Sustainable biomass production under CO2 conditions and effective wet microalgae lipid extraction for biodiesel production

Sustainable biomass production under CO2 conditions and effective wet microalgae lipid extraction for biodiesel production

Journal Pre-proof Sustainable biomass production under CO2 conditions and effective wet microalgae lipid extraction for biodiesel production M. Lakshm...

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Journal Pre-proof Sustainable biomass production under CO2 conditions and effective wet microalgae lipid extraction for biodiesel production M. Lakshmikandan, A.G. Murugesan, Shuang Wang, Abd El-Fatah Abomohra, P. Anjelin Jovita, S. Kiruthiga PII:

S0959-6526(19)34268-4

DOI:

https://doi.org/10.1016/j.jclepro.2019.119398

Reference:

JCLP 119398

To appear in:

Journal of Cleaner Production

Received Date: 4 July 2019 Revised Date:

2 November 2019

Accepted Date: 19 November 2019

Please cite this article as: Lakshmikandan M, Murugesan AG, Wang S, Abomohra AE-F, Jovita PA, Kiruthiga S, Sustainable biomass production under CO2 conditions and effective wet microalgae lipid extraction for biodiesel production, Journal of Cleaner Production (2019), doi: https://doi.org/10.1016/ j.jclepro.2019.119398. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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Bi omas s

Ce l lr upt ur eandl i pi dbodi e sr e l e as e

1

Sustainable biomass production under CO2 conditions and effective wet microalgae

2

lipid extraction for biodiesel production

3

M. Lakshmikandan

4

Anjelin Jovita b, S. Kiruthiga b

5

a

6

b

7

Sundaranar University, Alwarkurichi 627 412, Tamil Nadu, India

8

c

a,b

, A.G. Murugesan b, Shuang Wang a*, Abd El-Fatah Abomohra

a,c*

, P.

School of Energy and Power Engineering, Jiangsu University, Jiangsu 212013, China Sri Paramakalyani Centre of Excellence in Environmental Sciences, Manonmaniam

Botany Department, Faculty of Science, Tanta University, Tanta 31527, Egypt

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

*Correspondence

24

[email protected](Shuang

25

Abomohra)

Wang);

[email protected](Abd

1

El-Fatah

26

Abstract

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The freshwater microalga Chlorella vulgaris MSU AGM 14 were cultured at different CO2

28

conditions (up to 8%) with 100 µmol photons m-2 s-1 to evaluate biomass and lipid

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productivity. For effective extraction of intra cellular lipids, a novel method based on mild

30

pressure (1-2.5 kg/cm2) with short period (5-15 min) of heat shock (50-70˚C) were studied

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for the wet biomass. The transesterified lipids were qualitatively and quantitatively analysed

32

by using Gas Chromatography coupled with Mass Spectrometry (GC-MS). The higher CO2

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aeration (8%) significantly increased the biomass productivity (23%) when compared with

34

control and CO2 (4%) aeration. Total lipid production (93%) acquired by conventional

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extraction procedure showed enhanced production simultaneously. The maximum lipid

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recovery (0.225g g-1 dw) was obtained at a pressure of 2 kg/cm2 and heating for 10 min at

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60˚C. The transesterified lipids showed that oleic acid (C18:1 - 51.62%) was the main

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component in both conventional and suggested lipid extraction process. The suggested

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extraction process showed significant increase in biodiesel yield by 26.7%. The energy

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outputs of biodiesel by conventional and suggested extraction process were 417.7 and 533.6

41

MJ ton-1, respectively. The overall results indicated that 8% of CO2 induced the biomass and

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lipid productivity by 94% and 54.8%, respectively, when compared with control. In addition,

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the suggested mild pressure with heat shock extraction process further enhanced the lipid

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recovery by 21% which serves as a cost-effective lipid extraction process for microalgae.

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46

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Keywords: Biodiesel; Microalgae; Chlorella vulgaris; Mild pressure; Heat shock; Lipid

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extraction

2

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1. Introduction

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Millions of years ago dead organic matter were been transformed into black gold by natural

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events like prolonged period of time and pressurized geothermal process. So far, crude oil is

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one of the most significant sources of energy on the planet. Since petroleum is a part of the

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global economy with limited reservoirs (Hamilton, 2009; He et al., 2010); Jones et al. (2004),

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experts are predicting that the end of petroleum era is near. But footsteps remain in the

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environment due to toxic pollution leading to climate change (Prasad and Kumari, 1987;

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Wang et al., 2017). The search for alternative fuel will tread on renewable energy resources

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peculiarly without damaging the bionomical balance (Elsayed et al., 2018; Yuan et al., 2019).

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Biodiesel gives some hope for renewable and clean energy with reduced carbon footprint.

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Predominately, biodiesel is produced from plant oil (canola, sunflower, soybean and rapeseed

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oil) by alkali-based transesterification process (Meher et al., 2006). But using food crops for

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biodiesel production has never been the right solution for fuel crisis problems since they

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interfere with food production for high grade arable land, initiating the food demand leading

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to hike in the food price (Chisti, 2007; Lin et al., 2011; Silitonga et al., 2013). The true fact is

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global oblige for food crops and products is awaited to double within 5 decades and in case of

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transportation fuels it is still more worsened (Hill et al., 2006).

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In such challenging situations, microalgae has attracted greater attentions for various forms of

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biofuel production like biodiesel (Abinandan et al., 2019; Jain et al., 2019), bioethanol

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(Sanchez Rizza et al., 2019), biohydrogen (Lakshmikandan and Murugesan, 2016a, b),

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bioelectricity (Powell et al., 2009) and biomethane (Raheem et al., 2018), since microalgae

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cultivation has positive environmental impact and doesn’t compete with food supplies.

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Furthermore, microalgae possess important advantages like higher photosynthetic efficiency

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leading to 10-50 times more significant CO2 mitigation when compared to terrestrial floras

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(Anto et al., 2019; Mondal et al., 2017). Prominent quantity of biomass and lipid production 3

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rate in oil bearing perennial crops like Jatropha, biomass productivity is about 4.1 ton ha-1 y-1

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whereas microalgae owes to two fold higher capability with less land demand and efficient

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non-edible oil source (Mathimani and Mallick, 2019; Mathimani et al., 2018). In addition,

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higher growth rate on harsh environmental conditions and its ability to grow on wastewater

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with various biorefinery applications are the significant features of microalgae (Abomohra et

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al., 2017; Nascimento et al., 2014). At normal environmental conditions, microalgae can

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accumulate appreciable quantity of lipids (structural lipids) but certain specific environmental

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stress conditions will stimulate the elevated level of lipid accumulation in microalgae (Tu et

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al., 2015; Widjaja et al., 2009). High CO2 concentration and length of photoperiod were

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reported to play a significant role in elevated biomass recovery and lipid content of

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microalgae (Che et al., 2019; Lv et al., 2010; Wahidin et al., 2013). From this phenomenon,

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CO2 and photoperiod-based microalgal lipid enhancement has been investigated widely on

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higher productivity and adaptive nature microalgal species.

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In the current study, eukaryotic green microalga Chlorella vulgaris was investigated as it is

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very commonly obtained in both fresh and seawater with high photosynthetic efficiency and

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lipid productivity (Battah et al., 2015). The crucial characters are; they are strongly resistant

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to any harsh conditions, and are invaders with high biomass productivity within a quick

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period of time (Ebrahiminezhad et al., 2014; Frumento et al., 2013; Safi et al., 2014). The

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crucial challenge of lipid extraction is selecting suitable cell disruption method to obtain

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maximum productivity. Several cell disruption techniques have been employed to recover

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lipids from the whole cells. Cell disruption techniques are grouped into mechanical and non-

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mechanical process; mechanical extraction method includes bead milling, sonication, high

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pressure homogenization, oil expeller, autoclaving, grinding and microwave techniques

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(Günerken et al., 2015; Mathimani et al., 2017; Postma et al., 2015). The commonly used

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non-mechanical extraction processes are chemical based Soxhlet, supercritical fluids and 4

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enzymatic extractions. Peralta-Ruiz et al. (2013) reported that 85% total energy inputs of

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biodiesel production has been shared by lipid extraction process itself. Conventional lipid

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extraction process requires microalgal biomass dewatering before lipid extraction process and

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85% of total energy input were exhausted by drying process (Patil et al., 2012). The crucial

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part of cell disruption is to increase the disruption rates, minimize damage of targeted product

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and lower the capital cost (Yap et al., 2015). High pressure based mechanical cell disruption

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methods are promising techniques for complete disruption of cells (Carullo et al., 2018).

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However, the high energy consumption, non-targeted intracellular compounds disruption and

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difficult downstream process make it more complicated to the economic viability. The

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applications of mild pressure in microalgae has been rarely investigated and are mainly

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focused on encouraging the growth and lipid accumulation (Praveenkumar et al., 2016).

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Lorenzen et al. (2017) reported that the significance of pressure (12 MPa) and heat (20˚C)

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combination increased higher efficiency of lipid extraction from dried biomass. In the present

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investigation mild pressure with heat shock were used to disrupt the wet cells of C. vulgaris

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to maximise the lipid recovery and reduce energy consumption in pressure-based cell

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disruption process.

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With the above perspective in view, detailed investigations were carried out to optimize cell

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growth and lipid productivity at higher CO2 concentrations and different photoperiod

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conditions. The effect of wet cell disruption by novel method based on mild pressure and heat

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shock treatment were then thoroughly investigated. The variations between conventional

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solvent extraction and the suggested extraction were analysed by GC-MS. Finally, microalgal

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cultivation, harvest cost, energy input and output of biodiesel from conventional and

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suggested extraction process were evaluated.

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2. Materials and Methods

5

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2.1. Microalgal strain and inoculum preparation

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Chlorella vulgaris MSU-AGM 14 (accession number KM189121) was obtained from

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Department of Environmental Science, Sri Paramakalyani Centre of Excellence in

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Environmental Sciences, Manonmaniam Sundaranar University, Tirunelveli, India. The

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microalga was grown in the Algae Culture Broth (Hi-Media, Mumbai) composed of NaNO3,

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1g; K2HPO4, 0.25g; MgSO47H2O, 0.513g; NH4Cl, 0.05g; CaCl22H2O, 0.02g; FeCl3, 0.003g

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per liter of deionized water at pH of 7.0±0.2. Cultures were incubated at 27±1˚C with

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continuous illumination (100 µmol photons m-2 s-1) and 4% CO2 aeration. When the cultures

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reached the exponential phase, inoculum was introduced into 30 litres cylindrical photo

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bioreactor for microalgal biomass accumulation. As per experimental setup, different

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photoperiod [light (L): dark (D) cycles] (24L, 18L+6D and 12L+12D) and different CO2

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supply (4% and 8%) were applied. Atmospheric air (with 0.04% CO2) was used as control.

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Microalgal growth was monitored every 12 h by using spectrophotometer (Au-2701,

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Systronics) at 680nm (OD680). After 12 days of incubation (stationary phase), cultures were

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harvested by centrifugation (Eppendorf 5804R, Germany) for 10 min at 3000×g to obtain the

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wet microalgal biomass. The cumulative biomass (g L-1 day-1) and lipid productivity (mg L-1

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day-1) were calculated by using the following Eq. (1) Biomass / Lipid productivity = (DWt - DWo)/t

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(1)

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Where DWt and DWoare the final and initial dry weights of the sample, respectively; while t

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is the time of harvest.

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2.2. Cell disruption and lipid extraction

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Approximately 11.2 g (adequate to 1g of dry weight) of harvested wet microalgal biomass

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were placed between two preheated (50˚C, 60˚C and 70˚C) annealed glass plates (2 mm

6

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thickness) at different pressured conditions (0.98, 1.47, 1.96 and 2.45 bar) and different

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exposure times (5, 10 and 15 min). After mild pressure with heat shock treatment, lipids were

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extracted by hexane: isopropanol (3:2 v/v) (Bian et al., 2018) and were kept in ice bath for 15

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min. For control, 1g of dried microalgal sample was used for cell disruption and lipid

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extraction process following the Bligh and Dyer (1959) method. The solvent extracts were

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filtered by using Whatman filter paper no.1 and were allowed to evaporate to obtain the total

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lipids which were stored at -20˚C in 1 mL chloroform for further analysis.

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2.3.Transesterification of sample

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In 10 mL screw caped glass tube, 25 mg of lipid sample was weighed and mixed with 1.5 mL

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of 0.5 N methanolic NaOH (Nascimento et al., 2012). The solution was mixed gently, and

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were sealed after nitrogen gas flushing, and were incubated in oven at 100˚C for 15 min.

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After heat treatment, samples were allowed to cool at room temperature, followed by addition

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of 2 mL of 12% boron trifluoride in methanol. Then the samples were sealed after nitrogen

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gas flushing. To initiate a transesterification reaction, samples were placed at 100˚C for 45

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min. After the transesterification, samples were allowed to cool at room temperature.

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Approximately 2 mL of isooctane were added into each sample and were shaked vigorously

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for 3 min. After mixing, the samples were immediately treated with 5 mL of saturated sodium

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chloride solution followed by gentle agitation. After mild centrifugation the isooctane layer

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was separated and were transferred into a clean glass tube and were stored at -20˚C for

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further analysis.

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2.4. Fatty acid analysis

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The fatty acid composition of the prepared fatty acid methyl esters (FAMEs) were assessed

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by Gas Chromatography (GC- Clarus 680 – Perkin Elmer) linked with Mass Spectrometry

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(MS- Clarus SQ8T - Perkin Elmer) equipped with capillary column Elite-5 MS (30 m 7

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length and 0.25 mm - internal diameter) and a flame ionization detector. Helium was used as

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a carrier gas at a flow rate of 1.3 mL min-1. The electron ionization mode (70 eV) was

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selected for mass spectrometry with full scan mode (range: 30–300 Da). The identification of

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fatty acid components were based on their retention times, abundance and fragmentation

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patterns comparison with NIST spectral library 2.0 g (2011).

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2.5. Determination of biodiesel properties

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Molecular characteristics of FAMEs describes the parameters of biodiesel quality such as

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saponification value (SV), cetane number (CN), iodine value (IV), long chain saturated factor

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(LCSF), cold filter plugging point (CFPP), allylic position equivalents (APE), bis- allylic

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position equivalents (BAPE), kinematic viscosity (υ), density (ρ) and higher heating value

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(HHV) (Islam et al., 2013; Talebi et al., 2013).

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The empirical Eq. (2-4) was applied to calculate the SV, IV and CN depending on the FAME

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profile. SV =

IV =

CN = 46.3 +

560 × ℎ 254 × ℎ

(2) ×

(3)

5458 − (0.225 × (&) (4) %&

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Where Ni is the percentage of ith FAME, Di is the number of double bonds in ith FAME,

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MW is the molecular weight.

8

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The degree of unsaturation (DU) were calculated based on the mass fraction of mono

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(MUFA) and poly unsaturated fatty acids (PUFA) by using the following Eq. (5). The LCSF,

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CFPP, APE and BAPE were calculated by applying the following Eqs. (6-9) DU =

+,- + (2 × .+,-) (5)

/0%, = (0.1 × 016: 0) + (0.5 × 018: 0) + (1 × 020: 0 (6) 0,.. = (3.1417 × /0%,) − 16.477 (7) APE =

( 78 × -98 ) (8)

BAPE =

(;78 × -98 ) (9)

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Where apn and bpn are the allylic and bis-allylic positions, respectively, in specific fatty acid;

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while Acn is the mass percentage of each fatty acid.

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FAMEs υ, ρ and HHV were calculated by using the following Eqs. (10-12) and final value of

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each factors were obtained by summation of all FAMEs fuel properties. ln(υ? ) = −12.503 + 2.496 × ln( @? = 0.8463 +

4.9

AA&? = 46.19 −

?

?)

− 0.178 × (10)

+ 0.0118 × (11)

1794 ?

− 0.21 × (12)

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Where Mi is the molecular weight; while N is thenumber of double bonds in ith fatty acid.

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2.6. Statistical analysis

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The results of means were calculated by using three replicates and were described as mean ±

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standard deviation. The significance of differences (p < 0.05) were analyzed using one-way

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analysis of variance (ANOVA) complied by least significant difference (LSD) using SPSS

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(IBM, v.20).

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3. Results and Discussion

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3.1. Microalgal growth and lipid accumulation

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C. vulgaris MSU AGM 14 were cultivated at two different percentages (4% and 8%) of CO2

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aeration for 16 days and were monitored for cell growth and intracellular lipid accumulations.

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While providing 8% aeration, cells have grown very well when compared to 4% and control

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(Fig.1). Both samples reached their stationery phase on day 10. Chan et al. (2010) reported

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that increasing CO2 aeration elevated the growth of microalgae in certain species such as B.

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braunii, C. vulgaris and Scenedesmus sp. Similarly de Morais and Costa (2007) confirmed

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that C. vulgaris can actively grow under high CO2 conditions. The microalgal production and

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productivity (Fig. 2) also evidenced enhanced production of biomass and lipids at elevated

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CO2 aeration when compared with control (0.04%). The cultures with 8% CO2 aeration

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produced 0.79±0.04 g L-1 of biomass and the productivity indicated a maximum significant

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amount of 0.064±0.003 g L-1 day-1 which represented 23% higher than that of the control.

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Whereas lipid production recorded 186±11 mg g-1 and productivity reached to 11.89±0.48 mg

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L-1 day-1, representing 93.96% significant increase over the control (Fig. 2b). In the present

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study, the effect of photoperiod and CO2 aeration on C. vulgaris MSU AGM 14 biomass and

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lipid accumulation were also evaluated (Table 1). The maximum biomass (1.44±0.02 g L-1

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dw) were observed at 24 h illumination with 8% CO2 aeration, with total lipids of 0.18±0.02

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g g-1. Whereas at 12 h L+12 h D illumination with 8% CO2, maximum lipid content of

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0.2±0.02 g g-1 were recorded with dry weight of 1.37±0.03 g L-1. These results revealed that

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microalgae require both light and dark conditions for effective lipid production. In addition, 10

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increased CO2 aeration proved to be one of the important factors for the elevation of lipid

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accumulation in microalgae. Previously, Chan et al. (2010) reported that the total lipid

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content of C. vulgaris were 11.92% of the dry weight, while the present study revealed that at

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suitable photoperiod with elevated CO2 condition C. vulgaris MSU AGM 14 has the potential

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to produce 20% of total lipids. The present investigation clearly represents that CO2 condition

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and photoperiod has significant impact on biomass and lipid accumulation of microalgae.

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3.2. Mild pressure with heat shock treatment

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Effective extraction of lipids from microalgal cells depends upon cell disruption technique

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and selection of extraction process (Halim et al., 2012; Prabakaran and Ravindran, 2011).

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There are several cell disruption techniques employed to extract intracellular lipids from

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microalgal cells. Previously, some research works explained significant advantages of mild

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pressure treatment on lipid accumulation during growth. Praveenkumar et al. (2016) reported

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that mild pressure treatment (10-15 bar) induces accumulation of neutral lipid in Chlorella

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sp., and few reports revealed that pressure tolerance ability differs from strain to strain.

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Seckbach (1971) reported that microalga Cyanidium caldarium can tolerate mild (1- 10 bar)

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pressures for 19 days at room temperature. While elevating the pressure significantly, growth

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has been inhibited with change in pigmentation. In addition, several studies revealed that high

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pressure can inhibit the growth or cause lethal damage to cells by affecting the normal

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functions of cell organelles (Mañas and Mackey, 2004; Robey et al., 2001). However, there is

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a gap in literature about the effect of pressure combined with heat shock on lipid extraction.

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In this study, wet thin layered C. vulgaris MSU AGM 14 was disrupted by mild pressure

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along with heat shock. The designed experiment showed good response in extracting the lipid

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content from the selected strain (Fig. 3). Results confirmed that 2 kg/cm2 (1.96 bar) pressure

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and 60˚C heat treatment with 10 min exposure time proved maximum effective extraction of

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0.225±0.013 g g-1 total lipid. Overall, experiment demonstrated that exposure time of 11

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microalgae in selected temperature is more important for effective extraction. When

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compared with control, the present study showed 12.5% additional recovery of total lipid.

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3.3.Composition of biodiesel

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The successful conversion of total lipids into FAME was examined by GC-MS (Table 2).

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Three most abundant methyl esters were determined in the biodiesel of the selected strain.

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Among them octadecenoic acid methyl ester (oleic acid) showed the highest relative content

250

in the pressurised sample (51.62%) when compared to other methyl esters. The other two

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methyl esters such as hexadecenoic acid methyl esters (14.53%) and octadecanoic acid

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methyl ester (12.08%) possessed considerable relative contents in the biodiesel. Similarly,

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Gao et al. (2010) reported that biodiesel of Chlorella protothecoides showed higher relative

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content for these three methyl esters. Mathimani et al. (2015) also reported that palmitic,

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oleic and linolenic acids are the major methyl esters of Chlorella sp. BDUG 91771biodiesel.

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The suggested lipid extraction process evidenced no significant differences were reported in

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the SFAs, MUFAs and PUFAs ratios with the control. The good quality biodiesel possesses

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certain significant properties like heat of combustion, ignition quality, viscosity, cold filter

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plugging point, lubricity and importantly oxidative stability which are defined by the

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structure of its FAME components. The productive amount of oleic acid content represents its

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enhanced oxidative stability for longer storage (Knothe, 2005). In addition, higher oxidizing

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tendency of biodiesel was evaluated by the presence of degree of unsaturation on FAMEs

263

(Hoekman et al., 2012). Ramos et al. (2009) also described that higher degree of unsaturation

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(>137) does not meet the European biodiesel standards. But in the present study, biodiesel of

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C. vulgaris MSU AGM 14 showed within the limit (86.13 and 86.71) of unsaturation. Allylic

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positions of biodiesel plays a crucial role in autoxidation/oxidation of unsaturated FAME.

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The linear relationship of oxidizability on bis- allylic position equivalents and allylic position

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equivalents has capablity to induce non-uniform oxidation in biodiesel (Iyer, 2017). In this 12

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study, biodiesel of C. vulgaris MSU AGM 14 showed the presence of lower value allylic

270

position equivalents and bis- allylic position equivalents. These calculated allytic position

271

results have evidenced that FAME composition of biodiesel possess reasonably good

272

oxidation stability. The combustion behaviour of biodiesel has been estimated based on

273

cetane number (Koley et al., 2018). Depending upon fuel cetane number, engines ignition

274

delay time may vary, shorter ignition time has been recorded in higher cetane numbered fuel

275

(Islam et al., 2013). Overall, the suggested lipid extraction process showed slight neglectable

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changes in all studied parameters with those of the control (Table 3).The resemblance

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analyses with standard biodiesel EN14214 and ASTM D6751-02 indicated that higher cetane

278

number and moderate quantity on degree of unsaturation. Higher viscosity and density of

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biodiesel may create many problems like fuel pump failure, carbon deposit, ring sticking and

280

poor atomization. Whereas, density values of biodiesel were within the range of standard

281

while the kinetic viscosity were slightly below the range. As reported by Yang et al. (2014)

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suitable amount of viscosity and density was observed in higher concentrations of oleic,

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palmitoleic and palmitic acid containing biodiesel. The determination of FAME derived HHV

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(39.22 and 39.46MJ kg-1) showed 14% less than the conventional petroleum derived diesel

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(46MJ kg-1). Thus, further studies are required to improve the biodiesel quality with respect

286

to the HHV of C. vulgaris lipids.

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3.4. Economic assessment

288

The economic feasibility of the present study was evaluated following Wang et al. (2019)

289

energy calculations. The system was designed to cultivate 30L of algal culture with energy

290

consumption of 77.65 kWh per cycle. Totally 24 cycles were performed to acquire 1 kg of the

291

dry biomass by utilizing 1863.72 kWh kg-1 of input energy and the estimated cost of expenses

292

was 140.27 USD including chemicals used for cultivation purpose (Fig. 4). In addition,

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microalgae harvest required 438.3 kWh kg-1 of energy with 26.38 USD kg-1 of dry biomass 13

294

recovery. Whereas extraction of lipids recorded that the suggested process required 13.7%

295

more expense to perform when compared to conventional method. However, almost 13% of

296

the effective lipid yield and elevated total FAMEs level carried out the attention to economic

297

significance. In addition, energy outputs of conventional and suggested methods were

298

calculated based on calorific value (HHV) of FAME and were recorded as 417.7 and 533.6

299

MJ ton-1 respectively. Despite higher energy consumption of the suggested extraction

300

method, it showed 27.7% higher energy output than the conventional extraction method. In

301

addition, the possible improvement in cultivation and harvesting process will encounter

302

higher cost and energy consumption in economical point of view. The large-scale cultivation

303

system and low energy consuming harvesting process like flocculation and bio-flocculation

304

allows wide possibility for feasible biodiesel production.

305

Conclusion

306

C. vulgaris MSU AGM 14 lipid production has been promoted by simultaneous increase of

307

biomass by higher CO2 conditions of microalgae. The increased amount of total lipid yield

308

(22.5 %) has been extracted by employing mild pressure and heat shock treatment. FAMEs

309

analysis of C. vulgaris MSU AGM 14 lipid evidenced the presence of high content of

310

monounsaturated and saturated fatty acids. In addition, degree of unsaturation, iodine value,

311

cold filter plugging point, kinematic viscosity and density of biodiesel strengthened the

312

appropriate characteristics of fuel standards. The overall results revealed that C. vulgaris

313

MSU AGM 14 is a potential strain for biodiesel production. The novelty of this study reveals

314

that mild pressure and heat shock cell disruption evidenced 27.7% elevated energy output,

315

which could reduce the total cost of energy consumption and increase the lipid recovery from

316

the selected strain.

317

Acknowledgement

14

318

The authors sincerely acknowledge National Natural Science Foundation of China (No.

319

51676091), China Postdoctoral Science Foundation (2019T120408, 2018M630529),

320

Foundation of State Key Laboratory of Coal Combustion (FSKLCCA1904), the Six talent

321

peaks project in Jiangsu Province (XNY-007, 2018) for the financial support. Authors also

322

thank Jiangsu University, Zhenjiang, China and Sri Paramakalyani Centre of Excellence in

323

Environmental Sciences, Manonmaniam Sundaranar University for providing facilities

324

required to carry out this work.

325

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Figures and tables captions

508

Figure 1: Growth curve of Chlorella vulgaris at different percentages of CO2 with confocal

509

microscopy images stained with Nile red to visualize the intracellular lipids.

510

Figure 2: Microalgal biomass and total lipid production (a) and productivity (b) of C.

511

vulgaris MSU AGM 14 at different percentages of CO2. The same series with the same letter

512

showed insignificant difference (at P<0.05).

22

513

Figure 3: Optimized extraction of total lipids from C. vulgaris MSU AGM 14 by suggested

514

mild pressure and heat treated method. The horizontal dashed line represents the lipid

515

recovery by the conventional extraction method.

516

Figure 4: Cultivation, harvest and extraction process cost and energy output for biodiesel

517

produced from microalgae C. vulgaris MSU AGM 14.

518

Table 1: Selection of suitable photoperiod lengths for optimizing the growth and lipid

519

productivity.

520

Table 2: Composition of biodiesel produced from different lipid extraction process.

521

Table 3: Resemblance between calculated properties of conventional and suggested

522

extraction processed biodiesel with biodiesel standards.

23

TABLES

Table 1. Selection of suitable photoperiod lengths for optimizing the growth and lipid productivity. Illumination time

Biomass CO2

(Hrs at 100 µmol -2 -1

photons m s )

18L+6D

12L+12D

24L

dry

Total lipid

weight

(gg-1 dw)

-1

Biomass

Lipid

productivity (gL-

Productivity (gL-

1

day-1)

1

day-1)

(%)

(gL )

Control

0.66±0.02a

0.12±0.02a

0.052±0.003a

0.006±0.002a

4%

0.69±0.05a

0.15±0.04ab

0.054±0.004ab

0.008±0.003a

8%

0.79±0.04b

0.19±0.02b

0.064±0.003b

0.012±0.002b

Control

0.49±0.05a

0.14±0.03a

0.038±.004a

0.005±.002a

4%

0.93±0.02b

0.17±0.05ab

0.075±.003b

0.013±.004b

8%

1.37±0.03c

0.2±0.02b

0.111±.002c

0.022±.003c

Control

0.38±0.05a

0.13±0.02a

0.029±.002a

0.004±.002a

4%

0.91±0.02b

0.16±0.04ab

0.074±.003b

0.012±.003b

8%

1.44±0.02c

0.18±0.02b

0.117±.003c

0.021±.003c

L- Light; D- Dark, values are mean ± SD, n=3. The same parameter at the same light regime with the same letter showed insignificant difference (at P<0.05).

Table 2. Composition of biodiesel produced from different lipid extraction process. Fatty Acid Methyl Esters

Relative content Control

Mild pressure and heat treated

Myristic acid (C14:0)

-

1.40

Palmitic acid (C16:0)

7.82

6.78

Palmitoleic acid (C16:1n-7)

15.11

14.53

Stearic acid (C18:0)

12.57

12.08

Oleic acid (C18:1n-9)

50.66

51.62

α - Linolenic acid (C18:3n-3)

10.18

10.24

γ - Linolenic acid (C18:3n-6)

-

0.04

Arachidic acid (C20:0)

2.98

3.27

Saturated fatty acids (%)

23.37

23.53

Monounsaturated fatty acids (%)

65.77

66.15ns

Polyunsaturated fatty acids (%)

10.18

10.28ns

Total FAMEs (%)

99.32

99.96ns

Unidentified (%)

0.68

0.04

ns

Showed insignificant difference with the corresponding control (at P<0.05).

Table 3.Resemblance between calculated properties of conventional and suggested extraction processed biodiesel with biodiesel standards. Biodiesel standards Properties

ASTM D6751-02

Degree of unsaturation

Mild pressure Conventional

EN 14214

and heat treated

-

-

86.13

86.71

-

-

201.48

203.04

Iodine value (I2100g )

NA

≤120

88.50

89.06

Cetane number

≥47

≥51

53.48

53.14

-

-

10.05

9.99

NA

≤5/≤-20

15.1

14.91

-

-

71.02

72.18

-

-

26.32

27.10

-

≤12

10.18

10.28

1.9-6.0

3.5-5.0

1.35

1.35

Density (g cm-3)

NA

0.86-0.90

0.86

0.87

Higher heating value (MJ kg-1)

NA

NA

39.22

39.46

-1

Saponification value (mg KOHg ) -1

Long chain saturated factor Cold filter plugging point (ºC) Allylic position equivalents (APE) Bis- allylic position equivalents (BAPE) C18:3 (%) Kinematic viscosity (mm2s-1)

FIGURES

Figure 1. Growth curve of Chlorella vulgaris at different percentages of CO2.

Figure 2. Microalgal biomass and total lipid production (a) and productivity (b) of C. vulgaris MSU AGM 14 at different percentages of CO2. The same series with the same letter showed insignificant difference (at P<0.05).

0.25

5 min 15 min

10 min

-1

Lipid recovery (g g dw)

0.20

0.15

0.10

0.05

o

70C +C N 70C +1 70C +1. 5 70C +2 70C +2. 5

60C +C N 60C +1 60C +1. 5 60C +2 60C +2. 5

50C +C N 50C +1 50C +1. 5 50C +2 50C +2. 5

0.00

2

Temperature ( C) + Pressure (kg/cm ) Figure 3. Optimized extraction of total lipids from C. vulgaris MSU AGM 14 by suggested mild pressure and heat-treated method. The horizontal dashed line represents the lipid recovery by the conventional extraction method.

Figure 4.Cultivation, harvest and extraction process cost and energy output for biodiesel produced from microalgae C. vulgaris MSU AGM 14.

Highlights: •

Elevated CO2 (8%) promoted biomass (23%) and lipid productivity (94%).



Mild pressure with heat shock evidenced 12.5% additional recovery of total lipids.



Suggested extraction increased 1.96% of PUFA and 0.58% of MUFA recovery.



FAMEs of suggested process proved 27.8% elevated energy output.

Declaration of interests ☐The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

No conflict of interest to declare