Document Type

Thesis - Open Access

Award Date


Degree Name

Master of Science (MS)


Biology and Microbiology

First Advisor

William R. Gibbons


Main limitations of dried distillers’ grains with solubles (DDGS) as meal for monogastric animals is the presence of high fiber content, reduced fat content, and poor amino acid balance. This causes a reduced economic value of DDGS for these species due to its low inclusion rates. The goal of this theses was to create new strains of a fungus, Aureobasidium pullulans, with enhanced cellulase production and use it to optimize an enzymatic saccharification, pretreatment, and fungal conversion process to enhance the nutritional value of DDGS. Various combinations of enzymatic saccharification, physical/chemical pretreatments, and mutant strains of fungi were investigated to hydrolyze DDGS fiber into carbohydrates which are metabolized by the fungi into single cell protein. Cellulose is the most abundant renewable carbon source found in nature and is of great interest to various industries, including the food industry. Generally, microorganisms can be used to convert cellulosic materials into higher protein content, which can subsequently be used for animal feed. However, without the aid of cellulase, the cellulose structure is resistant to degradation. Genome shuffling was used to improve cellulase production from Aureobasidium pullulans Y-2311-1. One strain developed via genome shuffling (A. pullulans GS23) displayed the largest increase in total cellulase activity, which was a 6-fold increase compared to the wild type strain. One of the strains created for the starting mutant population by methyl methanesulfonate (MMS) had a 3- fold increase compared to the wild type strain. A. pullulans GS23 also had an increase in exoglucanase and β-glucosidase activity compared to the wild type strain (6.95-fold and 1.52-fold increase, respectively). The crude protein amount of A. pullulans GS23 had a 1.04-fold increase compared to the wild type strain after 5 days of fermentation. Various commercial enzymes were also tested on DDGS to help break down the fibers along with various physical/chemical pretreatments to improve the overall fiber digestibility. But the high cost associated with the use of commercial enzymes is a challenge with the industrial application of enzymes in feed application. This experiment was a two-step process where commercial enzymes were tested on untreated DDGS to down select the enzyme that performed well at low dosages. Then the DDGS was treated by various physical and/or chemical pretreatments and tested with the best enzyme with even lower dosages. Lower enzyme dosages on pretreated DDGS was tested versus the untreated DDGS because the pretreatment would have liberated more sugars, resulting in less enzymes to be used. It was found the commercial enzyme that performed well at low enzyme dosages was Viscozyme L. When this was incorporated with the best pretreated DDGS, dilute acid, with 1 mg/g of Viscozyme L, it had an increase of ~280% in total sugars as compared to untreated DDGS with a Viscozyme L dosage of 2 mg/g. A combination of enzymatic saccharification and/or dilute acid pretreatment of DDGS was conducted with the three fungal strains to evaluate what strain and treatment performed the best. The fungal strains tested was wild type A. pullulans Y-2311-1 and two mutant strains (GS and MMS) with enhanced cellulase production created from the wild type strain. GS A. pullulans on the enzyme untreated DDGS had the highest protein level with a percent increase of 13.59%. While MMS A. pullulans on enzyme untreated DDGS performed well in increasing the fat content, with a percent increase of 27.07%, and decrease the fiber, with a percent decrease of 13.89%. Overall, GS A. pullulans on the enzyme untreated DDGS performed the best in improving the raw DDGS feedstock. The GS strain still improved the fat content and decreased the fiber content. Most of the minerals had higher levels than the MMS strain and all the amino acids analyzed were also higher than the MMS strain, except for tryptophan which was the same. Lysine levels were also higher in the GS strain than the MMS strain with 0.66% and 0.43% respectively. The dilute acid pretreatment released high levels of sugars and decrease the fiber content but with the creation of inhibitors, fungal fermentation could not be used to further improve the DDGS protein levels.

Library of Congress Subject Headings

Distillers feeds.
Proteins in animal nutrition.
Yeast-like fungi.
Fungal metabolites.


Includes bibliographical references (pages 125-149)



Number of Pages



South Dakota State University


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