Conversion of Mixed Waste Office Paper to Ethanol by Genetically Engineered Klebsiella oxytoca Strain P2

Abstract

Unsorted, mixed waste office paper (MWOP) is an excellent substrate for conversion into fuel ethanol using a recombinant strain of Klebsiella oxytoca which ferments cellobiose and cellotriose to ethanol at near theoretical yields, eliminating the need for supplemental β‐glucosidase. This organism was tested with commercial fungal cellulase in optimized simultaneous saccharification and fermentation experiments (SSF) using MWOP as a substrate (pH 5–15.2, 35 °C). Similar rates and yields were obtained with dilute acid‐pulped (hydrolysis of hemicellulose) and water‐pulped MWOP on a dry weight basis although viscosity was reduced by the acid pretreatment. In simple batch fermentations, 40 g/L ethanol was produced after 48–72 h with 100 g/L MWOP and 1000 filter paper units (FPU) of cellulase/L, a yield of 550 L of ethanol/metric ton. Cellulase usage was further reduced by recycling SSF residues containing bound enzymes in multistage fermentations. This approach reduced the requirement for fungal cellulase while retaining rapid ethanol production and high ethanol yield. In our optimal design, broths containing an average of 39.6 g/L ethanol were produced in three successive stages with an average fermentation time of 80 h (567 FPU of fungal cellulase/L; 6.1 FPU/g of substrate). This represents a yield of 0.426 g of ethanol/g of substrate, 539 L/metric ton, 129 gal/U.S. ton. MWOP contains approximately 90% carbohydrate. Thus the combined efficiency for saccharification and fermentation to ethanol was 83.3% of the theoretical maximum.

Ethanol from Biomass Hydrolysates by Efficient Fermentation of Glucose and Xylose – A Review

“…The PET operon was genetically stabilized in K. oxytoca M5A1 to create strain P2 by integrating the operon along with a chloramphenicol acetyl transferase ( cat ) marker at the site of the chromosomal pyruvate formate lyase ( PFL ) gene . Strain P2 was tested successfully on various feedstocks including mixed office paper obtaining ethanol yields of 0.426 g g −1 , sugarcane bagasse with ethanol yields of 0.43 g g −1 , corn fiber with the yield of 0.35 g g −1 , and sugar beet pulp with ethanol yields of 0.12 g g −1 (Fig. d).…”

Advances in consolidated bioprocessing systems for bioethanol and butanol production from biomass: a comprehensive review

“…Previously, recombinant strains of K. oxytoca containing Z. mobilis pdc (pyruvate decarboxylase) and adhB (alcohol dehydrogenase) genes have been developed. These strains are able to direct the metabolism of pyruvate to ethanol, but for efficient ethanol production they need cellulase to be added to the cellulosic materials, which imposes additional cost on the ethanol production process (Ohta et al, 1991b;Doran and Ingram, 1993;Bothast et al, 1994;Doran et al, 1994;Brooks and . Ingram, 1995;Golias et al, 2002).…”

Production of recombinant bacterial endoglucanase as a co-product with ethanol during fermentation using derivatives ofEscherichia coli KO11

“…General processes for the conversion of this material into ethanol include the hydrolysis of hemicellulose into a mixture of hexose and pentose sugars using dilute sulfuric acid (Asghari et al, 1996) and the hydrolysis of cellulose using enzymes (Brooks and Ingram, 1995). Lignin, a phenolic polymer, can be burned as boiler fuel.…”

“…In the 1970s, Gulf Oil developed a simultaneous saccharification and fermentation process (SSF) for ethanol production from cellulose in which both enzymatic hydrolysis and fermentation occur together in a single vessel (Gauss et al, 1976). Improvements to this process have been made, such as the development of mutant strains of Trichoderma longibranchiatum that produce high levels of secreted cellulases (Nidetzky et al, 1995), genetic engineering of ethanologenic bacteria which utilize cellobiose (Wood and Ingram, 1991), discovery of cellobiose utilizing yeasts (Freer, 1991), and recycling of cellulase enzymes during SSF (Brooks and Ingram, 1995). Despite these improvements, however, the cost of commercial cellulases remains as a major barrier to commercialization.…”