Quick test for ethanol-adapted Synechocystis cultures

Figure 9: Quick test reference picture for distinguishing between WT and JL2.JL2 (A) and WT (B) under 0 % [v/v], 2 % [v/v] and 3 % [v/v] ethanol in six-well plates with 5 mL cultures held under ~ 40µE m⁻² s⁻¹ light and mild shaking after an incubation period of 24 h.

In order to distinguish between JL2 strains and WT for ongoing experiments a quick test system for the cultures has been established. Before starting a new experiment, a time frame of seven days, in which no ethanol was in the medium, was held. New experiments with ethanol adapted strains started with a standardized quick test in which JL2 cultures were compared to the WT. The test is based on a 24 h stress experiment in which the different stains were spiked with different ethanol concentration in 6-well plates with a 5 mL culture volume. The results were evaluated visually compared to a reference (Fig.9). Growth analysis in 6-well plates showed a clear visual difference at the different tested concentrations of ethanol. With 2 % [v/v] ethanol treated WT Synechocystis cultures exhibit, in contrast to JL2 cultures, a strong agglomeration effect, which became stronger at a concentration of 3 % [v/v] ethanol in the media. At this concentration, JL2 cells started to exhibit a slightly similar effect. All cultures exhibited increasing growth retardation with rising ethanol concentrations. All these factors have been used as a confirmation before the experiments.

Ethanol adaptedSynechocystis appeared to have different sedimentation properties (Fig.9), generally a marker for impairment with pili, and thus slightly backing the observation of the impairment of ethanol with pili like structures. In order to pin point a change of ethanol adaptation PilA7 was analyzed on the Northern blot level. No significant difference between JL2 and WT cultures after the treatment with 2 % [v/v] ethanol (data not shown) was observed, validating the impairment of ethanol with the corresponding gene for PilA7 but giving no further information concerning the sedimentation property differences. The observed agglomeration ofSynechocystis cultures in 6-well-plates after ethanol treatment (Fig.9) could further highlight the ethanol dependent impairment on cellular appendices.

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2 Results and Discussion

2.3.4 Properties of an ethanol-adapted strain under production condition

In order to determine, whether an elevated tolerance against ethanol leads to an increased ethanol production, cultures of pre-adapted Synechocystis, which show the desired attitude (JL2) were conjugated with the ethanol production construct (chapter 3.2.6.4.) and compared to the corresponding WT control strain (Fig.10). The growth advances to WT of the JL2 strains under external ethanol (Fig.8) can be also be seen under ethanologenic conditions (Fig.10A). After 24 h the growth superiority of JL2 producer compared to a WT producer is already visible and increases successively with the onset of the time. The comparison study revealed that JL2 strains which are used as the production platform for ethanol showed further favorably attributes. Ethanologenic JL2 strains showed an elevated Chl a content under all tested time points and conditions, and further generated more ethanol. A relative percentile surplus (Fig.10B) of Chl a content of ethanologenic JL2 producer strains was observed. It shows a constant elevation with slight decrease in the first four days of ethanol production.

The effect gets successively more visible and vivid with the onset of the production time and reaching over 60 % of the level of the ethanologenic WT strain. Parallel conducted control experiments with non- ethanologenic JL2 strains compared to a WT strain with no ethanol in the media showed a constant Chla content under the same conditions and with 2 % [v/v]

ethanol in the media, like in ethanologenic conditions, a higher Chl a content of JL2 was observed (data not shown). In the first days, the Chlacontent surplus is comparable between non-ethanologenic conditions and ethanologenic conditions and increases successively on a prolonged time frame reaching the highest point of the experiment up to nearly 100 % surplus of the Chla content of the compared WT. Ethanol measurements in media of ethanologenic cultures depicted an increased ethanol production under the tested conditions, which reached a relative percentile production surplus of over 150 % of the JL2 strain. It can be stated that a pre-adaptation to laboratory conditions and ethanol containing media leads to a significant increase in ethanol production under unchanged cultivation conditions.

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Figure 10: Growth properties and relative percentile surplus of Chla content and ethanol generation of ethanologenic JL2 strain compared to an ethanologenicSynechocystis strain derived from a WT.Comparison of growth properties (A) of pre-adapted ethanologenic Synecho-cystis strains (JL2) (circles) vs. ethanologenic WT (squares) via optical density measurements at 750 nm (OD₇₅₀) and relative percentile surplus of Chla(B) content of ethanologenic cultures (tri-angles) and non-ethanologenic cultures (squares) depict with the relative ethanol production surplus of JL2 to WT in dotted lines. The error bars denote standard deviations of duplicates under standard laboratory conditions.

2.3.5 Properties of an ethanol-adapted strain under high salinity conditions

High salt stress experiments with an increasing concentration of sea salts have been conducted (Fig.10). Under expected up-scaled production environment, conditions can change due to evaporation in the direction of higher salinity. Also, the question if acquired tolerance towards one stress (ethanol) can lead to new effects in other stress conditions (high salt stress) was addressed.

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Figure 11: Growth properties (A) and relative Chl a content surplus in percent on day 9 (B) of pre-adapted Synechocystis cultures (JL2) compared to WT cultures. Circles mark WT, squares JL2 cultures. Seawater salts concentrations are depict by solid lines for 1 fold seawater concentration, big dotted lines for 2 fold of seawater concentration, small dotted lines for 2,5 fold of seawater concentration and solid lines with empty symbols for 3 fold of seawater concentration de-notate as 1xSW, 2xSW, 2,5xSW and 3xSW respectively. The error bars dede-notate standard deviations of duplicates under standard laboratory conditions with 40 mL Erlenmeyer flasks.

Although JL2 cultures showed no clear growth advantages in higher salt concentrations, but rather show a salt concentration specific growth change compared to the WT (Fig.11A), the observed phenotype under ethanol stress with its elevated Chl a content cannot only be seen under the former tested condition but is manifesting also with rising salt concentrations (Fig.11B). The experiment also indicated that both used strains are very tolerant to high salt conditions and can at least survive under high salt stress with 2.5 fold seawater salts for 9 days. All in all high salinity experiments are indicating that the observed phenotype of JL2 is seen under different condition and that both stresses bear similarities to each other.

2.4 Impairment of ethanol with the pigment