Evaluation of the ethanol tolerance for wild and mutant Synechocystis strains by flow cytometry

Abstract

Flow cytometry was used to evaluate the effect of initial ethanol concentrations on cyanobacterial strains of Synechocystis PCC 6803 [wild-type (WT), and ethanol producing recombinants (UL 004 and UL 030)] in batch cultures. Ethanol recombinants, containing one or two metabolically engineered cassettes, were designed towards the development of an economically competitive process for the direct production of bioethanol from microalgae through an exclusive autotrophic route. It can be concluded that the recombinant Synechocystis UL 030 containing two copies of the genes per genome was 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.

Keywords: Enzymatic activity; Ethanol; Flow cytometry; Membrane permeability; Synechocystis wild and mutant strains; Tolerance.

Fig. 1

Construction of UL 004 (a single cassette strain) and UL 030 (a double cassette strain). The Genbank assession of the Synechocystis adhA was Gen Bank: AP012205.1 (nt 3,530,233 to 3,531,243), CDS: BAK51882.1; The GeneBank assession of the Zymomonas pdc was Gene Bank HM235920.1 (nt 493.2199) CDS: ADK13058.1.

Fig. 2

Flow cytometric controls – WT Density plots concerning CFDA staining (a, b), PI staining (c, d), FSC/SSC signals (e, f), autofluorescence FL3 signal, detecting Chorophyll signal (g, h), autofluorescence FL4 signal, detecting phycocyanin signal (i, j). a), c), e), g), i) – Cells collected from a WT exponential growing culture b), d), f), h), j) – WT dead cells treated with ethanol 70% (5 min incubation) The percentages of cells in each quadrant, calculated by the Flowing Software, are displayed in the density plots a), b), c) and d). PCEA correspond to the proportion of cells stained with CFDA, thus with enzymatic activity; therefore it is read in the upper left quadrant of the plots FL1 versus FSC-H (Fig. 2a) and b)). PCIM correspond to the proportion of cells not stained with PI, thus with intact membrane; therefore it is read in the bottom left quadrant of the plots FL2 versus FSC-H (Fig. 2c) and d)).

Fig. 3

Evolution of optical density readings at 730 nm during WT (a), UL 004 (b) and UL 030 (c) batch cultures, in the presence of increasing initial ethanol concentrations. OD₇₃₀ averages resulted from two independent replicates (n = 2) and were affected by a relative error not exceeding 15%.

Fig. 4

PCEA evolution during WT (a), UL 004 (b) and UL 030 (c) batch cultures, in the presence of increasing initial ethanol concentrations. Error bars correspond to the standard deviation resulted from two independent replicates (n = 2).

Fig. 5

PCIM changes during WT (a), UL 004 (b) and UL 030 (c) batch cultures, in the presence of increasing initial ethanol concentrations. Error bars correspond to the standard deviation resulted from two independent replicates (n = 2).

Fig. 6

Density plots SSC/FSC and photos concerning flow cytometric analysis and optical microscope observations of cells collected after 70 h, from WT, UL 004 and UL 030 batch cultures at 0 g/l (control), 30 g/l and 40 g/l of ethanol. WT SSC/FSC density plots: a)–c) WT optical microscope photos (1000× magnification): d)–f) UL 004 SSC/FSC density plots: g)–i) UL 004 optical microscope photos (1000× magnification): j)–) UL 030 SSC/FSC density plots: m)–o) UL 030 optical microscope photos (1000× magnification): p)–r)

Fig. 7

Optical observations (1000×) of WT (a) and UL 004 (b) cells harvested from batch cultures at initial 30 g/l ethanol and UL 004 (c) cells at 40 g/l ethanol (t = 400 h).