Flow cytometry was used to
evaluate the effect of initial ethanol concentrations on cyanobacterial strains
of Synechocystis PCC 8063 [wild-type (WT),
and ethanol producing recombinants (UL 004 and UL 030)] in batch
cultures. Ethanol recombinants, containing one or two metabolically engineered
casettes, were designed towards the development of an economically competitive
process for the direct production of bioethanol from microalgae through an
exclusive autotrophic route.
The three Synechocystis strains behaved
differently in the presence of ethanol. The biomass concentration was reduced by
25% relatively to the wild type at initial ethanol concentrations of 10 g.L-1,
15 g.L-1 and 20 g.L-1 for the WT, UL 004 and UL 030
strains, respectively. For the WT strain, the proportion of cells with
enzymatic activity (PCEA) progressively decreased as the ethanol concentration
increased and the culture aged, while UL 004 PCEA showed a pronounced
reduction only for initial ethanol concentrations above 10 g.L-1,
relatively to the control. UL 030 PCEA was always above 80 % for initial ethanol
concentrations in the range of 0-20 g.L-1. For all the strains, the
proportion of cells with intact membrane (PCIM) followed similar profiles to the
PCEA profiles.
The cyanobacteria
morphology was also affected by the presence of ethanol. For WT and UL 004
strains, as the initial ethanol concentration increased and the cultures aged,
the cells aggregated and formed filamentous structures. UL 030 cells also
aggregated with the ethanol concentration increase and age, but in a lesser
extent, and did not form filaments.
It can be concluded that the recombinant Synechocystis
strains tested (UL 004 and UL 030) are more tolerant to the presence
of ethanol than the WT strain, and the most efficient ethanol producer (UL 030
containing two copies of the genes per genome) was also the most tolerant to
ethanol. Nevertheless, to implement a production process using recombinant
strains, the bioethanol produced will be required to be continuously extracted
from the culture media via a membrane-based technological process for example
to prevent detrimental effects on the biomass. The results presented here are
of significance in defining the maximum threshold for bulk ethanol
concentration in production media.