(177o) Butanol Production from Hydrolysate of Jerusalem Artichoke Tubers By Clostridium Acetobutylicum

Butanol production from hydrolysate of Jerusalem
artichoke tubers by Clostridium acetobutylicum

Lijie
Chen*, Youduo Wu,

School
of Life Science and Biotechnology, Dalian University of Technology, No.2
Linggong Road, Dalian, China. ljchen@dlut.edu.cn

Recently, biobutanol,
as one of the alternative advanced biofuels with superior fuel properties, have
attracted worldwide attention on clostridial ABE fermentation based on various
low-cost and substainable feedstocks. However, the major problems associated
with butanol production from fructose-based feedstocks Jerusalem artichoke (JA)
tubers include insufficient capability of fructose utilization, unexpected lag
phase for metabolic physiology of Clostridium acetobutylicum, which
seriously hinder butanol production, yield and productivity. Zn2+ has been demonstrated to be the key
factor responsible for the improved fructose-based ABE fermentation and play
pleiotropic roles in global levels corresponding to the pH/ORP fluctuations,
metabolic flux redistribution and changes in transcription, implying that
extracellular and intracellular cues might be also of great importance for
regulating fructose-based ABE fermentation. Based on the Zn2+-mediated response, bioprocess engineering
strategies of pH and ORP control, transcriptional analysis for butanol stress
followed by genetic engineering manipulation have been investigated from the
viewpoint of efficient butanol production from glucose/fructose mixture or JA
tubers hydrolysate.

Significant
improvements on ABE fermentation from glucose/fructose mixture were observed
with initial pH controlled at 5.5 to OD620 1.0
or pH controlled above 4.9, which resulted in increased butanol production and
yield to 10.6 g/L, 0.19 g/g and 11.4 g/L, 0.20 g/g. Furthermore, the ORP
controlled above -460 mV also led to significantly improved butanol production
and yield up to 13.2 g/L and 0.26 g/g. Importantly, as high as 12.0 g/L butanol
was achieved from JA tubers hydrolysate together with butanol productivity and
yield of 0.21 g/L/h and 0.22 g/g due to the combined pH and ORP control
strategies, compared to those of 5.4 g/L, 0.08 g/L/h and 0.16 g/g in the
control. Moreover, fructose PTS related operon fru and central carbon
metabolism associated with ATP and NADH supply have been facilitated by ORP
controlled above -460 mV through Q-RT-PCR and LC-ESI/MS analysis.

Global
transcriptional analysis was further conducted to elucidate the Zn2+-mediated response on butanol stress.
Expression levels of multiple genes involved in glucose/fructose transport (glcG,
fruC and fruD) and central carbon metabolism associated with
glycolysis, acidogenesis and Solventogenesis were differentially up-regulated.
Genetic engineered strains were further constructed by overexpressing glucose
PTS gene glcG, fructose PTS genes fruC and fruD in C. acetobutylicum
respectively. Especially, glcG and fruC overexpression led to
improved ABE fermentation from glucose/fructose mixture with butanol production
increased to 12.7 and 13.1 g/L. Furthermore, fruC overexpression
contributed to 8.0 g/L butanol produced from JA tubers hydrolysate while little
difference observed for glcG overexpression.