Phosphate-Uptake

5.3.3.1 Background, Principle and Aim

Algae cells can accumulate excessive phosphorus, beyond their immediate requirements (Currie and Kalff 1984). The assimilated P is stored in the cells primarily as polyphosphate (Rhee 1973; Tilman and Kilham 1976). This mechanism is known as luxury consumption. The growth rate of an algae cell is proportional to its amount of internal polyphosphate stor- age, also known as cell quota. Various environmental factors, such as light (Chisholm and Stross 1976; Rivkin and Swift 1982), temperature (Goldman and Carpenter 1974; Goldman 1979; Gotham and Rhee 1981), pH (Lawry and Jensen 1979), and other factors (Rigby et al. 1980) are known to influence the rate of P-uptake. The developed SAM-X model (see chap- ter 6) is based on P-limited algae growth realized via the internal cell quota approach. Each alga species in the model is characterized by parameters related to P-uptake kinetics, such as minimum internal P concentrations and maximum P-uptake rates.

Many sources in literature are available (see chapter 6.4.1 and 11.3) to obtain values for typ- ical P-uptake rates and minimum cell quotas for different algae species. D. subspicatus and

P. subcapitata are well investigated in literature, whereas C. terricola and C. pyrenoidifera

were not studied to the same extent. In case it was not possible to find literature values for parameters for the respective species, it was necessary to obtain specific values, or at least ranges, for related species, genus or families. The claim to verify literature values motivated to the performance of P-uptake experiments with D. subspicatus and C. pyrenoidifera. Four short-term P-uptake tests (each two for D. subspicatus and C. pyrenoidifera) were performed by spiking phosphate into starving algae cultures in order to investigate P-uptake kinetics of algae cells. Additionally, the aim was to determine related parameter values (maximum P- uptake rates, vmax and minimum cell quotas, qmin) by own experiments. The determined val-

ues were compared with the ranges found in literature. In addition, four long-term P-uptake observations were done; one for each species. This was realized by additional measure- ments of P dynamics during four capacity tests. The experimental data was used to verify the developed model for simulations of long-term growth and P-uptake kinetics of the algae.

Static Algae Tests

Material and Methods 47

5.3.3.2 Preparation and Setup 5.3.3.2.1 Short-term Tests

In total, four short-term P-uptake experiments with D. subspicatus (test 1 and 2) and C. pyre-

noidifera (test 3 and 4) were performed using the standard phosphomolybdate reaction with

ascorbic acid as the reducing agent. The method is photometric determination as molyb- denum blue after acidic hydrolyses and oxidation at 100-120°C, where the photometrical measurement of dye intensity was performed at 880 nm. The technique is based on the measurement of ortho-phosphate. The digestion of both dissolved organic as well as particu- late phosphorus compounds was therefore required to determine the total P content. An un- filtered sample was acquired in order to include all solid matters in the digestion process. Digestion was performed by heating the sample with peroxodisulfate and sulfuric acid. The detailed method, phosphate measurements and all needed calculations are described by ISO (2004). Rating curves with a phosphate standard were determined prior to test start. Blank values for comparison with the rating curves were measured two times during each test (Figure 5-14 and Figure 5-15).

Figure 5-14: Rating curves with phosphate stand-

ard (tests with D. subspicatus) Figure 5-15: Rating curves with phosphate stand-ard (tests with C. pyrenoidifera)

All materials were rinsed with hydrochloric acid prior to test start in order to avoid impurities with residuals of phosphate. The biomass was quantified by measurement of optical density using a photometer with a 5 cm cuvette at 720 nm wavelength (D. subspicatus) or by using a 1 cm cuvette at 680 nm wavelength (C. pyrenoidifera). Calculation of cell numbers was per- formed by using the rating curve described in chapter 5.1.8.

y = 0.4721x R² = 0.9997 0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.000 0.200 0.400 0.600 0.800 1.000 1.200 Phos pha te c onc ent ra tion (m g∙ L -1) Extinction

Measured Rating points (test 1) Rating points (test 2) Linear (Measured)

y = 0.4514x R² = 0.9901 0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.000 0.200 0.400 0.600 0.800 1.000 1.200 Phos pha te c onc ent ra tion (m g P∙ L -1) Extinktion

Measured Rating points (test 1) Rating points (test 2) Linear (Measured)

D. subspicatus

Starving cultures of D. subspicatus were cultivated for 5 days in 2.5 L Erlenmeyer flasks at 20°C and 35±10 µE∙m-2_∙s-1 _{in P-free OECD 201 medium. Test duration was 10 hours (test 1)} and 2 days (test 2). Samples were taken 90 minutes (test 1) and 30 minutes (test 1 and 2) prior to test start. At test start (t0), a nominal concentration of 1.2 mg P∙L-1 (test 1) and 1.4 mg P∙L-1 _{(test 2) was spiked into flasks containing the starving cultures. Samples of the cultures} were taken every 20-30 minutes and treated according to the method for phosphate determination. At each time point, a sample of 80 mL was taken from the algae culture and divided into subsamples of 10 mL for measurements of optical density, 10 mL for digestion of total P, and 60 mL for further measurements. The 60 mL sample was filtrated using P-free filters. 40 mL of the filtrated sample was then prepared for ortho-phosphate measurements. The filters were digested to obtain data of the filtered algae and the P content of the cells.

C. pyrenoidifera

Two starving populations were cultivated in P-free WARIS-H medium at 20°C and 55±10 µE∙m-2_∙s-1 _{for 25 days. Both tests were performed on two consecutive days. Before} spiking phosphate into the starving cultures, samples were taken, 85 and 69 minutes (test 1) and 75 minutes (test 2) prior to test start. At test start (t0) a nominal concentration of 0.8 mg P∙L-1 _{(test 1) and 0.5 mg P∙L}-1 _{(test 2) was spiked into one of the starving cultures. In both} tests, samples were taken every 20 minutes and treated the same way as for the tests with

D. subspicatus. An overview of the performed P-uptake experiments is given in Table 5-9. Table 5-9: Overview of performed short-term P-uptake experiments

Test Species Duration _(d) Light intensity _(µE∙m-2_∙s-1₎ Temperature _(°C) P in medium _{(mg P∙L}-1₎

1 D. subspicatus 0.35 35±10 20±1 0, 1.2 at t0

2 D. subspicatus 2 35±10 20±1 0, 1.4 at t0

3 C. pyrenoidifera 0.35 55±10 20±1 0, 0.8 at t0

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Material and Methods 49

5.3.3.2.2 Long-term Tests

The long-term P-uptake in algae cells was measured during one capacity test for each spe- cies (chapter 5.3.2, test 2 for D. subspicatus and P. subcapitata, test 4 for C. terricola and C.

pyrenoidifera). At specific sampling days for D. subspicatus and P. subcapitata, an additional

sample of 2 mL was taken from each control batch. For C. terricola and C. pyrenoidifera, also an additional sample of 2 ×1.5 mL was taken from each batch and 1 µL of Lugol’s iodine (so- lution of elemental iodine and potassium iodide in water) was added to each aliquot. These three samples were pooled, diluted with water in a ratio of 1:10, and divided into two speci- mens. One was used for determination of total phosphate and the other one was centrifuged with 30000 rpm for 10 minutes and used for ortho-phosphate determination. The samples were prepared for total PO4 and ortho-PO4 measurement according to the method described in the safety data sheet for the LCK 348 phosphate test (Hach Lange GmbH 2006). The prin- ciple is similar to the method used in the short-term P-uptake tests: phosphate ions react with molybdate and antimony ions in an acidic solution to form an antimonyl phosphomolybdate complex, which is reduced by ascorbic acid to phosphomolybdenum blue. Calculations of ortho-P and total-P were also done according to the above mentioned method.