Mass cultivation of new algae Tetraselmis straiata BBRR 1 under open raceway ponds for biodiesel and biocrude production

Annakkili Baskara Boopathy1,2,3, Thanasekaran Jayakumar3, Muthu Ganasan Rajaram1, Natarajan Mohan1, Chinnasamy Senthil4, Subramanian Nagaraj1, Ramasamy Rengasamy1, Manjunath Manubolu5, Joen-Rong Sheu3*, Chao-Chien Chang6,7,8* 1Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai-600 025, Tamil Nadu, India; E-Mail: annakkilibaskar@gmail.com (A.-B.B); raja.ram091@gmail.com (M.-G.R); jnmohan08@gmail.com (N.M); nagalilly@gmail.com (S.N); profrrengasamy@yahoo.com (R.R)


Introduction
In the process of modernization and development, human being always needed energy, which increased the dependency on the available fossil fuel sources.The exhaustive use of fossil fuel sources has raised the serious concerns worldwide not only about energy security but also for negative impact on environment.India's growing demand for petroleum-based fuel has created challenges for the country's energy security, as almost 90% of its crude oil requirement is imported from oil producing countries.Algae have been widely used for fuel production because of their high photosynthetic efficiency, high biomass production, and fast growth [1].Microalgae as biomass has potential chemical composition such as proteins, lipids and carbohydrates [2].
Many algae accumulate substantial amounts of non-polar lipids, mostly in the form of tri acyl glycerol or hydrocarbons, and these levels may reach up to 20-50% of dry cell weight.Algae can also grow in saline, brackish and coastal seawater with slight struggle [3].Microalgae can provide an alternative biofuel feedstock due to their rapid growth rate, greenhouse gas fixation ability (net zero emission balance) and high production capacity of lipids and do not compete with human and animal food crops.Moreover, they will grow on non-arable land and saline water [4].
Microalgae cultivation is a promising methodology for solving some of the future problems of biomass production (i.e.renewable food, feed and bioenergy production).There is no doubt that in conjunction with conventional growth systems, novel technologies must be developed in order to produce the large-scale sustainable microalgae products [5].The most conservative scenario contains algae oil from microalgae grown in open ponds on non-arable land filled with salt water [6].Many algae are exceedingly rich in oil, which can be converted into biodiesel.The oil content of some microalgae exceeds 80% of the dry weight of algae biomass [7].Aquatic biomasses present an easy adaptability to grow in different conditions either in fresh or marine water or in a wide range of pH.This makes the aquatic biomass more adaptive or an enhanced CO2 fixation to afford a high biomass production.CO2 is usually bubbled from beneath at rates for optimum uptake by microalgae cells [8].Microalgae are traditionally considered as good source of fatty acids [3], and the fatty acids in microalgae are suitable for biofuel synthesis [9].The fatty acid profiles of microalgae has been well established [10,11].Microalgae have a strong capacity to produce lipids, which can be easily converted to biodiesel.
The most cost effective cultivation system for mass culture production of Tetraselmis straiata (T.straiata) BBRR1 is to grow in an outdoor open airway pond.However, trials on cultivating T. straiata in non-axenic systems often fail due to thriving of other green algal and cyanobacterial contaminants, and hence it relatively slow downs the growth of T. straiata.
Therefore, methodological or technological breakthrough to control the growth of these contaminating species is awaited to minimize the oil production cost using microalgae.The aim of the present study is to mass cultivate the alga T. straiata isolated from the saltpans bodies Kovelong, Chennai, Tamil Nadu, India in the open raceway pond.The growth performance in terms of biomass, pigments, lipids, proteins and carbohydrates were analyzed.Carotenoids are linked with numerous health benefits, such as hindrance of age-related macular degeneration, cataract, some cancers, rheumatoid arthritis, muscular dystrophy and cardiovascular problems.
Since T. straiata holds substantial amounts of carotenoids, this species may also have health benefits for human beings.In the present attempt, water samples collected from the saltpans (60 ppt) Kovelong, Chennai, Tamil Nadu, India, were brought to the laboratory and segregated by using spread plate technique (Figure 1a,b.) on f/2 agar medium.Four different isolates of quardriflagellate alga, Tetraselmis were isolated and identified as, Tetraselmis chuii, T. gracilis, T. straiata and T. tetrathele based on their morphological features of colour, colony, morphology and cell size [12].Tetraselmis is a euryhaline microalga and commonly present in saltpans and marine environments [13,14].The colonies T. chuii Butcher is a green four-flagellated alga, ovoid body shape with a distinct curve (cell length: strains, the f/2 medium is the most commonly used medium [15].

Dry biomass and total lipids in Tetraselmis species
The four different species of microalgae Tetraselmis were taken for the growth analysis under laboratory conditions revealed that the isolate of T. straiata showed a maximum biomass and lipid content of 0.58 ± 0.021 g L -1 (Table 1) and 151 ± 3 mg L -1 (Table 2), respectively, and followed by T. chuii, T. tetrahele and T. gracilis (Figure 2a, b).Huerlimann et al. [16] observed that T. gracilis showed a maximum biomass of 0.35 g L -1 , which was 8.0% less than the test alga, T. straiata and the specific growth rate of 0.25.Arkronrat et al. [17] reported a maximum and a specific growth rate of 0.16 in Tetraselmis sp., which was 36% less than the present alga, T. straiata.In the present study, the isolate of T. straiata Butcher had maximum lipid content than the other three isolates.Thirty-day-old culture of T. straiata Butcher was harvested and analysed the biochemical parameters.T. straiata had maximum lipid content of 33.60% dry weight and lipid productivity of 29.14 mg L -1 day -1 (Table 3).Huerlimann et al. [16] had found only 18.6 mg L -1 day -1 of lipid productivity on Tetraselmis sp., which was 37% less than the present isolate T. straiata.
The morphology of alga within in each chemical competition could vary in relation to age and culture conditions [18].As the morphological heterogeneity of the alga makes the identification difficult, molecular tools like PCR play a vital role in confirming the systematic position of the experimental alga.In the present study, a partial 18S region of the ribosomal RNA gene was isolated and amplified with the specific primers.The sequence was found 99% similarity with T. straiata JQ315813 KMMCC 1157 strain.The obtained sequence was compared with the existing sequences in the NCBI database by the BLAST algorithm homology (sequence identity) confirmed with a close relationship of the isolated candidate T. straiata.
Based on the classical taxonomy as well as molecular taxonomy, the test alga was identified and confirmed as T. straiata Butcher BBRR1 (Figure 3).The sequence was submitted in the GenBank, NCBI and the Accession Number is KP317837.

Phytochemicals, lipids, biomass and fatty acids of T. straiata BBRR1
The isolate T. straiata BBRR1 performed well than the rest of three isolates in the of 0.05 ± 0.004 mg L -1 and 0.03 ± 0.002 mg L -1 of chlorophyll a and b were increased during the growth period and they raised up to 6.13 ± 0.005 mg L -1 and 5.23 ± 0.003 mg L -1 on 15 th and 18 th day, respectively.The concentration of chlorophyll a and b were recorded maximum on 15 th day and reached a plateau thereafter (Figure 4 a, b), although the productivity in terms of biomass continued to increase until day 15.This data indicated that the cells continued to grow resulting on the net increase in the biomass concentration up to day 15.Similarly, the initial concentration of 0.15 ± 0.002 mg L -1 of total carotenoid content was increased up to 56.5 ± 0.23 mg L -1 on 18 th day (Figure 4c).The initial biomass of 0.12 ± 0.01 g L -1 was increased up to a maximum of 0.95 ± 0.06 g L -1 on Modified CFTRI-ABRR I medium, which was 28% higher than control on 15 th day (Figure 5).Based on the total biomass productivity, the lipid production and comparative biochemical analysis was calculated in T. straiata BBRR1 cultivated under laboratory and open raceway pond as shown in Tables 3 and 4. Daily biomass productivity for the T. straiata BBRR1 cultivated under open raceway pond was observed about 0.063, and 0.050 g L -1 d -1 , specific growth rates of 0.45 and 0.39, division rates of 0.64 and 0.56 and generation times of 1.55 and 1.79 in the Modified CFTRI-ABRR I medium and f medium (control), respectively (Table 5).The present isolate, T. straiata BBRR1 showed an aerial biomass productivity of 8.83 g L -1 m 2 d -1 in Modified CFTRI-ABRR I medium.Similarly, Fon-Sing and Borowitzka [19] reported that the alga recorded average biomass productivity of 8.3 g L -1 m 2 d -1 in Tetraselmis sp.
The alga had a maximum total lipid of 152 ± 39 mg L -1 on 18 th day, which was 19% more than control (f medium).Total lipid percentage / ash -free biomass of the sample in Modified CFTRI-ABRR I medium was 27.50%, as against 29.76% in control on 18 th day.The organism showed lipid productivity of 8.43 and 7.06 mg L -1 d -1 when it was grown in Modified CFTRI -ABRR I medium and control medium, respectively.Tetraselmis was the most suited for the mass cultivation due to fast growth, ease of culture and relatively high lipid content.Rodolfi et al. [11] reported Tetreselmis and Nannochloropsis showed high lipid productivity and found to be the promising marine species for biomass production.
Nutrients management could change biochemical constituents of algae reported by Hsieh and Wu [20].During the study period, the lipid content in T. straiata BBRR1 increased due to growth limiting factors like nutrient deficiency in the lack of nitrogen and phosphate sources.
Pernet et al. [21], Li et al. [22] and Arumugam et al. [23] also reported that the biomass and lipid content of microalgae were affected at different cultivation and nutrients conditions.The culture in the open system was observed periodically under compound microscope, and the end of the study the algal biomass in the pond was harvested and dried.The different parameters such as lipids, proteins, and carbohydrates were similar to the observations made under laboratory conditions and open raceway ponds (Table 4).
The alga T. straiata BBRR1 produced 15.2% of lipid content on its dry weight basis under open raceway pond.The total content of lipids varies from 1-85% of the dry weight with values higher than 40% being typically reached under nutrient limitation studied by Regan [24].
The fatty acid composition of hexane extracted FAMEs was analysed by GC-MS.The isolate T. straiata BBRR1 revealed the presence of 33.14 % of Palmitic acid, methyl ester, followed by benzenedicarboxylic acid, oleic acid, arachidonic acid, eicosapentaenoic acid and 9-Octadecenoic acid methyl ester.
Based on the results obtained in the present study, the volumetric productivity of biomass were 0.089 g L -1 d -1 in Modified CFTRI-ABRR I medium.This study also indicates that T.
straiata BBRR1 has the potential to produce 48.72 dry tons of biomass ha -1 year -1 .In the view of shrinking fossil fuel sources, algae mass cultivation for biofuel production is gaining importance day by day.However, the cost of production of algal biomass needs to be reduced below $200 dry tons to make algae as an attractive biomass feedstock for biofuel production.
However, the cost of growth medium used for mass cultivation of T. straiata BBRR1 was estimated as $53,000.Similarly, the cost of the cultivation media required to produce 1 ton of algal biomass through open raceway ponds was estimated as $1128 t -1 .As the estimated cost of growth medium used for mass cultivation of T. straiata BBRR1 was higher than the selling cost of biofuel algae, future research efforts are needed to reduce the nutrients cost through the use of municipal, agricultural and industrial waste streams rich in organic and inorganic nutrients which are renewable in nature.

Isolation of Tetraselmis spp.
The algal samples collected from the saltpans of Kovelong, Chennai, Tamil Nadu, India were brought to the laboratory and the colonies were segregated based on their morphological features using compound microscope [12].A single colony of the four different isolates was picked up with the help of micropipette.After thoroughly washed with sterile f/2 medium for five times, the isolates was subjected to grow in the f/2 agar medium [15].The culture medium was kept at 24 + 1 o C in thermostatically controlled room, illuminated with cool fluorescent lamps at irradiance of 30 µEm -2 s -1 , under 12/12 light dark cycle.

Laboratory studies
Experiments were conducted in 500 mL Erlenmeyer flasks with 270 mL of sterile f/2 medium and inoculated with 30 mL of optimally grown four different isolates of T. straiata separately.The experimental studies were carried out for a period of 21 days.The growth parameters such as cell number, biomass [25], the levels of different pigments such as Chlorophyll a, Chlorophyll b and carotenoids [26], total carbohydrates [27], proteins [28] and lipids [29] were recorded at every 3 days interval during the study period.It revealed that T.
straiata Butcher showed good growth and lipid productivity than the rest.Therefore, this alga was chosen for further investigation.

Molecular characterization
The genomic DNA samples of T. straiata were isolated from the lyophilized algal biomass using the PCR kit obtained from GENEI, Bangalore, India.RNA contamination was eliminated by digesting the extract with 10 µg of RNase-A for 30 min at 37°C.The amount of DNA of the sample was quantified by measuring its absorbance at 260 nm in a spectrophotometer.The optical density (OD) of 1.0 corresponds to 50 µg/mL of double stranded DNA [30] and the purity of DNA was determined at 260 and 280 nm.DNA solution that had a value of 1.8 obtained from the data recorded at 260 nm/ 280 nm was considered as pure.
In the present study, the 18S rRNA gene region of the T. straiata isolates were subjected for the amplification of the primers (GENEI, Bangalore, India) as described by Richards et al. [31].The forward primer 5'-GTAACCCGTTGAACCCCATT -3' and reverse primer 5'- CCATCCAATCGGTAGTAGCG -3' were used as described by Liu et al. [32].Polymerase chain reaction (PCR) was performed in a ABI thermal cycler (ABI) using a PCR program with initial denaturing at 95℃ for 5 min, followed by 35 cycles of denaturation at 95℃ for 1 min, annealing at 58℃ for 55 seconds and extension at 72℃ for 50 seconds and a final extension at 72℃ for 10 min.The PCR products were separated through agarose gel electrophoresis.The purified PCR products were separated using 1.4 % agarose gels and stained with 0.5 µg/mL ethidium bromide and the gel was viewed and captured using Vilber Loumart Gel Documentation system.
Sequences were determined by the chain termination method with the use of Dye Deoxy Terminator Cycle Sequencing Kit (Perkin Elmer Applied Bio -system, UK) using an AV1377 automated DNA sequencer.

Familiarization of T. straiata Butcher BBRR1 for outdoor condition
The isolate of T. straiata BBRR1 grown under laboratory condition produced high biomass concentration.This experiment was conducted for a period of 21days in batch mode.At every 3 days interval, pH, biomass, pigments, total carbohydrates, proteins and lipids were analyzed and recorded.Microscopic analysis was carried out daily to check the purity of the culture.The average temperature and light intensity were ranged from 28°C to 35°C and 42000 and 52000 lux, respectively.At the end of the study period, the algal biomass was harvested and analyzed for different parameters.

Biomass harvest of T. straiata BBRR1
In open raceway ponds, the biomass of T. straiata BBRR1 was harvested by switch off the paddle wheel for a period of 24 h.The biomass was collected after the medium was drained off.
The algal cells settled at the bottom in the open raceway pond was harvested after 12 h through autoflocculation on 21 day.The biomass in the open raceway pond was washed with ground water and the cells were allowed to settle.This process was repeated for 3 times in order to remove the excess salt in the algal biomass.The washed algal cells were spread on white plastic sheet and dried over the sun light for 3 h followed by oven drying at 60°C for 8 h.

Biomass estimation
Twenty five millilitres of algal culture was taken and washed thrice with 25 mL of isotonic solution containing 0.65 M ammonium formate [25] in order to remove excess salt.Pre weighed Whatman GF/C glass microfiber filters (1.2 µm) was used to obtain the biomass after filtration in the moisture analyser (Mettler Toledo HR83).Then, the filter along with the biomass was placed in the moisture analyzer and its final dry weight was recorded after drying at 100˚ C for ~8 minutes.Dry weight was calculated after subtracting the filter weight and expressed the values as g L -1 .

Extraction of algal oil and fatty acid analysis
Ten grams of dried algal biomass was treated with hexane 1:5 (w/v) and extracted algal oil by using Soxhlet apparatus at the boiling temperature of the solvent for 8 h.The solvents containing the algal oil were filtered through Whatman GF/C filter paper.The solvents were recovered using rotary evaporator at the respective boiling temperature.The algal oil was recorded gravimetrically and expressed the values as ash-free biomass.

Acid transesterification
Ten gram of total lipid was extracted from the alga by using chloroform and methanol (2:1) and 0.6 mL g -1 of sulphuric acid.The reaction mixture was kept at 90˚C in a water bath for 40 min and intermittently mixed.Then, it was allowed to cool at room temperature, and added 2.0 mL g -1 of distilled water and mixed for 45 sec.After phase separation the solvent layer was used for all the analyses.Identification of FAME was made by matching their recorded spectra with the data bank mass spectra of NIST library V 11 provided by the instruments software.

Statistical analysis
Throughout the study period, triplicates were maintained for each experiment.The results were expressed as Mean ± standard deviations.Data were analysed statistically using Origin Pro.v 8.0 for Windows.

Conclusion
This study envisaged on researching the feasibility and sustainability of microalgae as biofuel feedstock to meet the energy crisis.In the present attempt, four different species of laboratory conditions and was scaled up to 150 L in an open pond.It was then transferred to the raceway pond containing 1350 L Modified CFTRI -ABRR I medium.The initial concentrations Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 19 October 2018 doi:10.20944/preprints201810.0449.v1 yield and lipid content was scaled up to1 and 2 L in flasks for 2 weeks and further scaled up to 10 L in transparent carboys for 2 weeks.The 15 L culture grown under laboratory condition was used as seed material, inoculated in 135 L medium in a 1.0 m 2 of 150 L capacity mini open raceway pond (Length 2.2 m; Width 0.50 m; Depth 0.26 m) floors are coated with FRP (Fibre-Reinforced Polymer) material and incubated for 2 weeks.The raceway ponds are provided with paddle wheel system for the aeration at 10 rpm.The culture was mixed with paddle wheel system during daytime.The alga in the pond was subjected for adaptation to open air condition for 2 cycles (sub-culturing at an intervals of 2 weeks).During the experimental period the evaporation of water was compensated with chlorine treated bore well water.The raceway ponds were protected from dust on the top with transparent polythene sheet.Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 19 October 2018 doi:10.20944/preprints201810.0449.v13.5.Mass culture of T. straiata BBRR1 in open raceway pond This experiment was conducted in a concrete raceway pond 10.0 m 2 of 1500 L capacity (Length 7 m; Width 1.5 m; Depth 0.26 m) floors are coated with reinforced polymer.The inoculum was raised in the following order: Optimally grown 15 L of T. straiata BBRR1 culture obtained from the laboratory condition was inoculated into 1.0 m 2 raceway pond with a biomass concentration of 0.06 g L -1 contained 135 L medium and kept for 10 days.Then the culture (150 L) was inoculated to 10.0 m 2 raceway pond contained 1350 L of basal (f/2) medium and the culture height in the pond was maintained at 15 cm level.The algal culture was mixed with paddle wheel system during day time to prevent settling and enhance dissolved CO2

Preprints
(www.preprints.org)| NOT PEER-REVIEWED | Posted: 19 October 2018 doi:10.20944/preprints201810.0449.v1contained biodiesel of fatty acid methyl ester (FAME) was collected and transferred to a pre weighed glass vial.The solvent was evaporated using liquid N2, and quantified the amount of biodiesel gravimetrically.The fatty acid composition of the sample was analysed by GC-MS.FAME was analyzes by GC-MS (Agilent 6890 gas chromatograph, 15 m Alltech EC -5 column (250 μ I.D., 0.25μ film thickness).A JEOL GCmate II bench top double-focusing magnetic sector mass spectrometer operating in electron ionization (EI) mode with TSS-2000 1 software

Figure 2 .
Figure 2. (a) Dry Biomass and (b) total lipids of Tetraselmis spp. at different intervals.

Figure 4 .
Figure 4. (a) Chlorophyll a (b) Chlorophyll b and (c) total carotenoids of T. straiata BBRR1 in Modified CFTRI ABRR I medium at different intervals in 10.0 m 2 open raceway

Figure 5 .
Figure 5. Dry biomass of T. straiata BBRR1 in Modified CFTRI ABRR I medium at different intervals in 10.0 m 2 open raceway pond

preprints.org) | NOT PEER-REVIEWED | Posted: 19 October 2018 doi:10.20944/preprints201810.0449.v1 contaminations
Tetraselmis belong to Chlorophytes were isolated as T. chuii Butcher, T. gracilis (Kylin) Butcher, T.straiata Butcher and T. tetrathele (G.S.West) Bucher.Among these isolates, T. , it can be used as a novel biomass feedstock to produce various biofuels.This work revealed the potential of T. straiata BBRR1 for biofuel production and could match the demand of energy in future.

Table 2 .
Total lipid content of T.tetrahele, T.gracilis, T.chuii and T.straiata grown in laboratory condition.

Table 3 .
Biomass, lipid productivity and lipid percentage of different Tetraselmis spp.

Table 4 .
Comparative biochemical analysis of T. straiata BBRR1 grown under laboratory and open raceway pond.

Table 5 .
Daily productivity, specific growth rate, divisions per day and generation time of T.

Table 6 .
Lipid profile of T. straiata BBRR1 grown under open raceway pond