A novel method to harvest Chlorella sp. via low cost bioflocculant: Influence of temperature with kinetic and thermodynamic functions

Accepted Manuscript A novel method to harvest Chlorella sp. via low cost bioflocculant: Influence of temperature with kinetic and thermodynamic functions Richa Kothari, Vinayak V. Pathak, Arya Pandey, Shamshad Ahmad, Chandni Srivastava, V.V. Tyagi PII: DOI: Reference:

S0960-8524(16)31565-6 http://dx.doi.org/10.1016/j.biortech.2016.11.050 BITE 17296

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

19 September 2016 11 November 2016 12 November 2016

Please cite this article as: Kothari, R., Pathak, V.V., Pandey, A., Ahmad, S., Srivastava, C., Tyagi, V.V., A novel method to harvest Chlorella sp. via low cost bioflocculant: Influence of temperature with kinetic and thermodynamic functions, Bioresource Technology (2016), doi: http://dx.doi.org/10.1016/j.biortech.2016.11.050

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A novel method to harvest Chlorella sp. via low cost bioflocculant: Influence of temperature with kinetic and thermodynamic functions Richa Kotharia*,Vinayak V. Pathaka,Arya Pandeya,Shamshad Ahmada,Chandni Srivastavaa,V.V. Tyagib a

Bioenergy and Wastewater Treatment Laboratory, Department of Environmental Sciences, Babasaheb Bhimrao Ambedkar University, Lucknow (U.P.), India b

Department of Energy Management, Shri Mata Vaishno Devi University, Katra (J&K) India

Abstract In this study, harvesting efficiency (HE) of bioflocculant (egg shell) was observed with variation in flocculent concentrations (0-100mgL-1), temperature (30°C, 35°C 40°C, 45°C and 50°C) and variable contact time (0-50 minutes). It was found maximum (≈95.6%) with 100 mgL-1 bioflocculant concentration whereas influence of temperature was also observed with optimized concentration of bioflocculant (100 mgL-1) at 40°C (≈ 98.1%) and 50°C (≈99.3%), in 30 minutes of contact time. Significant changes in algal cell structures were also analyzed after exposure to various temperatures with microscopy, SEM (Scanning electron microscopy) and EDS (Energy dispersive X-ray spectroscopy) images with and without bioflocculant. The experimental data was found to be a good fit with pseudo-second order kinetic model. The thermodynamic functions such as ∆G (Gibbs free energy), ∆H (Enthalpy), ∆S (Entropy) were also determined. The negative value of ∆G and positive value of ∆H and ∆S shows the spontaneous and endothermic nature of flocculation process. Keyword: Bioflocculant, low-cost, biomass harvesting, kinetic models, thermodynamic functions.

*

Corresponding author: [email protected]

1

1. Introduction Cost effective harvesting technology is a bottleneck in the research and development of algal based bioenergy and value-added compounds production at commercial level (Zhu et al., 2013; Min et al., 2011). Currently, various harvesting methods are in practiec for harvesting of algal biomass from their suspension such as centrifugation, gravity sedimentation, natural and pressurized filtration, chemical flocculation, electro-flocculation, and vacuum filtration, etc. (Chen et al., 2011; Zhang et al., 2011; Uduman et al., 2010). Among these filtration process with centrifugation, was reported with the highest yield in terms of percentage solid content i.e. 22%. However, pressurized filtration process was found with more efficiency (i.e. 27%) than centrifugation (Vandammea et al., 2012). The other centrifugation processes range between 1.5 to 18% regarding separation efficiency. The major drawback with centrifugation and pressurized filtration process is high energy usage i.e. 8 kw-m-3 in compare to all above said harvesting process. In general, chemical flocculants have been known for commercial applications (Wu et al.,2012). Flocculation or coagulation, gravity sedimentation, and flotation are quite an inexpensive approach to harvest algal cells. The co-cultivation of some fungal strains along with algal cells promotes the algal biomass flocculation. The main drawback regarding this method is that it requires long culture time and not relevant for harvesting of all microalgal biomass (Knuckey et al., 2006). Therefore, flocculation is an advanced method to harvest algal biomass regarding harvesting efficiency, operation economics, and technological feasibility (Liu et al., 2013; Vandamme et al., 2012; Papazi et al., 2010). There are some inorganic salts like ferric chloride, aluminum sulfate, multivalent metal salts (multivalent aluminium salts) act as a legend to flocculate algal cells and make it settle down (Wan et al., 2015;Udhaya et al., 2014; Letelier-Gordo et al., 2014). These chemicals are highly efficient regarding harvesting algal biomass, but the main shortcoming related to these chemicals is their high cost that makes the techniques economically unviable. Therefore, selection of a cost effective harvesting method is critical, although, chemical coagulants have been widely used for harvesting of algae from water (Wyatt et al., 2012). Their use for harvesting become problematic as they reduce the quality of algal biomass, water and increase the complexity in lipid extraction and its conversion process. Hence, application of bioflocculant can be cost effective and efficient option for biomass harvesting in an eco-friendly method (Ndikubwimana et al., 2014; Ahmad et al., 2013). Recently, Jeong (2015) have reported egg shell efficiency as bioflocculant and achieved harvesting efficiency up to 98%. The use of waste eggshell (boiled/unboiled) as a coagulating or flocculating 2

material is immensely advocated to harvest algal cells as it is economically very cheap, nontoxic and non-corrosive as in the case of chemicals and very easy to handle and easily available. Egg shells have the characteristic feature of biodegradability, biocompatibility, significant adsorption properties, and noteworthy flocculation ability. It possesses tremendous cationic charge density and can easily adsorb and destabilize the negative charges present over microalgal cells. The main composition of egg shell is calcium carbonate i.e. 95%, followed by the calcium phosphate, magnesium carbonate, and soluble and insoluble proteins covers remaining 5% (Jeong, 2015). Harvesting of microalgae using bio-flocculants has a great potential to decrease microalgae biomass production costs at commercial level. Here, in the present work attempts has been made to investigate the efficiency of bioflocculant i.e. egg shell for harvesting of algal biomass from its culture suspension. The obtained experimental data was also evaluated for kinetics and thermodynamic studies. Furthermore, effects on cell structure were also analyzed with temperature variations. 2. Material and methods 2.1. Culture conditions for algal growth The culture of Chlorella sp. was acquired from National Collection of Industrial Microorganism (NCIM), Pune, India and cultured in BG-11’s nutrient growth medium. The BG-11 medium was prepared by addition of 100 mL of stock solution of NaNO3 (15gL-1), 10 mL of each stock solution of K2HPO4 (4.0 gL-1); MgSO4.7H2O (7.5 gL -1); CaCl2.2H2O (3.6 gL-1); Citric acid (0.60 gL-1); Ammonium ferric citrate green (0.60 gL-1); EDTANa2 (0.1 gL1

); Na2CO3 (2 gL-1) and 1 mL of each micronutrient solution (H3BO3, 2.86 gL-1; MnCl2.4H2O,

1.81gL-1; ZnSO4.7H2O, 0.22 gL-1; Na2MoO4.2H2O, 0.39 gL-1; CuSO4.5H2O,

0.08 gL-1;

Co(NO3)2.6H2O, 0.05 gL-1). The pH of the growth medium was maintained upto 7.2. The culture of alga was grown in one liter Erlenmeyer flask provided with white fluorescent cool light (12:12 hr) at optimum temperature 25±2ºC. Algal cultures were manually agitated to provide homogenous nutrient distribution as well as to avoid wall growth and sedimentation. Biomass growth was measured by taking optical density at 665 nm by using UV visible spectrophotometer (HALO-DB 20, Thermo- Scientific). 2.2. Preparation of bioflocculant and analysis of bioflocculant HE with different concentration, temperature and time 3

The waste egg shells remained after peeling of boiled/unboiled egg was collected from local market due to ease to availability. Egg shells were washed with distilled water and dried at 40°C in an oven. Dried egg shells were grind to obtain the fine powder and sieved manually using micro sieve. The eggshell powder (100 mg) was dissolved in 10 ml of 0.1 molL-1 acid solution with continuous stirring for 30 min. The acid solution was then diluted to 100 ml using deionised water to make a final eggshell concentration (bioflocculant) of 1000 mgL-1. Harvesting of algal biomass was performed by applying various concentrations of bioflocculant i.e. egg shell solution (0 to 100 mgL-1), within variable contact time (0 to 50 minute), and temperature (i.e. 30°C, 35°C, 40°C, 45°C, 50°C). The selected concentration of bioflocculant was added and homogenised on 300 rpm (rotation per minute), using a mini orbital shaker (VWR Advanced Orbital Shaker, Model 15000). The supernatant was taken to measure the optical density (OD) at 665 nm to determine the extent of algal biomass. The harvesting efficiency (HE, %) of bioflocculant was determined by following equation: HE (%) = [1− {ODa665 (t)/ ODa665 (t0)} / {ODb 665 (t) ODb665 (t0)} × 100]..... (eq. 1) Where, ODa665 (t0) = Initial turbidity of algal cell suspension prior to addition of bioflocculant. ODa665 (t) = Turbidity of algal cell suspension prior to addition of bioflocculant at time t. ODb665 (t0) = Initial turbidity of algal cell suspension after addition of bioflocculant. ODb665 (t) = Turbidity of algal cell suspension after addition of bioflocculant at time t. 2.3. SEM–EDS Analysis The SEM-EDS (SEM model: JSM- 6490LV, Make: JEOL, Japan) analysis was applied to study the surface morphology and cell wall composition of the algal cell. Dried algal cells covered with thin layer of gold using a sputter coater has been tested in SEM-EDS unit to know the changes in cell surface and composition with bioflocculant at different concentration and temperature with time. 2.4. Kinetic model Kinetic study by pseudo-second order model is mainly used for adsorption process (Nuhoglu and Malkoc, 2009). However, various authors have used this model to evaluate the rate of reaction other than adsorption process such as biomass production and oil production 4

(Maurya et al., 2014). Therefore, surface binding of bioflocculant with respect to the time can be also evaluated by following this model. The rate of pseudo-second order reaction usually depends on binding of flocculants on the surface of algal biomass, which can be expressed by following equation: t/qt= 1/K2 qe2+t/q …………………………… (eq. 2) Where, K2 = Rate constant (gmg-1min-1); qe = Amount of biomass (mgg-1) flocculated at equilibrium; qt = Amount of biomass (mgg-1) flocculated at time t. Initial variables such as qe and qt are quantified by following equation: q = (Ci-Cf)*V /m ……………………………. (eq.3) Where, q = Bio-flocculation capacity; Ci, = Initial algal biomass concentration; Cf = Biomass concentration after flocculation; V = Volume of the solution (L); m = Amount of bioflocculant (mg L-1). The pseudo-second order kinetic model is expressed by plot between t/q versus t. Kinetic variables such as K2 (rate constant), h (initial flocculation rate) and qe (calculated bioflocculation capacity) can be calculated from the slope and intercept of the straight line equation. 2.5. Determination of thermodynamic functions The feasibility of flocculation process over different ranges of temperature can be determined through deployment of Eyring and Arrhenius equations. Both of these equations i.e. Eyring, provide the value of various thermodynamic functions such as enthalpy, entropy and Gibb’s free energy and Arrhenius helps in analysing the magnitude of activation energy for the harvesting of algae at different temperatures. 2.5.1. Eyring equation The effect of temperature on time-dependent flocculation of algal biomass can be expressed by the thermodynamic parameters. Eyring type plot between lnK2/T versus 1/T was assessed to calculate the thermodynamic parameters. Eyring equation is used in chemical kinetics to describe the variance of the rate of reaction with temperature. The change in enthalpy (∆H), entropy (∆S), and Gibbs free energy (∆G) after adsorption of bioflocculant has been investigated in flocculation process at different temperature by using Van't Hoff equation (Yao et al., 2010; Shivaraj et al., 2001). The thermodynamic parameters such as standard free energy changes (∆G), the standard enthalpy changes (∆H) and the standard entropy change 5

(∆S) is obtained from experiments at various temperatures using the following equations 4 and 5: Intercept = [ln (kb/h) + ∆S/R] Slope = [-∆H/R]

………………….. (eq. 4)

……………………….............. (eq. 5)

Where, kb= Boltzmann constant; h= Plank’s constant; and R= Gas constant. The slop and intercept of strait line equation is used to calculate the thermodynamic parameters i.e. ∆H and ∆S. The Gibb’s free energy (∆G) (Equation 6) has been calculated by the obtained value of ∆S and ∆H for different temperature in Calvin. ∆G= ∆H-T∆S............. ……………………. (eq. 6) Where, ∆G = Gibbs free energy; ∆H = Enthalpy; ∆S = Entropy. The above said equation had been applied to know the mathematical relationship between bioflocculant adsorption by the algal cell at different temperature and time. Thermodynamic parameters obtained from Van’t Hoff graph, which is also known as Eyring type equation is the most widely accepted models have been taken into consideration to know the rate of reaction after absorbance of bioflocculant by the algal cell. 2.5.2. Arrhenius equation Arrhenius equation has been used to investigate the activation energy i.e. maximum energy required to start the chemical reaction after incorporation of bioflocculant. The Arrhenius equation expresses that flocculation is a function of temperature in pseudo-second order rate constant. The Arrhenius equation can be expressed by the following equation: Slope = -Ea/R

……………………….. (eq. 7)

Where, -Ea = Arrhenius activation energy; R = Gas constant which is equal to 8.314 j mol -1 k-1. 3. Results and discussion 3.1. Growth of microalgae The growth pattern of Chlorella sp. in BG-11 medium of test solution were observed and found that during the first 7 days, the microalgal growth was swift; afterwards it conquered almost a constant speed. The biomass productivity was obtained higher than attained by Min 6

et al., (2011) 0.035 µgml-1day-1. The present study demonstrates that significant biomass productivity in terms of specific growth rate with 0.84 µgml-1day-1 in compared to the specific growth rate reported by Zhu et al., (2013) (0.296 µgml-1day-1) of algal biomass productivity. The maximum biomass concentration was obtained 3.38 gL-1 which is higher than 2.25 gL-1 as reported by Choi et al., (2015) for Chlorella vulgaris in JM medium. Although, growth of Chlorella sp. was also observed with FOG’s growth medium in our previous studies (Kothari et al., 2012, Pathak et al., 2015). Here, in present study we select the BG 11 for Chlorella just to investigate the comparative assessment in terms of growth rate with our previous studies and also cited by other researchers in their studies. 3.2. Effect of selected bioflocculant concentration and its HE The harvesting of algal biomass from its suspension was observed by applying different concentration of bioflocculant ranges from the 0 to 100 mgL-1 with different time intervals (minutes). The harvesting efficiency was found to be increase with increase in the bioflocculant concentration and contact time. The maximum harvesting efficiency (95.6%) was obtained with bioflocculant concentration of 100 mgL-1 at 45 minute of time interval, while with 20 mgL-1 bioflocculent concentration, 66.5% of harvesting efficiency was found within same contact time. Harvesting efficiency of control (without addition of bioflocculant) was found relatively lower (15.6%) in comparison to harvesting efficiency obtained with bioflocculant. Change in the turbidity of algal suspension with different bioflocculant dose and time interval is shown in fig. 1. The harvesting efficiency of egg shell was found higher than the harvesting efficiency of chemical flocculants such as Al2(SO4)3 and ZnCl2 reported by Papazi et al., (2010). The authors have achieved harvesting efficiency of 60% for Chlorella minutissima by addition of 1 gL−1 of Al2(SO4)3 and ZnCl2 in, respectively, 1.5 and 6 h. However, present bioflocculation process is based on cheap and easily available waste egg shell, which is more effective and economically viable in compare to the traditional harvesting methods such as centrifugation, filtration, and flotation that have been applied to various algal species (Vandammea et al., 2012) as cited in literature. In the case of chemical flocculation, although significant separation efficiency was reported by Udhaya et al., (2014), but sometimes it damages algal cell structure. On the other hand, microalgae flocculated with chemical flocculants have already reported for commercial level application, but their frequent release in aquatic environment may cause toxicity to the aquatic organisms. Recently, Pirwitz et al. (2015), investigated the flocculation of Dunaliella sp. by using FeCl3, NaOH and Electrolysis and observed impairment of cell surface with NaOH and electrolysis 7

based flocculation. Thus, use of bio-waste as bioflocculant seems to be significant for algal harvesting process with no toxic effects in comparison to chemical based flocculants. 3.3. Effect of temperature The efficiency of egg shell solution (100 mgL-1) to flocculate algal biomass over different temperature (30°C, 35°, 40°C, 45°C, 50°C) was also investigated to get more critical findings of harvesting efficiency of bioflocculant (figure 2). The highest harvesting efficiency was obtained (≈99.3%) with maximum temperature 50°C, whereas, the lowest harvesting efficiency was obtained (≈97.09%) at room temperature (30°C), with the use of bioflocculant concentration of 100 mgL-1. The rate of flocculation was found to be increase with increase in temperature, it is due to the influence of temperature on Floc formation (Joudah, 2014). Some researchers had already reported that flocculation can also be induced by changing the culture conditions by applying temperature changes (Lei et al., 2015). The findings of present study are also found in conformity with study on effect of temperature to harvest algal biomass by using bioflocculant conducted by Salim et al. (2012 & 2011). Hence, temperature plays a significant role to enhance the rate of reaction and binding capacity of bioflocculant with algal biomass. The experimental data also exposed that there is reduction in time as the temperature increases. At 40°C and 50°C harvesting of algal biomass has taken place approximately in half an hour. To support the experimental data regarding harvesting efficiency of algal biomass, kinetics and thermodynamics model has been applied and described in further section by this study. 3.4. SEM-EDS analysis SEM-EDS analysis was performed on algal cell harvested from control and bioflocculant (100 mgL-1).

The microscopic images of cell suspension showed a greater extent of

aggregation of algal cell with bioflocculant in compare to control. The SEM analysis of algal cells harvested from control and cell suspension treated with bioflocculant showed resemblance in surface morphology of algal cells. On the other hand, marked effect of temperature (30°C, 40°C, 50°C) on surface morphology of algal cell was observed. The results obtained from the SEM micrograph showed a normal and smooth shape of algal cell surface prior to the flocculation process while after treatment with bioflocculant, algal cell shows deposition of calcium with minute changes in cell surface at 40°C, however, major changes in cell surface were observed with flocculation process at 50°C temperature. Hence, this experiment shows that temperature has a marked effect on cells surface structure due to 8

its effect on binding of bioflocculant with cell surface. A slight whitening of the algal cell has been obtained after treatment with bioflocculant at each temperature. It is due to the calcium deposition of the egg shell, which is confirmed and supported by EDS analysis. Similar to the present study, Zheng et al. (2012) observed no disintegration in cell surface of Chlorella vulgaris flocculated by poly glutamic acid at room temperature. Another study conducted by Choi et al. (2015) also found that egg shell solution has non-toxic effect on algal cell during the harvesting process. The distinguished difference due to use of bioflocculant on the algal cells have been completely visualized with the help of microscopic images and SEM-EDS analysis in this study. 3.5. Kinetic model and thermodynamic functions Experimental data for flocculation of algal biomass by using bioflocculant was found best fitted with pseudo-second order kinetic’s model. The plot t/q versus t was found significant over all temperature and shows high extent of correlation (R2 = >90) as illustrated in Fig. 3. The rate constant derived from straight line equation was found increasing with increase in temperature and ranges from (0.002-0.041 mgg-1 ) as shown in Table 1. It clearly confirms that effective flocculation can be achieved at higher temperature. The values of initial flocculation rate i.e. ‘h’ also resemble the same observation and found to be increase with increase in temperature. The maximum value of ‘h’ was 8.33 mg g-1min-1 at 50°C. The feasibility of flocculation process at different temperature was observed through evaluation of thermodynamic variables (Fig. 4) with Eyring plot (lnK2/T verses 1/T) and Arrhenius plot (lnK verses 1/T). The ∆H and ∆S values have been calculated from the slope and intercept of Eyring plots. The positive value of ∆H shows the endothermic nature of adsorption, and there may be a possibility of physical adsorption. In the case of physical adsorption, an increase in temperature of the system will enhance the bioflocculent adsorption up to some extent. The positive value of ∆S shows an increased disorder and randomness in bioflocculant adsorption by algal culture. The negative value of ∆G depicts that adsorption is highly favourable for flocculants i.e. the reaction is spontaneous as given in Table 2. The positive value of ∆H indicates that interaction of flocculant by algal biomass is an endothermic process, and the positive value of ∆S reveals that, there is an increase in randomness during adsorption process (Nuhoglu et al., 2009). Activation energy was obtained from the Arrhenius plot (lnK verses 1/T) and the calculated value is 113.04 kjmol-1. Activation energy is the minimum required energy to start a chemical 9

reaction. Therefore, at 50°C harvesting efficiency was obtained maximum. But, Chlorella sp. grow well at 25-30°C and can tolerate up to 40°C for few hours only. Although, at 50°C cell structure of Chlorella shows some variations like cell wall deformation and cell disruption, and change in surface structure up to some extent. Hence, it is concluded that at 40° C temperature with bioflocculant (100 mgL-1) is highly efficient to harvest algal biomass from the suspension. 4. Conclusion The present study reveals that optimized concentration (100 mg/L) of bioflocculant provides a potential option to harvest the Chlorella sp. with maximum harvesting efficiency without temperature (95.6%) and with temperature (98.1% at 40°C) without any cell surface structural deformities in the exposure time of 30 minutes. Compared to inorganic flocculants, eggshell as bio-flocculent is of zero cost and does not impart harmful impact on the algal cell. The experimental data is proven significant by the pseudo-second order kinetic model and thermodynamic functions. Hence, results are very much helpful in low-down the capital cost involves in use of chemical flocculants for harvesting at large scale. Acknowledgement: We would like to thank Department of Environmental Microbiology and Director, University Science Instrumentation Centre (USIC), Babasaheb Bhimrao Ambedkar University to providing the instrumentation facilities for our research work. References: 1. Ahmad, H., Rajab, A., Azni, I., Norhafizah, A., 2013. Production and characterization of a bioflocculent produced by Aspergillus flavus. Bioresour Technol. 127,489-493. 2. Chen, C.-Y., Yeh, K.L., Aisyah, R., Lee, D.-J., Chang, J.-S., 2011.Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: a critical review. Bioresour. Technol. 102(1),71–81. 3. Choi, H.J., 2015. Effect of optical panel distance in a photobioreactor for nutrient removal and cultivation of microalgae. World J Microbiol Biotechnol. 30(7) 20152023. 4. Jeong, C.H., 2015. Effect of eggshells for the harvesting of microalgae species. Biotechnol & Biotechnolog Equip, 29, 666-672. 10

5. Joudah Rasha Azeez.,2014. Effect of Temperature on Floc Formation Process Efficiency and Subsequent Removal in Sedimentation Process. J. Engg Develop, 18,(4)176-87. 6. Knuckey, R.M., Brown, M.R., Robert, R., Frampton, D.M.F., 2006. Production of microalgal concentrates by flocculation and their assessment as aquaculture feeds. Aquacult. Eng. 35 (3), 300–313. 7. Kothari R , Vinayak V. Pathak, Virendra Kumar, D.P. Singh, 2012.Experimental study for growth potential of unicellular alga Chlorella pyrenoidosa on dairy waste water: An integrated approach for treatment and biofuel production, Biores Technol (116),466–470 8. Lei, X., Chen, Y., Shao, Z., Chen, Z., Li, Y., Zhu, H., Zhang, J., 2015. Effective harvesting of the microalgae Chlorella vulgaris via flocculation-flotation with bioflocculant. Bioresour technol. 198, 922-925 9. Letelier-Gordo, C.O., Holdt, S.L., De Francisci, D., Karakashev, D.B., Angelidaki, I., 2014. Effective harvesting of the microalgae Chlorella protothecoides via bioflocculation with cationic starch. Bioresour. Technol. 167, 214–218. 10. Liu, J., Tao, Y., Zhang, Y., Li, A., Sang, M., Zhang, C., 2013. Freshwater microalga harvested via flocculation induced by pH decrease. Biotechnol Biofuels, 6, 98. 11. Maurya, R., Ghosh, T., Paliwal, C., Shrivastav, A., Chokshi, K., Pancha, I., Ghos, A., Mishra, S., 2014. Biosorption of Methylene Blue by De-Oiled Algal Biomass: Equilibrium, Kinetics and Artificial Neural Network Modelling. PLoS ONE 9(10): e109545. doi:10.1371/journal.pone.0109545. 12. Min, M., Wang, L., Li, Y., Mohr, M.J., Hu, B., Zhou, W., Che, P., Ruan, R., 2011.Cultivating Chlorella sp. in a pilot scale photobioreactor using centrate wastewater for microalgae biomass production and wastewater nutrient removal. Appl. Biochem. Biotehnol. 165,123-137. 13. Ndikubwimana, T., Zeng, X., Liu, Y., Chang, J.S., Lu, Y., 2014. Harvesting of microalgae Desmodesmus sp. F51 by bioflocculation with bacterial bioflocculant. Algal Res. 6,186–93. 14. Nuhoglu, Y., Malkoc, E., 2009. Thermodynamic and kinetic studies for environmentally friendly Ni (II) biosorption using waste pomace of olive oil factory. Bioresour Technol. 100, 2375–2380. 15. Papazi, A., Makridis, P., Divanach, P., 2010. Harvesting Chlorella minutissima using cell coagulants. J Appl Phycol 2, 349-355. 11

16. Pathak V.V, Richa Kothari, A.K. Chopra, D.P. Singh., 2015. Experimental and kinetic studies for Phycoremediation and dye removal by Chlorella pyrenoidosa from textile wastewater, J Environ Manag 163, (1)270–277. 17. Pirwitz K, Rihko-Struckmann L, Sundmacher K., 2015.Comparison of flocculation methods for harvesting Dunaliella. Bioresour Technol 196,145–152. 18. Salim S., Bosma, R., Vermuë, M.H., Wijffels, R.H., 2011. Harvesting of microalgae by bio-flocculation. J Appl Phycol. 23(5), 849–855. 19. Salim S., Vermue, M.H., Wijffels, R.H., 2012. Ratio between auto- flocculating and target microalgae affects the energy-efficient harvesting by bio-flocculation. Bioresour Technol. 118,49-55. 20. Shivaraj, R., Namasivayam, C., Kadirvelu, K., 2001. Orange peel as an adsorbent in the removal of acid violet from aqueous solution. Waste Management. 21,105. 21. Udhaya, R., Benedict Bruno, L., Sandhya, S., 2014. Evaluation of chemical flocculation-electro flocculation for harvesting of halotolerant microalgae. Int J Environ Sci. 4, 899-905 22. Uduman, N., Qi, Y., Danquah, M.K., Forde, G.M., Hoadley, A., 2010. Dewatering of microalgal cultures: a major bottleneck to algae-based fuels. J. Renew. Sustain. Energy 2 (1), 012701. 23. Vandamme, D., Foubert, I., Fraeye, I., Muylart, K., 2012. Influence of organic matter generated by Chlorella vulgaris on five different modes of flocculation. Bioresour Techno. 124, 508-511. 24. Wan, C., Alam, M.A., Zhao, X.Q., Zhang, X.Y., Guo, S.L., Ho, S.H., Chang, J.S., Bai, F.W., 2015. Current progress and future prospect of microalgal biomass harvest using various flocculation technologies. Bioresour. Technol. 184, 251–257. 25. Wu, Z., Zhu, Y., Huang, W., Xhang, C., Li, T., Zhang, Y., Li, A., 2012. Evaluation of flocculation induced by pH increase for harvesting microalgae and reuse of flocculated medium. Bioresour Technol, 110,496-502. 26. Wyatt, N.B., Gloe, L.M., Brady, P.V., Hewson, J.C., Grillet, A.M., Hankins, M.G., Pohl, P.I., 2012. Critical conditions for ferric chloride-induced flocculation of freshwater algae. Biotechnol Bioeng. 109, 493-501. 27. Yao, Y., Xu, F., Chen, M., Xu, Z., Zhu, Z., 2010. Adsorption behavior of methylene blue on carbon nanotubes. Bioresour Technol. 101, 3040-3046.

12

28. Zhang, Y., Tian, J., Nan, J., Gao, S., Liang, H., Wang, M., Li, G., 2011. Effect of PAC addition on immersed ultrafiltration for the treatment of algal-rich water. J. Hazard. Mater. 186 (2–3), 1415–1424. 29. Zhu, L., Ang, Z., Shu, Q., Takala, J., Hiltunen, E., Feng, P., Yuan, Z., 2013. Nutrient removal and biodiesel production of freshwater algae cultivation with piggery wastewater. Water Res. 47, 4294-4302. 30. Zheng H., Gao, Z., Yin J., Tang, X., Ji, X., Huang, H., 2012. Harvesting of microalgae by flocculation with poly (glutamic acid). Bioresource Technology 112, 212–220.

13

Fig. 1. HE of Chlorella sp. with different concentrations (mgL-1) of bioflocculant (Page no. 7) Fig. 2. HE of Chlorella sp. at different temperatures (°C) with 100 mgL-1 concentration of bioflocculant (Page no. 8) Fig. 3. Pseudo-second order rate constant at different temperatures (°C) i.e. (a) 30; (b) 35; (c) 40; (d) 45; (e) 50. (Page no. 9) Fig. 4. Thermodynamic functional plots at various temperatures (°C) with Eyring (Ey) and Arrhenius plot (Ea) equations

(Page no. 9)

1.6 1.4

Absorbence (665)

1.2 1

0mgL¯¹ 20mgL¯¹

0.8

40mgL¯¹ 0.6

60mgL¯¹ 80mgL¯¹

0.4

100mgL¯¹

0.2 0 0

5

10

15

20

25 30 35 Time (minute)

40

45

50

55

Fig. 1. HE of Chlorella sp. with different concentrations (mgL-1) of bioflocculant

14

1.6

Absorbance(665 nm)

1.4 1.2 1

(T )30˚C

0.8

(T) 35°C

0.6

(T )40˚C

0.4

(T) 45°C

0.2

(T) 50°C

0

0

5

10

15

20

25

30

35

40

45

50

Time (minute)

Fig. 2. HE of Chlorella sp. at different temperatures (°C) with 100 mg/L concentration of bioflocculant

15

Fig. 3. Pseudo-second order rate constant at different temperatures (°C) i.e. (a) 30 (b) 35 (c) 40 (d) 45 (e) 50

16

1/T 0.0031

0.00315

0.0032

-2

0.00335 0 Y(Ey) = -22217x + 61.05 R² = 0.93 -2 Y(Ea) = -13597x + 38.65 R² = 0.91 -4

-3

-6

-4

-8

-5

-10

-6

-12

-7

-14

In(k)

-1

0.00325

0.0033

In(K2/T)

0.00305 0

Fig.4. Thermodynamic functional plots at various temperatures (°C) with Eyring (Ey) and Arrhenius plot (Ea) equations

17

Table-1: Value of variables obtained with pseudo-second order model

(Page no. 9)

Table-2: Thermodynamic functions of bioflocculant adsorption by Chlorella sp. (Page no. 9)

18

Table-1: Value of variables obtained with pseudo-second order model Pseudo-second order model K2 (mg/g)

q e (mg/g)

h (mg g-1min-1)

R2

30

0.0023

21.739

1.064

0.97

35

0.003

19.23

1.109

0.99

40

0.01

16.667

2.789

0.99

45

0.01

14.925

3.319

0.99

50

0.041

14.285

8.333

0.99

Temperature (°C)

Table-2: Thermodynamic functions of bioflocculant adsorption by Chlorella sp. Thermodynamic parameters

Values (KJ/mol)

Enthalpy, ∆H

184.7121

Entropy, ∆S

1.613157

Activation energy, Ea

113.04

Gibbs free energy, ∆G

Temp.(Kelvin)

∆G

303

-304.074

308

-312.14

313

-320.206

318

-328.272

323

-336.338

19

Highlights •

Low-cost bioflocculant based algal biomass harvesting

•

Effect of different concentrations of bioflocculant on algal biomass

•

Effect of temperature with optimized concentration on harvesting efficiency

•

Kinetics and thermodynamic functions to support the experimental data

20