** Development of a Sustainable and Efficient Harvesting Technique**

**4.2 Materials and Methods .1 Microalgae as an adsorbent**

*T. obliquus* KMC24 was used as an adsorbent in this study. The microalgae culture was
maintained in BG 11 medium. The biomass productivity was determined using the equation
(3.1) as mentioned in the methodology section of chapter 3.

**4.2.2 ** **Eggshell-derived bioflocculant as an adsorbate **

The waste eggshells (boiled and unboiled) were collected from the canteens of IIT Guwahati hostels and were dried in a hot air oven for 24 h at 60 °C after washing with distilled water several times. The dried eggshells were ground to fine powder using a mixer grinder.

The powdered eggshell was then sieved manually using a 325-mesh sieve. The bioflocculant
was extracted from the eggshell powder by using 10 mL of 0.1 mol L^{-1} HCl solution to dissolve
100 mg of the powder with continuous stirring for 35 min on a magnetic hot plate stirrer

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(Tarsons digital spinot). The resulting solution was further filtered through a 0.2 μm PTFE
syringe filter, and a final bioflocculant concentration of 1000 mg L^{-1} was obtained by diluting
the filtrate to 100 mL with distilled water [333]. This flocculant extract was used as a stock
solution for subsequent use in the experiments.

**4.2.3 ** **Optimization of bioflocculant concentration, pH, and temperature **

Batch flocculation experiments were performed in 250 mL conical flasks containing
150 mL BG-11 medium. The microalgal cells were harvested in their early stationary phase,
i.e., on the 14^{th} day of cultivation, when the DCW (dry cell weight) was maximum, i.e., 2.35 g
L^{-1}. Different concentrations of eggshell-derived bioflocculant solution (0–140 mg L^{-1}, at an
increment of 20 mg L^{-1}) was applied to determine the influence of bioflocculant concentration
on harvesting of microalgal biomass at room temperature 22°C and pH 7.

Harvesting efficiency was further enhanced by optimizing pH values (2, 4, 6, 8 and 10) of the culture medium at room temperature 22°C using optimal bioflocculant concentration.

The pH values were maintained using 0.5 mol L^{-1} NaOH and 0.5 mol L^{-1 }HCL. The effect of
temperature on harvesting efficiency was also studied by varying the temperature (25°C, 35°C,
45°C, 55°C) of the culture medium. The temperature optimization experiment was carried out
at an optimal pH value and bioflocculant concentration. The culture temperature was
maintained using a shaking water bath (EQUITRON, MEDICA INSTRUMENT MFG. CO.).

All the above studies were carried out within the variable contact time (0–40 min).

**4.2.4 ** **Harvesting efficiency of microalgal biomass **

To determine the harvesting efficiency of the bioflocculant under various parameters, 2 mL of the supernatant was collected from the center of the algal suspension, and the optical density (OD) was measured in every 5 min of interval for 40 min. The optical density was determined at 750 nm by a UV-visible spectrophotometer. The harvesting efficiency (HE, %) of bioflocculant was calculated from the following equation [334]:

HE(%) =^{𝑂𝐷}^{750}^{(𝑡}^{0}^{)−𝑂𝐷}^{750}^{(𝑡)}

𝑂𝐷750(𝑡0) × 100% (4.1)

where, 𝑂𝐷_{750}(𝑡_{0}) and 𝑂𝐷_{750}(𝑡) are the OD of microalgal cell suspension measured at time
zero and 𝑡, respectively.

**4.2.5 ** **Analytical methods **

Microscopic images of the treated (with bioflocculant) and untreated (without bioflocculant) microalgal cells were captured under optical microscope (Axio Scope.A1, Zeiss, TH-2569_156151002

102 | P a g e USA) to examine their morphological changes. The floc size of microalgae was considered as an evaluation mark for determining the flocculation efficacy of the bioflocculant. The bigger the size of the floc, higher the harvesting efficacy of the bioflocculant [334]. The morphology of the samples was determined by FESEM (Zeiss Sigma-300 Field Emission Scanning Electron Microscopy). Elemental components were determined by using energy dispersive spectroscopy (EDX) coupled with FESEM. The zeta potential values were recorded in triplicates using a Zeta potential analyzer (Malvern Zetasizer ZEN 3690), and the mean values were calculated in millivolts (mV) [334].Soluble calcium concentration was determined by Systronics flame photometer (model no. 128).

The lipid content (Lcontent, %) of the dry biomass was determined by the protocol, as reported earlier [335]. Transesterification and FAME analysis were carried out using the protocol mentioned in the methodology section of chapter 3.

**4.2.6 ** **Determination of adsorption kinetics**

The mechanism of flocculation-sedimentation process can be determined by estimating
the adsorption rate. Kinetic models such as pseudo-first order equation (4), pseudo-second
order equation (5) and intra-particle diffusion models equation (6) were used to study the
adsorption kinetics of eggshell-derived bioflocculant by* T. obliquus *KMC24 cells. Many
researchers have employed these kinetic models to evaluate the flocculation of microalgae by
bioflocculants [56,334]. The high cationic charge density of eggshell enables it to be strongly
adsorbed onto the negatively charged microalgal cells, which consequently causes them to
flocculate due to charge neutralization.

Thus, flocculation of *T. obliquus* KMC24 by eggshell-derived bioflocculant can be
considered as an adsorption process where bioflocculants are adsorbed over the microalgal
cells. Therefore, these most commonly used kinetic models were employed to assess the
binding of bioflocculants over microalgal cell surface with regard to time. The bioflocculant
adsorption over microalgal cells 𝑞_{𝑡} (mg g^{-1}) at time 𝑡, was determined from the following
equation [336]:

𝑞_{𝑡} =^{(𝐶}^{0}^{−𝐶}^{𝑡}^{)𝑉}

𝑀

(4.2)

where, 𝑞_{𝑡} is the quantity of microalgal biomass (mg g^{-1}) flocculated at time 𝑡 by the flocculant,
𝐶_{0} (mg L^{-1}) and 𝐶_{𝑡} (mg L^{-1}) are the microalgal cell concentration in the culture medium at time

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ln(𝑞_{𝑒}− 𝑞_{𝑡}) = 𝑙𝑛 𝑞_{𝑒}− 𝐾_{1}𝑡
(4.3)

𝑡
𝑞𝑡= ^{1}

𝐾2𝑞𝑒2+ ^{1}

𝑞𝑒𝑡

(4.4)

𝑞_{𝑡} = 𝑘_{𝑖𝑛𝑡}𝑡^{1/2}+ 𝐶_{𝑖}

(4.5)

where, 𝑞_{𝑡} and 𝑞_{𝑒} are the quantity of microalgal biomass (mg g^{-1}) flocculated at time t and
equilibrium respectively, 𝑡 (min) represents the contact time, while 𝐾_{1} (min^{-1}), 𝐾_{2} (g mg^{-1 }min^{-}

1) and 𝑘_{𝑖𝑛𝑡} (mg g^{-1} min^{-1/2}) are the rate constants. 𝐶_{𝑖} represents the intercept (mg g^{-1}).

**4.2.7 ** **Determination of adsorption thermodynamics **

The spontaneity of the adsorption of eggshell-derived bioflocculant onto the microalgal cells was assessed using the Van’t Hoff equation. The Gibbs free energy (ΔG) change is associated with the equilibrium constant (Kc) for biosorption of eggshell-derived bioflocculant over microalgal cells by the following relationship:

∆𝐺° = −𝑅𝑇𝑙𝑛𝐾_{𝐶}
(4.6)

where, T is the absolute temperature in Kelvin and R is the gas constant (8.314 J mol^{−1 }K^{−1}).

The Gibbs’ free energy equation is as follows:

∆𝐺° = ∆𝐻° − 𝑇∆𝑆°

(4.7)

where, ΔH and ΔS (J mol^{-1} K^{-1}) is the change in enthalpy and entropy respectively. Combining
equation (7) and (8), we get:

𝑙𝑛𝐾_{𝐶} = −^{∆𝐻°}

𝑅𝑇 +^{∆𝑆°}

𝑅

(4.8)

A plot of 𝑙𝑛𝐾_{𝑐} vs 1 𝑇⁄ is a linear graph, whose intercept and slope yields ∆𝑆° 𝑅⁄ and ∆𝐻° 𝑅⁄
respectively.

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**4.2.8 ** **Activation energy analysis **

The activation energy of the system was determined using the Arrhenius equation [337].

The activation energy was calculated to determine the minimum energy required for starting the chemical reaction once the bioflocculant is incorporated. Flocculation is expressed as a function of temperature in the pseudo-second order rate constant according to the Arrhenius equation (Equation 10) [56]:

𝑘 = 𝐴𝑒^{−𝐸𝑎}^{𝑅𝑇} (4.9)
where, 𝐸_{𝑎}(J mol^{−1}) is the Arrhenius activation energy, 𝐾 is the rate constant, *T* (Kelvin) is the
absolute temperature, *A *is the pre-exponential factor and *R* is the universal gas constant.

Alternatively, the above equation can be expressed in its natural logarithm as
𝑙𝑛𝑘 = −^{𝐸}^{𝑎}

𝑅𝑇+ 𝑙𝑛𝐴 (4.10)

**4.2.9 ** **Recycling of harvested medium **

The culture medium recovered after harvesting of microalgal cells using eggshell-
derived bioflocculant was investigated for recyclability for the next cultivation cycle. After
flocculation-sedimentation process, the spent medium was separated from the settled
microalgal flocs by gravity. The pH of the harvested medium was neutralized, and the BG-11
medium was added before recycling the medium. The concentration of the inoculum was
adjusted to have similar starting cell density in the recycled culture medium for the growth
studies. The growth of *T. obliquus* KMC24 cells in the recycled culture medium was
determined by recording the OD of the medium at 750 nm daily.