Process for Preparing Value-Added Products from Microalgae Using

Research Article Cite This: ACS Sustainable Chem. Eng. 2017, 5, 10019-10028

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Process for Preparing Value-Added Products from Microalgae Using Textile Effluent through a Biorefinery Approach Sourish Bhattacharya,†,⊥,# Sumit Kumar Pramanik,‡,# Praveen Singh Gehlot,§,⊥,# Himanshu Patel,†,⊥ Tejal Gajaria,∥,⊥ Sandhya Mishra,*,§ and Arvind Kumar*,§ †

Process Design and Engineering Cell, CSIR−Central Salt and Marine Chemicals Research Institute, Bhavnagar−364002, India Analytical Division and Centralized Instrument Facility, CSIR−Central Salt and Marine Chemicals Research Institute, G. B. Marg, Bhavnagar, Gujarat−364002, India § Salt & Marine Chemicals Division, CSIR−Central Salt and Marine Chemicals Research Institute, Bhavnagar−364002, India ∥ Marine Biotechnology and Ecology Division, CSIR−Central Salt and Marine Chemicals Research Institute, Bhavnagar−364002, India ⊥ Academy of Scientific and Innovative Research (AcSIR), Bhavnagar−364002, India

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‡

S Supporting Information *

ABSTRACT: A model was designed for effective utilization of textile effluent as the nutrient medium for the production of high-value products from Chlorella variabilis through a greener approach. Biomass productivity of 74.96 ± 2.62 g/(m2/d) with lipid yield of 20.1 ± 2.2% (wrt dry biomass) was obtained using textile effluent as the nutrient source. A novel integrated process is developed based on detergent (sodium dodecyl sulfate) hydrolysis to convert the carbohydrates present in microalgal biomass to reducing sugars for microbial fermentation, while making available lipids for downstream processing of γ-linolenic acid, leaving the protein rich fragment behind. Our experimental data showed that from 495 g of microalgal biomass, 109.4 g total lipids was extracted containing 34.65 g γ-linolenic acid, and 1.3 g pure ε-polylysine from 36.68 g of reducing sugars. A two-step efficient green process was developed for recovering ε-polylysine using ethylammonium nitrate having 74% recovery. In addition to value-added products, CSIR-CSMCRI’s Chlorella variabilis (ATCC PTA 12198) can remediate 100% of aluminum, 82.72% boron, 45.66% calcium, 100% cobalt, 14.5% potassium, 0.1% magnesium, 42.18% sodium, 100% nickel, and 100% iron. A total decrease of 78.17% total phosphate and 25.22% total inorganic phosphate with respect to total phosphate present in the effluent was observed. KEYWORDS: Textile effluent, Chlorella variabilis, Unreacted dyes, Bicarbonate, γ-Linolenic acid

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INTRODUCTION Industrialization has an important function in the growth of the country. Textiles are an important and fast-growing industrial sector which is also essential for any growing economy. The textile industry uses various types of raw materials such as cotton, woollen, and synthetic fibers. The textile industries may be categorized into two groups, i.e., dry and wet fabric industries. Solid wastes are generated in dry fabric industries, whereas wet fabric industries utilize a lot of water generating large amounts of wastewater containing salts and unreacted dyes.1 However, the final disposal of this effluent in solid form (sludge) or liquid form is still a challenge, as, even after treatment through conventional techniques, it does not decolorize and detoxify the dye effluents.2 However, textile dyes make water toxic3 and unsafe for human and animal consumption. At the same time, they cause an imbalance within the aquatic ecosystem and can serve as mutagenic agents which is harmful to the environment.4−7 Simultaneously, the © 2017 American Chemical Society

discharge of effluents pollutes rivers, affecting soil properties as well as the growth of plants along with biodiversity.8−10 Various researchers have performed studies about reduction in dye concentration as well as remediation of salts present in the textile effluent through microalgae. Chia and Musa were able to reduce concentrations of indigo dye present in textile effluent by growing Scenedesmus quadricanda ABU12 in the effluent.11 Furthermore, removal at a concentration of 17.5% with respect to total dye color and chemical oxygen demand (COD) level was done utilizing Chlorella vulgaris.12 Simultaneously, 73% of the total dye present in textile effluent was collected from the Karur unit, Tamil Nadu.13 Prabina and Kumar developed a process for decolorizing the dye present in textile effluent by growing a microalgal consortium in the Received: June 16, 2017 Revised: August 30, 2017 Published: September 6, 2017 10019

DOI: 10.1021/acssuschemeng.7b01961 ACS Sustainable Chem. Eng. 2017, 5, 10019−10028

Research Article

ACS Sustainable Chemistry & Engineering effluent.14 The consortium consists of Anabaena sp., Nostoc sp. and Chlorella sp. which, when grown in the effluent, removed 90% of dye. Microalgae such as Chlorella, Scenedesmus, and Ankistrodesmus species possess the potential to biodegrade organic pollutants present in oil mill wastewater and paper industry wastewater.15−19 Simultaneously, microalgae also have the potential to remediate heavy metals through their accumulation by physical adsorption, ion exchange and chemisorption, covalent bonding, surface precipitation, redox reactions, or crystallization on the cell surface.20−27 Microalgae play a key role in nutrient removal through assimilating it as they require high nitrogen and phosphorus for proteins, nucleic acid, and phospholipid synthesis inside the cell.28−32 Another important application of microalgae is in pollutant removal as certain green microalgae and cyanobacteria can use toxic recalcitrant compounds as carbon, nitrogen, sulfur, or phosphorus sources for its growth.33,34 Chlorococcum vitiosum has the potential to remediate COD at a concentration of 13% along with complete removal of the heavy metals.35 In-addition to the reduction of COD and dye concentration in the textile effluent, researchers have succeeded in cultivating microalgae in high rate algal ponds (HRAP). Chlorella vulgaris was cultivated in HRAP (dimensions 1 m × 0.5 m × 0.3 m; agitation 15 rpm using paddle wheels) using textile effluent containing Supranol Red B3W dye for generation of 106.7 mg/ L biomass.36 However, it has been reported that live algae as well as nonviable algae (dried algal mats) have been used in the reduction of dyes present in effluent.37 The mechanism involves both biosorption and bioconversion. Maurya et al.37 reported utilizing a nonviable microalgal mat for reducing methylene blue dye. Simultaneously, nonviable Spirogyra biomass can be utilized as an important biosorbent for removal of Synazol dye present in wastewater.38,39 Chlorella vulgaris can remove around 69% of the color through converting mono-azodyes such as tectilon yellow 2G to aniline.40 Such potentiality of microalgae for removing reactive dyes present in the effluent can be done through manipulating the microalgal growth. In the present study, a model was designed for effective reduction of chemicals as well as residual dyes present in the textile effluent. The chemicals present in the textile effluent are utilized as the nutrient for the growth of Chlorella variabilis, and at the same time, the unreacted dyes reduced through microalgae making the effluent safer for discharge. One of the major challenges related with the algal biofuel production in a biorefinery approach is improving biomass utilization for net energy gain providing an economically viable and scalable process for deriving commercially important coproducts through a greener route. Laurens et al. demonstrated an integrated technology based on moderate temperature and low pH (controlled microwave pretreatment) to convert the carbohydrate in wet algal biomass to soluble sugars for fermentation, while making lipids more accessible for downstream extraction and leaving a protein-enriched fraction behind.41 However, there have been several hydrolysis techniques being developed by researchers in the past few decades, but the most common and green process available for hydrolysis of carbohydrates from, e.g., cellulosic biomass, microalgal biomass is the use of enzymes.42 Researchers have developed few low-cost ionic liquids for hydrolysis of lignocellulosic biomass;43 the economics of the scalable processes are still in question. However, ionic liquids can be

used for the extraction and purification of high-value biopolymers from algal sources. This work aimed to investigate the potential of carbohydrate containing biomass of Chlorella variabilis grown mixotrophically using textile waste as a substrate for ε-polylysine production along with microalgal lipids containing γ-linolenic acida nutraceutical. To the best knowledge of the authors, no published work exists wherein the hydrolysis of Chlorella biomass was performed using sodium dodecyl sulfate (SDS)a detergentand extraction and purification of ε-polylysine performed using an ionic liquid, e.g. ethylammonium nitrate. Furthermore, the microalgal biomass grown using textile effluent along with critical nutrients yielded 74.96 ± 2.62 g/ (m2/d) with a total lipid yield of 20.1 ± 2.2%.

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EXPERIMENTAL SECTION

Culture Maintenance of Microalga Chlorella variabilis PTA 12198. Oleaginous CSIR-CSMCRI’s Chlorella variabilis (ATCC PTA 12198) was isolated from Diu, India (N 20° 41.341′; E 70° 53.734′) and was maintained as a monoalgal culture in modified Zarrouk’s media44 as per the work of Bhattacharya et al.45 Inoculum Development. The inoculum was initially prepared in 10L carboys (Zarrouk’s medium, 25 ± 5 °C) from a 1L Erlenmeyer flask (Zarrouk’s medium, 700−800 l×, 25 ± 5 °C) in the CSMCRI-P2 medium as per Bhattacharya et al. The air temperature during the daytime was 46 ± 3 °C and during the night was 35 ± 3 °C during May of 2016. Microalgal Cultivation. CSMCRI’s terrace (21° 75.92783′ N; 72° 14.41304′ E; elevation 121 ft) was chosen as the mass cultivation site. The cultures were agitated manually three times a day. Effluent collected from the textile industry was diluted with tap water in the proportion of 3:7 due to high alkalinity of the effluent. One plastic tank having area 1.1 m × 1.1 m with depth 0.085 m was used for the cultivation of Chlorella variabilis (ATCC PTA 12198) utilizing textile effluent. The availability of abundant sunlight and prevailing high temperature conditions during the day were the other factors favorable for the selected strain. The Chlorella variabilis was grown in the tank using 40% textile effluent with tap water. The previously described inoculum raising tank was supplemented with CSMCRI-P2 medium consisting of grams per liter NaHCO3 5; NaNO3 1.2; K2HPO4 0.25; K2SO4 0.25; NaCl 1.0; CaCl2 0.04; Na2EDTA 0.08; MgSO4.7H2O 0.1; FeSO4.7H2O 0.01. Mass Cultivation. The cultivation was carried out during the peak summer season (June 2016) in Gujarat, India, with a 46 ± 3 °C ambient air temperature. The water temperature range was 43 ± 3 °C during the entire cultivation. The desired culture for the mass cultivation needed was initially grown in inoculum raising tank with an area of 0.35 m2 each. The mass cultivation tank was monitored regularly by measuring the OD at 540 nm using UV−visible spectrophotometer (Cary Bio 50, Varian Inc., USA) and biomass yield was measured by measuring gravimetrically the dry biomass obtained after centrifugation of the culture of C. variabilis. The open cultivation of Chlorella variabilis (ATCC PTA 12198) was carried out from the 13th to 18th of June 2016 having average solar irradiation of 4.8 kWh/(m2/d). The air temperature during day time was 46 ± 3 °C, and during the night it was 43 ± 3 °C during June 2016. A cell concentration of 2 g/L (wet basis) was used to inoculate the tank with an area of 1.2 m2. The biomass yield and total lipid yield were monitored on a regular basis. The agitation of the ponds was done manually three times a day. The textile effluent collected from western overseas, Jetpur, Gujarat. The textile effluent consists of 0.2 g/L inorganic carbon and 4.3 g/L organic carbon in the form of sodium and ammonium bicarbonate having a pH of 12.11 ± 1.2 which was utilized for the growth of Chlorella variabilis. The composition of textile effluent obtained from the textile mill at Jetpur, Rajkot, Gujarat, contains (ppm) total dissolved solids 8.13, ammonium ion 5.8, nitrate 24.1, salinity 7.7, 10020


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ACS Sustainable Chemistry & Engineering aluminum 52, boron 1200, cobalt 60, calcium 894, chromium 4, iron 0.5, potassium 2300, magnesium 441, manganese 1, sodium 5368, zinc 1, lead 2 and nickel 16.1. Due to high alkaline concentration, the effluent was mixed with tap water at a proportion of 4:6 ratio i.e 40 L of textile effluent is mixed with 60 L fresh water, followed by addition of P2 medium containing (g/L) K2HPO4 0.25, NaNO3 1.2, K2SO4 0.25, NaCl 1.0, MgSO4.7H2O 0.2, EDTA 0.08, CaCl2 0.04, FeSO4 0.01. In addition to components of P2 medium, media also consists of aluminum 2.08 g, boron 40 g, cobalt 2.4 g, calcium 2.679 g, iron 0.02 g, potassium 92 g, magnesium 17.64 g, manganese 0.04 g, sodium 214.72 g, zinc 0.04 g, lead 0.08 g, nickel 25.764 g, and chromium 0.16 g which came directly from the effluent being added. Thereafter, 40 L of Chlorella variabilis culture was added to the medium as an inoculum (Figure 4). Lipid Estimation. The lipid content was quantified gravimetrically from the sun-dried biomass.46 The microalgal lipid was extracted three times to obtain clear extracts using 10 mL of chloroform and methanol (1:2 v/v) in 1 g biomass. The pooled extracts were filtered to remove the cell debris. The filtered extract was evaporated under vacuum to dryness at 55 °C using a Büchi rotary evaporator. Fatty Acid Profiling. The Chlorella variabilis methyl esters (CvME) were obtained from the lipid using 1 mL of 1% sodium hydroxide in methanol, followed by heating at 55 °C for 15 min. Thereafter, 5% of 2 mL methanolic HCl was added with heating at 55 °C for 15 min.47 The prepared FAMEs were then separated by adding 1 mL of hexane to the reaction mixture. The FAMEs containing hexane were analyzed by a GC-2010 gas chromatograph coupled with a mass spectrometer (GC-MS QP-2010, Shimadzu, Japan). The FAMEs were analyzed through a gas chromatography mass spectrometer (GC-2010 twinned with a GC-MS QP-2010) from Shimadzu (Japan). An RTX-5-fused silica capillary column (30 m × 0.25 mm, 0.25 μm) maintained a flow rate of 1 mL/min and a precolumn pressure of 49.7 kPa with Helium as a carrier gas. The column temperature regime was 40 °C for 3 min, followed by an increase at a rate of 5 °C/min up to 230 °C, and then maintained at 230 °C for 40 min. The injection volume and temperature were 1.0 μL and 240 °C, respectively, with a split ratio of 1/30. The mass spectrometer operated in electron compact mode with 70 eV of electron energy. The ion source and the interface temperature were set at 200 °C. The peaks were compared with the standards with respect to their retention times (Standard FAME Mix C4−C24; SigmaAldrich) by GC-MS postrun analysis and quantified by area normalization. Elemental Composition of Biomass. The elemental (C, H, N, S) composition (%) of dried biomass (90 °C for 24 h in the oven) was analyzed by the CHNS analyzer (elementarvario Micro), and sulfanilamide was used as a reference standard.48 The measured values of the standard had 150 °C through thermogravimetric analysis (TGA). Moisture Analysis. The moisture content of the ionic liquid was measured using Karl Fisher (KF) titrator (890 titrando, MetrohmSwitzerland). Synthesized EAN was found to be having only 1.27% moisture content. NMR. Chemical shift (δ) values (ppm) are the following: 1.15 (t, 3H), 2.85 (m, 2H), 7.76 (s, broad due to N, 3H) are shown in Figure 3. Downstream Processing of ε-PL. Ammonium Sulfate Precipitation. In order to precipitate the total protein present in the supernatant after separating the biomass, 100 mL of the supernatant

energy efficiency =

energyproducts energyinputs

× 100% (4)

Considering nonrenewable energy sources for the production of highvalue products from microalgae involving the energy required for running equipment and considering energy outputs in products, the energy efficiency can be estimated as

energy efficiency =

energyproducts nonrenewable energyinputs

× 100% (5)

Figure 3. NMR spectrum of synthesized EAN in DMSO-d6 at 200 MHz. 10022


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ACS Sustainable Chemistry & Engineering

Chlorella variabilis due to the presence of large quantity of inorganic and organic nutrient sources present in it. The biomass productivity of 74.96 ± 2.62 g/(m2/d) with lipid yield of 20.1 ± 2.2% (wrt dry biomass) was obtained (Figure 2). Carbon fixation rate was around 141 g/(m2/d). The possible reason may be presence of both organic and inorganic carbon source present in textile effluent used for growth of the Chlorella variabilis. The cultivation was carried out during the summer of 2016, i.e., during the month of June, as biomass productivity of Chlorella variabilis is maximum during that period.45 Also, during the cultivation, there was an increase in pH from the initial day to final day which indicates an increase in the growth of the biomass may be due to bicarbonate uptake. However, biomass productivity varies with light intensity, nutrient supplementation, etc. Benemann, Goebel, and Weissman (1988) obtained 30 g/(m2/d) biomass productivity using Chlorella sp. (Chlorophyceae),58−60 whereas, Liang et al. found 11.2 g/(m2/d) biomass productivity in open ponds using Chlorella vulgaris.61 The lipid productivity was found to be highest on the sixth day (production age 144 h). The maximum total lipid yield of 20.1 ± 2.2% (wrt dry biomass) was obtained. The fatty acids present in the microalgal oil are 8.2% C17:1, 3.3% C15:0, 32.61% C18:3n6, 12.56% C18:0, and 43.4% C16:1. Carbon Utilizing Percentage by Chlorella variabilis from the Culture Medium. During the initial day of cultivation, i.e., just after the addition of the inoculum, 430 g total organic carbon and 20 g total inorganic carbon was present in the media. However, 379.17 g total carbon was utilized by Chlorella variabilis after 3 days and 388.935 g of total carbon was utilized after 6 days wherein maximum biomass was obtained. In total, 86.43% of total carbon was utilized by the Chlorella variabilis with respect to the total carbon present in the medium (Table 1). Bioremediation in Textile Effluent. Reduction of elements present in textile effluent through its accumulation by Chlorella variabilis was observed. It was observed that using Chlorella variabilis (ATCC PTA 12198), the microalgae can remediate 96% of aluminum, 82.72% boron, 45.66% calcium, 98% cobalt, 14.5% potassium, 0.1% magnesium, 42.18% sodium, 94% nickel, and 90% iron present in the textile effluent (Figure 5). Simultaneously, the decrease of 78.17% total phosphate and 25.22% total inorganic phosphate was observed in the effluent through utilizing microalgae Chlorella variabilis (Table 2). However, in most of the reports, bioremediation of textile effluent was carried out using algal consortium or with bacterial isolate,62,63 whereas our process deals with unialgal strain. Also, the present study demonstrates a sustainable model for producing value added products along with bioremediation of textile effluent. Decrease in Dye Concentration during Cultivation of Microalgae. Figure 6 illustrates the decrease in concentration of dye present in the textile effluent during the cultivation of microalgae. However, it is also clear just seeing through the naked eye that there is a decrease in the color from the

Figure 4. (a) 40% textile effluent media prior to inoculation. (b) Cultivation of Chlorella variabilis in open tanks using 40% textile effluent.

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RESULTS AND DISCUSSIONS Mass Cultivation of C. variabilis using Textile Effluent in Open Tanks. The textile effluent was used for growing Table 1. Carbon Utilization Percentage by Chlorella variabilis days 0 3 6

total inorganic carbon present in the media (g)

total oraanic carbon present in the media (g)

total carbon present in the media (g)

total carbon utilized (g)

carbon utilization %

20 ± 0.8

430 ± 3.6

450 ± 2.8

838.2788 ± 3.8 379.17 ± 2.4 388.935 ± 4.2

80.96966695 ± 3.3 84.26455796 ± 2.6 86.43417105 ± 3.5

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Table 3. Total Residual Sugars Obtained from SDS Mediated Hydrolysis of Microalgae

Table 2. Phosphate Utilization by the Microalgae Chlorella variabilis (ATCC PTA 12198) day

total phosphate (mg/L) 11.45 10.76 9.90 8.95 78.17

± ± ± ± ±

1.2 1.4 0.8 0.13 0.7

inorganic phosphate (mg/L) 8.76 6.98 4.53 2.21 25.22

± ± ± ± ±

total sugars (mg) per gram of dry biomass of Chlorella variabilis

1 3 5 7 10

3.68 ± 0.86 41.856 ± 2.6 74.1 ± 6.86 70.8 ± 5.33 68.6 ± 6.4

were obtained from chemical hydrolysis. The microalgal hydrolysate obtained from SDS mediated hydrolysis was subjected to 100 g/L alumina for complete SDS removal and the residual hydrolysate was directly subjected to microbial fermentation for production of ε-polylysine. After complete submerged fermentation having 36 h production age and agitation 220 rpm, fermentation broth was centrifuged to obtain the supernatant containing εpolylysine at a scale of 1 L. The extracellular production of εpolylysine was found to be 1.76 mg/mL. Overall, 1.76 g εpolylysine was produced extracellularly in the fermentation broth utilizing 34g total reducing sugars having 5.18% carbon utilization efficiency. Extraction and Purification of ε-PL using EAN. The supernatant obtained after centrifugation was subjected to 40% ammonium sulfate saturation precipitated 7% of the total protein present in the supernatant; 60% ammonium sulfate saturation precipitated 76.48% of the total protein present in the supernatant; 80% ammonium sulfate saturation precipitated 38.77% of the total protein present in the supernatant. Therefore, 60% ammonium sulfate saturation was considered further for precipitating ε-PL for recovery of maximum ε-PL. The extracellular production of ε-polylysine in fermentation broth was found to be 1.76 mg/mL i.e., 1.76 g of ε-PL was present in the fermentation broth containing microalgal hydrolysate. After ammonium sulfate precipitation, 1.35 g of crude ε-PL was obtained. The pure 1.3 g ε-PL was found to be precipitate as a light yellow powder which was recovered by decanting the residual EAN and drying the precipitate at 60 °C to obtain pure ε-PL powder (Figure 7). Characterization of Isolated ε-PL Using 1H NMR. The peaks showing peptide linkage between α-carboxyl group and the ε-amino group, confirming the structure as ε-polylysine as per the work of Bhattacharya et al.64 Scale up Potential for Generation of Microalgal Biomass by Phytoremediation of Textile Effluent through Biorefinery Approach. The mass balance analysis of γ-linolenic acid production from Chlorella variabilis was studied. In the current context, textile effluent was supplementing the carbon and nitrogen source for the growth of Chlorella variabilis. From 495 g of microalgal biomass, 109.4 g total lipids can be extracted containing 34.65 g γ-linolenic acid. After lipid extraction, SDS mediated hydrolysis of spent microalgal biomass yielded 36.68 g of reducing sugars and protein rich biomass was left containing 9.65g total proteins. The microbial fermentation using obtained hydrolysate containing 36.68 g fermentable sugars along with medium components was carried for obtaining 1.3 g pure ε-polylysine (Figure 8). The energy efficiency calculations were done considering all energy inputs during γ-linolenic acid and ε-polylysine production which includes the energy requirement for process

Figure 5. Reduction of elements in the textile effluent through Chlorella variabilis (PTA 12198).

0 2 4 6 % reduction

concentration of SDS (w/v)

0.8 1.1 0.7 1.05 0.9

Figure 6. Decrease in dye concentration during cultivation of microalgae.

supernatant collected from the initial day culture and the culture having maximum biomass productivity on the sixth day (Figure 6). SDS Mediated Hydrolysis of Deoiled Microalgal Biomass. The total carbon present in spent microalgal biomass was 8% wrt dry spent biomass. For substituting chemical route for hydrolysis of microalgal biomass to a greener route for obtaining microalgal hydrolysate as carbon and nitrogen source, various concentrations of SDS was used. However, the hydrolysis reaction carried out at ambient temperature for 10h using 5% (w/v) SDS yielded 74.1 mg reducing sugars per gram dry microalgal spent biomass (Table 3). Simultaneously, the saccharification yield was also compared through chemical and enzymatic route. After completion of the hydrolysis of dried spent microalgal biomass (obtained after oil extraction) through chemical and enzymatic route, it was found that 25.22 mg reducing sugars were obtained per gram dried spent microalgal biomass through enzymatic hydrolysis and 20 mg of reducing sugars per gram dried spent microalgal biomass 10024


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Figure 7. Extraction and purification of ε-polylysine using ionic liquid ethylammonium nitrate.

Figure 8. Mass balance fluxogram showing green process for microalgal biomass as energy feedstock through biorefinery approach.

Table 4. Economic and Energetic Inputs Used for the Green Metric Calculations economic evaluation input

cost

energetic evaluation input

value

ref

nutrient salt for microalgal cultivation 250 L tank for cultivation electricity cost total microalgal biomass manpower cost

Rs 1.3 per kg biomass Rs 2250 Rs 991 495 g Rs 2500

K-fertilizer nitrate fertilizer phosphate fertilizer HHV microalgae HHV dry Bacillus licheniformis biomass HHV crude reducing sugars

8.036 MJ fossil/kg K 216.956 MJ fossil/kg NO3 6.650 MJ fossil/kg P 16.31 MJ/kg 23.13 kJ/g 16.3 MJ/kg

65 65 65 45 66 67

water cost water pump (2) chemicals

Rs 2.4 Rs 9000 2291.15

equipment, utilities, and fossil energy input. Table 4 shows all the energy inputs including energy accumulated in the product,

and Table 6 shows green metrics value involving E-factor and material efficiency. The analysis of E-factor and material 10025



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efficiency calculated using the real values are in considerable ranges. The cost of γ-linolenic acid and ε-polylysine production were also analyzed and mentioned in Table 5. The revenues

INR

revenue

INR

depreciation (5 y) raw materials utilities labor and other

409 2292.5 991 2500

microalgal oil γ-linolenic acid ε-polylysine protein powder

139 45000 133.776 5670

*Tel.: +91-278-2567760. E-mail: [email protected] (S.M.). *E-mail: [email protected] (A.K.). ORCID

Tejal Gajaria: 0000-0001-5329-6624 Sandhya Mishra: 0000-0002-2412-4927 Author Contributions #

These authors contributed equally to the work.

Notes

The authors declare no competing financial interest.

Table 6. Metrics of Microalgal γ-Linolenic Acid and εPolylysine Evaluated in This Work

a

metric

real value

E-factor material efficiency total energy input γ-linolenic acid production cost ε-polylysine production cost

4.8 × 10−3,a 0.995 × 10−3,a 123.9 kWh 158 INR/g 1453 INR/g

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ACKNOWLEDGMENTS S.B. and S.M. would like to acknowledge CSIR for providing financial support through CSC 0105 and 0203. S.K.P. acknowledges SERB (PDF/2015/000745) for financial support. The authors gratefully acknowledge Dr. Pankaj Pathak, Assistant Professor, Environmental Science and Engineering Department, Marwadi Education Foundation, Rajkot, for arranging the textile effluent from Jetpur. P.S.G. would like to acknowledge UGC for SRF. S.B. acknowledges Kaumeel Chokshi for analyzing the phosphate content in the effluent and in the samples. The authors would like to thank ADCIF, CSIR-CSMCRI, Bhavnagar, for the help during the analysis of the effluent and biomass. BDIM is acknowledged for providing PRIS number CSIR-CSMCRI-152/2016.

Calculated considering the output of residual nutrients as a waste.

from selling the γ-linolenic acid at costs of 1500 and 103 INR per gram along with protein powder and residual algal oil is generating a net gain of 44752 INR. However, the present process is at a demonstration scale, and the energy and cost calculations may vary at pilot scale and manufacturing scale. Based on the experimental data, it can be stated that based on material efficiency and economic assessment, the developed process may be feasible and has a good scope for its scalability at 1 ton scale utilizing the textile effluent as a nutrient medium for producing γ-linolenic acid and ε-polylysine.

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REFERENCES

(1) Chia, A. M.; Lombardi, A. T.; Melao, M. G. G.; Parrish, C. C. Lipid composition of Chlorella vulgaris (Trebouxiophyceae) as a function of different cadmium and phosphate concentrations. Aquat. Toxicol. 2013, 128, 171−182. (2) Puvaneswari, N.; Muthukrishnan, J.; Gunasekaran, P. Toxicity Assessment and microbial degradation of Azo dye. Indian J. Exp. Biol. 2006, 44, 618−626. (3) Robinson, T.; Mcmullan, G.; Marchant, R.; Nigam, P. Remediation of dyes in textile effluent: a critical review on current treatment technologies with a proposed alternative. Bioresour. Technol. 2001, 77, 247−255. (4) Rocha, A. A., Ed. Algae and the environment: a general approach; Sociedade Brasileira de Ficologia: São Paulo, 1992. (5) Knasmüller, S.; Zöhrer, E.; Kainzbauer, E.; Kienzl, H.; Colbert, B.; Lamprecht, G.; Schulte-Hermann, R. Detection of mutagenic activity in textiles with Salmonella typhimurium. Mutat. Res., Genet. Toxicol. Test. 1993, 299, 45−53. (6) Jäger. Research Feasibility Study, Hydrotox GmbH, report to the European Commission, 1998. (7) Mathur, N.; Bhatnagar, P.; Sharma, P. Review of the mutagenicity of textile dye products. Universal J. Environ. Res. Technol. 2012, 2 (2), 1−18. (8) Srivastava, S. K.; Kumar, R.; Srivastava, A. K. Effect of textile industry effluents on the biology of river Tons at Mau (U.P.) I Physicochemical characteristics. Poll. Res. 1994, 13 (4), 369−373. (9) Raj, G. B.; Patnaik, M. C.; Subbaiah, V. V. Heavy metal pollution in soil and fodder (paragrass) irrigated with sewage and effluent water all along Musi River. Proc. Acad. Environ. Biol. 1997, 6 (2), 177−182. (10) Khandelwal, S. Impact of dyeing industries, wastewater on vegetation of Luni catchment area: A case study through remote sensing technique. J. Environ. Poll. 1996, 3 (2), 77−78. (11) Chia, M. A.; Musa, R. I. Effect of indigo dye effluent on the growth, biomass production and phenotypic plasticity of Scenedesmus quadricauda (Chlorococcales). An. Acad. Bras. Cienc. 2014, 86 (1), 419−428.

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CONCLUSION One of the major environmental issues with the textile industry sector is the disposal of their effluent containing unreacted dyes and high concentration of salts. Most of the textile effluents consist of a high concentration of bicarbonate salts which is an important substrate for the growth of Chlorella sp. In the present study, Chlorella variabilis was grown in open tanks at a scale of 100 L using 40% textile effluent for generating microalgal biomass containing γ-linolenic acid which is an important nutraceutical and generally added into the cooking oils. A total of 495 g microalgal biomass was generated containing 34.65 g γ-linolenic acid. A 36.68 g portion of fermentable sugars was extracted from the deoiled microalgal biomass for preparing 1.3 g ε-polylysine which has various biomedical applications in the pharmaceutical sector.

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AUTHOR INFORMATION

Corresponding Authors

Table 5. Cost and Revenues of Microalgal γ-Linolenic Acid and ε-Polylysine cost per batch

Research Article

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S Supporting Information *

The Supporting Information is available free of charge via the Internet at The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acssuschemeng.7b01961. 1 H NMR spectrum in chloroform-d and 13C NMR spectrum in chloroform-d. The obtained microalgal PUFA fraction of Chlorella variabilis after silver-silica gel column chromatography was found to be γ-linolenic acid after its characterization through 1H NMR and 13C NMR (PDF) 10026


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Process for Preparing Value-Added Products from Microalgae Using

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