Essay: Solar driven mass cultivation and extraction of lipid from Chlorella variabilis: A case study.

ABSTRACT
Chlorella variabilis grown in hypersaline environment (3-8 ��Be) in open solar salt pans during the summer season at temperature 45��3��C showed promising results towards development of renewal energy. A total of 746.23 Kg dry biomass generated during the cultivation period with average calorific value of 3885 kCal/kg and an average biomass productivity of 34.59 g/m2/d (dry basis) was achieved with total lipid content of 22.59 �� 0.9% (w/w) on dry basis. A total of 771.63 m2area was used for cultivation with 360 m3 culture volume. The average calorific value of lipid obtained was 9288 kCal/kg. The total energy input of 33.12 MJ for lipid extraction from one kg of microalgal biomass could be easily sufficed from solar energy to improve energy output to input ratio of extraction process. The fatty acid profile of the lipid extracted from C. variabilis showed its suitability for the biodiesel production. Further, unmodified diesel motor vehicle was run successfully using the produced B100 biodiesel. Hence, marking the entire process a solar driven cost effective and energy efficient leads towards sustainable development of microalgae based biofuel for future commercialization.
Keywords: Mass cultivation; Solar salt pans; Chlorella variabilis; Solar Extraction; Biodiesel

1. Introduction
Microalgae, as a source of biofuel is having many advantages over traditional biofuel crops including the potential to be grown on the marginal land, the use of water sources not suitable for agriculture (e.g. high salt content could be tolerated). Potentially, microalgae can provide fuels in several distinct forms (Kr��ger and M��ller-Langer, 2012) microalgal biomass can be utilized for combustion; utilizing crude algal oil for direct combustion for its usage in other transportation fuels such as diesel, gasoline and jet fuel (kerosene) (Patidar et al., 2015); preparation of biogas through anaerobic digestion of the biomass (Zamalloa et al., 2011; Markou et al., 2013); biohydrogen; bioethanol via fermentation of carbohydrates derived from algae (Matsumoto et al., 2003; Ho et al., 2013); and bioethanol produced directly through algal photosynthesis (Williams, 2009; L�� et al., 2011). Basically as compared to the terrestrial crops, which take a season to grow and contain a maximum of about 5% of dry oil content, microalgae grow quickly and contain high oil content (Chisti, 2007).
For microalgal biofuel production at commercial scale, biomass generation is a major bottleneck being faced. It is therefore necessary to make the process economically feasible and to ensure an uninterrupted supply of biomass for consumer markets (Brennan et al., 2010). Aside from carbon, in the form of carbon dioxide, microalgae require nitrogen, which is associated with their primary metabolism. Lipids are synthesized as a reserve material during the nutritional stress conditions. Phosphorus is a major nutrient for growth of microalgae and should be added to the medium in the form of phosphates. In addition to these components, trace metals such as Mg, Ca, Mn, Zn, ad Cu, and vitamins are added to the culture medium for higher productivity (Liu et al., 2011; Mata et al., 2010; Lardon et al., 2009). Higher temperatures accelerate microalgae metabolism while lower temperatures lead to inhibition (Mendes and Vermelho, 2013). Microalgae may be cultivated in open systems or closed photobioreactors.for mass cultivation; Closed system e.g. in photobioreactors, at operational volumes of 50-100L or even at higher volumes, evenly distribution of light is not possible(Chen 1996, Pulz 2001). Large scale open ponds can be built of plastic, concrete and bricks. (Oswald, 1992; Tredici, 2004). Nowadays, most commonly used open system is the ‘raceway pond’, an oval form resembling a car-racing circuit (Lee, 2001; Bhowmick et al.2014; Chisti, 2007). Basically these cultivation systems require relatively low construction and operating cost for which pilot systems can be made on degraded and non agricultural lands/coastal land(Chen, 1996; Tredici, 2004). Open ponds have several advantages over closed systems e.g. cost effective, lesser energy to operate, utilizes direct solar energy for growth of microalgae, lesser maintenance (Brennan et al., 2010). Additionally, the open systems are economically advantageous, but have problems of contamination from protozoa, viruses, other species of algae, fungi, and airborne micro-organisms, besides not allowing the control of the water temperature and light. In the last few years, various strains of Chlorella studied with much focus in open cultivation for CO2 sequestration in order to reduce he effects of global warming (Kleinheinz & Keffer, 2002, Yanagi et. al.,1995).
There are various reports on the effect of biomass productivity of Chlorella sp. affected by culture depth. Fang et al., 2013 reported increase in the biomass productivity due to increase in the culture depth from 2-10 cm in open ponds from 8.41-11.22 g/m2/d. One of the key factor in increasing biomass productivity is optimal depth and optimal cell density (Fang et al., 2013). Nowadays, Chlorella sp. is getting global attention for biofuel production due to its biomass productivity, oil content/fatty acid profile, adaptation to various physico-chemical parameters ,autoflocculating capability, etc. (Doucha et al., 2009, Hartig et al., 1988, Hsieh et al., 2009, Liang et al., 2009, Lv et al., 2010, Wang et al., 2013). It is very difficult to select a particular culture technology in terms of the consistent biomass productivity as mass culture system varies with different geographical locations, culture strategies (batch, continuous or semi- continuous systems), salinity, etc. (Chaumont, 1993). According to Weissmann et al., 1988, biomass productivities ranges from 10-50g/m2/d with an average around 20g/m2/d.
At present there is very less literature available on mass cultivation of microalgae in solar salt pans using sea water as the medium. Therefore, we decided to prepare a model for mass cultivation of halotolerant microalgae (Chlorella variabilis) in open solar salt pans using sea water as the growth medium.
2. Material & methods
2.1. Experimental set up
2.1.1. Organism
Oleaginous CSIR-CSMCRI’s Chlorella variabilis (ATCC PTA 12198) was grown in CSMCRI’s – modified Zarrouks’ medium consisting of g/l NaHCO3 25, NaNO3 2.5, K2HPO4 0.5, 1.0 K2SO4, 1.0 NaCl, 0.04 CaCl2, 0.08 Na2EDTA, 0.2 MgSO4.7H2O, 0.01 FeSO4.7H2O, 1 ml of A5 trace metal solution consisting of g/l H3BO3 2.86, (NH4)6Mo7O24 0.02, MnCl2.4H2O 1.8, CuSO4.5H2O 0.08 and ZnSO4.7H2O 0.22 and 1ml of B6 trace metal solution consisting of mg/ NH4NO3 22.96, K2Cr2(SO4)4.24H2O ‘ 96, NiSO4.7H2O ‘ 47.85, Na2SO4.2H2O ‘ 17.94, Ti(SO4)3 40, Co(NO3)2.6H2O 49.48.
2.1.2. Inoculum development
The inoculum was initially prepared in 20 L carboys from 1L shake flask. Thereafter the inoculum was further scaled up to the open plastic tank with dimensions 1.1 x 1.1 m each having 10 cm depth.
2.1.3. Cultivation site
The institute’s own Experimental Salt Farm was chosen as the cultivation site. Proximity to the seacoast also helped us in agitating the ponds during night due to the prevailing wind movement. Additionally, during the summer season, due to lesser wind movement, the cultures were agitated manually every 2 hours using a perforated hollow pipe (Fig. 1). Proper agitation ensured homogeneous mixing of the nutrients, proper gas exchange between the liquid bulk and the atmosphere and uniform exposure of each cell to sunlight for proper photosynthesis to take place. Seawater was easily available for our cultivation with which we avoided the use of freshwater. The seawater also had the added advantage of being rich in some nutrients such as nitrate, phosphate, sodium chloride, magnesium. Potassium, etc. The availability of abundant sunlight and prevailing high temperature conditions during the day were the other factors which were favorable for the selected strain.
2.1.4. Pond preparation
Preparation of nine solar salt pans with dimensions of 6 x 3 m each, six pans with dimensions of 15 x 6 m each & nine pans of 35 x 4 m each at the Experimental Salt Farm. Plastic (LDPE) liners were spread in each of them to prevent seepage of cultures. The total area covered under cultivation was 771.63 m2. A culture depth of 28 – 30 cm was maintained in all the pans. The total culture volume in the cultivation facility was 360 m3 (360,000 L) having 771.63 m2 total cultivation area.
2.1.5. Culture media
Chlorella variabilis was grown in each of the ponds using pristine seawater. Initially, two of the ponds (6 x 3 m each) were supplemented with 5 g/l sodium bicarbonate, 1.2 g/l sodium nitrate and 0.0125 g/l ferrous sulphate to acclimatize the algae. All the chemicals used were of commercial grade. These two ponds were used as inoculum for the entire cultivation. The inoculum ponds were allowed to acclimatize and were further used for inoculating all the other ponds. The culture volume transferred in each case was 10% (v/v).
2.2. Mass cultivation
The cultivation was carried out during peak summer season in Gujarat, India in the months of April -June 2012. The outdoor temperature during the cultivation was 45��3 ��C. and the water temperature range throughout the cultivation was observed to be 40 �� 3��C. The culture needed was first grown in two inoculum raising ponds with an area of 18 m2 each (Fig. 1). The ponds were monitored regularly by measuring the pH, OD at 540 nm and biomass yield. Cell concentration of 5g/l (wet basis) was used to inoculate 7 more tanks with an area of 18 m2 each and 3 tanks with an area of 90 m2 each. PH, OD at 540 nm, biomass yield and environmental parameters were regularly monitored. The ponds were agitated manually (thrice a day) using a hollow pipe tied with strings at its ends up to 18 days.
After 20 days of cultivation, it was observed that the biomass settled automatically at the bottom of the ponds. The supernatant from each pond was transferred into an empty tank for charging the next batch as the inoculum and the settled biomass was collected and sun-dried.
Environmental parameters were monitored regularly using a multi-parameter probe (PCD 650, Thermo Fisher Scientific, USA) and optical density was measured at 540 nm using a UV-Visible spectrophotometer (Cary Bio 50, Varian Inc., USA). Environmental parameters studied included pH, electrical conductivity, total dissolved solids (TDS), total salinity, dissolved oxygen (DO) and nitrate ion concentration. The available seawater was analysed for its nutrient status determination
2.3. Estimation of biomass productivity
Biomass productivity was calculated using the following relation:

BP (g/m2/d) =

Additionally, individual productivities for each of the ponds were calculated separately using the same relation
Carbon fixation rate were calculated using the formula given below:
Carbon fixation rate = BP x 1.88 (Wang et al. 2008; Chisti et al. 2007)
2.4. Calorific value and proximate composition of microalgal biomass
The calorific value or heat of combustion was measured by using a bomb calorimeter following ASTM D 240. The measurements were done in the triplicate using 1 g each of the sample. The moisture content and ash content were determined gravimetrically by following AOAC 925.04 and AOAC 938.08 methods. The 10 g samples were taken for drying at 105 ��C until constant weight and difference is calculated to be the moisture content. The water free biomass was there after burnt at 500 ��C for 5 h and ash content was determined. Later the Carbon, Nitrogen, Hydrogen and Sulfur content was analyzed using the CHNS analyzer.
2.5. Lipid estimation
Lipid content was quantified gravimetrically from the sun-dried Chlorella variabilis biomass using Bligh and Dyer [Bligh and Dyer, 1959]. The microalgal lipid was extracted thrice, after which the clear extracts were observed, by treating one gram biomass with 10 ml of Chloroform and Methanol (1:2 v/v). The extracts were pooled and filtered for removal of cellular debris. The pooled extract was evaporated under vacuum to dryness at 55 ��C using B��chi rotary evaporator.
2.6. Conventional electricity run solvent extraction of non-polar lipids using Soxhlet
The solid phase extraction of Chlorella variabilis biomass was carried out using n-hexane, a non-polar solvent. The microalgal biomass (10 g) was packed in a cellulosic thimble and placed inside the extraction vessel of the Soxhlet. The solvent was placed inside the collecting vessel of the apparatus. The extraction was run for 16 h with the total of 20 refluxes. The extracted lipid was quantified gravimetrically and later re-dissolved to analyze the fatty acid composition. The fractions were taken further for fatty acid analysis through GC/MS.

2.7. Solar energy mediated Soxhlet extraction of non-polar lipids
The setup of Soxhlet was assembled as represented in the Fig. 3 and tracking was done so that the solar radiation focus at the center of the parabolic trough depicted in the figure to heat the collecting vessel. The condenser maintained at 10 ��C was placed over the extraction vessel containing biomass filled cellulosic thimbles clamped over the collecting vessel, which was circulated with the cooling water provided by the chiller running on the solar panels. The focus of the parabolic trough was at 80��C. The extracted lipid was determined gravimetrically by evaporating the solvent. Further, the lipid was analyzed through GC/MS for fatty acid profile.
2.8. Fatty acid profiling
The lipid was transmethylated to fatty acid methyl esters (FAMEs) using 1 ml of 1% NaOH in MeOH, followed by heating at 55 ��C for 15 min. Thereafter, 2 ml of 5% methanolic HCl was added with heating at 55 ��C for 15 min (Carreau and Dubacq, 1978). The FAMEs prepared were then separated by adding 1 ml of hexane to the reaction mixture. The FAMEs containing hexane were analyzed by a GC-2010 gas chromatograph coupled with mass spectrometer (GC’MS QP-2010, Shimadzu, Japan). The FAME s were analyzed through a gas chromatography mass spectrometer (GC-2010 twinned with a GC’MS QP-2010) of Shimadzu (Japan). RTX-5 fused silica capillary column (30 m x 0.25 mm, 0.25 ��m) maintained flow rate of 1 ml/min and a pre-column pressure of 49.7 kPa with Helium (99.9% purity) as a carrier gas was used. The column temperature regime was 40 ��C for 3 min, followed by ramping at the rate of 5 ��C/min up to 230 ��C and then maintained at 230 ��C for 40 min. The injection volume and temperature were 1.0 ��L and 240 ��C, respectively with the split ratio of 1/30. The mass spectrometer operated in electron compact mode with 70 eV of electron energy. The ion source and the interface temperature were set at 200 ��C (Kumari et al., 2010). FAME peaks were compared with the standard FAME peaks with respect to their retention times (Standard FAME Mix C4’C24; Sigma Aldrich) by GCMS Post-run analysis and quantified by area normalization
2.9. Comparison of the energy consumption in oil extraction
The energy of oil was calculated by using bomb calorimeter as described above while the energy used in extraction of oil from the microalgae was calculated using an energy meter across the conventional soxhlet apparatus.
3. Results & discussions
3.1. Growth in open mass cultivation
The biomass productivity was found to be maximum during Summer’ 2012 having 45 g/m2/d with average biomass productivity of 32.45 g/m2/d,(Table 1). During Summer’ 2012 (April ‘ June’ 2012), there was a peachy increase in the pH from the initial day of inoculation upto the day of harvesting i.e the 21st day which clearly indicated that the increase in the growth of the biomass which may be due to bicarbonate uptake (Table 2). It was found that the optical density and pH were directly proportional to each other i.e. in the ponds as the optical density increased, the pH also increased. The concentration of dissolved CO2 was found to decrease up to 9th day and thereafter it was found to be stable. It was observed that the initial CO2 concentration present during the initial day was 10 ppm which was in decreasing order till the 9th day (1 ppm) and thereafter the dissolved CO2 concentration was stable till the day of harvesting i.e on 21st day (Fig. 2). These changes may be attributed due to two main causes; Chlorella variabilis favors free CO2 over bicarbonate when free CO2 concentration were comparatively higher up to 10 ppm in initial growth period and, increasing pH lowers down CO2 concentration due to conversion of CO2 in to bicarbonate on successive growth. It was noticed that dissolved free CO2 was limiting after 9th day of growth period. It was found that total dissolved inorganic phosphate uptake by Chlorella variabilis was 16.08 ��M during Summer’ 2012 on 21st day (Table 2). There was significant decrease in total dissolved solids on each successive day of the growth. Nitrate uptake rate was 19.05 ppm was observed during Summer’ 2012 due to maximum photosynthetic rate of Chlorella variabilis in comparatively higher temperature to build amino acids, proteins and enzymes for metabolism. Nitrate uptake rate is function of temperature in many strains and increases on high temperature (Reay et al. 1999).
3.2. Calorific value and proximate composition of microalgal biomass
Lipids are synthesized as a reserve material during the nutritional stress conditions. Higher temperatures accelerate microalgal metabolism while lower temperatures lead to inhibition (Mendes and Vermelho, 2013; Chokshi et al., 2015). It is very difficult to select a particular culture technology in terms of the consistent biomass productivity as mass culture systems varies with different geographical locations, culture strategies (batch, continuous or semi continuous systems), salinity, etc. (Chaumont, 1993).The average aerial biomass productivity in the present study was 34.59 g/m2/d. and maximum was 45 g/m2/d whereas total lipid content of the sundried C. variabilis biomass was quantified and found to be 22.59 �� 0.9% (w/w) on dry basis. The average calorific value of biomass and lipid are 3885 kCal/kg and 9288 kCal/kg, respectively which are also evident from Zemke et al. 2010. The C. variabilis biomass on an average composed of 48.95%, 6.183% and 0.25%, Carbon, Hydrogen and Sulphur respectively.
3.3. Lipid extraction using conventional Soxhlet
Lipid extraction from 1 kg of sun-dried C. variabilis biomass, packed into the cellulosic thimbles, was done in a regular Soxhlet apparatus of capacity 10 L at 80�� C temperature for 16 hrs with 5 L n-hexane. 4 kWh of energy was used in heating the hexane in collector vessel for 18 hour extraction time, after which no lipid extract was visibly seen in the extract collected in flask. The extract was filtered and filtrate was evaporated to yield solvent free lipid. The lipid obtained was 10.53 �� 0.07% (w/w).
3.4. Solar energy mediated lipid extraction in Soxhlet apparatus
Solar energy based non-polar lipid extraction was performed with the Soxhlet extractor equipped over the centre of parabolic solar concentrator as shown in Fig1. Lipid was extracted from 1 kg sun-dried microalgae C. variabilis, packed inside cellulosic thimbles and placed within the Soxhlet column over the 10 L round bottom flask with 5 L of n-hexane and adjusted at the focus of the parabolic solar concentrator. The assembly was cooled by a condenser maintained at 10 ��C by the chiller ran on solar photovoltaic panels. The soxhlet was run for 18 h after which the pooled extract was filtered and evaporated to obtain the solvent free 9.48 �� 0.68 % (w/w) lipid on the dry basis.
3.5. Comparison of the energy consumed during the extraction
The energy inputs calculated by energy meters installed across chillers and heating devices, it was found that 4 kWh of energy was consumed in heating device for 16 h run of heating mantle in heating hexane, while 5.2 kWh of energy was consumed in chilling operations for maintaining condenser at 10 ��C. The total energy input was calculated to be 33.12 MJ for extracting lipids from one kg of microalgal biomass while the energy value of oil extracted was found to be 3.27 MJ. The conventional soxhlet apparatus proves to be 11 times more energy intensive while in solar energy mediated solar extraction devoid of energy inputs and gives the positive energy balance.
3.6. Fatty acid composition of non polar lipid extracted through conventional soxhlet extraction
Total lipid content of 22.59 �� 0.9% (w/w) on dry basis was obtained from the biomass cultivated during summer’ 2012. The fatty acids present in the microalgal oil are 0.4% of C14:0, 12.1% of C16:0, 1% of C16:1, 1% of C16:2, 4.2% of C18:0, 29.4% of C18:1, 45.7% of C18:2, 4.8% of C18:3 and 1.4 % of C22:0 (Table 3). However, the degree of unsaturation of the extracted non polar lipid was 39% which was quite low. The overall fatty acid composition of non-polar lipid extracted from the Chlorella variabilis biomass was found suitable for making engine worthy biodiesel due to presence of lower degree of unsaturation (Mishra et al., 2012; Yu et al., 2012).
4. Cconclusion
Mass cultivation of microalgae is of vital importance in the backdrop of their increasing economic importance as well as the need for large-scale production of various commercially important compounds from microalgae. In the studies done here, it was revealed that we have identified the best strain of Chlorella which is halo-tolerant as well as thermo-tolerant with auto-settling capability as it was found growing between 3-8 Be’ salinity having maximum biomass productivity of 45 g/m2/d during summer season. Also this process is based on the microalgal mass cultivation in open solar salt pans utilizing the unused land and seawater of the salt works makes over all cultivation cost effective as compared to the conventional raceway ponds. Drying of the auto-settled biomass was done utilizing natural solar energy which has also proved to be an important tool in the development of extraction process which is highly energy intensive. Therefore, this case study is one of the best for microalgal mass cultivation and extraction of lipids utilizing less energy where we have used sun, sea and shore in a most efficient manner.

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