Triggering of fatty acids on Tetraselmis sp. by ethyl methanesulfonate mutagenic treatment

Accepted Manuscript Triggering of fatty acids on Tetraselmis sp. by ethyl methanesulfonate mutagenic treatment

S. Dinesh Kumar, Kang Sojin, P. Santhanam, B. Dhanalakshmi, S. Latha, Min S. Park, Mi-Kyung Kim PII: DOI: Reference:

S2589-014X(18)30021-5 doi:10.1016/j.biteb.2018.04.001 BITEB 22

To appear in: Received date: Revised date: Accepted date:

2 March 2018 1 April 2018 3 April 2018

Please cite this article as: S. Dinesh Kumar, Kang Sojin, P. Santhanam, B. Dhanalakshmi, S. Latha, Min S. Park, Mi-Kyung Kim , Triggering of fatty acids on Tetraselmis sp. by ethyl methanesulfonate mutagenic treatment. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Biteb(2017), doi:10.1016/j.biteb.2018.04.001

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ACCEPTED MANUSCRIPT Triggering of fatty acids on Tetraselmis sp. by ethyl methanesulfonate mutagenic treatment S. Dinesh Kumar1, 2, Kang Sojin3, P. Santhanam2, B. Dhanalakshmi4, S. Latha5, Min S. Park6 and Mi-Kyung Kim1* 1

Marine Planktonology & Aquaculture Lab., Department of Marine Science, School of

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MCK Biotech Co. Ltd., Daegu R&D Fusion Center, Daegu -42713, South Korea.

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College of Natural Sciences, Keimyung University, Daegu-42601, South Korea

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Marine Sciences, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India.

PG and Research Department of Zoology, Nirmala College for Women (Autonomous),

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Coimbatore-641018, Tamil Nadu, India Department of Petrochemical Technology, Anna University (BIT Campus),

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Tiruchirappalli –620 024, Tamil Nadu, India Center for Microalgal technology and Biofuels, Institute of Hydrobiology,

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Chinese Academy of Science, Wuhan 430072, China

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* Corresponding author: [email protected]: [email protected]

Corresponding Author:

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CEO/Professor

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Dr. Mi-Kyung Kim

MCK Biotech Co. Ltd., #533, Daegu R&D Fusion Center Daegu -704 948, South Korea. E-mail: [email protected] Tel: +82538130725 Fax : +82538130726

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ACCEPTED MANUSCRIPT Abstract The present study aimed to triggering of growth, lipid and fatty acids production of oceanic microalgae Tetraselmis sp. for biofuel production under mutagenic treatment with ethyl methanesulfonate (EMS). The maximum optical density (0.51±0.02), cells concentration (138±6.9× 104 cells mL-1), biomass (0.63±0.03 g L-1), chlorophyll ‘a’

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(3.91±0.19 mg L-1) were found in DKMK06 in first generation which was treated with EMS

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at 50 µmol mL-1 for 60 min. DKMK06 was exhibited the highest lipid productivity

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(48±0.9%) during first generation and DKMK05 during second and third generations and percentages of lipid were 46.5±1.1 and 41±1.1%. The same trend has been observed in

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FAME contents and highest saturated and mono unsaturated fatty acid combinations were found in DKMK05 in 2nd generation (42.49%) followed by third generation (41.5%) which

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were higher than the first generation produced by DKMK06 (39.98%).

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Keywords: Tetraselmis sp.; ethyl methane sulfonate; mutants; lipid; FAME; microalgae.

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1. Introduction

In current scenario, the modern globe extremely depends on overseas sources to fulfill

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their requirements to avoid the petroleum products scarcity and this might be a main reason for crude oil price rising (Teo et al., 2014). Nowadays, modern researchers are seeking an

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alternate and environment friendly fuel source to overcome this problem. After many attempts, biodiesel is considered as alternate and low cost fuel energy for this systemized world without harming environment (Rajinders, 2006). But, biofuel obtained from oil crops, waste oil and fat couldn’t fulfill the demand of alternative fuel source to petroleum oil (Yusuf, 2007). To overcome this issue, microalgae are treated as possible better source for obtaining biofuel by researchers. Because, they have the ability to generate large quantity of triacylglyceride (TAG), large biomass within a short period of generation cycle and resilience capacity in stress conditions (Qiang et al., 2008). 2

ACCEPTED MANUSCRIPT While producing algal oil, we need to produce the oil-rich algal biomass with low cost of production. Nowadays, genetically modified algae by genetic engineering could be reducing the production cost (Roessler et al., 1994). Among many genetic engineering methods, random mutagenesis termed as an easy and possible method to solve the problems like homologous recombination and gene inactivation (Lee et al., 2014). Random

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mutageneses consisting many advantages include easy to process and there is no need of huge

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information about organisms used for mutagenic treatment. Particularly, interfered with ethyl

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methane sulfonate (EMS) mediated random mutagenesis is a promising and powerful techniques to promote point mutation by inflecting DNA like A-T to G-C (Mobini-Dehkordi

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et al., 2008). So for several attempts have been made for selecting random mutagenesis for enhancing lipid in microalgae. Augustine et al. (2014) tried to enhance the biomass and lipid

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productivity of Nannochloropsis sp. by EMS at different growth phases. Chlamydomonas reinhardtii was able to produce higher lipid while treating with EMS and its efficiency was

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confirmed with proteomic studies (Lee et al., 2014). Kawaroe et al. (2015) confirmed that 0.1

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µ M of EMS has increased the Dunaliella sp. cell size three times larger than the control cells. Nannochloropsis sp. has the ability to produce higher lipid and biomass under nitrogen

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starved condition when treated with EMS, finally its volumetric production was also increased (Anandarajah et al., 2012). Beachama et al. (2015) made an attempt on random

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mutagenesis on Nannochloropsis salina CCAP849/3 with the help of EMS and UV and they got result as FAME increase nearly 156% from the wild type. Wang et al. (2016) has been tried to prove the nitrosoguanidine as a better mutagen candidate than the EMS for enhancement of lipid productivity of Nannochloropsis oceanica. The lipid productivity enhancement of water surface floating microalgae Botryosphaerella sp. and Chlorococcum sp. using two chemical mutagen ethyl methane sulfonate

and 1-methyl-3-nitro-1-

nitrosoguanidine was performed by Nojima et al. (2017). Two rounds mutagenisis has been

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ACCEPTED MANUSCRIPT performed in Chlorella vulgaris using EMS to enhanced lipid content 67% than the wild type (Sarayloo et al., 2017). These attempts have proven that EMS mutagenesis enhanced the biomass, lipid as well as FAME contents in microalgae which are the essential properties of biodiesel. But these studies failed to prove the stability of EMS on the microalgal cells for

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continuous enhanced production. Most of the studies concluded that EMS treatment of microalgae had big changes in

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their DNA and the output has been revealed in their growth, biomass and biochemical

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composition. But, there are no studies discussed about reversion (back mutation), its dealing with microalgae reverse to stable condition or the status of alga after the first generation.

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During genetically modified stage or next stage, growth and biomass production were lower

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than the first generation. In this stage, the present study dealing with isolation and characterization of mutant strains that were produced after mutating the wild type Tetraselmis sp. up to three generations and evaluate their growth and biomass productivity. This work is a

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first attempt to enhance the growth, biomass and lipid production of oceanic microalgae Tetraselmis sp. with the help of chemically induced mutants from wild type. The study also dealt with growth and biochemical variations in Tetraselmis sp. in respect to generation wise

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after chemical mutation of the parent strain.

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2. Materials and methods 2.1.Strain and culture conditions Oceanic microalgae Tetraselmis sp. was obtained from Center for Marine Bioenergy Consortium, Inha University, Seoul, South Korea. The stock culture of Tetraselmis sp. was maintained in multi room light-emitting diode (LED) chamber (HST-120LE-4, Hanbaek St. Co., South Korea). The culture has been fertilized with Walne medium (Walne. 1970) under the 25ºC of temperature. The photoperiod (PP) of 12L:12D were maintained with 150µ mol m-2 s-1 of photosynthetic photon flux intensity (PPFI). The stock culture were maintained in 4

ACCEPTED MANUSCRIPT 250 mL of round bottom conical flask containing 200 mL of sterilized sea water fertilized with Walne medium. The total indoor culture methods were followed according to Perumal et al. (2014). After 5–8 the maximum exponential phase was obtained. 2.2.Mutagenesis treatment

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The mutagenesis treatment was carried out by ethyl methane sulfonate (EMS) (Sigma-Aldrich, USA) as mutagenic agent and the methods were adopted from Anandarajah

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et al. (2012) with slight modification. To find a suitable strain for further experiments and

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successful strain improvement, we determined an appropriate concentration of EMS for

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treating parent culture. After treating parent culture, the survival rates of parent culture was counted by microscope. As first step, 1ml of centrifuged parent culture (107cells/ml) was

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obtained in 16 screw cap tubes (20ml, Pyrex, USA) and added various concentrations of EMS (25, 50, 75 and 100µmol mL-1). The samples were kept for incubation at treatment duration

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(30, 60, 90 and 120 minutes) and the tubes were subjected to gentle agitation. The treatments

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were stopped at appropriate time intervals (30, 60, 90 and 120 min) by adding 1ml of sodium thiosulphate (7% w/v). The treated cells were washed three times with double distilled water

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for removing EMS from the cells and the tubes were incubated in dark place over night prior to agar plating. On next day, the treated parent culture was subjected to agar plating fertilized

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by f/2 medium and culture was maintained as described in Perumal et al. (2014). The plated cells were monitored for 14 days and fast growing colonies were picked and inoculated in to 50 ml round bottom conical flasks filled with 30ml of sterilized seawater fertilized with Walne medium. The plated strains were washed thrice by adding 1ml of sodium thiosulphate (7% w/v) and again subjected to agar plating fertilized with f/2 medium. This section was treated as a second generation and the same procedure has been followed up to three generations after 14 days. These strains were cultured for 14 days in sterilized sea water contain Walne medium and finally lead to crude spectrophotometric (UV visible 5

ACCEPTED MANUSCRIPT Spectrophotometer, 3200, X-MA, Human Corporation, South Korea) analysis for finding variations between EMS treated cells and wild type cells. 2.3.Selection of mutant strains All the treated strains were grown in agar plates, fast growing colonies were named

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with EMS concentration and treatment time and the list of strains were DKMK01 (25µmol mL-1 of EMS: 30 min), DKMK02 (25µmol mL-1 of EMS: 60 min), DKMK05 (50µmol mL-1

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of EMS: 30 min), DKMK06 (50µmol mL-1 of EMS: 60 min), DKMK10 (75µmol mL-1 of

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EMS: 60 min), DKMK12 (75µmol mL-1 of EMS: 120 min) and DKMK14 (100µmol mL-1 of

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EMS: 60 min) and their parent wild type Tertaselmis sp. was also included for further experiments. The strains were initially cultured in 100mL of round bottom conical flasks

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under the conditions mentioned in section 2.1. Cultures at exponential phase were used as inocula and spectrophotometric analyses. This inoculum transferred to the other glass

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2.4.Experimental setup

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columns for further experimental studies.

Based on their survival rate and fast colony forming ability in agar plates, the mutant

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strains selection has been made. Among the seven mutants’ strains grown in the agar plate,

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the three strains (DKMK05, DKMK06 and DKMK10) selected for further experiments with control (wild type). The selected strains were grown in 500 mL of round bottom conical flasks filled with 400mL of sterilized seawater fertilized with Walne medium under 25ºC of temperature, 12L:12D of photoperiod (PP) and 150 µ mol m-2 s-1 of photosynthetic photon flux intensity (PPFI) in FINEPCR orbital shaker and whole experiment has been carried out in light-emitting diode (LED) chamber (HST-120LE-4, Hanbaek St. Co., South Korea). Sampling was made every three days and analyzed for cell density, optical density, biomass, chlorophyll ‘a’, total lipid and fatty acid methyl esters. 6

ACCEPTED MANUSCRIPT 2.5.Evaluation of growth Tetraselmis sp. growth was observed by analyzing the absorbance of algae sampled at 680 nm (OD680) in a UV visible Spectrophotometer (3200, X-MA, Human Corporation, South Korea). The cell concentration was calculated by counting the cells using

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hemocytometer with proper dilution. 2.6.Evaluation of biomass

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Biomass was estimated daily by the gravimetric method according to Richmond et al.

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(2003) with minor modifications. In brief, 10 mL of Tetraselmis sp. culture was filtered onto

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a pre-weighed (W0) glass fiber (GF/C) filter paper (pore size 0.45μm) and the filter paper was dried over 100ºC for 24 h. The weight of algal cells along with filter paper was estimated

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(W1). The biomass concentration of Tetraselmis sp. was calculated using following formula: 𝑊1 − 𝑊0 10/1000

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𝐵𝑖𝑜𝑚𝑎𝑠𝑠 (𝑔 𝐿-1) =

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2.7.Evaluation of chlorophyll ‘a’,

To estimation of chlorophyll ‘a’ done by according to Wellburn (1994) method with

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minor modification. In short, 1 mL of algae sample was centrifuged at 5000 ×g for 10 min, after that supernatant was discarded and 1mL of methanol was added in to the pellet. Then

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the sample was incubated at 60 ºC for 50 min in water bath and lysate was removed by centrifuging the sample at over 5000 ×g for 6 min. The sample absorbance was measured at 470, 653, and 666 nm and chlorophyll ‘a’ concentration was estimated using the following formula: Chla = 15.65 A666 – 7.34 A653 Note : A666, A653, and A470 are optical density value of 666 nm and 653 nm and 470 nm respectively. 7

ACCEPTED MANUSCRIPT 2.8.Extraction and quantification of total lipid Total lipid content was estimated by Bligh and Dyer (1959) method with slight modification. Briefly, 100 mg of lyophilized algal biomass was homogenized by mortar and pestle after adding 15 mL of chloroform:methanol (2:1) mixture. The homogenized algal

continued up to three cycle.

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sample was filtered by Whatman no.1 filter paper and these extraction and filtration were By using hexane, mortar-pestle and filter paper were washed

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and all the filtrates were shifted to round bottom flask through sodium sulfate for removing

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moisture content. Then the solvent was transferred to rotary evaporator for evaporation. After completing evaporation process, the lipid was re-dissolved with 10 mL of hexane and shifted

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to dry pre-weighed test tube. After evaporation of hexane by using evaporator, the lipid

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content was estimated gravimetrically according to following equation: BWAE (g) − BEBE (g) × 100 WDAC (g)

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Total Lipid (%) =

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Beaker weight after extraction

BWBE

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Beaker weight before extraction

WDAC

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Weight of the dry algal cell

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BWAE

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2.9.Preparation of fatty acid methyl esters (FAME) The FAME content was determined by the direct transesterfication method (Na et al., 2011). The Tetraselmis sp. cells were harvested by centrifugation (3800×g, 10 min) and washed thrice with distilled water and freeze dry over 4 days in a lyophilizer. 10mg of dried cells were mixed vigorously for 10 min after adding 2 mL of freshly prepared methanol: chloroform mixture (2:1 v/v). Then sample mixed with 1ml of chloroform and methanol, 0.3ml of 95% sulfuric acid and 1ml of internal standard heptadecanic acid (500 mg/L), after adding, sample was mixed well for 5 min and incubated in water bath over 100ºC for 10 min 8

ACCEPTED MANUSCRIPT with the help of closed tubes After adding 1ml of distilled water, sample was cooled at room temperature and centrifuged at 4000×g for 10 min. The lower organic phase was filtered and extracted by using syringe with 0.2µm filter paper. The FAME samples were analyzed by gas chromatograph (Shimadzu, GC2014, Japan) with flame ionization detector (FID). Twenty microliters of each sample was injected into FAMEWAX column (Restek, USA) (30 m × 32

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mm ID × 25 mm film thickness). The temperature of the program was as follows: initial

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140ºC with 5 min hold; ramp 2ºC/min to 230ºC with a 5 min hold. Column flow was set at

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22.2 mL/min. The instrument condition was as follows: nitrogen as carrier gas; FID set at 260ºC, and split ratio of 10:1. The run time for a single sample was 55 min. Each sample was

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analyzed in triplicates, and FAME identification was done by comparison with standard

3. Results and discussion 3.1.Selection of strains

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certificate, Supelco FAME mix C4 e C24 (Bellefonte, PA, USA).

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Among the 16 numbers of mutagen treatments, 7 EMS treated strains (25 µmol mL-1

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of EMS: 30 min, 25 µmol mL-1 of EMS: 60 min, 50 µmol mL-1 of EMS: 30 min, 50 µmol mL-1 of EMS: 60 min, 75 µmol mL-1 of EMS: 60 min, 75 µmol mL-1 of EMS: 120 min and

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100 µmol mL-1 of EMS: 60 min) were successfully grown in agar plates. Fig. 1 shows the

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spectrophotometric results of EMS treated strains. The spectrophotometric analysis revealed that, after first generation the gene modification reversion of mutation was noticed in EMS treated strains and the strains have been back to normal condition, since no peak was found in first generation strains. The presence of peak in second and third generation strains and distance between each strains revealed that they have been use 2nd 3rd generation for uniformity back to normal condition (back mutation). After treating with EMS, the survival rate of EMS treated strains have been counted. Among the 16 mutant strains tested, the lowest survival rate (1.9%) was found in DKMK10 (75 µmol mL-1 of EMS: 60 min) and 9

ACCEPTED MANUSCRIPT followed by DKMK06 (50 µmol mL-1 of EMS: 60 min) and DKMK05 (50 µmol mL-1 of EMS: 30 min) and the percentage of survival was 2 and 3 generations respectively (Fig.2). Among the 16 strains tested, low surviving (DKMK05, DKMK06 and DKMK10) strains have been subjected to further experiments for evaluation of biomass, lipid, and FAME production. Their low survival rates clearly demonstrate that the high radiation exposure used

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for mutation made a great impact on nonfunctional genes in microalgae (Eisenstadt et al.,

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1994). The strains which have higher survival rates demonstrate that the EMS does not

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modified the genes. 3.2.Growth

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The present study dealt with physiological consequences of changes in the genetics of mutant strains, and we correlated the growth, biomass, chlorophyll ‘a’, total lipid and FAME

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contents for the mutant strains DKMK05, DKMK06, DKMK10 and their parent wild type (control). At the same time, we found the genetically modified microalgae by EMS make the

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impact on the growth and biomass production by generation wise in EMS treated microalgae.

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Fig. 3a-c shows the growth curve under three generations of the mutant Tetraselmis sp. Usually, the optical density of algae in batch culture started increasing from day 1 to 14. In

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the present study the sampling were made every 72 hours interval for analyses and totally eight sampling has been made including initial (0 day). From the overall trend of growth

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curves, maximum growth was observed in DKMK06 (0.51±0.02) in first generation and DKMK05 in 2nd (0.52±0.02) and 3rd (0.49±0.02) generations. Higher growth rates in 1st, 2nd and 3rd generations in Tetraselmis sp. explains that the DKMK06 has showing its maximum growth in 1st generation only, whereas DKMK05 showing its growth in 2nd and 3rd generations. It seems that 50 µmol mL-1 of EMS and 30 min treatment mutagenized strains should take longer time for their growth output. The lowest growth curves were observed in wild type in 1st generation (0.38±0.01), DKMK06 in 2nd 10

ACCEPTED MANUSCRIPT (0.35±0.02) and 3rd (0.32±0.01) generations. The results demonstrate that the after 1st generation DKMK06 strain growth was declined compared to wild type it might be due to EMS effects on cells and possibly algal cells processed to reversion (back mutation). Fig. 4 d-f shows cells density and the result explained the similar trend observed in growth curve.

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The highest cells density (138±6.9×104 cells mL-1) of 1st generation strains were obtained in DKMK06 and 2nd and 3rd generations were observed in DKMK05 strains and the cell density

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were 241±12×104 cells mL-1 and 198±9.9×104 cells mL-1 respectively. The cell density results

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clearly explained that DKMK05 can produce more cell density than DKMK06 and wild type Tetraselmis sp. The prominent cell production was observed in DKMK05 strain and the cell

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density were 42% higher in 2nd generation and 30% higher in the 3rd generation. From the

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results of growth curve (OD and cell density) we observed that the DKMK05 better strain than the DKMK06 for continuous cells production. In all three generations, second best cell production was observed in DKMK10 than the other strains (DKMK06 and wild type). In

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more than that highest cell density and optical density were found in DKMK05 (50 µmol mLof EMS: 30 min) than DKMK06 (50 µmol mL-1 of EMS: 60 min) and DKMK10 (75 µmol

mL-1 of EMS: 60 min), it clearly explains that higher EMS concentration and treatment time

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can kill the cells or reduce the growing ability. On the other hand, excess addition of EMS

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and treatment time can reduce the acidity (pH) of culture medium (Fig. 3a-c) and water color has been changed due to bleaching. Due to pH changes (increase or decrease beyond the limit), photosynthesis rate also be change. Because, microalgal photosynthesis rate depends on carbon availability and water pH (Kawaroe et al., 2015).

3.3.Biomass Fig. 4a-c shows the volumetric biomass productivity of wild type and three mutants Tetraselmis sp. during three generations. From the results we confirmed that in all three 11

ACCEPTED MANUSCRIPT generations among the four strains tested, any one mutant strain has higher biomass productivity than the wild type Tetraselmis sp. In the first generation, DKMK06 has dominant biomass production from 6th day to end of the experiment (21st day). Total biomass production of the 21st day Tetraselmis cells of the DKMK05, DKMK06, DKMK10 and wild type in first generation were 0.56±0.03, 0.63±0.03, 0.56±0.02 and 0.42±0.02g L-1

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respectively and the highest biomass yield was observed in DKMK06 strain. According to

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Sarayloo et al. (2017), these results revealed that the random mutagenesis by using EMS has

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been targeting broad changes of nucleotide right through the genome with help of chemical mutagenic agents can be used to improve the biomass production capacity of Tetraselmis sp.

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while grown on a pilot scale in a FPBR. Like optical and cell density, in second and third generation highest biomass productivity were observed in DKMK05 mutant and the biomass

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concentration were 0.57±0.03, 0.53±0.02g L-1 respectively. Wild type and DKMK10 obtaining biomass yield after DKMK05 in second and third generations. Among the three

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generations tested, the maximum biomass productivity (0.57±0.03) was achieved by

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DKMK05 in second generation and the productivity ratio in generation wise 1st (DKMK06) > 2nd (DKMK05) > 3rd (DKMK05). From these results we understood that compared to higher

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concentration of EMS treatment strain (DKMK10), lower or moderate concentration of EMS treated strains (DKMK06 and DKMK05) have produced maximum biomass. The similar

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trend has been observed by Doan and Obbard (2012) and they stated that high concentration of EMS or long treatment period did not enhance the volumetric biomass productivity in microalgae.

3.4.Chlorophyll ‘a’ In the present study we estimate the photosynthetic ability of mutant strains we choose cellular chlorophyll ‘a’ (Chl ‘a’) were used as a tool for all the four strains. Fig. 4d-f 12

ACCEPTED MANUSCRIPT shows the daily chlorophyll ‘a’ production of Tetraselmis mutant (DKMK05, DKMK06 and DKMK10) with wild type (WT), and the maximum chlorophyll ‘a’ production was observed in DKMK06 mutant at first generation and the concentration was 3.91±0.2mg L-1 whereas lowest concentration was recorded in wild type Tetraselmis sp. in same generation and the concentration was 2.61±0.1 mg L-1. The chlorophyll ‘a’ concentration was dominated by

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DKMK10 from day 6 to day 12. After day 12, DKMK10 was slightly decreased and

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DKMK06 was increased up to day 21. Compared to wild type, 33.2% higher production of

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chlorophyll ‘a’ was achieved by DKMK06 mutant in first generation. While come to second and third generation for chlorophyll production maximum production ability was shifted from

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DKMK06 to DKMK05 and the concentration of chlorophyll ‘a’ were 2.06±0.1 and 1.98±0.09 mg L-1. In 2nd and 3rd generations, DKMK05 produced 6.1 and 11.11% chlorophyll ‘a’ than

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the wild type. In the present study we found the positive correlation between biomass and chlorophyll ‘a’ production. When biomass production was increased chlorophyll ‘a’ also

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increased proportionally and the results were supported by previous researchers (Anandarajah

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et al., 2012).

Table 1 compares chlorophyll ‘a’ and biomass content between wild type and three

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mutant strains grown in three generations which disclose that the DKMK06 in first

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generation and DKMK05 in second and third generations were comparably higher than the wild type Tetraselmis sp. But chlorophyll ‘a’ production of DKMK05 was lower than the wild type in second and third generations.

3.5.Total lipid Generally, total quantity of lipids produced by microalgal cells is an another important factor for efficient biodiesel production. While coming to biodiesel production

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ACCEPTED MANUSCRIPT from microalgae need to optimize the two important factors like the amount of lipid produced per cell and the number of cells per unit of culture per unit of time (Wang et al., 2009). The total lipid composition of wild type and three mutant strains results were provided in Fig. 5. The total lipid production of wild type, DKMK05, DKMK06 and DKMK10 over the culture period of 21 days were 35±0.7, 42±0.84, 48±0.96 and 46±0.92% respectively. Among the

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four strains including wild type tested, the highest lipid composition was observed in

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DKMK06 in first generation.

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The same trend has been obtained in second and third generations, mutant strains comparably higher in the lipid production than the wild type Tetraselmis sp. In second and

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third generations, the maximum lipid production was 46.5±1.16 and 41.1±1.17%. In all three

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generations, the lowest lipid production was observed in wild type Tetraselmis and the results clearly explained that the EMS significantly enhances the total lipid production in microalgae. Compared to wild type, DKMK06 strain in first generation and DKMK05 strain

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in second and third generations produced total lipid more than 13, 12.5 and 9.2 %. Generally, dry cell weight was inversely proportional to lipid content and biomass production were inversely related, because cells can use considerable amount of energy for their growth rather

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than lipid accumulation (Converti et al., 2009; Liliana et al., 2009). Compared to other

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studies, the present study yields the higher amount of total lipid by chemically mutant strains (Table 3). All the three strains has been produced 1.37, 1.20 and 1.31 times greater than the wild type Tetraselmis sp. The escalating of lipid production occurred due to inducing heavyion in algal cell wall by EMS mutagenic treatment (Wang et al., 2016).

3.6.Fatty acids

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ACCEPTED MANUSCRIPT In Table 2, the fatty acid composition of three mutant strains and wild type Tetraselmis strain analyzed at end of experiment (21 days). Fatty acids less than one percentage are mentioned in table as others. The major fatty acids of the present study were C14:0 (Myristic acid), C16:0 (Palmitic acid), C16:1 (Pamitoliec acid), C18:0 (Stearic acid), C18:1-3n (Alpha-Linolenic acid) and C18:3n3 (Linolenic acid). In all three mutant and wild

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type strains, the highest fatty acids levels were maintained by C16:0 and it ranged from

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17.16±0.45 to 20.03±0.55%. The same trend has been observed in second and third

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generations also and the ranges were 18.02±0.43-25.45±0.59% and 17.29±0.31-24.32±0.56%. Mostly, microalgae synthesis fatty acids from esterification to glycerol- based membrane

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lipids. But such a favorable conditions, microalgae can change their biosynthesis pathways for lipid production towards the accumulation of the triacylglyceride. (Anandarajah et al.,

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2012). Occurrence of TAG also important for producing high quality and quantity of biofuels, it will happen when oleaginous microalgae faced the favorable conditions or gene

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modifications in their environment. When some positive mutations occurred in the

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biosynthesis pathway of fatty acids, the significant changes will happened in the quantity of C16:0. It’s might be due to upregulation of Δ6 desaturase and elongase along the omega-6

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pathway, but more and more biosynthesis-related enzymes were confirmed to contribute to

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this variation (Beachama et al., 2015). Saturated (C16:0) and mono unsaturated (C18:1) fatty acids are play an important role while producing high quality of biodiesel from the microalgae. The quality of biodiesel is depends on their percentage of availability in total FAME (Leonardi et al., 2011). Among the four strains in first generation, DKMK06 produced high quantity of SFA and MUFA (39.98%) compared to wild type, DKMK05 and DKMK10 were 31.2, 34.5 and 31.39% respectively. Fig. 6 confirmed that the SFA and MUFA were high compared to other fatty acids and these results were supported by Leonardi et al. (2011). The high amount of SFA 15

ACCEPTED MANUSCRIPT and MUFA were produced by DKMK05 in second and third generations and the percentages were 42.49 and 41.5 and these concentrations were higher than the first generation. However the tested three mutant strains and wild type Tetraselmis sp. in three generations exhibit various type of growth, biomass, chlorophyll ‘a’, total lipid and same

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trend of fatty acids synthesis. Noticeable changes were observed in accumulation of biomass, chlorophyll and total lipid productions were found in the tested mutant strains as well as in

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generation wise too. Division of cells and total lipid producing ability of the genetically

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modified mutant strains were significantly higher than the wild type. Among the seven mutant strains, three strains (DKMK05, DKMK06 and DKMK10) were selected from

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chemical mutagenesis of wild type Tetraselmis sp. and DKMK06 for first generation and

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DKMK05 for second and third generations were considered as best candidates for sustainable microalgal biofuel production. The conventional methods of mutagenesis for mutate the parent strain to improve the strain ability to produce large amount of biomass and total lipid.

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If the mutant strain tested in large scale and their production were satisfactory means will form the parent strain to the further mutation.

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4. Conclusion

Microalgae are the potential candidate that can produce high quantity of lipids,

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proteins and carbohydrates in short period of time and their high value biomass can be associated with animal feed, chemicals and biofuel productions. Among three selected strains, DKMK05 and DKMK06 can be used as parent strains for further experiments to increase lipid production. To produce a high quality biofuel, it is important to select lipid rich microalgal strains and growing them under optimum environmental conditions for maximum biomass and lipid production for valuable and sustainable bioenergy source.

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ACCEPTED MANUSCRIPT Acknowledgements Authors (SDK and MKK) are thankful to the National Marine Bioenergy R & D Consortium, Ministry of Ocean & Fisheries, South Korea for providing financial supports. One of the authors (SDK) thanks the UGC (Post-Doctoral Fellowship (Ref. No. F./311/2017/PDFSS-2017-18-TAM-13681 dated 19.06.2017), Govt. of India for providing

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fellowships.

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ACCEPTED MANUSCRIPT Legends to figures Fig. 1. Spectrophotometric confirmation of EMS mutagenized Tetraselmis sp. strains Fig. 2. Cells to colonies conversion rate of Tetraselmis sp. under various EMS concentration and treatment duration.

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Fig. 3. Optical density (a-c) and cell count (d-f) of mutant and wild type Tetraselmis strains in various generations

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sp. mutant with wild type in three generations

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Fig. 4. Volumetric biomass productivity (a-c) and chlorophyll ‘a’ (c-d) curves of Tetraselmis

Fig. 5. Total lipid composition of wild type and mutant strains of Tetraselmis sp. in three

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generations

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Fig. 6. Major fatty acid composition (%) of wild type and mutant strains of Tetraselmis sp. in

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three generations

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ACCEPTED MANUSCRIPT Legends to table Table 1

Final (21st day) productivity of biomass and chlorophyll ‘a’ content of three mutants and their parent wild type Tetraselmis sp. during three generations of treatment

Table 2

Fatty acid composition (%) of three mutants and parent wild type Tetraselmis sp.

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The total lipid production of chemically mutant strains by early workers

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Table 3

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during three generations of treatment

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ACCEPTED MANUSCRIPT Fig. 1

1st generation

2nd generation

3rd generation

Presence of peak in all strains

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EMS treated

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Fig. 2

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ACCEPTED MANUSCRIPT Fig. 3 (a) 1st generation

(b) 2nd generation

(c) 3rd generation

T P

I R

C S U

N A

(d) 1st generation

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(e) 2nd generation

D E

T P E

C C

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Fig. 4

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(f) 3rd generation

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(b) 2nd generation

(c) 3rd generation

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C S U

N A

(e)1st generation

(f) 2nd generation

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(g) 3rd generation

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2ndgeneration

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3rdgeneration

ACCEPTED MANUSCRIPT Table . 1 Tetraselmis sp. strain

First generation

Second generation

Third generation

Biomass (g L-1)

Chlorophyll ‘a’ (mg L-1)

Biomass (g L-1)

Chlorophyll ‘a’ (mg L-1)

Wild type

0.42±0.02

2.61±0.13

0.54±0.02

2.03±0.10

DKMK05 DKMK06 DKMK10

0.56±0.02 0.63±0.03 0.56±0.03

2.99±0.14 3.91±0.20 3.18±0.18

0.57±0.03 0.50±0.01 0.51±0.02

2.06±0.10 1.99±0.09 2.00±0.11

N A

D E

M

T P E

C C

A

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Chlorophyll ‘a’ (mg L-1)

0.27±0.01

1.76±0.08

0.53±0.02 0.37±0.01 0.42±0.03

1.98±0.09 1.75±0.08 1.76±0.06

T P

I R

C S U

Biomass (g L-1)

ACCEPTED MANUSCRIPT Table 2 Total Fatty acids (%) Wild type

DKMK05

DKMK06

DKMK10

C14:0

2.15±0.04

1.87±0.04

1.89±0.05

1.92±0.05

C16:0

18.43±0.37

18.14±0.42

20.03±0.55

17.16±0.45

C16:1

5.46±0.11

4.56±0.10

5.53±0.15

C18:0

8.26±0.17

6.86±0.16

6.13±0.17

7.57±0.20

C18:1-3 (n)

11.25±0.23

15.91±0.37

19.95±0.55

14.23±0.37

C18:3n3

10.95±0.22

14.35±0.33

13.56±0.38

15.96±0.41

Total

56.50±1.13

61.69±1.42

67.09±1.86

61.26±1.59

Others

43.50±0.87

38.31±0.88

32.91±0.91

38.74±1.01

C14:0

1.67±0.03

1.85±±0.04

1.72±0.04

1.95±0.05

C16:0

21.57±0.32

25.45±0.59

21.29±0.53

18.02±0.43

C16:1

5.40±0.08

6.57±0.15

4.80±0.12

6.45±0.15

C18:0

7.06±0.11

6.38±0.15

6.45±0.16

6.92±0.17

C18:1-3 (n)

16.06±0.24

17.04±0.39

16.09±0.40

16.91±0.41

C18:3n3

12.15±0.18

13.85±0.32

13.45±0.34

12.10±0.29

Total

63.91±0.96

71.14±1.64

63.80±1.60

62.35±1.50

Others

36.09±0.54

28.86±0.66

36.20±0.91

37.65±0.90

1.58±0.03

1.79±0.04

1.75±0.03

1.83±0.03

21.23±0.38

24.32±0.56

20.37±0.39

17.29±0.31

5.27±0.09

5.38±0.12

4.98±0.09

6.11±0.11

6.57±0.12

6.12±0.14

6.12±0.12

6.83±0.12

C18:1-3 (n)

15.99±0.29

17.18±0.40

15.87±0.30

15.98±0.29

C18:3n3

12.08±0.22

11.99±0.28

14.11±0.27

12.85±0.23

Total

62.72±1.13

66.78±1.54

63.20±1.20

60.89±1.10

Others

37.28±0.67

33.22±0.76

36.80±0.70

39.11±0.70

C16:1 C18:0

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C16:0

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C14:0

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Second generation

Third generation

T

First generation

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4.42±0.11

ACCEPTED MANUSCRIPT Table 3

S. No 1. 2. 3. 4. 5. 6. 7. 8.

Strain

Mutagen

Type of mutation

Nannochloropsis salina CCAP849/3 Nannochloropsis oceanic (LAMB003)) Botryosphaerella sp. (AVFF007) Chlorococcum sp., (FFG039) Chlorella vulgaris (UV715-EMS25) Tetraselmis sp. (DKMK06) Tetraselmis sp. (DKMK05) Tetraselmis sp. (DKMK10)

ethyl methanesulfonate

Single random mutagenesis Single random mutagenesis Single random mutagenesis Single random mutagenesis Two round random mutagenesis Single random mutagenesis Single random mutagenesis Single random mutagenesis

ethyl methanesulfonate ethyl methanesulfonate ethyl methanesulfonate Ultra violet and ethyl methanesulfonate ethyl methanesulfonate

D E

ethyl methanesulfonate

T P

ethyl methanesulfonate

E C

T P

1.37 % (↑) 1.20 % (↑)

Present study

1.31 % (↑)

Present study

I R

C S

1.10 % (↑) 1.11 % (↑) 2.13 % (↑)

Note: (↑) denotes the mutants strains X number of times greater than the wild type (without mutant) strains

C A

31

Reference Beacham et al., (2015) Wang et al., (2016) Nojima et al., (2017) Nojima et al., (2017) Sarayloo et al., (2017) Present study

1.29 % (↑)

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A

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Lipid productivity (%) 1.30 % (↑)

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Highlights

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IP CR US AN M ED PT CE



Tetraselmis sp. is a potential stout microalgal with high lipid content. Influence of ethyl methane sulfonate on fatty acid production was investigated. Growth and biomass production were significantly increased while the strains were chemically mutated The mutant strains DKMK06 and DKMK05 contains rich FAME on all three generations

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  

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