Enhanced accumulation of alpha-linolenic acid rich lipids in indigenous freshwater microalga Desmodesmus sp.: The effect of low-temperature on nutrient replete, UV treated and nutrient stressed cultures

Accepted Manuscript Enhanced accumulation of alpha-linolenic acid rich lipids in indigenous freshwater microalga Desmodesmus sp.: the effect of low-temperature on nutrient replete, UV treated and nutrient stressed cultures P.V. Sijil, R. Sarada, V.S. Chauhan PII: DOI: Reference:

S0960-8524(18)31555-4 https://doi.org/10.1016/j.biortech.2018.11.028 BITE 20680

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

6 September 2018 5 November 2018 8 November 2018

Please cite this article as: Sijil, P.V., Sarada, R., Chauhan, V.S., Enhanced accumulation of alpha-linolenic 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 (2018), doi: https://doi.org/10.1016/j.biortech. 2018.11.028

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Enhanced accumulation of alpha-linolenic acid rich lipids in indigenous freshwater microalga Desmodesmus sp.: the effect of low-temperature on nutrient replete, UV treated and nutrient stressed cultures P.V. Sijil 1,2, R. Sarada 1,2, V.S. Chauhan 1,2* 1

Academy of Scientific and Innovative Research (AcSIR), CSIR - Central Food

Technological Research (CFTRI) Campus, Mysuru - 570 020, India 2

Plant Cell Biotechnology (PCBT) Department, CSIR - CFTRI, Mysuru - 570 020, India.

*Corresponding author E-mail: [email protected]

Postal Address: Dr. Vikas Singh Chauhan, Sr. Principal Scientist, Plant Cell Biotechnology (PCBT) Department, CSIR - Central Food Technological Research Institute (CFTRI), Mysuru - 570 020, Karnataka, India Phone: +91-821-2516501

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Introduction The alpha-linolenic acid (ALA), an essential omega-3 polyunsaturated fatty acid (PUFA), has been attributed with various health benefits (Barceló-Coblijn and Murphy, 2009). The Food and Nutrition Board of the Institute of Medicine (IOM) (now National Academy of Medicine), USA has recommended an Adequate Intake (AI) of ALA for adult male and female as 1.6 g and 1.1 g, respectively (Institute of Medicine, 2005). The major source of ALA are plants (Barceló-Coblijn and Murphy, 2009). The global demand for omega-3 fatty acids has been increasing significantly over the last two decades necessitating the search for additional sources (Adarme-Vega et al., 2012). The microalgae could be an excellent source of PUFA rich lipids (Chisti, 2007) as they are known to accumulate high content of lipids rich in PUFA, have higher photosynthetic efficiency and surface area productivity compared to crop plants, have simple nutritional requirements and do not require agricultural land. Microalgae are known to accumulate lipids under various growth-limiting environmental and abiotic stress conditions as a part of their survival mechanism (Lu et al., 2017). The stress conditions adversely affect the photosynthetic apparatus and other macromolecules of microalgae, leading to the growth retardation and further assimilation of carbon towards lipids (Kamalanathan et al., 2016). The depletion of nutrient like nitrogen and phosphorus leads to accumulation of lipids in microalgae, mainly the tri acyl glycerol (TAG) consisting of saturated fatty acids (SFAs) and mono unsaturated fatty acids (MUFAs) (Paliwal et al., 2017). The UV radiation has also been shown to induce the lipid accumulation in different microalgal cultures (Guihéneuf et al., 2010; Sharma et al., 2015). Exposure of microalgae to lower temperatures causes the reduction in the membrane fluidity by solidification of saturated lipids in the cell membrane and disturbs the

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intracellular metabolism (Flaim et al., 2014). Freshwater and marine microalgae have been shown to accumulate PUFA as an adaptive mechanism to maintain membrane fluidity when exposed to low temperatures (Lu et al., 2017). The indigenous microalgae isolated from natural waters could serve as potential candidates for production of PUFA rich lipids as they adapt to various environmental stresses for their survival and growth. The studies on the accumulation of ALA rich lipids in indigenous freshwater microalgae have remained limited. Vidyashankar et al. (2015) reported the indigenous freshwater microalgae Scenedesmus dimorphus, Chlorella sp. and Chlorococcum sp. to accumulate 17 to 26% lipids with ALA content being 28 to 30% of total fatty acids. Acetylcholine treatment enhanced the ALA content in Chlorella spp. with maximum value of 31% of total fatty acids (Parsaeimehr et al., 2015). An enhanced ALA content of 20% of total fatty acids has been reported in UV irradiated Chlorella sp. (Wong et al., 2007). Lowering the cultivation temperature of Nanochloropsis oculata from 25 to 15 °C enhanced the ALA fraction of total fatty acids from 10 to 18% (Converti et al., 2009). In the present study, the indigenous freshwater microalgae were studied for their ALA, accumulating potential. The independent effect of low temperature, UV treatment, and nutrient stress and effect of low temperature on UV treated and nutrient stressed cultures was studied. 2. Materials and methods 2.1. Microalgal cultures The four indigenous microalgae used in the study were identified based on their morphological features (Philipose, 1967) and cell dimensions, enumerated using a light microscope (Olympus BX51, Japan) fitted with a camera and imaging software (ProgRes

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C5, Germany). The morphological features and geographical locations of the natural habitat of three indigenous microalgae used in the study, viz., Scenedesmus dimorphus CFR 1-05/FW, Desmodesmus sp. CFR 1-01/FW (reported as Scenedesmus sp.) and Oocystis pusilla CFR 6-01/FW, have been reported (Vidyashankar et al. 2015). The Chlorella sp. CFR 3-05/FW used in the study was unicellular with round cells (4 to 5 µm), isolated from water body at CSIR-NEERI campus, Nagpur, India (Geographical coordinates: 210 08’25”N, 790 05’56”E). The identification of Desmodesmus sp. CFR 101/FW was confirmed using ITS-2 gene sequencing and the sequences has been submitted to NCBI (http://www.ncbi.nlm.nih.gov) nucleotide databank (accession no., MG019910.1). The microalgae were maintained by regular subculturing every 20 days allowing the cultures to achieve a final biomass concentration of about 0.5 g L-1 from an initial biomass concentration of about 0.1 g L-1. 2.2. Culture conditions, measurement of growth and pigments The microalgal cultures were maintained in nutrient replete sterile Bold’s basal medium (BBM) at 25 ± 1 °C under the cool white fluorescent light of 30 µE m-2 s-1 intensity with 16:8 h light and dark cycle and were referred to as nutrient replete cultures. The purity of the cultures was ensured by repeated subculturing and microscopic observation. The optical density (OD) of cultures was measured at regular intervals at 560 nm (UV-VIS Spectrophotometer SP 3000, Optima, Tokyo). The culture aliquots were drawn and harvested at different ODs by centrifugation at 5000 rpm for 10 minutes (model Sorvall Legend X1R, Thermo Scientific, Osterode, Germany) and freeze dried (Cool safe 55-4 pro, Scanvac, Denmark). A Linear regression was used to establish the correlation between OD (x) and biomass (dry cell weight g L-1) (y) for each of the cultures: y = 0.5729 x - 0.0504 (R2 = 0.9831) for Desmodesmus sp; y = 0.4505 x + 0.0189 (R2 = 0.9842) for

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Scenedemus dimorphus; y = 0.4853 x - 0.061(R2 = 0.991) for Oocystis pusilla and y = 0.4035 x + 0.0076 (R2 = 0.9914) for Chlorella sp. The specific growth rate (µ) was calculated by the equation, µ = 1/t × ln (Xm/X0) where ‘Xm’ and ‘X0’ are the concentrations of biomass (g L-1) at the end and beginning of a batch culture, respectively, and ‘t’ is the duration of the batch culture. Doubling time (D) was calculated by the equation D = 0.693/µ. The microalgal cultures were allowed to reach stationary phase (three weeks) and the biomass yield (Y) (g L-1) was calculated by the equation Y= Xm-X0 and the biomass productivity (P) (g L-1d-1) by the equation P = XmX0/T2-T1, where ‘T2-T1’ represent the incubation period of an experiment with ‘T 1’ and ‘T2’ being the initial (day 0) and final day (in number) of incubation respectively(Converti et al., 2009; Vidyashankar et al., 2015). The culture aliquots were centrifuged and the obtained pellet was extracted with methanol for spectrophotometric estimation of pigments using Lichtenthaler's equations (Lichtenthaler, 1987). 2.3 Low temperature stress, UV treatment, and Nutrient stress studies The microalgae were incubated at 5 °C and 15 °C using temperature controlled incubator shakers (Scigenics, India). For the UV treatment studies, the microalgal cultures with cell density of 1.58 × 107 cells/mL (0.03 g L-1 biomass) were exposed to UV-C radiation (254 nm). An aliquot of 40 mL of culture was spread into a thin layer on a Petri plate (15 cm diameter), placed inside a UV chamber and was irradiated for different periods of time viz., 20 min, 40 min, and 60 min, translating to UV dosage of 6, 12 and 18 J cm-2, respectively. The UV treated microalgal cultures were centrifuged, the cell pellets were inoculated in fresh BBM medium (40 mL) and kept overnight in the dark. The UV treated cultures were subcultured for five cycles under normal growth conditions. These

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recovered cultures obtained after subculturing were studied for growth, lipid content and fatty acid profile for evaluating the stability and the long-term effect of the of UV treatment on the microalgal culture. The UV treated cultures were also subjected to lowtemperature stress. For nutrient stress studies, the microalgae were inoculated in the nitrogen-deficient medium (N-), phosphorous deficient medium (P-) and the medium deficient in both the nitrogen and phosphorus (N-P-). The microalgal cultures subjected to various treatments were harvested after the 21 days of incubation. The light intensity and photoperiod during the studies were maintained as described for the normal growth conditions. The biomass obtained was freeze-dried (Cool safe 55-4 pro, Scanvac, Denmark) for analytical studies. 2.4. Measurement of Photosystem II (PS II) related parameters The aliquots of microalgae (2 mL) were kept in the dark for 15 min for dark adaptation. The PS II related parameters were analysed by measuring the chlorophyll fluorescence of the dark adapted microalgal cultures (Markou et al., 2017) using a portable PAM fluorometer (AquaPen-C AP-C 100, Photon Systems Instruments, Czech Republic) (Markou et al., 2017; Mathur et al., 2011). The photosynthetic parameters measured were i) Fv/ Fm: The maximum photosynthetic efficiency (quantum yield), ii) ABS/ RC: Total number of photons absorbed by reaction centres (average antenna size), iii) DI 0/RC: Total dissipation of un-trapped excitation energy from all reaction centres (dissipation energy). 2.5. Lipid extraction and Fatty Acid Methyl Ester (FAME) analysis The total lipid was extracted from a known quantity of freeze-dried biomass (100 mg) with chloroform and methanol (2:1 ratio), and the fatty acids were esterified as described by Vidyashankar et al. (2015). The Fatty Acids Methyl Esters (FAME) were

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dissolved in HPLC grade n-hexane and injected in GC (Shimadzu 2010 plus, Japan) equipped with flame ionisation detector (FID). A poly (dimethyl) siloxane capillary column (30 m × 0.32 mm ID × 0.25 µm film thickness) (Rtx-1, Restek Inc.USA) was used for FAME separation. 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. The nitrogen gas 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 C-24 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 relative percentage composition of total FAME (Vidyashankar et al., 2015). 2.6. 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 discussion 3.1. Effect of low temperature on nutrient replete cultures of freshwater microalgae 3.1.1. Effect on growth, lipid and fatty acid profile Nutrient replete cultures of four indigenous freshwater microalgae, viz., Chlorella sp., Desmodesmus sp., Oocystis pusilla and Scenedesmus dimorphus, were first evaluated for their response to low-temperature stress (15 °C and 5 °C). The incubation at low temperature affected the growth of microalgal cultures (Fig. 1 and Table 1). The Oocystis pusilla culture showed about 18 to 22% lower specific growth rate at low temperature than 7

the control cultures (maintained at 25 °C). The doubling time increased by 2.2 to 2.8 h and the biomass yield reduced by 24% to 37% compared to control. The biomass productivity reduced by 23% to 35% compared to control. The specific growth rate of Scenedesmus dimorphus, Desmodesmus sp. and Chlorella sp. reduced by 39% to 48% at low temperatures than the control with 5 to 10 h increase in the doubling time. The biomass yield and biomass productivity showed a reduction of 48% to 62% than control. The lowtemperature incubation reduced the chlorophyll and carotenoid content of all the cultures (Table 1). The low temperature has been known to adversely affect the growth of other microalgae, e.g., a reduction in cultivation temperature from 20 to 15 °C led to a 53% reduction in specific growth rate in Nannochloropsis oculata (Converti et al., 2009). The lipid content of the control cultures of microalgae maintained at 25 °C was in the range of 18 to 22% w/w (Table 1). On incubation of cultures at 5 °C, the Scenedesmus dimorphus and Desmodesmus sp., showed a lipid content of 34% w/w, a significant increase of 1.84 and 1.52 fold, respectively, compared to control. The Chlorella sp. and Oocystis pusilla cultures incubated at 5 °C showed a 1.45 fold increase in lipid content than control. The increase in the lipid content of microalgal cultures incubated at 15 °C was not significant. An increase of 1.2 to 1.4 fold in lipid content with values in the range of 31 to 35% (w/w) has been reported for Scenedesmus sp. cultures when grown at 20 oC and 10 oC (Xin et al., 2011). The low temperatures have a growth limiting effect and also reduce the membrane fluidity in microalga (Flaim et al., 2014) leading to an enhanced accumulation of lipids as an adaptive response (Lu et al., 2017). Therefore, lower temperature can be used as a potential tool for enhancing lipid accumulation in microalgae. The varied increase in the lipid content of microalgae seen in the present study indicates that adaptive response to lower temperatures in microalgae varies from species to species.

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The biomass productivity and the lipid content affect the lipid productivity of microalgal cultures (Table 1). In control cultures maintained at 25 oC, the higher biomass productivity and lipid content of Desmodesmus sp. and Chlorella sp. resulted in correspondingly higher lipid productivity (7.07 and 7.6 mg L -1 day-1, respectively) compared to Scenedesmus dimorphus and Oocystis pusilla (4.8 mg L-1 day-1 and 4.7 mg L-1 day-1, respectively). At 15 °C, the Scenedesmus dimorphus, Desmodesmus sp. and Chlorella sp. cultures showed about a 50% reduction in biomass productivity leading to a 45% to 56% reduction in lipid productivity. However, at 5 °C these cultures showed only 29 to 35% reduction in lipid productivity than their corresponding control cultures. Though the reduction in biomass productivity at 5 °C was more than 50% for these microalgae, a significant enhancement in their lipid content over control cultures compensated for the effect of reduced biomass productivity and manifested in comparatively better lipid productivity than at 15 °C. The Oocystis pusilla showed no significant increase in the lipid content or decrease in biomass productivity at lower temperatures and this led to an insignificant change in its lipid productivity. The results suggest that microalgae with an ability to accumulate higher lipid content under stress can minimize the reduction in lipid productivity when the biomass productivities are adversely affected by the stress. The low-temperature stress has been shown to enhance the unsaturation of fatty acids in microalgae as an adaptive mechanism to maintain the membrane fluidity (Lu et al., 2017). In the present study, the Desmodesmus sp, Chlorella sp., and Oocystis pusilla showed an enhanced unsaturation of fatty acids at low temperature (Fig. 1). The ALA fraction of total fatty acids showed an enhancement of 39 to 44%, an increase of 1.3 to 2.4 fold compared to control cultures. The Desmodesmus sp. cultures incubated at 5 °C showed the highest percentage (44%) of ALA fraction of fatty acids. This translated to an

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ALA content (w/w) in the biomass of 15%, an increase of 2.74 fold than the control cultures. This increase in ALA fraction of total fatty acids in Desmodesmus sp. at low temperature was accompanied by a reduction in palmitic acid (C 16:0) and linoleic acid (C 18:2) fractions of total fatty acids. The palmitic acid fraction of total fatty acids reduced by about 26% and 8% compared to control at 15 oC and 5 oC respectively. The linoleic acid fraction of the total fatty acid showed about 96% reduction at 5 oC and about 29% reduction at 15 oC compared to control. The PUFA enhancing effect of low-temperature has been reported for various microalgae. However most of these studies are concentrated on eicosapentaenoic acid (EPA) (C 20:5) and docosahexaenoic acid (DHA) (C 22:6) with a few reports on ALA (Paliwal et al., 2017). Cultivation of Nanochloropsis oculata at lower temperature (15 °C) enhanced the ALA fraction of total fatty acids to 18% (Converti et al., 2009). The Scenedesmus sp. grown at 10 oC showed a higher ALA fraction of total fatty acids (Xin et al., 2011). The expression patterns of key genes involved in the fatty acid biosynthesis of Haematococcus pluvialis showed a significant change under low-temperature cultivation (Lei et al., 2012). The culture exhibited 8.9 fold increase in gene expression of omega-3 fatty acid desaturase which converts linoleic to alpha-linolenic acid at low temperature. Similarly, there was an increase in gene expression of biotin carboxylase (4.8 fold), Acyl carrier protein (ACP) (2.6 fold), Acyl-ACP thioesterase (3fold), stearoyl-ACP-desaturase (2.5 fold) under low temperature (Lei et al., 2012). Therefore the increase in ALA content in Desmodesmus sp. under low temperature in the present study might also be due to the changes in the expression patterns of key genes involved in the fatty acid biosynthesis pathway.

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Therefore, enhancement in lipid accumulation, ALA fraction of total fatty acids and ALA content in biomass at low temperature, could be used as the criteria for selection of the candidate microalga for the production of ALA-rich lipids. From the results discussed above, the Desmodesmus sp. was selected as the candidate microalga as it showed higher lipid accumulation (34% w/w) with a significant enhancement in ALA fraction of total fatty acids (44%), and higher ALA content in biomass (15% w/w) at low temperature. 3.1.2. Effect on photosynthetic parameters of Desmodesmus sp. At low-temperature, microalgae can maintain normal metabolism and photosynthetic activity by a synergistic interaction and mutual coordination between the pigments, lipids, fatty acids and pigment-protein complexes which maintain the stability of thylakoid membrane (Yi-Bin et al., 2017). The PS II plays a major role in cold stress in microalgae and the variations of PSII activity reflect the physiological status of microalgae cells indicating the stress response (Markou et al., 2017). The low-temperature adaptation and physiological health of Desmodesmus sp. was studied by monitoring the PSII changes in cultures incubated at 5 °C. The photosynthetic efficiency (QY or Fv/Fm), antenna size (ABS/RC) and energy dissipation (DI0/RC) were studied and compared with control cultures incubated at 25 °C for 15 days period, and the data are presented in Fig. 2. The antenna size increased by 2.68 fold at 5 °C which could be due to the change in the number of light harvesting complexes or due to the inactivation of reaction centre (RC) in response to the low-temperature stress. The increase in the dissipation energy by 4.3 fold at 5 °C suggests a reduction in the active RC and also the loss of connectivity between PSII heterogeneous units (Mathur et al., 2011). The increased antenna size and dissipation energy at 5 °C were accompanied by a reduction in photosynthetic efficiency (QY) to 0.32, a 51% reduction compared to control cultures. It has been shown that the photosynthetic

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efficiency is inversely proportional to the photosystem antenna size. A photosynthetic efficiency lower than 0.5 indicates that microalgal cells are experiencing the physiological stress and are not in good health (Ramanna et al., 2014). Therefore, the photosynthetic parameters indicated that the Desmodesmus sp. cultures experienced photosynthetic stress at 5 °C. This physiological stress may have led to the enhanced accumulation of lipids, a well-known response of microalgae to physiological stress (Guschina and Harwood, 2009). 3.2. Effect of UV treatment on Desmodesmus sp. 3.2.1. Recovery of growth and photosynthetic parameters after UV treatment The effect of UV treatment on Desmodesmus sp. was studied by exposing the culture to UV radiation for three different time intervals, 20 min (UV 20 min), 40 min (UV 40 min) and 60 min (UV 60 min). The UV treated cultures were inoculated in fresh growth medium for recovery. The photosynthetic parameters of the microalgal culture after the UV treatment indicated that the physiological health of microalga was adversely affected (Fig. 3). The UV treated cultures showed 6 fold increase in average antenna size and 18 fold increase in energy dissipation compared to control and the photosynthetic efficiency of the cultures reduced to zero. It is generally recognized that UV radiation degrades photosynthetic pigments and proteins of chloroplasts, especially the D1 protein of the photosystem II (PSII) (Guihéneuf et al., 2010). It has also been shown to induce photo inhibition of the biosynthesis of pigments in microalgae (Guihéneuf et al., 2010). These effects are manifested in terms of increased antenna size, increased dissipation energy and reduced photosynthetic efficiency in the present study. The photosynthetic parameters started showing recovery from 3rd day onwards, and by the 7th day of incubation under normal growth conditions, the QY, antenna size and energy dissipation were similar to

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control cultures and remained so throughout the study period. The UV treated cultures showed a recovery in growth after five days reaching a biomass concentration of about 0.15 g L-1 on 15th day under normal growth conditions (Fig. 3A). 3.2.2. Growth, lipids and fatty acid profiles of UV treated Desmodesmus sp. after subsequent subculturing The UV treated Desmodesmus sp. cultures were subsequently subcultured for five cycles, and these recovered cultures were further studied to understand the long-term effect of UV treatment. The UV treated cultures showed improved growth parameters than control culture (Fig. 4 and Table 2). The specific growth rate of UV treated cultures increased by 1.19 to 1.29 fold than control cultures (0.072 µ day-1) with a reduced doubling time of 7.4 to 8.1 h (1.5 to 2.2 h less than control). The recovered UV 40 min and UV 60 min cultures showed a 1.4 fold increase in biomass productivity and 1.36 to1.38 fold increase in biomass yield respectively. The chlorophyll and carotenoid content of the recovered UV treated cultures were similar to control cultures. The UV radiation causes random mutation by damaging the ultrastructure of nuclear and chloroplastic DNA and also affects the enzymes and biochemical pathways (Guihéneuf et al., 2010). The changes observed in the growth parameters of UV treated microalgal culture may be attributed to these effects of UV. Also, the improved growth parameters of the recovered UV treated cultures after repeated subculturing shows that the UV radiation effected changes were stable. The UV 60 min, UV 20 min and UV 40 min cultures showed a significant increase of 1.6 fold (37% w/w), 1.41 fold (33% w/w), and 1.35 fold (31% w/w), respectively, in their lipid content compared to control (UV untreated) cultures (Table 2). The higher

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biomass productivity and increased lipid content resulted in higher lipid productivities in UV treated cultures. The UV 40 min and UV 60 min cultures showed lipid productivity of 12.16 and 14.83 mg L-1 day-1, respectively, which was 1.8 to 2.1 fold higher than the control cultures. The UV 40 min and UV 60 min cultures showed a 1.21 to 1.26 fold increase in the ALA fraction of total fatty acids compared to control (Fig. 4). There was no significant change in the profile of other fatty acids of microalgal lipids. An increase in lipid content and ALA fraction of total fatty acids in UV40 min and UV60 min cultures led to 1.7 fold (10% w/w) and 1.92 fold (11% w/w) increase in ALA content of biomass, respectively. The UV20 showed 1.44 fold increase in the ALA content of biomass mainly due to an increase in the lipid content. Therefore, the UV treatment resulted in enhanced ALA content of biomass by enhancing the lipid content and/or the ALA fraction of total fatty acids. It has been suggested that the exposure to UV radiation reduces the capacity of microalgae to absorb inorganic nutrients like nitrogen, phosphate, and dissolved inorganic carbon (Guihéneuf et al., 2010). This may subject microalgae to a condition similar to nutrient stress, known to induce lipid accumulation (Guihéneuf et al., 2010). An enhancement in unsaturated fatty acids by UV irradiation has been observed in different microalgal cultures (Sharma et al., 2015; Sharma and Schenk, 2015). The UV irradiation led to a 1.8 fold increase in ALA (20% of total fatty acids) in Chlorella sp. (Wong et al., 2007) and a 1.3 fold increase in ALA in Pavlova lutheri (Guihéneuf et al., 2010). In contrast, the Odontella aurita and Chlamydomonas sp. showed a reduction in ALA content after UV irradiation indicating that the changes in the fatty acid profile are species-specific (Guihéneuf et al., 2010; Wong et al., 2007). The PUFA are known to have antioxidant effects as they absorb the reactive oxygen species leading to their reduction in cells.

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Therefore, the increase in PUFA in microalgae can be attributed as a defence response of microalgae against UV generated cellular reactive oxygen species (Sharma et al., 2015; Sharma and Schenk, 2015). 3.2.3. Effect of low temperature on growth, lipids and fatty acid profile of UV treated Desmodesmus sp. The incubation at low temperature (5 °C) and UV treatment improved the total lipid and ALA content of Desmodesmus sp. cultures independently. The UV treated microalgal cultures, which were recovered and maintained under normal growth conditions through regular subculturing, were incubated at 5 °C for 21 days to study the effect of low temperature on UV treated cultures. The growth parameters of recovered UV treated cultures incubated at 5 °C were comparable to the control cultures incubated at 5 °C. However, the lipid and ALA content of the cultures showed significant differences (Table 2). The specific growth rate of control cultures and UV 20 min, UV 40 min and UV 60 min cultures on incubation at 5 oC reduced by about 41 to 45% compared to their corresponding cultures at the normal growth temperature of 25 oC (Table 2). The UV 20 min, UV 40 min, and UV 60 min cultures on incubation at 5 °C, showed a lipid content (w/w) of 49%, 62% and 59%, respectively, which was (i) 2.11, 2.67 and 2.55 fold, respectively, higher than the control cultures incubated at normal growth temperature; (ii) 1.5, 1.98, 1.60 fold, respectively, higher than the corresponding UV treated cultures incubated at normal growth temperature; and (iii) 1.43, 1.81, 1.73 fold, respectively, higher than control cultures incubated at 5 °C.

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The control culture at 5 °C showed a reduced lipid productivity compared to the corresponding cultures at normal growth temperature due to the reduced biomass productivity (Table 2). However, UV treated cultures incubated at 5 °C showed the lipid productivity of 7.3 to 9.3 mg L-1 day-1, which was comparable to the control cultures at the normal growth temperature of 25 °C. This suggests that the increase in lipid content of UV treated culture at low temperature could compensate for the loss of biomass productivity induced by low temperature, thereby enabling the UV treated cultures to attain the lipid productivities comparable to control. The UV 20 min, UV 40 min and UV 60 min cultures incubated at 5 °C showed a respective increase of 1.27, 1.63 and 1.50 fold in ALA fractions of total fatty acids than control cultures at the normal growth temperature of 25 °C (Fig. 4). When compared to their corresponding cultures incubated at normal growth temperature, the increase was 1.24 to 1.29 fold. Also, the ALA fractions of total fatty acids of 5 °C incubated UV 40 min, and UV 60 min cultures were comparable to control culture incubated at 5 °C (Fig. 4). The ALA content (w/w) of the biomass of UV 20 min, UV 40 min and UV 60 min cultures incubated at 5 °C increased by (i) 2.68, 4.36 and 3.83 fold, respectively, compared to control cultures incubated at 25 °C; (ii) 1.86, 2.56 and 1.99 fold, respectively, than their corresponding cultures incubated at 25 °C, and (iii) 1.074 fold, 1.747 fold and 1.53 fold, respectively, than control cultures incubated at 5 °C. The results suggest that the incubation of UV treated cultures at low temperature had an added effect leading to a significant increase in lipid content. Also, as the ALA fraction of total fatty acids of these cultures remained comparable to control cultures incubated at 5 °C, the overall ALA content of the biomass was higher compared to cultures experiencing the independent effect of UV treatment and low temperature.

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3.3. Effect of nutrient stress on Desmodesmus sp. 3.3.1. Effect of individual and synergistic stress of nitrogen and phosphorous deprivation on growth, photosynthetic parameters, lipids and fatty acid profile Nutrient stress is widely used as a tool for enhancing the accumulation of lipids in microalgae. Though several studies have been reported on individual effects of nitrogen and phosphorus stress on lipid accumulation in microalgae (Paliwal et al., 2017), the interest on the study of combined or synergistic stress effect of these two nutrients has been gaining importance in recent times (Arora et al., 2016; Kamalanathan et al., 2016). In the present study, the response of the Desmodesmus sp. to the individual stress of nitrogen deprivation (N-) and phosphorous deprivation (P-) and synergistic stress of nitrogen and phosphorus deprivation (N-P-) was studied. It was observed that while N- and N-Pcultures showed a 53% to 55% reduction in specific growth rate compared to control (nutrient replete cultures), the phosphorus deficiency (P-) alone did not affect the growth of microalgae (Table 3). A similar trend was observed for the biomass yield and biomass productivity which showed about 66 to 70% reduction in N- and N-P- cultures with no significant change in P- culture. It has been observed that while the nitrogen starvation significantly affects the microalgal growth, the effect of phosphorous starvation on growth is not significant (Kamalanathan et al., 2016; Arora et al., 2016). The Chlamydomonas reinhardtii showed a 30% reduction in specific growth rate under nitrogen starvation and 10 % reduction under phosphorous starvation (Kamalanathan et al., 2016). Similarly, Chlorella minutissima showed significant reduction in biomass productivity under nitrogen starvation but insignificant reduction under phosphorus starvation (Arora et al., 2016).

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The nitrogen deprivation adversely affected the photosynthetic apparatus with the N- and N-P- cultures showing around 2.50 to 2.64 fold increase in the antenna size and 3.67 to 3.87 fold increase in the dissipated energy than control cultures (Fig. 5). Most of the energy absorbed was dissipated as heat in N- and N-P- cultures resulting in around 37 to 39% reduction in their photosynthetic efficiency than the control cultures. The stress effect of nitrogen starvation on photosynthesis may be due to its role in the synthesis of components of photosynthetic apparatus (Kamalanathan et al., 2016) as nitrogen is required for biosynthesis of proteins and photosynthetic pigments (Cheng and He, 2014). These results suggesting the nitrogen starvation as the main stressor are similar to the observations made by Kamalanathan et al. (2016) for Chlamydomonas reinhardtii where phosphorus starvation did not show an adverse effect on photosynthetic parameters. This may be attributed to the ability of microalgae to sustain the demand of phosphorus when deprived, by utilising the internal storage pool of polyphosphates (Kamalanathan et al., 2016; Arora et al., 2016). The nitrogen and phosphorous starvation led to an enhanced accumulation of lipids in Desmodesmus sp. The lipid content (w/w) of N- cultures was 39%, and in the case of NP- cultures, it was 37%, an increase of about 1.66 to 1.74 fold than control cultures. The Pcultures of Desmodesmus sp. showed a lipid content (w/w) of 28%, which was 1.27 fold higher than control but 25% to 28% lower than N- and N-P- cultures (Table 3). Similar to the present study, the nitrogen starvation and the starvation of both the nitrogen and phosphorus was shown to increase the lipid content by 1.73 to1.78 fold in Chlorella minutissima reaching a maximum content of 48% and 49% respectively (Arora et al., 2016). According to Msanne et al., (2012), in the initial stages of nitrogen starvation the microalgal cells accumulate starch whereas at later stages the lipid accumulation occurs at

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the expense of assimilated carbon in the form of cellular components particularly proteins, starch and chlorophyll molecules. However, the metabolic pathway channelling of protein degradation to lipids in microalgae has remained largely unexplored. The phosphorus is required for the biosynthesis of nucleic acids, phospholipids, and membrane development etc. (Paliwal et al., 2017). The phosphorous starvation has been reported to increase the lipid content of various microalgae viz., Scenedesmus sp., Chlorella sorokiniana, Chlorella vulgaris and Chlorella ellipsoidea YJ1 (Wu et al., 2013). In the present study, the P starvation had only a minor stress effect on the growth and photosynthesis, resulting in a comparatively lower enhancement in lipid accumulation. The reduction in the biomass productivity (about 66-70%) of N- and N-P- cultures led to a 43 to 50% reduction in their lipid productivity. However, the biomass productivity comparable to control and an increased lipid content than control led to a 1.13 fold increase in the lipid productivity of P- cultures (Table 3). The N- and N-P- cultures showed a 26% and 48% reduction in their ALA fraction of total fatty acids, respectively (Fig. 5). It was reported that the nitrogen and phosphorous starvation enhance the saturated and mono unsaturated fatty acids in microalgal cultures (Arora et al., 2016). The Chlamydomonas reinhardtii exhibited a 37% reduction in ALA yielding 0.05µg/mg when both nitrogen and phosphorous was depleted in the medium (Yang et al., 2018). The escalation of TAG synthesis pathway in microalgae under nitrogen starvation has been attributed to a reduced content of polar lipids (mainly PUFA) (Paliwal et al., 2017). In the present study also, a cumulative increase in SFA and MUFA (mainly C 16:0 and C 18:1) could be attributed for a reduction in the ALA content of N- and N-Pcultures. However, the ALA fraction of total fatty acids in P- culture did not show any significant change and was comparable to control cultures. The maintenance of the ALA

19

fraction in P- cultures could be attributed to the availability of reserve/endogenous phosphorus, like stored polyphosphate (Kamalanathan et al., 2016; Arora et al., 2016), compensating for the non-availability of phosphorous from the growth medium. However, the ALA content of biomass in all the cultures (N-, N-P- and P-) was comparable to control cultures (Table 3) which could be attributed to higher lipid accumulation and/or maintenance of ALA fraction in total fatty acids in nutrient stressed cultures. 3.3.2. Effect of low-temperature on growth, lipids and fatty acid profile of nutrient stressed Desmodesmus sp. The nitrogen and phosphorous starvation, independently and synergistically, enhanced the total lipid content of Desmodesmus sp. with a varied fatty acid profile. The low-temperature stress led to an enhancement of the ALA fraction of total fatty acids in control culture and UV treated culture (see section 3.1 and 3.3 above). Therefore, the lowtemperature stress was combined with nutrient stress to explore its effect on enhancement of ALA-rich lipid in Desmodesmus sp. As described, both the low temperature and the nitrogen deficiency (N- and N-P-) stress individually led to a reduction of specific growth rate, biomass yield and biomass productivity in Desmodesmus sp. (see section 3.1.1 and 3.3.1). When the nitrogen deficiency (N- and N-P-) was combined with low-temperature stress, the adverse effect of these stresses on microalgal growth was multiplied. The growth of P- cultures, which had remained unaffected at 25 °C, too was adversely affected under low temperature. The nutrient deprived (N-, P- and N-P-) microalgal cultures failed to record any growth under low temperature, and during the period of study, there was 7 to 12% reduction in biomass concentration. The loss in biomass concentration had a negative effect on all the growth-

20

related parameters viz., specific growth rate, biomass yield and biomass productivity (Table 3). The total lipid content (w/w) of N-, P- and N-P- cultures incubated at 5 °C, was 41%, 40% and 42%, respectively, i.e., an increase of 3%, 40% and 13%, respectively, compared to their corresponding cultures maintained at normal growth temperature of 25 °C (Table 3). When compared to the lipid content of control cultures at 25 °C, the increase in the lipid content of N-, P- and N-P- cultures at 5 °C was 1.75 fold, 1.70 and 1.81 fold, respectively. It was observed throughout the study that the lipid content of Desmodesmus sp. increased by about 1.54 fold (from about 22% w/w to 33% w/w) when subjected to low-temperature stress by incubating at 5 °C. When compared with each other, the total lipid content of 5 °C incubated N-, P- and N-P- cultures did not show any significant difference (40% w/w to 42% w/w). Therefore, it appears that when the culture experienced the combination of low-temperature stress and nutrient stress, the low temperature played the role of the major stressor for inducing lipid accumulation and the effect of nutrient stress was minor and also did not appear to be nutrient specific. The ALA fraction of total fatty acids of N-, P- and N-P- cultures incubated at 5 °C were 33%, 23% and 25%, respectively, considerably lower than the 44% shown by the control (nutrient replete) culture incubated at 5 °C (Fig. 5). The ALA fraction of total fatty acids in N- cultures at 5 °C was 1.31, and 1.77 fold higher than control and N- cultures maintained at the normal growth temperature of 25 °C, respectively. In the case of Pcultures incubated at 5 °C, the ALA fraction of total fatty acids remained the same as in control cultures and P- cultures at 25 °C. The ALA fraction of total fatty acids of the N-Pculture incubated at 5 °C was about two-fold higher than the corresponding cultures maintained at 25 °C and comparable to the control culture maintained at 25 °C. This

21

suggests that while the lower temperature promoted the unsaturation of fatty acids in nutrient-replete conditions, the effect was not so pronounced with nutrient-depleted cultures and also the depletion of phosphorous and nitrogen affected the microalgal response to lower temperature differently. The increase of ALA fraction of total fatty acids in N- cultures and non- enhancement in P- and N-P- cultures at 5 °C suggests that phosphorous is required for ALA synthesis in the microalgal culture. Phosphorus is required for membrane development and for the synthesis of phospholipids which mainly contains PUFA. Therefore, phosphorous concentration in the growth medium can affect the overall PUFA content in microalgae (Guschina and Harwood, 2009; Paliwal et al., 2017). The non-availability of enough phosphorus in the growth medium obstructed the enhancement of ALA in phosphorous deficient cultures. However, the maintenance of the level of ALA fractions of total fatty acids in P- cultures at 5 °C to those observed in control and P- cultures at 25 °C could be attributed to the availability of reserve phosphorus, like stored polyphosphate in microalgal cultures (Arora et al., 2016). This reduction in ALA fraction of total fatty acids in nutrient-deprived cultures also resulted in reduced ALA content in biomass compared to control cultures at 5 °C. 4. Conclusion The low temperature, UV treatment and nutrient stress independently led to an enhancement in lipid accumulation in the indigenous freshwater microalga Desmodesmus sp. The low temperature (5 °C), as an independent stress, enhanced the lipid content to 34% (w/w) and ALA fraction of total fatty acids to 44%. The incubation of UV treated cultures at low temperature (5 °C) enhanced the lipid content to 62% (w/w) with ALA fraction being 42%. Therefore, the low-temperature stress alone or incubation of UV

22

treated cultures at low temperature could be used as a strategy to obtain microalgal biomass with ALA-rich lipids. Competing interest statement: Authors declare that they do not have any competing interests to declare. Acknowledgments PVS acknowledges the UGC, Govt. of India for the award of Senior Research Fellowship. Authors thank Director, CSIR-CFTRI for constant encouragement. References 1. Adarme-Vega, T.C., Lim, D.K.Y., Timmins, M., Vernen, F., Li, Y., Schenk, P.M., 2012. Microalgal biofactories: a promising approach towards sustainable omega-3 fatty acid production. Microb. Cell Factories 11, 96. 2. 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. Bioresour. Technol. 213, 79-87. 3. Barceló-Coblijn, G., Murphy, E.J., 2009. Alpha-linolenic acid and its conversion to longer chain n- 3 fatty acids: Benefits for human health and a role in maintaining tissue n- 3 fatty acid levels. Prog. Lipid Res. 48, 355-374. 4. Cheng, D., He, Q., 2014. Assessment of environmental stresses for enhanced microalgal biofuel production-an overview. Front. Energy Res. 2, 26. 5. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294-306. 6. Converti, A., Casazza, A.A., Ortiz, E.Y., Perego, P., Del Borghi, M., 2009. Effect of temperature and nitrogen concentration on the growth and lipid content of

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Nannochloropsis oculata and Chlorella vulgaris for biodiesel production. Chemical Engineering and Processing: Process Intensification 48, 1146–1151. 7. Flaim, G., Obertegger, U., Anesi, A., Guella, G., 2014. Temperature-induced changes in lipid biomarkers and mycosporine-like amino acids in the psychrophilic dinoflagellate Peridinium aciculiferum. Freshw. Biol. 59, 985-997. 8. Guihéneuf, F., Fouqueray, M., Mimouni, V., Ulmann, L., Jacquette, B., Tremblin, G., 2010. Effect of UV stress on the fatty acid and lipid class composition in two marine microalgae Pavlova lutheri (Pavlovophyceae) and Odontella aurita (Bacillariophyceae). J. Appl. Phycol. 22, 629-638. 9. Guschina, I.A., Harwood, J.L., 2009. Algal lipids and effect of the environment on their biochemistry, in: Lipids in Aquatic Ecosystems. Springer, pp. 1-24. 10. Institute of Medicine: Food and Nutrition Board, 2005. Dietary reference intakes for energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein, and amino acids (macronutrients). Washington, DC: National Academy Press. 11. Kamalanathan, M., Pierangelini, M., Shearman, L.A., Gleadow, R., Beardall, J., 2016. Impacts of nitrogen and phosphorus starvation on the physiology of Chlamydomonas reinhardtii. J. Appl. Phycol. 28, 1509-1520. 12. Lei, A., Chen, H., Shen, G., Hu, Z., Chen, L., Wang, J., 2012. Expression of fatty acid

synthesis genes and fatty acid accumulation in Haematococcus pluvialis under different stressors. Biotechnology for Biofuels 5, 18. 13. Lichtenthaler, H.K., 1987. [34] Chlorophylls and carotenoids: pigments of photosynthetic biomembranes, in: Methods in Enzymology. Elsevier, pp. 350–382. 14. Lu, Q., Li, Jun, Wang, J., Li, K., Li, Jingjing, Han, P., Chen, P., Zhou, W., 2017. Exploration of a mechanism for the production of highly unsaturated fatty acids in

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Scenedesmus sp. at low temperature grown on oil crop residue based medium. Bioresour. Technol. 244, 542-551. 15. Markou, G., Dao, L.H., Muylaert, K., Beardall, J., 2017. Influence of different degrees of N limitation on photosystem II performance and heterogeneity of Chlorella vulgaris. Algal Res. 26, 84-92. 16. Mathur, S., Allakhverdiev, S.I., Jajoo, A., 2011. Analysis of high temperature stress on the dynamics of antenna size and reducing side heterogeneity of Photosystem II in wheat leaves (Triticum aestivum). Biochim. Biophys. Acta BBA-Bioenerg. 1807, 2229. 17. Msanne, J., Xu, D., Konda, A.R., Casas-Mollano, J.A., Awada, T., Cahoon, E.B., Cerutti, H., 2012. Metabolic and gene expression changes triggered by nitrogen deprivation in the photoautotrophically grown microalgae Chlamydomonas reinhardtii and Coccomyxa sp. C-169. Phytochemistry 75, 50–59. 18. Paliwal, C., Mitra, M., Bhayani, K., Bharadwaj, S.V., Ghosh, T., Dubey, S., Mishra, S., 2017. Abiotic stresses as tools for metabolites in microalgae. Bioresour. Technol.244, 1216-1226. 19. Parsaeimehr, A., Sun, Z., Dou, X., Chen, Y.-F., 2015. Simultaneous improvement in

production of microalgal biodiesel and high-value alpha-linolenic acid by a single regulator acetylcholine. Biotechnology for biofuels 8, 11. 20. 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. Bioresour. Technol. 168, 127-135. 21. Sharma, K., Schenk, P.M., 2015. Rapid induction of omega-3 fatty acids (EPA) in Nannochloropsis sp. by UV-C radiation. Biotechnol. Bioeng. 112, 1243-1249.

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22. Sharma, K.K., Li, Y., Schenk, P.M., 2015. Rapid lipid induction in Chlorella sp. by

UV-C radiation. BioEnergy Res. 8, 1824-1830. 23. Vidyashankar, S., Venu Gopal, 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. 24. Wong, C.Y., Chu, W.L., Marchant, H., Phang, S.M., 2007. Comparing the response of Antarctic, tropical and temperate microalgae to ultraviolet radiation (UVR) stress. Journal of Applied Phycology 19, 689–699. 25. Wu, Y.-H., Yu, Y., Hu, H.-Y., 2013. Potential biomass yield per phosphorus and lipid accumulation property of seven microalgal species. Bioresour. Technol. 130, 599-602. 26. 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. 27. Yang, L., Chen, J., Qin, S., Zeng, M., Jiang, Y., Hu, L., Xiao, P., Hao, W., Hu, Z., Lei, A., 2018. Growth and lipid accumulation by different nutrients in the microalga Chlamydomonas reinhardtii. Biotechnol. Biofuels 11, 40. 28. Yi-Bin, W., Fang-Ming, L., Xiu-Fang, Z., Ai-Jun, Z., Bin, W., Zhou, Z., Cheng-Jun, S., Jin-Lai, M., 2017. Composition and regulation of thylakoid membrane of Antarctic ice microalgae Chlamydomonas sp. ICE-L in response to low-temperature environment stress. J. Mar. Biol. Assoc. U. K. 97, 1241-1249.

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Figure Captions Fig. 1. Growth and fatty acid profiles of nutrient replete cultures of microalgae at different temperatures, A and E- Scenedesmus dimorphus, B and F- Desmodesmus sp., C and GChlorella sp., and D and H- Oocystis pusilla Fig. 2. Photosynthetic parameters of Desmodesmus sp. at 25 °C and 5 °C, A- photosynthetic efficiency, B- average antenna size and C- energy dissipation Fig. 3. The recovery of growth (A), photosynthetic efficiency (B), average antenna size (C), and energy dissipation (D), of Desmodesmus sp. after UV treatment Fig. 4. Growth and fatty acid profile of recovered UV treated Desmodesmus sp. after subsequent subculturing, at different temperature, A- growth profile at 25 °C, B-fatty acid profile at 25 °C, C-growth profile at 5 °C, D-fatty acid profile at 5 °C Fig. 5. The effect of Nutrient stress on Desmodesmus sp. growth profile (A), photosynthetic efficiency (B) , average antenna size (C), energy dissipation (D), fatty acid profile (E), and fatty acid profile of nutrient stressed cultures incubated at 5 oC (F)

27

45

0.6

Biomass (g L-1)

Relative % of fatty acids

A

0.5 0.4 0.3 0.2 0.1

40 35

E

30 25 20

15 10 5 0

0 0

5

10

15

25

15

5

25

15

5

Incubation Temperature (°C)

Time (Days)

0.7

B

0.6

Biomass (g L-1)

Relative % of fatty acids

0.8

0.5 0.4 0.3 0.2 0.1 0 0

5

10

50 45 40 35 30 25 20 15 10 5 0

F

15

Incubation Temperature (°C)

Time (Days) 0.7

C

0.6

Biomass (g L-1)

Relative % of fatty acids

60

0.5 0.4 0.3 0.2

0.1 0

50 40 30 20

10 0

0

5

10

15

25

45

Relative % of fatty acids

0.6

D

0.5

15

5

15

5

Incubation temperature (°C)

Time (Days)

0.4

Biomass (g L-1)

G

0.3 0.2 0.1

40

H

35 30 25 20 15 10 5

0

0 0

5

10

15

Time (Days) 25 °C

15 °C

25

Incubation Temperature (°C) C-16:0

5 °C

C-18:0

C-18:1

C-18:3

C-18:2

28 Fig. 1. Growth and fatty acid profiles of nutrient replete cultures of microalgae at different temperatures, A and E- Scenedesmus dimorphus, B and F- Desmodesmus sp., C and G- Chlorella sp., and D and H- Oocystis pusilla

0.8

A

0.7

3.5

0.6

3

0.5

2.5

ABS/RC

Fv/Fm

4

0.4 0.3

2 1.5

0.2

1

0.1

0.5

0

B

0 1

3

5

7

9

Time (Days)

11

13

15

1

3

5

7

9

11

13

15

Time (Days)

3

2.5

C

DI0/RC

2

1.5

1

0.5

0

1

3

5

7

9

11

13

15

Time (Days) 25 °C

5 °C

Fig. 2. Photosynthetic parameters of Desmodesmus sp. at 2925 °C and 5 °C, A- photosynthetic efficiency, B- average antenna size and C- energy dissipation

0.8

0.5

A

B

0.7

0.4

0.6

0.35

Fv/Fm

Biomass (g L-1)

0.45

0.3

0.5

0.25

0.4

0.2

0.3

0.15

0.2

0.1

0.1

0.05 0

0 1

3

5

7

9

11

13

15

17

1

3

5

7

control

UV20min

UV40min

11

13

15

UV60min

9

8

8

7

C

7

D

6

DI0/RC

6 5

ABS/RC

9

Time (Days)

Time (Days)

4

5 4

3

3

2

2

1

1

0

0 1

3

5

7

9

11

13

15

UV20min

UV40min

3

5

7

9

11

13

15

Time (Days)

Time (Days) control

1

UV60min

Fig. 3. The recovery of growth (A), photosynthetic30efficiency (B), average antenna size (C), and energy

dissipation (D), of Desmodesmus sp. after UV treatment

40

0.9

A

35

Relative % of fatty acids

0.8

Biomass (g L-1)

0.7 0.6 0.5 0.4 0.3 0.2

B

30 25 20

15 10 5

0.1

0

0 1

3

5

7

9

11

Control

13

0.5

UV 40 min

UV 60 min

50

C

0.45 0.4

45

Relative % of fatty acids

Biomass(g L-1)

UV 20 min

Culture conditions

Time (Days)

0.35 0.3 0.25 0.2

0.15 0.1

D

40 35

30 25 20 15 10 5

0.05

0

0 1

3

5

7

9

11

13

control

Time (Days) control

UV 20 min

UV 40 min

UV 20 min

UV 40 min

UV 60 min

Culture conditions UV 60 min

16:0

18:0

18:1

18:3

18:2

31 Fig. 4. Growth and fatty acid profile of recovered UV treated Desmodesmus sp. after subsequent subculturing, at different temperature, A- growth profile at 25 °C, B-fatty acid profile at 25 °C, C-growth profile at 5 °C, D-fatty acid profile at 5 °C

0.7

0.8

A

0.6

0.6

0.5

0.5

0.4

Fv/Fm

Biomass (g L-1)

B

0.7

0.3

0.4 0.3

0.2

0.2

0.1

0.1 0

0 1

3

5

7

9

11

Time (Days)

control

N-

13

1

15

7

14

21

Time (Days)

P-

N-P-

control

4.5

N-

P-

N-P-

3

4 3.5

C

3

D

2

2.5

DI0/RC

ABS/RC

2.5

2

1.5

1.5

1

1 0.5

0.5 0

0 1

7

14

21

1

7

Time (Days) control

N-

P-

control

N-P-

E

Relative % of fatty acids

Relative % of fatty acids

21

N-

P-

N-P-

50

40 35

14

Time (Days)

30 25

20 15 10

45 40

F

35 30 25 20 15 10

5

5

0

0 control

N-

P-

N-P-

control

C-18:0

C-18:1

C-18:3

P-

N-P-

Culture conditons

Culture conditions C-16:0

N-

C-18:2

C-16:0

C-18:0

C-18:1

C-18:3

C-18:2

32 Fig. 5. The effect of Nutrient stress on Desmodesmus sp. growth profile (A), photosynthetic efficiency (B) , average antenna size (C), energy dissipation (D), fatty acid profile (E), and fatty acid profile of nutrient stressed cultures incubated at 5 oC (F)

Table 1. The effect of cultivation temperature on growth and biochemical characteristics of nutrient replete cultures of microalgae. Temp. Scenedesmus (oC) dimorphus Specific growth rate (µ day-1) 25 0.077±0.005b 15 0.040±0.005a 5 0.040±0.010a

Desmodesmus sp.

Chlorella sp.

Oocystis pusilla

0.075±0.005b 0.046±0.009a 0.040±0.004a

0.093±0.003b 0.051±0.007a 0.054±0.003a

0.073±0.004b 0.060±0.004a 0.057±0.002a

9.2±0.80b 15.0±3.17a 15.9±1.43a

7.5±0.20b 13.6±1.95a 12.9±0.63a

9.4±0.51b 11.6±0.71a 12.2±0.10a

0.380±0.02b 0.195±0.04a 0.183±0.02a

0.433±0.01b 0.172±0.02a 0.191±0.01a

0.316±0.01c 0.239±0.02b 0.198±0.01a

0.032±0.002b 0.016±0.003a 0.015±0.003a

0.036±0.001b 0.015±0.002a 0.015±0.001a

0.026±0.001c 0.020±0.002b 0.017±0.001a

22.09±3.08a 24.17±0.87a 33.64±1.00b

21.12±0.68a 24.08±1.01b 30.67±1.64c

18.05±1.46a 22.12±1.71b 26.12±1.11c

7.60±0.24c 3.37±0.14a 4.91±0.26b

4.69±0.38a 4.42±0.34a 4.44±0.19a

3.42±0.27a 9.64±0.71b 8.84±0.27b

4.51±0.63a 8.71±0.59b 10.20±0.76b

Doubling time (h) 25 15 5

9.08±0.71b 19.25±3.33a 18.10±4.67a

Biomass yield (g L-1) 25 0.318±0.03b 15 0.128±0.003a 5 0.119±0.02a Biomass productivity (g L-1 day-1) 25 0.026±0.023b 15 0.011±0.002a 5 0.010±0.002a Lipid content (%w/w) 25 15 5

18.64±1.97 a 23.99±0.67b 34.25±0.74c

Lipid productivity (mg L-1 day-1) 25 4.85±0.51c 7.07±0.99b a 15 2.64±0.07 3.87±0.14a b 5 3.42±0.07 5.05±0.15a Relative ALA content in biomass (% w/w) 25 3.76±0.02a 5.44±0.51a 15 6.33±0.20c 9.66±0.18b b 5 5.26±0.10 14.91±0.61c Total chlorophyll (µg mL-1) 25 3.05±0.25b 15 2.35±0.23a 5 2.47±0.08a Total carotenoid (µg mL-1)

15.56±0.73b 7.06±0.74a 6.01±0.30a

5.44±0.38 b 3.67±0.11 a 3.27±0.24 a

6.69±0.11b 7.69±0.69b 3.17±0.23a

1.28±0.05a 1.11±0.03a 1.28±0.14a

6.13±0.53b 2.45±0.21a 2.46±0.12a

2.7±0.27b 2.03±0.12a 2.03±0.21a

2.76±0.29b 2.82±0.14b 1.43±0.10a

25 15 5

Data represents mean ± SD of three replicates, mean values in each column of each section sharing common alphabets are statistically not significant at P < 0.05 by one way ANOVA.

Table 2. The growth parameters and biochemical characteristics of UV treated Desmodesmus sp. at 25 °C and 5 °C 5 °C

25 °C Parameters

Control

UV20min

UV40min

UV60min

Control

UV20min

UV40min

UV60min

Specific growth rate (µ day-1) Doubling time (h) Biomass yield (g L-1) 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)

0.072±0.004a

0.086±0.003b

0.093±0.003b

0.089±0.005b

0.039±0.005a

0.051±0.003a

0.051±0.006a

0.050±0.008a

9.60±0.59b

8.10±0.29a

7.4±0.26a

7.8±0.40a

17.9±2.29a

13.7±0.79a

13.5±1.75a

13.9±2.57a

0.365±0.02a

0.431±0.02b

0.498±0.01c

0.504±0.02c

0.171±0.02a

0.180±0.01a

0.178±0.02a

0.189±0.03b

0.030±0.002a

0.036±0.002b

0.042±0.001c

0.042±0.002c

0.015±0.003a

0.015±0.002a

0.015±0.002a

0.016±0.003a

23.15±2.16a

32.63±1.31b,c

31.21±1.00b

36.97±0.97c

34.18±1.39a

48.83±1.44b

61.93±3.40c

59.09±1.19c

7.04±0.66a

11.71±0.47b

12.96±0.42b

14.83±0.39c

4.98±0.50a

7.34±0.22b

9.19±0.51c

9.33±0.19c

5.97±0.35a

8.59±0.39b

10.16±1.11b,c

11.49±0.36c

14.92±0.99a

16.02±1.36a

26.05±1.53b

22.88±1.05b

16.39±1.18a

16.66±0.98a

17.11±0.90a

17.13±0.28a

6.56±0.51a

6.81±0.08a,b

7.74±0.45b,c

8.28±0.43c

6.36±0.51a

6.32±0.46a

7.23±0.28a

7.13±0.36a

2.39±0.19a,b

2.03 ± 0.17a

2.69 ± 0.34a,b

2.79 ± 0.29b

Total chlorophyll (µg mL-1) Total carotenoid (µg mL-1)

Data represents mean ± SD of three replicates, mean values in a raw sharing common alphabets are statistically not significant at P<0.05 by one way ANOVA.

Table 3. The growth parameters and biochemical characteristics of nutrient stressed Desmodesmus sp. cultures at 25 °C and 5 °C 5 °C

25 °C Parameters

Control

N-

P-

N-P-

Control

N-

P-

N-P-

Specific growth rate (µ day-1) Doubling time (h) Biomass yield (g L-1)

0.076±0.007a

0.034±0.003b

0.071±0.006a

0.036±0.005b

0.040±0.012

-

-

-

9.1±0.80a

20.5±1.60b

9.8±0.84a

19.7±2.78b

16.7±1.5

-

-

-

0.367±0.05a

0.104±0.01b

0.325±0.04a

0.124±0.02b

0.180±0.01

-

-

-

0.031±0.004a

0.010±0.001b

0.027±0.004a

0.010±0.001b

0.017±0.005

-

-

-

22.49±2.41a

39.03±1.19c

28.07±1.96b

37.26±2.18c

34.05±1.35a

40.53±2.16b

39.50±1.80b

42.10±2.44b

6.75±0.74a

3.38±0.10b

7.59±0.53a

3.85±0.22b

5.48±0.33

-

-

-

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) Total chlorophyll (µg mL-1) Total carotenoid (µg mL-1)

5.57±0.54a

7.17±0.39b

7.22±0.38b

4.92±0.50a

14.95±0.72b

13.21±0.89b

9.10±0.56a

10.57±0.49a

15.66±0.45c

9.73±0.22b

16.05±0.38c

7.05±0.13a

6.37±0.26a

6.32±0.37a

6.17±0.44a

6.10±0.54a

5.85±0.30b

3.27±0.23a

5.38±0.29b

3.87±0.11a

2.14±0.18

2.12±0.20a

2.01±0.11a

2.08±0.14a

Data represents mean ± SD of three replicates, mean values in a row sharing common alphabets are statistically not significant at P<0.05 by one way ANOVA. “-” denotes no positive values

HIGHLIGHTS 

Freshwater microalga Desmodesmus sp. produced ALA-rich lipids under low temperature, UV treatment and nutrient stress.



Low temperature led to a 1.8 fold increase in ALA fraction of total fatty acids.



UV treated cultures at low temperature showed a 2.6 fold increase in lipid content.



UV treated cultures at low temperature showed maximum ALA content of biomass.