A microalgal-based preparation with synergistic cellulolytic and detoxifying action towards chemical-treated lignocellulose.

Manuel Benedetti,Paolo Longoni,Michel Goldschmidt‐Clermont,Luca Dall’Osto,Natalia Herrero Garcia,Damar Lopez‐Arredondo,Simone Barera,Roberto Bassi,Zeno Guardini,Luis Herrera‐Estrella,David Bolzonella

doi:10.1111/pbi.13447

Abstract

SummaryHigh‐temperature bioconversion of lignocellulose into fermentable sugars has drawn attention for efficient production of renewable chemicals and biofuels, because competing microbial activities are inhibited at elevated temperatures and thermostable cell wall degrading enzymes are superior to mesophilic enzymes. Here, we report on the development of a platform to produce four different thermostable cell wall degrading enzymes in the chloroplast of Chlamydomonas reinhardtii. The enzyme blend was composed of the cellobiohydrolase CBM3GH5 from C. saccharolyticus, the β‐glucosidase celB from P. furiosus, the endoglucanase B and the endoxylanase XynA from T. neapolitana. In addition, transplastomic microalgae were engineered for the expression of phosphite dehydrogenase D from Pseudomonas stutzeri, allowing for growth in non‐axenic media by selective phosphite nutrition. The cellulolytic blend composed of the glycoside hydrolase (GH) domain GH12/GH5/GH1 allowed the conversion of alkaline‐treated lignocellulose into glucose with efficiencies ranging from 14% to 17% upon 48h of reaction and an enzyme loading of 0.05% (w/w). Hydrolysates from treated cellulosic materials with extracts of transgenic microalgae boosted both the biogas production by methanogenic bacteria and the mixotrophic growth of the oleaginous microalga Chlorella vulgaris. Notably, microalgal treatment suppressed the detrimental effect of inhibitory by‐products released from the alkaline treatment of biomass, thus allowing for efficient assimilation of lignocellulose‐derived sugars by C. vulgaris under mixotrophic growth.

Highlights

Lignocellulose, the most abundant organic carbon source on Earth, has great potential for conversion into renewable fuels
Biological treatments include the use of microbial cell wall degrading enzymes (CWDEs), which are currently obtained by culturing mesophilic fungi and bacteria with lignocellulolytic activities (Sanchez 2009)
As efficient cellulose hydrolysis requires optimized enzymatic activities ratios, we decided to produce transgenic C. reinhardtii strains that express independently each of the four hyperthermophilic CWDEs (HCWDEs): it allows to optimize the enzymatic cocktail for maximum yield and to keep loading each activity as low as possible

Summary

Introduction

Lignocellulose, the most abundant organic carbon source on Earth, has great potential for conversion into renewable fuels. Chemical pretreatments are harmful for the environment and negatively impact the rationale of using lignocellulose to produce cleaner forms of fuels Such treatments generate byproducts that inhibit the microbial fermentation of lignocellulose-derived sugars, reducing the conversion yield into biofuel-related compounds (Jo€nsson and Martın 2016). Biological treatments include the use of microbial cell wall degrading enzymes (CWDEs), which are currently obtained by culturing mesophilic fungi and bacteria with lignocellulolytic activities (Sanchez 2009). Such organisms secrete a wide array of CWDEs in low amounts, as they are strictly required for their own livelihood

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Conclusion