Intracellular composition measurements

2.14.1 Carbohydrate content determination

Carbohydrates were extracted from the microalgal cells using a variation of the method adapted from Myklestad and Haug (1972). An aliquot of the culture (10 ml) was centrifuged at 5000 rpm for ten minutes. The supernatant was discarded and the pellet rinsed with 10 ml 0.5 M ammonium bicarbonate, to remove any salt residue (Zhu and

Lee, 1997). Re-centrifugation followed at 5000 rpm for 10 minutes and the supernatant was decanted. The algal pellet was then evaporated to dryness in an oven set at 50-55 ºC.

Total carbohydrate was extracted by adding 80% sulphuric acid to the dried sample. This suspension was vortexed and incubated at 20 ºC for 20 hours.

The carbohydrate extract was then tested to determine the total carbohydrate content using the phenol-sulphuric acid method derived by Dubois et al. (1956). After incubation, the suspension was again vortexed thoroughly and a 100 µl aliquot was transferred to a well cleaned (washed and rinsed with distilled water) and dried test tube. A 500 µl aliquot of 4% phenol and 2.5 ml 96% sulphuric acid was added to the test tube and the resulting suspension was incubated at room temperature for 45 minutes. The absorbance of the solution was then read at 490 nm using a S2100 Diode Array Spectrophotometer (Biowave – Labotec). This absorbance value was used to determine the carbohydrate content from a previously constructed standard curve, using glucose as a standard (Figure 2.6).

y = 0.0107x + 0.0113 R² = 0.9898

0 0.2 0.4 0.6 0.8 1 1.2

0 20 40 60 80 100 120

Glucose concentration (ug)

Absorbance (490nm)

2.14.2 Colorimetric determination of intracellular nitrogen

Digestion of the microalgal cells was conducted using a variation of the method described by Lindner and Harley (1942). A 10 ml aliquot of the algal culture was centrifuged at 5000 rpm for 10 minutes. The cell pellet was rinsed with 10 ml 0.5 M ammonium bicarbonate and re-centrifuged. The pellet was then dehydrated in an oven at a temperature of between 50 to 55 ºC. The dried pellet was weighed and transferred to a digestion tube. A 2 ml aliquot of 96% sulphuric acid was added to the digestion tube and the suspension was heated on a heating block (HI 839800 COD REACTOR – 2008 Hanna instruments) that was maintained at 105 ºC. The heating process was carried out in a fume hood until the dried cell pellet was partially disintegrated and dissolved (no cell clumps were observed). The solution was then cooled in an ice bath and 0.5 ml 30%

hydrogen peroxide was added to the digestion tube. The resulting solution, in the digestion tube, was transferred to the 105 ºC heating block in the fume hood and was allowed to boil until it turned clear. The tube containing the digested cell solution was then allowed to cool in an ice bath and the solution was made up to 10 ml with distilled water.

A method developed by Dorich and Nelson (1983) was utilised for the nitrogen colorimetric assay. Reagents one and two were made up at least 24 hours prior to use.

For reagent one, 17 g sodium salicylate, 12.5 g sodium citrate and 12.5 g sodium tartrate was dissolved in approximately 375 ml de-ionized water. Thereafter, 0.06 g sodium nitroprusside was added to the mixture. The solution was then made up to 500 ml with de-ionized water. For solution two, 15 g sodium hydroxide was dissolved in approximately 375 ml de-ionized water in a volumetric flask and was allowed to cool in an ice bath. A 5 ml aliquot of sodium hypochlorite was then added to the liquid in the volumetric flask and the solution was made up to 500 ml with de-ionized water.

A 20 µl aliquot of the digested cell solution, obtained using the procedure outlined above, was transferred to a sterilized (autoclaved for 30 minutes at 121 °C) and well dried test tube. Reagent one (1 ml) was added to the test tube and the resulting solution was mixed well and left to incubate at room temperature for 15 minutes. Reagent two (1 ml)

was then added to the test tube, the contents were mixed well and allowed to incubate at room temperature for an hour to ensure full color development. Absorbance readings were then taken at 660 nm using a S2100 Diode Array Spectrophotometer (Biowave – Labotec). The absorbance readings were used to determine the intracellular nitrogen content using a standard curve that was constructed using ammonium sulphate as a standard (Figure 2.7).

y = 0.004x R² = 0.9924

0 0.05 0.1 0.15 0.2 0.25

0 5 10 15 20 25 30 35 40 45 50 55

Nitrogen Concentration (ug/ml)

Absorbance (660 nm)

Figure 2.7: Standard curve depicting nitrogen concentration with respect to absorbance readings at 660 nm. Error bars represent standard deviation (n = 3).

2.14.3 Colorimetric determination of intracellular phosphorous

A similar procedure, as depicted in Section 2.14.2, was used for the digestion of microalgal cells for the phosphorous assay. The single variation was the volume of the digestion mixture (sulphuric acid and hydrogen peroxide mix). Where 2.5 ml was used for the nitrogen digestion procedure only 0.3 ml was used for this method. A 0.2 ml aliquot of 96% sulphuric acid was added to the dehydrated algal pellet and heated as

digest solution was then made up to 10 ml with distilled water as in the previous nitrogen digestion process (Section 2.14.2).

A method developed by Dorich and Nelson (1983) was also utilised for the phosphorous colorimetric assay. This method required the use of molybdate reagent. For the preparation of molybdate reagent 4.3 g ammonium molybdate and 0.4 g antimony sodium tartrate were dissolved in 400 ml distilled water. A 54 ml aliquot of 96% sulphuric acid was then carefully added to the above solution. The resulting mixture was allowed to cool in an ice bath and thereafter made up to 1000 ml with distilled water.

A 0.5 ml aliquot of the cell digest, obtained using the protocol outlined above, was transferred to a sterilized (autoclaved for 30 minutes at 121 °C) and well dried test tube.

2 ml 1% ascorbic acid and 1.5 ml molybdate reagent was added to the test tube and the contents were mixed well using a vortex. An incubation period followed for an hour at room temperature for full color development. Absorbances of the resulting solution were then read at 820 nm using a S2100 Diode Array Spectrophotometer (Biowave – Labotec).

The phosphorous content was determined with the aid of the absorbance readings and a previously constructed standard curve using potassium dihydrogen orthophosphate as a standard (Figure 2.8).

y = 0.0792x R² = 0.977

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

0 1 2 3 4 5 6 7 8 9 10 11

Phosphorous Concentration (ug/ml)

Absorbance (820nm)

Figure 2.8: Standard curve depicting phosphorous concentration with respect to absorbance readings at 820 nm. Error bars represent standard deviation (n = 3).