CHAPTER 3: MICROALGAE PROCESSING FOR JET FUEL PRODUCTION
3.2 MATERIAL AND METHODS
3.2.1 Sampling and culture maintenance
To obtain pure Nannochloropsis sp. cells for cultivation and the rest of the experiment, new cells were cultivated on petri dishes. On these petri dishes, colonies of Nannochloropsis sp.
cells from a stock solution made up of Nannochloropsis sp. cells were spread in parallel lines crossing each other. Parallel lines were drawn in one direction to reduce the risk of contamination. These lines of cells in solution were spread on a viscous and thick mixture made of F/2 media and 15 g of agar added to the petri dishes presented in Figure 4. Thereafter, petri
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dishes with colonies of cells were kept in an incubator at 25 °C with the aim of generating pure Nannochloropsis sp. cells.
After 5 to 7 days, cells appeared as pellets on the surface of the thick mixture. Some cells were pure and others were not due to probable contamination of growth. To minimise contamination of pure cells on the petri dishes, pure cells were isolated and put on new petri dishes with the thick mixture of F/2 media-agar. Lastly, they were kept at 25 °C in the incubator again. After a period of 7 days, pure cells of Nannochloropsis sp appeared on the surface of the thick mixture. These pure cells or pellets were collected and added to F/2 media solution to run a cultivation batch in a photobioreactor, presented in the left part of Figure 5.
The photobioreactor produced the biomass under photosynthetic conditions for a cultivation period of 15 days. The stock solution was also collected for future use in case of sample loss or contamination or any other incident occurring during experiments.
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Figure 4: Cells of Nannochloropsis sp. on petri dishes for cultivation
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Figure 5: cultivation batch (left) and F/2 media solution (right) 3.2.2 Media preparation for species cultivation
The importance of F/2 media regarding algae biomass production is to provide nutrients effectively to microalgae cells during cultivation. This allows effective growth of cells under operating conditions defined by light, temperature, pH, and salinity. Microalgae need nitrates, phosphates, and trace elements including heavy metals for an effective growth. Media solutions such as F/2 are an ideal environment in which these elements are mixed and supplied to microalgae during cultivation. Nitrates allow the production of nucleic acid and proteins, and contribute greatly to synthesis and lipid production, while phosphates are acting as energy carriers.
The saline wastewater was prepared in the laboratory to suit the cultivation purposes in domestic wastewater which is rich in nutrients such as nitrates and phosphates. The salinity of wastewater was important because Nannochloropsis sp is a marine species that grows well in a saline environment.
F/2 media is prepared according to Guillard and Ryther’s (1962) method.[42] It is widely used with saline water or seawater for the cultivation of many species including marine species. In this study, the F/2 media preparation begins with 950 ml of saline water. This is prepared from tap water mixed with commercial salt at 30 ppt salinity, which is considered as the average
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salinity for seawater in which Nannochloropsis sp. can effectively grow. Defined quantities of NaNO3, NaH2PO4.H2O, Na2SiO3.9H2O, trace elements, and vitamins were added to saline water following Guillard and Ryther’s method.[42] The final volume is brought to 1 l by adding saline water or natural filtered seawater to the photobioreactor. Finally, the mixture is sterilised for 1 hour at 105 °C in an autoclave. The uptake of nutrients by microalgae cells takes place during cultivation, so microalgae cells grow as much as they absorb nutrients up to the time the growth process reaches the stationary phase. At this stage, harvesting can be undertaken and a new batch can be started.
3. 2.3 Cultivation of the species and growth under proficient monitoring of the system Cultivation of Nannochloropsis sp. aims to produce as much as biomass possible to be used for downstream processes.These processes need more biomass with high lipid content in order to generate more crude bio-oil. An effective, monitored system is required with defined operating conditions. Effective monitoring is very important because it can assist in the optimisation of the biomass production. In this study, cultivation was undertaken for a maximum of 15 days, as mentioned above, and is presented in Figures 6 to 10.
Experiments were carried out in a 1 l photobioreactor (see Figure 4). The salinity of 30 ppt was chosen with the aim to simulate species growth conditions in the marine environment.
Generally, the average seawater salinity favourable for species growth varies between 25 and 35 ppt. Nannochloropsis sp. being a marine species, it was essential to grow it under conditions of salinity similar to the marine environment. Concerning the pH, the culture was maintained at values equal to or slightly above 7. The biomass was growing at an ambient temperature between 15 °C and 35 °C with an average light intensity of 1000 lux using fluorescent light for 24 hours of illumination. CO2 was added daily to these batches and its volume was equivalent to 15 % of the photobioreactor.[43] The addition of CO2 was intended to boost the lipid content of the growing biomass. To expose the cells to maximum illumination, facilitate gas exchange, and provide effective mixing, biomass aeration was undertaken during cultivation. Growth data are summarised in table 6.
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Table 6: summary of growth data for Nannochloropsis sp biomass
Parameter Salinity Temperatu re range
Average pH
Light intensity (average)
CO2
addition
Cultivation cycle and mode
Value 30 ppt Controlled:
15,20,25,30 and 35 OC
Neutral 1000 lux 5 % of the total volume
15 days in a photobioreactor