Biochemical Engineering Journal 149 (2019) 107241

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular article

Effect of alginate concentration in wastewater nutrient removal using T

alginate-immobilized microalgae beads: Uptake kinetics and adsorption
studies
Shantanu Banerjeea, Palas Balakdas Tiwadea, Kumar Sambhava, Chiranjib Banerjeeb,
Soubhik Kumar Bhaumika,
⁎

a
Department of Chemical Engineering, Indian Institute of Technology (Indian School of Mines), 826004, Dhanbad, India
b
Department of Environmental Science and Engineering, Indian Institute of Technology (Indian School of Mines), 826004, Dhanbad, India

HIGHLIGHTS

• Effect of alginate concentration on nutrient removal was investigated.

• Uptake kinetics and adsorption of nutrients onto alginate beads was evaluated.
• 3% alginate concentration optimal both for nutrient removal and adsorption.
• Adsorption of NH +
and PO 3−
- P onto alginate matrix through hydrogen bonding.
• 3% alginate concentration bear optimal pore size for nutrient access.
4 4

ARTICLE INFO ABSTRACT

Keywords: Effect of alginate concentration on wastewater nutrient removal by alginate-immobilized microalgae bead
Alginate concentration systems (AIMS) of Chlorella vulgaris was investigated to delineate the underlying role of alginate matrix in the
Nutrient removal removal process. The study includes kinetic evaluation of nutrient removal in AIMS of different alginate con-
Uptake kinetics centration (1.5–4.5% w/v%) followed by an assessment of abiotic adsorption characteristics of blank alginate
Adsorption
beads systems (BABS) of same alginate concentrations. Results establish 3% alginate concentration as optimal
Charge-interaction
Pore morphology
both for nutrient removal in AIMS and adsorption in BABS. The adsorption data in the latter fitted well with
Langmuir isotherms, suggesting monolayer physisorption, which was later corroborated through Fourier-
Transform Infrared Spectroscopy (FTIR) and Energy Dispersive X-ray (EDX) analysis. A scheme for adsorption of
NH4+ - N and PO43−- P on alginate matrix through hydrogen bonding was illustrated. The optimal trends were
explained based on pore morphology and bead structural integrity.

1. Introduction synthetic polymers [3]. A popular immobilization medium is the algi-

Since its inception over a century ago, microalgae-based treatment
routes continue to be the most popular means for nutrient removal in
tertiary water treatment in order to prevent eutrophication. The re-
moval of secondary metabolites and pollutants is achieved through in
situ photosynthesis occurring ‘in parallel’ with the biological nutrient
assimilation [1]. The processes boost the oxygen levels and offer a sy-
nergistic advantage of algal biofuel production [1]. Algae-immobiliza-
tion techniques play a crucial role in harvesting the biomass without
the use of mechanical devices [2]. One popular method is the entrap-
ment that encloses the algal cells in a three-dimensional matrix of
nate gel, due to its lower toxicity, higher porosity, transparency, and
milder physical environment to the cells [4]. Alginate-bead based algal
nutrient removal has been studied with respect to various parameters
such as bead concentration, bead cell count, type of alginate material,
bead stability, manufacture cost etc. [5]. Some studies have highlighted
the role of alginate concentration on bead deterioration and nutrient
removal [6,7]. Yet the underlying physicochemical interaction of algi-
nate matrix with the nutrient species has not been exclusively ad-
dressed.
Compared to the free cells, the nutrient removal efficiency of algi-
nate-immobilized microalgae bead system (AIMS) is considerably high

⁎
Corresponding author at: Department of Chemical Engineering, Indian Institute of Technology (Indian School of Mines), Dhanbad, 826004, Jharkhand, India.
E-mail address: soubhikge@gmail.com (S.K. Bhaumik).

https://doi.org/10.1016/j.bej.2019.107241
Received 8 January 2019; Received in revised form 18 May 2019; Accepted 23 May 2019
Available online 24 May 2019
1369-703X/ © 2019 Elsevier B.V. All rights reserved.
S. Banerjee, et al.
Nomenclature
Ce Nutrient concentration at equilibrium(mg L−1)
Co Initial nutrient concentration(mg L−1)
De Effective diffusivity (mm2 h−1)
h Initial adsorption rate(mg mg−1 min−1)
k Kinetic constant (L mg−1 s−1)
m Mass of the adsorbent (mg)
P Volumetric productivity (mg L−3 h−1)
q Adsorption capacity(mg g−1)
qe,c Isotherm model calculated adsorption capacity(mg mg−1)
qe Adsorption capacity at equilibrium(mg g−1)
Qm Maximum monolayer coverage capacity (mg g−1)
r Radius of the bead (mm)
S Total substrate (nutrient) concentration at an instant ‘t’
(mg L−1)
S0 Initial substrate concentration (mg L−1)
[8]. Immobilized Chlorella vulgaris exhibits uptake rates 15% and 31%
higher than suspended free algae, for NH4+ - N and PO43−- P respec-
tively [9]. Nutrient removal by the same species in immobilized form is
reported to be over 95% for NH4+ - N ions and 99% for PO43−- P ions in
three days, compared to only 50% of NH4+ - N and 40% PO43−- P
removal by their free counterparts [10]. One factor responsible for
enhanced removal is the protective environment provided by the sur-
face to the indigenous microalgae species by acting as a physical barrier
against native microorganisms (through smaller pore size) in waste-
water while allowing unlimited access of nutrients from the wastewater
[11]. Hydraulic Retention Time (HRT) of nutrient removal is lower for
AIMS (0.3–0.8 days) compared to high rate algal ponds (HRAP) (2–6
days) [12], rendering AIMS highly suitable for continuous wastewater
treatment [13].
Other factors for enhanced nutrient removal in AIMS exist through
the possible chemical interactions between alginate matrix and nutrient
species. Alginate is shown to have a high affinity for cations because of
the carboxyl groups (COO−) [14]. Physical adsorption (through elec-
trostatic interaction) of NH4+ and PO43−- P ions on the free binding
sites present on alginate structure is anticipated to be a precursor step
before assimilation by the entrapped algae. The adsorption rates would
vary with the alginate concentration due to the proportionate con-
centration of binding sites available in the matrix. The hypothesis could
be cross-examined by analyzing the effect of alginate concentration in
the removal process in AIMS as well as in blank alginate bead systems
(BABS). The mechanism of binding of nutrient ions on the alginate
matrix would be explored along with the effects of alginate con-
centration on matrix pore morphology and bead integrity. Thus, algi-
nate concentration could be evaluated as a potential design optimiza-
tion parameter in nutrient removal using AIMS.
Herein, the effects of alginate concentration on wastewater nutrient
removal by immobilized Chlorella vulgaris were systematically analyzed
in AIMS and BABS. The nutrient removal of AIMS of different alginate
concentrations (w/v%): 1.5%, 2%, 3%, 4%, 4.5% was investigated ex-
perimentally. Kinetic parameters were evaluated through models for
biomass growth and nutrient removal with quantification of the diffu-
sional resistance in the matrix. To delineate the role of alginate, in-
dependent batch adsorption studies were conducted on BABS of the
same concentrations, and optimum conditions were evaluated. The
experimental data were fitted to adsorption isotherm and kinetic
models. The nutrient mechanisms were examined through Fourier-
Transform Infrared Spectroscopy (FTIR) analysis and Energy Dispersive
X-ray (EDX) analysis. The influence of alginate concentration on the
pore morphology and bead stability was explored. The optimum algi-
nate concentration was assessed based on nutrient removal efficiency,
adsorption capacity, pore restrictive nutrient uptake and bead stability.
2
Biochemical Engineering Journal 149 (2019) 107241
Sna Unassimilable substrate concentration (mg L−1)
t Time (h)
Va Volume of the adsorbate in solution (L)
X Momentary concentration of microorganisms (mg L−1)
X0 Initial biomass concentration (mg L−1)
Xm Final biomass concentration (mg L−1)
Y Microalgae yield coefficient; ratio of biomass amount
produced per amount of substrate consumed (mg mg−1)
Greek letters
μ Maximum specific growth rate (h−1)
μN Maximum specific NH4+ - N uptake rate (h−1)
μP Maximum specific PO43−- P uptake rate (h−1)
Ƞ Effectiveness factor
ϕ Thiele modulus
2. Experimental
2.1. Materials
2.1.1. Microalgae culture preparation
A stock suspension of unicellular microalgae Chlorella vulgaris was
cultured in TRIS-Acetate-Phosphate (TAP) medium and incubated at
27 ± 2 °C under controlled illumination (10,000 lx) with 16 h/8 h
light/dark photoperiods [15]. After the 12-day cultivation period, algal
cells were harvested by centrifugation at 3000 g-forces for 10 min. The
cell residues were washed and resuspended in deionized water resulting
in a concentrated algal suspension with a cell density of 106 cells mL−1.
2.1.2. Microalgae bead preparation
Algal suspension was mixed with sodium alginate solution in 1:1 v/
v ratio to prepare algal alginate suspension of (w/v %) 1.5%, 2%, 3%,
4%, 4.5%. The different suspension was allowed to drop precisely into a
calcium chloride solution (5.5%w/v%). After 12 h of hardening, Ca-
Alginate beads of diameter 2–5 mm were formed. The beads were
rinsed with deionized water and saline solution (0.85% NaCl) and fi-
nally starved in a fresh, sterile saline solution for about 3 days at
27 ± 2 °C under illumination of 12000 lx [16]. BABS of different al-
ginate concentrations were prepared in the same way using deionized
water in place of algal suspension.
2.1.3. Wastewater sampling
Wastewater was collected from a municipal secondary effluent
stream at Bekar bandh pond, Dhanbad, Jharkhand, India (23°48′1″N,
86°25′44″E). The composition of the wastewater is given as (mg L−1):
NH4+ - N, 66.23 and PO43−- P, 10.85.
2.2. Experimental procedure
2.2.1. Nutrient removal experiments
AIMS of different concentration along with control BABS (3%) and
free algae were treated with wastewater in conical flasks (1000 mL
capacity) for 5 days under continuous sparging (2 L min−1), controlled
temperature 27 ± 2 °C and illumination cycles of 16 h/8 h light/
darkness (10800 lx) (Fig. S1 in Supplementary material). pH was
maintained below 8.5 (except in aerated wastewater) to prevent phy-
sical process such as NH3 stripping and precipitation of PO43−- P, which
ensured that the removal occurred solely by algal uptake [10]. For
analysis, 25-mL samples were collected at increasing time intervals:
8 h–24 h.
S. Banerjee, et al. Biochemical Engineering Journal 149 (2019) 107241

2.2.2. Adsorption studies
Batch adsorption experiments of NH4+ - N and PO43−- P onto BABS
were conducted in 100 mL conical flasks on a thermostatic shaker at
120 rpm at 27 °C. The adsorption capacity of BABS was calculated from
the equation:
(Co Ce ) Va
qe =
(1)
m
Adsorption kinetics were evaluated as a function of contact time at
an initial nutrient concentration of 40 mg L−1 NH4+ - N and 20 mg L−1
PO43−- P at 7 pH. The effect of pH was examined for a range of 4 to 9
for an initial nutrient concentration: NH4+ - N: 15 mg L−1; PO43−- P:
5 mg L−1. For the same initial nutrient concentration and 7 pH, the
effect of adsorbent dosage (of 3% alginate beads) for a range of alginate
mass from 0.3 g to 1.5 g was evaluated. The effect of initial nutrient
concentration on sorption was determined using concentration of NH4+
- N: 9.17 mg L−1 to 60 mg L−1 and corresponding PO43−- P:
5.35 mg L−1 to 25 mg L−1 using 1000 mg sorbent dosage for 600 min.
The effect of calcium chloride concentration: (w/v%) 2%, 3%, 4%, 5%
and 6% was observed for adsorption of nutrients on alginate. The nu-
trient medium used for adsorption studies consisted of both NH4+ - N
and PO43−- P and their adsorption were studied simultaneously.
2.2.3. Alginate characterization
The removal process was characterized both in terms of charge
based interaction and morphological structure. The former was studied
through FTIR and EDX for samples before and after treatment with
wastewater. The morphological study was conducted based on a Field-
Emission Scanning Electron Microscope (FE-SEM) to characterize the
average pore size distribution. Finally, the durability and strength of the
alginate was studied through leakage experiments.
2.3. Analytical methods
chamber using a vacuum pump (Tarsons, USA) by decreasing pressure
slowly and steadily to 80 mm Hg for 24 h and were examined using an
FE-SEM (Supra 55, Carl Zeiss, Germany) at 10 kV. The diameter of the
random pores was measured using the integrated SmartSEM™ software.
Experiments were conducted to analyze the leakage of algal cells from
the AIMS into the aqueous medium. Leakage of cells was checked by
collecting 10 mL well-mixed medium samples and measuring their
turbidity (Turbiquant 3000 T, Merck, USA) at regular intervals.
2.3.4. Statistical analysis
Each experiment was performed in triplicates. The mean, con-
fidence interval and standard deviation values of the triplicates were
calculated. Data were analyzed using parametric one-way analysis of
variance (ANOVA) method. The statistical significance of the result was
evaluated using Student’s paired t-test. For probability p-value ≤ 0.05,
the effect of alginate gel concentration on nutrient removal and biomass
growth was considered significant, and otherwise not.
3. Kinetic model
3.1. Algae growth kinetics
The microbial growth is governed by Verhulst logistic kinetic model
equation [21]:
X0 Xm e µt
X=
Xm X 0 + X0 e µt (2)
The growth is characterized by productivity, which is defined as the
rate of change in biomass concentration from 10% higher than the in-
itial concentration to 90% of the final [22].
µ (0.9Xm 1.1X0 )
P=
ln ( 9(Xm 1.1X0 )
1.1X0 ) (3)

2.3.1. Nutrient and biomass measurement

Concentrations of NH4+ - N and PO43−- P were measured according 3.2. Nutrient removal kinetics
to standard analysis methods: 4500-NH3 F [17] and 4500-P E [17] re-
spectively using UV–vis spectrophotometer (ThermoFisher Scientific, The relation for nutrient consumption is given as [22]:
Massachusetts, USA). Biomass of AIMS was measured by immersing the
beads in 0.1 M sodium tri-citrate solution. The released cell con- S=
( X0
Y
+ S0 (S0) Sna) (
Sna S0 ( X0
Y
+ S0 ))e µt

centration was evaluated spectrophotometrically (Dry weight, mg L−1

= (OD680 ×11.62) ̶ 3.9695; R2 = 0.996). The chlorophyll concentra-
(S0 Sna ) (S (
0
X0
Y
+ S0 ))e µt
(4)
tion inside the AIMS was analyzed according to a previous study [18]. Where Y is the microalgae yield coefficient defined as:
Ten beads were dissolved in 5 mL of 90% acetone and kept in darkness
(Xm X0 )
for around 24 h. Chlorophyll a and Chlorophyll b concentration level Y=
(S0 Sna ) (5)
were measured using UV–vis spectrophotometer (ThermoFisher Scien-
tific, Massachusetts, USA). The internal diffusion coefficient (De) for
NH4+ - N and PO43−- P inside the beads was determined using effusion 4. Results and discussion
experiments according to a previous study [19].
4.1. AIMS uptake batch kinetics
2.3.2. FTIR and EDX analysis
FTIR analysis for characterization was performed using FTIR spec- 4.1.1. Growth
trophotometer (MB3000, ABB) equipped with a MIRacle three reflec- The growth of microalgae was analysed for a period of 120 h
tion diamond attenuated total reflectance device (Pike Technologies, (Fig. 1(a)). The algal cell count increased from an initial of 7 × 106 cells
USA). The elemental composition of the bead was evaluated with bead−1 to approximately 90 × 106 cells bead−1. Chlorophyll levels of
Energy-Dispersive X-ray Spectroscopy (EDX) using a scanning electron different AIMS increased almost linearly with time (Fig. 1(b)). The
microscope (Zeiss EVO50, Oberkochen, Germany). trend of decreasing final chlorophyll levels were in the order:
3% > 4% > 2% > 4.5% > 1.5%. The increase in cell count and levels
2.3.3. Pore size and leakage measurements of chlorophyll confirmed photosynthesis [23,24]. The growth of algae
Beads for Scanning Electron Microscopy (SEM) imaging were pre- in AIMS and free suspended system followed similar pattern, differing
pared using the method described in a previous study [20]. Two beads slightly in the final biomass concentration (Xm):
of each alginate concentration were fixed in 5% (v/v) glutaraldehyde 3% > 4% > 2% > 4.5% > 1.5% > free cells.
solution in 1 M HEPES buffer at pH 7.2. They were washed twice using
the same buffer and then dehydrated using increasing ethanol con- 4.1.2. Nutrient removal
centrations: 30% for 10 min, 50% for 30 min, 75% for 10 h, 100% for Fig. 2 shows the evolution curves of NH4+ - N and PO43−- P con-
30 min and 100% for 60 min at 4 °C. The beads were vacuum dried in a centration in free algae systems, AIMS, and control BABS along with bar

3
S. Banerjee, et al.
Fig. 1. (a) Evolution of biomass concentration in free and immobilized mi-
croalgae (symbol represent experimental values; lines represent model values)
(b) Evolution of chlorophyll concentration in different immobilized config-
urations.
plot correlating the % removal with HRT. In both the cases, % removal
by control experiments with BABS was minimal, establishing algal as-
similation as the primary mechanism of removal than chemical and
physical processes. Non-control of pH in aerated wastewater reflected
the enhanced removal of NH4+ - N and PO43−- P, due to increased pH
(> 8) resulting in ammonia stripping and phosphorus precipitation
[24].
4.1.2.1. Ammonium removal. In AIMS, the NH4+ - N concentration
reduced rapidly during the initial 24 h, slowed down between 36–72 h,
and almost settled to zero afterwards (Fig. 2a). For free cells, it
continued to decrease steadily although the % removal was
significantly lesser (28% difference in removal) compared to AIMS
within 48 h (Fig. 2b). After 48 h, the trend for highest NH4+ - N
removal using AIMS was (in decreasing order) as:
3% > 4% > 2% > 4.5% > 1.5% > free algae. However, net removal
after 120 h was nearly equal for all alginate concentrations (> 98%),
slightly differing from free algae (94.31%). The higher performance for
AIMS (3% Alginate) is also evident from the removal achieved in 8 h
HRT: 46.9% compared to 17.8% removal by free algae.
4.1.2.2. Phosphorus removal. The PO43−- P concentrations declined
sharply in all alginate concentrations within 8 h and eventually
stabilized (Fig. 2c). Removal after 16 h was observed in the following
trend (in decreasing order): 3% > 4% > 2% > 1.5% > 4.5% > free
algae. The net removal after 120 h was > 95% for all alginate
concentrations and 90.36% for free algae (Fig. 2d). Within 8 h, 86.2%
PO43−- P removal was achieved in AIMS (3% Alginate) compared to
4
Biochemical Engineering Journal 149 (2019) 107241
39.7% PO43−- P in free algae.
The results of 3% alginate are compared against existing literature
in Table 1. The % removal obtained using 3% alginate is higher com-
pared to other concentrations for specific retention time. The remark-
able feature is that for 3% AIMS, the NH4+ - N (at 8 h) and PO43−- P (at
4 h) removal was 12% more than its closest alginate concentration.
4.1.3. Kinetic parameters
The latest generic approach for modelling multi-nutrient removal
accounts for growth and removal kinetics for each nutrient exclusively.
Verhulst model [21] was adopted for algal growth kinetics while Pho-
tobiotreatment model (PhBT) [22] was used for removal kinetics of
individual nutrient.
4.1.3.1. Growth kinetics. Fitting Eq. 2, the different kinetic growth
parameters of microalgae for all the concentrations are summarized
in Table 2. No significant difference was found in the comparison of the
specific growth between concentrations (p > 0.05) [10].The values for
the specific growth rates compared well with other studies (0.03-
0.04 h−1) having relatively higher levels of NH4+ - N and PO43−- P
than others [8,22,25]. No influence of alginate matrix on the growth
characteristics was observed [10,24,26].
An optimum value of productivity was observed for 3% alginate
(7.10 mg L−1 h−1). This value was significantly different from the
productivity value of 4% alginate (6.68 mg L−1 h−1), with a p-value of
0.0462. The value of productivity compared well with that obtained in
previous studies (6.25 mg L−1 h−1) [25]. However, it was slightly
higher than that obtained by other researchers (4.365 mg L−1 h−1) [27]
and (4.16 mg L−1 h−1) [28] for Chlorella vulgaris. The higher value of
productivity was attributed to the higher initial nutrient concentration
in the wastewater.
4.1.3.2. Nutrient removal kinetics. Photobiotreatment (PhBT) model
[22] treats the growth of microalgae in the presence of nutrient-
substrate as an autocatalytic reaction. PhBT model (Eq. 4) is
applicable to simple reactive systems as in free algae. Nevertheless, it
can also be applied to AIMS for negligible diffusional resistances, as
confirmed through the estimates of the effectiveness factor : unity for
both NH4+ - N and PO43−- P (Table 3).
The summary of uptake rates is presented in Table 4. In the case of
PO43−- P, due to the fast uptake by algae, a model could not be ob-
tained. Hence, the kinetic data for PO43−- P were obtained using ex-
perimental values. The highest maximum specific uptake rate was ob-
served in 3% alginate: 0.137 h−1 and 0.47 h−1 for NH4+ - N and
PO43−- P respectively. The maximum specific NH4+ - N uptake rate was
similar to obtained in a study [22], specifically in the case of NH4+ - N
rich wastewater medium. The maximum uptake rate of PO43−- P was
much higher than that of NH4+ - N in each case [29]. This agrees with
previous studies that reported the maximum specific uptake rate for
phosphorus to be up to three-four times that of nitrogen [22].
A correlation of kinetics between NH4+ - N and that of PO43−- P
exists through the sequence in which they are required in the cell
metabolism: Demand for phosphorus depends on the level of nitrogen
imbibed [30]. P is stored in the ribosomal RNA and participates in the
synthesis of cellular molecules such as phospholipids and nucleic acids.
Being a precursor for other uptakes, uptake of NH4+ - N serves as the
rate-limiting step in the overall removal kinetics. Hence,understanding
the mechanism of NH4+ - N uptake is sufficient for overall kinetics.
4.2. Adsorption studies on BABS
4.2.1. Effect of contact time
The effect of time was observed from 0 to 720 min and is shown in
Fig. 3 (a)-(b). The equilibrium time was approximately 600 min for both
NH4+ - N and PO43−- P. The adsorption capacity increased to
4.97 mg g−1 for NH4+ - N and 2.16 mg g−1 for PO43−- P. The difference
S. Banerjee, et al. Biochemical Engineering Journal 149 (2019) 107241

Fig. 2. (a) Evolution of NH4+ - N uptake in free algae, AIMS and BABS (symbol represent experimental values; lines represent model values except for A and B) (b) %
NH4+ - N removal for AIMS and free algae (c) Evolution of PO43−- P uptake in free algae, AIMS and BABS (symbol and line both represent experimental values) (d) %
PO43−- P removal for AIMS and free algae.

Table 1
Nutrient removal efficiency of different alginate-immobilized microalgae nutrient removal systems.
Culture Microalgae Algae- Alginate Retention NH4+ - N PO43−- P References
condition concentration (%) time (d)
Initial Concentration Removal Initial Concentration Removal
(mg L−1) Efficiency (%) (mg L−1) Efficiency (%)

Batch C. vulgaris 3% 1 66 70 11 89 Present Work

Batch C. vulgaris (P. 2% 1 50 49 10 36 [53]
Putida*)
Batch C. vulgaris FACHB- 3% 1 49 98 13 100 [54]
30
Continuous C. vulgaris FACHB- 3% 1 49 97 13 87 [54]
30 (P. Putida*)
Batch S. obliquus 3% 1 10 30 1 28 [7]
Batch C. vulgaris 2% 1 66 60 11 83 Present work
Batch C. vulgaris 2% 2 – 80 – 53 [23]
Batch C. sorokiniana 2% 2 48 63 3 53 [11]
Batch C. vulgaris 2% 1 – – 39 4 [16]
Batch C. vulgaris 2% 1 37.5 - 47.5 40 – 94 [10]
Batch C. vulgaris 2% 1 9.8 33 35.7 1.5 [55]

* Co-immobilized microorganism.

among the adsorption capacities of different alginate concentrations was
appreciable for NH4+ - N but negligible for PO43−- P. Almost all alginate
concentrations reached equilibrium at the same time. The difference was
observed in adsorption capacities, which depended upon the number of
active sites available. 3% alginate showed relatively higher adsorption
capacity for both nutrients. As per these results, rest of batch adsorption
experiments on the physical properties were conducted for a time in-
terval of 600 min, to ensure that equilibrium was reached.
4.2.2. Effect of pH
The effect of pH on NH4+ - N and PO43−- P adsorption was eval-
uated (Fig. 3 (c)–(d)). Maximum adsorption capacity for NH4+ - N and
PO43−- P adsorption was observed at 7.0 pH (2.21 mg g-1) and at 9.0 pH
(1.9 mg g-1) tively, for 3% alginate. Guluronic and mannuronic acid
residues of alginate have pKa values of 3.38 and 3.65 respectively.
When the alginate is immersed in pH greater than its pKa, the hydrogel
becomes entirely negative charged through the COO− ions, which

5
S. Banerjee, et al. Biochemical Engineering Journal 149 (2019) 107241

Table 2
Kinetic parameters for microalgae growth.
Xo (mg L−1) Xm (mg L−1) μ (h−1) R2 P (mg L−1 h−1)

Free algae 144.33 ± 25.02 732.67 ± 16.16 0.036 ± 0.0004 0.980 5.25 ± 0.10
1.5% 143.00 ± 18.08 853.33 ± 41.63 0.037 ± 0.0010 0.992 6.13 ± 0.15
2% 137.33 ± 16.62 900.10 ± 30.00 0.038 ± 0.0007 0.988 6.53 ± 0.14
3% 143.67 ± 17.78 960.00 ± 45.82 0.039 ± 0.0010 0.986 7.10 ± 0.17
4% 144.67 ± 13.01 911.67 ± 43.10 0.038 ± 0.0009 0.982 6.68 ± 0.19
4.5% 139.00 ± 12.52 891.33 ± 47.59 0.037 ± 0.0008 0.987 6.39 ± 0.22

Values represent means of triplicates; ± shows standard deviation from mean.

Table 3
Diffusion-Reaction model parameters: for NH4+ - N and PO43−- P uptake.
Bead (% alginate) Radius (mm) k × 10−5 (L mg−1 h-1) k × 10−3 (L mg−1 h-1) De (mm2 h−1) ϕ × 10−4 ϕ × 10−3 η

NH4+ - N PO43−- P NH4+ - N PO43−- P NH4+ - N PO43−- P NH4+ - N PO43−- P

1.5% 2.080 9.47 ± 1.43 8.21 ± 1.71 1.5191 0.9630 1.00 ± 0.07 1.17 ± 0.13 0.999 0.999
2% 2.175 10.4 ± 1.70 8.00 ± 1.50 1.3242 0.9384 1.31 ± 0.10 1.36 ± 0.13 0.999 0.999
3% 2.210 10.9 ± 1.62 8.05 ± 1.74 1.2483 0.9035 1.30 ± 0.09 1.31 ± 0.15 0.999 0.999
4% 2.250 10.11 ± 2.14 7.24 ± 1.54 1.1460 0.8826 1.44 ± 0.15 1.40 ± 0.15 0.999 0.999
4.5% 2.375 10.3 ± 1.90 7.22 ± 1.15 1.0297 0.8821 1.78 ± 0.16 1.61 ± 0.13 0.999 0.999

Values represent means of triplicates; ± shows standard deviation from mean.

Table 4
Kinetic parameters for NH4+ - N and PO43−- P removal.
So (mg L−1) Sna (mg L−1) μN (h−1) R2 Y k × 10−5 (L mg−1 h−1)

NH4+ - N Free algae 66.23 ± 3.52 3.76 ± 1.57 0.061 ± 0.0004 0.975 13.85 ± 0.79 7.06 ± 0.13
1.5% 66.23 ± 3.52 0.89 ± 0.17 0.095 ± 0.0053 0.969 15.53 ± 0.71 9.47 ± 1.43
2% 66.23 ± 3.52 0.06 ± 0.09 0.112 ± 0.0073 0.983 16.34 ± 0.78 10.40 ± 1.70
3% 66.23 ± 3.52 0.06 ± 0.02 0.137 ± 0.0075 0.986 19.05 ± 0.79 10.90 ± 1.62
4% 66.23 ± 3.52 0.15 ± 0.003 0.119 ± 0.0080 0.977 18.02 ± 0.77 10.11 ± 2.14
4.5% 66.23 ± 3.52 0.21 ± 0.02 0.107 ± 0.0090 0.966 15.84 ± 0.91 10.34 ± 1.90

So (mg L−1) Sna (mg L−1) μP (h−1) R2 Y k × 10−3 (L mg−1 h−1)

PO43−- P Free algae 10.85 ± 1.03 6.53 ± 0.66 0.24 ± 0.022 0.972 3.10 ± 0.26 8.10 ± 2.07
1.5% 10.85 ± 1.03 1.98 ± 1.00 0.27 ± 0.025 0.961 3.83 ± 0.76 8.21 ± 1.71
2% 10.85 ± 1.03 1.90 ± 0.88 0.34 ± 0.053 0.984 4.76 ± 0.63 8.00 ± 1.50
3% 10.85 ± 1.03 1.50 ± 0.94 0.47 ± 0.046 0.984 6.42 ± 1.53 8.05 ± 1.74
4% 10.85 ± 1.03 1.81 ± 0.94 0.35 ± 0.044 0.975 5.58 ± 1.39 7.24 ± 1.54
4.5% 10.85 ± 1.03 2.21 ± 0.77 0.28 ± 0.012 0.965 4.61 ± 0.54 7.22 ± 1.15

Values represent means of triplicates; ± shows standard deviation from mean.

attract the oppositely charged cation NH4+ - N [31]. As the pH in-
creases above 4 up to 7, the COO− ions in the matrix increases which
leads to strong interactions between the cationic NH4+ at 7 pH [8]. In
the case of PO43−- P, the adsorption capacity increased as the pH in-
creased from 6 onwards. At pH higher than 7.0, the calcium ions inside
the alginate favoured the precipitation of PO43- ions as calcium phos-
phate [10,24], hence, the adsorption of PO43−- P was higher at elevated
pH.
4.2.3. Effect of initial nutrient concentration
For both NH4+ - N and PO43−- P, at a fixed amount of adsorbent
dosage, with the increase in their initial nutrient concentration the
adsorption capacity increased but the % extent of adsorption decreased.
In the case of NH4+ - N, the capacity reached up to 6.47 mg g−1 for 3%
BABS. The increase in adsorption capacity can be linked to an increase
in affinity for charge interactions for an increase in cations (NH4+) and
the enhanced concentration gradient. For PO43−- P the increase in
concentration led to an increase of adsorption capacity by
1.328 mg g−1. The enhanced concentration gradient at higher PO43−- P
concentrations increases the amount adsorbed onto alginate.
4.2.4. Effect of adsorbent dosage
The % adsorption extent increased with increase in the sorbent
(alginate) mass in the solution for NH4+ - N and decreased for PO43−- P
(Fig. S2 in Supplementary material). In the case of NH4+ - N, the in-
crease in alginate mass increases the number of active sites which leads
to an increase in adsorption. For PO43−- P, the adsorption decreased
due to the increasing repulsion forces (COO− ions) which are similar to
Wan’s result [32]. Adsorption capacity for both NH4+ - N and PO43−- P
decreased with increasing alginate dosage. The increase in adsorbent
dosage could possibly lead to unsaturation of active sites and ag-
gregation, which reduces the available total surface area available for
adsorption [33,34].
4.2.5. Effect of CaCl2 concentration
Cross-linking solution is an important parameter determining nu-
trient adsorption for a specific alginate concentration. For NH4+ - N,
increased CaCl2 concentration lead to increase in % NH4+ - N removal
(Fig. S3 in Supplementary material). Increase in CaCl2 concentration
promotes additional inter crosslinking inside alginate [35], which in-
creases the pore formation inside the matrix and the active COO− sites.
At lower CaCl2 (1–2 w/v%) concentration, NH4+ - N removal decreased
due to a lower degree of crosslinking (lower COO− sites). In the case of

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S. Banerjee, et al. Biochemical Engineering Journal 149 (2019) 107241

Fig. 3. Sorption studies experiment effect on BABS (a) Effect of contact time on NH4+ - N (b) Effect of contact time on PO43−- P (c) pH on NH4+ - N (d) pH on
PO43−- P (e) Initial nutrient concentration on NH4+ - N (f) Initial nutrient concentration on PO43−- P.

PO43−- P, the % removal declined with increasing concentration of
CaCl2 due to an increase in anionic charge due to the increase of COO−
sites. Therefore, an optimum concentration of 3% CaCl2 is desirable to
achieve proper crosslinking of the alginate matrix [7].
In the BABS experiments, 3% alginate concentration exhibited the
highest adsorption capacity for all cases. Hence, this concentration was
used to evaluate the adsorption isotherms and kinetics.
4.2.6. Adsorption isotherm models
The adsorption equilibrium data for 3% BABS was interpreted using
Langmuir, Freundlich and Redlich-Peterson (R − P) isotherm models
[36] (models described in Table S1 in Supplementary material). The
models were fitted in their non-linear form with the experimental data
in Fig. 4(a)-(b) with the fitted parameters listed in Table S2 (Supple-
mentary material). The best fit was observed for the Langmuir model
(NH4+ - N: R2 = 0.990 and PO43−- P: R2 = 0.974) followed by the
R − P model (NH4+ - N: R2 = 0.988 and PO43−- P: R2 = 0.973) and the
Freundlich model (NH4+ - N: R2 = 0.973 and PO43−- P: R2 = 0.958).
The value of exponent β in R − P approached unity for both NH4+ - N
and PO43−- P implying conformity to Langmuir isotherm model. This
implies that the adsorption of the nutrients occurs at specific homo-
genous sites as mono-layer adsorption onto alginate matrix and surface.

7
S. Banerjee, et al.
Fig. 4. Adsorption isotherms obtained through experimental data nonlinear fitting (a) for NH4+ - N (b) for PO43−- P. Adsorption kinetics of BABS using Pseudo first
order model and Pseudo-second order model (c) for NH4+ - N (d) for PO43−- P.
Fig. 5. FTIR spectra of AIMS and BABS, before and after adsorption.
The adsorption of NH4+ - N and PO43−- P onto alginate was mainly
because of electrostatic attraction between functional groups and the
nutrient ions in the aqueous solution as seen in other studies [37–39].
The maximum monolayer adsorption capacities calculated from Lang-
muir isotherm model of NH4+ - N and PO43−- P are 15.58 mg g−1 and
3.29 mg g−1, respectively.
8
Biochemical Engineering Journal 149 (2019) 107241
4.2.7. Adsorption kinetics
The adsorption process of N and P onto alginate was characterized
based on Lagergren pseudo-first-order model and Ho pseudo-second-
order model [40,41] (models described in Table S3 in Supplementary
material). The non-linear constants obtained from pseudo-first-order
and pseudo-second-order models were calculated from non-linear ki-
netic plots given in Fig. 4(c)-(d) and summarized in Table S4 (Supple-
mentary material). The pseudo-first order model (NH4+ - N: R2 > 0.996
and PO43−- P: R2 > 0.991) fitted the model better than pseudo-second-
order model (NH4+ - N: R2 > 0.98 and PO43−- P: R2 > 0.98). The
calculated values of qe,c from the pseudo-first-order model closely ap-
proximates the experimental qe values than the qe,c obtained from the
pseudo-second-order model. This implies that the adsorption process
for NH4+ - N and PO43−- P was physisorption. The high adsorption
capacity of 3% alginate BABS compared to other alginate concentra-
tions promotes nutrient removal, and this effect is amplified in the case
of AIMS due to the presence of microalgae.
4.3. Alginate characterization and mechanism investigation
4.3.1. FTIR and EDX analysis
The mechanism was explored from FTIR (Fig. 5) and EDX studies
conducted on AIMS and BABS before and after treatment with waste-
water. The FTIR spectra of untreated BABS and AIMS exhibited peaks at
3330 cm−1 (NH stretching), 1641 cm−1 (COO − asymmetric stretching
(strong)), and 1033 cm−1 (CO − stretching of alcoholic groups).
Spectra of treated AIMS and BABS beads revealed the predominant
peaks 3360 cm−1 (NH stretching and Hydrogen bond (broad);
S. Banerjee, et al. Biochemical Engineering Journal 149 (2019) 107241

Fig. 6. Mechanism for NH4+ - N and PO43−- P adsorption and algal assimilation on AIMS.

overlapping with OH stretching), 1641 cm-1 (COO asymmetric Supplementary material) showed the emergence of the peak for ni-
stretching), 1423 cm-1 (NH deformation vibration) and at 1035 cm-1 trogen and phosphorous after treatment. The nitrogen content (wt%)
(P − O stretching (strong)) [42–44]. The EDX analysis (Fig S4 in (N) increased from an initial value of 0% to 7.234% in BABS and

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S. Banerjee, et al. Biochemical Engineering Journal 149 (2019) 107241

Fig. 7. SEM images of pores in different alginate concentration beads (a) Typical morphological surface of a bead (b) 1.5% Alginate bead (c) 2% Alginate bead (d)
3% Alginate bead (e) 4% Alginate bead (f) 4.5% Alginate bead.

Fig. 8. Cell leakage from AIMS into a nutrient-deficient medium (a) Evolution of leakage concentration (turbidity) with time (b) Turbid solutions of AIMS, obtained
after the 5th day of the experiment.

4.118% in AIMS. Phosphorus content (wt%) increased from an initial
value of 0% to 5.095% in BABS and 1.295% in AIMS. The above ana-
lysis showed a similar behaviour of both AIMS and BABS during ad-
sorption of nutrients into the bead, except for the difference in N and P
content, ruling out any influence of microalgae on the adsorption of
nutrients onto alginate.
4.3.2. Mechanism of nutrient adsorption
The interaction of nutrient with the alginate structure is illustrated
in Fig. 6. An interesting decline in the peak at 3361 cm−1 to 3330 cm−1
is observed after the adsorption of nutrients. Hydrogen bonding causes
significant band broadening and lowering of the mean adsorption fre-
quency as noted in the region [44]. In the presence of an electrostatic
environment within the polymeric matrix, the NH4+ ion forms a weak
hydrogen bond with the carbonyl oxygen of the free COO− ions
[45,46]. The observations were in line with Etter’s rule, which states
that best hydrogen pair donor (NH4+, in our study) forms a hydrogen
bond with the best available hydrogen bond acceptor (COO−) [47]. In
case of PO43−- P, the emergence of a new peak at 1035 cm−1 is at-
tributed to P − O stretching, confirming the presence of PO43−- P being
adsorbed. The broadening of the peak suggests that the physical
interaction of the PO43− ion with OH is through hydrogen bonding
[48].
The above trends comply with the adsorption isotherm results as
well. Langmuir isotherm model assumes monolayer adsorption, char-
acterized by homogenous adsorption, i.e. all sites have equal affinity for
adsorbate [36]. The NH4+ ion adsorption is based on electrostatic at-
traction between the protonated amino group and the negatively
charged COO− ions. This interaction involves constant and equal ad-
sorption energy which agree with the correlation coefficients obtained
from Langmuir isotherm. In the case of AIMS, these ions (NH4+ and
PO43−), which are weakly bonded to the alginate structure, are as-
similated by the microalgae in the course of time. The assimilation
results in unoccupied vacancies in the alginate chain at COO− and OH
groups, which causes more uptake of nutrients from outside the algi-
nate bead environment. This process continues until the microalgae
limit the uptake of nutrients (possibly due to the stagnant phase) and
consequently saturating the bead with adsorbed nutrients.
4.3.3. Morphological studies
Apart from adsorption characteristics, the concentration of the al-
ginate gel affects the removal indirectly through varying pore size. SEM

10
S. Banerjee, et al.
images of BABS are shown in Fig. 7. The average surface pore size of
alginate in the decreasing order is as follows: 1.5% alginate
(0.542 ± 0.094 μm), 2% alginate (0.506 ± 0.087 μm), 3% alginate
(0.394 ± 0.076 μm), 4% alginate (0.240 ± 0.180 μm) and 4.5% algi-
nate (0.119 ± 0.180 μm). The increase in alginate gel concentration
causes the matrix to become denser and reduces the pore size, leading
to restrictive uptake of nutrients or other macro-molecules [49]. The
porosity of the matrix also decreases with increasing alginate con-
centration, which reduces the total void volume inside the matrix
[50,51].
4.3.4. Durability studies of alginate bead
The leakage tendency was examined through separate experiments
on AIMS conducted for 15 days (Fig. 8). Leakage increased with de-
creasing alginate concentration and was highest in 1.5% alginate. After
the 8th day, the 1.5% alginate beads lost their integrity while the 2%
and 3% were seen to disintegrate on the 11th day and 14th day, re-
spectively. Increasing alginate concentration increases the crosslink
density and solid volume fraction, resulting in higher mechanical
strength and structural integrity [52]. Higher alginate concentration
(> 3%) were observed to retain their integrity until the end of the
experiment, and the cell leakage was significantly lesser than lower
alginate concentration beads.
The findings indicate that a balance in the alginate concentration is
vital to obtain maximum nutrient adsorption and uptake and mini-
mizing the subsequent leakage of cells, which can result in higher nu-
trient removal. Alginate concentration of 3% was seen to be the best
among all concentrations, in terms of adsorption on alginate and pore
restrictive nutrient uptake. A previous study also reported 3% alginate
concentration to be an optimum for nutrient removal and lipid pro-
ductivity [7]. Since immobilization increases cost to wastewater treat-
ment, hence, for the overall process to be economical, the im-
mobilization process should be optimized. As such, the careful selection
of sodium alginate concentration used to prepare the bead is imperative
in enhancing the economic capability of the algal wastewater treat-
ment. Due to the advantage of obtaining higher removal in lesser HRT,
3% alginate shall be used as packings in packed bed reactor for con-
tinuous nutrient removal in our future application of this batch study.
5. Conclusion
The present study analyzes the effect of alginate concentration on
wastewater nutrient removal using alginate immobilized microalgae
beads. Within the alginate systems, AIMS of 3% alginate exhibited
optimal values for specific nutrient uptake rate and productivity.
Adsorption studies on BABS characterized the enhanced nutrient ad-
sorption by the immobilizing matrix. Elaborate FTIR and EDX analysis
on BABS confirmed the role of alginate in providing adsorption sites for
catalyzing the nutrient uptake. The optimal trends were explained
through elaborate morphology analysis that showed 3% to strike the
balance of adsorption on alginate and nutrient access.
Acknowledgement
CB highly acknowledges the Department of Science and Technology
(DST) Govt. of India for providing financial support as well as project
grant from INSPIRE Faculty award scheme (DST/INSPIRE/04/2014/
002322).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.bej.2019.107241.
11
Biochemical Engineering Journal 149 (2019) 107241
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