Document Type

Thesis - University Access Only

Award Date

2002

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

Abstract

Sclerotium glucanicum is a fungal organism that produces the exopolysaccharide, scleroglucan, that has industrial applications as a binding agent, as a drive agent in chemically enhanced oil recovery, and in enhancing immune response as a biological response modifier. Scleroglucan can be produced from a wide variety of carbon sources including many monosaccharides, disaccharides, and complex carbohydrates. Despite the diverse types of carbon sources that can be used to produce scleroglucan, industrial production has been limited due to costs of the defined medium and long fermentation times (120 h or longer). As a result of these factors, the primary objective of this study was to develop an inexpensive, industrially viable production medium which S. glucanicum could use to grow and produce scleroglucan. To accomplish this objective, condensed corn solubles (CCS) was evaluated as an alternative production medium. CCS is produced as a terminal by-product in corn ethanol production. Ethanol is considered the commercial product of this process while CCS is a comparatively low-value to negative value byproduct. Despite the low commercial value of CCS, it is relatively high in carbohydrates, proteins, minerals and vitamins. The levels of these components appear to make CCS a good candidate for a microbial growth medium. Previous studies using CCS as a growth medium for gum producing organisms, Sphingomonas paucimobilis and Aureorbasidum pullulans, have proven that successful acclimation and at least average gum production occur on the CCS medium. Acclimation of S. glucanicum to the CCS medium, composed of 240 g/L CCS and 0 g/L glucose, from the defined medium was performed in a series of shake flasks spanning 10 weeks. S. glucanicum was subcultured on a hybrid media prepared by blending progressively higher levels of CCS medium with lower levels of the defined medium. On the defined medium (Modified Wang's), S. glucanicum was able to produce 11 .3 g/L scleroglucan, and once acclimated to the CCS, gum levels were 12.0 g/L. Use of a CCS-based medium results in difficulties in quantifying cell biomass. Filtration is the method commonly used to recover the filamentous cell biomass, but the CCS solids also accumulate in the filter. Moreover, some of the CCS solids appear to be solubilized and incorporated into cell biomass, thus preventing one from simply subtracting baseline (0 h) CCS levels to calculate biomass. As a result, the second objective of this study was to develop a method to quantify cell biomass when using the CCS-based medium. Since S. glucanicum is a filamentous organism, viable cell count methods are not practical. Thus, I attempted to correlate the cell biomass to cell protein levels in a non-gum producing medium and later developed an alternative medium that would make cell quantification easier. Correlation of protein to dry weight, although seemingly successful in the defined medium, encountered the same problem as filtration. That is, protein in the CCS was incorporated into cell biomass over the fermentation run. As a result, an alternative filtered CCS medium was developed which allowed use of the filtration method to quantify biomass, without any of the problems that arose in using the unfiltered CCS medium. This medium was used in all subsequent trials. The primary way to boost scleroglucan yields is to optimize the carbon: nitrogen ratio. Prior studies have noted that limitation of nitrogen and/or phosphorus increased scleroglucan yields. Researchers have also noted that catabolite repression is the primary paradox in scleroglucan production. If initial glucose levels are too high, cell growth is inhibited, but low glucose levels result in a lack of sufficient glucose for scleroglucan formation. In the filtered CCS medium, CCS serves primarily as a source for nitrogen, and phosphorus, but also supplies some carbon. However, additional carbohydrate must be added for scleroglucan production. As a result of these factors, the third objective of this study was to determine the optimal glucose: CCS ratio. In shake flask trials, I initially evaluated a combination of glucose levels from 0 g/L to 40 g/L and CCS levels from 80 g/L to 360 g/L (wet weights pre-filtration). The optimal ratio found was at 1 g/L glucose to 8 g/L CCS. This ratio, though, did not hold at glucose levels over 40 g/L and less than 10 g/L and CCS levels over 360 g/L and under 120 g/L. The optimal formulations for scleroglucan production included: 30 g/L glucose: 240 g/L CCS, 15 g/L glucose: 160 g/L CCS, and 20 g/L glucose: 160 g/L CCS. At 30 g/L glucose: 240 g/L CCS and 20 g/L glucose: 160 g/L CCS however, 40 - 50% of supplemented glucose remained unutilized. To improve this fermentation efficiency and boost scleroglucan concentrations, bioreactor trials were attempted using the 20 g/L glucose: 160 g/L CCS formulation. Maximal scleroglucan concentration achieved in the filtered CCS medium was 26.5 g/L compared to 13.3 g/L in the Modified Wang's medium. Fermentation efficiency was also improved in the filtered CCS medium to 95.6% (83.9% in the defined medium). The final production costs for scleroglucan production were lowered from $3.36/kg scleroglucan in the defined medium to $0.37/kg scleroglucan in the CCS-based medium. Production levels and productivity, although improved, are still not competitive with the most widely used microbial exopolysaccharide, xanthum.

Library of Congress Subject Headings

Sclerotium (Genus) Corn products industry -- By-products Culture media (Biology)

Format

application/pdf

Number of Pages

114

Publisher

South Dakota State University

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