Chemostat measurements: Growth and competition experiments

To determine the effects of conductivity on the growth rate of P. salinarum, experiments were carried out in a continuous algal culturing system (chemostat) described by Shafik et al. (2001) (Figure 7). The experiment was carried out at 29±1°C, a Thermo Scientific AC150-A25 circulating bath was responsible to keep the distilled water at the specific temperature in the aquarium and also indirectly in the culturing vessel. Light intensity of 200 µmol m^-2 s^-1 with

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12:12 light:dark cycle was provided by daylight tubes of Tungsram (F74). Light intensity was measured before the experiments in distilled water. The aquarium was illuminated from one side and the wall of the other three part of the aquarium was covered by mirrors to provide homogenous illumination. During the experiment the light intensity inside the culturing vessel probably decreased according to the self-shading of P. salinarum cells. The cultures were aerated with sterilized air (obtained by passing air through a Millipore membrane with 0.2 µm pore size). The air supply was not only responsible for the supply of CO2 but also for the continuous mixing of the culture.

Subsamples from the main culture at an exponential phase were transferred into the culturing vessels. The cultures were first grown in the initial medium (M0) before carrying out tests on the performance of the species under different salt concentrations. Fourteen different culture media were used (Appendix 6) to test the effect of the different concentrations of chloride (NaCl) and carbonate forms (both Na2CO3 and NaHCO3) on the growth of P.

salinarum. Shifts between media of different concentrations were done when the cultures reached steady state, therefore, sample numbers from different media slightly differed. Effects of increasing the concentration of chloride and carbonate forms were examined separately in two different culture vessels with an approximate volume of 1000 mL. The growth medium was continuously added with a Masterflex L/S Variable-Speed Drive, at a flow rate of 160±15 mL d^-1. The flow rate was determined based on pilot studies, which were carried out both on P.

salinarum and L. fusiformis. The chosen experimental conditions were found to be optimal for the photosynthesis of P. salinarum in the present study and are similar to conditions in the species’ natural habitat.

Changes in biomass were monitored by measuring the optical density of the samples at 750 nm with spectrophotometer (Metertech SP-8001) and/or by checking the samples under microscopy if it was necessary. Samples were taken three times a week. Growth rate (𝜇) were calculated using the formula of Shafik et al. (1997) as previously described by Novick and Szilard (1950) and Monod (1978):

𝜇 =𝑙𝑛(𝐴₁− 𝐴₀) 𝑡₁− 𝑡₀ + 𝐷,

Where A1 is the absorbance of the culture at time t1, A0 is the absorbance of the culture at time t0 and D is the dilution rate.

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Figure 7 Graphical illustration of the continuous culturing system (chemostat), after Shafik et al. (2001)

Competition experiment

Growth and competition of the two species were examined in the above described chemostat (Figure 7). Same experimental setup was used in the case of the mixed culture as previously described in the case of P. salinarum. The only difference was the flow rate, which was increased to 285±18 mL d^-1. Higher flow rate was applied because of the huge differences between the growth rate of L. fusiformis and P. salinarum (in pilot studies L. fusiformis was found to grow very fast even under ~500 mL d^-1 dilution rate but and P. salinarum disappeared from the cultivating vessel at such high rates).

In the competition experiment, the mixture of the above described monoalgal cultures was applied. The initial biomass concentration ratio in the mixed culture of the two species was 90:10 L. fusiformis: P. salinarum µg chlorophyll a L^-1 in a cultivating chamber with an approximate volume of 1 L. Samples were taken three times a week to estimate population sizes by counting individual number according to Utermöhl (1958).

To examine the effect of the rapidly changing environment (increase and decrease of conductivity) on the species’ growth and co-occurrence, the concentration of NaHCO3 was increased to 60 g L^-1 and Na2CO3 to 16 g L^-1 in M0 medium. Mixed cultures were grown for the first 30 days in M0 medium, then for 30 days in the medium with increased conductivity, and after that for further 30 days in M0 medium again. The changes were monitored in the concentration of carbonate forms by measuring the conductivity at each sampling time.

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The growth of both species was separated into sections according to the experimental setup and growth rates were calculated for each section separately. The growth curve of L.

fusiformis and P. salinarum was divided into three phases: phase I was the initial growth in the initial M0 medium, in phase II the level of conductivity was increased and in phase III biomass was recorded after the return to the initial medium. Growth rates were calculated according to the formula provided by Sprouffske and Wagner (2016) with the addition of dilution, since continuous culture was used:

𝑁_𝑡= 𝐾

1 + (𝐾 − 𝑁₀ 𝑁₀ ) 𝑒^−𝑟𝑡

+ 𝐷,

where Nt is the number of cells at time t, N0 gives the population size at the beginning, K is the maximum possible population size in a particular environment, or the carrying capacity, r is the growth rate if there were no restrictions imposed on total population size and D is the dilution rate.

5.2.4 Statistical analysis

To determine whether temperature and/or light intensity treatments had a statistically significant effect on the photosynthetic activity of L. fusiformis and P. salinarum, two-way analysis of variance (ANOVA) was conducted.

Because of the unequal variances and sample sizes, Welch's t-test was applied to determine whether the difference in average growth rates under carbonate dominated and chloride dominated media were significant. Spearman rank correlation coefficient was used to assess the relationship between growth rate and conductivity in carbonate and chloride dominated media. In case of both main media types, a one-way analysis of variance (ANOVA) was performed to test whether the modification of carbonate and chloride content (indicated by the different medium subtypes, Appendix 6) affects significantly the growth rates of P.

salinarum. Subsequently, Tukey’s post hoc multiple comparison tests were conducted between each pair of carbonate and chloride dominated media.

To determine whether temperature and/or light intensity treatments had a statistically significant effect on the photosynthetic activity of L. fusiformis and P. salinarum, a two-way analysis of variance (ANOVA) was carried out. Statistical analyses were carried out using R statistical computing environment (R Core Team 2018).

59 5.3 Results