‘The influence of algal growth phases and its density effects on the photosynthetic rate in Chlorella, Synechocystis and Synechococcus under LED lighting’

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‘The influence of algal growth phases and its

density effects on the photosynthetic rate in

Chlorella, Synechocystis and Synechococcus under

LED lighting’

Bachelor Thesis by Andes Vreeken Student number: 10150463 Supervisors: Dr. J.M. Schuurmans &

Dr. J.C.P. Matthijs July 3rd, 2015

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Abstract

The present day depletion of fossil fuels is leading to a lot of research effort to find renewable energy sources. With the ability to genetically engineer cyanobacteria such as Synechocystis and Synechococcus to produce a variety of products, these new sources might have been found. Now that new possibilities have been presented, it is of utmost importance that a maximum efficiency in both prodcut formation as well as biomass yield on light is established so that they are able to compete with fossil fuels.

Increasing the efficiency can be done in several ways, such as lighting optimization or photosystem adaptation. However, the effects of population density on photosynthetic rate have never really been studied with this application in mind. Therefore, this research will focus on the influence of algal growth phases and the population density on the photosynthetic rate in three species commonly used in the biotechnology; Synechocystis PCC6803, Synechococcus PCC7942 and Chlorella sorokiniana.

This will be investigated by measuring the oxygen production in samples of the three species. Several different densities and light inputs will be tested. The measurements will be done with LED lights, as they are an energy efficient light source with high future potential. The aim of this study is to contribute to existing culturing programs in order to keep the product formation efficiency as high as possible.

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1. Introduction

Global problems such as fossil fuel depletion, greenhouse gas emissions, ecosystem destruction and water pollution have been hot topics for the past decades. Every year several conferences are held to set up rules how we can slow down this rapid degradation of this planet. Most of the time global agreements fail to receive the majority of votes. In a rare occasion such as the Kyoto Protocol (1997), global agreements are made and deadlines are set. Unfortunately the deadlines often get pushed further into the future, because the countries do not adapt fast enough to achieve the set agreements. Ofcourse innovations have been made in a lot of sectors, but it takes time to get these innovations distributed over the whole world. However, one of these innovations has gained increased interest and is thought to become very influential; the genetic modification of cyanobacteria, which are more commonly known as algae. These Genetically Modified Organisms (GMO’s) have had their DNA modified so that they produce certain products such as ethanol and isoprene (Deng & Coleman, 1999, Lindberg et al., 2010). The use of these genetically modified algae is therefore not only limited to biofuels, but can also be used in other sectors that require high-value products. Additionally, if algae become the main source of biofuel production, feedstocks will no longer be needed for fermentation to create ethanol. This will in its turn reduce the pressure on ecosystems, and will also have a positive effect on the prices of the food market, as competition is reduced (Parmar er al. 2011). Lastly, when growing algae becomes more common, effects on the atmosphere will become visible as algae fix carbon from carbondioxide. For these reasons, genetically modifying and growing algae has gained increased interest over the past years, as it seems to be an interesting renawable energy and prodcut source. However, a lot of adaptations and improvements can still be made to this technique, as culturing algae can be managed in different ways.

1.1 Lighting

One of the adaptations has been made in the field of lighting. Instead of lighting the algae with normal white light, research has shown that algal cultures can be grown under monochromatic LED-light, which decreases the lighting costs significantly and opens windows to special lighting programs (Matthijs et al., 1996, Schuurmans et al., Submitted). Previous research by Matthijs et al. (1996), Vejrazka et al. (2013) and Schuurmans et al. (Submitted), has shown that further increasing the efficiency is possible by giving the cyanobacteria flashing light from the red emission spectrum. Flashing light has recently been tested on cyanobacteria, as a part of the light reaction (left side of figure 2) can be performed in the dark. Because LED-lights do not consume energy when they are turned off, flashing LED-lights are an energy efficient way of presenting light to cyanobacteria. This is a field of research that has gained increased interest, and promising results (such as an even higher growth efficiency than with continuous light) have already been achieved (Schuurmans et al. Submitted).

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1.2 Excess energy

Another important aspect with respect to the culturing of cyanobacteria, is the leaking away of excess energy, which is called energy dissipation. This can be explained as follows; when cyanobacteria grow in sunlight, the amount of light they receive fluctuates. Because photosynthetic receptors can become damaged due to the variation of light intensity, defense mechanisms have evolved in these cyanobacteria. When more light comes into the photosynthetic area than the system can handle, photons are dissipated and released as heat by flavor di-iron proteins and Organge Carotenoid Proteins (OCP) (Bersanini et al. 2014, Kirilovsky, 2007). In this way, the cyanobacteria are not using all the energy that is presented tothem.

This seems in contrast with studies in the late nineties, such as Qiang & Richmond (1994), Qiang & Richmond (1996) and Hu et al. (1998). They suggest that a combination of the highest lighting conditions and a high population density is preferable, as it would result in a maximum biomass production. However, present research on algal culturing disagrees with this statement. As stressed by Liu et al. (2015), the costs of biomass production should be kept as low as possible. Only then biofuels and other products synethesized by algae will be able to compete and replace fossil fuel products in the global system. When taking this and the energy dissipation into account, presenting continuous high lighting conditions to algae as Qiang & Richmond (1994,1996) and Hu et al. (1998) suggest, does not particularly seem the most energy efficient way of light management. Therefore, research to increase the photosynthetic efficiency of algae is crucial.

Figure 1: An overview of photosynthesis in a cell. The energy of the incoming light is transfered to ATP and NADPH and produces oxygen. In the Calivn cycle, the ATP and NADPH is used to reduce CO2 to organic compounds (Mihael Fofic,

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1.3 Population density

A final, but key point in the culturing of cyanobacteria is the population density of the algae. In the studies of Qiang & Richmond (1994,1996) and Hu et al. (1998), population density was a condition that should be kept optimal in relation with the light input. Yet, with the idea of energy efficiency in mind, this condition can also be approached in another way. The studies of Vejrazka et al. (2013), Schuurmans et al. (Submitted) and Bersanini et al. (2014) have shown that new optimum values of light input and decreases in energy dissipation are possible for different species of cyanobacteria. Though, new optimum population density values that correspond with the new optimum light values have yet to be found.

Han et al. (2015) suggest that different culture conditions might be needed for the different algal growth phases, because populations become denser as the population grows. High density populations that are not exposed to enough light, may cause reduced efficiency which is not desirable (Li et al., 2008). If a good combination of density- and light-management can be found, photosynthesis efficiency could possibly be kept at a higher rate for longer periods of time. Therefore, this study will investigate what the influence of algal growth phase and its related density properties is on the photosynthetic rate of Chlorella sorokiniana,

Synechocystis spp. PCC 6803 and Synechococcus spp. PCC 7942, combined with different

intensities of light. If algal growth phase and its related density properties influences the photosynthetic rate significantly, then adjustments to the methods of culturing can be made to be able to keep a high photosynthetic rate. These two strains of cyanobacteria and one strain of green alga are commonly used in the biotechnological sector, and will therefore serve as the main focusing species of this research.

Algae harvest light for photosynthesis with the use of antenna complexes. It is expected that the amount of antenna produced by the photo harvesting complex is density dependant, and that dense populations therefore contain algae with high amounts of antenna. For this reason, it is hypothesized that diluting a population from a late algal growth phase to an early growth phase, will result in a high photosynthetic rate. Though, exposing such a dense and light sensitive (due to the high amounts of antenna produces) population to a lot of light over longer periods of time, might damage the photosystem. This problem is known, but still in need of more research.

This study will however focus on the analysis of the photosynthetic rate of the three species by measuring the oxygen production of a sample. This is due to the fact that oxygen is a reactant of photosynthesis (see figure 1 above), and it is therefore commonly accepted that when an organism is producing relatively much oxygen, the photosynthetic rate of that organism is high at that particular moment. The oxygen production of the three species will be measured under continuous LED light.

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2. Materials and methods

2.1 Bacterial and algal strains

In this research, Synechocystis spp. PCC 6803, Synechoccus spp. PCC 7942 and Chlorella

sorokiniana are used. They are grown as a batch culture in a stove at 30°C on a BG-11

mineral medium which is supplemented with 5 mM of Na2CO3. The plate in the stove rotates at 100 rounds per minute and the algae are presented continuous fluorescent white light with an intensity of 50 µmoles∙m-2∙s-1. The first batch of the three species (hereafter refered to as “species” batch 1) was made by grafting the species in a sterile 2L shaking flask at an OD750 value of 0,05. A week later, the second batch of the three species (hereafter refered to as “species” batch 2) was made in the same way. With a week of growth difference between the two batches, the OD750 values from the different batches would vary enough from each other to be able to dilute the OD750 of one batch to the OD750 value of another and see interesting results.

2.2 LED lighting

Two LED lights will be used for this study, which will be placed on two sides of the study setup. Both these lights are supplied by © Philips Lighting NV Eindhoven, and are seperately tunable with software supplied by the University of Amsterdam (UvA). The same tuning settings as the research of Schuurmans et al. (Submitted) were used. These settings consist of: duty cycle DC (which means the fraction of time of the total cyclus time that is spent in the light) and intensity (0 to 580 µmoles∙m-2∙s-1). Lighting during the oxygen experiments was chosen to be with deep red monochromatic (659nm) light.

The exact amount of light that the LED lights emit at each timestep, is determined by a timetable that can be read by the LED lighting software. This timetable is made by measuring the amount of light each one of the LED’s exactly emit (in µmoles∙m-2∙s-1) when a certain intensity is asked from the LED’s. After adding these two values, a graph is made of which the trendline formula is then calculated. This formula is in the y = ax + b form, of which the x value for both the LED’s then can be calculated. When these values are filled in for both the LED’s, together the LED’s now emit the exact light intensity that is required for the tests. These values are then converted into LED lighting software compatible format.

2.3 Oxygen measurements

The amount of oxygen that is produced by the species in question will be measured equal to the oxygen measurements of Schuurmans et al. (Submitted). Samples will be placed in small full glass transparent double walled vessels with an internal sample chamber of 3,5 ml (UvA, Amsterdam). Three vessels will be used simultaneously, which will be illuminated by LED lamps on both sides of the vessels to minimize shading effects. The measurements will be recorded using Firesting Optodes (Pyroscience, Germany), which are calibrated by flushing them with N2 gas to set the 0% value for dissolved oxygen in water and by air to set the 100% value for dissolved oxygen in water. Before the recording is started, the percantage of oxygen in the three samples is brought back to below 20% by flushing them with N2 gas. The oxygen production during illumination will be recorded for three minutes for each condition.

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2.4 Cell counts

The collected samples will be tested for cell amount and cell biovolume using the CASY 1 TTC cell counter with a 60 µm capillary (Schärfe Systems GmbH, Reutlingen, Germany). Samples were diluted 1111 times in Casyton before counting, with 3 replicates per sample. condition. Before counting, a blank count with only Casyton was done and substracted from the total count of the sample.

2.5 Absorption spectra

The optical density (OD) at 680 nm and 750 nm were measured every day with the Pharmacia MkII Photospectrometer (Pharmacia Biotech, Sweden). The measured values were used to create a growth curve for each of the species. Secondly, the OD750 values were used to determine in which algal growth phase the species was situated at that moment (see table 1 below). Thirdly, the values were used to make the desired dilution samples with help of the following formula: 𝑂𝐷1 × 𝑉1 = 𝑂𝐷2 × 𝑉2. In this formula the OD1 (optical density) and V1 (volume) values presented the values before diluting the sample and the OD2 and V2 values the desired dilution values.

2.6 Chlorophyll content

Chlorophyll a was extracted by using 90% acetone and adding DMSO to the sample and letting it rest for 10 minutes at 4°C. For Chlorella sorokiniana chlorophyll a as well as chlorophyll b content had to be extracted. This was done by adding 90% methanol to the sample, subsequently bead beating it for one minute and then letting it rest for 10 minutes in a waterbath of 75°C. This procedure was then repeated again, except for the bead beating, which was only done for twenty seconds during the second procedure. Afterwards, the supernatant of all the species samples was measured at 664nm in the photospectrometer. Chlorophyll a content was then calculated by the following formula: [Chla] (mg/L) = 12.76*[A664] (Porra et al., 1989).

2.7 Slopes and maximum values

To be able to study the results in a different way than merely analyzing the graphical aspect of the PI-curves, version 3.2 of the statistical software ‘R’ was used (Hornik, 2015). The calculated oxygen values from the PI-curves were loaded into the program. By using the following formula: 𝑃 = 𝑃𝑚 × 𝑡𝑎𝑛ℎ(𝑎 × _𝑝𝑚𝐼 ), the hyperbolic tangent is fitted for the values that were loaded in. This fit gives the value of Pmax (pm), which describes the maximum value of oxygen production the sample could theoretically reach. The Pmax value thus shows the potential that the specific sample has. The fit also gives the value of a, which is a value for the slope of the PI-curve and tells us what the affinity for light for this sample is. These two values van also be presented graphically and shed a different light on the findings.

Growth phase: OD750 value:

Early growth 0.30 - 0.50

Mid growth 0.50 - 0.80

Late growth 0.80 - 1.00

Early stationary 1.00 - 1.30

Late stationary 1.30 - 1.50

Table 1: Algal growth phases and their corresponding OD750 values. This table is used for the dilution experiments, as it enables a simple classification and easier references.

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0 0.2 0.4 0.6 0.8 1 1.2 0 4 5 6 7 10 14 OD 750 Time (days) 2d 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 4 7 10 11 12 13 14 17 21 OD 750 Time (days) 2e 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 4 5 6 7 10 14 OD 750 Time (days) 2f

3. Results

3.1 OD750

The OD750 values of the three species were measured every day after the populations were grafted. Measurements from batch 1 were taken from the 22th of May (t=0 is at 22th of May) untill June 5th. After June 5th, only Synechocystis PCC 6803 from batch 1 was measured for its OD750 value, as this species was used for the dilution experiments. Measurements from batch 2 were taken from the 29th of May (t=0 at 29th of May) untill June 12th. This was done to see how much the batches had grown in the past day. If the OD750 value of batch 1 had reached another growth phase (see table 1), dilution experiments were done on that day. Additionally, if the OD750 values of batch 2 had increased 1,5 times or more, a photo-irradiance (PI) curve of the species was made. The graphs of the OD750 values of both batches for the three species can be seen in figure 2 below.

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 4 7 10 11 12 13 14 OD 750 Time (days) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 4 5 6 7 10 14 OD 750 Time (days) 2b 0 0.2 0.4 0.6 0.8 1 1.2 0 4 7 10 11 12 13 14 OD 750 Time (days) 2c 2a

Figure 2: The OD750 values over time of Chlorella sorokiniana of batch 1 (2a) and batch 2 (2b), Synechococcus PCC7942 batch 1 (2c) and batch 2 (2d) and Synechocystis PCC6803 batch 1 (2e) and batch 2 (2f).

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Unfortunately, in none of the six figures the OD750 values have formed the typical growth curve for algae, also known as the ’S-Curve’. These figures however do give a proper overview of all the OD750 values that have been measured during the experiment. Additionally, it can serve as a graphic overview to see from which part of the growth curve dilutions have been made and if the algae where in a high or low growth phase at that day.

3.2 CASY counts

CASY counts were done when a PI-curve of the sample was made. As mentioned before, a PI-curve of a sample from batch 2 was made when the OD750 value had increased 1,5 times or more, to avoid results that do not differ significantly from one another. Unfortunately, the basis on which CASY counts were done was not regularly enough to gain enough measurements. Besides that, on the second day of measurements the CASY counter seems to have given results that were not in line with the other three measurements. After extrapolating a value for the second day from the other CASY results, a more logical value was found. However, a meaningfull figure such as the figures from the OD750 measurements could regrettably not be made, due to a lack of values.

3.3 Chlorophyll extraction

If the conditions for PI-curve measurements and cell counting were met, a chlorophyll extraction from the sample was also done. For this reason, the amount of data for meaningfull chlorophyll figures was also not sufficient. Nonetheless, the chlorophyll extractions were still essential for this research, as the value after measuring the chlorophyll sample for optical density was used for calculating the actual chlorophyll content in the dilution experiments. This results in a PI-curve of the sample that is corrected for the dilution that the sample received.

3.4 Oxygen measurements without dilution

When combining all the PI-curves of different OD750 values of one species, clear differences between the different densities were not found. This is in contrast with what was expected; after a population is grafted and the lag phase is over, a population is often found to grow exponentially in the early growth phase. In the mid and late growth phase, the population does not grow exponentially anymore, but the population still increases in number. Due to the fact that for a high growth rate a high photosynethetic rate is needed, it was expected that oxygen production would be high in with low densities and decreasing when the population density increases.

The acquired PI-curves were also used for the calculation of Pmax and alpha values as described in the Materials and Methods section. Combining all the Pmax values resulted in figure 3 below. The same was done for all the alpha values, which resulted in figure 4 below. On the x-axis the measurement numbers represent the data on which a sample was taken, with 1,2 .. 6 being respectively 22nd, 29th of May, 2nd, 4th, 8th and 12th of June. Not all Pmax values could be calculated due to problems with fitting the formula on the graph. This was probably caused by the long series of low values of some PI-curves. The large standard error bars were caused by the same problem, as some values had te be deleted to be able to make the fit. Unfotunately, this resulted in larger standard errors.

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-20 -10 0 10 20 30 40 4 7 4 6 10 14 Pm ax Time (days) 0 0.05 0.1 0.15 0.2 0.25 0.3 4 7 4 6 10 14 Pm ax Time (days)

It was expected that high Pmax values would occur in the late stages of the growth curve, because the photo harvesting complexes of the algae are expected to have a lot of antenna to catch the little light that is presented to them. However, figure 3 shows that only

Synechocystis PCC6803 follows this expected pattern. Even when the standard errors are

taken into account, Chlorella sorokiniana and Synechococcus PCC7942 do not follow the expected pattern. Reasons for the cause of these fluctuations in oxygen production have unfortunately remained unknown during this research.

Figure 3: The Pmax values for all three species combined. The columns of Chlorella sorokiniana are coloured blue, those of Synechococcus PCC7942 are coloured red and the colour of the columns of Synechocystis PCC6803 is green. The first two clusters of columns are Pmax values from batch 1, the other four clusters are Pmax values of batch 2.

Figure 4: The alpha values for all three species combined. The columns of Chlorella sorokiniana are coloured blue, those of Synechococcus PCC7942 are coloured red and the colour of the columns of Synechocystis PCC6803 is green. The first two clusters of columns are alpha values from batch 1, the other four clusters are alpha values of batch 2.

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-15 -10 -5 0 5 10 15 20 25 30 0 50 100 150 200 O 2 ( µ mo le s·mi n -1·mg [C h lA ] -1)

Light intensity (µmoles·m-2_·s-1₎

For the alpha values, a same pattern was expected as with the Pmax values. So with

increasing population density, the slopes of the PI-curves would increase in steepness. This seems like a logical expectation, as the amount and intensity of light that can be absorbed by the algae, decreases as the population becomes denser. Unfortunately, the same fluctuations occured in the alpha values and as mentioned before, the cause has remained unknown. Again

Synechocystis PCC6803 was the only species that showed somewhat of the pattern that was

expected. The increase is over time is present, but not convincingly. Because Synechocystis PCC6803 shows the least fluctuations over time, this species was chosen to serve as the main species for the dilution experiments.

For this subsection of this paper it is difficult to make well founded conclusions, however a small amount of data of Synechocystis confirm the expected results a little; potential (Pmax) and slope (alpha) increase in late growth phases. Based on this conclusion, the dilution experiments were done when batch 1 Synechocystis PCC6803 was in the late growth and early stationary phase.

3.5 Oxygen measurements after dilution

It was chosen to dilute Synechocystis PCC 6803 from batch 1 back to three different algal growth phases (table 1), to see what effects the density properties have on the oxygen production of this species. Six dilutions were made and measured for their oxygen production; twice a dilution to the early growth phase was made, twice a dilution to the mid growth phase and twice a dilution to the late growth phase. After analyzing the PI-curves (figure 5, 6 and 7) to see what growth phase dilution resulted in high oxygen production, the Pmax and alpha values were calculated and combined to form the figure 8 and 9.

Figure 5: PI-curves of the dilutions to OD750 0.3055 (early growth phase). The red squares form the PI curve of the dilution from OD750 0.89 to 0.3055. The green triangles show the PI-curve of the dilution from OD750 1.035 to 0.3055. The blue diamonds represent the normal PI-curve of the sample with OD750 0.3055.

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-10 -5 0 5 10 15 0 50 100 150 200 O 2 ( µ mo le s·mi n -1·mg [C h lA ] -1)

Light intensity (µmoles·m-2_·s-1₎

-15 -10 -5 0 5 10 15 0 50 100 150 200 O 2 ( µ mo le s·mi n -1·mg [C h lA ] -1)

Light intensity (µmol·m-2_·s-1₎

The three PI-curves that are presented above, are the PI-curves of the diluted samples and the original samples combined in one figure. Each figure shows the results of the diluted and original samples of one specific growth phase. It can be seen that in general, most of the diluted samples perform better than the original samples. As far as can be concluded from these combined PI-curves, both samples that were diluted to an OD750 value of 0.3055 (early growth), and one sample that was diluted to an OD750 of 0.65 showed the most promising results. They show the greatest difference in oxygen production.

Figure 6: PI-curves of the dilutions to OD750 values 0.58 and 0.65 (mid growth phase). The red squares form the PI curve of the dilution from OD750 0.81 to 0.58, while the green triangles show the normal PI-curve of the sample with OD750 0.58. The purple crosses show the PI-curve of the other dilution that was measured; from OD750 0.975 to 0.65. The blue diamonds represent the normal PI-curve of the sample with OD750 0.65.

Figure 7: PI-curves of the dilutions to OD750 value 0.87 (late growth phase). The red squares form the PI curve of the dilution from OD750 1.25 to 0.87. The green triangles represent the PI-curve of the dilution from OD750 1.26 to 0.87. The normal PI-curve of the sample with OD750 0.87 is shown by the blue diamonds.

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-15 -10 -5 0 5 10 15 20 25 30 35 40 Pm ax Dilution

Graphically inspecting the three figures above can already lead to some conclusions, but they would be more interesting and trustworthy if these conclusions were supported in another way. For this reason, the Pmax and alpha values for these PI-curves were calculated as explained in the Materials and Methods section. The Pmax values of the diluted and original samples were combined in figure 8 below.

The first and fourth measurements of the original Pmax values have very big standard errors due to a difficult PI-curve that was hard to fit. Nonetheless, three dilutions show a significant difference between the original and diluted potential value; early stationary phase diluted to late growth phase, early stationary diluted phase to early growth phase and late growth phase diluted to mid growth phase. Yet, diluting from the early stationary phase to the early growth phase shows the biggest difference in potential values. It is important to note that these values can only be reached theoretically, as they are the product of a hyperbolic tangent, of which the end point lies in the infinite. Looking at the alpha values (slopes of the PI-curves) can show which diluted sample has the most affinity for light and will therefore grow fastest under low light intensities.

Figure 8: Pmax values of the PI-curves of diluted Synechocystis PCC6803 samples. The red columns show the Pmax values that the original sample to which OD750 value was diluted had. The blue columns show the Pmax values from the samples that where diluted from a specific growth phase. For the x-axis, the first number indicates from which growth phase was diluted, and the number behind the arrow indicates to which growth phase was diluted.

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0 0.05 0.1 0.15 0.2 0.25 0.3 A lp h a Dilution

Simularly to the figure with Pmax values, the figure for alpha values above also shows three cases in which the difference between the original alpha value and the diluted alpha value is significant. Both the dilutions to the early growth stage show the biggest alpha values and thus have the most affinity for light, which will cause a high growth rate at the low light intensities.

In the end, from these dilution experiments it can be concluded that diluting a sample from late growth or early stationary phase to an earlier growth phase will result in higher oxygen production and thus photosynthetic rate. However, the growth rate that is diluted to is a very important factor, and different levels of significance are seen. The dilutions to the different growth phases will now be assessed taking all three (Oxygen production, Pmax and alpha)

figures in consideration.

Diluting from the early stationary to late growth phase does show a significant difference in one of the Pmax values, but does not differ significantly from the original values for alpha values and has a minimal difference in oxygen production. This dilution does not offer the highest result that is possible, and is therefore not the best method for increasing the photosynthetic rate.

Diluting from the late growth to mid growth phase resulted in two fairly different results. Allthough there was a difference in the OD750 value that was diluted from as well as the value it was diluted too (0.975 to 0.65 and 0.81 to 0.58), both samples occurred in the same growth phases according to table 1. This could indicate a wrong classification of the growth phase table, but it could also be daily variation in oxygen production. It is hard to point out a specific cause for the different values that were found for these two samples, though repeating the experiment could show which one of the two samples was acting different. Whilst one of the samples showed a significant higher pmax, alpha and oxygen values, the results were not of the highest significancy that was seen during this study. Diluting samples from the late

Figure 9: Alpha values of the PI-curves of diluted Synechocystis PCC6803 samples. The red columns show the alpha values that the original sample to which OD750 value was diluted had. The blue columns show the alpha values from the samples that where diluted from a specific growth phase. For the x-axis, the first number indicates from which growth phase was diluted, and the number behind the arrow indicates to which growth phase was diluted.

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growth to mid growth phase therefore does not offer the best method for increasing the

photosynthetic rate.

Diluting from the early stationary and late growth phase to the early growth phase resulted in high Pmax and alpha values. The oxygen production also showed a clear difference between the diluted and the original values, but the dilution from the early stationary phase excelled the most. Although the standard errors in the Pmax figure are very big, again the Pmax value of the dilution from the early stationary growth phase excells and is big enough to overcome this error and differ significantly from the original Pmax value. In figure 9, both dilution values are a tenfold or more bigger than the original values, which indicates a high affinity for light. The dilution from the early stationary phase shows the highest affinity for light though. In conclusion, both the dilutions from the late growth and early stationary phase performed better in every way, except for the Pmax value of the late growth phase dilution. However, the early stationary phase dilution gained the highest values in all three analyzed sectors, and therefore offers the best method for increasing the photosynethetic rate of Synechocystis PCC6803.

4. Conclusion

The research question of this study was the following; ‘What is the influence of algal growth

phase and its related density properties on the photosynthetic rate of Chlorella sorokiniana, Synechococcus PCC7942 and Synechocystis PCC6803?’

After the interpretation of the Pmax, alpha and oxygen production figures, it can be concluded that the algal growth phase influences the photosynthetic rate of Synechocystis PCC6803the most if a sample is diluted from the late stationary phase to the early growth phase. This can only be concluded for Synechocystis PCC6803 as this was the only species that dilution experiments were done with. Besides this, it can also be concluded that when following the growth curve, the potential oxygen production (pmax) and affinity for light (alpha) of

Synechocystis PCC6803 increase with an increasing population density. Unfortunately this

can again only be concluded for Synechocystis PCC6803, as the Pmax and alpha values of the other values fluctuated too much to be able to draw properly founded conclusions.

Based on the findings of this research, it is advised to dilute a Synechocystis PCC6803 population from a late stationary phase (OD750 1.00 – 1.30) to an early growth phase (OD750 0.30 – 0.50) if a high photosynthetic rate needs to be ensured.

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5. Discussion

The results of this study were promosing; significant higher slopes and potential values were seen for three out of the six experiments. However, the experiment in which the sample was diluted from OD750 1.035 (early stationary phase) to OD750 0.3055 (early growth phase) was by far the best. The sample showed a very high affinity for light for the lower light intensities, but also a very high potential oxygen production. As it was already suggested in the introduction of this study, this effect is probably caused by the high amount of antenna that were produced by the photo harvesting complex. Producing a lot of antenna is a reaction of the photo harvesting complex to the low availability of light; it is crucial for the algae that the very small amounts of light the reach the lower situated cells are harvested. So by having a lot of antenna, the chance is bigger that the cell can pick up some photons. When these cells are diluted and suddenly placed in a very light environment, all these antenna harvest photons which causes the photosynthetic rate and therefore oxygen production to increase dramatically due to the large amounts of energy that are available. This is an effect that this study was hoping to find; having a high photosynthetic rate while presenting the algae low intensities of light. Though, in this study only one experiment with a dilution from the early stationary phase to the early growth phase was done. As the algae in this study sometimes had a doubtly low oxygen production on one day, and a very high oxygen production the on other day, this might have caused a bias in the results. Therefore it is unfortunately not possible to draw and strong and well founded conclusions from these findings, as the values could also be lower when tested again, which could result in other dilutions being more effective. Nonetheless, the fact that the slopes and Pmax values were higher in three out of six dilutions can not be denied. Future research on this subject can aim for even higher Pmax and alpha values, whilst taking into account that due to the fluctuating nature of algae (as found in this research) experiments in dulpo or triplo are highly preferred.

Another important aspect is that the experiments in this study only had a duration of half an hour. When the algae still have a lot of antenna active due to the former low light conditions, diluting them to very light conditions causes large amounts of photons to be harvested by the algae. At first this might result in a high photosynthetic rate, but less thought has been paid to the long term effects. It is not farfetched to think that the photo harvesting complex can only handle such high amounts of light for a short period of time, as it adjusted itself in this way because the lighting conditions were low. When the light intensities become high again, the photo harvesting complex is assumed to adjust itself to these new conditions. However, if it keeps getting flooded with light for longer periods of time when it is still in its ‘low-light’ stage, damaging of the system is not unthinkable. A possible aim for future research could therefore be to study what the long term effects of diluting and lighting samples is. This would result in knowledge about how long the algae can handle the high amounts of light or how fast they adjust there photosystem and what the consequences of a damaged photosystem are. There is however also another way of approaching this problem; with the knowledge gained by this study, is it possible to trick the photo harvesting system to keep the high amounts of antenna while keeping the photosynthetic rate as high as this study has found? In conclusion, this study has only revealed a small part of the possibilities that are possible with algal cultures. The research that has been proposed in this discussion are merely suggestions, but it does show how interesting and understudied this research topic is.

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6. Acknowledgdements

I would like to thank Merijn Schuurmans in particular for advising and helping me on a daily basis during my thesis research and experiments. Additionally, I would like to thank Hans Matthijs for being my second supervisor and the master students Sanne Ypenburg, Joris Solleveld and Bregje Brinkmann for helping and advising me during my lab work.

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