Finding a viable method to recover oil from the corn ethanol industry's co-products has considerable economic prospects for ethanol bio-refineries. This study examined the effects of enzymes and ethanol on oil recovery from dried distillers' grains with solubles (DDGS) and oil distribution in the whole stillage (WS). Protease and cellulase enzymes were tested either individually or in combination with the heavier fractions of DDGS and resulted in 18-20% more oil than the original DDGS. More than 90% of the oil was recovered from the heavier fraction of DDGS using ethanol at 30ºC with 30% solid loadings. Ethanol addition also improved oil partition in WS's liquid fraction by 17–20%. Overall, enzymes and ethanol treatments showed a positive effect on oil recovery from DDGS and WS. Ethanol bio-refineries may use these findings to recover oil as no significant changes are required in the ethanol plant's design.

Doctor of Philosophy / Department of Grain Science and Industry / Praveen V. Vadlani / Distillers Dried grain with Solubles (DDGS) is a co-product of grain-based ethanol and is primarily used as livestock feed. With increasing production of DDGS, it is imperative to produce value-added products and/or find new applications of DDGS to help sustain the biofuel industry. Carotenoids are expensive yet essential feed additives. Since animals cannot synthesize carotenoids and animal feeds including DDGS are generally poor in carotenoids, about 30-120 ppm of total carotenoids is added to animal feed to improve animal health. The objectives of this study were to 1) produce carotenoid (astaxanthin and β-carotene)-enriched DDGS by Phaffia rhodozyma and Sporobolomyces roseus monoculture and mixed culture submerged fermentation of whole stillage, 2) optimize fermentation media by response surface methodology (RSM) and mixture design followed by validation, 3) evaluate the nutritional profile of carotenoid-enriched DDGS, 4) improve carotenoid production by the use of precursors, and 5) develop carotenoid-enriched feeds namely, wheat bran, rice bran and soybean products. Carotenoid-enriched DDGS was produced from both monoculture and mixed culture fermentation with yields ranging from 17-233 µg/g. Upon media optimization, astaxanthin and β-carotene yields, especially in P. rhodozyma were enhanced by 177% and 164% to yield 98 and 275 µg/g respectively. Nutrition profiling of the carotenoid-enriched DDGS showed that the secondary fermentation resulted in low fiber, protein and %N and enhanced fat. Fiber was reduced by 77% and 66% by P. rhodozyma and S. roseus respectively, whereas the crude fat increased by 80% in mixed culture fermentation. Additionally, abundant vaccenic acid, a monounsaturated fatty acid was seen in S. roseus and mixed culture fermented DDGS. Vaccenic acid is a precursor of conjugated linolenic acid which is known to confer numerous health benefits. Fermentation of milo DDGS, wheat bran, rice bran and soybean products also resulted in carotenoid enrichment, with the best astaxanthin yield of 80 µg/g in rice bran, and best β-carotene yield of 837 µg/ g in soy flour. Precursors like mevalonic acid, apple pomace and tomato pomace increased carotenoid yield in DDGS and other substrates, with the yield increment depending on the substrate. Mevalonic acid resulted in the best astaxanthin and β-carotene yield increment by 140% and 236% resulting in 220 µg/g and 904 µg/g respectively in corn DDGS. Apple pomace and tomato pomace resulted in 29% carotenoid yield increment. Numerous studies thus far have used cheap agricultural substrates to produce carotenoids especially astaxanthin using P. rhodozyma with the intent of extracting the carotenoids for use in animal feed. However, by fermenting the animal feed directly, carotenoid-enriched feed can be produced without the need for extraction. By this simple yet novel carotenoid value addition, premium feeds or feed blends can be developed. Apart from carotenoid enrichment, low-fiber DDGS can help expand the market base of DDGS for use in non-ruminant feeds. Carotenoid value addition of DDGS can not only help sustain the biofuel industry but can also capture the aquaculture feed base which heavily relies on astaxanthin supplementation.

A significant by-product of bioethanol plants is whole stillage, commonly used to produce animal feed due to its nutritious value, has a potential to be used to produce various value-added products while eliminating a costly process step is an alternative approach. In this study, production and separation of additional ethanol, fungal biomass and enzyme were successfully achieved with the cultivation in membrane bioreactors in batch process condition. Process optimization studies regarding fermentation and filtration conditions were carried out. Up to 10.4 g/l ethanol per litre of used whole stillage can be produced in simultaneous saccharification and fermentation (SSF) condition without any pH adjustment and additional pretreatment step. Also, 50% diluted whole stillage provided 87% higher ethanol production comparing to non-diluted medium. Moreover, 71 % higher biomass production was obtained with the filtrate of 50% diluted whole stillage comparing to 25% diluted one. Considering the achieved results, a two-stage cultivation using SHF (Separate Hydrolysis and Fermentation) strategy in membrane bioreactors for separation of ethanol, lignin-rich stream, protein-rich fungal biomass and enzymes was proposed. The present thesis showed that the integration of filamentous fungi with membrane bioreactors can increase the range of products that can be produced from whole stillage.

Establishing a commercial, lignocellulose-based, second-generation ethanol process has received several decades of attention by both researchers and industry. However, a fully economically viable process still remains a long-term goal. The main bottleneck to this achievement is the recalcitrance of lignocellulosic feedstocks, although there are several other factors, such as the huge investment required for second-generation ethanol facilities. An intelligent alternative solution discussed in this thesis is an integrated approach using firstgeneration ethanol plants for second-generation processes. Wheat is the major feedstock for first-generation ethanol in Europe; therefore, wheat-based lignocellulose waste, such as wheat straw, bran, and whole stillage fiber (a waste stream from first-generation wheat-based ethanol plants) was the primary focus of the integration model in this thesis. Since the major share of first-generation ethanol plant economics focuses on the animal feed DDGS (Distillers’ dried gains with solubles), the integration of lignocellulose should be designed in order to maintain DDGS quality. An ethanol-producing edible filamentous fungus, Neurospora intermedia, a potential protein source in DDGS, was considered for use as the fermenting microbe. The morphological and physiological aspects of this fungus were studied in the thesis, leading to the first report of fungal pellet development. An alternative approach of using dilute phosphoric acid to pretreat lignocellulose, as it does not negatively affect fungal growth or DDGS quality, was demonstrated in both the laboratory and on a 1m3 pilot scale. Furthermore, the process of hydrolysis of pretreated lignocelluloses and subsequent N. intermedia fermentation on lignocellulose hydrolysate was also optimized in the laboratory and scaled up to 1 m3 using an in-house pilot-scale airlift bioreactor. Fungal fermentation on acid-pretreated and enzyme-hydrolyzed wheat bran, straw and whole stillage fiber resulted in a final ethanol yield of 95%, 94% and 91% of the theoretical maximum based on the glucan content of the substrate, respectively. Integrating the first- and second-generation processes using thin stillage (a waste stream from first-generation wheat-based ethanol plants) enhanced the fungal growth on straw hydrolysate, avoiding the need for supplementing with extra nutrients. Based on the results obtained from this thesis work, a new model for integrated first- and second-generation ethanol using edible filamentous fungi processes that also adds value to animal feed (DDGS) was developed.