Outdoor cultivation of Chlorella pyrenoidosa in paddy-soaked wastewater and a feasibility study on biodiesel production from wet algal biomass through in-situ transesterification

Biomass and Bioenergy 143 (2020) 105853

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Outdoor cultivation of Chlorella pyrenoidosa in paddy-soaked wastewater and a feasibility study on biodiesel production from wet algal biomass through in-situ transesterification J. Umamaheswari a, M.S. Kavitha b, S. Shanthakumar a, * a b

Department of Environmental and Water Resources Engineering, School of Civil Engineering, Vellore Institute of Technology (VIT), Vellore, 632014, India CO2 Research and Green Technologies Center, Vellore Institute of Technology (VIT), Vellore, 632014, India

A R T I C L E I N F O

A B S T R A C T

Keywords: Phycoremediation Rice mill wastewater Bio-diesel production Chlorella pyrenoidosa Algal technology Single-step transesterification

Sustainable resources management, incorporating energy markets and resources such as electricity, fossil fuels, renewable and sustainable energy capital is essential for society to understand production and conversion of various forms of energy, their current as well as future supply. Waste-to-energy (WTE) or energy-from-waste (EFW) is a well-identified transitional technology which could prevent complete depletion of renewable re­ sources. In our present study, the selected microalgae (Chlorella pyrenoidosa) was cultured in paddy-soaked wastewater (PWW) using outdoor raceway ponds of 50 L capacity where biotransformation of nutrients (NH3–N removal: 75.89 ± 0.69%; PO4–P removal: 73.71 ± 0.75%; yield co-efficient YN: 6.12 mg biomass/mg of N; YP: 7.77 mg biomass/mg P) has occurred with better growth and biochemical composition (dry biomass weight: 1.56 ± 0.11 g/L; chlorophyll: 15.57 ± 0.14 mg/L; specific growth rate (SGR): 0.42/d; lipids: 27.47 ± 1.41% biomass; carbohydrates: 23.77 ± 1.00% and protein: 46.12 ± 3.55%). Further, the obtained algal lipid was identified for a wide range of fatty acid methyl esters (FAME) and consequently brought forward to in-situ single-step transesterification by optimizing reaction conditions. Central composite design (CCD) of response surface methodology (RSM) has given optimized conditions of sample amount: 2 g (wet); methanol sulphuric acid volume: 3 mL; and hexane volume: 4 mL, under the reaction temperature of 90 ◦ C for maximum biodiesel conversion (46.54% of algal lipids). The outcome of our current research may add value to the application and development of WTE technology for sustainable energy conservation.

1. Introduction Globally almost 500 million metric tonnes (MMT) of paddy is pro­ cessed to meet rice per capita demand of 53.7 kg/year [1]. United states department of agriculture (USDA) estimated that 70.41% of South Asia’s total rice production is from India (2019–2020) which is nearly one-fourth of global rice production [2]. Rice milling industries are the oldest agro-processing industries with gross revenue of more than ₹255 billion per annum (based on 2016 report). These massive industries process more than 100 million tonnes per year (118 MMT for 2019–2020) and supply rice for greater than 60% of the population in India. The primary source of wastewater from these industries is from paddy soaking as it requires a water quantity of 30% more than of its weight in addition to water requirement for initial washing and cleaning of paddy. Hence, a considerable amount of wastewater was released to

nearby land and water bodies. Due to less knowledge and unawareness of the treatment system among small and medium scale industrialists, those initiate nutrient pollution (pollution due to excess nutrients) [3–5]. Moreover, fermentation of organic load, phenols and natural sugars from a starch granule of rice, phytic acid from rice bran, and leaching of ammonia from paddy during prolonged soaking resulted in the high level of BOD, COD, ammoniacal nitrogen and phosphates in wastewater thus can be utilized as a source of nutrients for algal growth [6]. Phycoremediation is an efficient and economic bioremediation technique, widely adopted in various industrial wastewater treatment, promotes extraction of value-added products and thereby supports sustainable wastewater management [7–10]. Several researchers investigated use of microalgae to remediate the wastewater [7,11–13]. Nutrients or pollutants present in wastewater can be utilized by

* Corresponding author. E-mail address: [email protected] (S. Shanthakumar). https://doi.org/10.1016/j.biombioe.2020.105853 Received 25 May 2020; Received in revised form 22 October 2020; Accepted 26 October 2020 Available online 3 November 2020 0961-9534/© 2020 Elsevier Ltd. All rights reserved.

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Fig. 1. (a) Experimental Setup - Outdoor culture; (b) Raw PWW (c) PWW after Inoculation with Chlorella pyrenoidosa at Initial Day of Culture.

catalyst lead to experimental accidents and health risks and, hence, optimization assists in the utilization of required quantities (solvents, catalyst, etc.) for maximizing biodiesel yield. Most of the algal biomass used for in-situ transesterification was cultured under controlled con­ ditions in a standard growth medium such as tris-acetate-phosphate medium (TAP) and walne’s medium etc [26,28,29]. Hence, our present research builds on previously available literature on phycoremediation of nutrient-rich wastewater and applying the concept of sustainable waste management followed by an eco-friendly production of biodiesel, representing the novel data on (i) growth per­ formance of selected microalgae, Chlorella pyrenoidosa under the out­ door condition with PWW as growth medium by using raceway ponds of 50 L working volume, (ii) assessment of treatment efficiency based on NH3–N and PO4–P removal in the adopted system, (iii) analysis of biochemical and fatty acid methyl ester (FAME) properties of obtained algal biomass, and (iv) development of effective biodiesel production method from wet algal biomass using single-step in-situ trans­ esterification by employing central composite design (CCD) of response surface methodology (RSM) for optimization of reaction conditions.

microalgae for their growth metabolism and, hence, biotransformation assists in the accumulation of high lipids in its cells [14,15]. Wastewater-grown algae with significant algal lipid productivity and fatty acid methyl ester (FAME) compounds, further identified algal species as a source of biofuel or biodiesel [16–20]. Biodiesel, an alternative renewable fuel, found to be a replacement of conventional or fossil diesel. Almost half a century back, i.e. during the energy crisis of the 1970s, green technology was started focussing more on bioenergy potential of algae. Nowadays, algal oil derivative was recognized as third-generation biofuel. It holds many advantages over other bio feedstocks, such as plant-derived (first generation) and vege­ table or animal waste-derived (second generation) biofuels. Usually, application of food crops like corn, sugarcane and vegetable oil was used to produce biodiesel and bioethanol. Usage of food crops for fuel gen­ eration created a struggle towards food demand and increased the food crop price. This paved an approach for second-generation fuel which utilizes nonedible oil, waste cooking oil and other lignocellulosic waste for biofuel production. Utilization of second-generation feedstock overcame the difficulties faced by first-generation feedstock, still, feedstock availability was a great challenge to biofuel plant. Thus, thirdgeneration biofuel production from algae was showing more significant promises towards fuel generation [21,22]. Furthermore, algae have the potential of highest photosynthetic efficiency and strong ability to withstand against any sort of surrounding environment [23]. Most of Chlorella species were identified for biofuel production in addition to other algal species such as Nostoc sp. Scenedesmus and Spir­ ulina sp [23]. High natural lipids (as triacylglycerols) in algae promotes biofuel properties [24,25]. Chlorella sp. MJ 11/11 yields about 24.6% (w/w) of lipids with a high composition of free fatty acids, triglycerides and esters [26]. Further, single-step transesterification, by adding algal lipids together with solvent (methanol), catalyst under specific tem­ perature and reaction time, provided the highest biodiesel conversion (~95%) [27]. In general, conventional transesterification was performed in two steps, (i) triacylglycerides (TAG) were extracted from algal lipids by solvent extraction method and (ii) further TAG were converted to fatty acid methyl esters (FAME) in the presence of solvents (mono hydroxy alcohol) and catalysts. Still, storage and handling of excess solvents,

2. Materials and methods 2.1. Phycoremediation of PWW 2.1.1. The algal strain used in the study Chlorella pyrenoidosa, freshwater green algae were used in the study. C. pyrenoidosa was procured from National Collection of Industrial Mi­ croorganisms (NCIM), National Chemical Laboratory (NCL), Pune, India and initially cultured in standard blue-green (BG11) medium [30,31]. After the growth period of seven days, centrifuged (Research centrifuge: REMI R24; 5000 rpm for 10 min) biomass was used on a percentage basis (v/v) and 30% inoculum (30 mL of algal culture per 100 mL of wastewater) with 3.1 × 106 cells/mL cell density was utilized for out­ door culture in raceway ponds. Raceway pond cultured microalgae were collected further by centrifugation (5000 rpm for 10 min) and harvested wet algal biomass was used for single-step in-situ transesterification process.

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Table 1 Paddy-soaked Wastewater (PWW) Characteristics before and after Outdoor Culturing of Chlorella pyrenoidosa and their percentage removal. Description

Before treatment

After treatment

% reduction

Remarks

pH Turbidity (NTU) Total alkalinity mg/L (as CaCO3)

6.0 288 ± 2 580 ± 5

8.60 51 ± 3 900 ± 14

-(55.16 ± 1.52)

110 ± 3 7255 ± 108 355 ± 88 6900 ± 20 250 ± 2 158 ± 5 211.50 ± 4.95 2250 ± 36 680 ± 16 2900 ± 24 265.30 ± 1.84

0 3012 ± 10 7±7 3005 ± 17 70 ± 4 35 ± 4 55.64 ± 2.43 960 ± 30 220 ± 10 400 ± 10 63.97 ± 1.73

100 58.48 ± 98.42 ± 56.45 ± 72.01 ± 77.91 ± 73.71 ± 57.34 ± 67.66 ± 86.21 ± 75.89 ±

0.68 2.23 0.17 1.95 2.59 0.75 0.92 1.00 0.58 0.69

Negative indicates the total alkalinity increases as the pH of the medium increases complete removal of hardness

Total hardness mg/L (as CaCO3) Total solids, mg/L Suspended solids, mg/L Dissolved solids, mg/L Chloride mg/L Sulphate mg/L Phosphorous as PO4 3− mg/L COD mg/L BOD at 27 ◦ C mg/L TOC mg/L Ammoniacal Nitrogen (NH3– N) mg/ L Naphthalene μg/L Fluorene μg/L Phenanthrene μg/L Anthracene μg/L Benzo(b)fluoranthene μg/L Benzo(a)pyrene μg/L

29 ± 0.4 123 ± 0.6 126 ± 0.2 4 ± 0.29 63 ± 0.7 47 ± 0.8

25 ± 0.6 93 ± 0.5 70 ± 2 3 ± 0.0 34 ± 0.5 31 ± 0.5

13.81 ± 24.39 ± 44.45 ± 24.60 ± 46.03 ± 34.04 ±

1.24 0.05 2.12 7.73 0.27 0.08

2.1.2. Analysis of wastewater Paddy-soaked wastewater (PWW) from the rice mill, located in Tir­ uvannamalai district (12.4918◦ N, 79.1097◦ E), Tamil Nadu, India, was utilized for present experimental study. The collected wastewater was analysed for its various physicochemical characteristics such as pH, turbidity (NTU), total alkalinity (mg/L as CaCO3), total hardness (mg/L as CaCO3), total solids (mg/L), suspended solids (mg/L), dissolved solids (mg/L), chlorides (mg/L), sulphates (mg/L), phosphorous as PO34 (mg/ L), chemical oxygen demand (COD mg/L), biological oxygen demand (BOD at 27 ◦ C mg/L), total organic carbon (TOC mg/L), ammoniacal Nitrogen (NH3–N mg/L) and poly-aromatic hydrocarbons (PAHs as μg/ L). American Public Health Association (APHA) and Bureau of Indian Standards [32,33] are standard methods adopted for physicochemical

analysis. Wastewater sample was tested in triplicates for better accuracy. 2.1.3. Experimental setup Experiments were performed in a pair of identical raceway ponds, having ~75 L working volume with a freeboard of 10 cm. A typical, elliptical-shaped raceway made up of acrylic material with overall di­ mensions of 1.40 m length x 40 cm width x 25 cm height and hexagonal paddle wheel with six segmental partitions, mounted on a working platform (rectangle steel table) was used in the study (Fig. 1(a)). Ex­ periments were conducted with 50 L working volume (inclusive of inoculum) in the outdoor condition under optimal photosynthetically active radiation (PAR) of ~832–922 W/m2 (3826–4240 μmol/m2/s)

Fig. 2. pH and Growth Measurements during Culture period (a) pH (b) Dry biomass (c) Chlorophyll-a (d) Cell density. 3

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Fig. 3. Removal percentages of (a) NH3–N and (b) PO4–P during culture period.

between the temperature range of 33–37 ◦ C. Re-circulation of biomass in raceway ponds was carried out by providing revolving speed of 10–15 rpm to paddle wheels which were further connected to the common shaft of 5 HP motor (Fig. 1(a)). Fig. 1(b) and (c) indicated that raceway ponds with 50 L of PWW each, before and after inoculums, respectively. Standard measurements were taken for growth indicators as well as nutrient depletion namely, (i) biomass dry weight, (ii) cell density, (iii) chlorophyll content, (iv) ammoniacal nitrogen, and (v) phosphates concentration. All the experiments were performed in duplicates (one sample per raceway).

of exponential phase (t1) [36–38]. Cells per mL of culture were deter­ mined by using a haemocytometer (Neubauer improved, Marienfeld, Germany). 2.1.5. Biochemical composition Three important biochemical compositions of algae such as lipids, protein and carbohydrates were measured in the treatment system by applying appropriate harvesting method, usually by centrifugation. Bligh and Dyer method of extraction was used for determination of lipids in which chloroform and methanol were used as solvent at the ratio of 2:1 and gravimetry was applied for lipids measurement [15,39]. Further, FAME analysis was performed for extracted lipids to identify its fatty acid profile (Programmable Gas chromatograph, GC 1100, Mayura Analytical LLP., India). Hot alkali aid method of extraction and lowry assay was adopted for protein measurement and a standard calibration curve was plotted with make use of bovine serum albumin (BSA), pro­ cured from Sigma Aldrich [40]. Carbohydrates determination was car­ ried out based on the anthrone method of experiments after employing a suitable cell disruption method [40]. Lipids, protein, and carbohydrates, measured in the treatment system, were expressed on a percentage basis in respective of dry biomass as given in below equation (4).

2.1.4. Growth measurements in the treatment system Biomass productivity (g/L/d) of cultured algae was determined from equation (1) as given below [34]. Biomass productivity =

w1 − w0 t1 − t0

/ / g L d

(1)

where, w0 – biomass weight measured at the initial day (t0); w1 – biomass weight measured at the final day (t1). Chlorophyll–a content of harvested microalgae was estimated from spectrophotometry (Benchtop UV visible spectrophotometer; HACH DR:6000; India) by measuring distinct optical densities, after processing centrifuged biomass with ethanol as solvent [35]. The formula used for

) / / ( / / Lipids protein carbohyrates mg l− 1 ( ) X 100 Lipids protein carbohyrates (%) = Dry biomass mg l− 1

2.1.6. NH3–N and PO4–P removal efficiency and yield Nutrient removal, NH3–N and PO34 -P removal was estimated on a percentage basis (Equation (5)) and yield obtained in treatment system as per mg of N or P was calculated as the ratio between the difference in initial and final biomass and the difference in N, P concentration (Equation (6)) [41–43].

the estimation of chlorophyll-a content is illustrated in equation (2). Chlorophyll − a = (9.90 × OD660 )– (0.77 × OD642.5 )

mg / L

(4)

(2)

where, OD660, OD642.5 = optical densities of cultures measured at 660 nm and 642.5 nm, respectively. Specific growth rate (μ) usually represents cells per mL of culture relate to the time interval and was determined from equation (3) as provided below. ( ) ln N1 N0 / Specific growth rate (μ) = per day or d (3) t1 − t0

N or P removal (%) =

YN ​ or ​ YP =

(Inital concentration − Final concentration) X 100 Initial concentration

Final Biomass − Initial Biomass ) ( (N0 or P0 ) − Nf or Pf

(5) (6)

where, No = Initial N concentration; Nf, = Final N concentration; Po = Initial P concentration; Pf = Final concentration of P in the treatment system.

where N0 is the number of cells per mL of culture at the start of expo­ nential phase (t0) of growth curve obtained by plotting the natural logarithm of the number of cells (ln (N)) in y-axis and growth period (t in days) in x-axis; and N1 is the number of cells per mL of culture at the end 4

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volume or as per coded value. Then, the reaction mixture was kept in a water bath to retain the desired reaction temperature for 60 min. After this in situ transesterification process, reaction vials could stand for an hour as residuals can settle at the bottom. Then, the mixture was filtered, and the residual was washed twice with hexane and distilled water for further extraction. This mixture was centrifuged at 5000 rpm for 15 min and transferred to a separation funnel. Biodiesel extracted from the process was measured gravimetrically. All experiments were conducted in a magnetic stirrer system with specific constant rotation speed (500 rpm) and temperature control [28]. Biodiesel yield was determined by using formula (equation (7)) as given below: Biodiesel ​ yield ​ (%) =

Table 2 Biochemical composition and presence of fatty acids in harvested Chlorella pyrenoidosa biomass. Description

Biochemical composition

1 2 3 4

Lipids (% biomass) Carbohydrates (% biomass) Protein (% biomass) Hydrocarbons Hexane 2(3H)-Furanone 1-Amino-3-fluorobenzene Fatty acids/esters Hexadecanoic acid/7-hexadecenoic acid, methyl ester, (z)-/hexadecanoic acid, 15-methyl-, methyl ester Butanoic acid, 3, 3-dimethyl – methyl ester Nonanoic acid methyl ester Hexanoic acid 6, 9, 10 octadecenoic acid (z)-, methyl ester/oleic acid Tridecanoic acid methyl ester Tetradecanoic acid, 12-methyl-, methyl ester 2- Propenoic acid 3-dimethylamino – methyl ester Ethyl homovanillate Valeric acid/pentanoic acid Alcohol 2-(Trimethylsilyl)cyclohexanol Acetic acid Amines Tridodecylamine Ketones 1,8-Dibromooctane 4-Methyloctane Methylolacetone Others n-Heptyl chloride 9-Decen-1-yl acetate Goitrin

27.47 ± 1.41 23.77 ± 1.00 46.12 ± 3.55

5

6 7 8

9

(7)

2.2.1. Optimization by central composite design (CCD) Optimization of operating conditions by considering coded variables of wet algal biomass between 0.5 (− 1) and 2 (+1) grams, 5% H2SO4 in a methanol solution of 1.5 (− 1) to 7.5 (+1) mL, hexane volume of 1 (− 1) to 5 mL (+1) and under reaction temperature between 45 (− 1) and 105 ◦ C (+1), were designed based on CCD of RSM. Four-level of vari­ ables in experiments will have 30 experimental runs and effect on in­ dependent factors and the interaction effect between dependent factors were analysed with quadratic equation (8) as mentioned below.

Fig. 4. Lipids measurement during culture period.

Sl. No

Algal biomass (g) ​ x Lipid content (%) Weight ​ of ​ biodiesel ​ (g)

Y =a0 + a1 X1 + a2 X2 + a3 X3 + a4 X4 + a12 X1 X2 + a13 X1 X3 + a14 X1 X4 + a23 X2 X3 + a24 X2 X4 + a34 X3 X4 + a11 X12 + a22 X22 + a33 X32 + a44 X42 +e (8)

present present present

where, Y is the response as biodiesel yield (%), a0 is offset coefficients, a1, a2, a3 and a4 are linear coefficients, a12, a13, a14, a23, a24 and a34 are interaction coefficients, a11, a22, a33 and a44 are quadratic coefficients and X1, X2, X3 and X4 are sample amount, methanol - sulphuric acid volume, hexane volume and reaction temperature, respectively [44].

present present present present present present present present present present

3. Results and discussion 3.1. Outdoor culturing of Chlorella pyrenoidosa in paddy-soaked wastewater 3.1.1. Characteristics of paddy-soaked wastewater (PWW) Analysed physicochemical characteristics of collected paddy-soaked wastewater (PWW) are presented in Table 1. pH was measured as 6 (<7), represented acidic (slightly) nature of wastewater. Total solids (TS), total dissolved solids (TDS), biological oxygen demand (BOD), chemical oxygen demand (COD), ammoniacal nitrogen and phosphates values measured for PWW were detected as greater in amount when compared to effluent standards prescribed by Central Pollution Control Board (CPCB) [45]. This is mainly because of persistent soaking, organic matter present in rice husk, and starch substances of rice bran, which enhance the gelatinization behaviour of rice. Ammoniacal nitrogen (>250 mg/L), phosphates (>200 mg/L), total organic carbon (~3000 mg/L) are nutrient suppliers for the growth of microalgae (Table 1).

present present present present present present present present present

2.2. In-situ transesterification

3.1.2. Growth characteristics of microalgae Initially, microalgae Chlorella pyrenoidosa was cultured in PWW under outdoor condition. Daily measurements were taken for growth characteristics and Fig. 2 shows the graphical representation of pa­ rameters observed during the culture period.

Transesterification experiments were conducted in a 20 mL quartz vials with air-tight caps which act as small reactor. Wet algal biomass centrifuged from raceway ponds having ~80% moisture content was used for single-step transesterification process. Experiments were designed by considering effects on biomass volume, solvents used, catalyst employed and temperature. With designed experimental con­ ditions, centrifuged algal biomass of desired quantity was mixed with an acidic catalyst of 5% H2SO4 in methanol mixture and hexane of desired

3.1.2.1. pH in the growth medium. The pH of growth medium increased with an increase in the growth period and recorded between 6.0 and 8.5 (Fig. 2(a)). Normally, different algal culture has different pH range, 5

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Table 3 Design of experiments and experimental responses in in-situ transesterification. Run

Sample amount (g)

Methanol and Sulphuric acid (ml)

Hexane volume (ml)

Temperature (◦ C)

Experimental yield (%)

Predicted yield (%)

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

2 2 0.5 1.5 2 2 2.5 1 1.5 1.5 1.5 1 1.5 1.5 2 1.5 1 1.5 1.5 2 1 1 1.5 1 1.5 1.5 1 2 1 2

6 6 4.5 4.5 3 3 4.5 3 4.5 4.5 4.5 6 4.5 4.5 3 1.5 3 7.5 4.5 3 6 6 4.5 6 4.5 4.5 3 6 3 6

2 4 3 3 2 2 3 4 1 3 3 2 3 3 4 3 2 3 3 4 4 4 5 2 3 3 2 2 4 4

90 90 75 75 90 60 75 60 75 105 75 90 75 45 60 75 60 75 75 90 60 90 75 60 75 75 90 60 90 60

17.78 26.79 24.79 32.65 28.95 20.77 33.3 23.98 31.72 14.4 32.65 20.29 32.48 20.24 30.36 27.53 23.4 25.18 32.65 46.54 30.07 14.67 38.09 42.28 32.15 30.41 20.12 34.13 30.89 27.09

19.53 25.24 25.72 32.17 28.09 20.90 33.01 23.37 32.27 14.49 32.17 18.88 32.17 20.78 30.00 28.06 23.18 25.29 32.17 47.40 29.16 15.68 38.17 42.56 32.17 32.17 20.00 32.85 30.40 28.35

Table 4 ANOVA analysis of the obtained RSM model. Source

Sum of squares

Df

Mean Square

F ratio

p-value prob > F

Model X1-Sample amount X2-Methanol sulphuric acid X3-Hexane X4-Reaction temperature X1 X2 X1 X3 X1 X4 X2 X3 X2 X4 X3 X4 X21 X22 X23 X24 Residual Lack of Fit Pure Error (e) Cor. Total

1593.25 79.68 11.50 52.24 59.31 55.32 79.34 107.49 184.89 420.56 104.19 13.47 51.72 16.02 361.82 20.67 16.78 3.89 1613.91

14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 10 5 29

113.80 79.68 11.50 52.24 59.31 55.32 79.34 107.49 184.89 420.56 104.19 13.47 51.72 16.02 361.82 1.38 1.68 0.78

82.60 57.83 8.34 37.92 43.05 40.15 57.59 78.02 134.20 305.25 75.63 9.78 37.54 11.63 262.62

<0.0001 <0.0001 0.0113 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0069 <0.0001 0.0039 <0.0001

significant

2.16

0.2046

not significant

mainly based on its photosynthetic reaction which requires CO2, mostly fixed by an enzyme, rubisco (Ribulose-1,5-bisphosphate carboxylase/ oxygenase). Prerequisite CO2 was primarily obtained from the conver­ sion of bi-carbonates (HCO−3 ) to CO2 and OH− . Further, released OH− was absorbed by H+ in the growth medium, leads to an inevitable in­ crease in pH in the treatment system [46]. Effluent from cassava ethanol fermentation was remediated by C. pyrenoidosa in a tubular photo­ bioreactor in which pH was observed between 5.0 and 7.5 [47] whereas it was slightly high (6.5–8.5) in wastewater from soybean processing industries. A similar study on culturing of C. pyrenoidosa in unsterilized piggery wastewater with a cost-effective culturing method to enhance biofuel production (i.e.) by applying air sparging and permitting simu­ lated flue gas, recorded the pH values between 6 and 9.5 [38]. Another study with dairy wastewater treatment by C. pyrenoidosa, observed pH

range of 6.0–8.5, which supports our present study [48]. 3.1.2.2. Biomass dry weight. Biomass dry weight is an important growth factor of algal culture and everyday measurements reveal the identifi­ cation of optimum biomass from the culture system. Fig. 2(b) shows recorded dry biomass values during the culture period. Maximum volumetric algal biomass productivity of 0.26 ± 0.02 g/L/d was ob­ tained in the current research with raceway ponds of 50 L working volume. Horizontal flow raceway ponds have more area of illumination provided efficient biomass productivity in PWW treatment system. Similar research on cultivation of microalgae consortium in raceways with the working volume of 500 L, using untreated carpet industry effluent as a growth medium, documented the volumetric biomass productivity of 0.057 ± 0.001 g/L/d [20]. Amongst various influencing 6

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rate of 0.42/d in our present work which is ~50–60% of values obtained from flask scale study (500 mL working volume) on piggery wastewater (unsterilized) with C. pyrenoidosa using sparging air and simulated flue gas [38]. However, conventional outdoor culturing in raceway ponds with regular mixing or recirculation of biomass provided a significant outcome with a less operational cost. 3.1.3. Treatment efficiency Percentage removal on NH3–N and PO4–P in outdoor culture were recorded as 75.89 ± 0.69% and 73.71 ± 0.75% with the yield coefficient of 6.12 mg biomass/mg of N (YN) and 7.77 mg biomass/mg P (YP), respectively (Fig. 3(a) and (b)). This is mainly due to N/P ratio of growth medium used in outdoor culture is ~1, whereas it was ~2.5 in indoor culture as reported by Aslan and Kapdan (2006) [41]. Further, a rapid increase in N-removal and slower increase P-removal were recorded in the study. A faster consumption of N was because of microalgae assimilates nitrogen sources and convert them into amino acids (protein) which will be useful in the construction of their cell walls and thereby helps in reproduction. Phosphates help in microalgal metabolism, by transferring across the plasma membrane of algal cells by the process, called phosphorylation which involves the production of ATP from ADP along with stored energy. Hence, the phosphates uptake process required a longer time when compared to N-consumption [35, 57,58]. Also, there was a gradual increase in lipid content for microalgal growth and maximum lipid content of 27.47 ± 1.41% of biomass was obtained at 7 days in the treatment system (Fig. 4). Physicochemical characteristics of PWW before and after outdoor phycoremediation process and their percentage removal were high­ lighted in Table 1. There is nearly complete removal (98.42 ± 2.23%) on total suspended solids in the treatment system whereas it was ~60% removal on total solids and total dissolved solids. About 72% removal on chlorides, 78% removal on sulphates were attained after the treatment. COD, BOD and TOC removal was recorded as ~60%, ~70% and ~90% respectively. Similarly, there is a minimal removal percentage on pol­ yaromatic hydrocarbons, observed during the study. Crater and Lie­ vense (2018) discussed the fit-falls, and ideologies of scaling up the industrial microbial processes [59]. Even though slightly less treatment efficiency (N, P removal) was obtained from the current work, but revealed the possibilities of implementing present technology to indus­ trial level at rice mill industries.

Fig. 5. Predicted values versus actual values of biodiesel yield.

parameters, working volume, temperature, illumination and irradiation through outdoor culturing are predominant factors which enhance biomass productivity [23]. Research work on flask-scale (250 mL in 500 mL conical flasks) cultivation of C. pyrenoidosa, in diluted piggery wastewater, observed the maximum biomass productivity of 0.04 g/L/d [49]. Another study on batch/fed-batch flask culture of C. pyrenoidosa in high nutrient soybean wastewater has given optimum biomass produc­ tivity of 0.64 g/L/d [50]. Thus, biomass potential hinges on nutrient availability of growth medium i.e. wastewater where microalgae grow or cultivate [51]. 3.1.2.3. Chlorophyll-a content. Chlorophyll is a nitrogen-rich pigment available in plants and green algae. This intracellular nitrogen pool further utilized for cell growth and biomass production [52]. Addi­ tionally, it supports microalgal growth metabolism as it absorbs light energy and converts into chemical energy by photosynthesis [53]. Chlorophyll content of PWW grown algal culture, increased with time and reached its maximum (15.57 mg/L) in 7 days (Fig. 2(c)) (10.02 ± 0.87 mg/g of biomass). Higher nitrogen treatment enhances the higher rate of chlorophyll restoration and decreases when there is a depletion in the nutrient supplement [52]. A study on effective production of algal biomass, lipid content and chlorophyll by culturing Chlorella sp. in aquaculture wastewater, provided maximum chlorophyll content of 7.12 ± 0.03 mg/g of biomass under an axenic condition which is ~70% lesser than our current research [54]. Another flask-scale lab study for nutrient removal of rice mill (paddy soaked) wastewater by C. pyrenoidosa reported the highest chlorophyll content of 5.55 mg/L (~50% of present study) [55].

3.1.4. Lipid measurement Lipid measurements during the culture period are presented in Fig. 4. As the number of cells increased, there is an increase in lipid production and reached the highest at greater cell concentration (7th day). Lipids play an important role in algal metabolism and growth cycle. Specific lipids act as imperious structural components for biological membranes. Further, it poses pathways in cell signalling and assists in sensing the change in ambient environmental condition. Usually, the lipid content of microalgal species was reported between 20 and 50% of dry biomass [17]. In our present study, the optimal lipid content of 27.47 ± 1.41% was obtained which identifies better production of lipids from the wastewater (PWW) treatment system (Fig. 4). 3.2. Biochemical composition and presence of fatty acids in algal culture

3.1.2.4. Cell density. Cell density or number of cells per mL of culture can also be a measure of biomass concentration described as direct growth measurement in the treatment system [56]. Fig. 2(d) represents the estimated number of cells per mL of culture during the culture period. As the number of cells increased with increase in biomass and chlorophyll content and reaches its maximum value at one point of time and from there declination in cell growth i.e. cell depletion starts, resulted in dead cells in the treatment system. It leads to a slight decrease in chlorophyll and dry biomass (Fig. 2(b) and (c)). Maximum cells per mL of (8.59 ± 0.13)x106 was quantified at a specific growth

Biomass productivity and chlorophyll content mainly support algal growth with 27.47 ± 1.41% of lipids, 23.77 ± 1.00% of carbohydrates and 46.12 ± 3.55% of protein (Table 2). Light intensity plays a vital role in an algal culture which depends on spectral quality and photoperiod in addition to culture depth and density of algal culture [60]. Adequate lighting in the outdoor culture provides more biomass dry weight as well as chlorophyll content, thereby the production of wealthy algal biomass. Other than some common hydrocarbons as a by-product of solvents, various kind of acids, alcohols and ketones were measured in the treatment system. Most common saturated fatty acids, hexadecenoic 7

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Biomass and Bioenergy 143 (2020) 105853

Fig. 6. 3D Response surface plots for biodiesel yield from the cultivation of C. pyrenoidosa in PWW with the independent parameters; sample amount (A); methanol sulphuric acid (B); hexane volume (C) and reaction temperature (D) (a) A&B (b) A&D (c) A&C (d) B&C (e) B&D and (f) C&D.

acid and other fatty acid methyl esters such as butanoic acid, octade­ cenoic acid/oleic acid and tetra-decanoic acid were identified in har­ vested algal lipids, supports biodiesel properties to proceed further for in-situ transesterification (Table 2).

variables and responses at a 95% confidence level. Further, the statis­ tical significance of the model was validated from the co-efficient of determination (R2). The R2 value was observed as 0.9872, showed only 1.28% of the variability in response was not explained by the attained model. It was evident that predicted values are very close to experi­ mental values which further confirmed the statistical significance of the model (Fig. 5 and Table 4). Optimum biodiesel conversion of 46.54% was recorded in the present study where direct in-situ transesterification was taken place when 2 g of wet algal biomass was mixed with a reaction mixture of 3 mL methanol sulphuric acid solution and 4 mL of hexane under reaction temperature of 90 ◦ C in 60 min. A study on response surface methodology-based optimization of in situ transesterifications of algal biomass with methanol, acidic (H2SO4) and base (NaOH) catalyst revealed that acidic catalyst provided the maximized biodiesel yield in 60 min [61]. With the similar conditions i.e. methanol, the acidic catalyst with an hour reaction time, maximum yield was achieved from our current research. Moreover, the available fatty acids, their structure, and percentages, obtained from gas chromatography mass spectrometry (GCMS) spectrum (Fig. 7) were presented in Table 5. From the data, it was revealed that PWW produced algal lipids was characterized with greater than 40% of palmitic acid, nearly 36% of oleic acid with least

3.3. In-situ transesterification and evaluation of biodiesel yield by optimization 3.3.1. Optimization of biomass yield With the experimental design values presented in Table 3, the opti­ mized RSM model in terms of coded variables to calculate biodiesel yield was given in the below equation (9). Y = 32.17 + 1.82*X1 − 0.69*X2 + 1.48*X3 − 1.57*X4 − 1.86*X1 X2 + 2.23*X1 X3 + 2.59*X1 X4 − 3.40*X2 X3 − 5.13*X2 X4 + 2.55*X3 X4 − 0.70*X12 − 1.37*X22 + 0.76*X32 − 3.63*X42

(9)

The significance of the obtained model was justified by ANOVA analysis. Table 4 shows ANOVA results for the obtained quadratic model for biodiesel yield. Larger F ratio fulfils level of significance, p-value (Prob.>F) as <0.05. There is a statistical relationship between selected 8

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Biomass and Bioenergy 143 (2020) 105853

A study on biodiesel production from marine algae, Scenedesmus sp. through in-situ transesterification process associated with acidic catalyst reported that acidic catalyst (H2SO4) was ~2.5-fold more efficient (48.41 ± 0.21%) than alkaline catalyst (NaOH) with optimized reaction temperature of 70 ◦ C; reaction time of 10 H; catalyst amount of 5% and biomass to solvent ratio of 1:15 [28]. Almost similar observation (with 46.54% biodiesel yield) was made from our present study with a high­ light of making use of wastewater (PWW) grown algae. Microwave irradiation was mostly adopted for better extraction of wet algal biomass compared to the conventional method [29]. Moreover, simple wet green algae in-situ transesterification process with catalyst-free approach i.e. microwave mediated supercritical ethanol condition also proved to be an efficient method of production of ethyl ester [62]. A single-step transesterification process was applied for biodiesel conversion from microalgae, Chlorella sp. MJ 11/11, provided maximum lipid conversion (95%) with 4 M catalyst concentration, 1:5 algal biomass/methanol ratio, 65 ◦ C reaction temperature, 7 H reaction time and 90 min biomass drying duration [27]. Nearly 50% of the result was obtained from our current research with the lowest possible operating conditions with similar Chlorella sp. (Chlorella pyrenoidosa), cultivated in paddy-soaked wastewater can be fruitful in the sustainable WTE conversion process.

Fig. 7. Gas chromatography mass spectrometry (GCMS) spectrum of PWW produced algal lipids.

3.4. Outcome, future perspective, and economic viability Table 5 The available fatty acids, their structure, and percentages, obtained from gas chromatography mass spectrometry (GCMS) spectrum. S.no

Fatty acids

Structure

Percentage

1 2 3 4 5

Butanoic acid Myristic acid Palmitic acid Oleic acid Linoleic acid

C4:0 C14:0 C16:0 C18:1 C18:2

1.67% 6.35% 42.67% 35.63% 4.38%

An initiative has been taken to identify renewable bioenergy from industrial wastewater (Paddy-soaked wastewater) and thereby to ensure sustainable wastewater management and WTE conversion. Within this view, phycoremediation technology was adopted for PWW treatment and outdoor cultivated microalgae was examined for its FAME charac­ teristics. Significant lipid profile encouraged us to go for further in-situ transesterification process. A single-step transesterification process with optimized operating conditions provided maximized biodiesel yield which is nearly 60% of the standard algal in-situ transesterification process. Algae usage for biofuel production has numerous advantages compared to oil crops. Algal oil also has comparable properties as petrodiesel [63]. Even though biofuel from algae has similar properties compared to petro-diesel, downstream processing of algae for biofuel generation is expensive compared to petro-diesel [64]. Biorefinery way of biodiesel production includes cultivation and harvesting of algae, lipid extraction and finally conversion of lipid into biodiesel production. Cost of biofuel from algae, usually based on methods used and varies from 150 $ to 6000 $/tonne [65–67]. Open pond cultivation is usually employed for algal cultivation since closed system demands high oper­ ating cost [68]. The requirement of water for open pond system and dewatering of large open ponds further increases the operating cost [69]. Centrifugation technique for algal harvesting also accounts for high cost [70]. Increase in lipid content has a positive influence on biofuel cost [71]. Possible way of biofuel production from microalgae is feasible without exhausting the available resources. Thus, the current research focuses on the production of biodiesel by utilizing paddy-soaked wastewater and thereby to study the possibilities of reducing cost for biofuel production. Further innovation and promotion on this WTE concept provide insight to adapt microalgae as a profitable fuel crop.

possible proportions of myristic acid (~6%), linoleic acid (~4%), and Butanoic acid (~2%). 3.3.2. The interaction effect between the parameters 3D surface plots provided the screenshot on the effect of biodiesel yield by considering chosen reaction conditions such as the amount of wet biomass (Sample amount: A), catalyst (methanol sulphuric acid: B), solvent (Hexane volume: C) and reaction temperature (D). Biodiesel yield increased with increase in sample amount i.e. the quantity of wet biomass and reached its maximum at the extraction of 2 g sample. The conversion was comparatively less (17.78%) when a high volume of catalyst (6 mL) was used (Run. No. 1). Once the catalyst amount was reduced to 3 mL then biodiesel conversion was increased to 46.54% (Run. No. 20) (Fig. 6(a)). Even though the biodiesel yield was slightly higher at its initial temperature of 60 ◦ C (Run No. 24), it increased with increase in sample amount and obtained the maximum value at 90 ◦ C (Fig. 6(b)). Sample amount and volume of hexane used for extraction were found to be directly proportional to each other. As the conversion was initially less at initial values of biomass weight, hexane volume and increased with an increase in both values as presented in Fig. 6(c). In contrast, the yield was optimum when extraction carried out with the reaction mixture of less amount of catalyst and more volume of hexane (Fig. 6 (d)). Despite being methanol sulphuric acid as an acidic catalyst in the esterification process, 90% of biomass yield was accomplished at 60 ◦ C, compared to conversion at 90 ◦ C (Fig. 6(e). However, the amount of catalyst used signified the esterification process at a higher temperature. Further, high temperature enhanced the conversion process thereby inhibited the catalytic poisoning through water vapour [44]. Conversion of biodiesel increased with increase in reaction temperature as well as hexane volume and reached its optimum at the temperature of 90 ◦ C and with hexane volume of 4 mL as depicted in Fig. 6(f).

4. Conclusion Outdoor cultivation of C. pyrenoidosa in paddy-soaked wastewater and subsequent in-situ single-step transesterification was investigated in the present assessment. Better phycoremediation (PWW treatment) ef­ ficiency with greater than 75% of NH3–N and more than 70% of PO4–P removal was achieved in the treatment system. Raceway ponds pro­ duced microalgal culture, was characterized with rich in biochemical composition, fatty acids, and esters. Consequently, single-step trans­ esterification process with optimized reaction conditions supports pro­ cess intensification and thereby better biodiesel yield (46.54%). The 9

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Biomass and Bioenergy 143 (2020) 105853

present study suggested that PWW cultured C. pyrenoidosa was desirable feedstock for biodiesel production and thereby helps in the future development of sustainable wastewater management and energy conservation.

[14]

[15]

CRediT authorship contribution statement

[16]

J. Umamaheswari: Methodology, Investigation, acquisition of data, Writing - original draft, preparation, final approval of the version to be submitted. M.S. Kavitha: interpretation of data, Data curation, Vali­ dation, Writing - review & editing, final approval of the version to be submitted. S. Shanthakumar: Conception and design of the study, Su­ pervision, Funding acquisition, Writing - review & editing, final approval of the version to be submitted.

[17] [18] [19]

Declaration of competing interest [20]

There are no conflicts to declare. Acknowledgement

[21]

The authors acknowledge the financial support provided by the Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India under the grant: YSS/ 2015/000527.

[22] [23]

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