The Elemental Composition of Synechocystis sp. PCC6803 under Nutrient Limiting Conditions

Bachelor Thesis Scheikunde

The Elemental Composition of Synechocystis sp. PCC6803

under Nutrient Limiting Conditions

door

Marian Blom

19 Juli 2014

Molecular Microbial Physiology Group

Verantwoordelijk docent Prof. Dr. K. J. Hellingwerf Begeleider

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Abstract:

The dependency on limited sources of certain chemicals can be reduced by using phototrophs to produce these or comparable chemicals. The model organism, Synechocystis sp. PCC6803, is a good candidate as phototroph, but before applying this on large scale more research is needed on the nutrient requirements and elemental composition of Synechocystis.

In this study Synechocystis is grown in Erlenmeyers in BG-11 medium with different concentration of phosphate or nitrate to determine the yield on phosphate and nitrate. The cultures grown with lower nitrate and phosphate concentrations were limited on other factors instead of nitrogen or phosphate. Except for Synechocystis grown in BG-11 medium with 0.88mM nitrate, which reached an OD730 ~1 before the end of log phase.

Another experiment was performed in which Synechocystis was grown in a photo-bioreactor in chemostat under light limited conditions in low-NO3-BG-11 medium (0.79mM).

Nitrogen limitation was achieved using 0.32mM NaNO3. Additionally, turbidostat around the

same OD730 was compared with chemostat. The number of photons absorbed per fixed CO2

was determined, which was 16.9photons/CO2 for chemostat under light-limited conditions.

This increased to 26.5photons/CO2, more than twice the amount in nitrogen-limiting

conditions. The mass percentage of carbon and nitrogen of the dryweight were measured. The C/N ratio was relatively high for all experiments and this is probably due to excess of nitrate in regular BG-11 used in literature. The biomass yield of the chemostat with 0.79mM nitrate was about 2.5 times higher than the yield on 0.32mM.

Summary (in Dutch)

Door het opraken van fossiele brandstoffen en andere chemicaliën is er veel aandacht voor duurzamere manieren om deze chemicaliën te verkrijgen. Een voorbeeld hiervan is het gebruiken van fotosynthetische bacteriën om biobrandstof te produceren. Een geschikte bacterie hiervoor is Synechocystis sp. PCC6803, omdat deze bacterie relatief makkelijk te modificeren is. Voordat dit toegepast kan worden op grote schaal moet er nog veel onderzoek gedaan worden naar onder andere de precieze hoeveelheid nitraat en fosfaat dat deze bacterie nodig heeft.

Dit werd gedaan door Synechocystis te laten groeien in BG-11 medium (een oplossing met benodigde stoffen om te groeien) met een lagere concentratie nitraat of fosfaat dan in de standaard BG-11 medium. Uit het experiment bleek dat de bacteriën waarschijnlijk eerder andere voedingsstoffen te kort kwamen voordat ze nitraat of fosfaat gelimiteerd werden, met uitzondering van de laagste concentratie nitraat, 0.88mM.

Ook werd er een experiment gedaan in een photo-bioreactor(PBR), een apparaat waarmee je de groeicondities precies kunt aanpassen. Hierin groeide Synechocystis in medium met minder nitraat. De inhoud van de PBR werd constant verdund door er medium in te pompen, en ondertussen gaat er automatisch eenzelfde hoeveelheid celsuspensie uit de PBR. Op een gegeven moment groeien de cellen net zo snel als ze door verdunning uit de PBR spoelen en zal de hoeveelheid bacteriën (ongeveer) gelijk blijven. Tijdens het experiment werd de licht intensiteit verhoogd waardoor de optische dichtheid ook steeg. Hieruit bleek dat ze light-gelimiteerd waren en niet nitraat-/stikstof-gelimiteerd. Door een medium te gebruiken met 2,5 half keer minder nitraat werden ze wel stikstof-gelimiteerd. Het percentage stikstof in het drooggewicht van bacteriën in beide experimenten werd bepaald en was lager dan in de literatuur, die standaard BG-11-medium gebruiken dat een grote overdosis aan nitraat bevat. Daarnaast werd ook bepaald hoeveel fotonen de bacteriën absorberen per CO2 die deze bacteriën vastleggen. Voor de licht-gelimiteerde bacteriën was

dat minder fotonen in vergelijking met stikstof-gelimiteerde. Daarentegen was de biomass opbrengst ongeveer 2,5 keer hoger bij de licht gelimiteerde bacteriën.

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Introduction

In order to decrease the carbon footprint and the dependency on fossil fuels, a greener and more sustainable production of important chemicals and biofuels is needed. Therefore, a lot of research is done on the use of photoautotrophic cells as “factories” for the production of these chemicals. They are an attractive host because these organisms can use sunlight and CO2

instead of fossil fuels.[1]

An example of such an organism is the Synechocystis sp. PCC6803, a cyanobacterium, which is a model organism and is frequently studied, partly because this photoautotroph can grow under a wide range of conditions.[2-8] For example, Synechocystis can grow heterotrophical. In addition, this is also the first photosynthetic organism of which the entire genome sequence was determined.[9]

Another benefit is that its genome relatively easy to modify and understand compared with that of green algae, making it more suitable for producing certain chemicals efficiently by modifying the genome.

However, before this can be applied on industrial scale, the yields have to improve. Next to improving expression of heterologous pathways, the growth conditions to optimize yield need to be determined for large scale use. The BG11 medium, which is generally used to grow cyanobacteria did not change much since the modification made by Stanier et al. in 1971.[10] This medium might not be optimized for Synechocystis sp. PCC6803. The actual nutrient requirements of Synechocystis sp. PCC6803 need to be specified since an excess of certain nutrients can lead to unnecessary storage compounds in the cells and to metabolic imbalance. To simulate and monitor such a conditions advanced photo-bioreactors(PBR’s) are used.

In this study, the nitrogen and phosphor requirements of Synechocystis sp. PCC6803 will be analyzed by growing them in batch in Erlenmeyers. The elemental composition with respect to carbon and nitrogen will be determined from bacteria grown in a PBR under nitrogen-limiting conditions in chemostat and turbidostat.

Materials and Methods

Erlenmeyers

Synechocystis sp. PCC6803 was grown in 25ml standard and modified BG-11 medium with 5 mM NaHCO3 in a 100ml Erlenmeyer placed in an orbital shaker type 44 (INNOVA) at

120rpm and 30 ºC.[10] The orbital shaker had a custom light panel of red and blue light for the experiments under red and blue light. The experiments were monitored overtime by measuring the OD730 and OD687 using a UV/Visible spectrophotometer (Lightwave II diode

array).

Synechocystis was grown in an incubator with red (636nm) light ~100μE/m2/s in BG-11 medium with nitrate concentrations of 9.6mM, 4.8mM, 2.4mM,1.2mM and 35.3mM. A reference Erlenmeyer was grown in the cool fluorescent white light incubator. In addition, Synechocystis was grown in the BG-11 medium with the standard concentration of 17.6mM nitrate and lower concentrations of 0.88mM, 1.76mM, 3.53mM, 7.06mM and 0mM in an incubator with red (636nm) and blue (445nm, 10% of red) light at ~80μE/m2/s. In order to determine the yield on phosphate Synechocystis was grown in BG-11 medium with phosphate concentrations of 0.023mM, 0.092mM, 0.161mM and 0.23mM in the cool fluorescent white light incubator with light intensities between 22 and 33μE/m2/s.

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Photo-Bioreactor

Preparation: Inoculum

The Photo-Bioreactor was inoculated using a pre-culture of Synechocystis sp. PCC6803 grown in 25ml BG-11 medium in the cool fluorescent white light incubator.

Photo-Bioreactor set up

For this experiment the Photo-Bioreactor (Photon Systems Instruments) with a cultivation vessel of 1L was used to control growth conditions. The PBR is described in Nedbal et al. 2008.[11] Figure 1 shows an abstract representation of the PBR and the use of the different in and outlets and other features. The OD730 and OD697 of the cell suspension were regularly

measured throughout the experiments on a benchtop photospectrometer and the temperature was kept on 30 ºC. The volume of the microbial suspension was 966.13mL.

Figure 1. Photo-bioreactor set up. This figure shows an abstract representation of the PBR. The cuvette that

contains the microbial suspension is closed with a steel lid that accommodates the tubing connectors and the pH and temperature sensor. The use of de different in and outlets for gas and medium are shown. It also shows where optical measurements were carried out and stirring took place.

The aeration through the PBR was 300ml/min with 0.30% CO2 & 99.70% N2 and the dilution

rate was 1.58x10-5/s. The BG-11 medium was used with a nitrate concentration of 0.79mM. The light intensity of the red (636nm) light was 90μE/m2/s and the blue (455nm) light was 15μE/m2/s. Chemostat was assumed when the OD720 had reached a steady state. This was

repeated, but with a higher red light intensity of a 100μE/m2/s. After chemostat was reached, the photosynthetically active radiation (PAR) was measured in photons of 400-700nm waveband in μmol/m2/s using a quantum sensor (LI-COR). This was measured over the length of the cuvette, 6.1cm, opposite to the LED lights. To determine the absorption of the cell suspension the PAR of BG-11 medium was used as blanc. Afterwards, samples were taken to determine the dryweight and its composition.

Another experiment was carried out with BG-11 medium with a concentration of 0.32mM nitrate. In this experiment the dilution rate was 1.68x10-5/s and the aeration was 300ml/min with 0.50% CO2 & 99.50% N2. The intensities of the red and blue light were

100μE/m2/s and 15μE/m2/s. Once chemostat was reached the PAR was measured and samples were taken. The experiment was repeated under the exact same conditions only in turbidostat with thresholds of ±2%.

5 Dryweight determination and composition

The dryweight was determined by drying 40ml samples over filters and measuring the weight. For the determination of the elemental composition, two volumes of 50ml were centrifuged (20min, 4250rpm, 4 ºC) after which the supernatant was discarded. The two samples were combined and washed 3x with MQ H2O. They were cooled in liquid nitrogen and stored at

-20ºC or directly put on the freeze dryer. After freeze drying for at least 24 hours the amount of nitrogen and carbon as a percentage of dry weight was determined. This was measured by using a vario el cube (Elementar), in which the samples are combusted and the released gasses are analyzed using purge & trap gas chromatography.

Glycogen Assay

Samples of 2ml were centrifuged (14000rpm, 4ºC, 5min) and the pellets were put in the -20 ºC freezer for at least 24hours. Then 200μl KOH 5.35M was added and they were hydrolyzed for 90min at 95ºC in a thermo shaker and afterwards 600μl ethanol was added. Then it was cooled on ice for 2hours and centrifuged (140000rpm, 4ºC, 5min) and washed 2x with ethanol. Afterwards 300μl acetate buffer (200mM, pH5.2) and 50μl amyloglucosidase (10mM) in acetate buffer were added and the samples were incubated overnight under constant agitation at 55ºC. Then the glucose concentration was determined using the D-fructose/D-glucose assay kit (megazyme). This assay was carried out according to the manufactures instructions on a 10times smaller scale in 96well plate using 50μl of supernatant.

Results and Discussion

Batch

The Synechosystis grown at different nitrate concentrations in the red incubator turned yellowish green after a short time. This color was different in comparison with the green control group in the white incubator. This was probably due the difference in light in the two incubators. Therefore, the experiment was repeated with a lower light intensity and with 10% blue light. The results of the Synechosystis grown in the red and blue incubator are shown in graph 1.

Graph 1. Growth of Synechocystis in BG-11 medium with nitrate concentrations of 0.88mM, 1.76mM, 3.53mM,

0,03125 0,0625 0,125 0,25 0,5 1 2 4 8 0 31 60 91 121 152 182 213 244 274 305 335 O D 730 time in hours 0.88mM 0.88mM 1.76mM 1.76mM 3.53mM 3.53mM 7.06mM 7.06mM 0mM 17.6mM

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7.06mM, 17.6mM and 0mM in ~80uE/m2/s red(636nm) + 10% blue(445nm) light. The OD730 is shown over

time for the different concentrations on a log scale.

Culture grown at 0.88mM shows a log phase which ends after ~50 hours with an OD730 of ~1,

as seen in graph 1. This end implies that the bacteria stopped growing exponentially because they were nitrogen limited. To get the exact values more measurements are needed around the end of the log phase. The bacteria grown with higher concentrations of nitrate stopped growing exponentially around the same time and OD730. This was in contradiction with the

expectation that the bacteria would stop growing exponentially at different OD730, due to the

different amount of nitrate available in the media. This suggests that the bacteria were limited on another factor, such as carbon dioxide. Noticeable is that the Synechocystis without any nitrogen added to the medium still showed some growth. This might be, because the Synechocystis still contains nitrate itself at the start of the experiment and some nitrate might be brought over from the medium of the original source of the bacteria.

Figure 2. Synechocystis grown in Erlenmeyers in in ~80uE/m2/s red(636nm) + 10% blue(445nm) light are shown in two pictures. The first picture shows the erlenmeyers at T= 74 hours and with BG-11 medium containing nitrate concentrations of (starting from the left) 17.6mM, 7.06mM, 3.53mM, 0.88mM and 1.76mM. The second picture shows the erlenmeyers at T= 188 hours and with medium containing nitrate concentrations of (starting from the left) 17.6mM, 7.06mM, 3.53mM, 1.76mM and 0.88mM.

Another observation was the change of color of the microbial suspension over time. After 74 hours the Erlenmeyers with a nitrate concentration of 0.88mM and 1.76mM had a yellow color, instead of the green color in the other Erlenmeyers. The Erlenmeyer containing 3.53mM was still green after 74 hours, but after 188 hours it was yellow as seen in Figure 2. Thus, this change in color is related to the lack of nitrogen. Since chlorophyll is responsible for the green color, the results suggest that the cells might have a lower amount of chlorophyll per cell if they are nitrogen limited. This could be, because chlorophyll contains nitrogen.[12] The amount of chlorophyll per cell can be calculated to see if it corresponds with the previous suggestion. The OD730 and OD687 are needed, because at 730nm the scatter of the cells is

measured which relates to the number of cells and the chlorophyll peak is at 687nm which relates to the amount of chlorophyll. The formula that was used is (OD687-OD730

)*10.186-0.08 and this value was divided by the OD730 to get a value that relates with the amount of

chlorophyll per cell. This formula is based on a linear relation between the difference in OD687-OD730 and measured chlorophyll concentration in and acetone extraction. In graph 2

the results of these calculations are shown and in the appendix are the OD687 measurements

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Graph 2. The OD730 andOD687 of Synechocystis grown in batch was used to give an indication of the amount of

chlorophyll per cell using the formula, ((OD687-OD730)*10.186-0.08)/OD730. The values from the formula are

shown over time in the graph for Synechocystis grown in BG-11 medium with nitrate concentrations of 0.88mM, 1.76mM, 3.53mM, 7.06mM, 17.6mM or 0mM in ~80uE/m2/s red(636nm) + 10% blue(445nm) light.

The results are not in line with the expectation of a decrease over time which is larger at lower concentrations. Thus, this formula might not apply under the circumstances of this experiment. (The negative value below 0.3 of the red line is probably caused by some mistake made, while measuring the OD. For example an air bubble in the sample could influence the measurement.)

The reason for the yellowish color is probably partly a result of the low nitrate concentration. The high light intensities could also play a part in the color change. This corresponds with the observation shown in figure 3. This figure shows that the Synechocystis grown in regular BG-11 medium in the white light incubator is green while the culture grown in the red blue incubator appears to be more yellowish green.

Figure 3. Synechocystis grown in regular BG-11 medium: the two Erlenmeyers on the left in ~80uE/m2/s red(636nm) + 10% blue(445nm) light for 17days and the two Erlenmeyers on the right grown with light intensities between 22 and 33μE/m2/s in the white light incubator for 30days. The Erlenmeyer marked with a red cross was contaminated.

Graph 3 shows the results of Synechocystis grown in BG-11 medium with different phosphate concentrations in the white light incubator. The Synechocystis grown in medium with the lowest concentration (0.023mM) phosphate ended the log phase around the same point as

0,3 0,5 0,7 0,9 1,1 1,3 1,5 1,7 1,9 2,1 23 54 82 113 143 174 204 235 266 296 327 (( O D687 -O D730)* 10.186 -0.08) /O D730 time in hours 0.88mM 0.88mM 1.76mM 1.76mM 3.53mM 3.53mM 7.06mM 7.06mM 0mM 17.6mM

8 Synechocystis grown with other concentrations. This suggests that phosphate wasn’t the limiting factor that stopped the culture from exponentially growing. Noticeable is that at the last measurements the OD730 value is in order of increasing phosphate concentration with one

exception. This exception is the Synechocystis grown in medium containing 0.092mM phosphate that reaches an OD730 of 11.05 and this could be because of a pipetting error.

Graph 3. Growth of Synechocystis in BG-11 medium with phosphate concentrations of 0.023mM, 0.161mM,

0.092mM and 0.23mM. Grown with light intensities between 22 and 33μE/m2/s in white light incubator. The OD730 is shown over time for the different concentrations on a log scale.

In this experiment the culture grown in Erlenmeyers with the lowest concentration of phosphate (0.023mM) turned yellowish as shown in figure 4. This color change seemed less than the color change of the Synechocystis grown in the red and blue incubator with different nitrate concentrations.

Figure 4. Synechocystis grown in Erlenmeyers in medium containing phosphate concentrations of (starting from

the left) 0.23mM, 0.161mM, 0.092mM and 0.023mM. The Erlenmeyer marked with a red cross was contaminated.

Photo-Bioreactor

The culture grown in the PBR reactor with a nitrate concentration of 0.79mM reached a stable OD730 around 0.67. Synechocystis grown in the same nitrate concentration with a light

intensity of a 100μE/m2/s instead of 90μE/m2/s reached a stable OD730 of 0.822 ± 0.003.

Thus, the culture was light limited instead of nitrogen limited.

0,015625 0,03125 0,0625 0,125 0,25 0,5 1 2 4 8 16 0 31 60 91 121 152 182 213 244 274 305 335 366 397 425 456 O D 730 time in hours 0.023mM 0.023mM 0.092mM 0.092mM 0.161mM 0.161mM 0.230mM 0.230mM

9 The chemostat with a nitrate concentration of 0.32 was nitrogen limited, this was based on the lower OD730 of 0.337±0.006 in comparison with the previous chemostat with

almost the same conditions except for nitrate concentration. In table 1 the OD730 and OD687

are shown and the results of chlorophyll calculations as explained before. Out of this calculation it seems that the turbidostat had the lowest amount chlorophyll per cell, but since the applicability of this formula is questionable and the large deviation no conclusions could be made.

OD730 OD687 ((OD687-OD730)*10.186-0.08)/OD730

① 0.822 ± 0.003 1.029 ± 0.033 2.47 ± 0.41 ② 0.337 ± 0.006 0.415 ± 0.007 2.11 ± 0.49 ③ 0.338 ± 0.003 0.411 ± 0.003 1.96 ± 0.03

Table 1. OD730 and OD687 for Synechocystis ① in chemostat and light limited with nitrate concentration of

0.79mM, ②in chemostat nitrogen limited with a nitrate concentration of 0.32mM, ③ in turbidostat with nitrate concentration of 0.32mM

The dryweight (g/ml) was determined and the results are shown in table I, II and III of the appendix. With these results and the results of the PAR measurements the amount of photons per fixed CO2 could be calculated. The results for the PAR measurements were 89.92 photons

μmol/(s*m2) absorbed for the chemostat with a nitrate concentration of 0.79mM and 58.34 for the chemostat with a nitrate concentration of 0.32mM.

The formula below was used to calculate amount of photons per fixed CO2 for the

chemostats with light intensities of a 100μE/m2/s and nitrate concentrations of 0.79mM and 0.32mM. It was expected that the amount of absorbed photons per fixed CO2 of the light

limited chemostat (0.79mM nitrate) would be lower than that of the nitrogen limited chemostat (0.32mM nitrate). The absorbed photons per fixed CO2 was calculated using the

formula shown below.

The data needed for this calculation includes the dryweight (g/ml) shown in table I, II and III of the appendix. The results were 16.9 for the light limited and 26.5 photons per fixed CO2 for

the nitrogen limited.

The mass percentage of nitrogen and carbon of the dryweight was also determined and is shown in table 2. C[ % ] N[ % ] C/N[ m/m ] ① 44.28 ± 0.40 7.14 ± 0.16 6.22 ± 0.31 ② 45.17 ± 0.72 7.54 ± 0.13 5.99 ± 0.03 ③ 44.56 ± 0.19 7.69 ± 0.02 5.80 ± 0.03 R 49.8 12.5 3.98

Table 2. The mass percentage of carbon and nitrogen of Synechocystis ① in chemostat and light limited with

nitrate concentration of 0.79mM, ②in chemostat nitrogen limited with a nitrate concentration of 0.32mM, ③ in turbidostat with nitrate concentration of 0.32mM and the reference by Kim et al. 2010.[8]

The results of the glycogen assay and calculations are shown in the table below and are based on data shown in table IV and graph I of the appendix. The results are in glucose per gram dryweight, since the glycogen was broken down to glucose and then the amount of glucose was determined. Thus, these results also include the glucose that was present in the cells. The chemostat and turbidostat have concentrations that come close to culture grown in BG-11 with 5mM Glucose by Miao et al.[13] In another article it is stated that cultures grown

10 in medium with 5mM glucose contain four times more glycogen per gram wet weight than cells grown in normal BG-11 without 5mM glucose.[14] This could possibly indicate that the cells grown in the PBR did storage extra glycogen.

Glucose gram/ gram dry weight ② 0.192 ± 0.024

③ 0.168 ± 0.043

R Glucogen: 0.185 ± 0.001 (BG11 + 5mM glucose)

Table 3. The glucose (gram) per gram dryweight after glycogen is broken down to glucose of Synechocystis

grown in, ② hemostat nitrogen limited with a nitrate concentration of 0.32mM, ③ in turbidostat with nitrate concentration of 0.32mM and the glycogen (gram) per gram dryweight of the reference by Miao et al. 2003.[13]

Conclusion

Except for the Synechocystis grown with a nitrate concentration of 0.88mM, the cultures grown in batch were limited on other factors. The experiments could be repeated with lower concentrations of nitrate or phosphate in order to ensure limitation on phosphor or nitrogen. In addition, the OD has to be measured more frequently to get a more precise value for the point at which the exponential growth ends and the bacteria are limited. CO2 was suggested as

the limiting factor instead of nitrogen in the experiment. To confirm this is extra NaHCO3

could be added and this should be compared with the previous results. As explained is the formula used to give an indication of the amount of chlorophyll per cell not the most ideal method under these circumstances. For future experiments, another option could be extracting the chlorophyll from the cell and counting the cells to obtain the chlorophyll/cell.

The cultures grown in the PBR under light-limiting conditions absorbed less photons per fixed CO2 than the culture grown in chemostat under nitrate-limiting conditions. This was

expected, since bacteria are usually the most efficient with the limiting factor.

The elemental composition of Synechocystis in literature differs from the composition of Synechocystis grown in a PBR with medium with lower concentrations of nitrate. The C/N ratio was significantly higher in all the experiments done in this thesis compared to literature.[8] _{This could be due the excess of nitrogen that could be stored in the cell when}

Synechocystis was grown under nitrate sufficient conditions as done in literature.[8]

The biomass yield in dryweight (mg/ml) of the nitrogen limited culture in chemostat in BG-11 medium with a nitrate concentration of 0.32mM was 0.0570±0.0055 mg/ml. For the light limited culture grown in medium with a nitrate concentration of 0.79mM a yield of 0.1489 ± 0.0055 was reached. The yield on 0.76mM is about 2.5times that of culture grown in BG-11 with 0.32mM nitrate. This is in line with the expectation of a linear relation between the concentration nitrate and the yield, but the second chemostat was light limited and might have produced more biomass if more light was available. Thus, before concluding a linear relation between biomass and nitrate concentration, more yields of nitrogen limited cultures grown in chemostat at different concentrations need to be obtained.

Additionally, a control is needed with the regular concentration nitrate of BG-11 medium in turbidostat with an OD730 around 0.337. The results of this experiment are needed

to confirm the suggestion that extra storage of glycogen took place based on the glycogen assay. In addition, this experiment would be a good reference for the elemental composition. This experiment can also be used to compare to the growth rate with the culture grown in turbidostat with 0.32mM nitrate in order to say something about the efficiency.

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References

[1] Angermayr, S. A.; Paszota, M.; Hellinwerf, K. J. Engineering a cyanobacterial cell factory for production of lactic acid. Appl environ microbial, 2012, 78(19), 7098-7106 [2] Kanesaki, Y.; Suzuki, I.; Allakhverdiev, S.I.; Mikami, K.; Murata, N.; Salt stress and

hyperosmotic stress regulate the expression of different sets of genes in Synechocystis sp PCC 6803. Biochem Biophys Res Commun, 2000, 290(1), 339–348

[3] Kempf, B.; Bremer, E.; Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments. Arch Microbiol , 1998, 170(5), 319–330 [4] Mikami, K.; Kanesaki, Y.; Suzuki, I.; Murata, N. The histidine kinase Hik33 perceives

osmotic stress and cold stress in Synechocystis sp PCC 6803. Mol Microbiol,2002, 46(4), 905–915

[5] Ogawa, T.; Kaplan, A. Inorganic carbon acquisition systems in cyanobacteria. Photosynthesis Res, 2003, 77(2–3), 105–115

[6] Ohkawa, H.; Price, G.D.; Badger, M.R.; Ogawa, T. Mutation of ndh genes leads to inhibition of CO2 uptake rather than HCOmath image uptake in Synechocystis sp strain PCC 6803. J Bacteriol, 2000, 182(9), 2591–2596

[7] Suzuki, I.; Kanesaki, Y.; Mikami, K.; Kanehisa, M.; Murata, N. Cold-regulated genes under control of the cold sensor Hik33 in Synechocystis. Mol Microbiol, 2001, 40(1), 235–244.

[8] Kim, H. W.; Raveerderm V.; Zhou, C.; Harto, C.; Rittmann, B.E. Photoautotrophic nutrient utilization and limitation during semi-continuous growth of Synechocystis sp. PCC6803 Biotechnology & bioengineering, 2010, 106(4) 553-563

[9] Kaneko, T.; Sato, S.; Kotani, H.; Tanaka, A.; Asamizu, E.; Nakamura, Y.; Miyajima, N.; Hirosawa, M.; Sugiura, M.; Sasamoto, S.; Kimura, T.; Hosouchi, T.; Matsuno, A.; Muraki, A.; Nakazaki, N.; Naruo, K.; Okumura, S.; Shimpo, S.; Takeuchi1, C.; Wada1, T.; Watanabe, A.; Yamada, M.; Yasuda, M.; Tabata1, S. Sequence Analysis of the Genome of the Unicellular Cyanobacterium Synechocystis sp. Strain PCC6803. II. Sequence Determination of the Entire Genome and Assignment of Potential Protein-coding Regions. DNA research, 1996, 109-136

[10] Stanier, R.Y.; Kunisawa, M.M.; Cohen-Bazir G. Purification and properties of unicellular blue-green algae (order Chlorococcales). Bacteriol Rev, 1971, 35, 171–201 [11] Nedbal, L.; Trtilek, M.; Cerveny, J.; Komarek, O.; Pakrasi, H.B.; A photobioreactor

system for precision cultivation of photoautotrophic microorganisms and for high-content analysis of suspension dynamics. Biotechnology & Bioengineering, 2008, 100(1), 902-909

[12] Fleming, I. Absolute Configuration and the Structure of Chlorophyll. Nature, 1967, 216 (5111), 151–152

[13] Miao, X.; Wu, Q.; Wu, G.; Zhao, N. Sucrose accumulation in salt-stressed cells of agp gene deletion-mutant in cyanobacterium Synechocystis sp. PCC 6803. Microbiology letters, 2003, 218(1), 71-77

[14] Yoo, S.; Catherine, K.; Spalding, M.; Jane, J. Effects of growth condition on the structure of glycogen produced in cyanobacterium Synechocystis sp. PCC6803. int. J. Biological Macromolecules, 2007, 40, 498-504

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Appendix

Filter Weight (gram) before filtration Average

I 0.0715 0.0714 0.07145

II 0.0716 0.0714 0.0715

III 0.0714 0.0717 0.07155

Control 0.0712 0.0712 0.0712

Filter Weight (gram) after filtration Average

I 0.0772 0.0775 0.07735

II 0.0778 0.0778 0.0778

III 0.0775 0.0775 0.0775

Control 0.0713 0.0713 0.0713

Filter

Average dryweight (gram) (before - after

minus “weight”of the control) of 40mL sample

I 0.00580

II 0.00620

III 0.00585

Avarge of I, II & III,

minus the control 0.00595 ± 0.000218 In mg/ml (minus control) 0.1489 ± 0.0055

Table I. Dryweight measurements of culture grown in chemostat in BG-11medium with 0.79mM nitrate.

Sample Weight (gram) before filtration Average

I 0.0719 0.072 0.0723 0.0722 0.0721 II 0.0717 0.0717 0.0719 0.0719 0.0718 III 0.0714 0.0715 0.0718 0.0719 0.07165 Control 0.0718 0.0718 0.0722 0.0723 0.072025

Sample Weight (gram) after filtration Average

I 0.0741 0.0746 0.07435

II 0.074 0.0743 0.07415

III 0.0739 0.0743 0.0741

Control 0.072 0.0722 0.0721

Sample

Average dryweight(gram) (before - after minus

“weight”of the control) of 40mL sample

I 0.00218

II 0.00228

III 0.00238

Avarge of I, II & III,

minus the control 0.00228 ± 0.000218

In mg/ml 0.0570 ± 0.0055

13

Filter Weight (gram) before filtration Average

I 0.0712 0.0716 0.0714

II 0.0715 0.0719 0.0717

III 0.0712 0.0713 0.07125

Control 0.0717 0.0718 0.07175

Filter Weight (gram) after filtration Average

I 0.0744 0.074 0.0742

II 0.0746 0.0742 0.0744

III 0.0724 0.0738 0.0731

Control 0.0717 0.0712 0.07145

Filter

Average dryweight (gram) (before - after minus

“weight” of the control) of 40mL sample

I 0.00310

II 0.00300

III 0.00215

Avarge of I, II & III,

minus the control 0.00275 ± 0.000522

In mg/ml 0.0688 ± 0.013

Table III. Dryweight measurements of culture grown in turbidostat in BG-11medium with 0.31mM nitrate.

Graph I. Results of the Glycogen assay. In this graph the calibration curve is shown and the absorption of

culture grown in BG-11medium with 0.31mM nitrate in chemostat (orange dot), and turbidostat, (green dot).

Chemostat Average Deviation

∆A 340nm 0.23572 0.29529 0.30636 0.22815 0.27513 0.3048 0.274242 0.034692 mg glucose/ml 0.0555105 0.069551 0.07216 0.053726 0.064799 0.071792 0.064590 0.008171

Turbidostat Average Deviation

∆A 340nm 0.27804 0.36561 0.20498 0.24626 0.3667 0.21925 0.28014 0.071143 mg glucose/ml 0.0654851 0.086125 0.048265 0.057995 0.086382 0.051629 0.06598 0.016756

Table IV. Results of the Glycogen assay. In this table the ∆A 340nm for culture grown in BG-11medium with

0.31mM nitrate in chemostat and turbidostat are shown. In addition, the concentration glucose for these absorption values according the calibration curve are shown.

y = 4,2428x + 0,0002 R² = 0,9997 -0,1 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 0,0000 0,0500 0,1000 0,1500 0,2000 0,2500 ∆ A 340nm

14

Graph II. Growth of Synechocystis in BG-11 medium with nitrate concentrations of 0.88mM, 1.76mM,

3.53mM, 7.06mM, 17.6mM and 0mM in ~80uE/m2/s red(636nm) + 10% blue(445nm) light. The OD687 is

shown over time for the different concentrations on a log scale. 0,01 0,1 1 10 0 31 60 91 121 152 182 213 244 274 305 335 O D 687 time in hours

N-limited growth OD687

0.88mM 1.764mM 1.764mM 3.528mM 3.528mM 7.056mM 7.056mM 0mM 17.64mM