Nichole Bauer

Document Type

Thesis - University Access Only

Award Date


Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology


Research to improve cellulosic ethanol production is important t o help reduce our dependency on fossil fuels and reduce carbon dioxide emissions. The goal of this project was to evaluate different strategies to reduce enzyme usage in converting cellulose to ethanol. Cellulose degrading enzymes are a major component of cellulosic ethanol production costs, so reducing enzymes use would improve the economics of this emerging industry. Kraft pulp, an intermediate in paper production, was used to represent a fractionated cellulose feedstock. Trials were conducted in either a 5 L submerged bioreactor (80 - 900 rpm) or a horizontal, solid state bioreact or with a paddle – type agitator (0- 60 rpm). Based on the literature, an average enzyme dosage for cellulose (34 FPU/g glucan) and glucosidase (135 CBU/g glucan) was calculated, and these were set as the 100% dosages. Enzyme dosages of 1- 133% were tested in submerged saccharification trials (at 50°C), using Novozyme Celluclast 1.5 (Cellulase) and Novozyme 188(~glucosidase), in a 4.8% (dm) kraft pulp slurry. Novozymes recommended dosages are at the low end of this spectrum, at 12g /g glucan (35% dosage) for cellulase and 1. 2g / g glucan (0. 9% dosage) for~- glucosidase. Saccharification trials showed a typical dosage response. The 133% enzyme dosage achieved the highest sugar concentration (~59 g/L glucose) and saccharification rate (2.45 g / L/ h), with a specific rate of 2. 2 x 10-4 g glucose / unit enzyme/h . the 10% enzyme dosage showed the highest specific saccharification rate (2. 9 x 10-4 g glucose/unit enzyme / h). Simultaneous saccharification and fermentation (SSF) trials (at 35°C) in the submerged bioreactor using Saccharomyces cerevisiae or Candida molischiana were conducted to compare enzyme dosages of 33, 67, 100, and 133% at 14% solids loading. Ethanol titer and productivity were similar for trials with 67% or higher of the normal enzyme dosage, while 33% enzyme dosage was much lower. Subsequent trials evaluated fedbatch SSF in the submerged bioreactor using S. cerevisiae, where additional kraft pulp was added to achieve 14% total solids loading. In control trials, enzyme dosages were maintained at either a 133% or 67 %, by adding additional enzymes when kraft pulp was fed into the bioreactor. For treatments, buffer was added instead which reduced the final enzyme dosages to 33% and 17% of the literature average. Ethanol production was similar for all trials except the 17% dosage trial. Therefore, this fed- batch approach reduced net enzyme dosage to 33% of the literature average (11 FPU cellulase/g glucan and 45 CBU ~glucosidase/g glucan). Fed- batch saccharification trials then were conducted in a solid state bioreactor to increase solids to (34. 8%), while reducing enzyme dosage to 19% of the literature average. Feedback inhibition of the enzymes was observed, due to the low levels of glucose recovery yield of 35% (maximum glucose concentration of 296. 13 g/L) To prevent enzyme inhibition, a fed - batch simultaneous saccharification and fermentation (SSF) was also completed within a solid state bioreactor at 34. 8% solids loading and a final enzyme dosage of 19%. Ethanol yields averaged only 20% of the theoretical maximum, even though abundant levels of glucose were available (64. 2 g/L). While water and ethanol loss occurred via evaporation through the lid (during sampling and kraft pulp addition) and leaking through the agitator seals, this could not account for the low yield. It was postulated that osmotic stress on the yeast (due to low water activity may have caused the reduced metabolic activity. To test this, and the potential of nitrogen limitation, we tested variable levels of water and looked at addition of yeast extract. By adding water to the fermented kraft pulp slurry to reduce the overall solids loading to 27. 8%, the ethanol yield rose to 47.85%, while reaching an ethanol titer of 57. 8 g / L. Adding yeast extract also resulted in a significant boost to ethanol yield.

Library of Congress Subject Headings

Cellulose -- Biodegradation.
Biomass conversion.
Ethanol fuel industry.



Number of Pages



South Dakota State University