Assessment of combination of pretreatment of Sorghum durra stalk and production of chimeric enzyme (β-glucosidase and endo β-1,4 glucanase, CtGH1-L1-CtGH5-F194A) and cellobiohydrolase (CtCBH5A) for saccharification to produce bioethanol

Abstract

Optimization of pretreatment and saccharification of Sorghum durra stalk (Sds) was carried out. The chimeric enzyme (CtGH1-L1-CtGH5-F194A) having β-glucosidase (CtGH1) and endo β-1,4 glucanase activity (CtGH5-F194A) and cellobiohydrolase (CtCBH5A) from Clostridium thermocellum were used for saccharification. Chimeric enzyme will save production cost of two enzymes, individually. Stage 2 pretreatment by 1% (w/v) NaOH assisted autoclaving + 1.5% (v/v) dilute H₂SO₄ assisted oven heating gave lower total sugar yield (366.6 mg/g of pretreated Sds) and total glucose yield (195 mg/g of pretreated Sds) in pretreated hydrolysate with highest crystallinity index 55.6% than the other stage 2 pretreatments. Optimized parameters for saccharification of above stage 2 pretreated biomass were 3% (w/v) biomass concentration, enzyme (chimera: cellobiohydrolase) ratio, 2:3 (U/g) of biomass, total enzyme loading (350 U/g of pretreated biomass), 24 h and 30 °C. Best stage 2 pretreated Sds under optimized enzyme saccharification conditions gave maximum total reducing sugar yield 417 mg/g and glucose yield 285 mg/g pretreated biomass in hydrolysate. Best stage 2 pretreated Sds showed significantly higher cellulose, 71.3% and lower lignin, 2.0% and hemicellulose, 12.2% (w/w) content suggesting the effectiveness of method. This hydrolysate upon SHF using Saccharomyces cerevisiae under unoptimized conditions produced ethanol yield, 0.12 g/g of glucose.

Abbreviation: Ct-Clostridium thermocellum, Sds-Sorghum durra stalk, TRS-Total reducing sugar, HPLC-High performance liquid chromatography, RI-Refractive index, ADL-acid insoluble lignin, GYE-Glucose yeast extract, MGYP-Malt glucose yeast extract peptone, SHF-separate hydrolysis and fermentation, OD-Optical density, PVDF-Poly vinylidene fluoride, TS-total sugar, FESEM-Field emission scanning electron microscopy, XRD-X-ray diffraction, FTIR-Fourier transform infra-red spectroscopy and CrI-Crystallinity index.

Keywords:

• CHIMERA
• cellobiohydrolase
• enzyme loading
• 2 stages pretreatment

Acknowledgements

This project was funded by DBT PAN-IIT, grant no. BT/EB/PAN IIT/ 2012 by Department of Biotechnology (DBT), Ministry of Science and Technology, India to Prof. Arun Goyal, Department of Biosciences and Bioengineering, Indian Institute of Technology (IIT) Guwahati. The authors acknowledge the use of FTIR spectrophotometer obtained through the Indo-Finnish project grant (BT/IN/Finland/08/AG/2011) from DBT, Ministry of Science and Technology, Government of India. The authors also acknowledge the Central Instrument Facility at IIT, Guwahati, for the provision of XRD and FESEM facilities and Department of Biosciences and Bioengineering, IIT Guwahati for HPLC facility.

Disclosure statement

The authors declare no conflict of interest.

Additional information

Funding

This project was funded by DBT PAN-IIT, grant no. BT/EB/PAN IIT/2012 by Department of Biotechnology (DBT), Ministry of Science and Technology, India to Prof. Arun Goyal, Department of Biosciences and Bioengineering, Indian Institute of Technology (IIT) Guwahati.