Process for Preparing Value-Added Products from Microalgae Using

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Process for Preparing Value Added Products from Microalgae Using Textile Effluent Through Biorefinery Approach Sourish Bhattacharya, Sumit Kumar Pramanik, Praveen Singh Gehlot, Tejal Gajaria, Himanshu Patel, Sandhya Mishra, and Arvind Kumar ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b01961 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 7, 2017

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ACS Sustainable Chemistry & Engineering

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Process for preparing value added products from microalgae using textile

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effluent through biorefinery approach

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Sourish Bhattacharya1,5#, Sumit Kumar Pramanik2#, Praveen Singh Gehlot3,5#,

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Himanshu Patel1,5, Tejal Gajaria4,5, Sandhya Mishra3*, Arvind Kumar3*

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Research Institute, Bhavnagar-364002, Bhavnagar - 364002, India.

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Marine Chemicals Research Institute, G. B. Marg, Bhavnagar, Gujarat - 364002,

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India.

Process Design and Engineering Cell, CSIR- Central Salt and Marine Chemicals

Analytical Division and Centralized Instrument Facility, CSIR-Central Salt and

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Research Institute, Bhavnagar-364002, India.

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Chemicals Research Institute, Bhavnagar-364002, Bhavnagar - 364002, India.

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Academy of Scientific and Innovative Research (AcSIR), Bhavnagar - 364002, India.

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#

These authors contributed equally to the work.

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*Corresponding Authors. Tel: +91-278-2567760. Email: [email protected] (Dr.

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Sandhya Mishra); [email protected] (Dr. Arvind Kumar)

Salt & Marine Chemicals Division, CSIR- Central Salt and Marine Chemicals

Marine Biotechnology and Ecology Division, CSIR- Central Salt and Marine

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Abstract

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A model was designed for effective utilization of textile effluent as the nutrient

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medium for the production of high-value products from Chlorella variabilis through

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greener approach. Biomass productivity of 74.96±2.62 g/m2/d with lipid yield of

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20.1±2.2% (w.r.t. dry biomass) was obtained using textile effluent as the nutrient

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source. A novel integrated process based on detergent (sodium dodecyl sulphate)

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based hydrolysis to convert the carbohydrates present in microalgal biomass to

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reducing sugars for microbial fermentation, while making available lipids for

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downstream processing of γ- linolenic acid, leaving protein rich fragment behind. Our

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experimental data showed that from 495 g of microalgal biomass, 109.4 g total lipids

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was extracted containing 34.65 g γ- linolenic acid, 1.3 g pure ε-polylysine from 36.68

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g of reducing sugars. A two-step efficient green process was developed for recovering

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ε-polylysine using ethyl ammonium nitrate having 74 % recovery. In addition to value

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added products, CSIR-CSMCRI’s Chlorella variabilis (ATCC PTA 12198) can

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remediate 100% of aluminium, 82.72% boron, 45.66% calcium, 100% cobalt, 14.5%

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potassium, 0.1% magnesium, 42.18% sodium, 100% nickel and 100% iron. A total

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decrease of 78.17 % total phosphate and 25.22% total inorganic phosphate with

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respect to total phosphate present in the effluent was observed.

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Keywords: Textile effluent, Chlorella variabilis, unreacted dyes, bicarbonate, γ-

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linolenic acid.

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Introduction

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Industrialization renders an important functioning in the growth of the country. Textile

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industry is an important and fast growing industrial sector which is also essential for any

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growing economy of the country. Any particular textile industry uses various types of raw

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materials such as cotton, woollen and synthetic fibres.

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categorized into two, i.e., dry and wet fabric industry. Solid wastes are generated in dry fabric

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industries, whereas wet fabric industries utilize lot of water generating a lot of waste water

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containing salts and unreacted dyes1. However, the final disposal of this effluent in solid

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form (sludge) or liquid form is still a challenge, as even after treatment through conventional

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techniques, it doesn’t decolorize and detoxify the dye effluents2. However, the textile dyes

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make water toxic3 and unsafe for human and animal consumption. At the same time, it causes

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an imbalance within the different aquatic ecosystem and can serve as the mutagenic agent

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which is harmful to the ecosystem4-7. Simultaneously, the discharge of effluents pollutes

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rivers, affecting soil properties as well as the growth of plants along with the biodiversity8-10.

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Various researchers have performed various studies about reduction in dye concentration as

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well as remediation of salts present in the textile effluent through microalgae. Chia and Musa,

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2013 was able to reduce concentration of indigo dye present in the textile effluent by growing

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Scenedesmus quadricanda ABU12 in the textile effluent. Furthermore, removal at a

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concentration of 17.5% with respect to total dye colour and chemical oxygen demand (COD)

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level was done utilizing Chlorella vulgaris12. Simultaneously, 73% of the total dye present in

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textile effluent collected from Karur unit, Tamil Nadu13. Prabina & Kumar, 2013 developed a

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process for decolorizing the dye present in the textile effluent by growing microalgal

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consortium in the effluent14. The consortium consists of Anabaena sp., Nostoc sp. and

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Chlorella sp. which when grown in the effluent, removed 90% of dye.

The textile industries may be



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Microalgae such as Chlorella, Scenedesmus and Ankistrodesmus species possess potential to

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biodegrade organic pollutants present in oil mill wastewater and paper industry wastewater15-

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accumulation by physical adsorption, ion exchange and chemisorption, covalent bonding,

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surface precipitation, redox reactions or crystallization on the cell surface20-27. Microalgae

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play a key role in nutrient removal through assimilating it as they require high nitrogen and

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phosphorus for proteins, nucleic acid and phospholipids synthesis inside the cell28-32. Another

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important application of microalgae is in pollutant removal as certain green microalgae and

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cyanobacteria can use toxic recalcitrant compounds as carbon, nitrogen, sulphur or

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phosphorous source for its growth33,34. Chlorococcum vitiosum has the potential to remediate

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COD at a concentration of 13% along with complete removal of the heavy metals35.

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In-addition to the reduction of COD and dye concentration in the textile effluent, researchers

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have succeeded in cultivating microalgae in high rate algal ponds (HRAP). Chlorella vulgaris

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was cultivated in HRAP (dimensions - 1m x 0.5m x 0.3m; agitation 15 rpm using paddle

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wheels) using textile effluent containing Supranol Red B3W dye for generation of 106.7 mg/l

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biomass biomass36. However, it has been reported that live algae as well as non-viable algae

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(dried algal mats) have been used in the reduction of dyes present in effluent37. The

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mechanism involves both biosorption and bioconversion. Maurya et al., 2014 reported

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utilizing non-viable microalgal mat for reducing methylene blue dye. Simultaneously, non-

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viable Spirogyra biomass can be utilized as an important biosorbent for removal of Synazol

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dye present in wastewater39,40. Chlorella vulgaris can remove around 69% of the colour

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through converting mono- azo- dyes such as tectilon yellow 2G to aniline40. Such potentiality

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of microalgae for removing reactive dyes present in the effluent can be done through

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manipulating the microalgal growth. In the present study, a model was designed for effective

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reduction of chemicals as well as residual dyes present in the textile effluent. The chemicals

. Simultaneously microalgae also have the potential to remediate heavy metals through its


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present in the textile effluent are utilized as the nutrient for the growth of Chlorella variabilis,

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and at the same time, the unreacted dyes reduced through microalgae making the effluent

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safer for its discharge.

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One of the major challenges related with the algal biofuel production in a biorefinery

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approach is improving biomass utilization for net energy gain providing economically viable

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and scalable process for deriving commercially important co-products through a greener

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route. Laurens et al., 2015 demonstrated an integrated technology based on moderate

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temperature and low pH (controlled microwave pretreatment) to convert the carbohydrate in

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wet algal biomass to soluble sugars for fermentation, while, making lipids more accessible

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for downstream extraction and leaving a protein-enriched fraction behind41. However, there

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are several hydrolysis techniques being developed by the researchers for past few decades,

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but the most common and green process available for hydrolysis of carbohydrates from, e.g.,

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cellulosic biomass, microalgal biomass is the use of enzymes42. Researchers have developed

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few low-cost ionic liquids for hydrolysis of lignocellulosic biomass43, its economics about the

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scalable process is still in question. However, ionic liquids can be used for the extraction and

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purification of high-value biopolymers from algal source.

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This work aimed to investigate the potential of carbohydrate containing biomass of Chlorella

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variabilis grown mixotrophically using textile waste as a substrate for ε-polylysine

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production along with microalgal lipids containing γ-linolenic acid - a nutraceutical. To the

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best knowledge of the authors, no published work exists wherein the hydrolysis of Chlorella

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biomass was performed using sodium dodecyl sulphate (SDS) – a detergent and extraction

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and purification of ε-polylysine performed using ionic liquid, e.g. ethyl ammonium nitrate.

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Furthermore, the microalgal biomass was grown using textile effluent along with critical

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nutrients yielded 74.96±2.62 g/m2/d with total lipid yield of 20.1±2.2%.



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Experimental Section

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Culture maintenance of microalga Chlorella variabilis PTA 12198

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Oleaginous CSIR - CSMCRI’s Chlorella variabilis (ATCC PTA 12198) was isolated from

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Diu, India (N 20° 41.341'; E70°53.734') and was maintained as a monoalgal culture in

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modified Zarrouk’s media44 as per Bhattacharya et al., 201645.

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Inoculum development

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The inoculum was initially prepared in 10L carboys (Zarrouk’s medium, 25±5 °C) from a 1L

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Erlenmeyer flask (Zarrouk’s medium, 700-800 lux, 25±5 °C) in the CSMCRI-P2 medium as

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per Bhattacharya et al., 2016. The air temperature during daytime was 46±3°C and during the

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night was 35±3 °C during May’ 2016.

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Microalgal cultivation

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CSMCRI’s terrace (21°75.92783’ N; 72°14.41304’ E; Elevation 121 ft.) was chosen as the

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mass cultivation site. The cultures were agitated manually three times a day.

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collected from textile industry diluted with tap water in the proportion of 3:7 due to high

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alkalinity of the effluent. One plastic tank having area 1.1m x 1.1 m with depth 0.085m was

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used for the cultivation of Chlorella variabilis (ATCC PTA 12198) utilizing textile effluent.

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The availability of abundant sunlight and prevailing high temperature conditions during the

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day were the other factors favourable for the selected strain.

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The Chlorella variabilis was grown in the tank using 40% textile effluent with tap water. The

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previously described inoculum raising tank was supplemented with CSMCRI-P2 medium

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consisting of g/l NaHCO3 5; NaNO3 1.2; K2HPO4 0.25; K2SO4 0.25; NaCl 1.0; CaCl2 0.04;

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Na2EDTA 0.08; MgSO4.7H2O 0.1; FeSO4.7H2O 0.01.

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Mass cultivation

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The cultivation was carried out during the peak summer season (June’ 2016) in Gujarat, India

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with a 46±3°C ambient air temperature. The water temperature range was 43 ± 3ºC during the 6 ACS Paragon Plus Environment

Effluent

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entire cultivation. The desired culture for the mass cultivation needed was initially grown in

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inoculum raising tank with an area of 0.35 m2 each. The mass cultivation tank was monitored

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regularly by measuring the OD at 540 nm using UV-visible spectrophotometer (Cary Bio 50,

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Varian Inc., USA) and biomass yield was measured by measuring gravimetrically the dry

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biomass obtained after centrifugation of the culture of C. variabilis.

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The open cultivation of Chlorella variabilis (ATCC PTA 12198) was carried out from 13th to

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18th June’ 2016 having average solar irradiation of 4.8 KWh/m2/d. The air temperature during

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day time was 46±3°C and during the night was 43±3 °C during June’ 2016.

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A cell concentration of 2g/L (wet basis) was used to inoculate the tank with an area of 1.2 m2.

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The biomass yield and total lipid yield were monitored on a regular basis. The agitation of the

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ponds was done manually three times a day.

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The textile effluent collected from western overseas, Jetpur, Gujarat. The textile effluent

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consists of 0.2 g/L inorganic carbon and 4.3 g/L organic carbon in the form of sodium and

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ammonium bicarbonate having a pH of 12.11±1.2 which was utilized for the growth of

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Chlorella variabilis. The composition of textile effluent obtained from the textile mill at

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Jetpur, Rajkot, Gujarat contains (ppm) total dissolved solids 8.13, ammonium ion 5.8, nitrate

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24.1, salinity 7.7, aluminium 52, boron 1200, cobalt 60, calcium 894, chromium 4, iron 0.5,

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potassium 2300, magnesium 441, manganese 1, sodium 5368, zinc 1, lead 2 and nickel 16.1.

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Due to high alkaline concentration, the effluent was mixed with tap water at a proportion of

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4:6 ratio i.e 40 L of textile effluent is mixed with 60L fresh water, followed by addition of P2

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medium containing (g/L) K2HPO4 0.25, NaNO3 1.2, K2SO4 0.25, NaCl 1.0, MgSO4.7H2O

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0.2, EDTA 0.08, CaCl2 0.04, FeSO4 0.01. It addition to components of P2 medium, media

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also consists aluminium 2.08 g, boron 40 g, cobalt 2.4 g, calcium 2.679 g, iron 0.02 g,

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potassium 92 g, magnesium 17.64 g, manganese 0.04 g, sodium 214.72 g, zinc 0.04 g, lead

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0.08 g, Nickel 25.764 g and chromium 0.16 g which came directly from the effluent being 7 ACS Paragon Plus Environment


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added. Thereafter, 40L of Chlorella variabilis culture added to the medium as an inoculum

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(Fig. 4a, 4b).

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Fig. 4a 40% textile effluent media prior to inoculation.

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Fig. 4b Cultivation of Chlorella variabilis in open tanks using 40% textile effluent.

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Lipid estimation

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The lipid content was quantified gravimetrically from the sun-dried biomass46. The

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microalgal lipid was extracted three times to obtain clear extracts using 10 ml of Chloroform

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and Methanol (1:2 v/v) in one gram biomass. The pooled extracts were filtered to remove the

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cell debris. The filtered extract was evaporated under vacuum to dryness at 55 °C using a 8 ACS Paragon Plus Environment

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Büchi rotary evaporator.

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Fatty acid profiling

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The Chlorella variabilis methyl esters (CvME) were obtained from the lipid using 1 mL of

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1% sodium hydroxide in methanol, followed by heating at 55 °C for 15 min. Thereafter, 5%

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of 2 ml methanolic HCl was added with heating at 55 °C for 15 min47. The prepared FAMEs

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were then separated by adding 1 ml of hexane to the reaction mixture. The FAMEs

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containing hexane were analyzed by a GC-2010 gas chromatograph coupled with a mass

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spectrometer (GC–MS QP-2010, Shimadzu, Japan). The FAMEs were analyzed through a

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gas chromatography mass spectrometer (GC-2010 twinned with a GC– MS QP-2010) from

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Shimadzu (Japan). An RTX-5-fused silica capillary column (30 m x 0.25 mm, 0.25 µm)

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maintained a flow rate of 1 mL/min and a pre-column pressure of 49.7 kPa with Helium as a

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carrier gas. The column temperature regime was 40 °C for 3 min, followed by an increase at a

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rate of 5 °C/min up to 230 °C, and then maintained at 230 °C for 40 min. The injection

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volume and temperature were 1.0 µL and 240 °C, respectively, with a split ratio of 1/30. The

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mass spectrometer operated in electron compact mode with 70 eV of electron energy. The ion

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source and the interface temperature were set at 200 °C. The peaks were compared with the

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standards with respect to their retention times (Standard FAME Mix C4–C24; Sigma Aldrich)

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by GCMS post-run analysis and quantified by area normalization.

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Elemental composition of biomass

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The elemental (C, H, N, S) composition (%) of dried biomass (90O C for 24 h in the oven)

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was analysed by the CHNS analyzer (elementarvario Micro) and, sulphanilamide was used as

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a reference standard48. The measured values of the standard had 150 ºC through thermogravimetric analysis (TGA).

C . However, EAN was found to be showing a good thermal stability at a temperature

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Fig. 2 DSC of synthesized Ethyl ammonium nitrate.

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Moisture analysis

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The moisture content of the ionic liquid was measured using Karl Fisher (KF) titrator (890

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titrando, Metrohm-Switzerland). Synthesized EAN was found to be having only 1.27%

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moisture content.

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NMR 13 ACS Paragon Plus Environment


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Chemical Shift (δ) value in ppm, 1.15 (t, 3H), 2.85 (m, 2H), 7.76 (s, broad due to N, 3H).

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

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Downstream processing of ε-PL

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Ammonium sulphate precipitation

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In order to precipitate the total protein present in the supernatant after separating the biomass,

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100 ml of the supernatant was subjected to 40 %, 60 % and 80 % saturated ammonium

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sulphate solution at 4 oC.

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Salting out

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Ammonium sulphate and other salts were removed through dialysis (5 KDa membrane) by

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using 0.1M tris HCl buffer solution to obtain crude ε-PL.

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Purification of ε-polylysine using Ethyl ammonium nitrate (EAN)

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The crude ε-PL extract obtained after ammonium sulphate precipitation was subjected to

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lyophilization for obtaining the dry powder containing ε-PL. The dried material containing ε-

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PL was completely dissolved in 10 ml EAN and stirred at 80 rpm for 15 min. at 60 °C to


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dissolve completely 1.35 g ε-PL present in the crude extract and kept immediately at -20 °C

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for 4 h for precipitation of ε-PL. Finally, the ionic liquid (EAN) was decanted, and the

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precipitate was dried for obtaining pure ε-PL.

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Green Metrics

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With reference to waste utilization for producing value added products, the greenness of the

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developed process was estimated as per Lukasik et al., 201357. The E-factor can be calculated

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as

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E − factor =

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Material efficiency can be calculated as

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! " = $%&'()*+,#

#

………….. Equation (1)


313

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-'.. *& /+*01().

= -'.. *& /+*01().,-'.. *& 2'.)3.


315

316

Basically, energy efficiency is having an important impact in all major biochemical processes

317

and the evaluation of new and existing processes. The energy efficiency can be calculated as

318

per below mentioned equation.

319

4!5" ! " =

$63+789:;?@ $63+78AB9=?@

× 100%


320



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 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Considering non-renewable energy sources for the production of high-value products from

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microalgae involving the energy required for running equipment and considering energy

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outputs in products, the energy efficiency can be estimated as,

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4!5" ! " = G*6%+3632'HI3 363+78

$63+789:;?@

× 100% ………….. Equation (5) AB9=?@

325

Results and Discussions

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Mass cultivation of C. variabilis using textile effluent in open tanks

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The textile effluent was used for growing Chlorella variabilis due to the presence of large

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quantity of inorganic and organic nutrient sources present in it.

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The biomass productivity of 74.96±2.62 g/m2/d with lipid yield of 20.1±2.2 % (w.r.t. dry

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biomass) was obtained (Fig. 2). Carbon fixation rate was around 141 g/m2/d. The possible

331

reason may be presence of both organic and inorganic carbon source present in textile

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effluent used for growth of the Chlorella variabilis. The cultivation was carried out during

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summer’ 2016, i.e., during the month of June’ 2016 as biomass productivity of Chlorella

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variabilis is maximum during that period45. Also, during the cultivation, there was an

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increase in pH from the initial day to final day which indicates an increase in the growth of

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the biomass may be due to bicarbonate uptake. However, biomass productivity varies with

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light intensity, nutrient supplementation, etc. Benemann, Goebel, and Weissman (1988)

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obtained 30 g/m2/d biomass productivity using Chlorella sp. (Chlorophyceae)58-60, whereas,

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Liang et al., 2013 found 11.2 g/m2/d biomass productivity in open ponds using Chlorella

340

vulgaris61.

341

The lipid productivity was found to be highest on the 6th day (production age 144h). The

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maximum total lipid yield of 20.1±2.2% (w.r.t. dry biomass) was obtained. The fatty acids


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present in the microalgal oil are 8.2% C17:1, 3.3% C15:0, 32.61% C18:3n6, 12.56% C18:0

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and 43.4% C16:1.

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Carbon utilizing percentage by Chlorella variabilis from the culture medium

346

During the initial day of cultivation, i.e., just after the addition of the inoculum, 430g total

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organic carbon and 20 g total inorganic carbon was present in the media. However, 379.17g

348

total carbon was utilized by Chlorella variabilis after 3 days and 388.935g of total carbon

349

was utilized after 6 days wherein maximum biomass was obtained. In total, 86.43% of total

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carbon was utilized by the Chlorella variabilis with respect to the total carbon present in the

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medium (Table 1).

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Table 1 Carbon utilization percentage by Chlorella variabilis

353 354

Bioremediation in textile effluent

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Reduction of elements present in textile effluent through its accumulation by Chlorella

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variabilis was observed. It was observed that using Chlorella variabilis (ATCC PTA 12198),

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the microalgae can remediate 96% of aluminium, 82.72% boron, 45.66% calcium, 98%

358

cobalt, 14.5% potassium, 0.1% magnesium, 42.18% sodium, 94% nickel and 90% iron

359

present in the textile effluent(Fig. 5). Simultaneously, the decrease of 78.17 % total

360

phosphate and 25.22% total inorganic phosphate was observed in the effluent through 17 ACS Paragon Plus Environment


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utilizing microalgae Chlorella variabilis (Table 2). However, in most of the reports,

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bioremediation of textile effluent was carried out using algal consortium or with bacterial

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isolate62-63, whereas our process deals with unialgal strain. Also, the present study

364

demonstrates a sustainable model for producing value added products along with

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bioremediation of textile effluent.

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367

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Table 2 Phosphate utilization by the microalgae Chlorella variabilis (ATCC PTA 12198) Day

Total phosphate (mg/L)

Inorganic Phosphate (mg/L)

0

11.45±1.2

8.76±0.8

2

10.76±1.4

6.98±1.1

4

9.90±0.8

4.53±0.7

6

8.95±0.13

2.21±1.05

% Reduction

78.17±0.7

25.22±0.9

369

370

371

372 373

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

374

Decrease in Dye concentration during cultivation of microalgae


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375

Fig. 6 illustrates the decrease in concentration of dye present in the textile effluent during the

376

cultivation of microalgae. However, it is also clear just seeing through the naked eye that

377

there is a decrease in the colour from the supernatant collected from the initial day culture

378

and the culture having maximum biomass productivity on the 6th day (Fig. 6).

379 380

Fig. 6 Decrease in dye concentration during cultivation of microalgae

381

Sodium dodecyl sulphate (SDS) mediated hydrolysis of deoiled microalgal biomass

382

The total carbon present in spent microalgal biomass was 8% w.r.t. dry spent biomass. For

383

substituting chemical route for hydrolysis of microalgal biomass to a greener route for

384

obtaining microalgal hydrolysate as carbon and nitrogen source, various concentrations of

385

SDS was used. However, the hydrolysis reaction carried out at ambient temperature for 10h

386

using 5% (w/v) SDS yielded 74.1 mg reducing sugars per gram dry microalgal spent biomass

387

(Table 3). Simultaneously, the saccharification yield was also compared through chemical

388

and enzymatic route. After completion of the hydrolysis of dried spent microalgal biomass

389

(obtained after oil extraction) through chemical and enzymatic route, it was found that 25.22

390

mg reducing sugars were obtained per gram dried spent microalgal biomass through

391

enzymatic hydrolysis and 20 mg of reducing sugars per gram dried spent microalgal biomass

392

were obtained from chemical hydrolysis. The microalgal hydrolysate obtained from SDS

393

mediated hydrolysis was subjected to 100 g/l alumina for complete SDS removal and the



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 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

394

residual hydrolysate was directly subjected to microbial fermentation for production of ε-

395

polylysine.

396

Table 3 Total residual sugars obtained from SDS mediated hydrolysis of microalgae Concentration of SDS (w/v)

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

1

3.68±0.86

3

41.856±2.6

5

74.1±6.86

7

70.8±5.33

10

68.6±6.4

397 398

After complete submerged fermentation having 36 h production age and agitation 220 rpm,

399

fermentation broth was centrifuged to obtain the supernatant containing ε-polylysine at a

400

scale of 1L. The extracellular production of ε-polylysine was found to be 1.76 mg/ml.

401

Overall, 1.76 g ε-polylysine was produced extracellularly in the fermentation broth utilizing

402

34g total reducing sugars having 5.18% carbon utilization efficiency.

403

Extraction and purification of ε-PL using EAN

404

The supernatant obtained after centrifugation was subjected to 40 % ammonium sulphate

405

saturation precipitated 7 % of the total protein present in the supernatant; 60 % ammonium

406

sulphate saturation precipitated 76.48 % of the total protein present in the supernatant; 80 %

407

ammonium sulphate saturation precipitated 38.77 % of the total protein present in the

408

supernatant. Therefore, 60 % ammonium sulphate saturation was considered further for

409

precipitating ε-PL for recovery of maximum ε-PL. The extracellular production of ε-

410

polylysine in fermentation broth was found to be 1.76 mg/mL i.e., 1.76 g of ε-PL was present 20 ACS Paragon Plus Environment

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411

in the fermentation broth containing microalgal hydrolysate. After ammonium sulphate

412

precipitation, 1.35 g of crude ε-PL was obtained. The pure 1.3 g ε-PL was found to be

413

precipitate as a light yellow powder which was recovered by decanting the residual EAN and

414

drying the precipitate at 60 °C to obtain pure ε-PL powder (Fig. 7).

415 416

Fig. 7 Extraction and purification of ε-polylysine using ionic liquid Ethyl ammonium nitrate

417

Characterization of isolated ε-PL using 1H NMR

418

The peaks showing peptide linkage between α-carboxyl group and the ε-amino group,

419

confirming the structure as ε-polylysine as per Bhattacharya et al., 201664.

420

Scale up potential for generation of microalgal biomass by phytoremediation of textile

421

effluent through biorefinery approach.

422

Mass balance analysis of γ- linolenic acid production from Chlorella variabilis was studied.

423

In the current context, textile effluent was supplementing the carbon and nitrogen source for

424

the growth of Chlorella variabilis. From 495 g of microalgal biomass, 109.4 g total lipids can

425

be extracted containing 34.65 g γ- linolenic acid. After lipid extraction, SDS mediated

426

hydrolysis of spent microalgal biomass yielded 36.68 g of reducing sugars and protein rich

427

biomass was left containing 9.65g total proteins. The microbial fermentation using obtained 21 ACS Paragon Plus Environment


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 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

428

hydrolysate containing 36.68 g fermentable sugars along with medium components was

429

carried for obtaining 1.3 g pure ε-polylysine (Fig. 8).

430 431

Fig. 8 Mass balance fluxogram showing green process for microalgal biomass as energy

432

feedstock through biorefinery approach.

433

The energy efficiency calculations were done considering all energy inputs during γ-

434

linolenic acid and ε-polylysine production which includes the energy requirement for process

435

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

436

energy accumulated in the product and table 6 shows green metrics value involving E-factor

437

and material efficiency. The analysis of E-factor and material efficiency calculated using the

438

real values are in considerable ranges. The cost of γ- linolenic acid and ε-polylysine

439

production were also analyzed and mentioned in table 5. The revenues from selling the γ-

440

linolenic acid at the cost of 1500 INR per gram and 103 INR per gram along with protein

441

powder and residual algal oil is generating a net gain of 44752 INR. However, the present

442

process is at a demonstration scale, and the energy and cost calculations may vary at pilot

443

scale and manufacturing scale. 22 ACS Paragon Plus Environment

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444

Based on the experimental data, it can be stated that based on material efficiency and

445

economic assessment, the developed process may be feasible and has a good scope for its

446

scalability at 1ton scale utilizing the textile effluent as a nutrient medium for producing γ-

447

linolenic acid and ε-polylysine.

448

Table 4 The economic and energetic inputs used for the green metric calculations Economic evaluation input

Cost

Nutrient salt for microalgal cultivation 250L tank for Cultivation Electricity cost

Rs. 1.3 per Kg Biomass

Total microalgal biomass Manpower cost

495gm

Water cost Water pump (2) Chemicals

Rs. 2250 Rs. 991

Rs. 2500

Energetic evaluation input K-Fertilizer

Nitrate Fertilizer Phosphate Fertilizer HHV microalgae HHV dry Bacillus licheniformis biomass HHV crude reducing sugars

Value

ref

8.036 MJfossil (kg K)−1

65

216.956 MJfossil (kg NO3)−1 6.650 MJfossil (kg P)−1 16.31 MJ/kg

65

23.13 kJ/g

66

16.3 MJ kg−1

67

Rs. 2.4 Rs. 9000 2291.15

449

450

Table 5 The cost and revenues of microalgal γ- linolenic acid and ε-polylysine Cost per batch

INR

Revenue

INR

Depreciation (5 years) Raw materials Utilities Labour and other

409

Microalgal oil

139

2292.5 991 2500

γ- linolenic acid ε-polylysine Protein powder

45000 133.776 5670

451 23 ACS Paragon Plus Environment

65

45


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 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

452

Table 6 Metrics of microalgal γ- linolenic acid and ε-polylysine evaluated in this work Metric

Real value

E-factor

4.8 х 10-3 a

Material

0.995 10-3 b

efficiency Total energy

123.9 KWH

input γ- linolenic acid

158 INR per

production cost

gram γlinolenic acid

ε-polylysine

1453 INR per

production cost

gram

453

a,b

454

Conclusion

455

One of the major environmental issues with the textile industry sector is the disposal of their

456

effluent containing unreacted dyes and high concentration of salts. Most of the textile

457

effluents consists of a high concentration of bicarbonate salts which is an important substrate

458

for the growth of Chlorella sp. In the present study, Chlorella variabilis was grown in open

459

tanks at a scale of 100L using 40% textile effluent for generating microalgal biomass

460

containing γ- linolenic acid which is an important nutraceutical and generally added into the

461

cooking oils. A total of 495 g microalgal biomass was generated containing 34.65 g γ-

462

linolenic acid. 36.68 g of fermentable sugars was extracted from the deoiled microalgal

463

biomass for preparing 1.3 g ε-polylysine which has various biomedical application in the

464

pharmaceutical sector.

Calculated considering the output of residual nutrients as a waste


Page 24 of 36

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465

Associated content

466

Supporting Information

467

1

468

signals from the fatty acid, δ 3.24 (single peak from methylic hydrogen of ester), δ 2.36

469

(triplet from methylenic group between olefinic hydrogen); δ 1.89 (triplet from methylenic

470

groups in δ position with respect to carbonylic group); δ 1.65-1.62 (multiplet from

471

methylenic group in both sides of olefinic hydrogen); δ 1.22-1.20 (multiplet from methylenic

472

group on δ position with respect to carbonylic group); δ 0.97-0.86 (signal from methylenic

473

groups in fatty acid chain); and δ 0.48 (triplet from terminal methyl group).

474

H NMR spectrum in chloroform-d, multiplet peaks at δ 5.34.98-4.89 belong to olefinic

13

C NMR spectrum in chloroform-d, δ 174.10, 130.15, 128.07, 51.37, 34.08, 31.57, 29.62,

475

29.39, 29.20, 27.21, 25.65, 24.97, 22.61, 14.07.

476

Obtained microalgal PUFA fraction of Chlorella variabilis after Silver-silica gel column

477

chromatography was found to be γ-linolenic acid after its characterization through 1H NMR

478

and

479

http://pubs.acs.org.

480

AUTHOR INFORMATION

481

*Corresponding Authors

482

Tel/Fax: +91-278-2567760.

483

Email: [email protected] (Dr. Sandhya Mishra);

484

Email: [email protected] (Dr. Arvind Kumar).

485

ORCID ID Sandhya Mishra: 0000-0002-2412-4927

486

ORCID ID Arvind Kumar: 0000-0001-9236-532X

487

ORCID ID Praveen Singh Gehlot: 0000-0002-5569-8296

13

C NMR. The Supporting Information is available free of charge via the Internet at



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Page 26 of 36

488

ORCHID ID Sourish Bhattacharya: 0000-0001-7257-1994

489

Notes

490

The authors declare no competing financial interest.

491

Acknowledgements

492

SB and SM would like to acknowledge CSIR for providing financial support through CSC

493

0105 & 0203. SKP acknowledges SERB (PDF/2015/000745) for financial support.

494

authors gratefully acknowledge Dr. Pankaj Pathak, Assistant Professor, Environmental

495

Science and Engineering Department, Marwadi Education Foundation, Rajkot for arranging

496

the textile effluent from Jetpur. PSG would like to acknowledge UGC for SRF. SB

497

acknowledges Kaumeel Chokshi for analyzing the phosphate content in the effluent and in

498

the samples. The authors would like to thank ADCIF, CSIR-CSMCRI, Bhavnagar for the

499

help during the analysis of the effluent and biomass. BDIM is acknowledged for providing

500

PRIS number CSIR-CSMCRI – 152/2016.

501

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TOC Graphic 600 DPI with 9 X 15 cm

706 707

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Synopsis: A sustainable model was designed for effective utilization of textile effluent as

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a nutrient medium for the production of high-value products from Chlorella variabilis

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through greener approach.

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Process for Preparing Value-Added Products from Microalgae Using

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