International Biodeterioration & Biodegradation xxx (2016) 1e8

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International Biodeterioration & Biodegradation

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A study of coagulating protein of Moringa oleifera in microalgae bio-

flocculation
Siti Hajar Abdul Hamid a, Fathurrahman Lananan a, Helena Khatoon b, Ahmad Jusoh c,
Azizah Endut a, d, *
a
East Coast Environmental Research Institute, Universiti Sultan Zainal Abidin, 21300 Kuala Terengganu, Malaysia
b
School of Fishery Science and Aquaculture, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Malaysia
c
School of Ocean Engineering, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Malaysia
d
Faculty of Innovative Design and Technology, Universiti Sultan Zainal Abidin, 21300 Kuala Terengganu, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Moringa oleifera is characterized by high coagulation properties, low cost and low toxicity hence is very
Received 17 November 2015 promising to be utilized as an alternative coagulant to recover microalgae biomass from its suspension
Received in revised form system. Hence, this study was performed with the objective to investigate the potentiality of M. oleifera
26 March 2016
as coagulant agent in harvesting microalgae and to investigate the effect of zeta potential in its
Accepted 28 March 2016
coagulation-flocculation activity. Bradford protein assay was applied for rapid and accurate determina-
Available online xxx
tion of protein concentration in the M. oleifera seed powder and protein powder. The flocculation ac-
tivities were determined at isoelectric pH by computing the flocculation efficiency in terms of microalgae
Keywords:
Moringa oleifera
biomass recovery and removal percentage at various coagulant dosages. It was observed that the protein
Coagulating protein concentration was 211.71 mg g
1 mL
1 in M. oleifera seed powder and 188.16 mg g
1 mL
1 in protein
Zeta potential powder which yielded 97% and 78% of biomass recovery, respectively at the dosage of 10 mg L
1. Result
Bio-flocculation showed that M. oleifera seed derivatives supersede chemical coagulant, alum which yielded 34% of
Chlorella sp. biomass recovery at the same dosage. M. oleifera seed powder and protein powder was proven to be
highly promising bio-coagulant and suitable alternative to the chemical coagulant in environmentally-
sustainable harvesting of microalgae biomass.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction techniques was often performed to pre-concentrate the algae

biomass.
Currently, the most commonly used separation methods are There are two steps for the recovering of algal biomass in a
filtration and centrifugation (Teixeira et al., 2012). However, filtra- commercial-scale processing before undergoing downstream pro-
tion is only effective for microalgal cells, which are relatively large, cessing (Lam and Lee, 2012; Molina Grima et al., 2003; Sharma
such as Arthrospira sp. but is unable to separate the biomass from et al., 2013). First, traditional harvesting method was the bulk
the cultivation medium for cells of smaller dimensions (Papazi harvesting which known as primary harvesting. The purpose of this
et al., 2010). The most successful techniques for microalgae primary harvesting is to separate microalgae from suspension via
biomass harvesting were centrifugation, filtration and flocculation. sedimentation, flocculation and floatation. The second step is the
Commercial systems mainly use centrifugation for harvesting, but thickening processing which known as secondary dewatering to
it is an expensive and energy intensive operation (Granados et al., concentrate the microalgae slurry after bulk harvesting. Normally,
2012). Hence, it was suitable only for the harvesting of biomass the thickening process was performed by using centrifugation and
with high-value products. For low-value products, a pre- filtration. An optimal harvesting technique should be independent
concentration step was necessary. In practice, a combination of of the cultured species, less energy consumption and few chem-
icals. It was also important that the harvesting technique was not
cause any damage to the valuable products during extraction pro-
* Corresponding author. East Coast Environmental Research Institute, Universiti
cess. Brennan and Owende (2010) had summarized the advantages
Sultan Zainal Abidin, 21300 Kuala Terengganu, Malaysia. and disadvantages of conventional harvesting techniques from the
E-mail address: [email protected] (A. Endut).

http://dx.doi.org/10.1016/j.ibiod.2016.03.027
0964-8305/© 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Abdul Hamid, S.H., et al., A study of coagulating protein of Moringa oleifera in microalgae bio-flocculation,
International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.03.027
2 S.H. Abdul Hamid et al. / International Biodeterioration & Biodegradation xxx (2016) 1e8

previous studies (Table 1). contribute to the environmental problem such as eutrophication
Microalgae harvesting technique were including centrifugation, and algal blooms. Recovery of the microalgae biomass from broth
filtration, sedimentation, chemical flocculation and floatation. In has been claimed to contribute 20e30% to the total cost of pro-
centrifugation, solid-liquid separation process was driving by a ducing the biomass (Gudin and Thepenier, 1986; Molina Grima
much greater force (gravity) to promote accelerated settling of et al., 2003). Hence, the microalgae biomass harvesting issue was
microalgae cells. This technique can be used for almost all types of exclusively focused in order to promote effective aquaculture
microalgae reliably and without difficulty (Pires et al., 2012). wastewater treatment for sustainable downstream processing of
However, centrifugal recovery is only feasible if the metabolite microalgae.
content in the targeted biomass is a high-value product. This is due Harvesting of microalgae biomass requires the minimum of two
to the high energy consumption is required during the separation solid-liquid separation steps. According to Molina Grima et al.
process. The large-scale biomass recovery become a problematic (2003), microalgae biomass can be harvested through the process
due to the high power consumption which increases the produc- of centrifugation, filtration or gravity sedimentation. These pro-
tion costs. In addition, the coagulation-flocculation required to be cesses may be preceded by a coagulation-flocculation step. But,
followed by flotation-sedimentation and finally dewatering step by centrifugal recovery of the biomass was only feasible for high-value
centrifugation were performed for low-cost biomass harvesting products such as Spirulina sp. to reimburse the high maintenance
(Schenk et al., 2008). cost. On the other hand, filtration recovery was unsatisfactory
Coagulation-flocculation was used to aggregate the microalgae because it was relatively slow and unsuitable for the biomass re-
cells and increase the effective ‘‘particle’’ size, thus enhancing covery in large-scale volumes. Filtration recovery was unsatisfac-
biomass recovery. Coagulants such as aluminum sulfate (alum), tory due to the exposure of the harvested biomass towards
ferric sulfate, ferric chloride, ferrous sulfate, sodium aluminate, iron membrane fouling and its suitability to only large-cell-sized
salts and chitosan has been used for the recovery of microalgae via microalgae. Hence, it required regular membrane changing and
coagulation-flocculation and was demonstrated successfully to maintenance which may contributed to the harvesting cost.
achieve the goal (Molina Grima et al., 2003). However, coagulation- Among the aforementioned methods, coagulation-flocculation
flocculation by metal salts may be unacceptable if harvested was considered to be an effective and convenient process, which
biomass is to be used for aquaculture purposes, animal feed or allows rapid treatment of large quantities of microalgae (Oh et al.,
organic fertilizer. High aluminum concentration does not caused 2001). Various methods of coagulation-flocculation can be used
effects only upon fish, but also birds and other higher animals in the to aggregate the microalgal cells to increase the effective particle
food chain that consumed the contaminated fish and insects. Ac- size and hence ease the sedimentation process for biomass recov-
cording to De-Bashan and Bashan (2004), another negative envi- ery. Microalgal cells carry a negative charge that prevents aggre-
ronmental effect of aluminum is that its ions can react with gation of cells in suspension (Molina Grima et al., 2003). Therefore,
phosphates, which causes total phosphate to be less available to the surface charge can be neutralized or reduced by the addition of
water organisms. It was reported that the major component of alum coagulants such as multivalent cations and cationic polymers to the
and acrylamide could lead to human health implications, such as culture. The use of biological based coagulant would assist sus-
involvement in Alzheimer's disease and the cause of cancers tainable aquaculture practices. The objectives of this study are (i) to
(Ahmad et al., 2011; Hamid et al., 2014). Therefore, an alternative of investigate the potentiality of Moringa oleifera seed as coagulant
environmentally friendly harvesting approach need to be devel- agent for freshwater Chlorella sp. biomass harvesting and (ii) to
oped completely not only to ease microalgae biomass recovery but study the effect of zeta potential and isoelectric of microalgae
also to preserve our natural environment. suspension toward coagulation-flocculation. As comparison to the
Besides that, the treated medium was also not suitable to be efficiency of coagulation-flocculation process, alum was selected as
reused because of the deterioration of water quality due to the control for the M. oleifera derivatives because it had been widely
addition of coagulants. Hence, the exploration of potential natural utilized as standard flocculation reagent in the water and waste-
coagulant is crucial for sustainable wastewater treatment utilizing water treatment protocols. This study had successfully elucidated
microalgae. The utilization of suspended microalgae culture in the potentiality of M. oleifera plant derivatives in harvesting
biological treatment would then lead to the requirement for the Chlorella sp. from the water column which is absence in current
effective separation process. Releasing treated water to the water research.
body without proper recovery of microalgae biomass could

Table 1
Harvesting techniques and their respective advantages and disadvantages (Brennan and Owende, 2010).

Harvesting technique Advantages Disadvantages

Centrifugation  High harvesting efficiency  High capital and operational costs

 Suitable for most algae types  Cell damage
 Rapid separation process  Difficult bulk harvest
 Easy to operate
Filtration  Water and nutrition reuse  Fouling
 Wide variety of filter and membrane types available  Slow process
 Suitable for large algal cells
Sedimentation  Low power consumption  Algal species specific
 Low requirement for skilled operators  Slow sedimentation rates
 Useful as pre-concentration step  Low cell recovery
Chemical flocculation  Wide range of flocculants available  Chemical contamination
 Ease to use  Removal of flocculants
 Highly sensitive to pH level
 Flocculants may be algal specific species
Floatation  Prone to harvest in mass culture  Algal species specific

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 S.H. Abdul Hamid et al. / International Biodeterioration & Biodegradation xxx (2016) 1e8 3

2. Methodology polymer were carried out based on Kwaambwa and Maikokera

(2007). Dried de-oiled M. oleifera powder was used for the
2.1. Chlorella sp. cultivation extraction of coagulant protein polymer. The extraction was per-
formed by adding 3% (w/v) sodium chloride (NaCl) solution and this
The microalgae culture used in this study was freshwater suspension was continuously agitated for 12 h in orbital shaker at
Chlorella sp., which was isolated from the African catfish, Clarias controlled temperature of 25 ± 2  C. The extract was filtered with
gariepinus pond of the Institute of Tropical Aquaculture, Universiti Whatman filter No.44 (11 cm diameter, 0.4 mm) and brown colored
Malaysia Terengganu. Freshwater suspended microalgae, Chlorella of NaCl extract was collected. It was further heated by using hot-
sp. was selected in this study due to its robustness to wide range of plate stirrer (IKA C-MAG HS7, Australia) in such a way that no white
temperature, salinity and illumination conditions (Lananan et al., precipitation is formed at the bottom of solution. The heated crude
2013). A water sample was collected using plankton nets with the protein extract solution was then poured into the dialysis tube and
mesh size of 20 mm. Water was sampled from fish pond and filtered submerged completely for 12 h in beaker containing cold water
through Whatman GF/C filter paper with 0.45 mm pore size to kept in an ice bath to maintain constant temperature of 2 ± 2  C.
obtain concentrated algae paste. Then, the volume of concentrated After completion of the dialysis procedure, the salt present in the
algae paste was re-suspended in fresh culture media to promote crude brown protein was osmotically extracted into the sur-
growth of a particular species in isolation process. The water rounding water solution leaving white protein extract inside the
sample was left to bloom before further used in isolation process. dialysis tube. Subsequently, the extracted white protein was
The primary stock of monoculture was up-scaled gradually starting transferred from the tube into sterile glass petri plates by rinsing it
from 5 ml to 100 ml. After the culture blooms, the culture was with sterile deionized water. The isolated protein was then soaked
transferred into 250 ml with the addition of culture media. It was with cold acetone in a homogenizer to remove lipid from the
cultivated for 8 days to produce secondary culture for further up- extracted protein polymer. This process is known as the delipida-
scaling. Normal air filtered with 40 mm air filter (Sartorius Stedim tion procedure, where the protein was dried at room temperature
Midisart® 2000 In-Line, USA) was provided as sterile aeration to to form fine protein powder.
prevent any bacterial contamination. Microalgae cultures were
maintained at room temperature (25 ± 2  C), illuminated with
white fluorescent light for 24 h photoperiod at 4100 lux (Hagner
2.4. Preparation of aluminum sulfate (alum) stock solution
EC-1, Indonesia). The microalgae were cultivated until the early
stationary phase of growth before undergoing coagulation-
Stock solution of alum (10 mg L
1 Al3þ) supplied by Merck was
flocculation process.
prepared by dissolving 1 g of dry solid in 99 mL of deionized water.
Then, the solution was mixed by using magnetic stirrer to ensure all
2.2. Preparation of M. oleifera seed powder and oil extraction
solids dissolve. A fresh solution was prepared every day for reliable
results by ensuring constant flocculant activity and reducing
The collection and processing of M. oleifera were prepared at
oxidation degradation. In this study, alum was used as control
Aquaculture Engineering and Water Quality Laboratory, Institute of
coagulant for the comparison with natural coagulants of M. oleifera
Aquaculture Tropical, Universiti Malaysia Terengganu. Dry pods of
seed powder and protein powder in coagulation-flocculation of
M. oleifera were collected within the region of Kuala Terengganu,
freshwater microalgae, Chlorella sp.
Malaysia. Dry pods were unshelled to obtain the seed. The bark
enveloping the seeds in the pods was removed manually and only
qualified seeds were selected to further extraction. Good quality
seed were identified from those, which were not rotten, old, 2.5. Zeta potential analysis
infected with diseases, brownish and dried once opened (Katayon
et al., 2006). Prior to the preparation of coagulant, the dry pods of The analysis of z-potential was performed using Zeta-Meter
M. oleifera were dried in 24 h at controlled temperature of System 3.0þ with unitron microscope and Type GT-2 cell (ZM3-
40e50  C. The temperature for drying process was ensure not D-G, USA) instrument. The z-potential measurement was per-
exceed 60  C to prevent the protein content from damage. M. formed using a technique of electrophoresis. A high quality ste-
oleifera seed powder was obtained from raw seed which were reoscopic microscope was used to observe colloidal particle inside a
grounded using laboratory mill and sieved through 600 mm stain- chamber called an electrophoresis cell. Electrodes placed in each
less steel sieve to ensure the distribution of fine powders. The end of the chamber were connected to a power supply which
powder form of these coagulants were stored in air-tight container creates an electric field across the chamber. Charged colloids will
and protected from light to avoid oxidation and degradation of its move in the field and their velocity and direction were related to
properties. Ethanol-based oil extraction procedure based on their z-potential. The electrophoresis cell holds the sample for
(Kwaambwa and Maikokera, 2007) was performed on the fine M. viewing under the microscope. It consists of two electrode cham-
oleifera seed kernel powder. Ninety-five-percent of ethanol was bers connected by an optically polished electrophoresis tube, 10 cm
added in 1:10 ratio (1 g of seed powder and 10 mL of ethanol) to long and 4 mm in diameter. Each cell was engraved with its relative
form suspension. Then, the solution was mixed using magnetic cell size and k factor. The cell size is needed to exactly align the cell
stirrer for 10 min to extract the active ingredients. After that, su- under the microscope while the k factor was used to preset the
pernatant from the extraction was separated by centrifugation Zeta-Meter System 3.0þ for specific conductance measurement.
(3000 rpm; 15 min) using bench-top centrifuge (Zentrifugen Uni- The electrophoresis cell of this Zeta-Meter System 3.0þ was fabri-
versal 1200, Germany). The settled powder which are de-oiled seed cated from fused quartz and Teflon that could handle both aqueous
was dried at room temperature for 24 h. system and organic solvents. The Zeta-Meter System 3.0þ first
measured the electrophoretic mobility (EM) of the particles, which
2.3. Purification of M. oleifera coagulation polymer as protein was expressed as microns/second per volt/centimeter
powder (mcm s
1 V
1). The first term, microns per second was simply a
velocity measurement, while the second term was the expression
The procedures for the purification of M. oleifera coagulation of electrical field strength.

Please cite this article in press as: Abdul Hamid, S.H., et al., A study of coagulating protein of Moringa oleifera in microalgae bio-flocculation,
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2.6. Braford protein analysis interaction between these factors. Although useful for multi-
factorial statistical determination, two-way ANOVA lacked the
Prior to the coagulation-flocculation test, each of the M. oleifera determination capability to point treatment with the most signif-
seed derivatives prepared was subjected to the determination of icant reading and rank them to the least significant. One-way
crude protein content. Bradford protein assay was used for the ANOVA was then employed to determine the significance within
rapid accurate determination of protein concentration. Among the each factor based on the result of two-way ANOVA. OriginLab Pro
several methods available, the spectroscopic Bradford assay was 9.1™ was used as the main software for the purpose of graphical
commonly used because it was considered as fast and sensitive method of analysis.
(Bradford, 1976). The Bradford protein assay was a simple proce-
dure for determination of total protein concentration in solutions 3. Results and discussion
that depends upon the change in absorbance based on the pro-
portional binding of the dye Coomassie Blue G-250 to proteins. A 3.1. Effect of protein concentration in M. oleifera seed derivatives on
set of standard was prepared from a stock solution of protein with biomass recovery
known concentration. The Bradford values obtained from the
standard was used to construct a standard curve. Then, this stan- Fig. 1 shows the protein concentration and the biomass recovery
dard curve was used to determine the protein concentration in M. percentage of Moringa oleifera seed derivatives from the pre-
oleifera samples. The standard curve of Bovine Serum Albumin liminary coagulation-flocculation assay. The highest concentration
(BSA) was prepared within the range of 0e1 mg mL
1. The Bradford of protein content was found in M. oleifera seed powder which is
reagent was a light brown in color. It was filtered through What- 211.71 mg g
1 mL
1 compared to seed coat (171.06 mg g
1 mL
1) and
man No.1 paper before use to rid of reagent of blue components. cupule (183.97 mg g
1 mL
1) for M. oleifera primary derivatives. In
Stock solution with concentration of 1 mg mL
1 was prepared by M. oleifera secondary derivatives, the protein concentration was
dissolving 1 g of BSA in 1000 mL of 150 mM NaCl solution. 150 mM 149.45 and 177.84 mg g
1 mL
1 for de-oiled seed and seed oil
NaCl solution was prepared by dissolving 8.775 g crystalline NaCl in respectively. The protein concentration for M. oleifera tertiary seed
1 L deionized water and stored frozen in 1 mL aliquots for quick use. derivatives were observed at 170.42 mg g
1 mL
1 in protein-lipid
NaCl solution was used to dissolve the standard and any unknown and 188.16 mg g
1 mL
1 in M. oleifera protein powder. The alum
protein samples to be tested. Due to the tendency of protein to was not included in the determination of crude protein content. All
absorb water during storage, it was important to obtain the exact
concentration of BSA by measuring its absorbance at 280 nm before
use. Therefore, the measurement was carried out by using dual-
beam spectrophotometer (Shimadzu UV-1800, Japan) to ensure
that 1 mg mL
1 solution of BSA have an absorbance of 0.66 at
wavelength 280 nm. After obtain the exact concentration,
100 mL mL
1 of BSA solution was of 595 nm. All measurements in
this procedure was carried out by using the quartz cuvette to avoid
color reagent stains on the cuvette.

2.7. Coagulation-flocculation assay

The Jar Test was performed to optimize the coagulation process

and to determine the effect of adsorption on the coagulant mech-
anism. The test was performed in triplicates to ensure quality
assurance and repeatability readings. Coagulation-flocculation and
sedimentation experiments prepared were performed in glass
beakers 0.12 m height by 0.09 m diameter (h/D ¼ 1.33) using a
250 mL microalgae volume. Standard sedimentation procedure in
jar and leaching test equipment (JLT-60, France) was used to
determine the efficiency of coagulation with various coagulation
parameters. The test rig has six space to accommodate 1000 mL
flocculation beaker simultaneously. Each beaker was equipped
with a paddle which the speed can be adjusted in the range of
20e400 rpm. A rapid stirring period of 5 min at 150 rpm followed
by a slow stirring period of 15 min at 10, 20, 30, 40 and 50 rpm were
carried out to initiate particle coagulation and flocculation. The
rapid mixing procedure allowed the microalgae biomass to be
mixed homogenously and in contact with the coagulant. However,
slow mixing allowed the suspended microalgae cells to collide with
sufficient energy to overcome the energy barrier and stick together
to form larger colloid.

2.8. Statistical analysis

Two-way ANOVA analysis was selected initially for experiments

with two interacting factors such as in flocculation assays which
involved various flocculant inoculation dosages and various mixing Fig. 1. Protein concentration and biomass recovery percentage of M. oleifera seed
rates. It could distinguish the significance within each factors and derivatives and alum (control).

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of these protein concentration was found in 1 g of each coagulants.

The preliminary coagulation-flocculation was performed in order
to investigate the efficiency of each M. oleifera seed derivatives as
coagulants.
In the coagulation-flocculation assay of primary M. oleifera de-
rivatives (Table 2), it was observed that only the seed powder was
able to recover microalgae biomass effectively with 97% biomass
recovery. On top of that, cupule and seed coat had less than 5% and
10% biomass recovery, respectively. The presence of coagulating
protein in the M. oleifera seed powder had contributed to the high
microalgae biomass recovery as compared to cupule and seed coat.
Although the protein concentration observed in both cupule and
seed coat is quite high, the characteristic of its non-flocculating
protein does not contributed to the coagulation-flocculation pro-
cess. The positive charge of protein molecules neutralized the
negatively charged microalgae cells once the M. oleifera seed
powder were introduced to the microalgae suspension. The addi-
Fig. 2. Zeta potential as a function of pH of the aqueous dispersions of alum, M. oleifera
tion of M. oleifera seed powder as coagulant agent disturbs the seed protein and M. oleifera protein powder.
stability of microalgae suspension system (Ndabigengesere and
Narasiah, 1998; Teixeira et al., 2012). During the coagulation pro-
cess, the energy barrier between the adjacent microalgae cells will lead to lower aggregation and flocculation of the particles. In M.
reduced and increased the attraction force. Hence, the aggregation oleifera seed powder dispersion, the value of positively and nega-
of microalgae biomass occurred and become larger in floc size to tively z-potential were þ45.69 mV and
38.60 mV both at pH 2.35
ease sedimentation process. Furthermore, both coagulants in sec- and pH 10.12, respectively. These values also indicate the high
ondary derivatives were observed to have biomass recovery of less stability of the M. oleifera seed powder dispersion. However, the
than 5%. For tertiary derivatives, it was found that only M. oleifera capability of aggregation of the particles is higher compared to
protein powder could recover the microalgae biomass for up to 78%. alum. On the other hand, M. oleifera protein powder was exhibited
Less than 5% biomass recovery was observed in flocculation test the negative and positive z-potential values which are þ28.51 mV
using M. oleifera protein-lipid, which is the mixture of protein and at pH 2.57 and
15.93 mV at pH 10.2. Hence, M. oleifera protein
lipid. powder has the highest capability of agglomeration and floccula-
tion of particles compared to alum and M. oleifera seed powder.

3.2. Determination of isoelectric point (i.e.p.) of coagulants

Fig. 2 shows the z-potential as a function of pH of the aqueous 3.3. Coagulation, flocculation, sedimentation and decantation
dispersions of alum, M. oleifera seed protein and M. oleifera protein
powder. All these three coagulants can be characterized with an The stable suspension of microalgae biomass formed from
isoelectric point (i.e.p.) at the point z-potential is zero. In the aquaculture wastewater phytoremediation does affect the separa-
neighborhood of the i.e.p., the dispersion is unstable and the ability tion process. There are two factors which affect the stability of the
of aggregation or flocculation to form larger particles is higher. If microalgae in suspension. The first is an electric charge on the algal
more alkali are added to the suspension, then the particles will tend surface which cause the development of intercellular repulsion
to acquire a more negative charge. However, if acid is then added to forces (Chen et al., 2011). While the second factor is the tiny cell
the suspension, a point will be reached where the negative charge dimension and cell density, which close to that of medium cause
is neutralized. Any further addition of acid can cause a buildup of slow cell sinking rate. Both of the electric repulsion interactions
positive charge. Therefore, a z-potential versus pH curve will be between algal cells and cell interactions with the surrounding
positive at low pH and negative at high pH. The i.e.p. for all coag- water contribute to the stability of the algal in suspension. Most of
ulant samples which are alum, M. oleifera seed powder and M. the planktonic algae is characterized as negatively charged sur-
oleifera protein powder was indicated at the pH between pH 6.5 to faces. The intensity of that charge is a function of algal species, ionic
pH 7.5. At this pH range, the value of z-potential was near to zero. strength of the medium, pH and other environmental conditions.
Alum was very stable in acidic and alkali form due to the high value Fig. 3 shows the coagulation (a), flocculation (b), sedimentation (c
of positively (þ64.38 mV at pH 3.38) and negatively z-potential and d) and decantation (e and f) processes. A coagulation process
(
38.40 mV at pH 10.83). Higher values of positively and negatively occurs after the addition of coagulants following by the rapid
z-potential indicate the high stability level of that dispersion. This mixing via agitation.

Table 2
The flocculation activity of Chlorella sp. culture using different Moringa oleifera seed derivatives and chemical coagulant, alum.

M. oleifera derivatives Coagulants (10 mg L
1) Flocculation activity Flocculation efficiency

Primary Cupule/wings No <5%

Seed coat No <10%
Seed powder Yes >97± 2%
Secondary Seed oil No <5%
De-oiled seed No <5%
Tertiary Protein þ lipid No <5%
Protein powder Yes >78± 4%
Chemical Aluminum sulfate,Al2(SO4)3 >37± 4% Yes

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6 S.H. Abdul Hamid et al. / International Biodeterioration & Biodegradation xxx (2016) 1e8

Fig. 3. Observation on Chlorella sp. sedimentation and decantation properties.

3.4. Optimization of coagulation-flocculation assay these coagulants in terms of microalgae biomass recovery was
compared with conventional coagulant, alum. In fact, alum was
The optimization of coagulation-flocculation for microalgae used commercially in treating wastewater for the removal of
harvesting was done by manipulating the parameters such as turbidity as well as microalgae harvesting.
coagulant dosage, the pH of the microalgal suspension and rapid- The optimization of coagulation-flocculation assay was per-
slow mixing protocol. The lab-scale harvesting process was per- formed by defining the most important factor which is the z-po-
formed using Jar Test in order to optimize the coagulation- tential of the suspended microalgae. The analysis of z-potential lead
flocculation process. In this study, coagulants used were M. olei- to the determination of i.e.p. of the colloidal system. Without the
fera seed powder and M. oleifera protein powder where these two determination of z-potential value in the colloidal system, the
derivatives show significant biomass recovery percentage from the optimization of coagulation-flocculation process could be prob-
preliminary coagulation-flocculation assay. The performance of lematic. Otherwise, the dosage used may be high and not economic.

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 S.H. Abdul Hamid et al. / International Biodeterioration & Biodegradation xxx (2016) 1e8 7

Table 3
Current and reported harvesting methods for respective microalgae species.

Harvesting methods Microalgae species Types of Harvesting References

microalgae efficiencies (%)

Flocculation with M. oleifera seed Chlorella sp. Freshwater >97 ± 5 Current study
powder
Flocculation with M. oleifera protein Chlorella sp. Freshwater >78 ± 4 Current study
powder
Flocculation with ɣ-PGA Chlorella protothecoides, Chlorella vulgaris LIMCE 001 and Botryococcus Freshwater >95 Zheng et al.
braunii LIMCE 003 (2012)
Flocculation with ɣ-PGA Chlorella vulgaris, Nannochloropsis oculata LICME 002 and Phaeodactylum Marine >90 Zheng et al.
tricornutum (2012)
Flocculation with chitosan Thalassiosira pseudonana Marine 90 Heasman et al.
(2000)
Flocculation with Aluminum Chlorella minutissima Freshwater >90 Papazi et al.
Chloride (AlCl3) (2010)
Centrifugation Phaeodactylum tricornutum Marine 94 Heasman et al.
(2000)

This is due to the high energy barrier to be overwhelmed between freshwater Chlorella protothecoides flocculation with poly glutamic
the adjacent cells in a suspension. The determination of z-potential, acid (ɣ-PGA) was over 95%. Marine Chlorella vulgaris flocculation
however could reduce the consumption of coagulant and simplify utilizing ɣ-PGA also gives high efficiency, which exceeds 90%. This
the optimization process. The optimization of coagulation- finding was similar with the flocculation of marine microalgae
flocculation process was considering the pH, mixing rate and using chitosan which yield 90% efficiency (Heasman et al., 2000).
coagulant dosage. In addition, the performance of harvesting pro- The efficiency of biomass recovery via centrifugation method was
cess was determined by the biomass recovery percentage. 94%. Papazi et al. (2010) also reported that flocculation of fresh-
Table 3 shows the flocculation efficiency according to harvesting water Chlorella minutissima by using alum yield over 90% efficiency.
methods for respective microalgae species. Study conducted by The current study shows that the flocculation efficiency was over
(Zheng et al. (2012)) concluded that harvesting efficiency of 97% using M. oleifera seed powder in low dosage. 78% flocculation

Fig. 4. The reduction of zeta potential of Chlorella sp. with respect to the coagulant dosage.

Please cite this article in press as: Abdul Hamid, S.H., et al., A study of coagulating protein of Moringa oleifera in microalgae bio-flocculation,
International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.03.027
8 S.H. Abdul Hamid et al. / International Biodeterioration & Biodegradation xxx (2016) 1e8

efficiency was achieved by the flocculation using M. oleifera protein Education through the Fundamental Research Grant Scheme
powder. The major disadvantages of using ɣ-PGA as a coagulant (FRGS), project no. FRGS/1/2014/STWN01/UNISZA/02/2 and FRGS/
purposes are its cytotoxicity and not biodegradable (Dubruel et al., 1/2013/TK07/UMT/01/1 for the support of this study.
2003). Alum however, was known as a chemical coagulant that
could remain in harvested biomass and cause the limitation of
downstream processing for aquaculture feeds. Although chitosan
are natural coagulant, the derivation process of chitosan from chitin
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Please cite this article in press as: Abdul Hamid, S.H., et al., A study of coagulating protein of Moringa oleifera in microalgae bio-flocculation,
International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.03.027