Algal Growth

The light-scattering properties of C. reinhardtii cells have been used to measure the chlorophyll (Chl) concentration and the optical density (OD) of the algal culture. The chlorophyll (Chl) concentration was quantified using the classical method developed by Arnon (Arnon, 1949). A small sample of culture (10-20 ml) was removed under sterile conditions. A few microlitres (typically 200 µl) were extracted from this sample using a pipette and diluted in 80% acetone – this

gives a dilution factor (DF) of 5. The solution was placed in a mini-centrifuge and spun at 13,000 rotations per minute (rpm) for 5 minutes. At the end of this procedure, a white pellet of algal cells formed, indicating that all Chl had dissolved. A spectrophotometer was then used to measure the light absorbance of the Chl sample at the photosynthetically active wavelengths of 645 nm (OD645) and 663 nm (OD663). It is possible to calculate the Chl content from these absorbance measurements using Equations 5.06-5.08 (Arnon, 1949).

Chl a (µg·ml-1_{) = (12.7 · OD663 - 2.69 · OD645) · DF Equation 5.06}

Chl b (µg·ml-1_{) = (22.9 · OD645 - 4.68 · OD663) · DF Equation 5.07}

Total Chl (µg·ml-1_{) = (20.2 · OD645 + 8.02 · OD663) · DF Equation 5.08}

The Chl concentration units (µg·ml-1_{) are in fact identical to nutrient concentration units (mg·l}-1_). The main advantage of using Chl content as a measure of algal growth is that the results show a strong correlation with the health of the C. reinhardtii culture (Markov et al., 2006). Any sign of contamination or malnutrition can quickly be detected and steps can be taken to prevent further fouling of the culture (Siaut et al., 2011). On the other hand, Chl measurements are time-consuming and cannot be carried out in situ. No two algal cells are the same, nor will they have the same Chl concentration; there are strong variations in Chl concentration between algal cultures and even within a single culture. This implies that specific Chl concentration targets are difficult to reproduce and H2 production measurements are therefore often specified per unit of Chl (Tsygankov et al., 2002; Melis & Melnicki, 2006).

OD is a direct measure of the algal cell density. An OD probe consists of a light emitting diode (LED) and a photodetector, located a short distance apart (an OD probe with an optical path length of 10 mm was used in the Sartorius tubular PBR). Light scattering caused by algal cells reduces the photocurrent in the probe and generates an OD measurement in absorbance units (AU). The LED emits light in the near infra-red (IR) region of the spectrum (840-910 nm) to avoid interference from the photosynthetic Chl absorption peaks. In the absence of an OD probe, a spectrophotometer was used to measure OD. In this case, a small sample of C. reinhardtii was extracted and analysed under the appropriate wavelength: at 663 nm to capture the C. reinhardtii absorption maximum or at 875 nm to avoid photosynthetic absorption peak interference. OD measurements were much

quicker and simpler to carry out than Chl concentration measurements. More importantly, it was also possible to carry out OD measurements continuously and in situ, using OD probes that were readily available on the market (Akkerman et al., 2002). OD is therefore a better choice for measuring algal growth in PBRs, particularly in light of the need to scale up and automate the H2 production process. The main disadvantage of OD measurements is that they do not discriminate between green C. reinhardtii cells, dead cells, contaminants or impurities and hence may not be fully representative of the cell density of the C. reinhardtii culture (Melis & Melnicki, 2006). Since OD measurements are carried out at sub-micrometer wavelengths, the majority of light scattering actually takes place off the organelles of C. reinhardtii cells (the cell diameter is 5-10 µm). This leads to OD measurement errors during the exponential phase of algal growth; C. reinhardtii daughter cells are smaller and have less developed organelles than the mature cells and are therefore less likely to register in the OD measurements (Posten & Schaub, 2009).

For the purpose of this thesis, a correlation has been established between OD measurements at 663 nm and at 875 nm and the corresponding Chl concentration, calculated from Equation 5.08 (Figure 5.01a & 5.01c). These correlations enable a quick calculation of the Chl content without the need to continuously implement Arnon’s method; the relationships are given in Equation 5.09 & 5.10.

OD663 (mg·l-1 Chl) = 17.9 · OD663 (AU) Equation 5.09

OD875 (mg·l-1 Chl) = 28.0 · OD875 (AU) Equation 5.10

The OD measurements have also been correlated to a C. reinhardtii cell count carried out using a microscope (Figure 5.01b & 5.01d). The cell count involves manually counting the number of cells in a small square and multiplying this value by 250,000. It should therefore be used only as a very rough approximation of the cell density. Cell counts can be calculated using Equation 5.11 & 5.12.

OD663 (cells) = 2.6·107 · OD663 (AU) Equation 5.11

(a) (b)

(c) (d)

Figure 5.01: Optical density (OD) calibrations

(a) Relationship between OD and Chl content at 663 nm (b) Relationship between OD and Cell count at 663 nm (c) Relationship between OD and Chl content at 875 nm (d) Relationship between OD and Cell count at 875 nm

5.2.4. Biohydrogen Production

Biohydrogen production can be expressed in terms of a production yield (mlH2·l-1), a production rate (mlH2·l-1·h-1) or a photochemical efficiency (%). Given that H2 has a molecular mass of 2 Daltons and a density of 0.089 g·l-1 _{at standard ambient temperature and pressure (Hemmes et al.,} 1986), it is possible to calculate the unit conversions for H2 production yield given in Equation 5.13.

1 mmolH2·l-1 = 2 mgH2·l-1 = 22.5 mlH2·l-1 Equation 5.13

H2 production rates follow the same unit conversions as H2 production yields. To calculate the H2 production yield per unit of Chl (mlH2·mg-1Chl), it is necessary to simply divide the H2 production yield (mlH2·l-1) by the Chl content (mgChl·l-1). Photochemical efficiency can be calculated using Equation 5.14 (Akkerman et al., 2002).

Photochemical efficiency [%]= H2 production rate [l⋅ s −1_{]⋅ H}

2 energy content [J⋅l −1_]

Absorbed energy [J⋅ s−1] ⋅100

Equation 5.14

The H2 production rate is measured experimentally. The higher heating value for H2 of 142 J·l-1 is used as the H2 energy content (Hemmes et al., 1986). The absorbed energy depends on the irradiation incident on the PBR and on the active reactor surface area (Tamburic et al., 2011b).

Table 5.05: Important unit conversions

Variable Alternative unit Unit used in thesis

Light intensity 1 µE·m-2·_s-1 _{0.2 W}·_m-2

SO42- concentration 1 mM 0.096 mg·l-1 CH3COO- concentration 1 mM 0.059 mg·l-1 NH4+ concentration 1 mM 0.018 mg·l-1 PO43- concentration 1 mM 0.095 mg·l-1 pO2 concentration 1 mg·l-1 12% OD663 1 AU 17.9 mg·l-1Chl OD875 1 AU 28.0 mg·l-1Chl H2 yield 1 mmolH2·l-1 22.5 mlH2·l-1

5.3. Photobioreactors