maximum gellan concentration obtained from the CCS-based medium was approximately 12 g/L and fermentation efficiency was 90.6% , similar to that on the defined medium. This level of production was rather low, compared to other exolysaccharides, like xanthan, which are produced at levels of 146 g/L. Therefore, further research needs to be done to optimize gellan production on alternative media.

Library of Congress Subject Headings

Gelation Polysaccharides Distilling industries -- By-products

Format

application/pdf

Number of Pages

86

Publisher

South Dakota State University

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