Scale up and efficient bioethanol production involving recombinant cellulase (Glycoside hydrolase family 5) from Clostridium thermocellum

Saprativ P Das,Deepmoni Deka,Arabinda Ghosh,Debasish Das,Mohammad Jawed,Arun Goyal

doi:10.1186/2043-7129-1-19

Abstract

Abstract Background Lignocellulose degrading fungal enzymes have been in use at industrial level for more than three decades. However, the main drawback is the high cost of the commercially available Trichoderma reesei cellulolytic enzymes. Results The hydrolytic performance of a novel Clostridium thermocellum cellulolytic recombinant cellulase expressed in Escherichia coli cells was compared with the naturally isolated cellulases in different modes of fermentation trials using steam explosion pretreated thatch grass and Zymomonas mobilis. Fourier transform infrared (FT-IR) spectroscopic analysis confirmed the efficiency of steam explosion pretreatment in significant release of free glucose moiety from complex lignocellulosic thatch grass. The recombinant GH5 cellulase with 1% (w v-1) substrate and Z. mobilis in shake flask separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) trials demonstrated highest ethanol titre (0.99 g L-1, 1.2 g L-1) as compared to Bacillus subtilis (0.51 g L-1, 0.72 g L-1) and Trichoderma reesei (0.67 g L-1, 0.94 g L-1). A 5% (w v-1) substrate with recombinant enzyme in shake flask SSF resulted in a 7 fold increment of ethanol titre (8.8 g L-1). The subsequent scale up in a 2 L bioreactor with 1 L working volume yielded 16.13 g L-1 ethanol titre implying a 2 fold upturn. The rotary evaporator based product recovery from bioreactor contributed 94.4 (%, v v-1) pure ethanol with purification process efficiency of 22.2%. Conclusions The saccharification of steam exploded thatch grass (Hyparrhenia rufa) by recombinant cellulase (GH5) along with Z. mobilis in bioethanol production was studied for the first time. The effective pretreatment released substantial hexose sugars from cellulose as confirmed by FT-IR studies. In contrast to two modes of fermentation, SSF processes utilizing recombinant C. thermocellum enzymes have the capability of yielding a value-added product, bioethanol with the curtailment of the production costs in industry.

Highlights

Lignocellulose degrading fungal enzymes have been in use at industrial level for more than three decades
The recombinant cellulase isolated from E. coli BL21 cells transformed with full length gene CtLic26A-GH5CBM11 from Clostridium thermocellum was shown to have better cellulolytic activity in an efficient simultaneous saccharification and fermentation (SSF) process using Jamun (Syzygium cumini) leafy biomass as the substrate [10]
Steam explosion pretreatment was employed for delignification to increase the efficiency of hydrolysis of lignocellulosic biomass, thatch grass

Summary

Introduction

Lignocellulose degrading fungal enzymes have been in use at industrial level for more than three decades. The main drawback is the high cost of the commercially available Trichoderma reesei cellulolytic enzymes. Bacterial cellulases are potential candidates as they can withstand harsh conditions such as high temperature, sugar, salt and ethanol concentrations during lignocellulose degradation and can metabolize wide range of sugars improving the process of ethanol production [7]. The cellulosome of Clostridium thermocellum is known to have one of the highest rates of cellulose utilization till date reported, that displays 50-fold higher specific activity than the corresponding Trichoderma reesei system against crystalline cellulose [9]. The recombinant cellulase isolated from E. coli BL21 cells transformed with full length gene CtLic26A-GH5CBM11 from Clostridium thermocellum was shown to have better cellulolytic activity in an efficient SSF process using Jamun (Syzygium cumini) leafy biomass as the substrate [10]

Methods

Results

Conclusion