Metabolic engineering of a cyanobacterium to increase sucrose accumulation as an alternative feedstock for bioethanol

MACARENA PEREZ CENCI; LEONARDO CURATTI; MARÍA EUGENIA SANZ SMACHETTI; GRACIELA LIDIA SALERNO

San Luis

Congreso; XIII Congreso Argentino de Microbiología General; 2018

ASOCIACION CIVIL DE MICROBIOLOGIA GENERAL

Microalgae have great potential as a feedstock for bioethanol and other biofuels. However, the current cost of producing biofuels from microalgae biomass is still high to be able to envision massive and profitable commercialization in the near future. One of the main constraints is the pretreatment and saccharification processes necessary to make the insoluble carbohydrates accessible to microbial fermentation. Hence, we hypothesized that re-routing the carbohydrates? metabolic pathway towards sucrose accumulation would facilitate recovery and fermentation. In order to achieve that, we over-expressed the spsB gene, encoding a SPS enzyme and its putative promoter, in Anabaena sp. PCC 7120, downstream of the constitutive promoter of pDU1, increasing by 2-fold the SPS activity. Under standard growth conditions, there was no significant difference in doubling time with the wt strain. However, the spsB+ strain showed an increased tolerance to saline stress, displaying doubling times of 36 ± 11 h in comparison to 53 ± 10 h of the wt strain when cultivated in the presence of 80 mM NaCl, and 39 ± 6 h in comparison to 59.4 ± 0.8 hours of the wt strain in the presence of 120 mM NaCl. Sucrose content was 10- or 5-fold higher than the wt when subjected to 80 or 120 mM NaCl loading, reaching values of 7.65 ± 0,04 % (w/w) or 9 ± 3 % (w/w) of their dry weight in 48 h, respectively. To extract sucrose from the spsB+ strain, two different low-energy methods were assessed. In the first one, the collected biomass was air-dried, milled with 15% sand and extracted with water at room temperature, which allowed the recovery of 32 ± 9% of the total sucrose. The second method consisted in an extraction by microwaves at a of power 200 w, using 4 pulses of 2 min each, recovering 56 ± 7% of the total sucrose content. Sucrose-rich preparations obtained from both methods were fermented by S. cerevisiae. While the preparation obtained by drying and milling allowed an ethanol production of 91% of the maximum theoretical value, the preparation obtained by microwaves exposure was around 50% of the maximum theoretical value. This efficiency was partially improved by adding a nitrogen source to the sucrose-rich preparation. Hence, ethanol productivity was 26 ± 6 mg. L of culture-1 for the extract obtained via drying and milling and 12 ± 1 mg. L of culture-1 for the extract obtained via microwave and supplemented with a nitrogen source. These results present a promising base-line to continue investigating the use of genetically-modified cyanobacteria biomass as an alternative bioethanol feedstock.