Bioresource Technology 172 (2014) 32–40

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Potential of Monoraphidium minutum for carbon sequestration and lipid production in response to varying growth mode Shailesh Kumar Patidar a, Madhusree Mitra a,b, Basil George a, R. Soundarya a,b, Sandhya Mishra a,b,⇑ a b

Salt and Marine Chemicals Discipline, CSIR – Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India Academy of Scientific & Innovative Research (AcSIR), CSIR – Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Monoraphidium minutum remediated

flue gas.  Intermittent CO2 supply maximizes

lipid accumulation while growing autotrophically.  Max. biomass productivity was observed in mixotrophic growth.  Higher C/N of lyophilized biomass showed higher lipid accumulation.  Resultant biodiesel met with European biodiesel standard.

a r t i c l e

i n f o

Article history: Received 16 June 2014 Received in revised form 11 August 2014 Accepted 16 August 2014 Available online 23 August 2014 Keywords: Biodiesel Flue gas Intermittent CO2 supply Mixotrophic Monoraphidium minutum

a b s t r a c t Mixotrophic growth at flask level and, autotrophic–mixotrophic and autotrophic growth in photobioreactor by utilizing CO2/air/flue gas were checked for the isolated strain of Monoraphidium minutum from polluted habitat. Our study confirmed that it is a saturated fatty acid rich (30.92–68.94%) microalga with lower degree of unsaturation oil quality (42.06–103.99) making it potential biodiesel producing candidate. It showed encouraging biomass productivity (80.3–303.8 mgl 1 day 1) with higher total lipid (22.80–46.54%) under optimum glucose, fructose, microalgal biodiesel waste residue and sodium acetate fed mixotrophic conditions. The pH control by intermittent CO2, continuous illumination with 30% flue gas, and utilization of biodiesel glycerin were effective schemes to ameliorate either biomass productivity or % lipids or both of these parameters at photobioreactor scale (7.5 L working volume). The modulation of environmental variables (pH control, CO2 and organic substrates concentration) could augment % saturated fatty acids, such as C16:0. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Microalgae have maximum aerial/volumetric biomass productivity with reported maximum carbon fixation capability in whole plant kingdom. Many of the microalgae are investigated for their ⇑ Corresponding author at: Salt and Marine Chemicals Discipline, CSIR – Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India. Tel.: +91 278 2561354; fax: +91 278 2567562. E-mail address: [email protected] (S. Mishra). http://dx.doi.org/10.1016/j.biortech.2014.08.070 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

potential of carbon fixation and biofuel production. Green microalgae are attaining significant attraction in sustainable green technology for the future of bioenergy and environment. Out of the all microalgal classes, most of the Chlorophycean microalgae are known as lipid rich and having comparatively higher biomass productivity (Rawat et al., 2013). Monorphidium sp. is among the oleaginous Chlorophycean species having favorable growth rates and diverse metabolic lipid pathways (Bogen et al., 2013). However, nutrient uptake rate, light, pH, concentration and bioavailability of nutrients are important factors to affect lipid content, fatty acid

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composition and carbon sequestration (Fields et al., 2014; Yu et al., 2012; Kumar et al., 2010). Flue gas capturing by microalgae requires an understanding on optimum environmental conditions for the specific strain, which helps in establishing higher biomass and lipid yield (Fields et al., 2014). Many microalgae can grow mixotrophically being able to utilize organic carbon during photosynthesis while utilizing inorganic carbon (Chandra et al., 2014). In this study, nitrate, phosphate and carbon of the growing culture in medium with closed photobioreactor system is monitored at regular intervals under varying CO2/flue gas concentrations. Further, effect of pH control, photoperiod, and CO2 concentration also estimated on the biomass productivity and lipid content. Mixotrophic growth potential of Monoraphidium minutum with respect to biomass, total lipid content and lipid profiling at flask level was also carried out. 2. Methods 2.1. Culture maintenance M. minutum (Accn No.CCNM-1042, CSMCRI culture repository) isolated from contaminated freshwater lagoon from Hazira, Surat, India (21o6.129´N, 72o37.794´E), was propagated as monoalgal culture, maintained in Zarrouk’s media (Zarrouk, 1966) under autotrophic condition by providing 100 lmole m 2 s 1 light for 12:12 dark–light period at 28 °C temp. Detail of isolation and identification is given (Supplementary Text S1). 2.2. Experiments under flasks conditions Monoalgal culture of the late log phase was harvested by centrifugation at 8000 rpm and fresh pellet washed with sterile water twice followed by re-centrifugation. Inoculum in the form of fresh wet pellet (100 mg) was re-suspended in 100 ml autoclaved media for the experiments under sterile conditions. Mixotrophic growth experiments conducted in autoclaved media containing organic substrate (Glucose/fructose/microalgal biodiesel waste residue/ sodium acetate) in addition to Zarrouk’s media and parallel control was run in each experiment. The inorganic carbon source was sodium bicarbonate and organic carbon source was glucose (G)/fructose (F)/biodiesel glycerin (BG)/sodium acetate (SA) for mixotrophic experiments. All experimental sets were run in triplicates with control. The pH on the initial day adjusted to 8.5 by using autoclaved 0.3 M KOH and HCl. Each flask confronted to 100 lmole m 2 s 1 light, 25 °C temp. and 12:12 dark–light period. Only manual shaking was provided once in each day of the experiment. 2.3. Experiments under photobioreactor conditions The bioreactor (BioFlo/CelliGen 115; 14 L capacity) externally equipped with lamp (100 lmole m 2 s 1) and having process control option for pH, dissolved oxygen (DO), agitation, temperature, pump feed, aeration, antifoam and foam/level, was used for the experiments on M. minutum. The photobioreactor equipped with mass flow controller up to four ports of gas inlet and mixing control options. The photobioreactor was sterilized before each experiment by autoclaving. Inoculum was added to autoclaved media under sterile condition in the photobioreactor. The working volume was 7.5 L for each batch of the experiment and, initial optical density for each experiment was 0.5. The rotation per minute (rpm) of impeller throughout all the experiments was 200 rpm and temperature was controlled at 28 °C. The details of different experimental variables of each set are shown in Table 1.

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2.4. Growth, biovolume, biomass productivity and carbon sequestration rate In addition to OD measurement of culture at 540 nm by using UV–visible spectrophotometer (Varian Cary 50 Bio, USA), fluorescence based biovolume was measured using Fluoroprobe (bbe, Moldaenke GmbH, Germany) on each day of the experiment. There was a linear relationship between OD540 and cell dry weight (Supplementary Fig. 1). Culture harvesting was done by using centrifugation (8000 rpm for 10 min.) while cells were reached at stationary phase and, then microalgal wet biomass was lyophilized. Biomass productivity and carbon fixation rate was calculated by following equation: Biomass productivity (mg/L/day) = Total dry weight (mg)/Total culture volume (L)  Time interval of growth (day) Carbon fixation rate (mg/L/day) = Biomass productivity  % C of dried biomass on harvesting day (Zhao et al., 2014) 2.5. CO2 monitoring on headspace CO2 analyzer (Thermo Scientific, 410i, USA) employed for CO2 measurement. The sampling tube arranged to connect tightly to the vent over photobioreactor which open up to pass the excess gas of the headspace over culture environment. The flow for sampling the gas for analysis into analyzer system through silicone tube was 0.8 L/min and, readings were taken for three minutes after warm up of 1 min. continuously. This was done regularly after accomplishment of photosynthetic period of 12 h/24 h. 2.6. Carbon measurement from culture Total organic carbon and total dissolved organic carbon was analyzed by TOC analyzer (elementarLiqui TOC/TN) in an interval of 3 days during the growth till microalgae reached to the stationary phase. Dissolved fraction was analyzed after filtration of sample with 0.45 lM whatman filter papers and, suitable dilution was done using Milli-Q water (Millipore, USA) for analysis. The elemental (C, H, N, S) composition (%) of dried biomass (105 °C for 24 h in oven) was analyzed by the CHNS analyzer (elementarvarioMicro) and, sulphanilamide was used as a reference standard. The measured values of the standard had sodium acetate > glucose > fructose, while, for optimum biomass productivity was fructose > glucose > microalgal biodiesel residue > sodium acetate. After attaining optimum biomass productivity as well as % lipid, it was seen that higher organic substrate concentration diminished these both parameters. It was previously established that optimum C/N ratio increases lipid % as well as growth which was in accordance with our findings (Chen and Johns, 1991; Morales-Sánchez et al., 2013). Generally in microalgae organic carbon is assimilated into intermediates of the TCA cycle and it is finally oxidized to carbon dioxide (Liu et al., 2010), hence, response of BG was similar to optimum high CO2 concentration which increased % lipid.

3.2. Autotrophic/autotrophic–mixotrophic growth, biomass productivity and lipid content in Photobioreactor The biomass productivity (mg/L/day) of the different experimental conditions investigated in photobioreactor was 140.82, 137.4, 88, 68.25 and 29.47 for E, D, C, and B, respectively. Total lipid content (% cdw) was 42.44, 35, 34.97, 31.58, and 29.68 for A, C, D, E, and B, respectively as shown in Fig. 2. Similar trend for carbon fixation rate was observed (Supplementary Fig. 2).Time for attainment of stationary phase of microalgal cells was also varied under different experiments (Fig. 3). Intermittent CO2 supply for controlling the pH improved the lipid content, which was the highest among all the experiments conducted in photobioreactor (Fig. 2). The continuous CO2 purging reduced the biomass productivity (29.47 mg/L/day) and total lipid content (29.68%) comparatively to other photobioreactor conditions due to stress of acidic pH. Photosynthetic –mixotrophic growth mode (E) in photobioreactor increased biomass productivity (137.4 mg/L/day) in comparison of A, B and C, wherein same photoperiod (12 h) applied. It was achieved by uptake of glycerol in late log phase. The condition D (flue gas 30% with 24 h photoperiod on constant pH 8.0) showed higher biomass productivity (140.82 mg/L/day) and, attained stationary phase comparatively earlier on 15th day which was remarkable incentive of saving of time with 34.97% of total lipid content (cdw) (Figs. 2 and 3). Condition C was different than condition D in respect of flue gas concentration (10% flue gas = 0.075% CO2) and photoperiod (12 h). Here, M. minutum received almost 1/3rd of CO2, and ½ of the light hrs. in comparison to condition D for photosynthesis, hence, biomass productivity declined up to 35.95%, but, total lipid content remained unaffected. Increase in lipid content depends on microalgal strain, as in many cases microalgal total lipid content increased on high CO2 (Carvalho and Malcata, 2005; Fields et al., 2014) as well as continuous illumination but there are many examples of contradiction wherein reverse effect observed (Chen and Johns, 1991). In one of the previous studies on Chlorella sp. it was observed that total lipid

content of microalgae aerated with flue gas was slightly lower than that aerated with only CO2 enriched gas (Chiu et al., 2011). 3.3. OD.540, biovolume and organic carbon pool of the culture The OD.540 of condition E reached maximum up to 4.32 among all the conditions, followed by condition D, A, C, B where it reached up to 3.53, 3.44, 3.37 and 2.52, respectively as shown in Fig. 3. OD540 followed the same order of total biomass content but it was not reflected in biomass productivity due to difference in attained interval of time to reach stationary phase. DOD540 per day was the maximum in condition D because of higher division rate as well as higher chlorophyll synthesis rate of cells owing to higher photosynthesis on 24 h photoperiod (Bouterfas et al., 2006). Biovolume based on fluorescence on different days of the growth were correlated with OD540, and, correlation values were varied due to different organic carbon concentration and pigment ratio in different growth stages (Supplementary Fig. 3). This indicated that fluorescent pigment composition even in single strain varied on different nutrient conditions. The contrasting effect of fluorescence based biovolume in relation to O.D.540 observed in mixotrophic stage in autotrophic–mixotrophic growth mode. R value for A, B, C, D and E were 0.642, 0.9626, 0.6141, 0.9947 and 0.2045, respectively (Supplementary Fig. 3). Mixotrophic growth mode (after 10th day in condition E) showed declension in chlorophyll synthesis rate because of switch on utilization of glycerol for the growth where comparatively lower photosynthesis took place than other conditions (A, B, C and D). The relationship was found to be maximum linear for continuous photosynthetic mode on 24 h of illumination. In general dissolved organic carbon concentration increased with respect to each successive day of growth in the initial phase of the growth interval of 6 days, but the concentration varied after this in different experimental conditions as shown in Fig. 4. It was observed maximum in condition D due to continuous luxuriant growth followed by condition C and E. E condition was an exception when mixotrophic growth started. An abrupt change in dissolved organic carbon concentration observed on 10th day when glycerol was added, subsequently dissolved organic carbon concentration declined. It showed the affinity of net organic carbon utilization of M. minutum during mixotrophic growth mode. Culture growing in intermittent CO2 supply also balanced the dissolved organic carbon concentration after 6th day wherein the slight decrement in dissolved organic carbon concentration observed. This indicated that under intermittent CO2 supply M. minutum also utilized organic carbon. Total organic carbon concentration was in accord with the order of OD540 of the culture medium as shown in Fig. 4. 3.4. Headspace CO2 (outlet CO2) trend The trend of outlet CO2 (ppm) exhibited that, after 6th day of the growth except condition B in all of the experimental conditions (A, C, D and E), CO2 reached 90% of reduction compare to 1st day’s peak CO2 level (Supplementary Fig. 4). The reduction of outlet CO2 was due to dissolution of CO2 as well as by uptake of dissolved inorganic carbon by microalgae. The removal of CO2 in condition B was poor and it reached lately up to 94% on 15th day. The removal was less up to log phase due to acidic pH as it decreased from 8.0 to 5.4 (Data not shown). The shift towards acidic pH increased free CO2 concentration hence more outgassing as CO2 (g) was observed at outlet vent. Continuous purging without the pH control elevated free CO2 under acidic pH which hampered the cell division as well as bicarbonate uptake by microalgae in comparison to other conditions. There was comparatively higher free CO2 concentration defused through the plasmalemma of microalgal cell easily and it raised low pH in cytosol which was toxic for the M. minutum (Miyachi et al., 2003).

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Fig. 4. Trend of total organic carbon and total dissolved organic carbon under different experimental conditions in photobioreactor. Details of experimental code exhibited on X-axis shown in Table 1.

The peak values for outlet CO2 for all conditions were directly correlated to inlet CO2 concentration on day 1 after that consumption exhibited on each successive day of the experiment.

3.5. Dissolved inorganic phosphate and dissolved inorganic nitrate removal rate from the medium It was seen in all of the experimental conditions that both phosphate and nitrate were utilized effectively as growth occurred on each successive day as shown in Fig. 5. Microalgal cells phosphate uptake rate was less after reaching near to stationary phase eventually because of phosphate release by microalgae in the organic/ inorganic form due to death or stress metabolism of the cells, hence it was again available for the live cells for reutilization. The removal rate (ppm/day) for phosphate from the media was 11.18, 8, 11.53, 14.77 and 15.10 for A, B, C, D and E, respectively. The minimum phosphate removal was noticed in condition B due to less growth. Maximum phosphate removal was noticed in condition D and E with the highest biomass productivity as more No.

of cells require comparatively higher amount of phosphate for the growth and metabolism (Figs. 5 and 2). The nitrate removal rate was 90.71, 79.17, 90.90, 70.67, and 101.81 for A, B, C, D, E, respectively. The maximum removal rate was observed in condition E wherein, under photosynthetic mode nitrate was taken up effectively but after shifting to mixotrophic growth the nitrate removal rate was comparatively lower. The glycerol rich microalgal biodiesel residue fed microalgal biomass on photoautoheterotrophic condition (E-mixotrophic mode from day 10th to 18th) utilized less nitrate. Although glycerol is known to increase nitrate uptake but, in this case, utilization of excess KOH for pH adjustment on 10th day and onwards, ascended stress which released more amino acids, ammonium and nitrate, did not strengthen the net removal rate. The condition D (30% flue gas) had lower nitrate removal rate but actual uptake rate may be higher because flue gas containing NOx increased soluble nitrate along with nitrite and nitrous in the medium. The maximum biomass productivity under full photoperiod had higher photoautotrophic growth and utilized nitrate as well as nitrite. The combine nitrogen uptake (nitrate + nitrite) by M. minutum was the maximum under

Fig. 5. Trend of dissolved (inorganic nitrate, nitrite and phosphate) nutrients of the culture under different experimental conditions in photobioreactor. Details of experimental code exhibited on X-axis shown in Table 1.

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condition D (continuous illumination supported growth on 30% flue gas). Nitrite removal was also observed in flue gas experiments after reaching the peak concentration in initial growth stage. Monoraphdium sp. is capable of utilizing NO as well as other nitrogen species, and NO may diffuse through plasmalemma (Brown, 1996; Nagase et al., 2001).

Table 2 Mean CHNS composition and C/N of lyophilized microalgal biomass having optimum lipid yield. Details of code A, B, C, D and E are shown in Table 1.

3.6. Relevance of nutrient to biomass productivity and lipid accumulation The stress evolved during intermittent carbon supply into microalgal culture inducted lipid content due to acute effect of free CO2 for a short interval. The induction of lipid accumulation under this condition was the result of complex regulation. Limiting CO2 supply for some minutes to adjust pH with comparatively higher photon exposure for 12 h (higher ratio of photon to dissolved CO2 for photosynthesis), was probably limiting the efficiency of C-3 cycle and eliciting TAG biosynthesis (Sforza et al., 2012). When the pH increased up to 9.0, CO2 (1% CO2 + 99% air containing 500 ppm CO2) supplied to adjust the pH up to 8.0 which dissolved in the form of bicarbonate along with free CO2. Free CO2 diffuses through plasmalemma easily (Moroney and Somanchi, 1999) and, unutilized excess CO2 in C-3 cycle or recycled excess carbon provided metabolic energy or substrate for TAG biosynthesis (Sforza et al., 2012; Msanne et al., 2012). It was observed in Dunalliela in that carbon availability was directly correlated with increment of total phospholipid content as growth increased and, on higher CO2 effect of nitrogen limitation was more pronounced (Gordillo et al., 1998). It may be also possible in condition A that sudden increase in CO2 level under intermittent CO2 may evolved nitrate limitation in the cell and increased lipid content. The combination of the stressors has different impact on lipid accumulation due to different metabolic changes and, response may be seen on different stages of the cell cycle as studied in microalgae (Fields et al., 2014). Microalgae in response to exogenous inorganic carbon supply determine the photoautotrophic carbon flow for lipid accumulation wherein types and timing of the cell matters and, precursors affect enzymes involved in fatty acid pathway (Valenzuela et al., 2013). Moll et al. (2014) observed that supplementation of inorganic carbon during the microalgal cell growth intermittently, excelled nutrient limitation; hence, lipid accumulation increased. This study supported the hypothesis of lipid accumulation on intermittent CO2 accorded the results obtained. The three major macronutrients, C, N and P are interlinked for the metabolism of macromolecules synthesis hence, limitation of these nutrients at late log to stationary phase enhance lipid accumulation by

Experimental Condition/organic substrate

%C

%H

%N

%S

C/N

Control A B C D E G (Glucose) F (Fructose) Biodiesel glycerin SA (Sodium acetate)

44.97 45.07 44 46.06 45.94 45 44 42.97 45.43 45.7

6.45 6.61 7.2 7.51 6.80 7.1 6.86 7.04 6.73 7.13

8.62 5.6 8.6 8.14 8.05 8.01 6.22 8.08 5.48 4.2

0.01 0.01 0.04 0.41 1.29 0.02 0.01 0.01 0.01 0.4

5.21 8.05 5.12 5.66 5.71 5.62 7.07 5.32 8.29 10.88

lowering protein/starch synthesis (Fields et al., 2014).This was also observed in ultrastructure images of M. minutum. However, any effect of excessive nitrogen uptake (NO2, NO3, and NO) on lipid accumulation was not observed during flue gas remediation (condition C and E). This may be due to counterbalance of C/N in microalgal cell, wherein N2O emission, organic nitrogen, organic carbon utilization and releasement in dense culture may be one of the strategies (Neilson and Lewin, 1974; Weathers, 1984). Previously effect of CO2 supplementation and sparging interval have been shown to affect lipid as well as growth in mixed consortium of microalgal culture (Prathima Devi and Venkata Mohan, 2012). The elemental composition (% C, H, N and S) of lyophilized microalgal biomass differed under different experimental sets as shown in Table 2. The elemental composition of lyophilized microalgal biomass varied 42.97–46.06, 6.45–7.51, 4.2–8.62, and 0.01–1.29, respectively. C/N composition varied 5.12–10.88. It was observed that C/N ratio was higher than control (5.21) in each experimental set except condition B. Higher C/N in microalgal biomass in condition A of photobioreactor and, microalgal biodiesel glycerin and sodium acetate fed mixotrophically grown biomass confirmed that lipid accumulation was due to nitrogen limitation in the cell. Ultrastructural images (TEM) of the M. minutum exhibited the formation of enlarged lipid granules during stationary phase while lipid granules were tiny during lag phase (Supplementary Fig. 5). 3.7. Fatty acid composition of M. minutum under different experimental conditions Fatty acid composition varied under different experimental conditions (Table 3). The saturated fatty acid (%) increased under both purged CO2 and two stage autotrophic–mixotrophic experi-

Table 3 Fatty acids composition of Monoraphidium minutum during different experimental conditions on the harvesting day. Details of code A, B, C, D and E are shown in Table 1. Values shown in the table are mean of triplicates (mean ± standard deviation). Name of the fatty acids

% fatty acid composition PBR operated cultures

C16:0 C16:1 C:18 C18:1n9c C18:2n6c C18:3n3 SFA USFA MUFA PUFA DU⁄

Culture grown in flasks

A

B

C

D

E

Control

Glucose (G)

Fructose (F)

Sodium acetate (SA)

Biodiesel glycerin (BG)

48.94 ± 2.2 4.45 ± 0.8 ND 28.71 ± 0.9 12.78 ± 0.1 5.12 48.94 51.06 33.16 17.9 68.96

58.79 ± 1.7 7.46 ND 11.12 ± 0.2 13.41 ± 0.5 9.22 ± 0.5 58.79 41.21 18.57 22.63 63.84

38.48 ± 1.2 ND ND 28.65 ± 0.8 21.93 ± 0.4 10.95 38.48 61.52 28.65 32.88 94.4

66.36 ± 2.1 ND ND 25.22 ND 8.42 ± 0.1 66.36 33.64 25.22 8.42 42.06

44.26 ± 0.8 7.49 ND ND 35.47 12.78 ± 0.4 44.26 55.74 7.49 48.25 103.99

30.92 ± 0.9 ND 11.57 28.72 ± 1.5 28.80 ± 0.6 ND 30.92 57.52 28.72 28.8 86.32

63.30 ± 1.5 ND ND 17.99 ± 0.2 18.71 ± 1.4 ND 63.3 36.7 17.99 18.71 55.41

50.98 ± 1.1 1.81 ± 0.1 3.67 27.22 ± 0.4 4.86 ± 1.2 11.46 ± 1.2 52.79 45.35 29.03 16.32 61.67

40.47 ± 0.6 ND ND 24.69 ± 1.1 34.81 ± 0.2 ND 40.47 59.5 24.69 34.81 94.31

47.86 ± 1.2 21.08 ± 0.3 14.59 ± 0.4 7.34 ± 0.5 6.41 ± 0.8 2.73 68.94 37.55 28.41 9.13 46.68

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S.K. Patidar et al. / Bioresource Technology 172 (2014) 32–40 Table 4 Theoretically estimated biodiesel properties for fatty acids of Monoraphidium minutum. Details of code A, B, C, D and E are shown in Table 1. Biodiesel properties

A

B

C

D

E

Control

Glucose

Fructose

Sodium Acetate

Biodiesel glycerin

Saponification value (mg KOH/g fat) Iodine value (g I2/100 g) Cetane number Long chain saturated factor Cold filter plugging point (°C) Cloud point (°C) Allylic position equivalents Bis-allylic position equivalents Oxidation stability (hrs.) Higher heating value (mJ/kg) Kinematic viscosity (mm2/s) Density (g/cm3)

209.41 67.42 57.19 4.89 1.11 20.75 64.51 23.02 9.18 39.33 1.32 0.87

212.18 66.97 56.96 5.88 2 25.93 56.38 31.85 7.8 39.24 1.29 0.87

206.65 95.45 51.24 3.85 4.38 15.25 94.41 43.83 6.18 39.35 1.3 0.87

211.87 45.72 61.77 6.64 4.38 29.92 42.06 16.84 16.6 39.3 1.33 0.87

209.68 106.7 48.32 4.43 2.56 18.29 96.5 61.03 5.03 39.23 1.24 0.87

204.75 77.99 55.41 8.88 11.42 11.27 86.32 28.8 6.69 39.45 1.35 0.87

211.27 50.07 60.87 6.33 3.41 28.3 55.41 18.71 8.89 39.31 1.33 0.87

209.26 66.45 57.43 6.93 5.29 21.82 59.86 27.78 9.82 39.33 1.32 0.87

206.85 85.26 53.5 4.05 3.75 16.3 94.31 34.81 5.98 39.35 1.31 0.87

212.49 46.73 61.47 12.08 21.47 20.18 25.62 11.87 15.49 39.29 1.33 0.87

ments. Among photobioreactor experiments, it was observed that saturated fatty acid (66.36%) was the highest in D condition wherein purged CO2 was the maximum (2.36%) in the form of 30% flue gas with other pollutants like SO2 and NO2 etc. The highest CO2 purging along with continuous illumination (24 h) increased saturated fatty acid C16:0 which was followed by condition B (48.93%) wherein pH was not controlled with continuous purging. The condition C was having minimum SFA (38.48%) due to minimum CO2% purging but with the highest unsaturated fatty acids (61.52%). The conditions A and E were having moderate saturated fatty acids of 48.93% and 44.25%, respectively. However, M. minutum under both conditions had exhibited either comparatively higher lipid content or biomass productivity, which makes it ideal for biodiesel production. Interestingly, condition E had the highest PUFA (48.25%) in the form of C18:2n6c (35.47%) and C18:3n3 (12.78%). The augmentation of C18:2n6c and higher declension of C18:1n9c in condition E comparative to others showed that an uptake of glycerol under mixotrophic condition influenced it in stationary phase. The elevated CO2 and continuous illumination augmented saturated fatty acids in M. minutum was in accordance to the previous studies (Chiu et al., 2011; Guermazi et al., 2014) Flask level experiments of mixotrophic growth exhibited that microalgal biodiesel waste residue fed microalgal cells had the maximum level of saturated fatty acids (62.45%). Except sodium acetate fed microalgal biomass which was having nearly comparable saturated fatty acid% to the control, all of the other organic substrates (G, F and BG) augmented it. However increase in C:16 was noticed in all mixotrophic grown biomass compare to control (autotrophic growth utilizing inorganic carbon as sodium bicarbonate) as shown in Table 3. The variation in composition of fatty acids was possibly impact of cumulative effect of environmental variables which actually regulate metabolism depends on the availability and concentration of precursors for fatty acid synthesis (Fields et al., 2014). The lower degree of unsaturation of oil of M. minutum in present study indicated in all mixotrophic as well as autotrophic condition at photobioreactor and flask level were