Optimization of Cultivation Conditions of Native Microalga Scenedesmus quadricauda and Evaluation of Lipids for Enhanced Biodiesel Production

Abstract

Biofuels are considered to be among the primary alternatives to the use of fossil fuels. These fuels, made from feedstock or waste raw materials, have the advantage of being renewable and contributing much less to global warming. Microalgae are a promising biodiesel source. Microalgae, unlike traditional crops that are now used to make commercialized biodiesel, may be grown on non-agricultural land and has a greater capacity for growth and yield. Cultivation has been considered as a critical stage in the generation of biofuels. The goal of the present study is to learn that Scenedesmus quadricauda has a potential for biodiesel production in the near future. Optimization studies revealed that BG-11 medium, temperature of 25 °C, pH 7.0, glucose and sucrose (as carbon sources), static condition (for lipid accumulation) & shaking condition (for biomass yield), cultivation days of 18, 21, and 24 day, NaNO₃ dosing of 1.0 mM followed by 0.8 mM (on 5th day of cultivation), 3% yeast extract dosing, 3000 lx light intensity, photoperiod cycles of 24L/0D (for biomass yield) and 18L/6D (for lipid production) and 10 mM concentration of NaCl (salinity stress) can be regarded as best suited physio-biochemical parameters for efficient biomass and lipid yield from S. quadricauda. FTIR indicated presence of various stretching of carbohydrates and lipids that again is supporting biodiesel production capability of S. quadricauda. SEM showed that cells of S. quadricauda under stress conditions became fragmented separated from coenobium and were not so compactly arranged. Present optimization studies along with Nile red fluorescence, FTIR and SEM revealed that S. quadricauda could be a suitable candidate to produce good quality biofuel and that also in stress conditions.

Graphical Abstract

Introduction

The world economy now relies on fossil fuels to generate the energy indispensable for heating, lighting, and mobility. As the world's population and economy grow, so will the need for fossil fuels. As a result, competition for these finite resources will heat up in the coming years. The worldwide energy shortage and higher emissions of greenhouse gases, combined with rapidly rising prices and uncertainty about petroleum availability, have fueled the search for an alternative and green source of renewable energy for environmentally conscious growth, with the potential to eventually replace fossil-based fuels [1]. Based on life cycle research, algae have intriguing properties as a possible raw material for biofuel generation and can absorb substantial volumes of CO₂ [2]. Microalgae have a high photosynthesis capacity and a strong ability to adapt to harsh environments such as a high salinity level, heavy metals ion content, toxicant presence, and elevated CO₂ concentration. A total of three types of energy-producing plants (and biofuel) have been developed till date [3] viz. biofuels of the first generation made from edible feedstocks like corn, soybeans, sugarcane, rapeseed, etc. 1st type of biofuels were neglected by the world for causing food conflicts due to their use, which led to the development of 2nd generation biofuels [4]. Corn wastes, kernels of palm, lignocellulosic wastes, inedible oil, waste cooking oil and animal fats are used to produce second generation biofuels, but a challenge of consistent feedstock supply is also associated with them. Far better biofuel feedstock, micro- and macro- algae have emerged as third generation feedstocks. Microalgae's high lipid content makes it a promising biodiesel production option. Aside from that, microalgae are the primary source of oxygen on the world, and their CO₂ biosequestration via photosynthesis points to microalgae biodiesel as a promising carbon–neutral fuel. Microalgae biomass is emerging as a biodiesel source in the present context [5]. These microorganisms outperform traditional crops in terms of growth and productivity. Furthermore, they can be grown with wastewater, reducing pressure for freshwater and enhancing sustainability [5, 6]. The concept of biological refinery applied to cultures of microalgae is a critical differentiator. Following lipid extraction, microalgal biomass residues contains a number of high-value bioproducts. There are numerous species of microalgae that may produce fuel oil currently, and they are listed like Chlorella, Botryococcus Nannochloropsis, Nostoc, Scenedesmus, Hematococcus, Spirulina, Dunaliella, species etc. [7, 8]. Glycerolipids, sterols, hydrocarbons, and waxes are only a few of the lipid-like substances produced by microalgae. Glycerolipids are the most prevalent and well-studied class of microalgae lipids. These have a glycerol backbone with one, two, or three fatty acid (FA) groups attached. FAs are a prominent part of microalgae biomass, accounting for between 5 and 60% of cell dry weight. [9]. TAG fatty acids are being researched for use in the development of vehicle biofuels [10]. Microalgae lipids have a more diverse FAs composition than plant oils [11]. Microalgae primarily create fatty acids with chain lengths of 12, 16, and 18 carbons, however certain species can synthesize fatty acids with chain lengths of up to 24 carbon atoms. TAGs are mostly composed of saturated (SFAs) and monounsaturated fatty acids (MUFAs), such as C14:0 (MA, myristic acid), C16:0 (PA, palmitic acid), C16:1 (POA, palmitoleic acid), C18:0 (SA, stearic acid), and C18:1 (OA, oleic acid), but polyunsaturated fatty acids (PUFAs) are present. Some PUFAs prevalent in microalgae lipids, such as C16:2 (hexadecadienoic acid), C16:3 (hexadecatrienoic acid), C16:4 (hexadecatetraenoic acid), and EPA, are not found in plant oils. [9]. Lots of environmental factors like light, temperature, salinity, nutrients, carbon sources and CO₂ concentration etc. have been found affecting the production of lipids and yield of biomass in microalgae [12].

The present study was done to examine that how different microalgae constituents primarily the lipid production and biomass yields of isolated Scenedesmus quadricauda vary with different environmental conditions and growth phases. Additionally, there are variations among species and occasionally even between subspecies of identical species. In order to get the highest amount of lipid production from each particular microalga, systematic research is required to optimize the medium. Another important thing about Scenedesmus spp. is that it is also resistant to contamination by other organisms because of its excellent tolerance for salinity and excessive alkalinity [13]. Fluorescent dye Nile Red (NR) method is a lipid-selective method that has been used in vivo lipid staining and quantitative analysis of different lipids in microalgae [14]. Similarly, stained lipid bodies in Scenedesmus quadricauda cells were also demonstrated by using Nile Red (NR) method. FTIR spectroscopy is an important and simple technique that is helpful for assessing the relative concentration of macromolecules such as proteins, lipids and carbohydrates in the cells of different algal species [15]. In the present study, FTIR spectroscopy was applied to Scenedesmus quadricauda cells that were cultivated in different nutrient stress situations so that changes in infra-red spectra may be used as a technique for measuring species-specific constituent cell quotas. SEM (Scanning Electron Microscopy) was used to study the morphological characteristics and other cellular aspects of Scenedesmus quadricauda under both normal and nutrient stress conditions.

Materials and Methods

Isolation, Preliminary Identification and Experimental Conditions

Scenedesmus quadricauda used in this study was secluded from ponds (fresh water) of village Jakhod Khera in District Hisar of Haryana state of India. A trinocular microscope (Olympus CX-31) fitted with a digital camera was used for the initial identification of pure algal strains. Standard isolation and culturing methods were used to repeatedly streak and plate in BG-11 media (composition is shown in Table 1) at pH 7.01 to produce pure isolates of green microalgae. Purified Scenedesmus quadricauda from the petri-plates were continuously maintained in 250 mL flasks containing 100 mL of BG-11 (pH 7.5) medium followed by incubation for 14 days in continuous white fluorescent light intensity of 3000 lx at a temperature of 25 ± 3 °C in a culture room. To avoid contamination, the media and flasks were sterilized in the autoclave for 20 min at 121 °C.

Table 1.

Composition of different culture media

Ingredients (gL−1)	BG-11	Different culture media
		BBM (pH 6.6)	Chu#10 (pH 6.5)	HS-Chu#10 (pH 6.5)	Allen (pH 7.8)
NaNO3	1.5	0.25	–	–	–
K2HPO4	0.04	0.075	0.5	0.0025	–
MgSO4·7H2O	0.075	0.075	2.5	0.0125	0.247
CaCl2·2H2O	0.036	0.025	–	–	–
Citric acid	0.006	–	–	–	–
Ferric ammonium citrate	0.006	–	–	–	–
EDTA (disodium salt)	0.001	–	–	–	–
Na2CO3	0.02	–	2.0	0.010	–
KH2PO4	–	0.175	–	–	0.272
NaCl	–	0.025	–	–	–
Ca(NO3)2		–	4.0	0.02	–
Na2SiO3	–	–	2.5	0.0125	–
FeCl3	–		0.08	0.0004	–
(NH4)2SO4	–	–	–	–	1.32
CaCl2	–	–	–	–	0.055
Trace metals solution having composition (H3BO3 2.86; MnCl2·4H2O 1.81; ZnSO4·7H2O 0.222; NaMoO4·2H2O 0.39; CuSO4·5H2O 0.079; Co (NO3)2·6H2O 0.0494)	1 1 mL	–	–	–	–
Alkaline EDTA solution having composition (EDTA 0.05; KOH 0.031)	–	1 mL	–	–	–
Acidified Iron solution having composition (FeSO4 ·7H2O 0.00498; H2SO4 1–2 drops)	–	1 mL	–	–	–
Boron solution having composition (H3BO3 0.01142)		1 mL	–	–	–
Trace metals solution having composition (ZnSO4 ·7H2O 0.00882; MnCl2·4H2O 0.00144; MoO3 0.00071; CuSO4·5H2O 0.00157; Co(NO3)2·6H2O 0.00049)	–	1 mL	–	–	–
Trace metals solution having composition (H3BO3 0.00248; MnSO4·H2O 0.00147; ZnSO4·7H2O 0.00023; CuSO4·5H2O 0.0001;(NH4)6Mo7O24·4H2O 0.00007; Co(NO3)2·6H2O 0.00014)	–	–	–	1 mL	–
Vitamins solution having composition (Thiamine·HCl (Vitamin B1) 0.050; Biotin (Vitamin H) 2.5; Cyanocobalamin (Vitamin B12) 2.5)	–	–	–	1 mL	–
Trace metals solution having composition (Fe-Na- EDTA·3H2O 0.03016; H3BO3 0.00286; MnCl2·4H2O 0.00179; ZnSO4·7H2O 0.00022; CuSO4·5H2O 0.000079; (NH4)6Mo7O24·4H2O 0.00013; NH4NO3 0.000023)	–	–	–	–	1 mL

Analytical Methods used for Determination of Various Constituents

Chlorophyll concentration was determined spectrophotometrically at 650 and 665 nm using the hot extraction method [16]. The chlorophyll content was calculated using the following formula and was expressed as µgmL⁻¹:

$$\text{Chlorophyll}\left(\mu {\text{gmL}}^{-1}\right)=2.55\times {10}^{-2}{\text{OD}}_{650}+0.4\times {10}^{-2}{\text{OD}}_{665}$$

The Lowry method [17] was used to calculate protein concentration at 660 nm. Carbohydrate was assessed using the anthrone reagent method [18], and carbohydrate concentration was calculated afterwards using a standard curve established by sampling graded doses of glucose. Total lipids were extracted using a slightly modified technique of Bligh and Dyer [19] by combining methanol-chloroform (2:1.5 v/v) with the algal sample. Gravimetric analysis was used to quantify the dry weight of algal biomass, and growth was represented by means of dry weight in gL⁻¹. The weight difference of a certain volume of culture filtered using Whatman GF/C filters and dried at 105 °C for 12 h was used to calculate dry mass. The growth was monitored by measuring the dried biomass on pre-weighed filter sheets until a constant weight was achieved.

Optimization Studies to Increase Biomass and Lipid Content

Several physiological and biochemical indicators, including composition of growth medium, influence of temperature, pH, light intensity, light/dark cycles, cultivation time, shaking/static conditions, carbon sources, nitrogen sources, nutrient stress etc. were optimized to enhance the biomass and lipid content in Scenedesmus quadricauda.

The algal cultures were exposed to five different media compositions with varying pH levels, such as BG-11, Bold's Basal Medium (BBM), Chu#10, Half strength (HS) Chu#10, and Allen media (composition of all these media is given in Table 1) in the culture facility of the departmental laboratory at 25 ± 3 °C, in order to determine the best culture medium. In reagent bottles, all of the media had been made and sterilized separately. The culture was harvested after seven days for further analysis.

The ideal temperature for growth varies depending on the species, so experiments were carried out in three separate batches at each of the five temperatures listed below: 25, 27, 30, 33, and 35 °C in orbital shaker with constant 3000 lx illumination.

The current study used BG-11 medium to know the effects of pH variations ranging from 5.0, 6.0, 7.0, 8.0, and 9.0. The choice of carbon source to promote Scenedesmus quadricauda growth was made directly in BG-11 media containing 100 mL of media solution and amended with 1% (w/v) of ten various sources of carbon (Glucose, Lactate, Sucrose, Mannitol, Fructose, Glycerol, Methanol, Ethanol, Carbonate, and Bi-carbonate). After a period of 12 days, analysis and comparison of numerous parameters, including biomass, protein, chlorophyll, lipid, and carbs contents, were performed.

Scenedesmus quadricauda was grown in an orbital shaker and samples were harvested at regular intervals each on days 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 days for analysis of various growth parameters. Three flasks were employed for collecting the algal culture on each day of the analysis.

Effect of shaking and static conditions was also evaluated for Scenedesmus quadricauda and for shaking conditions experiments were carried out in Erlenmeyer flasks (250 mL) having sterile BG-11 culture medium (100 mL) with pH 7.5 inoculated with 1 mL fresh algal inoculum in BOD incubator cum shaker (at 30 °C and 120 rpm) with 3000 lx continuous white light for 14 days and control was also run parallel under static condition. Analysis of various growth parameters were performed after 14 days.

Media of Scenedesmus quadricauda was also supplemented with different sources of nitrogen for growth and lipid enhancement. Sodium nitrate (NaNO₃) was individually used as a source of nitrogen in BG-11 medium by performing experiments in Erlenmeyer flasks (250 mL) containing of BG-11 media (100 mL) supplemented with nitrate (0.2, 0.4, 0.6, 0.8, 1.0 mM) using 1000 mL stock solution of NaNO₃, similarly, the dosing of another nitrogen source i.e. yeast extract was done by adding different concentrations of yeast extract (0.5, 1.0, 1.5, 2.0, 2.5, 3.0%) to the BG-11 medium before autoclaving. The experiments were performed at 25 °C and at 120 rpm in BOD incubator cum shaker for 11 days. Control culture was run parallel in BG-11 medium. pH of the medium was adjusted to 7.5 prior to autoclaving using 1N NaOH/H₂SO₄.

Effect of nitrate dosing was also studied on the culture by administering 1 mM NaNO₃ in BG-11 medium at various growth phases. Nitrate administration was made in such a way that culture received nitrate dosing at 0th, 2nd, 5th, 9th and 11th day in separate set of flasks at 25 °C and 120 rpm for 12 days. The culture without nitrate was also run parallel as a control. The impact of the amount of light and the photoperiod cycling on the development of algal species were studied in order to optimize microalgal growth in bulk culture systems and lipid content, were studied in batch culture. BG-11 media was illuminated with continuous light of different intensities (400, 1000, 2000, 3000, 5000 lx) using fluorescent tubes at 25 °C and at 120 rpm in BOD incubator cum shaker. In order to find out the influence of duration of light and dark conditions (in hrs) on algal growth, experiments were conducted under three different photoperiod cycles (24L/0D, 18L/6D and 12L/12D) at 25 ± 3 °C in culture racks equipped with timer for adjustment of light dark cycles.

Scenedesmus quadricauda was cultivated in BG-11 media supplemented with varied doses of NaCl (1.0, 2.0, 4.0, 8.0, and 10.0 mM) to investigate the effect of salt. A suitable dose was prepared from a stock solution of 1000 mM NaCl. All experiments have been carried out in 250 mL Erlenmeyer flasks, each containing 100 mL of BG-11 medium modified with varied doses of salt as indicated above. The cultures were incubated for 15 days at 25 °C in a BOD incubator cum shaker at 120 rpm, with a control culture in BG-11 media running in parallel. The medium and flasks were sterilized in an autoclave for 20 min at 121 °C in order to prevent any contamination. All tests employed one ml ten days fresh Scenedesmus quadricauda inoculums. All of the preceding tests were carried out in triplicate.

Nile Red Fluorescence Assay for Demonstration of Potential Lipid Bodies

Preparation of Nile Red Solution

Mix 0.5 gm of Nile Red powder in 1 mL of acetone to make the stock solution. 0.05 mL of this baseline solution was mixed with 50 mL of a 75:25 glycerol/water combination. This prepared solution was used to label algal cells.

Procedure of Nile Red Staining

Nile red staining was used to identify intracellular droplets of lipid [20]. Microalgae cells (0.5 mL) were placed in micro centrifuge tubes (1.5 mL) and centrifuged at 1500 rpm for 10 min before being rinsed several times with physiological saline solution (0.5 mL). Microalgae cells were resuspended in 0.5 mL of saline solution, and the Nile red solution (0.1 mgmL⁻¹ in acetone) was added to cell suspensions (1:100 v/v) and incubated for 10 min. Microalgal cells were seen under a fluorescent microscope after staining. The microscopic images were captured using an inverted microscope (Leica DMIL LED, Leica Microsystems, Germany).

Fourier Transform Infra-Red (FTIR) Spectroscopy

FTIR spectrum analysis was performed to explain the change in functional groups discovered in Tri-acyl glycerol (TAG) fatty acids in Scenedesmus quadricauda as biomass was collected under both normal and stress circumstances, with stress conditions induced by altering the composition of BG-11 media. In the present study, two modified BG-11 media compositions were used i.e. media lacking nitrate, and media lacking phosphate. With these modified BG-11 media, one set of control was also run parallel in which BG-11 media of normal composition was used that was having both nitrate and phosphate as constituents. The spectra were obtained using a Perkin Elmer FTIR System (Spectrum BX) with a diffuse reflectance accessory and a 400–4000 cm⁻¹ range. The KBr backdrop disc approach was used to acquire all spectra in transmission mode.

The algae were incubated at 25 °C under shaker incubator without nitrate, without phosphate, and a control was also run parallel to obtain information specific to the functional group, as well as information on the interaction of the groups with other parts of the molecule and on the spatial properties of the groups by FTIR. After 14 days of development, the biomass was collected by centrifuging 50 mL of the culture at 8000–10,000 rpm and stored in a deep freezer (New Brunswick Scientific, England) at − 80 °C before lyophilization in 50 mL centrifuge tubes. The 48-h lyophilized samples were stored in deep freezer in 50 mL centrifuge tubes after being lyophilized in a lyophilizer (Christ Alpha 2–4 L D plus, Germany). The samples were pulverized with KBr in an agate pestle and mortar. Before examination, sample pellets were formed using a hydraulic pellet press (Thane, Maharashtra). The background acquired from the KBr disc was automatically eliminated from the KBr-prepared spectra of the sample discs. On the transmittance axis, all spectra were shown using the same scale.

Ultra Structural Analysis of Scenedesmus quadricauda by Scanning Electron Microscopy

Morphological features and other cellular details of Scenedesmus quadricauda was studied under normal as well as nutrient stress conditions by employing scanning electron microscopy by taking the help of method described by Fowke and coworkers [21]. Samples to be analyzed were first fixed in 2% glutaraldehyde + 0.2 M phosphate buffer (pH 6.8) for 12 h then dried in ascending series of alcohol upto 100%, sputter coated with gold and examined in a scanning electron microscope (Table top) Research Grade SEMTRAC, Japan. The SEM was used in the Department of Physics, Guru Jambheshwar University of Science and Technology, Hisar. The surface that was visible was coated with gold using a sputter coater apparatus, and its interior was scanned at × 10, × 500, × 1.0 k, × 2.0 k, and × 5.0 k magnifications.

Statistical Analysis

Using SPSS 16.0 software, the data for various optimization parameters were statistically analysed using analysis of variance (ANOVA) using Posthoc-Duncan LSD Alpha (0.05). The results were interpreted using standard errors of means [22]. Individual means were compared for statistical significance using least significance difference.

Results

Identification of Isolated and Purified Scenedesmus quadricauda Along with Nile Red Fluorescent Microscopic Examination

Microphotograph of Scenedesmus quadricauda is depicted in Fig. 1a. Nile red florescent microscopic examination revealed the presence of cellular neutral lipid bodies in the microalgae (Fig. 1b). Scenedesmus quadricauda cell containing lipids showed the yellow fluorescence under florescent microscope.

Fig. 1.

a Microscopic picture of Scenedesmus quadricauda × 100. b Fluorescence microscopic images of Scenedesmus quadricauda

Optimization Experiments to Improve Biomass Production and Lipid Content

Scenedesmus quadricauda produced significantly (P ≤ 0.05) greater biomass and significantly (P ≤ 0.05) greater overall carbohydrate and total chlorophyll amount in BG-11 medium than in all other media. (Table 2). BBM was found to be best suited medium regarding production of lipid content.

Table 2.

Effect of media, temperature, pH and light intensities (lux) on biomass (gL⁻¹), lipid (% dcw), protein (mgmL⁻¹), total carbohydrate (mgmL⁻¹) and total chlorophyll (µgmL⁻¹) contents of Scenedesmus quadricauda

Parameters	Media
	BG-11	Bold’s Basal Medium (BBM)	Half-Strength (HS) Chu#10 Medium	Chu#10 Medium	Allen Medium
Biomass (gL−1)	1.23C ± 0.06	0.91B ± 0.03	0.94B ± 0.04	0.92B ± 0.02	0.71A ± 0.01
Lipid (% dcw)	11.82B ± 0.44	14.33C ± 0.45	7.37A ± 0.38	6.36A ± 0.40	13.96C ± 0.18
Protein (mgmL−1)	0.030A ± 0.0020	0.029A ± 0.0032	0.026A ± 0.0031	0.025A ± 0.0031	0.024A ± 0.0011
Total carbohydrate (mgmL−1)	0.060C ± 0.0041	0.038A ± 0.0031	0.045AB ± 0.0048	0.056BC ± 0.0052	0.034A ± 0.0028
Total chlorophyll (µgmL−1)	12.81D ± 0.33	11.66C ± 0.26	10.69C ± 0.23	8.85B ± 0.48	6.45A ± 0.39

Parameters	Temperature
	25 °C	27 °C	30 °C	33 °C	35 °C
Biomass (gL−1)	2.45E ± 0.02	2.28D ± 0.01	1.30A ± 0.01	1.47B ± 0.04	1.61C ± 0.03
Lipid (% dcw)	15.45C ± 0.49	14.19BC ± 0.04	12.75B ± 0.46	9.30A ± 0.69	9.70A ± 0.63
Protein (mgmL−1)	0.058C ± 0.0032	0.046B ± 0.0008	0.034A ± 0.0015	0.029A ± 0.0015	0.028A ± 0.0015
Total carbohydrate (mgmL−1)	0.17B ± 0.0101	0.08A ± 0.0062	0.07A ± 0.0049	0.08A ± 0.0026	0.07A ± 0.0044
Total chlorophyll (µgmL−1)	25.49C ± 1.29	23.74C ± 0.95	20.69B ± 0.23	19.08AB ± 0.54	17.01A ± 0.77

Parameters	pH
	5.0	6.0	7.0	8.0	9.0
Biomass (gL−1)	1.44A ± 0.06	1.74B ± 0.03	2.01C ± 0.09	1.84BC ± 0.07	1.63AB ± 0.04
Lipid (% dcw)	14.10A ± 0.55	14.72A ± 0.56	15.99A ± 0.50	15.68A ± 0.82	14.62A ± 0.50
Protein (mgmL−1)	0.054B ± 0.0039	0.044B ± 0.0049	0.051B ± 0.0034	0.030A ± 0.0026	0.028A ± 0.0017
Total carbohydrate (mgmL−1)	0.075BC ± 0.0056	0.086BC ± 0.0060	0.090C ± 0.0052	0.059AB ± 0.0088	0.044A ± 0.0131
Total chlorophyll (µgmL−1)	17.33A ± 0.79	23.19B ± 1.26	21.66B ± 1.03	17.98A ± 0.98	15.11A ± 0.55

Parameters	Light intensities (lux)
	400	1000	2000	3000	5000
Biomass (gL−1)	0.77A ± 0.04	1.20B ± 0.02	1.37C ± 0.02	1.66D ± 0.04	1.28BC ± 0.04
Lipid (% dcw)	6.78A ± 0.33	10.78B ± 0.27	12.73C ± 0.05	14.67D ± 0.06	12.28C ± 0.36
Protein (mgmL−1)	0.046BC ± 0.0014	0.050D ± 0.0014	0.044C ± 0.0011	0.037B ± 0.0017	0.031A ± 0.0011
Total carbohydrate (mgmL−1)	0.042A ± 0.0017	0.050B ± 0.0017	0.059C ± 0.0014	0.067D ± 0.0014	0.058C ± 0.0014
Total chlorophyll (µgmL−1)	11.91A ± 0.28	18.81B ± 0.30	22.63C ± 0.32	25.47D ± 0.55	19.61B ± 0.59

A, B, C, D, E, F, G Means with unlike superscript in the row differ significantly (P ≤ 0.05)

Scenedesmus quadricauda yielded higher biomass and produced higher lipid, protein, total carbohydrate and total chlorophyll contents at 25 °C temperature followed by 27 °C. It was interesting to find that with increase in temperature above 27 °C the values of all the analytical parameters decreased in Scenedesmus quadricauda (Table 2). Microalgae showed higher biomass yield, total carbohydrate content, lipid and protein contents at pH 7.0 as compared to all other pH ranges (Table 2). Total chlorophyll content was found to be increased upto pH 6, although decreasing trend of total chlorophyll content was observed as pH range was increased beyond pH 6.0. When compared to all other light intensities, biomass production, lipid, total carbohydrate, and total chlorophyll levels were found to be considerably (P ≤ 0.05) greater in Scenedesmus quadricauda at 3000 lx (Table 2).

Scenedesmus quadricauda yielded significantly higher (P ≤ 0.05) biomass and produced higher lipid, protein, total carbohydrate and total chlorophyll contents when glucose and sucrose were used as a carbon sources as compared to all other carbon sources and control conditions (Table 3).

Table 3.

Effect of different carbon sources and cultivation time in days on biomass (gL⁻¹), lipid (% dcw), protein (mgmL⁻¹), total carbohydrate (mgmL⁻¹) and total chlorophyll (µgmL⁻¹) contents of Scenedesmus quadricauda

Parameters	Carbon source
	Control	Glucose	Lactate	Sucrose	Mannitol	Fructose	Bicarbonate	Methanol	Ethanol	Carbonate
Biomass (gL−1)	1.17A ± 0.01	1.65F ± 0.02	1.46D ± 0.02	1.63F ± 0.02	1.52DE ± 0.01	1.48D ± 0.00	1.30B ± 0.02	1.40C ± 0.02	1.57E ± 0.02	1.12A ± 0.01
Lipid (% dcw)	12.69C ± 0.24	15.89E ± 0.38	13.91D ± 0.03	5.28E ± 0.35	13.59D ± 0.31	11.38A ± 0.34	12.51BC ± 0.27	15.41E ± 0.16	12.42BC ± 0.12	11.76AB ± 0.04
Protein (mgmL−1)	0.030BC ± 0.0017	0.095G ± 0.0034	0.042DE ± 0.0015	0.189H ± 0.0017	0.062F ± 0.0014	0.046E ± 0.0026	0.021A ± 0.0017	0.029ABC ± 0.0043	0.037CD ± 0.0025	0.028AB ± 0.0030
Total carbohydrate (mgmL−1)	0.037AB ± 0.0017	0.048C ± 0.0014	0.041B ± 0.0005	0.037AB ± 0.0008	0.046C ± 0.0008	0.040AB ± 0.0011	0.054D ± 0.0011	0.040AB ± 0.0005	0.054D ± 0.0017	0.036A ± 0.0020
Total chlorophyll (µgmL−1)	23.27D ± 0.40	27.99F ± 0.13	23.57D ± 0.33	19.32C ± 0.22	19.97C ± 0.15	26.35E ± 0.26	12.64A ± 0.12	28.85G ± 0.19	29.27G ± 0.15	13.65B ± 0.38

Parameters	Cultivation time in days (Time course duration)
	3	6	9	12	15	18	21	24	27	30
Biomass (gL−1)	0.29A ± 0.03	0.94B ± 0.02	1.02B ± 0.05	1.31C ± 0.04	1.46CD ± 0.02	1.72EF ± 0.06	1.89F ± 0.04	1.83EF ± 0.06	1.65DE ± 0.11	1.52D ± 0.10
Lipid (% dcw)	3.15A ± 0.04	4.99B ± 0.02	7.23C ± 0.06	8.20D ± 0.05	9.40E ± 0.08	10.18F ± 0.02	13.48G ± 0.13	15.36H ± 0.06	13.54G ± 0.08	9.19E ± 0.09
Protein (mgmL−1)	0.012A ± 0.0005	0.022C ± 0.0008	0.034E ± 0.0012	0.074H ± 0.0011	0.053G ± 0.0005	0.040F ± 0.0011	0.030D ± 0.0005	0.022C ± 0.0014	0.018B ± 0.0005	0.014A ± 0.0008
Total carbohydrate (mgmL−1)	0.016A ± 0.0014	0.034BC ± 0.0023	0.045CD ± 0.0014	0.052DE ± 0.0028	0.063E ± 0.0010	0.140F ± 0.0152	0.061E ± 0.0020	0.043CD ± 0.0012	0.020AB ± 0.0011	0.016A ± 0.0012
Total chlorophyll (µgmL−1)	3.35A ± 0.07	9.60B ± 0.35	13.47C ± 0.25	13.89C ± 0.05	15.37D ± 0.33	17.57E ± 0.29	20.08F ± 0.01	32.17I ± 0.32	29.72H ± 0.34	26.91G ± 0.03

A, B, C, D, E, F, G, H, I: Means with unlike superscript in the row differ significantly (P ≤ 0.05)

Biomass yield, lipid, protein, total carbohydrate and total chlorophyll contents were found to be significantly (P ≤ 0.05) higher at 21st day, 18th day and 24th day of cultivation in Scenedesmus quadricauda (Table 3). Scenedesmus quadricauda yielded higher biomass and produced higher protein, total carbohydrate and total chlorophyll contents in shaking condition in comparison to static condition (Table 4). In contrast, lipid content was found to be greater under static conditions than in shaking conditions. Scenedesmus quadricauda exhibited higher biomass yield, total carbohydrate and total chlorophyll contents at photoperiod cycles of 24L/0D and exhibited higher lipid and protein contents at photoperiod cycles of 18L/6D and 12L/12D, respectively (Table 4).

Table 4.

Effect of different cultivation conditions and light & dark conditions on biomass (gL⁻¹), lipid (% dcw), protein (mgmL⁻¹), total carbohydrate (mgmL⁻¹) and total chlorophyll (µgmL⁻¹) contents of Scenedesmus quadricauda

Parameters	Different cultivation conditions
	Shaking condition	Static condition
Biomass (gL−1)	1.06	0.81
Lipid content (% dcw)	11.89	14.57
Protein content (mgmL−1)	0.059	0.033
Total carbohydrate content (mgmL−1)	0.083	0.047
Total chlorophyll content (µgmL−1)	13.56	5.91

Parameters	Light and dark conditions/Photoperiod cycles (in hrs)
	24L/0D	18L/6D	12L/12D
Biomass (gL−1)	1.75B ± 0.04	1.55A ± 0.03	1.47A ± 0.03
Lipid content (% dcw)	12.27B ± 0.08	14.34C ± 0.07	10.22A ± 0.05
Protein content (mgmL−1)	0.035A ± 0.0020	0.037A ± 0.0011	0.040A ± 0.0023
Total carbohydrate content (mgmL−1)	0.068B ± 0.0031	0.062AB ± 0.0023	0.058A ± 0.0023
Total chlorophyll content (µgmL−1)	24.74C ± 0.57	22.34B ± 0.34	18.91A ± 0.50

A, B, C, D: Means with unlike superscript in the row differ significantly (P ≤ 0.05)

Biomass yield and production of lipid, protein, total carbohydrate and total chlorophyll contents of Scenedesmus quadricauda was found to be significantly (P ≤ 0.05) higher at NaNO₃ dosing of 1.0 mM followed by 0.8 mM as compared to other NaNO₃ dosing rates and also in control condition (Table 5). All the studies that are above mentioned in this paragraph support our findings of higher biomass yield and production of lipid, protein, total carbohydrate and total chlorophyll contents of Scenedesmus quadricauda at higher rate of NaNO₃ dosing of 1.0 mM followed by 0.8 mM. Effect of NaNO₃ at various growth phases of Scenedesmus quadricauda revealed higher biomass yield, lipid, protein and total chlorophyll content at NaNO₃ dosing (1 mM) on 5th day of cultivation (Table 5).

Table 5.

Effect of NaNO₃ dosing (1 mM) at various growth phases in days, NaNO₃ dosing (mM), NaCl concentrations (mM) in BG-11 medium and yeast extract dosing (%) on biomass (gL⁻¹), lipid (% dcw), protein (mgmL⁻¹), total carbohydrate (mgmL⁻¹) and total chlorophyll (µgmL⁻¹) contents of Scenedesmus quadricauda

Parameters	NaNO3 dosing (1 mM) at various growth phases in days
	Control	2	5	9	11
Biomass (gL−1)	0.52BC ± 0.0072	0.67C ± 0.0046	0.49BC ± 0.0100	0.34AB ± 0.0034	0.18A ± 0.0081
Lipid (% dcw)	10.44A ± 0.041	12.77B ± 0.043	19.40E ± 0.097	14.64D ± 0.117	13.10C ± 0.072
Protein (mgmL−1)	0.020A ± 0.0023	0.025AB ± 0.0020	0.031C ± 0.0017	0.029BC ± 0.0008	0.028BC ± 0.0008
Total carbohydrate (mgmL−1)	0.081A ± 0.0011	0.090B ± 0.0005	0.095C ± 0.0020	0.100D ± 0.0008	0.110E ± 0.0011
Total chlorophyll (µgmL−1)	0.43A ± 0.007	5.42E ± 0.047	6.61F ± 0.101	3.44C ± 0.043	3.13B ± 0.085

Parameters	NaNO3 dosing (mM)
	Control	0.2	0.4	0.6	0.8	1.0
Biomass (gL−1)	0.93A ± 0.02	1.01B ± 0.02	1.16C ± 0.02	1.26D ± 0.02	1.35E ± 0.02	1.49F ± 0.00
Lipid (% dcw)	7.58A ± 0.05	7.73A ± 0.03	10.13B ± 0.06	11.39C ± 0.15	13.22D ± 0.10	14.64E ± 0.06
Protein (mgmL−1)	0.067A ± 0.0014	0.074A ± 0.0011	0.088B ± 0.0008	0.099C ± 0.0008	0.124D ± 0.0014	0.211E ± 0.0057
Total carbohydrate (mgmL−1)	0.079A ± 0.0020	0.085B ± 0.0008	0.090C ± 0.0017	0.096D ± 0.0014	0.102E ± 0.0014	0.108F ± 0.0011
Total chlorophyll (µgmL−1)	15.14A ± 0.06	15.25A ± 0.02	15.40B ± 0.02	15.52B ± 0.01	15.98C ± 0.05	16.47D ± 0.02

Parameters	NaCl concentrations (mM) in BG-11 medium
	Control	1.0	2.0	4.0	8.0	10.0
Biomass (gL−1)	1.26AB ± 0.027	1.21A ± 0.026	1.25AB ± 0.020	1.27B ± 0.012	1.29B ± 0.008	1.37C ± 0.014
Lipid (% dcw)	7.67E ± 0.058	7.45D ± 0.055	7.21C ± 0.063	6.02B ± 0.070	5.76A ± 0.050	7.75E ± 0.036
Protein (mgmL−1)	0.070C ± 0.0005	0.040A ± 0.0005	0.063B ± 0.0005	0.072D ± 0.0008	0.077E ± 0.0005	0.112F ± 0.0008
Total carbohydrate (mgmL−1)	0.034A ± 0.0003	0.036B ± 0.0005	0.039C ± 0.0005	0.040D ± 0.0003	0.042D ± 0.0005	0.047E ± 0.0005
Total chlorophyll (µgmL−1)	15.28C ± 0.020	16.29F ± 0.008	15.63E ± 0.029	15.50D ± 0.008	15.21B ± 0.012	14.50A ± 0.011

Parameters	Yeast extract dosing (%)
	Control	0.5	1.0	1.5	2.0	2.5	3.0
Biomass (gL−1)	1.05A ± 0.02	1.19B ± 0.00	1.32C ± 0.02	1.40D ± 0.01	1.54E ± 0.01	1.64F ± 0.01	1.85G ± 0.02
Lipid (% dcw)	12.15A ± 0.04	13.29B ± 0.03	13.94C ± 0.02	15.02D ± 0.05	15.97E ± 0.30	18.46F ± 0.02	16.38E ± 0.34
Protein (mgmL−1)	0.075A ± 0.0014	0.083B ± 0.0005	0.097C ± 0.0008	0.125D ± 0.0018	0.149E ± 0.0008	0.192G ± 0.0011	0.157F ± 0.0020
Total carbohydrate (mgmL−1)	0.081A ± 0.0011	0.083A ± 0.0005	0.090B ± 0.0005	0.095C ± 0.0020	0.100D ± 0.0008	0.110E ± 0.0011	0.079A ± 0.0012
Total chlorophyll (µgmL−1)	9.12A ± 0.05	14.06B ± 0.08	17.04C ± 0.11	19.06D ± 0.09	20.03E ± 0.33	21.23F ± 0.37	23.33G ± 0.32

A, B, C, D, E, F, G: Means with unlike superscript in the row differ significantly (P ≤ 0.05)

Scenedesmus quadricauda revealed significantly (P ≤ 0.05) higher yield of biomass along with higher production of lipid, protein and total carbohydrate at 10 mM concentration of NaCl in BG-11 medium (Table 5). Whereas, Scenedesmus quadricauda revealed significantly (P ≤ 0.05) higher production of total chlorophyll content at 1 mM concentration of NaCl in BG-11 medium (Table 5). Scenedesmus quadricauda exhibited significantly (P ≤ 0.05) higher biomass yield and higher total chlorophyll content at yeast extract dosing of 3% followed by 2.5% as compared to all other dosing rates and control condition (Table 5). Similarly, Scenedesmus quadricauda exhibited significantly greater (P ≤ 0.05) fat, protein, and total carbohydrate levels at a yeast extract dosage rate of 2.5% as compared to all other dosing rates and the control.

FTIR Spectroscopic Study in Normal as Well as Stress Conditions

In the present work we were interested particularly in two main functional groups viz. carbonyls (at ≈ 1740 cm⁻¹) and aliphatics (at ≈ 2800–3000 cm⁻¹) as they are widely present in lipids and carbohydrates. Table 5 showed that band pattern in FTIR spectra of different Scenedesmus quadricauda in normal as well as stress conditions. Typical band is also assigned to Scenedesmus quadricauda by taking into consideration already published band assignments as mentioned in literature. FTIR spectra of the entire freeze-dried biomass of Scenedesmus quadricauda are well depicted in Fig. 2. FTIR spectra of Scenedesmus quadricauda revealed excessive stretching of the typical bands in the stress (or nutrient deprived) conditions when compared with the typical bands of normal condition (Fig. 2).

Fig. 2.

FTIR spectra of Scenedesmus quadricauda grown under normal (A) and nutrient deprived/stress conditions (B*). (*See for excessive stretching of the typical bands in the stress (or nutrient deprived) conditions as compared to typical bands of normal condition)

Lipid-carbohydrate mainly v_as(CH₂) and v_s(CH₂) stretching was characterized by strong peaks of 2923 & 2852 cm⁻¹ for Scenedesmus quadricauda in both normal and nutrient deprived (stress) conditions (Table 6). Presence of strong peaks of 1152 cm⁻¹ & 1048 cm⁻¹ for Scenedesmus quadricauda revealed the characteristic presence of carbohydrate v(C–O–C) of polysaccharides in these algal strains in both normal and nutrient deprived (stress) conditions (Table 6).

Table 6.

Band pattern obtained in FTIR spectra of Scenedesmus quadricauda under both normal as well as stress conditions (nutrient deprived conditions)

Band	Main peak (cm−1) in different screened algal strains	Assignment of typical band to peaks as described in literature*	Wave number range (cm−1)
	Scenedesmus quadricauda
1	3305	Water v(O–H) stretching Protein v(N–H) stretching (amide A)	3029–3639
2	2923, 2852	Lipid-carbohydrate Mainly vas(CH2) and vs(CH2) stretching	2809–3012
3	2362	C≡N stretching, Nitriles	2300–2000
4	1710	Cellulose-Fatty Acids v(C=O) stretching of esters	1763–1712
5	1649	Protein amide I band Mainly v(C=O) stretching	1583–1709
6	1547	Protein amide II band mainly δ(N–H) bending and v(C–N) stretching	1481–1585
7	1384	Protein δs(CH2) and δs(CH3) bending of methyl Carboxylic Acid vs(C–O) of COO groups of carboxylates Lipid δs(N(CH3)3) bending of methyl	1357–1423
8	1242	Nucleic Acid (other phosphate-containing compounds) vas(> P=O) stretching of phosphodiesters	1191–1356
9	1152	Carbohydrate v(C–O–C) of Polysaccharides	1134–1174
10	1048	Carbohydrate v(C–O–C) of polysaccharides	980–1072

Band 1-Residual water, Band 1,5,6,7-Proteins, Band 2,4,7-Lipids, Band 4-Cellulose, Band 8,10-Nucleic acids, Band 2,9,10,11-Carbohydrates

Band pattern obtained in different screened algal strains was found to be similar in normal as well as stress conditions

*Band assignment based on findings of different researchers (Keller 1986, Stuart, 1997, Giordano et al. 2001, Benning et al. 2004, Dean et al. 2007, Duygu et al. 2012, Ponnuswamy et al. 2013)

The above mentioned stretchings of v_as(CH₂), v_s(CH₂) and v(C–O–C) indicated the presence of lipid and carbohydrates in all the Scenedesmus quadricauda and strongly favours the biodiesel producing capability of Scenedesmus quadricauda. Nucleic acids (other phosphate-containing compounds) v_as(> P = O) stretching of phosphodiesters was characterized by strong vibrations of 1242 cm⁻¹ for Scenedesmus quadricauda in both normal and nutrient deprived (stress) conditions (Table 6). These stretchings again are indicating towards the biodiesel producing capability of Scenedesmus quadricauda. These peaks are typical of the long chain fatty acid methyl esters found in biodiesel. Residual water was postulated in the region of 3305 cm⁻¹ due to water v (O–H) stretching in Scenedesmus quadricauda in both normal and nutrient deprived (stress) conditions (Table 6).

The close correlation between the peaks and the existence of band 2, 4 and 7 suggested that high lipid and carbohydrate as well as presence of nucleic acid in Scenedesmus quadricauda. Bands in the region of 2362 cm⁻¹ in Scenedesmus quadricauda is due to the C≡N stretching that showed the presence of the nitriles group in both normal and nutrient deprived (stress) conditions (Table 6). FTIR results show that there are different signatures for triglycerides and phospholipids in the FTIR spectrum of Scenedesmus quadricauda. From these results it can be inferred that Scenedesmus quadricauda isolated and identified in the present study have a good potential for biodiesel production and it is the sole objective of our study.

Ultra Structural Analysis by Using Scanning Electron Microscope (SEM)

Scanning electron micrographs of Scenedesmus quadricauda under normal as well as nutrient stress conditions is depicted in Fig. 3 at a potential of 10 kV and magnifications of × 10, × 500, × 1.0 k, × 2.0 k and × 5.0 k. Scanning electron micrographs of Scenedesmus quadricauda under normal as well as nutrient stress conditions shows characteristic colonies of cells, usually in pairs of two or four cells that are arranged in rows with two spines at each end of a chain, net like cell wall structure with ridges (Fig. 3C–F). These spines help the immobile cells to float at the water surface, where light and nutrients are in better supply.

Fig. 3.

Scanning electron micrographs of Scenedesmus quadricauda [A (× 10), C (× 2.0 k), E (× 5.0 k)—under normal condition; B (× 10), D (× 2.0 k), F (× 5.0 k)—under nutrient stress condition]

Under normal conditions, the cells of Scenedesmus quadricauda coenobium are compactly arranged in bundles and are non-fragmented (Fig. 3A, C, E), whereas in stress conditions, there is separation along with fragmentation of algal cells from coenobium, and these are not as compact as under normal conditions (Fig. 3B, D, F).

Discussion

Scenedesmus quadricauda cell containing lipids showed the yellow fluorescence under florescent microscope. These results are in congruence with findings of other scientists [23]. According to the findings of the current study, an increase in biomass leads to an increase in chlorophyll content in microalgae, which is consistent with the findings in the algal species Chlorella vulgaris, where a study reported higher chlorophyll production with increased biomass production [24]. Other researchers have shown that when algal biomass increases, so does the chlorophyll concentration of the algae [25]. Similar results of greater algal biomass yield in BG-11 media were obtained in a study that found BG-11 to be the optimal medium for green algae biomass production [26, 27]. The total chlorophyll content of the cells of algae was higher in BG-11 medium, indicating that the medium has a greater ability to support heterotrophic cultures. The greater lipid content in Scenedesmus quadricauda could be due to the low nitrate levels in BBM and Allen media, as greater lipid production in green algae Neochloris oleoabundans as well as other algal species at lower NaNO₃ and KNO₃ levels have also been reported [28]. Many algae species have been found to be capable of accumulating triacylglycerols that have elevated oleic acid content in nitrogen-deficient circumstances [29]. Some experts believe that nitrogen is one of the typically identified dietary limiting element that causes fat buildup [30].

Various scientists studied the physiological responses of microalgae in regard to temperature variations and revealed that microalgae have some difference in their adaptability towards temperature variations and temperature has been considered as one of the most important variables that affects algal growth as well as its chemical composition [31–33]. According to scientists, temperature have a significant impact on microalgae fatty acid composition as well as contents [34, 35]. The optimum temperature range for the growth of Scenedesmus spp was reported to be between 20 and 40 °C by many scientists [36], this is consistent with the outcomes of our investigation. A study used various pH values ranging from 6.5 to 8.5 to study the maximum growth and found similar results, i.e. algae at pH 7.4–8.0 elucidated a significant improvement in growth, thereby telling that alkaline pH is essentially required for microalgae growth [37, 38]. In a study it was also observed that there are higher rates of microalgae growth when glucose was used as substrate instead of other sources like sugars, phosphate of sugars, organic acids, and monohydric alcohols [39]. Basic difference in the composition of heterotrophic and autotrophic culture medium is supply of additional organic carbon source in heterotrophic cultures [40] that too we have undertaken in our present study. The effects of several carbon sources on biomass output and lipid production in a new lipid-rich microalga Scenedesmus sp. strain R-16 revealed that glucose was the ideal substrate producing highest biomass, fastest growth rate and highest total lipid [41], and these findings correspond with our findings. In algae, just two pathways, the Embden Meyerhof Pranas Pathway (EMP) and the Pentose Phosphate Pathway (PPP), have been discovered, as opposed to multiple other pathways employed by bacteria for aerobic glycolysis (glucose breakdown) [42].

According to different studies, light intensity, in addition to nutrients and temperature, plays an essential role in determining algal development and lipid production and it may also affect composition of fatty acid in algae [11]. These findings strongly support our study. The length, intensity, and overall quality of light are among the most significant parameters in photosynthetic organism success. Light intensity is known to alter the synthesis of many cell components. Synthesis of neutral lipids is also affected by light intensity variation and supply of carbon dioxide [11]. In the presence of saturating light, however, they synthesize sugars, lipids, and starch via the pentose phosphate route, which requires phosphate reduction, as is also the case in our study. Other investigators have noticed reduced yield of biomass, less lipid production, and greater protein synthesis at lower light intensities, which is similar to our findings [43]. There is paucity of literature particularly on the effect of cultivation time (days) on the growth parameters of different microalgae. Other investigators observed similar findings of increased lipid buildup in the static period in their review [44]. Scenedesmus quadricauda exhibited higher biomass yield, total carbohydrate and total chlorophyll contents at photoperiod cycles of 24L/0D and exhibited higher lipid and protein contents at photoperiod cycles of 18L/6D and 12L/12D, respectively. Temperature and light intensity, day length is the determinant factor on the microalgae biomass productivity in large-scale microalgae cultures [44] and these findings too, support our study. It was revealed in a study that the range of 625–675 nm (red) there was significant increase in cell volume along with occurrence of early division of nuclei in cells of Scenedesmus obliquus [45]. It is generally considered that algae showed a growth rate which is directly related to the duration of the light period and other researchers are also in agreement to this relationship [46].In a study, mixed effects of temperature (20 °C, 25 °C and 30 °C), nitrate concentration (0.5 mM and 2.0 mM), pH buffer, and bicarbonate addition (trigger) on biomass growth and lipid accumulation were investigated in the environmental alga PW95, classified as a Chlamydomonas-like species through morphological characterization and phylogenetic analysis (18S, ITS, rbcL). They revealed that without the use of a buffer or bicarbonate addition, the combination of higher temperature (30 °C) and lower nitrate level (0.5 mM) resulted in maximal daily biomass accumulation (5.30 106 cells/mL), high biofuel potential before and after nitrate depletion (27% and 20%, respectively), higher biofuel productivity (16 and 15 mg/L/d, respectively), and desirable fatty acid profiles (saturated and unsaturated C16 and C18 chains) [47].

According to a study, in order to boost biomass and algal growths, the media might be treated with nitrite, nitrate, and urea that has been proven to be the optimum nitrogen source [48] and these findings are in collaboration with our findings. Nitrogen is the most essential nutrient that affects both biomass yield and lipid production in many microalgae [30]. Effects of nitrogen on algal biomass and lipid accumulation have also been reported in many other algal strains like Scenedesmus dimorphous [49], Tetraselmis suecia, Skeletonema costatum and Thalassiosira pseudonana [50]. The rise in lipid accumulation could be attributed to algal cells' ability to develop under stress conditions, which enhances the growth of lipid content [51].

Our results of FTIR are in consonance with the findings of other scientists who selected four microalgae species viz. Scenedesmus spp., Chlorella spp., Rivularia spp. and Haematococcus spp. based on the potential of lipid compound and Chlorella, Scenedesmus & Jatropha FTIR transmittance spectra indicated the existence of hydroxyl, alkane, alkene, and carbonyl groups. They discovered bands at 3436 cm⁻¹ caused by O–H stretching vibrations in Scenedesmus spp. They discovered bands about 2681 cm⁻¹ that correspond to the symmetric C-H stretching vibration in Scenedesmus spp. Three unique bands were detected in the 1641, 1310, and 1000 cm⁻¹ regions, indicating an amount of esters within the processed samples [52]. Similar findings that are observed in the present study regarding SEM analysis were also observed by Ren and coworkers [53] who preliminary identified microalga as genus Scenedesmus on the basis of SEM. Recently, Nile Red was utilised in 2D fluorescence spectroscopy in conjunction with chemometric modelling to quantify the lipid content of the microalgae Scenedesmus spp. The method for lipid quantification utilizing anticipated linear models and 2D fluorescence showed to be robust and rapid when compared to the classic gravimetric method [54].

From these results it can be inferred that isolated and identified Scenedesmus quadricauda in the present study have a good potential for biodiesel production.

Conclusion

The present study suggests that the most effective optimization parameters to increase biomass yield and lipid accumulation along with enhanced fatty acid production is cultivation of Scenedesmus quadricauda in BG-11 medium, at a temperature of 25 °C, pH 7.0, glucose & sucrose (as carbon sources), static condition (for lipid accumulation) & shaking condition (for biomass yield), cultivation days of 18th, 21st and 24th day, NaNO₃ dosing of 1.0 mM followed by 0.8 mM, & that also on 5th day of cultivation, yeast extract dosing of 3%, light intensity of 3000 lx, photoperiod cycles of 24L/0D (for biomass yield) & 18L/6D (for lipid production) and 10 mM concentration of NaCl (salinity stress). It was also elucidated that Nile red fluorescence method can be used as a rapid method for determination of lipids in the native microalgae. Results of FTIR spectroscopy also indicated the presence of carbohydrates & lipids thereby making it as suitable strain for biodiesel production. Scanning electron micrographs revealed that cells of S. quadricauda under stress conditions became fragmented and the cells gets separated from coenobium and were not found compactly arranged. Our findings revealed this native microalga has biodiesel production capabilities.

Author Contributions

ASK and NRB conceptualized the research; ASK performed all the experiments and study; NRB helped in final analysis part and in the preparation of manuscript. The authors read and approved the final manuscript.

Declarations

Conflict of interest

We certify that we do not have any conflicting commercial objectives or personal ties that could appear to have affected the work disclosed in this paper.