‘Study of the possible mixotrophic
characteristics of Phaeocystis globosa’
By
Nina van Haastert, 12107913
28 May 2021, Amsterdam
Primary supervisors: Dr. Susanne Wilken,
Secondary supervisor: Dhr. Sebastiaan Koppelle,
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Scientific summary
A phenomenon termed mixotrophy describes that phytoplankton is not exclusively autotrophic, but that it can use photosynthesis as well as phagotrophy for nutrition during its life. Multiple studies found indications for mixotrophic characteristics in Phaeocystis globosa (P.globosa). Presumably, mixotrophy enhances the biological carbon pump which has consequences for current marine and climate models. Besides, mixotrophy might be an explanation for the success of the yearly P.globosa bloom, which has multiple negative consequences for fisheries and tourism. Investigating the possible mixotrophic characteristics of P.globosa is necessary to take appropriate measurements to combat climate change and to decrease the negative consequences for fisheries and tourism.
In this research it is investigated if P.globosa belongs to the mixotrophs using resource limiting conditions as a trigger. Vitamin B1, vitamin B12, nitrogen and phosphorous limiting treatments were created. When the growth rate of P.globosa was affected due to the limiting resource, the bacterial prey C.marina was added to the treatments. A flow cytometer was used to make an indication of P.globosa cells that had ingested C.marina.
The results suggest that in both resource limiting and resource rich conditions ingestion of C.marina takes place. The percentage of P.globosa that presumably ingest C.marina is low in all conditions. Therefore, the possible mixotrophic characteristic is expected to have little or no effect on scientific models and on the success of the P.globosa blooms. These results do not support the need to consider mixotrophy for policy plans to combat
change and to decrease the negative consequences for fisheries and tourism.
Images from confocal microscopy demonstrate that ingestion of C.marina by P.globosa cannot be proven. Therefore, it cannot be concluded yet that P.globosa belongs to the mixotrophs. However, strong indications for phagotrophy are observed. Further research is necessary to draw a conclusion about the possible mixotrophic characteristics of P.globosa.
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Table of content
1. Introduction………...4
2. Theoretical framework………...5
3. Methods & Materials……….6
3.1 Organisms and culture conditions………..6
3.2 Flow cytometer………...6
3.3 Tests………...6
3.3.1 Starvation test………...6
3.3.2 CellBrite Fix Membrane Stain test………7
3.3.3 Dilution test………..7 3.4 Experiments………...8 3.4.1 Preparations………..8 3.4.2 Measurements………..9 4. Results………...10 4.1 Tests……….10 4.1.1 Starvation test……….10
4.1.2 CellBrite Fix Membrane Stain test……….10
4.1.3 Dilution test………11
4.2 Onset of resource limitation……….12
4.2.1 Experiment 1………..12
4.2.2 Experiment 2………..13
4.3 Does resource limitation trigger phagocytosis in P.globosa? ………14
5. Discussion………16
5.1 Resource limitation………..16
5.1.1 Resource limitation in PgG(A) ………...16
5.1.2 Resource limitation in Pgimf………..16
5.1.3 Resource limitation compared………...16
5.2 Mixotrophy triggered by resource availability………16
5.3 Effect of mixotrophy………...18
5.3.1 Fishery and tourisms………..18
5.3.2 Biological carbon pump ………18
6. Conclusion………...19
7. References………...20
8. Acknowledgement………...23
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1 Introduction
Carbon occurs in various forms throughout its pathway on Earth (Post et al., 1990). An important part of the carbon cycle is the biological carbon pump, which absorbs atmospheric carbon dioxide (CO2) and stores it in the deep ocean (Cavan, Henson, Belcher & Sanders, 2017; Basu & Mackey, 2018). The pump is driven by autotrophic phytoplankton, which form the base of the marine food web. This web illustrates who eats who and describes how a community functions (Pimm, Lawton & Cohen, 1991). The autotrophic phytoplankton uses CO2 and sunlight to convert inorganic nutrients into organic nutrients for nutrition, a process called photosynthesis (Cavan et al., 2017; Townsend, 2012). Eventually, particles or dead organisms containing these nutrients will sink and will be stored in the deep ocean (Hülse, Arndt, Wilson, Munhoven & Ridgwell, 2017). Another place in the food web is taken by heterotrophic zooplankton. Some of them use phagotrophy for nutrition, which is a process whereby organisms ingest living prey (Wilken et al., 2019; Jones, 2000; Burkholder, Glibert & Skelton, 2008; Mitra et al., 2016).
A phenomenon termed mixotrophy describes that phytoplankton is not exclusively autotrophic, but that they can use photosynthesis as well as phagotrophy for nutrition during their life (Burkholder et al., 2008; Stoecker, Hansen, Caron & Mitra, 2017; Wilken et al., 2019; Ward & Follows, 2016; Jones, 2000; Flynn et al., 2013). This mixotrophy obscures the traditional distribution between autotrophs and heterotrophs (Wilken et al., 2019; Ward & Follows, 2016; Mitra et al., 2016). In this traditional distribution, energy is lost by metabolism processes during the energy transfer from autotrophs to heterotrophs (Townsend, 2012). Predictions state that the mixotrophic characteristics reduce this loss of energy resulting in a higher trophic level transfer efficiency and thus a higher biomass throughout the entire food web. This, together with the assumption that mixotrophy causes the biomass in the lower trophic levels to decrease due to hunting of autotrophs, is expected to cause an increase in global organisms size. It is predicted that this results in more sinking carbon and therefore an enhancement of the biological carbon pump (Caron, 2016; Ward & Follows, 2016).
Research demonstrates that mixotrophy is more common in phytoplankton than previously thought (Wilken et al., 2019, Ward & Follows 2016; Mitra et al., 2016; Mitra et al., 2014). Omitting mixotrophs from food web models by scientists produces unrealistic models that do not reflect nature as it functions in real life (Flynn et al., 2013). This leads to an increased chance of misunderstanding marine and climate systems. It is important to discover which species are mixotrophs to determine the effect on the biological carbon pump (Caron, 2016; Wilken et al., 2019). In this way, scientific systems will be more accurately represented, which is important for implementation of policy plans to combat climate change.
Phaeocystis is a well-studied global distributed marine haptophytes (Schoemann, Becquevort, Stefels, Rousseau & Lancelot, 2003.; De Rijcke, 2017; Mars Brisbin & Mitarai, 2019). One of the six Phaeocystis species, Phaeocystis globosa (P.globosa), has many negative consequences for fisheries and tourism (Schoemann et al., 2003; Riegman, Noordeloos & Cadée, 1992; Philippart et al., 2020; Verity et al., 2007). For example, P.globosa causes fish mortality, a decrease in growth and reproduction of fish, it gives off an unpleasant smell and it can cause foam along the coast, which results in dangerous conditions for water sports enthusiast (Schoemann et al., 2003; Peperzak, Colijn, Gieskes & Peeters, 1998; Philippart, 2020). Although P.globosa is traditionally characterized as phytoplankton, there are indications that the species has mixotrophic characteristics (Verity et al., 2007; Davidson & Marchant, 1992). Based on previous fieldwork and a genomic model, it is predicted that the PgG(A) strain of P.globosa from the North Sea is able to ingest bacteria (Koppelle, personal communication, 2021). This mixotrophic characteristic might be an explanation for the success of P.globosa blooms.
Investigating the possible mixotrophic characteristics of P.globosa is necessary to take appropriate measurements to combat climate change and to decrease the negative consequences for fisheries and tourism. Therefore, the research question is as follows:
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2 Theoretical framework
The success of P.globosa blooms
P.globosa has a polymorphic lifecycle: it has an unicellular and colonial stage throughout its life (Hamm, 2000; Mars Brisbin & Mitarai, 2019; Philippart et al., 2020; Wang, Wang & Smith, 2011). In the colonial stage a bloom is often formed, creating a matrix of polysaccharides with thousands of cells (Schoemann et al., 2003; Hamm, 2000; Mars Brisbin & Mitarai, 2019; Philippart et al., 2020). In the North Sea, P.globosa blooms yearly around April, when high light intensity and low nutrient concentrations favor the production of this matrix (Lancelot et al., 2005; Prins, Desmit & Baretta-Bekker, 2012; Philippart et al., 2020). The matrix ensures that the species is less susceptible to prey. Besides, it stores energy and nutrients that can be used afterwards, which makes the matrix a possible explanation for the high competitiveness of colonial P.globosa (Prins et al., 2012).
According to Peperzak et al. (1998), P.globosa does not require silica for growth. In contrast to diatoms, which consume nitrogen (N), phosphorous (P) and silica during their spring bloom just before the P.globosa bloom occurs. Due to this heavy uptake of nutrients by diatoms, silica eventually becomes limiting. This causes the diatoms to be outcompeted by P.globosa.
Also, the P concentration eventually becomes limited due to P uptake by diatoms. In this P limiting condition, P.globosa appears to be a good competitor as well (Prins et al., 2012). One explanation for this is phosphatic activity. During this process enzymes convert organic phosphate into inorganic phosphate, which P.globosa can use for growth (Prins et al., 2012; Lancelot et al., 2005; Veldhuis & Admiraal, 1987). Although this could explain the growth under P limiting conditions, Wang et al. (2011) demonstrate that a high concentration of P compared to silica is necessary for the success of P.globosa (Lancelot et al., 2007; Wang et al., 2011).
A high N concentration would also ensure the success of P.globosa (Lancelot et al., 2007; Wang et al., 2011). Overall, P.globosa appears to be a poor competitor for N and a surplus of N is necessary for the success of this Phaeocystis species (Gypens, Lacroix & Lancelot, 2007).
Research demonstrates that, besides nutrients, P.globosa needs vitamins for growth (Tang, Koch & Gobler, 2010). Spencer (1981) states that P.globosa is auxotrophic for both B1and B12. This means that the species cannot produce these vitamins itself and have to gain them from external sources in order to grow. Environmental conditions triggering phagotrophy
Limiting resource availability could be an advantage for mixotroph over autotrophic specialists (Li, Edwards, Schvarcz, Selph & Steward, 2021). By changing nutrition from photosynthesis to phagotrophy, mixotrophs can obtain nutrients and vitamins by ingesting prey, whereas autotrophs do not have this possibility (Wilken et al., 2019; Ward & Follows, 2016; Nagata, 2000). Besides, in nutrient limiting conditions, the highest concentration of nutrients is located in microbial prey, while the concentration in the water is significantly lower (Burkholder et al., 2008). Therefore, it is assumed that limiting resource conditions triggers phagotrophy in mixotrophs.
In recent decades, research on phagocytosis triggered by limiting resource availability mainly focused on nutrients such as P and N (Arenovski, Lim, & Caron, 1995; Carvalho & Granéli, 2010; Nygaard & Tobiesen, 1993). Research on limiting vitamins as a trigger is lacking or unusable. However, given that P.globosa require vitamins for growth, vitamin limiting conditions are expected to be a trigger (Sanudo-Wilhelmy, Gobler, Okbamichael & Taylor, 2006; Peperzak et al., 2000).
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3 Methods & Materials
This chapter describes the necessary background information and the description of the experiments. For reproduction of the experiments, a ‘Method & Materials extension’ has been created, in which the tests and experiments are explained step-by-step (Appendix I).
This research investigated if P.globosa has mixotrophic characteristics by conducting two experiments. The first experiment investigated the onset of resource limitation for various resource limiting conditions. The second experiment explored if this resource limitation triggered phagocytosis. This was done by adding bacterial prey to resource limiting treatments when the growth rate of P.globosa was affected.
3.1 Organisms and culture conditions
Strains of P.globosa were provided by NIOZ Texel and transferred twice a week in a 1:5 ratio in L1 medium (Appendix II). In experiment 1, the PgG(A) strain was used. The culture was incubated in a 12h light:12h dark cycle at 15°C and a light intensity of 70 umol photons m-2 s-1. In experiment 2, the Pgimf strain was used and stored in the incubator in which the light cycle was changed to 16h light:8h dark. Light cycle and strain switching was due to the inexplicable premature death of the PgG(A) strain after experiment 1.
As bacterial prey, strains of Dokdonia and Cobetia marina (C.marina) were used. They were provided and grown by the University of Amsterdam in Difco2216 medium (Appendix IV). The cultures were incubated at 23° C and with shaking 110 RPM min-1.
3.2 Flow cytometry
The Accuri C6 Flow Cytometer (FCM) counted the cells. This was done by a laser, which emitted blue light at a wavelength of 488 nm. The scattered light and fluorescence emitted from the cells is detected at different wavelength by using a 530/30 filter for channel FL1 and a 670 LP filter for channel FL3. P.globosa was identified by the fluorescence emission of chlorophyll a. The species was visible as a cluster with relatively high FL3-H values. A FL3-H threshold value was set on 80 000. The bacteria were stained with CellBrite Fix Membrane Stain (CellBrite). They were visible as a cluster with relatively high FL1-H values. A FL1-H threshold value was set on 10 000.
The cell count was used to calculate the growth rate of P.globosa in a specific condition between between two measurements. The following formula was used, in which Tn is the cell count on day number n, Tn-1 the cell
count the day before and T the amount of days of the time interval: ln(𝑇𝑛) − ln(𝑇𝑛−1)
𝑇
A One-Way ANOVA and TukeyHSD test were conducted to investigate in which limiting condition P.globosa had growth rates significantly different from the control treatment. A p-value of 0.05 was used as threshold value. When P.globosa has ingested bacteria, the P.globosa cells are expected to have both high FL3-H and FL1-H values.
3.3 Tests
Before the experiments could be conducted, a number of tests were performed. 3.3.1 Starvation test
The cell size of Dokdonia and C.marina had been attempted to decrease by starvation. This was done in order to have a small enough cell size for ingestion by P.globosa. First, the bacteria were grown for two days, as described above, to reach high enough concentrations. After, the Difco2216 medium was removed by centrifuging twice and resuspending in autoclaved Atlantic seawater. Next, the washed pellet was dissolved in 50 ml Atlantic seawater and stored in a 4° C fridge for starvation.
To investigate the shape and size after starvation, the bacteria were examined on the fluorescence microscope. Samples from both bacteria before and after nine days starvation were fixed with glycerol. After, the samples were diluted with Milli Q to a concentration of 106 cells ml-1. These dilutions were filtered with a 0.2µm
pore filter. Next, the filters with the bacteria were added to microscopy slides and stained with the fluorescent DNA stain DAPI. The slides were stored in a -20°C freezer. After one day, the slides were viewed under the fluorescence microscope.
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3.3.2 CellBrite stain test
The CellBrite stain can be added to living cells. This is beneficial since, heat-killed bacteria do not trigger feeding in some algae strains (Bock et al., 2021).
The test investigated whether the stain works on bacterial cells and which incubation time ensures a maximum signal from the stain. For this, the staining protocol was used (Appendix V). A sample was taken from Dokdonia and C.marina cultures. These samples were centrifuged, after which the pellet was resuspended in Phosphate-buffered saline (PBS). The sample was split into two samples. One 0.5 ml sample was fixed with 10 microliters glycerol. This sample was shaken and stored in a 4°C fridge for 30 minutes. After, a 10 times dilution was created using 2μ filtered TE buffer. 5 microliter of 200 times diluted Sybr Green 1 was added to this dilution. After the incubation time of 30 minutes, the dilution series was completed. The different dilutions with Sybr Green 1 stained bacteria were quantified on the FCM. Based on this results, the dilution necessary to reach a desired concentration of 1.0 * 109 cells ml-1 was discovered. Subsequently, the still living bacteria were stained with
CellBrite and diluted 104 times to achieve this 1.0 * 109 cells ml-1. After 15, 30, 60 minutes and three days, the
FL1- H signal from the CellBrite stained bacteria were measured on the FCM. 3.3.3 Dilution test
After experiment 1, PgG(A) did not grow properly and even died prematurely. This test was conducted to investigate whether over-diluting could be an explanation for this prematurely dead. First, the density of PgG(A) culture was determined by measuring samples of the mother culture. Based on this value, a 2, 10, 100 and 567 times dilution was created using the L1 medium (Table 1).
Table 1.The different dilution factors and the starting concentration of P.globosa in cells ml-1. The dilution were created using the L1 medium.
The starting concentration is based on three replicates, which is indicated with the error range.
Dilution Starting concentration P.globosa (cells ml-1)
2x 2.43*106 ± 5.29*104
10x 5.07*105 ± 8.08*103
100x 5.73*104 ± 1.22*104
567x 8.13*103 ± 1.52 *103
Daily, 150 microliters samples of the dilutions were measured on the FCM. Based on the cell count, the growth rate was calculated as described above. The experiment was finished when the growth rate of the dilutions were below zero (Figure 1).
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3.4 Experiments
3.4.1 Preparations Treatments
A B1 limiting, B12 limiting, N limiting, P limiting and resource rich treatment were created (Methods & Materials extension). During the experiments, the treatments were incubated as described above. In addition to the other treatments, a treatment without P.globosa (blank) was created in experiment 1 for the plate reader measurements. In experiment 2 no plate reader measurements were performed, because experiment 1 showed that no useful data was obtained from the optical density (OD) measurements.
Media
The treatments were created using several variations of the L1 medium (Table 3 & 7 Appendix I, Appendix II,). In experiment 1, Na2SiO3 had been omitted from the recipe. Besides, a basis of mixed seawater containing 80%
artificial seawater and 20% natural seawater collected at Texel was used. In experiment 2, Na2SiO3 had not been
omitted. In addition, mixed seawater containing 50% artificial seawater and 50% Atlantic seawater was used as a basis.
Approximately 250 ml medium for each treatment was created based (Table 3 & 7 Appendix I). A basis of mixed seawater was created. NaNO3, NaH2PO4 * H2O, Na2SiO3 * 9 H2O and the trace element solution were
added. The media were autoclaved, after which the 2µ filtered vitamins were added. For the vitamins, stock solutions according to the L1+ recipe were used (Appendix III). Next, the pH of all media was measured to ensure it had a pH value between 8.1 and 8.3. 3.7% 2µ filtered HCL or 2µ filtered NaOH was added to reach this pH.
To create the N and P limiting media they were added in line with the ratios of Maat & Brussaard (2016) (Table 2). To create B1 and B12 limiting media, the specific vitamin was omitted from the vitamin stock solution. Table 2. N:P ratios to create the N and P limiting conditions according to Maat & Brussaard (2016).
Treatment N (µM) P (µM)
N limitation 4 16
P limitation 400 1
P.globosa
A starting concentration of 2.0*104 P.globosa cells ml-1 in each treatment was desired. Experiment 1 showed that
the concentrations turned out lower. In order to be able to compare the experiments, a desired starting value of 1.0*104 P.globosa cells ml-1 was chosen for experiment 2.
The density of the P.globosa mother cultures was measured on the FCM. Based on these results, P.globosa was diluted using the specific limited medium to reach the desired starting concentration.
C.marina
In experiment 2, C.marina was used as bacterial prey. The species had a desired concentration of 5 million cells ml-1 after addition to the specific treatment. The starved C.marina was prepared for addition to the treatments the
same way as in the CellBrite test. A few aspects deviated from this test. After washing, C.marina was diluted to a concentration of 1.0 *109 and stained with CellBrite. Next, they were resuspended in the specific limited media.
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3.4.2 Measurements
Daily, a 150 microliters sample of all treatments were pipetted into a 96 well plate.First,the OD was measured in a plate reader at wavelengths of 680 and 730 nm. Measurements were corrected by subtracting the value of the blank from each measurement. Next, the growth rate was calculated using the FCM.Experiment 1 was
finished if the growth rates in all treatments were affected (Figure 2).One hour after the addition of C.marina in experiment 2, samples were taken. All samples were run through the FCM twice. The first time, the FL1-H value was set on 10 000 to visualise C.marina. The second time, the FL3-H value was set on 80 000 to visualise P.globosa and discover whether P.globosa cells have ingested C. marina.
To visualise P.globosa a FL3-H value was set on 80 000. To visualise C.marina cells a FL1-H value was set on 10 000.
Twenty-four hours later, ingestion by P.globosa was checked again (Figure 3).
Figure 4. Schematic overview of experiment 1.
Figure 2. Schematic overview of experiment 1.
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4 Results
This chapter describes the main results. An extension is created which contains all the data on which the figures and tables in this section are based (Appendix VI). For the raw flow cytometer data a google docs folder is made:
https://drive.google.com/drive/folders/15nq0_5ZhceOaa_FgdFP3KoK-lJCW2Prh?usp=sharing.
Note: Permission must be requested to view this data and the data is only available through the BD Accuri™ C6 program.
4.1 Tests
4.1.1 Starvation test
No large difference between the cell size of Dokdonia and C.marina before and after starvation was observed (Table 3). This indicates that starvation of nine days does not decrease cell size significantly. Dokdonia is relatively large and has an elongated cells which often occur in clusters, which could make ingestion difficult. C.marina is relatively small and has a rounded shape. Clusters hardly occur. Therefore, it is believed that C. marina can be easily ingested (Figure 4).
Table 3. Average cell size with standard deviation of Dokdonia and C.marina before and after nine days of starvation. Based on a minimal sample size of forty cells (Appendix VI).
Before starvation (µm) After nine days starvation (µm)
Dokdonia 1.98 ± 1.64 1.87 ± 1.17
C.marina <1 <1
4.1.2 CellBrite test
The results demonstrate that an incubation time of 15, 30, 60 minutes and three days had a similar FL1-H signal of ± 80 000 for both Dokdonia and C.marina (Figure 5 & 6). This means that the stain works on bacterial cells and that the signal does not fluctuate over time.
Before starvation After nine days starvation
Dokdonina
C.marina
Dokdonina
C.marina
Figure 4. Fluorescence microscopy photos with a hundred times magnification lens of Dokdonia and
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4.1.3 Dilution test
The dilution test demonstrates that most dilutions resulted in growth rates below zero (Figure 7). No significant differences between the various dilutions were observed. Therefore, diluting a large amount of times is most likely not the cause of the premature death of PgG(A). Because no explanation was found for the premature death of the PgG (A) strain, it was decided to perform experiment 2 with the Pgimf strain.
Figure 7 . Growth rates for the four different dilutions of P.globosa. Significant differences between growth rates of treatments are indicated with a capital letter.
Figure 5. Three figures, in which FL1-H is plotted against the counts for the first replicate of Dokdonia stained with CellBrite Fix Membrane stain in a 104 times dilution. The purple peak demonstrates the signal of Dokdonia after respectively 15, 30 , 60 minutes and three days of
incubation time of the CellBrite Fix Membrane stain. The black peak demonstrates the signal of the background. The background is absent in the last plot due to the changing FL1-H threshold value from 1000 to 8000.
Figure 6. Three figures, in which FL1-H is plotted against the counts for the first replicate of C.marina stained with CellBrite Fix Membrane stain in a 104 times dilution. The purple peak demonstrates the signal of C.marina after respectively 15, 30, 60 minutes and three days of
incubation time of the CellBrite Fix Membrane stain. The black peak demonstrates the signal of the background. The background is absent in the last plot due to the changing FL1-H threshold value from 1000 to 8000.
Dokdonia Dokdonia Dokdonia Dokdonia
C.marina C.marina C.marina C.marina
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4.2 Onset of resource limitation
The results of the plate reader measurements are included as an appendix in this report (Appendix VII). These results are not shown in the results section, because no usable data has come from the OD measurements. 4.2.1 Experiment 1
All starting concentrations were significantly lower than the desired starting concentration of 2.0*104 cells ml-1
(Figure 8,left).
Figure 8. Concentration in cells ml-1 (left) and growth rates (right) for the B1 limiting, B12 limiting, N limiting, P limiting and rich treatment
between the start of the experiment and day five (T0-T5), between day five and day six (T5-T6), between day six and day seven (T6-T7) and between day seven and day eight (T7-T8). Significant differences between growth rates of treatments are indicated with a capital letter. Over time, the number of cells ml-1 increases exponentially, until the growth is affected. Besides, variation in cell
abundance in a treatment increases with time. The growth curves will have a fairly stable value until the resource becomes limiting. Remarkable is that from day five to day six (T5-T6) the growth rates of the treatments are higher than on the other days.
For the B1 limiting treatment, the absolute number of P.globosa cells ml-1 are relatively high. The
higher absolute number is probably caused by the slightly higher starting concentrations in the B1 limiting treatment (Figure 7, left). The growth rate is affected on day eight (p-value = 0.0441). In the B12 limiting treatment, the P.globosa starting concentration was the highest of all treatments. However, the absolute values of P.globosa cells ml-1 on day eight (T8) are lower than in the B1 limiting and control treatment (Figure 8, right).
This can be explained by the fact that in the B12 limiting treatments P.globosa has slightly lower growth rates from day zero to day seven (T0-T7) and by the low growth rates from day seven to day eight (T7-T8) compared to the B1 limiting and control treatment (Figure 8, right). The growth rate is affected on day eight (p-value = 0.0122). In the N limiting treatment, a higher starting concentration than the control treatments was observed. However, P.globosa grow less well in the N limiting treatment. An explanation could be that the small amount of N in this treatment caused P.globosa to already be somewhat limited in its growth. The growth rate is affected on day eight (p-value = 0.0069). In the P limiting treatment, the growth rate is on average below zero between the start of the experiment and day five (T0-T5). These results indicate that P.globosa was dying and that the growth rate was affected before day five (T5). However, it is unclear what happened before day five (T0-T5).
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4.2.2 Experiment 2
All starting concentrations were higher than the desired starting concentration of 1.0*104 cells ml-1 (Figure 9, left).
Differences in absolute concentrations within the treatments increase with time (Figure 9, left). Besides, the results demonstrate that after the start of the experiment, P.globosa needs time to recover (Figure 9, right). The species may have experienced a brief temperature shock and became stressed from pipetting. All treatments need one day to recover except the P limiting treatment, which has a lag-phase of two days.
In the B1 and B12 limiting treatment, the growth rate of P.globosa is not affected in five days (B1: p-value = 0.8900, B12: p-p-value = 0.9943). Striking are the high absolute values of the B12 limiting treatment compared to the control treatment on day five (T5) (Figure 9, left). In the N limiting treatment, the growth rate is affected on day four (p-value = 0.0166). In the P limiting treatment, the growth rate differed significantly from the control treatment during the whole experiment. However, only after day four (T4) the growth rate in the P limiting treatment became significantly lower. This indicates that on day four the growth is affected in P limiting conditions (0.0442). In the N and P limiting treatment, the growth rate is still affected on day five (N: value = 0.0000, P: p-value = 0.0088).
Figure 9. Concentration in cells ml-1 (left) and growth rates (right) for the B1 limiting, B12 limiting, N limiting, P limiting and rich treatment
between the start of the experiment and day one (T0-T1), between day one and day two (T1-T2), between day two and day three (T2-T3), between day three and day four (T3-T4) and between day four and day five (T4-T5). Significant differences between growth rates of treatments are indicated with a capital letter.
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4.3 Does resource limitation trigger phagocytosis in P.globosa?
One hour after addition of C.marina to the different treatments, P.globosa cells that are assumed to ingest C.marina are observed (Figure 10, ‘P.globosa 1 hour after addition of C.marina’). The amount was determined by creating a range around the cells with a higher FL1-H value than the original P.globosa cluster. A new suitable range has been created for each measurement. Some P.globosa cells have a FL1-H value that is higher than the maximum FL1-H value of C. marina. This could indicate ingestion a cluster of C.marina (Figure 10, ‘P.globosa 1 hour after addition of C.marina’). It is also possible that these clusters are not ingested, but visible on the plot due to their relatively high FL3-H value. In this research, these cluster cells have been considered as ingested by P.globosa.
P.globosa without addition
of C.marina
P.globosa 1 hour after
addition of C.marina C.marina
B1 limiting treatment
B12 limiting treatment
N limiting treatment
P limiting treatment
Rich (control) treatment
Figure 10. FL3-H is plotted against FL1-H. Demonstrated is the cluster of P.globosa cells without addition of C.marina (left), the P.globosa cells after one hour of addition of C.marina cells (middle) and the C.marina cells stained with CellBrite Fix Membrane stain (right) for each treatment. The red range indicate the P.globosa cells that are assumed to have ingested C.marina. The green range indicate the P.globosa cluster that has not ingested C.marina. To visualise P.globosa a FL3-H value was set on 80 000. To visualise C.marina cells a FL1-H value was set on 10 000.
15 The percentage of P.globosa cells that have ingested C. marina was significantly higher in the N and P limiting treatment compared to the B12 limiting and control treatment (Figure 11). However, the number of added C.marina cells is also significantly higher (Figure 12). The absolute number of P.globosa cells that ingested C.marina is lower for these treatments, but the amount of P.globosa cells present was also lower (Table 5). Table 5. Cell count in cells ml-1 of P.globosa and of P.globosa that ingested C.marina. All values are based on four replicates. Variations of
the replicates are demonstrated with the error ranges.
No significant difference in ingestion by P.globosa between 1 hour or 24 hours after the addition of C.marina is observed (N: p-value = 0.0758, P:0.6298) (Figure 13).
Treatment P.globosa cells/ml *104 Ingested C.marina cells/ml *103
B1 limiting 7.07 ± 4.81 2.14 ± 0.71
B12 limiting 11.68 ± 2.27 3.18 ± 0.27
N limiting 2.79 ± 1.87 1.75 ± 1.05
P limiting 2.36 ± 1.85 1.38 ± 1.08
Rich (control) 5.21 ± 1.57 1.27 ± 0.20
Figure 11. Percentage of P.globosa cells that ingested C.marina after one hour of addition of C.marina. Significant differences between percentages of treatments are indicated with a capital letter.
Figure 13. Percentage of P.globosa cells that ingested C.marina after one and twenty-four hours of addition of
C.marina for the N and P limiting treatments. Significant differences between percentages of treatments are
indicated with a capital letter.
Figure 12. The amount of C.marina that is added to the various treatments. Significant differences between percentages of treatments are indicated with a capital letter.
Percentage of P.globosa cells that ingested C.marina in one hour Starting concentration of C.marina
16
5 Discussion
5.1 Resource limitation
It is important to note that all species eventually run into limitation, even if the species is perfectly adapted to limiting conditions. To make a statement about how well P.globosa is adapted to certain conditions, comparison with other species is necessary, which is beyond the scope of this research. Moreover, the exact amount of resources is unknown due to the use of natural seawater.
5.1.1 Resource limitation in PgG(A)
The growth rate of P.globosa in both the B1 and B12 limiting treatment was affected after eight days. However, P.globosa in the B12 limiting treatment has a lower growth rate than in the B1 limiting treatment. Besides, the absolute values on day eight are also lower in the B12 limiting treatment. This could be explained by the higher starting values in the B12 limiting treatment, which means that the resources are used up faster. It could also indicate a higher presence of B1 in the natural seawater. Another possibility is that PgG(A) is well adapted to B1 limiting conditions. Further research is needed to draw a conclusion. The affected growth seems to indicate that the PgG (A) strain of P.globosa was unable to produce the vitamins itself. This suggests that PgG(A) is B1 and B12 autotrophic, which is in line with the results of Spencer (1981). In the N limiting treatment, the growth rate was affected after eight days as well. However, it appears that during the eight days, P.globosa grows slower in the N limiting treatment than in the vitamin limiting treatments, even though this is not a significant difference. This could indicate that P.globosa already suffers from the N limiting conditions before the growth is significantly affected. In the P limiting treatment, the growth rate was affected after five days. This indicates that PgG (A) is more sensitive to P limiting conditions than to N limiting conditions.Although this can also be caused by the high N: P ratio in the P limiting treatment compared to the N limiting treatment. To conclude that P.globosa is actually a good competitor under P limiting condition, further research is needed in which multiple species are compared. 5.1.2 Resource limitation in Pgimf
In the B1 and B12 limiting conditions, the growth rate of the Pgimf strain was not affected within five days. In the N and P limiting conditions, the growth rate of the Pgimf strain was affected on day four. This indicates that Pgimf is more sensitive to the nutrient limitations than to the vitamin limitations. P.globosa has a lower end concentration in the P limiting conditions compared to the N limiting condition, while the start concentrations were the same. This can be explained by the longer recovery time in the P limiting condition, which may indicate a higher sensitivity to this condition.
5.1.3 Resource limitation compared
It is important to note that Pgimf and PgG(A) were not cultured in the same media and light cycle. This could explain the different behaviors between the strains.
In both strains, the growth rate of the Pgimf strain was not affected within five days in the vitamin limiting conditions. For the PgG (A) strain, the absolute cell count in the vitamin limiting conditions is 2,2 *105 cells ml-1
on the fifth day. For the Pgimf stain this value is 0,94*105 cells ml-1, while the starting values are on average
slightly higher. Differences in vitamin availability in the different natural seawaters could be an potential explanation. In addition, it is not known whether the B1 and B12 limiting conditions also affect the growth rate after eight days in the Pgimf strain. For the PgG (A) strain, the average absolute values in the P limiting condition on day five is lower than for the Pgimf strain. The cell size of both strain is equal and therefore not explains the difference in absolute values.Based these results, it seems that the PgG (A) is more sensitive to P limting condition. However, both strains do not grow well in the P limiting conditions. In the N limiting condition, the growth was affected four days earlier in the PgG(A) strain compared to the Pgimf strain. This shows that the Pgimf strain is more sensitive to N limiting conditions.
5.2 Mixotrophic characteristics of P.globosa
The results indicate that P.globosa ingest C.marina under resource limiting and resource rich conditions. However, the number of P.globosa cells that are assumed to ingest bacteria is higher under resource limiting conditions. This suggest that limiting conditions might trigger phagotrophy. Nonetheless, the different encounter rates of P.globosa and C.marina in the treatments could also be an explanation, since a higher concentration of C.marina is added to the nutrient limiting treatments compared to the resource rich conditions.
Confocal microscopy images demonstrate that most C.marina cells that are assumed to be ingested by P.globosa are actually not ingested but attached to the outside of P.globosa (Figure 14).This attachment could be
17
enhanced by an excess of carbon in nutrient limiting conditions, which is the results of the continued photosynthetically fixation of carbon by P.globosa. Some species discrete this dissolved organic carbon, which makes the cell surface more adhesive (S. Wilken, personal communication, 2021). This could also be an explanation for the higher percentage of P.globosa cells that ingest C. marina under resource limiting conditions.
Only three cases are found where P.globosa presumably ingested C.marina (Figure 15). This is a strong indication for mixotrophic characteristics of P.globosa, but determination is difficult due to unclear cell contours (Figure 15). Remarkable is that two of the three cases were found in resource rich conditions (Figure 15). Which suggests that N and P limiting conditions are not stronger triggers for phagotrophy.
Based on the FCM results, the percentage of P.globosa that seems to take up bacteria has a value between 2.09 - 10.6% with an average of 4.55%. The confocal microsopy images show that if ingestion actually takes place, this value would be lower. This small percentage is comparable to the haptophyte L.galbana and the chlorophyta N.rotuna under similar, but not entirely the same, conditions (Anderson, Charvet & Hansen, 2018). According to Anderson et al. (2018), this small percentage could be explained by the fact that cells are different from each other, that some cells are dividing or that the resources are not evenly distributed in the medium. As a result, the percentage could also differ greatly between measurements and individual experiments. In addition, the percentage of cells that take up bacteria appears to be species dependent. For example, more than 40% of a chlorophyta N.pyriformis population appears to ingest bacteria. According to Anderson et al. (2018), this difference in percentage could also depends on the strategy of the mixotroph. A low percentage indicates a survival strategy, while the species with a higher percentage probably use phagotrophy not only for survival but also for growth (Anderson et al., 2018).
Literature demonstrates that phagotrophy is a primitive characteristic of autotrophs, but that this characteristic has disappeared in some autotrophs due to a too high cost-benefit ratio (Raven, 1997; Wilken et al., 2019). The confocal microscopy images show a strong indication for phagotrophy. Although this cannot be concluded with certainty, P.globosa presumably belongs to the mixotrophs. This would indicate that the benefits
Figure 14 . C.marina cells (green) attached the surface of P.globosa (red). The dotted lines indicates the expected cell contours. Images are obtained by dhr. Sebastiaan Koppelle.
Figure 15 . The three cases where C.marina (green) is potentially ingested by P.globosa (red). The dotted lines indicates the expected cell contours. Images are obtained by dhr. Sebastiaan Koppelle.
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outweighed the costs of phagotrophy for this species. This suggests that the species is often in conditions where phagotrophy is beneficial. According to Raven (1997) and Avrahami & Frada (2020), phagotrophy is mainly beneficial in resource limiting conditions. However, twenty-four hours after addition of C.marina, the growth rates of P.globosa are still affected. Besides, the growth rates decreased to values below zero. This indicates that the possible phagotrophy does not help P.globosa to grow in one day. This means that P.globosa requires more time to utilize C.marina for growth or that phagotrophy provides no benefits in N and P limiting conditions.If the latter is the case, phagotrophy is expected to be beneficial in a condition that is not yet known. This could be in a limiting condition of a resource, which P.globosa only needs in little amounts. In this situation, the low ingestion rate is expected to be enough for survival or to grow properly.
5.3 Effect of mixotrophy
Based on the results, it cannot be concluded that P.globosa belongs to the mixotrophs. However, there are some strong indications for the mixotrophic characteristics of P.globosa.The following sections describe the effect of the mixotrophic characteristic assuming the indications are correct.
5.3.1 Fishery and tourisms
The results demonstrate that P.globosa does not grow properly after the addition of C.marina in nutrient limiting conditions. Therefore it is expected that phagotrophy does not contribute to the success of P.globosa in these circumstances. Accordingly, mixotrophy does not need to be included in policy plans to reduce the negative impact of P.globosa on fishing and tourism. However, other bacterial prey, prey density or limiting condition might trigger phagotrophy and explain the success of P.globosa.
5.3.2 Biological carbon pump
P.globosa is expected to only use phagotrophy as survival strategy. Therefore, the percentage of P.globosa that ingest bacteria is low. This suggest that the trophic level transfer efficiency does not increase significantly. Moreover, there will be little or no hunting of bacteria. As a result, the mean global organisms size and sinking carbon are not expected to increase. Assumably, no enhancement of the biological carbon pump will be observed.
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6 Conclusion
The results demonstrate that in both resource limiting and resource rich condition ingestion of bacterial prey takes place. However, the percentage of P.globosa that ingest C.marina is higher in the N and P limiting conditions.
Whether this is caused by the higher encounter rates, the nutrient limitation or stronger adhesiveness of these cells is not clear. Therefore, it cannot be concluded that N and P limitation is a trigger for phagotrophy in P.globosa.
The confocal microscopy images do not support the ingestion of C.marina by P.globosa. The majority of C. marina is attached to the outside surface of P.globosa and thus is not ingested. Only a few cases are observed in which C.marina is potentially ingested. Overall, it cannot be concluded that P.globosa belongs to the mixotrophs. However, some strong indications are observed.
If these indications are correct, P.globosa belongs to the mixotrophs. It is suggested <10% of P.globosa cells ingest C.marina. Therefore, it is expected that little or no enhancement of the biological carbon pump will be observed. Besides, it appears that potential phagotrophy does not cause the success of P.globosa in N and P limiting conditions. Based on these results, mixotrophy does not need to be included in policy plans to reduce the negative impact of P.globosa on fishing and tourism or to combat climate change.
Taking everything into consideration, it cannot be concluded that P.globosa has mixotrophic characteristics. However, indications for mixotrophy are observed. It is possible that different bacterial prey, P.globosa-prey ratio or a different condition might be a (stronger) trigger for phagotrophy in P.globosa. Further research is needed to draw a conclusion about the mixotrophic characteristics of P.globosa and the possible triggers.
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8 Acknowledgements
I would like to express my gratefulness to my supervisors Dr. Susanne Wilken and Dhr. Sebastiaan Koppelle for the guidance, discussions, encouragement and additional measurements during my internship at the Freshwater and Marine Ecology department of the University of Amsterdam. I am very grateful for the confidence you had in me and for motivating me when I encountered difficulties during the research.
I owe a deep sense of gratitude to Mw. Prof. dr. Corina Brussaard for sharing her helpful knowledge of Phaeocystis and for providing supplies for the experiments.
I would like to thank my fellow students Savannah Sarkis, Rocio Rodrigues and Shai Slomka de Oliveira for exchanging articles and knowledge about various subjects.
I thank profusely all the staff members of the laboratory of the University of Amsterdam , with a special thank you to Dhr. Dr. Merijn Schuurmans.
I would also like to thank the University of Amsterdam. I would not have been able to conduct this research without the trust and funding of the University of Amsterdam.
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Appendix I
Methods & Materials extension
'Study of the possible mixotrophic characteristics of
Phaeocystis globosa ‘
By
Nina van Haastert, 12107913
28 May 2021, Amsterdam
Primary supervisors: Dr. Susanne Wilken,
Secondary supervisor: Dhr. Sebastiaan Koppelle,
25
3.1 Organisms and culture conditions
The cultures were cultures in 50 ml plastic cultures flask. For each transfer, 20 ml L1 medium and 5 ml P.globosa culture was used. Before transferring, the cultures were mixed using a 5 ml automatic pipette. The cultures were kept sterile the whole research.
3.3 Tests
3.3.1 Starvation test
1. Two Erlenmeyers containing 50 ml Difco2216 were prepared
2. 200 microliters of Dokdonia was pipetted into the 50 ml Erlenmeyer containing Difco 2216 3. 200 microliters of C.marina was pipetted into the other 50 ml Erlenmeyer containing Difco 2216 4. The cultures were stored in an incubator with a temperature of 23°C and 110 RPM min-1
5. After one day, 200 microliters of Dokdonia in 50 ml Difco 2216 was pipetted into a new 50 ml Erlenmeyer containing Difco 2216
6. 200 microliters of C.marina in 50 ml Difco 2216 was pipetted into a new 50 ml Erlenmeyer containing Difco 2216
7. After two days, the cultures were transferred into 50 ml sterile tubes 8. These tubes were put into the centrifuge (22°C, 4000rcf, 10 minutes) 9. The supernatant was removed from the tubes
10. The pellets were resuspended into 25 ml Atlantic Seawater 11. The tubes were put into the centrifuge (22°C, 4000rcf, 10 minutes) 12. The supernatant was removed from the tubes
13. The pellets were resuspended into 25 ml Atlantic Seawater 14. The tubes were put into the centrifuge (22°C, 4000rcf, 10 minutes) 15. The supernatant was removed from the tubes
16. The pellets were resuspended into 50 ml Atlantic Seawater
17. Three 0.5 ml samples of each culture was taken ‘Before starvation’ 18. The samples were fixed with 10 microliters Glycerol
19. The tubes were stored into a 4°C fridge for nine days
20. After nine days, three 0.5 ml samples were taken ‘After nine days of starvation’ 21. The 0.5 ml samples were fixed with 10 microliters Glycerol
22. The sample was shaken and stored in a 4°C fridge for 30 minutes
22. The six samples, three from before and three from after nine days starvation were diluted ten times. For this, 450 microliter TE buffer and 50 microliter sample was used
23. The samples were stained with 5 microliter Sybr Green 1 stain 24. The samples were kept in the dark for 20 minutes
25. A dilution series was made of the sample
22. The samples, before and after starvation, were run through the flow cytometer (Settings on ‘Medium’, 50 microliters and a FL1-H threshold on 1000)
23. Based on these results the samples were diluted with Milli Q to a concentration of 106cells ml-1 (Table 1) 23. A filter protector was put on a filter
24. These protectors were wettened with a few drops Milli Q 25. The 0.2 25 mm filters were put on the filter protectors 26. 1 and 2 ml of the samples were put on the filters 27. The filter was closed with the lid
28. The vacuum pump was connected and turned on so that the samples were filtered and only the bacteria remained on the filter
29. Slides were prepared by adding 20 microliters DAPI on the slides and 20 microliters on the DAPI slide cover.
30. The filters with the remaining bacteria were put on the slides 31. The slide covers with DAPI were put on the filters.
32. The slides were stored in a -20°C freezer.
Table 1. Cells ml-1 demonstrated for Dokdonia and C.marina before and after nine days of starvation. Also the dilution is shown to reach a
concentration of 106 cells ml-1.
Cells ml-1 *109 Dilution
Dokdonia before starvation 4,72 4718
Dokdonia after nine days of starvation 13,44 12440
C.marina before starvation 0,58 582
26
3.3.2 CellBrite Fix membrane stain test
Before the Sybr Green 1 and CellBrite Fix Membrane stain can be used, they must thaw on ice in the dark. This will take a few minutes.
Dokdonia
1. A 1 ml sample was taken from the growing Dokdonia culture. 2. The sample was centrifuged (22° C, 3000 rfc, 10 minutes) 3. The supernatant was removed
4. The pellet was resuspended in 1 ml 2μ filtered Phosphate-buffered saline (PBS) 5. The in PBS resuspended sample was split into two samples (both 0.5 ml) 6. The 0.5 ml sample was fixed with 10 microliter Glycerol
7. The sample was shaken and stored in a 4°C fridge for 30 minutes
7. A 10 times dilution was created containing 450 microliter 2μ filtered TE buffer and 5 microliter of the fixed sample
8. 5 microliter of 200 times diluted Sybr Green 1 was added to this dilution 9. The dilution was stored in the dark and at room temperature for 30 minutes 10. A dilution series of a 100, 1000, 10 000, 100 000 dilution was created
11. The cell count was measured on the flow cytometer (‘Medium and limit set on 1 minute) 12. 0.1 milliliter of the other, still living, Dokdonia sample was taken
13. 1 microliter of 1000 times diluted CellBrite Fix Membrane Stain was added 14. Of this CellBrite Fix Membrane Stained sample, 5 microliters was taken
15. This 5 microliters was diluted 10 000 times to reach a concentration of 1.0 * 109cells ml-1 16. The dilution was stored in the dark and at room temperature
17. Samples were taken after 15, 30 and 60 minutes
18. These samples were measured on flow cytometer (‘Medium, limit set on 1 minute and FL1-H threshold on 1000)
19. The dilution was centrifuged (22° C, 3000 rfc, 10 minutes) 20. The supernatant was removed
21. The pellet was resuspended in 0.1 microliter of L1 medium 22. 3 samples of 0.5 ml were taken
23. 3 samples were fixed with 10 microliter of Glycerol
24. The sample was shaken and stored in a 4°C fridge for 30 minutes 24. The fixed samples were frozen with liquid nitrogen
25. The samples were stored in the -80⁰ Celsius freezer 26. After three days, the fixed sample were thawed
27. The fixed samples were measured on the flow cytometer (‘Medium, limit set on 1 minute and FL1-H threshold on 1000)
C.marina
1. A 1 ml sample was taken from the growing C.marina culture. 2. The sample was centrifuged (22° C, 3000 rfc, 10 minutes) 3. The supernatant was removed
4. The pellet was resuspended in 1 ml 2μ filtered PBS
5. The in PBS resuspended sample was split into two samples (both 0.5 ml) 6. The 0.5 ml sample was fixed with 10 microliter Glycerol
7. The sample was shaken and stored in a 4°C fridge for 30 minutes
7. A 10 times dilution was created containing 450 microliter 2μ filtered TE buffer and 5 microliter of the fixed sample
8. 5 microliter of 200 times diluted Sybr Green 1 was added to this dilution 9. The dilution was stored in the dark and at room temperature for 30 minutes 10. A dilution series of a 100, 1000, 10 000, 100 000 dilution was created
11. The cell count was measured on the flow cytometer (‘Medium and limit set on 1 minute) 12. 0.1 milliliter of the other, still living, 0.5 ml C.marina sample was taken
13. 1 microliter of 1000 times diluted CellBrite Fix Membrane Stain was added 14. Of this CellBrite Fix Membrane Stained sample, 5 microliters was taken
15. This 5 microliters was diluted 10 000 times to reach a concentration of 1.0 * 109cells ml-1
27 17. Samples were taken after 15, 30 and 60 minutes
18. These samples were measured on flow cytometer
19. The dilution was centrifuged (22° C, 3000 rfc, 10 minutes) 20. The supernatant was removed
21. The pellet was resuspended in 0.1 microliter of L1 medium 22. 3 samples of 0.5 ml were taken
23. 3 samples were fixed with 10 microliter of Glycerol
24. The sample was shaken and stored in a 4°C fridge for 30 minutes 24. The fixed samples were frozen with liquid nitrogen
25. The samples were stored in the -80⁰ Celsius freezer 26. After three days, the fixed sample were thawed
27. The fixed samples were measured on the flow cytometer (‘Medium and limit set on 1 minute) 3.3.3 Dilution test
1. Three samples of 150 ml of the mother culture of PgG(A) P.globosa were taken
2. These samples were measured on the flow cytometer (Settings ‘Fast” and ‘Limit’ set on 50 ml) 3. The average of samples were taken to calculate the average density of the culture
4. Based on the density, a 2,10,100 and 567 times dilution was created. The 567 dilution is based on a desired start concentration of 2,0*104P.globosa cells ml-1. The dilutions were made using the L1 medium with a basis of
80% Artificial Seawater and 20% Natural Seawater collected at Texel. Table 2 demonstrates the start concentration of each dilution.
Table 2.The different dilution factors and the starting concentration of P.globosa in these dilutions. The dilution were created using the L1 medium. The starting concentration is based on three replicates, which is indicated with the error range.
Dilution Starting concentration P.globosa (cells/ml)
2x 2,43*106 ± 5,14*104
10x 5,07*105 ± 8,35*103
100x 5,73*104 ± 1,21*104
567x 8,13*103 ± 1,52*103
5. Three replicates of each treatment were created
6. The well plates were stored in an incubator with a 12h light:12h dark cycle, a temperature of 15° C and a light intensity of 70 umol photons m-2 s-1
7. Before taking samples, each well plates was mixed gently by pipetting up and down 8. 150 microliter samples of each replicate of each treatment were taken
9. The samples were vortexed
10. Directly after vertexing, the samples were run through the flow cytometer. (Settings ‘Fast” and ‘Limit’ set on 50 ml)
11. The formula ‘(ln(Tn) – ln(Tn-1) )/ T’ was used to measure the growth rate between two days.
12. When the growth rate was above zero, no further action was needed and the daily measurements were performed again the next day. If the growth rate was below zero, the experiment was finished for this particular dilution
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3.4 Experiments
3.4.1 Preparations
3.4.1.1 Media Experiment 1
1. 250 ml of each treatment was made using ~250 ml of mixed seawater as a basis (80% Artificial Seawater and 20% Natural Seawater collected from Texel)
2. NaNO3, NaH2PO4 * H2O and the trace element were added (Table 3) 3. The bottles were autoclaved at 121° Celsius for 15 minutes.
4. The vitamin stocks were prepared (Table 4) 5. The vitamins were added (Table 3)
6. A 5 ml sample of each treatments was taken 7. The pH was measured
8. 3,7% 2μ filtered HCL or NaOH was added to the media to change the pH (Table 4) 9. The pH was measured (Table 5)
Table 3. Different treatments of the P.globosa cultures. Demonstrated is the variations of the L1 medium by showing what is added to the ~250 ml mixed seawater. (Vitamin values are according to the L1+ medium)
Treatment Condition ~250 ml mixed seawater +
1 B1 limitation • 0.25 ml NaNO3
• 0.25 ml NaH2PO4 * H2O • 0.25 ml trace element
• 0.25 ml B1 limiting vitamin solution
2 B12 limitation • 0.25 ml NaNO3
• 0.25 ml NaH2PO4 * H2O • 0.25 ml trace element
• 0.25 ml B12 limiting vitamin solution
3 N limitation • 0.256 ml 100x dilution of NaNO3
• 0.25 ml NaH2PO4 * H2O • 0.25 ml trace element • 0.25 ml rich vitamin solution
4 P limitation • 0.25 ml NaNO3
• 1.52 ml 100x dilution of NaH2PO4 * H2O • 0.25 ml trace element
• 0.25 ml rich vitamin solution Control Nutrient & Vitamin rich • 0.25 ml NaNO3
• 0.25 ml NaH2PO4 * H2O • 0.25 ml trace element • 0.25 ml rich vitamin solution
