Utilization of Scenedesmus obliquus biomass as feedstock for biodiesel and other industrially important co-products: An integrated paradigm for microalgal biorefinery

Algal Research 12 (2015) 328–336

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Utilization of Scenedesmus obliquus biomass as feedstock for biodiesel and other industrially important co-products: An integrated paradigm for microalgal biorefinery Reeza Patnaik, Nirupama Mallick ⁎ Agricultural and Food Engineering Department, Indian Institute of Technology Kharagpur, West Bengal 721302, India

a r t i c l e

i n f o

Article history: Received 13 May 2015 Received in revised form 4 September 2015 Accepted 11 September 2015 Available online xxxx Keywords: Algal refinery β-carotene Biodiesel Bioethanol Mixotrophy Scenedesmus obliquus

a b s t r a c t With an aim to design a microalgal biorefinery taking Scenedesmus obliquus as a model organism, a detailed sequential production protocol was developed for the first time for β-carotene, biodiesel, omega-3 fatty acids, glycerol and bioethanol from S. obliquus biomass. This research study not just projects S. obliquus as a feasible option for a microalgal biorefinery but also addresses the issue of economic and environmental sustainability by suggesting an optimized nutrient condition for maximizing benefits from the microalgal biomass. GC–MS technique has been used for the qualitative and quantitative analysis of the biodiesel obtained, and mass spectrophotometric technique has been used for quantitative analysis of the other co-products. The detailed process developed, yielded 0.06 g of β-carotene, 38 g of biodiesel, 2 g of omega-3 fatty acids, 3 g of glycerol and 17 g of ethanol from 100 g of S. obliquus biomass. A comparative analysis of the total light energy consumed and costs of nutrients incurred under autotrophic and mixotrophic modes has been presented. Additionally, more than twice the number of cultivation cycles per year, producing N 12 fold higher biodiesel yield and N4 fold higher yield of coproducts under optimized growth condition further highlights the significance of the strategy suggested. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Extensive research efforts are underway for expansion of the microalgal lipid production to a major industrial process. Though large scale microalgal production still remains a challenge, advances in the field of genetic engineering and bio-refining, present prospects to develop this process in an ecological and economical way in a few years [1]. Initiation of scale-up and commercialization of microalgal products on an industrial scale has been proposed by a few researchers in the recent past but a more careful step-by-step development has been cautiously stressed upon by few others [2]. There are nearly 300,000 species of microalgae providing vast opportunities for microalgal biofuel production but unfortunately only a few species are being currently used as biofuel feedstock today stressing the need for a detailed analysis of other potential microalgal biofuel feedstocks [3]. For a profitable microalgal industry, low cost production systems must be designed. Research studies have shown that feedstock cost accounts for a substantial portion of the total biodiesel cost [4]. Hence for a sustainable and economical microalgal industry procurement and production/cultivation of microalgae must be done at lower prices. Various nutritional factors and physical parameters are known to influence the growth and accumulation of bioactive compounds in ⁎ Corresponding author. E-mail address: [email protected] (N. Mallick).

http://dx.doi.org/10.1016/j.algal.2015.09.009 2211-9264/© 2015 Elsevier B.V. All rights reserved.

microalgae. Of the factors studied, nitrogen sources and their concentrations greatly affect the accumulation of various microalgal components. However, the microalgal growth compromised with under such conditions raises the cost of production with lower yields of the desired products from lower quantities of microalgal harvest [5]. In recent years, mixotrophy is evolving as an effective mode for cultivation and functional components accumulation in microalgae [6]. An earlier report from our laboratory [7] has shown that lipid accumulation in Scenedesmus obliquus could be boosted profoundly (40 fold) against control when previously grown in a medium supplemented with glucose followed by subjecting the biomass to optimized concentrations of nitrate, phosphate and sodium thiosulphate at the second phase. Optimizing the cultivation conditions maximizes the output thus making microalgal biofuels economically more competitive. Despite the high suitability of microalgae for biorefining owing to the diverse composition of their biomass, the co-production of valueadded products from microalgae is still a challenging issue [8]. Hence, mild extraction technologies that preserve the utility of the various cell components (like proteins, vitamins, lipids, carbohydrates, omega3 fatty acids) need to be explored [9]. The main purpose of this study is to ensure minimum cost of production and a positive energy balance through upgradation of nutrients and resources while maximizing the lipid yield from S. obliquus biomass and simultaneous valorization of the whole microalga through establishment of a biorefinery set-up. Therefore, this research study has focused

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on optimizing mixotrophic conditions for maximizing lipid yield for biodiesel production from Scenedesmus obliquus (Trup.) Kutz. (SAG 2763a) and on developing a detailed protocol for sequential production of β-carotene, biodiesel, omega-3 fatty acids, glycerol and bioethanol from the same biomass. A comparative analysis of the total light energy consumed and costs of nutrients incurred under autotrophic and mixotrophic conditions has also been presented.

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Experiment designing and data processing was done using Design Expert-software (version 7.1.1, Stat-Ease, Minneapolis, USA). Optimization was carried out using the Central Composite Rotary design (CCRD) to understand and analyze the effects of interaction of these four variables on the lipid content in the microalgal cells. Statistical analysis of the model was performed using analysis of variance (ANOVA). 2.5. Transesterification of lipids and analysis of biodiesel

2. Materials and methods 2.1. Experimental organism and growth conditions The green microalga, S. obliquus (Trup.) Kutz. (SAG 276-3a), preserved under sterile conditions, was cultured in 100 ml of N 11 medium contained in 250 ml Erlenmeyer flasks [10] without aeration. Light at an intensity of 75 μmol photon m− 2 s− 1 PAR was continuously supplied for a photoperiod of 14:10 h. The pH value was adjusted at 6.8 and temperature was set at 25 ± 2 °C. Shaking of the culture flasks was done, two to three times a day, to prevent sticking of the culture to the bottom of the flask. This was denoted as control culture. The effects of mixotrophy on biomass yield, and lipids and co-products accumulation in S. obliquus were scrutinized by supplementing various concentrations (0.04–2.0%) of dextrose, sodium salts of acetate, citrate and bicarbonate, glycerol and cysteine in N11 medium. The effects of interaction of the best concentrations of these carbon sources were also investigated. 2.2. Estimation of dry weight and specific growth rate Dry cell weight (dcw) was estimated as per the protocol of Rai et al. [11]. A noted volume of microalgal culture after centrifugation for 10 min at 5000 rpm was harvested and dried at 60 °C till a constant weight was reached at. The specific growth rate (μ) was calculated by using the following equation:

Transesterification of microalgal lipids was done using 82:4:1 molar ratio of methanol, hydrochloric acid and oil at 65 °C for 6.4 h reaction time [13], and was analyzed further by gas chromatography–mass spectrometry (GC–MS) for knowing the fatty acid methyl ester composition of the biodiesel sample. For analytical purposes, the GC-MS has a gas chromatograph (Autosystem XL) containing a PE-5 capillary column, composed of phenyl, methylpolysiloxane with the dimension 30 m × 0.25 mm × 0.25 μm and a Turbomass Gold Mass Spectrometer (Perkin- Elmer, Shelton, CT, USA). An internal standard was prepared using methylpentadecanoate. 2.6. Estimation of omega-3 fatty acids Omega-3 fatty acid content was estimated using a GC-MS following the procedure described above. 2.7. Extraction and estimation of β-carotene A noted volume of microalgal sample was centrifuged for 10 min at 5000 rpm, and the collected biomass was put in acetone. The mixture was kept overnight at 4 °C for complete extraction and was subsequently centrifuged for 5 min at 5000 rpm for separation of the extracted pigments. β-carotene analysis was done using thin layer chromatography [14], and was quantified spectrophotometrically by plotting a standard curve at an absorbance of 450 nm. 2.8. Estimation of carbohydrates and bioethanol production

ln ðn2 =n1 Þ μ¼ t2 −t1 where, n1 and n2 stand for the optical density (663 nm) of the culture suspension at the beginning (t1) and end (t2) of the selected time intervals. 2.3. Extraction and estimation of lipids Extraction and estimation of lipids was done according to the protocol suggested by Bligh and Dyer [12], as detailed in Mandal and Mallick [7].

The carbohydrate estimation was done by phenol-sulphuric acid method [15]. Acid hydrolysis of complex polysaccharides to simple sugars with 2 N concentrated sulphuric acid followed by fermentation with Saccharomyces cerevisae was carried out for bioethanol production from the dry biomass of S. obliquus. The conditions and concentrations of sulphuric acid for pre-treatment, and the fermentation process were previously standardized in our laboratory. Ethanol was separated from the fermented broth with Tri-n-butyl phosphate (TBP) and estimated by dichromate oxidation method following Seo et al. [16]. 2.9. Estimation of glycerol

2.4. Optimization of lipid accumulation Four variables, namely concentrations of acetate, citrate and nitrate, and incubation period (Table 1) that showed maximum influence on the lipid accumulation in S. obliquus were selected through the screening experiments conducted under various mixotrophic conditions. Table 1 Variables and levels of experimental design for response surface. Independent variable

Acetate (%) Citrate (%) Nitrate (g/L) Incubation period (days)

Coded symbol

Level −2 (−α)

−1

0

1

2 (+α)

A B C D

0.11 0.11 –0.425 0.5

0.14 0.14 0.05 7

0.17 0.17 0.525 13.5

0.2 0.2 1 20

0.23 0.23 1.475 26.5

Glycerol, obtained as a by-product of the transesterified product of S. obliquus lipids, was purified after separation from the biodiesel following the protocol of Xiao et al. [17]. For quantification of glycerol, the sample was treated with sodium metaperiodate and acetyl acetone. The yellow compound produced, exhibited a maximum absorbance peak at 410 nm. Glycerol concentration was calculated with the help of a standard curve. 2.10. Statistical analysis All the experiments were conducted in triplicate independent cultures to confirm their reproducibility. A window-based software MSTAT-C was used to perform Duncan's new multiple range test to analyze the difference between the yields of various products in control and selected treatments.

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3. Results and discussion 3.1. Effects of various carbon sources on biomass yield and lipid accumulation As observed in the growth pattern of S. obliquus under supplementations of various concentrations (0.04–2.0%) of carbon sources in N11 medium, biomass yield was enhanced by N2 fold under dextrose, acetate, citrate, bicarbonate and acetate + citrate supplementations for 14 days of incubation (Fig. 1A) as a consequence of higher specific growth rates (Table 2) under such conditions. This finds support from the reports published by Wang et al. [18] and Kong et al. [19] in which addition of organic carbon sources significantly influenced the growth of microalgae. The high rates of respiration under glucosesupplemented conditions [20], oxidation of acetate to form malate in glyoxysomes and citrate in mitochondria to provide carbon skeletons for biosynthesis under acetate and citrate available conditions [21], and the compensation of photorespiratory loss by active uptake of

bicarbonate [22] could be possible reasons for such higher biomass yields under the above conditions. Mixotrophic nutrition for increasing lipid accumulation in microalgae has been demonstrated by Kong et al. [19] and Hamed and Klock [23]. In this research article, supplementation of glycerol, acetate, citrate and cysteine was found to increase the lipid content in the S. obliquus cells, but the total lipid yield increased significantly in dextrose-, glycerol-, bicarbonate-, acetate- and citrate- supplemented cultures (Fig. 1B). The lipid yield being a product of the biomass yield and lipid content of the microalga, a rise or fall in either of the two or both the factors influences the yield of the extracted lipid eventually influencing the total lipid production rate. An observation of the dextrose-supplemented culture significantly validates this fact as the lipid yield rises by a significant value in comparison to the control culture due to increase in biomass yield despite a low value of the lipid accumulated in the microalgal cells. Contrary to this, in case of cysteine-, glycerol + acetate- and glycerol + citrate-supplemented cultures, a rise in lipid content could not contribute to a

Fig. 1. A Representation of maximum biomass yield of S. obliquus under individual and interactive carbon supplemented conditions. Values are mean ± SE of the data obtained from experiments carried out by using three independent cultures. Values inside the histograms show the concentrations of the carbon doses for maximum response. B Maximum lipid accumulation potential of S. obliquus under individual and interactive carbon- supplemented conditions. Values are mean ± SE of the data obtained from experiments carried out by using three independent cultures. Values inside the histograms show the concentrations of the carbon doses for maximum response.

R. Patnaik, N. Mallick / Algal Research 12 (2015) 328–336 Table 2 Specific growth rates of S. obliquus under different culture conditions. Culture condition

Specific growth rate (μ/day)

Control Dextrose (1.6%) Glycerol (1.6%) Acetate (0.3%) Citrate (0.3%) Bicarbonate (0.16%) Cysteine (0.04%) Bicarbonate + Acetate (0.16 + 0.3%) Acetate + Bicarbonate (0.16 + 0.08%) Bicarbonate + Citrate (0.16 + 0.3%) Citrate + Bicarbonate (0.16 + 0.08%) Glycerol +Acetate (0.16 + 1.2%) Acetate + Glycerol (0.6 + 0.3%) Glycerol + Citrate (0.16 + 1.6%) Citrate + Glycerol (0.6 + 0.16%) Acetate + Citrate (0.16 + 0.16%) Citrate + Acetate (0.16 + 0.16%)

0.26 ± 0.03 0.41 ± 0.06 0.39 ± 0.01 0.45 ± 0.05 0.44 ± 0.03 0.40 ± 0.02 0.28 ± 0.04 0.35 ± 0.01 0.39 ± 0.07 0.34 ± 0.05 0.39 ± 0.02 0.33 ± 0.06 0.30 ± 0.05 0.30 ± 0.04 0.24 ± 0.02 0.38 ± 0.07 0.36 ± 0.04

Values are mean ± SE of the data obtained from experiments carried out by using three independent cultures.

significant rise in the lipid yield of the microalga due to its retarded growth under such conditions. However, in case of acetate + citrate-supplemented vessels, a rise in both the biomass yield and lipid content by N2 fold, significantly raised the lipid yield by 4.5 fold in comparison to the control culture. Although the role of acetate and citrate in influencing lipid accumulation in microalgae was significantly demonstrated, a contradiction to the present study was found in Chlorella protothecoides, where acetate did not exert a positive impact on the deposition of lipids [24]. Similarly, Estévez-landazábal et al. [25] demonstrated that the uptake of acetate for stimulation of lipid accumulation required high concentrations of nitrate, whereas in this study, acetate assimilation under normal concentrations of nitrate was clearly evident. A balanced ratio of carbon to nitrogen in culture media for promoting lipid accumulation in the microalgal cells [26] possibly explains our findings. Nevertheless, species-specific dependence of microalgal lipid accumulation on the concentrations and interaction of carbon sources used cannot be ignored [27].

3.2. Optimization of lipid accumulation Considering the role of nitrate [7], and the contribution of acetate and citrate (Fig. 1B) towards accumulation of lipids in the green microalga, S. obliquus, optimization experiments using RSM to maximize the lipid accumulation in the microalgal cells were conducted in the presence of acetate, citrate and nitrate as a function of incubation period (Table 1). In recent years, RSM has evolved as a simple and reliable methodology for finding solutions to complex parameter design problems. The results of the central composite rotary design (CCRD) for analyzing the effects of interaction of the four critical variables (concentrations of acetate, citrate, nitrate, and incubation period) on lipid accumulation in S. obliquus have been presented in Table 3. The range of the experimental values for lipid accumulation under different combinations of the factors, has shown a variation between 22.6 and 56.2% (dcw) while the results predicted by the model are in the range of 23.4–59.8% (dcw). The results of the F-test demonstrated a high significance of the chosen variables in yielding the response which was confirmed by the low probability value of the F-test (data not shown). Additionally, the tested model was found to have a good fit which was proven by the value of coefficient of determination (R2). The R2 value nearer to 1 (0.941), validated the accuracy of the model.

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Table 3 Central Composite Rotary Design matrix with actual and predicted response for lipid accumulation. Variables

Response

Run

A

B

C

D

Actual

Predicted

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

0.17 0.23 0.14 0.2 0.11 0.14 0.17 0.14 0.2 0.14 0.17 0.17 0.14 0.17 0.17 0.2 0.14 0.2 0.2 0.17 0.14 0.17 0.17 0.2 0.2 0.14 0.17 0.2 0.17 0.17

0.17 0.17 0.14 0.2 0.17 0.2 0.17 0.2 0.14 0.14 0.17 0.17 0.2 0.17 0.17 0.2 0.14 0.14 0.14 0.17 0.14 0.17 0.11 0.2 0.2 0.2 0.17 0.14 0.23 0.17

0.53 0.53 0.05 1 0.53 1 0.53 1 1 1 0.53 0.53 0.05 0.53 –0.42 (0) 1 1 0.05 1 0.53 0.05 0.53 0.53 0.05 0.05 0.05 0.53 0.05 0.53 1.48

13.5 13.5 7 7 13.5 7 0.5 20 20 20 13.5 13.5 7 13.5 13.5 20 7 7 7 13.5 20 26.5 13.5 7 20 20 13.5 20 13.5 13.5

55.2 45.9 49.4 35.9 46.2 37.6 24.3 33.6 34.1 30.9 55.8 54.7 43.1 54.7 36.2 34.2 33.4 43.7 35.7 54.9 27.2 42.8 48.7 42.7 27.9 22.6 54.9 24.5 49.3 33.2

59.8 47.7 54.2 33.6 49.8 37.9 25.7 33.9 34.7 32.2 54.3 56.9 49.3 52.9 56.4 33.7 38.9 47.7 37.5 55.6 27.5 43.1 48.4 42.9 29.2 23.4 55.6 24.4 49.3 33.1

The application of response surface methodology generated the resulting regression equation in which the two-factor model terms, AD, BD and CD, which did not significantly fit the model (P N 0.05) were omitted. An experimental relationship between the response (lipid content) and the variables in coded units has been drawn in this equation: Y ðlipid contentÞ ¼ þ29:05–0:29A−0:05B−2:46C−5:66D þ 0:95AB þ 0:76 AC þ0:48BC−2:42A2 −1:90B2 −2:52C2 −3:68D2

Y denotes the lipid content expressed in percent dry cell weight, and A, B, C and D denote the test variables (concentrations of acetate, citrate and nitrate, and days of incubation, respectively). The significance of the individual variables was ascertained by student's t-test and their corresponding P values (data not shown). All the model terms, linear (A, B, C and D), quadratic (A2, B2, C2 and D2) and two-factor (AB, AC and BC) were found to fit well at P b 0.05. The residual values of the individual responses did not demonstrate much variation between the observed and fitted values of the individual responses, indicating excellent adequacy of the regression model. Response surface plots are a diagrammatic representation of the main and the interactive effects of any two variables at a time, keeping the other variables fixed at a single level (for eg. at zero level). These response surface plots facilitate an easier interpretation of the results through a three dimensional view (Fig. 2). The highest point within the space of the response surface diagram indicates the maximum predicted value of the response i.e. lipid content. As shown in Fig. 2A, lipid accumulation in S. obliquus increases with increasing concentrations of both acetate and citrate. Similarly, Fig. 2B and Fig. 2C also demonstrate higher lipid accumulation under increased concentrations of acetate and citrate respectively, at fixed concentrations of the other two respective variables. As demonstrated in Fig. 2B and Fig. 2C, increased nitrate concentrations have not been found to have a

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Fig. 2. 3D Response surface graphs showing the interactive effects of the critical variables on lipid accumulation potential of S. obliquus.

positive influence on the lipid content of the microalgal cells. Concentric contour circles towards the middle of the response surface plot in Fig. 2A indicated requirement of equivalent proportions of acetate and citrate for rise in lipid accumulation in S. obliquus, whereas elongated contours along the ‘x’ axis in Fig. 2B, C demonstrated a correspondence towards higher concentrations of acetate (Fig. 2B) and citrate (Fig. 2C) against comparatively lower concentrations of nitrate for obtaining the optimum response. The optima in the three response surface plots were found within the space of the boundary suggesting selection of an ideal range of variables for optimization of lipid accumulation in S. obliquus. The optimal values of the four variables were found to be 0.17% of acetate, 0.17% of citrate, 0.4 g/L of nitrate, and an incubation period of 9 days for accumulation of 56.4% of lipid against a predicted value of 59.8% (dcw). Lipid accumulation up to 56.4% (dcw), 4.5 fold against control, was observed (Table 4).

3.3. Characterization of the transesterified product (biodiesel) of S. obliquus lipids Fatty acid profile of the biodiesel obtained from S. obliquus grown in acetate- and citrate-supplemented cultures, showed an increase in palmitic acid methyl ester content up to 46.8% under acetate supplementation, while that under the control condition was 40.1% (Table 5). Citrate supplementation however, brought a rise in the oleic acid content. Linoleic acid content showed a marginal rise when acetate and citrate were supplemented simultaneously. In contrast, linolenic acid content showed a decline under acetate and citrate supplementations indicating increasing oxidative stability of the transesterified product. The culture conditions optimized for enhanced lipid accumulation also showed a significant rise in the saturated + mono-unsaturated fatty acid methyl ester content of the biodiesel obtained. The biodiesel

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333

opti-

Table 4 Lipid content of S. obliquus before and after optimization. Variable

Before optimization

After optimization

Lipid content (% dcw) Before optimization

Acetate (%) Citrate (%) Nitrate (g/L) Incubation period (days)

0 0 1.0 21

0.17 0.17 0.4 9

12.5 ± 0.81

After optimization Predicted

Experimental

59.8

56.4 ± 0.55

Table 5 Relative % of fatty acid methyl esters (FAMEs) in S. obliquus biodiesel under selected mixotrophic and optimized conditions.

Control Acetate (0.16%) Citrate (0.16%) Acetate (0.16%) + Citrate (0.16%) Optimized condition

Biodiesel yield (%weight of oil)

Palmitic acid (% FAME)

Stearic acid (% FAME)

Oleic acid (% FAME)

Linoleic acid (% FAME)

Linolenic acid (% FAME)

Omega-3 fatty acids (% FAME) EPA

DHA

65.7 ± 2.5a 66.4 ± 2.6b 67.2 ± 1.4c 67.7 ± 2.5d 68.3 ± 1.6d

40.1 ± 0.8a 46.8 ± 1.1b 35.8 ± 1.2c 40.1 ± 0.7a 45.3 ± 0.4b

Nil Nil 5.2 ± 0.2b 9.9 ± 0.5a 8.8 ± 0.4a

34.9 ± 0.8a 31.1 ± 0.2a 38.8 ± 0.3b 30.4 ± 0.6a 27.1 ± 0.7a

14.1 ± 0.2a 13.1 ± 0.5a 13.1 ± 0.3a 17.0 ± 0.8b 15.1 ± 0.2a

10.9 ± 0.2b 6.6 ± 0.6a 7.1 ± 0.2a 0.2 ± 0.4c 1.2 ± 0.1d

Nil 0.9a Nil 1.3a 2.2b

Nil 1.5a Nil 1.1a 0.3b

EPA: Eicosapentanoic acid, DHA: Docosahexanoic acid. Peak areas with less than 0.1% were considered to be negligible. Values are mean ± SE of the data obtained from experiments carried out by using three independent cultures. Values within a column followed by different letters are significantly different from each other (P b 0.05; Duncan's new multiple-range tests). A separate analysis was done for each column.

yield relative to the weight of the lipids was 65–68%. The presence of higher proportions of mono-unsaturated methyl oleates and saturated methyl palmitate has been known to improve the oxidative stability in biodiesel [28]. The European biodiesel standard EN 14214 [28] suggests lower quantities of linolenic acid, below 12% of the total FAME, as ideal for a good quality biodiesel. 3.4. Accumulation of various co-products in S. obliquus 3.4.1. Omega-3 fatty acids production As shown in Table 5, the presence of pharmaceutically and nutraceutically important PUFAs, i.e. omega-3 fatty acids in the acetate-, acetate + citrate- supplemented and the optimized conditions were observed. The omega-3 fatty acids produced under acetate- and acetate + citrate-supplemented conditions contained comparable proportions of eicosapentanoic acid (EPA) and docosahexanoic acid (DHA), while the relative percentage of EPA was higher under the optimized condition. EPA content in S. obliquus was 0.9 and 1.25% in the acetateand acetate + citrate-supplemented conditions, respectively, while the percentage of DHA under similar growth conditions was 1.5 and 1.05%, respectively. Biodiesel sample from S. obliquus under the optimized condition contained 2.2% EPA and only 0.3% DHA. The role of acetate in influencing the presence of omega-3 fatty acids in microalgae [29], explains the reported findings. However, the fatty acid composition did not show much variation in the tested organic carbon sources [18]. 3.4.2. β-carotene production The effects of selected mixotrophic and the optimized conditions on the β-carotene production potential of S. obliquus were investigated. Though cellular β-carotene accumulation did not show any significant rise under exogenous carbon supplementations, more than 3 fold rise in the yield was recorded in acetate- and citrate-supplemented cultures (Fig. 3A). Similarly, a 2 fold rise in total carotenoids was observed in the acetate- and citrate-supplemented cultures although the cellular carotenoid accumulation increased marginally against control (Fig. 3B). The

mized condition also showed a 2 fold rise in the β-carotene yield against control, but the total carotenoid accumulation depicted a declining trend. Thus the increase in the β-carotene yield could be attributed to the rise in biomass yield under the mixotrophic conditions. A positive correlation (r = 0.96) between the biomass and β-carotene yield in S. obliquus further supports the above explanation.

3.4.3. Bioethanol production Individual supplementation of acetate, and the interactive supplementation of acetate + citrate were found to raise carbohydrate content N50% dcw on the 14th day of incubation, while a carbohydrate content of 44% (dcw) was recorded in the citrate-supplemented condition (Table 6). N11 medium with the optimized concentrations of acetate, citrate and nitrate accumulated carbohydrate only up to 30% (dcw) against 22% control. Under carbon available conditions, higher quantities of 3-phosphoglycerate, the substrate for carbohydrate synthesis are formed due to enhanced carboxylase activity of the enzyme Rubisco. An analogy in this regard can be found in report by Abreu et al. [30] in which mixotrophic nutrition for accumulation of high amounts of carbohydrates has been substantially justified. However, a reduction in carbohydrate content under the optimized condition demonstrated that nitrate concentration in the medium could also regulate the carbohydrate accumulation in the test microalga. Carbohydrates like starch and cellulose in chloroplasts and cell walls respectively [31], are not readily fermentable for ethanol production by microorganisms. Hence, hydrolysis of the complex polysaccharides is recommended. In this research report, the acid pre-treatment of S. obliquus cells with 2 N sulphuric acid before being subjected to fermentation with Saccharomyces cerevisae has been demonstrated, which finds support from the findings of Markou et al. [32], where a concentration lower than 5 N sulphuric acid for pre-treatment of microalgal cells has been suggested. Maximum bioethanol yield of ~ 20 g was obtained from 100 g of dry biomass under the selected mixotrophic and the optimized conditions against 10 g with N 11 grown biomass (control) (Table 6).

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Fig. 3. A β-carotene content/yield (mg/g and mg/L) of S. obliquus under control and selected carbon supplemented conditions. Values are mean ± SE of the data obtained from experiments carried out by using three independent cultures. The values inside the histograms denote the concentrations of the carbon doses for maximum response. B Total carotenoid content/yield (mg/g and mg/L) of S. obliquus under control and selected carbon supplemented conditions. Values are mean ± SE of the data obtained from experiments carried out by using three independent cultures. The values inside the histograms denote the concentrations of the carbon doses for maximum response.

3.4.4. Glycerol production Glycerol produced during glycerolipid metabolism in microalgae can be recovered through purification during the transesterification of the microalgal oil to biodiesel [33]. The glycerol content in the above selected samples was analyzed and expressed on the basis of percent weight of biodiesel as well as percent of dry cell weight. The amount of glycerol obtained in this study, through purification during biodiesel conversion was ~8% of the weight of the biodiesel in accordance with the reports of Crooks [34] and OECD/FAO [35], while the amount of glycerol contained in the S. obliquus biomass was ~3% per cell dry weight (data not shown) under the selected mixotrophic conditions. Comparatively, a lower quantity of glycerol could be recovered during transesterification of the biodiesel sample obtained under the control condition (Table 7). Varieties of impurities are known to be contained in the crude glycerol obtained from the transesterified microalgal oil, lowering the recovered purified yield. Purification of this crude glycerol through saponification

releases the combined glycerol from the glycerides thus enhancing the quantity of the glycerol produced [17]. The purification protocol of Xiao et al. [17] was successfully applied in this study. Table 7 Glycerol production as a by-product of transesterification from the selected samples. Culture condition

Glycerol content (% weight of biodiesel)

Glycerol yield (g/L)

Control Acetate (0.16%) Citrate (0.16%) Acetate (0.16%) + Citrate (0.16%) Optimized condition

6.3 ± 0.36a 8.4 ± 0.22b 7.9 ± 0.15c 8.2 ± 0.27c 8.5 ± 0.42b

0.02 ± 0.003c 0.18 ± 0.02a 0.17 ± 0.03a 0.11 ± 0.01b 0.06 ± 0.002a

Values are mean ± SE of the data obtained from experiments carried out by using three independent cultures. Values within a column followed by different letters are significantly different from each other (P b 0.05; Duncan's new multiple-range tests). A separate analysis was done for each column.

Table 6 Maximum biomass yield, carbohydrate content and bioethanol yield in S. obliquus under the selected mixotrophic and the optimized conditions. Culture condition

Biomass yield (g/L)

Carbohydrate yield (g/L)

Carbohydrate content (% dcw)

Bioethanol yield (g/L)

Bioethanol content (% dcw)

Control Acetate (0.16%) Citrate (0.16%) Acetate (0.16%) + Citrate (0.16%) Optimized condition

1.35 ± 0.04a 3.13 ± 0.29b 3.14 ± 0.24b 2.31 ± 0.23c 1.92 ± 0.08d

0.29 ± 0.02c 1.62 ± 0.21a 1.38 ± 0.19b 1.27 ± 0.24a 0.58 ± 0.09d

22. 2 ± 1.34a 52.1 ± 4.37b 44.2 ± 2.49b 55.1 ± 5.53c 30.2 ± 1.79d

0.13 ± 0.01a 0.68 ± 0.05b 0.62 ± 0.03c 0.53 ± 0.03d 0.34 ± 0.05c

10 ± 1.15b 22 ± 2.20a 20 ± 1.17a 23 ± 1.24a 18 ± 1.19c

Values are mean ± SE of the data obtained from experiments carried out by using three independent cultures. Values within a column followed by different letters are significantly different from each other (P b 0.05; Duncan's new multiple-range tests). A separate analysis was done for each column.

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335

Fig. 4. Schematic representation of a detailed process for sequential production of biodiesel and other industrially valuable co-products from S. obliquus biomass.

3.5. Microalgal refinery Microalgae today seem to be the most potential renewable substitute for biodiesel production. To ensure the economic and environmental feasibility and sustainability of this energy resource, valorization of the microalgal biomass and a systematic evaluation of the environmental aspects through interpretation of the results is essential. This study considerably advocates an optimized nutrient condition for the sequential production of biodiesel along with industrially valuable co-products from S. obliquus biomass (Fig. 4) and clearly defines and describes the main objective of this research work thus identifying the boundaries and environmental effects to be reviewed for life cycle impact assessment in the future. The harvested wet biomass of S. obliquus was treated with acetone for β-carotene extraction, and then dried at 60 °C followed

by lipid extraction using a binary solvent system (chloroform-methanol). The de-fatted biomass was then subjected to acid hydrolysis for the breakdown of the complex polysaccharides to simple monomers for bioethanol production through fermentation by S. cerevisae. 100 g of S. obliquus biomass under the optimized condition yielded 0.06 g of β-carotene, 38 g of biodiesel, 2 g of omega-3 fatty acid, 3 g of glycerol and 17 g of bioethanol. The optimized mixotrophic culture condition suggested in this research paper is a more preferrable option not just for the higher yield of products but also for reducing the production cost as is well illustrated in Table 8. When S. obliquus was grown under the optimized conditions with 4 times higher monetary investment in nutrients, a N 6 fold rise was observed in the biodiesel yield in comparison to the normal autotrophic condition, and a ≥ 2 fold rise was observed for β-carotene,

Table 8 A comparative analysis of the total light energy consumed and costs of nutrients incurred under autotrophic and mixotrophic culture conditions. Culture condition

Incubation period (days)

Biomass yield (g/L)

β-carotene yield (mg/L)

Biodiesel yield (g/L)

Omega-3 fatty acid yield (g/L)

Glycerol yield (g/L)

Bioethanol yield (g/L)

Total light energy consumption (KWh/m2)

Cost of nutrients incurred (Rs/100 L)

Autotrophy (control) Mixotrophy (optimized)

21

1.35

0.58

0.11

Nil

0.02

0.13

9.71

9

1.92

1.22

0.73

0.02

0.06

0.34

4.16

113.00 (1.83)⁎ 431.00 (6.98)⁎

⁎ Cost of nutrients incurred in USD.

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omega-3 fatty acids, glycerol and bioethanol in just 9 days of incubation period, thus reducing the light energy consumption to more than half the energy consumed for autotrophic growth of the same microalga. Additionally more than twice the number of cultivation cycles per year, leaving 30 days for cleaning and maintenance of the cultivation system with N 12 fold increase in biodiesel yield and N4 fold increase in βcarotene, omega-3 fatty acids, glycerol and bioethanol production under optimized growth condition further highlights the novelty and significance of the strategy suggested. 4. Conclusion From the results of this research study, it can be confidently concluded that the green microalga, Scenedesmus obliquus (Trup.) Kutz. (SAG 276-3a) has in it sufficient accumulation of lipid and other important functional components which can be further boosted in the presence of only 0.17% of acetate and citrate without compromising with the biomass yield, thus making it a potential biofuel feedstock. Furthermore, mixotrophy as a mode of nutrition should be preferred over autotrophy not just as a significant stimulant of lipid accumulation in S. obliquus but also as a more sustainable and economically viable process for industrial production of biofuel from microalgal feedstock as is clearly shown in this research paper. Additionally recycling of solvents used during the production process can be encouraged making this approach a more economic and environmentally sustainable option. Hence, the detailed protocol for sequential production of industrially important products described for the first time in this research study can be categorically considered for application in a microalgal biorefinery. Acknowledgments Financial support from NFBSFARA, the Indian Council of Agricultural Research, New Delhi, India, is highly acknowledged. The authors are also thankful to Mr. Mahesh Kumar and Mr. Bhanendra Singh for their kind help during standardization of protocols for β-carotene and bioethanol, respectively, and to Mr. Sourav Kumar Bagchi for his useful suggestions during the preparation of this manuscript. References [1] P.J.l.B. Williams, L.M.L. Laurens, Microalgae as biodiesel and biomass feedstocks: Review and analysis of the biochemistry, energetic and economics, Energy Environ. Sci. 3 (2010) 554–590. [2] V. Budarin, A.B. Ross, P. Biller, R. Riley, J.H. Clark, J.M. Jones, D.J. Gilmour, W. Zimmerman, Microalgae biorefinery concept based on hydrothermal microwave pyrolysis, Green Chem. 14 (2012) 3251–3254. [3] Y.C. Sharma, B. Singh, J. Korstad, A critical review on recent methods used for economically viable and eco-friendly development of microalgae as a potential feedstock for synthesis of biodiesel, Green Chem. 13 (2011) 2993–3006. [4] T.M. Mata, A.A. Martinsa, N.S. Caetano, Microalgae for biodiesel production and other applications: a review, Renew. Sust. Energ. Rev. 14 (2010) 217–232. [5] R.P. John, G.S. Anisha, K.M. Nampoothiri, A. Pandey, Micro and macroalgal biomass: a renewable source for bioethanol, Bioresour. Technol. 102 (2011) 186–193. [6] R.H. Wijffels, M.J. Barbosa, An outlook on microalgal biofuels, Science 329 (2010) 796–799. [7] S. Mandal, N. Mallick, Microalga Scenedesmus obliquus as a potential source for biodiesel production, Appl. Microbiol. Biotechnol. 84 (2009) 281–291. [8] Y. Chisti, Biodiesel from microalgae beats bioethanol, Trends Biotechnol. 26 (2008) 126–131. [9] L. Wang, J.G. Wang, J. Littlewood, H.B. Cheng, Co-production of biorefinery products from Kraft paper sludge and agricultural residues: opportunities and challenges, Green Chem. 16 (2014) 1527–1533.

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