Second-generation bioethanol production from phytomass after phytoremediation using recombinant bacteria-yeast co-culture

Sustainable management of harvested hyperaccumulating plants used in phytoremediation of toxic metals is considered quite challenging. The biomass of hyperaccumulators can be an attractive feedstock for bioethanol production, while removal of toxic metals and efficient ethanol conversion systems are necessary to ensure safe and optimum bioethanol production. This study aimed to develop a compatible and efficient consolidated bioprocessing system (CBP) to produce bioethanol from phytomass of arsenic (As)-hyperaccumulator Pteris vittata. A recombinant kefir yeast Kluyveromyces marxianus (KR7 strain) contained six cellulase genes and two recombinant Bacillus subtilis (Type 1 and Type 2 strains) carrying cellulosomal genes in different arrangements were used, and the saccharification and fermentation of P. vittata phytomass were investigated through one-step (KR7 strain only) and co-culture (B. subtilis + KR7 strain) methods. The results of reducing sugar showed that KR7 strain possesses higher saccharification ability than both B. subtilis strains. While the co-culture of B. subtilis Type 2 strain with KR7 strain gave the highest ethanol yield, which achieved 48.5% ethanol yield from P. vittata phytomass after 3 d cultivation. In addition, simple pretreatment by shaking P. vittata with 1% of HNO3 efficiently removed more than 80% of As from the phytomass, suggesting its applicability and feasibility for efficient As extraction to ensure efficient and non-toxic ethanol production. Our results confirm that the developed recombinant bacteria-yeast co-culture possesses a great potential for optimum bioethanol production from As-hyperaccumulators-derived phytomass via CBP.