Accepted Manuscript Biofuel potential of the newly isolated microalgae Acutodesmus dimorphus under temperature induced oxidative stress conditions Kaumeel Chokshi, Imran Pancha, Khanjan Trivedi, Basil George, Rahulkumar Maurya, Arup Ghosh, Sandhya Mishra PII: DOI: Reference:

S0960-8524(15)00003-6 http://dx.doi.org/10.1016/j.biortech.2014.12.102 BITE 14424

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

27 November 2014 29 December 2014 30 December 2014

Please cite this article as: Chokshi, K., Pancha, I., Trivedi, K., George, B., Maurya, R., Ghosh, A., Mishra, S., Biofuel potential of the newly isolated microalgae Acutodesmus dimorphus under temperature induced oxidative stress conditions, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2014.12.102

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Biofuel potential of the newly isolated microalgae Acutodesmus dimorphus under temperature induced oxidative stress conditions

Kaumeel Chokshiab, Imran Panchaab, Khanjan Trivedibc, Basil Georgea, Rahulkumar Mauryaab, Arup Ghoshbc, Sandhya Mishraab*

a

Discipline of Salt & Marine Chemicals, CSIR- Central Salt and Marine Chemicals Research

Institute, Bhavnagar - 364002, India b

Academy of Scientific & Innovative Research (AcSIR), CSIR- Central Salt and Marine

Chemicals Research Institute, Bhavnagar - 364002, India c

Discipline of Wasteland Research, CSIR- Central Salt and Marine Chemicals Research

Institute, Bhavnagar - 364002, India *Author for correspondence Email address: [email protected] Tel: + 91 278 256 5801 / 256 3805 Extn. 6160 Fax: + 91 278 256 6970 / 256 7562

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Abstract Lack of control over temperature is one of the major issues in large scale cultivation of microalgae. Therefore, it is important to evaluate the effects of cultivation temperature on the growth and physiology of microalgae. In the present study, freshwater microalgae Acutodesmus dimorphus was grown at different temperature in continuous and two stage cultivation. Results revealed that during continuous cultivation A. dimorphus grows better at 35ºC than at 25ºC and 38ºC. At 35ºC, A. dimorphus produced 22.7% lipid (containing 59% neutral lipid) and 33.7% carbohydrate along with 68% increase in biomass productivity (23.53 mg/L/day) compared to 25ºC grown culture. Stress biomarkers like reactive oxygen species, antioxidant enzymes like catalase and ascorbate peroxidase and lipid peroxidation were also lowest in 35ºC grown culture which reveals that A. dimorphus is well acclimatized at 35ºC.

Keywords: Microalgae, Temperature stress, Reactive oxygen species, Antioxidant enzymes, Biofuel

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1. Introduction Microalgae based biofuels is one of the potential alternatives to conventional fossil based fuels to meet the increasing energy demands and fuel crisis in the world. Because of their faster growth rate, higher photosynthetic efficiency and higher lipid and carbohydrate contents, microalgae can be considered as an alternative renewable biofuel production feedstock (Chisti, 2007). Besides biofuel, microalgae have the potential for production of value added compounds like fatty acids, pigments, antioxidants and metabolites with pharmacological applications (Converti et al., 2009). Further, de-oiled microalgal biomass can also be used for bioethanol, biogas, animal feed, fertilizer and biosorption of dyes (Maurya et al., 2014) making microalgal biorefinery by reducing the overall cost of microalgal based biofuel. For sustainable microalgal based biofuel production, currently, research is focussed on increasing the growth rate and biochemical properties of microalgae. Lipid and carbohydrate content in microalgae can be enhanced by chemical stimuli like nitrate (Pancha et al., 2014) and phosphate starvation, salinity stress, physical stimuli like changes in culture pH, temperature and light intensity or photoperiods (George et al., 2014). Among all cultural parameters, temperature is a very crucial factor for the growth and physiology of microalgae (Ho et al., 2014) as it directly influences the photosynthesis (Yun et al., 2010), biochemical composition (Converti et al., 2009) and many other physiological processes of microalgae. Finding the strain that can withstand range of temperatures is of key importance for large scale cultivation of microalgae. Many microalgae have the ability to grow over a wide range of temperatures; however, the response and adaptation of algae to different temperature stress depends on their origin (de Boer et al., 2005; Li and Qin, 2005). Therefore, it is important to evaluate the effects of environmental temperature on the growth of microalgae for their use in large scale cultivation.

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In any organism, fluctuating environmental conditions trigger a series of physiological processes and generation of reactive oxygen species (ROS) like hydrogen peroxide, singlet oxygen, peroxide, hydroxyl and superoxide radicals, which are highly reactive and cause damage to proteins, lipids, carbohydrates and DNA, ultimately resulting into programmed cell death (Alscher et al., 1997). Stress-induced ROS accumulation is counteracted by various enzymatic antioxidant scavengers such as catalase (CAT), ascorbate peroxidase (APX) and non-enzymatic low molecular metabolites such as proline, ascorbic acid etc. (Gill and Tuteja, 2010). According to recent report, oxidative stress is a mediator for increased lipid accumulation in microalgae (Yilancioglu et al., 2014) and oxidative stresstolerant microalgae are highly efficient for biofuel production (Osundeko et al., 2013; Zhang et al., 2013). Relationship among cultivation temperature, ROS level, specific growth rate and lipid content in the microalgae Scenedesmus sp. has been reported by Xin et al. (2011). In the present study, a freshwater microalgae Acutodesmus dimorphus was grown in temperature stress conditions in continuous and two stage cultivation. Various biochemical parameters like photosynthetic pigments, lipids, carbohydrate, protein along with nutrients consumption and stress biomarkers like hydrogen peroxide, antioxidative enzymes activities, proline content and lipid peroxidation were evaluated to examine the immediate response of A. dimorphus to the temperature stress as well as role of ROS in biofuel production potential of A. dimorphus.

2. Materials and methods 2.1. Microalgae and its growth condition The microalgae used in this study was isolated from an industrial effluents polluted stream in GIDC sector of Ankleshwar (21.618 N, 73.027 E), Gujarat, India. It was identified as Acutodesmus dimorphus based on the following morphological characteristics (John et al.,

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2011). Coenobia of 4 linearly or distinctly alternately arranged cells in a row; cells 4-5 µm wide and 18-22 µm long; broadly spindle shaped, tapering to slightly extended apices, with inner cells straight; marginal cells slightly outwardly curved but only in sub apical part. The culture was maintained in BG-11 medium at 25 ± 2ºC under 12:12 hours of light:dark period in CSIR-CSMCRI culture collection centre (Accession No.: CCNM 1045). Morphological observations of algal culture under experimental conditions were carried out using Leica DM IL LED inverted microscope, Leica Microsystems. 2.2. Experimental conditions The present study was focussed on growing microalgae A. dimorphus under various temperature stress conditions. In the first, continuous cultivation experiment, cells were grown at four different temperature viz. 25ºC, 35ºC, 38ºC and 40ºC throughout 15 days of growth period. In the second, two stage cultivation experiment, cells were first grown at 25ºC and then transferred at 35ºC and 38ºC after 6, 9 and 12 days of cultivation. During continuous cultivation at 40ºC, A. dimorphus cells did not grow after 3 days [as monitored from Optical Density (OD) at 750 nm] and therefore in continuous and two stage cultivation 25ºC, 35ºC and 38ºC were considered for the experiment. Light intensity (60 µmolm-2s-1) and photoperiod (12:12 hours of light:dark period) remained same throughout the study. All the experiments were carried out in triplicate in 1 L Erlenmeyer flasks containing 500 ml of BG11 media inoculated with 10% of actively growing culture of A. dimorphus. Flasks were manually shaken 3 times a day to avoid adherence of the cells to the surface of the flasks. 2.3. Determination of growth and biomass productivity Microalgal growth was monitored at regular interval of 3 days by measuring the OD at 750 nm. The biomass productivity (mg/L/day) was calculated according to equation P = (X2 − X1) / (t2 − t1), where X2 and X1 are the dry cell weight (mg/L) at time t2 and t1, respectively. 2.4. Determination of pigments, protein, proline and lipids

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Total pigments were extracted using methanol and chlorophylls and carotenoids contents were calculated according to equations described in Pancha et al. (2014). For the estimation of crude protein, total nitrogen content of microalgae was measured using a CHNS elemental analyzer (Perkin-Elmer Model 2 400, USA) and the protein content was calculated as described in Pancha et al. (2014). Proline was extracted with 3 % sulphosalicylic acid and estimated as described in Pancha et al. (2014). Lipid content was estimated gravimetrically using chloroform: methanol (1:2, v/v). Total lipid was further fractionated by column chromatography using chloroform/acetic acid (9:1, v/v) to collect neutral lipid, acetone/methanol (9:1, v/v) to collect glyco lipids and methanol to collect phospholipids as described in Pancha et al. (2014). 2.5. Determination of carbohydrate content The carbohydrate content was determined using the method reported by National Renewable Energy Laboratory (NREL), USA (Van Wychen and Laurens, 2013). Known quantity of lipid extracted biomass was digested using 72% (w/w) sulphuric acid by incubating at room temperature for 1 hr. The hydrolysate was then diluted with distilled water to bring the sulphuric acid concentration to 4% (w/w) and incubated at 121ºC for 1 hour. After centrifugation at 10,000 rpm for 5 min, the supernatant was used to determine total sugar content by phenol sulphuric acid method (Dubois et al., 1956). 2.6. Determination of hydrogen peroxide (H2O2) H2O2 content was determined according to Velikova et al., (2000). Algal cells were harvested by centrifugation and the cell pellet was homogenized in 0.1% w/v TCA solution. The homogenate was centrifuged at 10,000 rpm for 10 min and 0.5 ml of supernatant was added to 0.5 ml of 10 mM phosphate buffer (pH 7.0) and 1 M KI. Absorbance of solution was read at 390 nm. H2O2 concentration [µmol H2O2 g-1 fresh weight (FW)] in the sample was

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determined from a calibration curve prepared using known concentrations of H2O2. As H2O2 is a prevalent form of reactive oxygen species (ROS), we have collectively used the term “ROS” for H2O2 throughout the study. 2.7. Determination of lipid peroxidation Lipid peroxidation was determined in terms of Malondialdehyde (MDA) level (Hodges et al., 1999). Algal cells were harvested by centrifugation and homogenized in 2 ml of 80:20 (v:v) ethanol:water, followed by centrifugation at 10,000 rpm for 10 min. A 1 ml aliquot of supernatant was mixed with 1 ml of thiobarbituric acid (TBA) solution comprised of 20.0% (w/v) trichloroacetic acid, 0.01% butylated hydroxytoluene and 0.65% TBA. Samples were then mixed vigorously, heated at 95°C for 25 min, cooled and centrifuged at 10,000 rpm for 10 min. Absorbances of supernatants were read at 450 nm, 532 nm, and 600 nm. MDA was calculated using following formula: MDA (µmol g-1 fresh weight) = [6.45 x (OD532 – OD600)] – [0.56 x OD450] / fresh weight (g) 2.8. Determination of ROS scavenging enzymes Extracts for the determination of enzyme activities were prepared under cold conditions. Algal cells were harvested by centrifugation and homogenized in 50 mM phosphate buffer (pH 7.0) containing 1 mM EDTA, 0.05% (v/v) Triton X-100, 2% (w/v) polyvinylpyrrolidone and 1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 12,000 rpm for 25 min at 4ºC and the supernatant was used as crude extract. Total protein content was measured using bovine serum albumin as a standard (Bradford, 1976). CAT (EC 1.11.1.6) activity was determined spectrophotometrically using crude enzyme extract, 3% H2O2 and phosphate buffer (pH: 7.0). Decrease in absorbance at 240 nm was recorded up to 150 seconds and CAT activity was calculated using an extinction coefficient of 0.0436 mM-1 cm-1 (Aebi, 1984). One CAT unit was defined as the enzyme amount that transforms 1 µmol of H2O2 per minute.

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APX (EC 1.11.1.11) activity was evaluated by the change in absorbance at 290 nm due to ascorbate oxidation and calculated using an extinction coefficient of 2.8 mM-1 cm-1 (Nakano and Asada, 1981). One APX unit was defined as the enzyme amount that transforms 1 µmol of ascorbate per minute. 2.9. Determination of residual nitrate and phosphate concentrations Residual nitrate concentration in the culture medium was estimated using salicylic acid and sodium hydroxide (Cataldo et al., 1975). Residual phosphate concentration in the culture medium was estimated using ascorbic method (Grasshoff et al., 1999). 2.10. Statistical analysis All experiments were carried out in triplicates and data presented is mean values of three independent replicates. Data were further analyzed using one-way analysis of variance (ANOVA) using InfoStat, 2012. The mean values were compared with LSD test and a significant difference was considered at p

Biofuel potential of the newly isolated microalgae Acutodesmus dimorphus under temperature induced oxidative stress conditions.

Lack of control over temperature is one of the major issues in large scale cultivation of microalgae. Therefore, it is important to evaluate the effec...
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