Strategies for enhancement of alpha-linolenic acid rich lipids in Desmodesmus sp. without compromising the biomass production

Journal Pre-proofs Strategies for enhancement of alpha-linolenic acid rich lipids in Desmodesmus sp. without compromising the biomass production P.V. Sijil, Vinaya R. Adki, R. Sarada, V.S. Chauhan PII: DOI: Reference:

S0960-8524(19)31445-2 https://doi.org/10.1016/j.biortech.2019.122215 BITE 122215

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Bioresource Technology

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1 August 2019 26 September 2019 26 September 2019

Please cite this article as: Sijil, P.V., Adki, V.R., Sarada, R., Chauhan, V.S., Strategies for enhancement of alphalinolenic acid rich lipids in Desmodesmus sp. without compromising the biomass production, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.122215

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Strategies for enhancement of alpha-linolenic acid rich lipids in Desmodesmus sp. without compromising the biomass production P.V. Sijila,b, Vinaya R. Adkia , R. Saradaa,b and V.S. Chauhana,b⁎ a

Plant Cell Biotechnology (PCBT) Department, CSIR-Central Food Technological

Research Institute (CFTRI), Mysuru - 570 020, India b

Academy of Scientific and Innovative Research (AcSIR), Ghaziabad - 201 002, India

*Corresponding author E-mail: [email protected]; [email protected] Address for correspondence Dr. Vikas Singh Chauhan, Sr. Principal Scientist, Plant Cell Biotechnology (PCBT) Department, CSIR - Central Food Technological Research (CFTRI), Mysuru - 570 020, Karnataka, India, Phone: +91-821-2516501 Abstract The indigenous freshwater microalga Desmodesmus sp. produces ALA rich lipids (about 23%). The phytohormones (DAH and KIN; 0.5 mg L-1) increased the biomass yield and lipid content of microalga by 1.4 to 1.7 fold. Mixotrophic cultivation (500 mM glucose and 100 mM sodium acetate) enhanced the biomass yield and lipid content by 1.8 to 2.7 fold. The sodium azide (1.0 mM) led to a 1.5 fold and 1.7 fold enhancement in the lipid content and ALA fraction of total fatty acids, respectively without affecting the biomass yield. The low temperature (5 °C) as the second stage of cultivation enhanced the ALA fraction of total fatty acids by 1.2 to 1.5 fold for untreated, phytohormone supplemented and mixotrophic cultures, without affecting the 1

biomass yield. These cultivation strategies could, therefore, be used for enhancement of ALA rich lipids in microalgae without compromising the biomass yield. 1. Introduction The omega-3 fatty acids, such as alpha-linolenic acid (ALA), are associated with health benefits viz, cardioprotection, anti-inflammation, neuroprotection, pre, and postnatal brain development, etc. (Barceló-Coblijn and Murphy, 2009; Doughman et al., 2007). Plant sources such as flaxseed, rapeseed, and soybean oils are the major sources of ALA (Barceló-Coblijn and Murphy, 2009); however, additional sources are needed to meet the increasing global demand. In recent years, the microalgae have emerged as an important sustainable source of omega-3 polyunsaturated fatty acids (PUFA) rich lipids (Sun et al., 2018). Sijil et al., (2019) have described an indigenous microalga Desmodesmus sp., which accumulates higher content of ALA rich lipids under stress conditions. The microalga subjected to nitrogen depletion and low temperature (5 °C) stress showed an enhancement of 1.7 and 1.5 fold in its lipid content, respectively with the low temperature leading to 1.8 fold increase in ALA fraction of total fatty acids (Sijil et al., 2019). However, the enhancement in lipid content and/or ALA fraction of total fatty acids under these stress conditions was associated with reduced growth and biomass productivity. The abiotic stress-based strategy to induce the enhancement in lipid content in microalgae has been shown to suffer from the drawback of reduced growth and biomass productivity in various microalgae e.g., Scenedemus sp. under salinity stress (Pancha et al., 2015), Chlorella minutissima under nitrogen starvation (Arora et al., 2016) and Nannochloropsis oculata under low temperature cultivation (Converti et al., 2009). Therefore, it is important to develop the strategies to enhance the lipid content of microalgae without compromising the biomass production. The

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phytohormones, sodium azide and mixotrophy have been recognized for their lipid enhancing effect on microalgae without adversely affecting the growth (Singh et al., 2016, Rai et al., 2013). The two-stage cultivation has also been suggested for enhanced lipid production without loss in biomass. In two-stage cultivation strategy, the culture in the first stage is allowed to grow under favourable conditions for maximum biomass production and then subjected to stress (e.g., nutrient limited conditions) for enhanced lipid accumulation (Singh et al., 2016). The present study was focused on the production of ALA rich lipids in Desmodesmus sp. without compromising the biomass production. The effect of phytohormones, mixotrophic cultivation, and sodium azide was studied on growth, lipid content and ALA fraction of total fatty acids of the microalga. The low temperature (5 °C) stress which had previously shown significant enhancement in ALA fraction of total fatty acids (Sijil et al., 2019), was studied for its possible use as an ALA fraction enhancing second stage of two-stage cultivation. 2. Materials and methods 2.1. Microalgal culture maintenance and measurement of growth and pigments The cultures of the indigenous microalga Desmodesmus sp. CFR 1-01/FW described earlier by Sijil et al., (2019) were maintained at 25±1 °C under the cool white fluorescent light of 30 μEm-2 s-1 intensity with 16:8 h light: dark cycle in sterile Bold’s basal medium (BBM). The purity of the culture was ensured by repeated subculturing, and microscopic observation using a light microscope (Olympus BX51, Japan) fitted with a camera and imaging software (ProgRes C5, Germany).

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The optical density (OD) of cultures was measured at regular intervals at 560 nm (UV-VIS Spectrophotometer SP 3000, Optima, Tokyo, Japan) to monitor the growth. The freeze-dried biomass was obtained at different ODs, and a linear regression was used to establish the correlation between OD (x) and biomass (dry cell weight g L-1) (y) for the culture: y=0.5729x−0.0504 (R2=0.9831) as described previously (Sijil et al., 2019). The specific growth rate (μ day-1) was calculated by the equation, μ=1/t×ln (Xm/X0), where ‘Xm’ and ‘X0’ are the biomass concentrations (g L-1) at the end and beginning of the batch culture, respectively, and ‘t’ is the duration of the batch culture. The equation D=0.693/μ was used to calculate the doubling time (D). The biomass yield (Y) (g L-1) was calculated by the equation Y=Xm-X0 and the biomass productivity (P) (g L-1 day-1) by the equation P=Xm-X0/T2-T1, where ‘T2-T1’ represents the incubation period of an experiment with ‘T1’ and ‘T2’ being the initial (day 0) and final day (in number) of incubation, respectively (Converti et al., 2009; Vidyashankar et al., 2015). The pellets obtained by the centrifugation of culture aliquots were extracted with methanol for spectrophotometric estimation of pigments using Lichtenthaler's equations (Lichtenthaler, 1987). 2.2. Effect of phytohormones, mixotrophy, sodium azide, and two-stage cultivation The effect was studied on growth parameters, photosynthetic parameters, pigments, lipid content, and fatty acid profile. An initial biomass concentration of about 0.23 g L-1 and a culture volume of 100 mL in BBM medium was used for all the experiments. The effect of phytohormones was studied by allowing the cultures to grow individually with two different phytohormones, viz, kinetin (6-Furfurylaminopurine) (KIN) and Diethyl Aminoethyl Hexanoate (DAH) at concentrations of 0.1, 0.5, 1 and 5 mg L-1. A stock of 100 mg L-1 of the phytohormone was prepared by dissolving KIN in

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0.1 M NaOH and DAH in distilled water. The effect of mixotrophy was studied by allowing the cultures to grow individually with two different organic carbon sources viz, glucose, and sodium acetate at concentrations of 50, 100, and 500 mM. The effect of sodium azide was studied by allowing the cultures to grow with 0.1, 0.5, 1, and 5 mM sodium azide. A stock of 100 mM sodium azide was prepared in distilled water. The concentrations of phytohormones, organic carbon source, and sodium azide were selected based on the preliminary experiments and literature survey. The effect of low temperature as the second stage of the two-stage cultivation was studied by allowing one set of cultures to grow for 12 days at 25 °C followed by incubation at 5 °C for 12 days. Another set of cultures remained at 25 °C for the whole duration of the study (i.e. 24 days) and served as control (single-stage cultivation). 2.3. Measurement of photosystem II (PS II) related parameters The aliquots (2 mL) of cultures were kept in the dark for 15 min and the chlorophyll fluorescence of these dark-adapted cultures were measured using a portable Pulse Amplitude Modulated (PAM) fluorometer (AquaPen-C AP-C 100, Photon Systems Instruments, Czech Republic) to analyse the PSII related parameters (Markou et al., 2017; Mathur et al., 2011). The photosynthetic parameters measured were (i) Fv/ Fm: the maximum photosynthetic efficiency (quantum yield or QY); (ii) ABS/ RC: total number of photons absorbed by reaction centres (average antenna size) and (iii) DI0/RC: total dissipation of un-trapped excitation energy from all reaction centres (dissipation energy). 2.4. Lipid extraction and fatty acid methyl ester (FAME) analysis

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The extraction of total lipids was carried out by chloroform and methanol (2:1 ratio) from a known quantity of freeze-dried biomass (100 mg), and the fatty acids were esterified as described by Vidyashankar et al., (2015). After dissolving in HPLC grade n-hexane, the fatty acid methyl esters were injected in GC (Shimadzu 2010 plus, Japan) equipped with flame ionisation detector (FID). The FAME separation was carried out by a poly (dimethyl) siloxane capillary column (30 m×0.32 mm ID×0.25 μm film thickness) (Rtx-1, Restek Inc. USA). A temperature program of 120 °C (5 min hold) to 280 °C (10 min hold) at a ramp rate of 5 °C min-1 was used. Nitrogen was used as a carrier gas, and the injection port temperature was set at 220 °C. The fatty acids were identified by comparing their retention times with standard FAME mixture (C-8 to C24 FAME mix, SUPELCO) and confirmed by the fragmentation pattern by GC MS (Perkin Elmer, Turbomass Gold, Mass spectrometer, USA). The fatty acids composition was expressed as a relative percentage composition of total FAME (Vidyashankar et al., 2015). 2.5. Statistical analysis The data were expressed as the mean ± SD of three replicates, which represent three independent experiments. One way ANOVA followed by Tukey Kramer multiple comparison post hoc tests at a significance level of P < 0.05 was used for statistical analysis of the difference between the groups. 3. Results and discussions 3.1. Effect of phytohormones, DAH and KIN on Desmodesmus sp. 3.1.1 Effect on growth, pigments and photosynthetic parameters

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The phytohormone supplementation improved the growth profile of Desmodesmus sp. (Fig. 1). The supplementation of cultures with 0.5 to 5 mg L-1 DAH showed 1.3 to 1.4 fold increase in specific growth rate than control (0.068 µ day-1) with 1.0 mg L-1 DAH supplementation showing a maximum increase of 1.7 fold in biomass yield and 1.6 fold in biomass productivity. The cultures supplemented with 1 and 5 mg L-1 KIN showed a maximum increase of 1.5 fold in the specific growth rate and 1.8 to 1.9 fold in the biomass yield and biomass productivity. The doubling time of the phytohormone supplemented cultures reduced by 2.4 to 3.3 h than control (10.2 h). The growth parameters of the cultures supplemented with 0.1 mg L-1 DAH and KIN were comparable to control. The total chlorophyll and carotenoid content of the phytohormones supplemented cultures were either comparable to control or did not show any significant change (Table 1). Kinetin is a type of cytokinin hormone that promotes cell division (Park et al., 2013) and the newly discovered phytohormone, the DAH, also has a similar effect (Salama et al., 2014). Therefore, the primary role of cytokinin in cell division could be responsible for the enhancement in the growth of KIN and DAH supplemented cultures (Salama et al., 2014). The exogenous application of cytokinin to the microalgae increase the cell growth by stimulating the key enzymes involved in the nitrogen assimilation and amino acid synthesis like NADH-dependent glutamate dehydrogenases (Piotrowska and Czerpak, 2009; Renuka et al., 2017). The growth-enhancing effect of each phytohormone is observed over an optimum range of concentration and concentrations, higher or lower than the optimum, are ineffective due to the homeostatic regulation of intra and extracellular phytohormone levels (Renuka et al., 2017). In the present study, 0.5 mg L-1 DAH and 1.0 mg L-1 KIN appeared to be the optimum concentrations for the

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microalgal growth as the concentrations higher than these did not show any further significant enhancement in growth. The growth-enhancing effect of KIN and DAH in the present study was similar to the results reported for other microalgae. Park et al., (2013) have reported 1ppm kinetin as the optimum concentration for the growth of Chlamydomonas sp. and Salama et al., (2014) have reported 10-5 M as the optimum concentration of DAH for Scenedesmus obliquus. The PSII parameters of the cultures were monitored to study the effect of the phytohormone on the photosynthetic apparatus of the microalga. The variations in PSII parameters reflect the physiological status of microalgal cells (Markou et al., 2017). There was no perceptible difference in the initial maximum photosynthetic efficiency (Fv/Fm) of the control, and phytohormone (DAH and KIN) supplemented cultures at all the concentrations with values ranging from 0.67 to 0.69. At the end of the cultivation period, the maximum photosynthetic efficiency of the control culture reduced to 0.62. However, for DAH supplemented cultures (0.1 and 0.5 mg L-1) the values were 0.64, and for KIN supplemented cultures (all the tested concentrations), it was in the range of 0.64 to 0.68 (Fig.1). This result indicates that the phytohormone supplemented cultures were able to overcome the aging-related reduction in maximum photosynthetic efficiency to various degrees, depending on the concentration. The cultures supplemented with 0.1 mg L-1 DAH showed about 9% reduction in average antenna size and 19% reduction in the dissipation energy, compared to control at the end of the cultivation period. The culture supplemented with other concentrations of DAH showed a minor difference of 4% in antenna size and 8% in dissipation energy compared to control. At the end of the cultivation period, the KIN supplemented cultures showed a 7 to 14% reduction in the

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average antenna size than control. The culture supplemented with 5 mg L-1 KIN showed a maximum reduction of 27% in dissipation energy compared to control (Fig 1). It has been shown that maximum photosynthetic efficiency is inversely proportional to the photosystem antenna size (Ramanna et al., 2014). Therefore, a general trend of higher values of maximum photosynthetic efficiency and reduced antenna size in phytohormone supplemented cultures in the present study suggests an improved PSII activity of Desmodesmus sp. A recent study by Renuka et al., (2017) also showed that the maximum photosynthetic efficiency of the phytohormone supplemented Acutodesmus obliquucs culture remained high. 3.1.2. Effect on lipid content and fatty acid profile The DAH and KIN supplementation (0.5 to 5 mg L-1) enhanced the lipid content of Desmodesmus sp. The enhancement in the lipid content and biomass productivity by phytohormone supplementation also led to an enhancement in lipid productivity. The supplementation with 0.5 mg L-1 DAH and KIN showed a maximum increase of 1.7 fold and 1.5 fold in lipid content, respectively than control (23%) and 2.5 fold increase in lipid productivity compared to control (5.69 mg L-1 day-1) cultures (Table 1). As the biomass productivity and lipid content of 0.1 mg L-1 DAH and KIN supplemented cultures were comparable to control, the lipid productivity of these cultures was also comparable to control. The lipid enhancing effects of DAH and KIN have also been reported for other microalgae. The DAH supplementation at 10-7 M enhanced the lipid content from 28 to 33% in Chlorella ellipsoidea and 28 to 30% in Scenedesmus quadricauda (Jiang et al., 2015). The kinetin supplementation at 1.0 mg L-1 enhanced the lipid accumulation to 39% in Acutodesmus obliquus (Renuka et al., 2017).

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The lipid enhancing effect of exogenous supplementation of microalgal cultures with phytohormones could be attributed to a combination of factors. Phytohormones are known to mimic lipid biosynthesis inducing stress conditions and also induce the fatty acid synthesis to improve the fluidity and permeability of cell membranes for enhanced absorption of phytohormones (Contreras-Pool et al., 2016; Jiang et al., 2015; Renuka et al., 2017). They are also known to influence the expression pattern of various genes involved in the lipid biosynthesis pathway of microalgae (Jusoh et al., 2015a, 2015b). The ALA fraction of total fatty acids of DAH and KIN supplemented cultures were comparable to control (27%) with values in the range of 25 to 27% (DAH supplemented cultures) and 24 to 29% (KIN supplemented cultures) (Fig 1). However, an enhanced lipid accumulation led to an enhancement in the ALA content of the biomass of 0.5 and 1.0 mg L-1 DAH (1.6 and 1.5 fold) and KIN (1.4 and 1.3 fold) supplemented cultures, compared to control (6.2%) (Table 1). The enhanced lipid productivity of 0.5, 1, and 5 mg L-1 DAH and KIN supplemented cultures led to a corresponding increase in ALA productivity. The increase was 1.6 to 2.5 fold for DAH supplemented cultures and 2 to 2.4 fold for KIN supplemented cultures, compared to the control (1.47 mg L-1 day-1) (Fig.1). The DAH and KIN supplemented cultures showed an increase of 1.3 to 1.7 fold in their oleic acid fraction than control (9%) and a decrease of 28 to 58% in their stearic acid fraction than control (7%). The phytohormone supplementation has been reported to affect the fatty acid profile of microalgae, with a varied response shown by different microalgae. Polyunsaturated fatty acid content of Scenedesmus obliquus increased by 56% to 59% at 10-5 M of indole-3-acetic acid (IAA) and DAH supplementation (Salama et al., 2014). The DAH supplementation enhanced the saturated fatty acids in Chlorella ellipsoidea and

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unsaturated fatty acids in Scenedesmus quadricauda (Jiang et al., 2015). In the present study, the phytohormone supplemented Desmodesmus sp. cultures showed a comparable ALA fraction, an increase in oleic acid fraction, and a decrease in the stearic acid fraction of total fatty acids compared to control. The present study; therefore, further emphasizes that the effect of phytohormone on the fatty acid profile of microalgae varies from species to species. 3.2. Effect of mixotrophic cultivation on Desmodesmus sp. 3.2.1. Effect on growth, pigments and photosynthetic parameters The mixotrophic cultivation with sodium acetate and glucose led to an enhancement in the growth profile of Desmodesmus sp. (Fig. 2). The supplementation of cultures with 100 mM and 50 mM of sodium acetate and glucose was most effective showing a maximum increase in specific growth rate of about 1.5 fold (sodium acetate) and 2 fold (glucose) than control; a reduction of 3.2 to 3.3 h (sodium acetate) and 4.9 to 5 h (glucose) in the doubling time than control and an increase of 1.7 to 1.8 fold (sodium acetate) and 3 fold (glucose) in biomass yield and biomass productivity than control (Table 2). The sodium acetate supplemented cultures showed 18 to 24% reduction in total chlorophylls and 24 to 34% reduction in total carotenoids. For the glucose supplemented cultures, the reduction in total chlorophylls and carotenoids was 28 to 32% (Table 2). Microalgae under mixotrophic cultivation, grow with both light and organics as energy sources, with CO2 and organic carbon as carbon sources, i.e., they can perform photosynthesis and acquire exogenous organic nutrients (Zhan et al., 2017). Therefore, under mixotrophic cultivation, the microalga is not solely dependent on

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photosynthesis, and light is not the limiting factor for growth (Kong et al., 2011). This may be the reason for the observed increase in growth and decrease in photosynthetic pigments, i.e., total chlorophyll and carotenoid content in Desmodesmus sp. in the present study. The results of the present study are in agreement with previous studies reported for other microalgae. A reduction in chlorophyll and carotenoid content in other microalgae such as Phaeodactylum tricornutum and Chlorella vulgaris has been reported when supplemented with organic carbon (Kong et al., 2011; Liu et al., 2009). The mixotrophic cultivation of Chlorella vulgaris with sodium acetate at 1 g L-1 showed an increase in biomass yield from 0.21 g L-1 to 0.45 g L-1 with an increase in biomass productivity from 0.04 g L-1 day-1 to 0.07 g L-1 day-1 (Kong et al., 2011). Cultivation of Nannochloropsis sp. with 30 mM glucose showed a 1.4 fold increase in the biomass yield (Xu et al., 2004). In another study, the biomass concentration of Chlorella protothecoides progressively increased from 10 to 80 g L-1 glucose supplementation, however, then decreased at 100 g L-1, which might have been due to the substrate inhibition (Shi et al., 1999). A slight reduction in the growth parameters of Desmodesmus sp. observed in the present study at higher concentration of 500 mM sodium acetate and glucose might also have been due to the substrate inhibition. As the microalgal culture was not solely dependent on photosynthesis for growth under mixotrophy, changes in the photosynthetic parameters were observed. By the end of the cultivation period (on 12th day), the sodium acetate supplemented cultures showed 44 to 52% reduction in maximum photosynthetic efficiency; 2.3 to 2.5 fold increase in average antenna size and 3.1 to 3.6 fold increase in dissipation energy than control cultures (Fig. 2). The glucose supplemented cultures showed 63 to 75% reduction in the maximum photosynthetic efficiency; 3.2 to 4.2 fold increase in relative

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antenna size and 4.9 to 6.6 fold increase in dissipation energy than control on 12th day (Fig 2). The mixotrophic cultures of Desmodesmus sp. supplemented with glucose and sodium acetate showed changes in photosynthetic parameters from 3rd day and 9th day of the cultivation period, respectively. The results suggest that glucose is the preferred source of organic carbon compared to sodium acetate under mixotrophic cultivation. The results obtained in the present study were in agreement with an earlier study where mixotrophic cultivation of Phaeodactylum tricornutum with glucose and acetate led to 9 to 16% reduction in maximum photosynthetic efficiency (Liu et al., 2009). 3.2.2. Effect of mixotrophy on lipid content and fatty acid profile The mixotrophy enhanced the lipid content of Desmodesmus sp. with a maximum increase of 1.8 fold than control (23%) at 100 mM sodium acetate and 500 mM glucose supplementation (Table 2). The enhanced lipid content and/or biomass productivity contributed to enhanced lipid productivity of the cultures. A maximum increase of 3.3 fold and 4.8 fold in lipid productivity than control (5.44 mg L-1 day-1) was observed at 100 mM sodium acetate and 500 mM glucose, supplementation respectively. Mixotrophic cultivation provides the energy and material for lipid biosynthesis. The glucose supplementation improves the lipid biosynthesis by enhancing the acetylCoA (Acetyl Coenzyme A) and NADPH in the media (Wan et al., 2011). Similarly, the acetate, as acetyl-CoA, is involved in the fatty acid chain elongation and redirected to lipid accumulation (Ramanan et al., 2013). It has been reported that the mixotrophic cultivation increases the expression of various enzymes (like acetyl-CoA carboxylase and acetyl-CoA synthetase etc.) involved in the lipid biosynthesis pathways leading to

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enhanced lipid accumulation in microalgae (Ramanan et al., 2013; Wan et al., 2011). The lipid content of the Chlorella vulgaris increased from 8% to 18% by 20 g L-1 glucose supplementation (Kong et al., 2011). Similarly, supplementation of Chlorella pyrenoidosa with 1% sodium acetate enhanced the lipid content from 3 to 13%, reaching a maximum lipid productivity of 16.6 mg L-1 day-1 (Rai et al., 2013). The fatty acid profile of the Desmodesmus sp. underwent a significant change under mixotrophy. The mixotrophic cultivation with sodium acetate showed 28 to 42% decrease in ALA fraction and 1.4 fold increase in the linoleic acid fraction of total fatty acids than control at all the tested concentrations (Fig. 2). Similarly, the mixotrophic cultivation with glucose showed 23 to 35% reduction in ALA fraction with 1.6 to 1.8 fold increase in the linoleic acid fraction of total fatty acids than control at all the tested concentrations (Fig. 2). Even though the ALA fraction of total fatty acids of sodium acetate and glucose supplemented cultures decreased, the increased lipid content at specific concentrations meant an increase in the relative ALA content of biomass of those cultures. The relative ALA content of biomass of cultures supplemented with 500 mM sodium acetate and glucose increased by 1.2 to 1.3 fold, respectively than control (5.87%) (Table 2). The enhanced lipid productivity of mixotrophic cultures also enhanced the ALA productivity of Desmodesmus sp. The ALA productivity of the culture increased by 1.9 fold and 1.8 fold than control (1.36 mg L-1 day-1) at 100 and 500 mM sodium acetate, respectively. The increase in ALA productivity was 3.4, 2.9, and 2.7 fold than control at 500, 50 mM and 100 mM glucose supplementation, respectively. At 50 and 100 mM sodium acetate, the oleic acid fraction was 1.6 to 1.8 fold higher than control, whereas, at 500 mM sodium acetate, stearic acid fraction

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showed a 2.7 fold increase than control. In glucose supplemented cultures, there was no significant change in the profile of other fatty acid fractions compared to control. The mixotrophic cultivation has been reported to affect the expression of various fatty acid biosynthetic pathway genes (Ramanan et al., 2013; Wan et al., 2011), which might lead to change in the fatty acid profile of microalgae. Similar to the present study, changes in the fatty acid profile have been reported for mixotrophic cultures of other microalgae. Mixotrophic cultivation of Chlorella pyrenoidosa with sodium acetate led to a decrease of linolenic acid fraction from 38% to 12% with an increase in palmitic, oleic and linoleic acid fractions (Rai et al., 2013). Similarly, the mixotrophic cultivation of Nannochloropsis sp. with 30 mM glucose exhibited a decrease of omega-3 polyunsaturated fatty acid eicosapentaenoic acid from 18% to 15% with an increase in palmitic, palmitoleic and oleic acid fractions (Xu et al., 2004). 3.3. Effect of sodium azide on Desmodesmus sp. 3.3.1. Effect on growth, pigments and photosynthetic parameters The Desmodesmus sp. culture was supplemented with 0.1, 0.5, 1 and 5 mM sodium azide. The higher concentration of 5 mM sodium azide inhibited the growth of the culture (Fig.3). However the specific growth rate, doubling time, biomass yield, biomass productivity, total chlorophyll and carotenoid content of 0.1 to 1.0 mM sodium azide treated cultures were comparable to control (Table 3). Yahya et al., (2018) have reported that 2 µM sodium azide led to 30% and 8% reduction in the specific growth rate of Desmodesmus maximus and Chlorella pyrenoidosa respectively and 2% increase in the specific growth rate of Acutodesmus obliquus. In another study, 20 µM sodium azide showed a minimal effect on the growth of Chlorella desiccata (Zalogin and Pick,

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2014b). The sodium azide treatment leads to growth inhibition in microalgae by affecting the respiration system as one of its targets. However, different sensitivity shown by different microalgal species in their growth response to sodium azide, could be due to the different levels of permeability to azide and capacities to neutralize azide (Yahya et al., 2018; Zalogin and Pick, 2014a, 2014b). At growth inhibitory concentration of 5 mM, the maximum photosynthetic efficiency of the Desmodesmus sp. culture reduced from 0.65 to zero on sixth day (Fig. 3). The reduced maximum photosynthetic efficiency was accompanied by an increase in average antenna size (10.9 fold) and dissipation energy (3.7 fold) than control on sixth day. The adverse impact of growth inhibitory concentration of sodium azide on PSII subsequently led to the death of the culture. In contrast, at lower concentrations of sodium azide (0.1 to 1.0 mM), the effect on photosynthetic parameters was not so pronounced. The culture showed 4 to 9% reduction in maximum photosynthetic efficiency, 14 to 18% increase in average antenna size and 25 to 35% increase in energy dissipation at 0.1 to 1.0 mM sodium azide supplementation, compared to control culture (Fig 3). The data suggests that the growth inhibitory effect of azide at specific concentrations could also be attributed to its adverse effect on PSII. The azide is known to induce oxidative stress by inhibiting oxidative evolution of PS II (Singh et al., 2016). 3.3.2. Effect on lipid and fatty acid profile The supplementation of Desmodesmus sp. cultures with 1.0 and 0.5 mM sodium azide showed an enhancement in lipid content by 1.5 fold and 1.1 fold, respectively, compared to the control cultures (22%) (Table 3). The increased lipid content resulted in a corresponding increase in lipid productivity compared to the control (5.74 mg L-1 day-

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1

). The lipid content of the cultures supplemented with 0.1 mM sodium azide was

comparable to control (Table 3). Similar to the results obtained in the present study, the 2 μM azide

treatment enhanced the lipid contents of Acutodesmus obliquus from 31 to

51% and that of Desmodesmus maximus from 33 to 43% (Yahya et al., 2018). Sodium azide at 20 mM induced a maximum lipid accumulation (60 to 70%) in Chlorella desiccate (Zalogin and Pick, 2014a, 2014b). Azide is a well-known metabolic inhibitor in plants and algae, with many potential sites of action (Zalogin and Pick, 2014b). Zalogin and Pick (2014b) have suggested that the enhanced accumulation of lipid in microalgae may be due to the nitrogen stress caused by azide which lowers the nitrogen assimilation by reducing the activity of nitrate reductase enzyme. However, as in the case of growth, a varied response is seen in microalgae to the lipid enhancing effect of azide and one of the reasons could be different relative sensitivities of nitrate reductase to azide in different species (Yahya et al., 2018; Zalogin and Pick, 2014b). The ALA fraction of total fatty acids at 0.1 to 1.0 mM sodium azide treatment was in the range of 40 to 44%, an enhancement of 1.6 to 1.7 fold than control (25%) (Fig.3). Coupled with this, the oleic acid fraction of total fatty acids showed a 63 to 79% reduction than control (14%), and the linoleic acid fraction showed a 28 to 44% reduction than control (15%) at these concentrations. The enhanced lipid content and ALA fraction of total fatty acids contributed to the enhancement of the ALA content of the biomass of sodium azide treated cultures. The ALA content of 1.0 mM azide treated cultures showed the maximum ALA content of biomass of 15%, a 2.6 fold increase compared to control. The ALA content of biomass at 0.5, and 0.1 mM sodium azide treatment showed an increase of 1.9 and 1.6 fold, respectively, than control (5.6%). The

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increased lipid productivity and /or ALA fraction of total fatty acids resulted in a similar fold enhancement in ALA productivity than control (1.46 mg L-1 day -1) (Table 3). The exact mechanism by which sodium azide alters the fatty acid profile of microalgae is not clear. Azide induces oxidative stress in plants by inhibiting various metabolic pathways (Singh et al., 2016). The PUFA are known to serve as antioxidants in microalgal cells due to their high ability to absorb the reactive oxygen species (ROS) (Sharma et al., 2015). This affinity to ROS has been shown to enhance the PUFA content of microalgae (Sharma et al., 2015). Therefore, the increase in PUFA (ALA) by azide treatment observed in the present study might have been a defensive response of microalgae to the oxidative stress. Similar to the present study, the azide treatment exhibited an enhancement in C 18:3 PUFA in Desmodesmus maximus and Acutodesmus obliquus (Yahya et al., 2018). However, in Chlorella pyrenoidosa, no significant difference was observed in C 18:3 PUFA (Yahya et al., 2018). The results of the present study and the earlier studies, therefore, indicate that the PUFA enhancing effect of azide on microalgae is not universal and vary from species to species. 3.4. Effect of low-temperature incubation as the second stage of cultivation on biomass yield, lipid, and fatty acid profile of Desmodesmus sp. Two-stage cultivation method, where higher biomass production is achieved in the first stage which is then followed by a lipid accumulating second stage, has been suggested to overcome the challenge of lower biomass yield during stress-induced lipid accumulation (Singh et al., 2016; Vidyashankar et al., 2013). The available literature shows that lipid enhancing strategies generally use nutritional and salinity stress in the second stage of cultivation (Singh et al., 2016). In Scenedesmus dimorphus, a two-stage

18

cultivation with nitrogen stress and salinity stress as the second stages enhanced the lipid content from 13% to 23% without significantly affecting the biomass content (Vidyashankar et al., 2013). However, the PUFA enhancing conditions are not well reported as second stage cultivation strategy. The low-temperature stress has been previously shown to enhance the lipid and PUFA (ALA) content in Desmodesmus sp. (Sijil et al., 2019). Therefore, in the present study, the incubation of the culture at low temperature (5 °C) was studied for its potential to serve as the lipid and PUFA (ALA) enhancing second stage of cultivation. Two sets of the cultures treated/supplemented with each of the effective lipid enhancing concentrations of phytohormones (0.5 mg L-1 DAH), organic carbon (500 mM glucose) and sodium azide (1.0 mM), as described in the previous sections were taken for the study. Two sets of the cultures without any treatment (untreated) were also included. One set of the cultures was subjected to two-stage cultivation i.e., 12 days at 25 °C followed by incubation at 5 °C for 12 days. Another set of cultures remained at 25 °C for the whole duration of the study (i.e. single stage cultivation for 24 days) and served as control for corresponding two stage cultures. The biomass yield of the single stage cultivated control cultures or cultures subjected to the two stage cultivation remained unchanged at the end of the cultivation period (24 days) (Fig.4). The second stage cultivation at low temperature, therefore, did not affect the biomass yield. At the end of the cultivation period (24 days), the set of the cultures subjected to two-stage of cultivation showed a lipid content of 36, 38, 39 and 49% for untreated, DAH supplemented, azide treated and mixotrophic (glucose) cultures, respectively, which were 83%, 4%, 12%, and 16% higher than the corresponding single-stage cultivated control cultures (Fig.4). In their study to understand the mechanism of lipid

19

accumulation in microalgae under low temperature, Xin et al., (2011) have experimentally shown that the increase in the lipid content in Scenedesmus sp. LX1, at 20 and 10 oC was correlated with the increase in reactive oxygen species (ROS) level. In a recent study using the integrated transcriptome, proteome and fatty acid profiling, Xing et al., (2018), have shown that the low temperature stress in the heterotrophic cultures of microalga Auxenochlorella protothecoides promoted the chloroplast fatty acid synthesis by enhancing the expression of the plastidial acetyl-CoA carboxylase (ACCase) and type-II fatty acid synthase. At the end of the cultivation period (24 days), the ALA fraction of total fatty acids of the two-stage cultivated DAH supplemented and untreated cultures was 44% and 34%, an increase of 1.5 fold and 1.3 fold than the corresponding single stage cultivated control cultures, respectively. The ALA fraction of total fatty acids of mixotrophic (glucose supplemented) cultures was 23%, showing a 1.2 fold increase than the corresponding single-stage cultivated control culture (Fig.4). The low-temperature stress as the second stage of cultivation was, therefore, able to enhance the ALA fraction of total fatty acids of the mixotrophic cultures which otherwise tend to reduce in the single-stage cultivation (Fig. 2). The ALA fraction of total fatty acids (44%) of the two-stage cultivated DAH cultures was similar to the values shown by 1.0 mM sodium azide treated single-stage cultures. The enhancement of the lipid content and the ALA fraction of the total fatty acids contributed to the enhancement of the ALA content of the biomass of the two stage cultivated untreated, DAH and glucose supplemented cultures. The ALA content of the biomass of the two-stage cultivated DAH supplemented cultures was about 17%, an enhancement of 1.5 fold than the corresponding single stage cultivated control

20

cultures. The ALA content of the biomass of the two-stage cultivated untreated and mixotrophic (glucose) cultures was about 12%, showing a 1.4 to 1.5 fold increase compared to the corresponding single-stage cultivated control cultures (Fig 4). As reported earlier (Sijil et al., 2019), the increase in the PUFA content is one of the major cold stress adaptation of microalga Desmodesmus sp. used in the present study. The increase in the ALA fraction of total fatty acids of cultures incubated to lowtemperature as the second stage of cultivation, therefore, suggests that the low temperature can be a selective stress for modulating the fatty acid profile. Use of lowtemperature stress as second stage cultivation is also advantageous as it did not affect the overall biomass yield. However, the two-stage cultivated sodium azide treated cultures behaved differently than other cultures in the present study showing a 37% reduction in ALA fraction of total fatty acids and 29% reduction in ALA content of biomass, compared to the corresponding single-stage cultivated control cultures. This appears to be an effect of combining the metabolic stress caused by sodium azide with low temperature stress. In the earlier study with the Desmodesmus sp., the microalga showed 1.5 to 1.8 fold increase in the lipid content under nutritional stress (nitrogen starvation) and lowtemperature stress (5 oC), with ALA fraction of total fatty acids increasing by 1.8 fold under low-temperature (Sijil et. al 2019). However this was characterised by a significant reduction of 47 to 73% in growth parameters resulting in 29 to 52% reduction in lipid productivity (Sijil et al., 2019). The strategies used in the present study were able to enhance the lipid content by 1.5 to 2 fold without compromising the growth, reflecting in the increased lipid productivity. The azide treatment and two-stage cultivation (low temperature as second stage) of DAH and glucose supplemented

21

cultures significantly enhanced the ALA fraction of total fatty acids without any loss in biomass yield.

4. Conclusion The lipid content of Desmodesmus sp. enhanced by 1.5 to 1.8 fold by phytohormones (DAH and KIN, 0.5 mg L-1), mixotrophy (500 mM glucose and 100 mM sodium acetate) and sodium azide (1.0 mM). The biomass yield under these conditions increased or remained unaffected. The single-stage (25 °C) azide cultures and the two-stage (25 and 5 °C) DAH cultures showed a maximum ALA fraction of total fatty acids (44%). The maximum ALA content of biomass (17%) was obtained in two-stage DAH cultures. These strategies show the potential to enhance the ALA rich lipids in microalgae without compromising the biomass yield. Competing interest statement: Authors have no competing interests to declare. Acknowledgments The authors acknowledge the CSIR Mission Project (HCP 0019) for the support. PVS acknowledges the UGC, Govt. of India for the award of Senior Research Fellowship. Authors thank Director, CSIR-CFTRI for constant encouragement. References 1. Arora, N., Patel, A., Pruthi, P.A., Pruthi, V., 2016. Synergistic dynamics of nitrogen and phosphorous influences lipid productivity in Chlorella minutissima for biodiesel production. Bioresource Technology 213, 79–87.

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16. Piotrowska, A., Czerpak, R., 2009. Cellular response of light/dark-grown green alga Chlorella vulgaris Beijerinck (Chlorophyceae) to exogenous adenine-and phenylurea-type cytokinins. Acta Physiologiae Plantarum 31, 573–585. 17. Rai, M.P., Nigam, S., Sharma, R., 2013. Response of growth and fatty acid compositions of Chlorella pyrenoidosa under mixotrophic cultivation with acetate and glycerol for bioenergy application. Biomass and Bioenergy 58, 251–257. 18. Ramanan, R., Kim, B.-H., Cho, D.-H., Ko, S.-R., Oh, H.-M., Kim, H.-S., 2013. Lipid droplet synthesis is limited by acetate availability in starchless mutant of Chlamydomonas reinhardtii. FEBS letters 587, 370–377. 19. Ramanna, L., Guldhe, A., Rawat, I., Bux, F., 2014. The optimization of biomass and lipid yields of Chlorella sorokiniana when using wastewater supplemented with different nitrogen sources. Bioresource Technology 168, 127–135. 20. Renuka, N., Guldhe, A., Singh, P., Ansari, F.A., Rawat, I., Bux, F., 2017. Evaluating the potential of cytokinins for biomass and lipid enhancement in microalga Acutodesmus obliquus under nitrogen stress. Energy Conversion and Management 140, 14–23. 21. Salama, E.-S., Kabra, A.N., Ji, M.-K., Kim, J.R., Min, B., Jeon, B.-H., 2014. Enhancement of microalgae growth and fatty acid content under the influence of phytohormones. Bioresource Technology 172, 97–103. 22. Sharma, K.K., Li, Y., Schenk, P.M., 2015. Rapid lipid induction in Chlorella sp. by UV-C radiation. BioEnergy Research 8, 1824–1830. 23. Shi, X.-M., Liu, H.-J., Zhang, X.-W., Chen, F., 1999. Production of biomass and lutein by Chlorella protothecoides at various glucose concentrations in heterotrophic cultures. Process Biochemistry 34, 341–347.

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24. Sijil, P.V., Sarada, R., Chauhan, V.S., 2019. Enhanced accumulation of alphalinolenic acid rich lipids in indigenous freshwater microalga Desmodesmus sp.: The effect of low-temperature on nutrient replete, UV treated and nutrient stressed cultures. Bioresource Technology 273, 404–415. 25. Singh, P., Kumari, S., Guldhe, A., Misra, R., Rawat, I., Bux, F., 2016. Trends and novel strategies for enhancing lipid accumulation and quality in microalgae. Renewable and Sustainable Energy Reviews 55, 1–16. 26. Sun, X.-M., Ren, L.-J., Zhao, Q.-Y., Ji, X.-J., Huang, H., 2018. Microalgae for the production of lipid and carotenoids: a review with focus on stress regulation and adaptation. Biotechnology for Biofuels 11, 272. 27. Vidyashankar, S., Deviprasad, K., Chauhan, V.S., Ravishankar, G.A., Sarada, R., 2013. Selection and evaluation of CO2 tolerant indigenous microalga Scenedesmus dimorphus for unsaturated fatty acid rich lipid production under different culture conditions. Bioresource Technology 144, 28–37. 28. Vidyashankar, S., VenuGopal, K.S., Swarnalatha, G.V., Kavitha, M.D., Chauhan, V.S., Ravi, R., Bansal, A.K., Singh, R., Pande, A., Ravishankar, G.A., 2015. Characterization of fatty acids and hydrocarbons of chlorophycean microalgae towards their use as biofuel source. Biomass and Bioenergy 77, 75–91. 29. Wan, M., Liu, P., Xia, J., Rosenberg, J.N., Oyler, G.A., Betenbaugh, M.J., Nie, Z., Qiu, G., 2011. The effect of mixotrophy on microalgal growth, lipid content, and expression levels of three pathway genes in Chlorella sorokiniana. Applied Microbiology and Biotechnology 91, 835–844.

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30. Xin, L., Hong-Ying, H., Yu-Ping, Z., 2011. Growth and lipid accumulation properties of a freshwater microalga Scenedesmus sp. under different cultivation temperature. Bioresource Technology 102, 3098–3102. 31. Xing, G., Yuan, H., Yang, J., Li, J., Gao, Q., Li, W., Wang, E., 2018. Integrated analyses of transcriptome, proteome and fatty acid profilings of the oleaginous microalga Auxenochlorella protothecoides UTEX 2341 reveal differential reprogramming of fatty acid metabolism in response to low and high temperatures. Algal Research 33, 16–27. 32. Xu, F., Hu, H., Cong, W., Cai, Z., Ouyang, F., 2004. Growth characteristics and eicosapentaenoic acid production by Nannochloropsis sp. in mixotrophic conditions. Biotechnology Letters 26, 51–53. 33. Yahya, N.A., Suhaimi, N., Kaha, M., Hara, H., Zakaria, Z., Sugiura, N., Othman, N., Iwamoto, K., 2018. Lipid production enhancement in tropically isolated microalgae by azide and its effect on fatty acid composition. Journal of Applied Phycology 30, 3063–3073. 34. Zalogin, T.R., Pick, U., 2014a. Azide improves triglyceride yield in microalgae. Algal Research 3, 8–16. 35. Zalogin, T.R., Pick, U., 2014b. Inhibition of nitrate reductase by azide in microalgae results in triglycerides accumulation. Algal Research 3, 17–23. 36. Zhan, J., Rong, J., Wang, Q., 2017. Mixotrophic cultivation, a preferable microalgae cultivation mode for biomass/bioenergy production, and bioremediation, advances and prospect. International Journal of Hydrogen Energy 42, 8505–8517.

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List of figures and tables Fig. 1. Effect of phytohormones (DAH and KIN) supplementation at different concentrations on Desmodesmus sp.: growth profile (A), maximum photosynthetic efficiency (B), average antenna size (C), energy dissipation (D) and fatty acid profile (E). Fig. 2. Effect of mixotrophic cultivation with different concentrations of sodium acetate and glucose on Desmodesmus sp.: growth profile (A), maximum photosynthetic efficiency (B), average antenna size (C), energy dissipation (D) and fatty acid profile (E). Fig. 3. Effect of different concentrations of sodium azide on Desmodesmus sp.: growth profile (A), maximum photosynthetic efficiency (B), average antenna size (C), energy dissipation (D) and fatty acid profile (E). Fig.4. Comparison of two-stage (cultivated for 12 days at 25 oC followed by incubation for 12 days at 5 oC) cultures of untreated, phytohormone (DAH) supplemented, sodium azide treated and mixotrophic (glucose supplemented) Desmodesmus sp. with their corresponding single-stage control cultures (cultivated for 24 days at 25 oC) at the end of cultivation period: biomass yield (A), total lipid content (B), fatty acid profile (C), and relative ALA content of biomass (D). Table 1. Effect of phytohormones (DAH and KIN) supplementation on growth parameters and biochemical characteristics of Desmodesmus sp. Table 2. Effect of mixotrophic cultivation with sodium acetate and glucose on growth parameters and biochemical characteristics of Desmodesmus sp. Table 3. Effect of sodium azide on growth parameters and biochemical characteristics of Desmodesmus sp.

28

29

0.9

0.8

0.8

Maximum photosynthetic efficiency (fv/fm)

A A

Biomass (g L-1)

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

5 Time (Days) 10 Control DAH 0.5 mg L¯¹ DAH 5.0 mg L¯¹ KIN 0.5 mg L¯¹ KIN 5.0 mg L¯¹

0.5 0.4 0.3 0.2 0.1

0

15

3

DAH 0.1 mg L¯¹ DAH 1.0 mg L¯¹ KIN 0.1 mg L¯¹ KIN 1.0 mg L¯¹

C

0.7

1.4

6

9

12

Time (days)

0.8

D

0.6

1.2

0.5

1

0.4

0.8

0.3

0.6

0.2

0.4

0.2

0.1

0

0 0

40

3

6

9

Time (Days)

12

25 20 15 10 5 0 18:0

18:1

18:2

18:3

Major fatty acids Control DAH 1.0 mg L¯¹ KIN 0.5 mg L¯¹

3

6

Control DAH 0.5 mg L¯¹ DAH 5.0 mg L¯¹ KIN 0.5 mg L¯¹ KIN 5.0 mg L¯¹

30

16:0

0

9

12

Time (Days)

E

35

Relative % of total fatty acids

0.6

Energy dissipation (DI0/RC)

Average antenna size (ABS/RC)

1.6

B

0

0

1.8

0.7

DAH 0.1 mg L¯¹ DAH 5.0 mg L¯¹ KIN 1.0 mg L¯¹

DAH 0.5 mg L¯¹ KIN 0.1 mg L¯¹ KIN 5.0 mg L¯¹

30

DAH 0.1 mg L¯¹ DAH 1.0 mg L¯¹ KIN 0.1 mg L¯¹ KIN 1.0 mg L¯¹

Maximum photosynthetic efficiency (fv/fm)

1.2

0.8

Biomass (g L-1)

Fig. 1. Effect of phytohormones (DAH and KIN) supplementation at different concentrations on Desmodesmus Bsize (C), energy dissipation (D) 0.7 A sp.: growth profile1 (A), maximum photosynthetic efficiency (B), average antenna 0.6 and fatty acid profile (E). 0.8 0.6 0.4

0.5

0.4 0.3 0.2

0.2

0.1 0

0 0 5 Time (Days) 10 15 Control Sodiun Acetate 50 mM Sodium Acetate 100 mM Sodium Acetate 500 mM Glucose 50 mM Glucose 100 mM Glucose 500 mM

0

9

12

6

Energy dissipation (DI0/RC)

6

C

5 4 3 2 1

5

D

4 3 2 1 0

0 0

3

6

9

12

Time (Days)

0

3

6

Control Sodium Acetate 100 mM Glucose 50 mM Glucose 500 mM

E

35 30

25 20 15 10 5 0 16:0

18:0

18:1

18:2

18:3

Majr fatty acids Control Sodium Acetate 100 mM Glucose 50 mM Glucose 500 mM

9

12

Time (Days)

40

Relative % total of fatty acids

6

Time (Days)

7

Average antenna size (ABS/RC)

3

Sodium Acetate 50 mM Sodium Acetate 500 mM 31 Glucose 100 mM

Sodium Acetate 50 mM Sodium Acetate 500 mM Glucose 100 mM

Maximum photosynthetic efficiency (fv/fm)

0.7

0.8

Biomass (g L-1)

Fig. 2. Effect of mixotrophic cultivation with different concentrations of sodium B acetate and glucose on 0.7 A profile (A), maximum photosynthetic efficiency 0.6 growth Desmodesmus sp.: (B), average antenna size (C), energy dissipation (D) and fatty acid profile (E). 0.6 0.5 0.4 0.3 0.2 0.1

0.5 0.4

0.3 0.2 0.1

0

0 0

5

10

15

0

Time (Days) Control 0.5 mM Sodium Azide 5.0 mM Sodium Azide

Energy dissipation (DI0/RC)

Average antenna size (ABS/RC)

C

8 6 4 2

12

1.8 1.6

D

1.4 1.2 1 0.8

0.6 0.4 0.2 0

0 0

3

6

9

0

12

Control 0.5 mM Sodium Azide 5.0 mM Sodium Azide

0.1 mM Sodium Azide 1.0 mM Sodium Azide

E

40 35

30 25 20 15 10 5 0 18:0

6

9

12

Control

0.1 mM Sodium Azide

0.5 mM Sodium Azide

1.0 mM Sodium Azide

5.0 mM Sodium Azide

50

16:0

3

Time (Days)

Time (Days)

Relative % of total fatty acids

9

2

10

45

6

0.1 mM Sodium Azide 1.0 mM Sodium Azide

14 12

3

Time (Days)

18:1

18:2

32 18:3

Major fatty acids Control

0.1 mM Sodium Azide

0.5 mM Sodium Azide

1.0 mM Sodium Azide

0.9 0.8

60

A

B

Lipid content (% w/w)

Biomass (g L-1)

Single stage 50 stage Fig. 3. Effect of different concentrations ofTwo sodium azide on Desmodesmus sp.: growth profile Two (A), maximum stage cultivation 0.7 Single stage cultivation photosynthetic efficiency (B), average antenna size (C), energy dissipation (D) and fatty acid profile (E). cultivation (control) cultivation 0.6 40 (control) 0.5 0.4 0.3 0.2

30 20 10

0.1 0

0

Cultivation for 24 days at Cultivation for 12 days at 25° C 25° C and 12 days at 5° C

Cultivation for 24 days at 25° C

Cultures

Cultures Untreated

DAH

Azide

Untreated

Glucose

Relative % of total fatty acids

45 40

Relative ALA content of biomass (%w/w)

50

C

35 30 25 20

15 10 5 0 16:0

18:0

18:1

18:2

18:3

Major fatty acids Single stage control untreated culture Two-stage untreated culture Single stage control DAH supplemented culture Two-stage DAH supplemented culture Single stage control Sodium Azide treated culture Two-stage Sodium Azide treated culture Single stage control glucose supplemented culture Two-stage glucose suppleneted culture

33

Cultivation for 12 days at 25° C and 12 days at 5° C

20 18 16

D

DAH

Azide

Single stage Cultivation (control)

Glucose

Two stage cultivation

14 12 10 8 6 4 2 0

Cultivation for 24 days at 25° C

Cultivation for 12 days at 25° C and 12 days at 5° C

Cultures Untreated Azide

DAH Glucose

Fig.4. Comparison of two-stage (cultivated for 12 days at 25 oC followed by incubation for 12 days at 5 oC) cultures of untreated, phytohormone (DAH) supplemented, sodium azide treated and mixotrophic (glucose supplemented) Desmodesmus sp. with their corresponding single-stage control cultures (cultivated for 24 days at 25 oC) at the end of cultivation period: biomass yield (A), total lipid content (B), fatty acid profile

34

Table 1. Effect of phytohormones (DAH and KIN) supplementation on growth parameters and biochemical characteristics of Desmodesmus sp. DAH (mg L-1)

Parameter Control 0.1

0.5

1.0

5.0

0.1

0.068±0.003a

0.069±0.004a

0.091±0.003b

10.2±0.5a

10.0±0.7a

7.6±0.3b

0.296±0.02a

0.297±0.02a

0.428±0.01b

0.494±0.02c,d

0.025±0.001a

0.025±0.002a

0.036±0.001b

0.041±0.001c,d 0.036±0.001b

0.024±0.001a

23.06±2.04a

24.12±1.07a,b

39.37±1.72d

33.42±2.14c

26.67±1.41a,b

23.88±1.41a,b

5.69±0.33a

5.97±0.42a

14.05±0.32d

13.75±0.44d

9.65±0.20b

5.73±0.24a

6.17±0.30a

6.36±0.21a

9.83±0.65c

8.97±0.35c,d

6.57±0.48a

6.89±0.14a,b

1.47±0.08a

1.57±0.05a

3.51±0.23d

3.69±0.14d

2 .38±0.17b

1.65±0.03a

15.10±0.43a

15.50±0.45a,b

16.72±0.43c

16.57±0.25c

16.18±0.37b,c

15.84±0.33a,b

5.53±0.23a

6.11±0.30a

5.54±0.24a

5.79±0.33a

6.09±0.30a

5.89±0.31a

Specific growth rate (µ day-1) Doubling time (h) -1

Biomass yield (g L )

0.097±0.001b,c 0.090±0.003b 7.1±0.1b

0.065±0.002a

7.7±0.2b

10.6±0.3a

0.434±0.01b

0.288±0.01a

Biomass productivity (g L-1 day-1) Lipid content (% w/w) Lipid productivity (mg L-1 day-1) Relative ALA content in biomass (% w/w) ALA productivity (mg L-1 day-1) Total chlorophyll (µg mL-1) Total carotenoid (µg mL-1)

Data represents mean ± SD of three replicates. The superscript (letters a, b, c and d) denote significant differences. The mean values in each row sharing a common superscript are statistically not significant at P < 0.05 by one way ANOVA

Table 2. Effect of mixotrophic cultivation with sodium acetate and glucose on growth parameters and biochemical characteristics of Desmodesmus sp. Sodium Acetate (mM)

Parameter

Control

Specific growth rate (µ day-1) Doubling time (h) -1

Biomass yield (g L ) -1

50

100

500

0.069±0.001a

0.102±0.003c

0.103±0.002c

0.092±0.001b

0.138±

10.0±0.2a

6.8±0.2c

6.7±0.2c

7.5±0.1b

5.0±0.

0.277±0.01a

0.467±0.01c

0.489±0.01c

0.416±0.01b

0.857±

0.023±0.001a

0.039±0.001c

0.041±0.001c

0.035±0.001b

0.071±

-1

Biomass productivity (g L day )

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Lipid content (% w/w) -1

-1

Lipid productivity (mg L day )

23.57±1.16a

23.50±1.09a

43.44±0.71d

39.54±1.05c

28.87±

5.44±0.03a

9.14±0.20b

17.70±0.05d

13.69±0.17c

20.61±

5.87±0.28c

3.39±0.28a

6.27±0.37c,d

7.08±0.17d,e

5.52±0

1.36±0.07a

1.32±0.11a

2.55±0.15b 11.42±0.69b

2.45±0.06b 11.68±0.25b

3.94±0

3.61±0.43b

3.58±0.32b

Relative ALA content in biomass (% w/w) ALA productivity (mg L-1 day-1) -1

Total chlorophyll (µg mL )

15.06±0.90 5.42±0.47a

Total carotenoid (µg mL-1)

a

12.32±1.36 4.12±0.18b

a

10.80± 3.89±0

Data represents mean ± SD of three replicates. The superscript (letters a, b, c, d, e, f and g) denote significant differences. The mean values in each row sharing a common superscript are statistically not significant at P < 0.05 by one way ANOVA

Table 3. Effect of sodium azide on growth parameters and biochemical characteristics of Desmodesmus sp.

Sodium Azide (

Control

Parameter

0.1

0.5

Specific growth rate (µ day-1)

0.066±0.002a

0.066±0.001a

0.064±0.001a

Doubling time (h)

10.5±0.3a

10.5±0.1a

10.8±0.2a

Biomass yield (g L-1)

0.311±0.02a

0.324±0.01a

0.313±0.01 a

Biomass productivity (g L-1 day-1)

0.026±0.001a

0.027±0.001a

0.026±0.001a

Lipid content (% w/w)

22.12±1.75a

22.42±1.51a

24.78±1.42a

Lipid productivity (mg L-1 day-1)

5.74±0.30a

6.05±0.24a,b

6.46±0.17b

Relative ALA content in biomass (% w/w)

5.62±0.24a

9.11±0.30b

10.60±0.48c

1.46±0.06a

2.45±0.082b

2.76±0.13b

14.95±1.09a

14.19±0.57a

14.20±0.77a

5.52±0.39a

5.33±0.21a

5.16±0.27a

-1

-1

ALA productivity (mg L day ) -1

Total chlorophyll (µg mL ) -1

Total carotenoid (µg mL )

Data represents mean ± SD of three replicates. The superscript (letters a, b, c and d) denote significant differences. The mean values in each row sharing a common superscript are statistically not significant at P < 0.05 by one way ANOVA

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HIGHLIGHTS 

Enhancement in ALA rich lipids was achieved without compromising the biomass



Phytohormones, mixotrophy and azide enhanced the lipid content by 1.5 to 1.8 fold



Azide increased the ALA fraction of total fatty acids by 1.7 fold



Low temperature as second stage of cultivation increased the ALA fraction



Two stage cultivation strategy increased the ALA content of biomass up to 17%

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Conflict of interest Authors declare that they do not have any conflict of interests to declare

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