Waste and Biomass Valorization

https://doi.org/10.1007/s12649-018-0421-8

ORIGINAL PAPER

Enzymatic Hydrolysate of Palm Oil Mill Effluent as Potential Substrate

for Bioflocculant BM-8 Production
Nurul Adela Bukhari1   · Soh Kheang Loh1 · Abu Bakar Nasrin1 · Jamaliah Md Jahim2

Received: 21 January 2018 / Accepted: 1 August 2018

© Springer Nature B.V. 2018

Abstract
Purpose  The aim of this study was to optimize the enzymatic hydrolysis of palm oil mill effluent (POME) to release ferment-
able sugar as a platform convertible to value-added product i.e. bioflocculant.
Methods  A Plackett–Burman design followed by a central composite design of response surface methodology was applied
to optimize the enzymatic hydrolysis of POME to release fermentable sugars. Subsequently the enzymatic hydrolysate of
POME was used to produce bioflocculant (BM-8) using POME-isolated Bacillus marisflavi NA8. The produced BM-8 was
then characterized and its potential in microalgae harvesting was explored.
Results  Pure substrate (100%, v/v) dosed with 1% (v/v) enzyme and agitated at 200 rpm yielded optimum fermentable sugar
from POME hydrolysate. Subsequently, the medium produced 9.72 g/L of BM-8 during 24 h of cultivation. The BM-8, which
composed primarily of polysaccharide (74%) and protein (25%) with 1% nucleic acid was found to be thermally stable and
able to withstand a wide pH range, with its optimum tolerance at pH 6. Additionally, the BM-8 was found efficient (90%
biomass recovery in 30 min) for precipitation of Chlorella vulgaris, thus suggesting its great potential as a flocculating agent.
Conclusion  The study demonstrated that POME hydrolysate may be used as a cheap and renewable substrate for BM-8
production. In addition, BM-8’s flocculating capability showed it potential to substitute hazardous chemical flocculants.
Graphical Abstract

Keywords  Enzymatic hydrolysis · Wastewater · Fermentation · Bioflocculant · Microalgae

1
* Nurul Adela Bukhari Energy & Environment Unit, Engineering & Processing
adela@mpob.gov.my Research Division, Malaysian Palm Oil Board (MPOB),
6, Persiaran Institusi, Bandar Baru Bangi, 43000 Kajang,
Soh Kheang Loh
Selangor, Malaysia
lohsk@mpob.gov.my
2
Department of Chemical and Process Engineering,
Abu Bakar Nasrin
Faculty of Engineering and Built Environment, Universiti
nasrin@mpob.gov.my
Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
Jamaliah Md Jahim
jamal@ukm.edu.my

13
Vol.:(0123456789)

Statement of Novelty
• Hydrolysis of palm oil mill effluent (POME) was opti-
mized by means of applying statistical tools to enhance
the release of reducing sugar for bioconversion into
value-added product i.e. bioflocculant BM-8.
• The BM-8 was produced using high yielding strain,
Bacillus marisflavi NA8 previously isolated from aer-
obically-treated POME, in a 5-L bioreactor.
• The physico-chemical properties of BM-8 was char-
acterized for the first time and it was found to be ther-
mally stable and able to withstand a wide pH range.
• BM-8 was found efficient for precipitating Chlorella vul-
garis, thus suggesting its great potential as a flocculating
agent to substitute the hazardous chemical flocculants.
Introduction
The palm oil industry is one of the leading industries in
Malaysia with the production of 18.43 million tonnes of
crude palm oil (CPO) from 5.7 million hectares of oil palm
planted area [1]. At the same time, palm oil mill effluent
(POME) nearly three times the quantity of CPO is gener-
ated as wastewater. More precisely, ~ 0.67 tonne of POME
is generated for each tonne of fresh fruit bunches (FFB)
processed. The FFB processed (85.84 million tonnes) thus
produced about 57.51 million tonnes of POME in 2016.
Due to its high chemical oxygen demand (COD) and bio-
chemical oxygen demand (BOD), POME is known to be
highly polluting if it is to be discharged to river stream
without any treatment. Through common POME treat-
ment system i.e. open ponding or digester tank, biogas
is released freely into the atmosphere. The biogas con-
stitutes 60–70% methane, 30–40% carbon dioxide and
trace amount of hydrogen sulphide. Its annual produc-
tion is estimated to be ~ 1713 million ­m 3 consisting of
674,368 tonnes of methane. However, the organic loading
of POME can be transformed into opportunities by recov-
ering the readily available nutrients for microbial fermen-
tation. In bioconversion, POME serves as an organic-rich
substrate for specific microorganisms to consume and grow
yielding biomass and some of the targeted bio-products.
One of the intended bio-products, bioflocculants, have
received great attention for scientific and biotechnological
consideration. Many studies have noted on the diversified
compositions and properties of bioflocculants from differ-
ent sources [2–5]. Their unique characteristics have con-
tributed to varying applications for bioremediation, e.g. in
various industrial processes to treat water and wastewater
[6, 7], heavy metals [8–10], toxic materials [11], and colour
13
Waste and Biomass Valorization
[12], in synthesis of nanoparticles [13], as potential emulsi-
fier [14], cell removal and biomass recovery i.e. microalgae
harvesting, etc. In recent years, microalgae have increas-
ingly been used as a potential biofuels feedstock but some-
how the progress hindered by high biomass harvesting cost
i.e. 20–30% of the total cost for biofuel production [15].
Applying flocculant in bulk harvesting of microalgae could
lower the cost [16]. It was shown earlier that bioflocculants
from Solibacillus silvestris and Bacillus agaradhaerens C9
W01 have a potential in harvesting microalgae Nannochlo-
ropsis oceanica DUT01 [15] and Chlorella minutissima
UTEX2341 [16], respectively. Recently, Li et al. [17–19]
proved that Streptomyces sp. hsn06, Aspergillus niger and
Shinella albus are highly efficient bioflocculants to har-
vest Chlorella vulgaris biomass. Bioflocculant, a promis-
ing alternative to synthetic flocculant as its name implies
is derived from microorganisms during growth; hence is
naturally occurring without involving chemicals. As such,
it is generally recognised as being a non-toxic, environmen-
tally-friendly and highly biodegradable substitute with no
secondary pollution [20, 21].
Although bioflocculants hold great potential, their com-
mercial production is still not economically viable. Numerous
researches have been carried out attempting to reduce their
production cost and increase yield and applicability [22].
Examples were (1) screening of high-yielding strains, (2)
genetic modification of microorganisms, (3) utilisation of low-
cost substrates, (4) optimization of fermentation conditions, (5)
improvement of bioreactor performance, (6) improvement in
downstream processing and (7) expanding the field applica-
tions of bioflocculants [16]. Previously, we have successfully
produced bioflocculant from POME in shake flasks using a
newly isolated high-yielding strain i.e. Bacillus marisflavi NA8
[23]. Under the conventionally-derived optimized conditions
using the enzymatically hydrolyzed POME, about 6.4 g/L of
bioflocculant was attainable [24]. Therefore, in this study, we
attempted optimizing the enzymatic hydrolysis of POME by
means of applying statistical tools to enhance the release of
reducing sugar. Subsequently, up scaling of bioflocculant from
the hydrolysed POME using the strain NA8 in a 5-L biore-
actor was conducted. The physico-chemical properties of the
obtained bioflocculant was characterized, and its potential in
microalgae harvesting was explored.
Materials and Methods
Raw Material
The fresh POME was sampled from Labu Palm Oil Mill
at Labu, Negeri Sembilan, Malaysia. The sample was pre-
served at 4 °C prior to analysis and further treatment. The
characteristics of raw POME were determined in accordance
Waste and Biomass Valorization

with the standard methods published by the American Public
Health Association [25, 26].
Bacterial Strain and Enzyme
The bioflocculant-producing strain, B. marisflavi NA8 was
previously isolated from the aerobically treated POME
[23]. The lignocellulase used was obtained from Universiti
Kebangsaan Malaysia (UKM) namely UKM-enzyme For-
mulation-3 (F3) with a total enzyme activity of 1493 U/mL.
The mixture of lignocellulases i.e. endoglucanase was pro-
duced in submerged fermentation process by a genetically
modified strain of Pichia pastoris. One unit of enzyme activ-
ity is defined as the quantity of enzyme to produce reducing
sugars equivalent to 1 µmol of glucose per minute from oil
palm empty fruit bunch as substrate under standard assay
conditions at pH 5.0 and 50 °C.
from POME were taken as the response for the respective
variable.
Three significant variables were identified from PBD as
the independent variables i.e. agitation (X1, rpm), enzyme
dosage (X2, %, v/v) and substrate concentration (X3, %, v/v)
which were then further examined via central composite
design (CCD). Both the Triton X-100 concentration and
incubation time were insignificant influencing factors as
identified, were set at 0.05% (v/v) and 18 h, respectively. The
RSC ­(Yi, g/L) was used as the dependent output variable.
A ­23-factorial CCD, with 6 central points leading to a total
number of 20 experiments was employed for the optimiza-
tion of POME hydrolysis. The second degree polynomials
(Eq. 1) were calculated based on the derived statistical data
to estimate the response, RSC ­(Yi).
Yi = b0 + b1 X1 + b2 X2 + b3 X3 + b11 X12 + b22 X22 + b33 X32
+ b12 X1 X2 + b23 X2 X3 + b13 X1 X3 (1)

Optimization of Enzymatic Hydrolysis of POME
The enzymatic hydrolysis was performed on raw POME
(100 mL) in a 250-mL screw capped flask. Initially, Plack-
ett–Burman Design (PBD) was used for variables screening.
Five independent variables (i.e. agitation, enzyme dosage,
incubation time, substrate concentration and Triton X-100
concentration) were selected and their conditions set in two
levels: (− 1) for low level and (+ 1) for high level (Table 1).
Twelve experimental runs were formed by the software.
All experiments were conducted in triplicate and the mean
values of reducing sugar concentration (RSC) released
where Yi is the predicted response (RSC), X1, X2, X3 are
independent variables, b0 is offset term, b1, b2, b3 are linear
effects, b11, b22, b33 are squared effects and b12, b22, b13 re
interaction terms.
All statistically and mathematically-derived results were
analyzed via PBD and CCD using Design Expert 9.0.5.1
Software (Statease, Inc., Minneapolis, MS, USA), and their
significance levels evaluated by variance analysis (ANOVA).
The proposed quadratic polynomial equation (Eq. 1) was
used to describe the mathematical relationship between the
response (RSC) and the independent variables. The RSC
was analyzed according to the dinitrosalicylic acid method

Table 1  PBD variable screening for enzymatic hydrolysis of POME

Run Variable Sugar concentration (g/L)
Agitation, X1 (rpm) Enzyme dos- Incubation Substrate Triton-X conc., X5 (%) Experimental Predicted
age, X2 (%, time, X3 (h) conc., X4 (%,
v/v) v/v)

1 100 (− 1) 1.0 (+ 1) 24 (+ 1) 100 (+ 1) 0.05 (− 1) 32.90 26.88
2 100 (− 1) 0.5 (− 1) 18 (− 1) 100 (+ 1) 0.05 (− 1) 32.54 26.88
3 200 (+ 1) 1.0 (+ 1) 18 (− 1) 100 (+ 1) 1.00 (+ 1) 15.92 26.88
4 100 (− 1) 0.5 (− 1) 24 (+ 1) 50 (− 1) 1.00 (+ 1) 18.01 26.88
5 200 (+ 1) 0.5 (− 1) 18 (− 1) 50 (− 1) 1.00 (+ 1) 34.20 26.88
6 200 (+ 1) 0.5 (− 1) 24 (+ 1) 100 (+ 1) 1.00 (+ 1) 17.74 26.88
7 100 (− 1) 1.0 (+ 1) 24 (+ 1) 50 (− 1) 1.00 (+ 1) 38.44 26.88
8 200 (+ 1) 0.5 (− 1) 24 (+ 1) 100 (+ 1) 0.05 (− 1) 18.99 26.88
9 100 (− 1) 1.0 (+ 1) 18 (− 1) 100 (+ 1) 1.00 (+ 1) 35.70 26.88
10 100 (− 1) 0.5 (− 1) 18 (− 1) 50 (− 1) 0.05 (− 1) 34.24 26.88
11 200 (+ 1) 1.0 (+ 1) 24 (+ 1) 50 (− 1) 0.05 (− 1) 20.86 26.88
12 200 (+ 1) 1.0 (+ 1) 18 (− 1) 50 (− 1) 0.05 (− 1) 22.98 26.88
Standardized effects 3.32 2.04 − 1.21 15.59 0.003167
Contribution (%) 4.22 1.60 0.56 93.02 3.84 × 10−6

13

[27]. A linear equation obtained from the glucose standard
curve was used to indicate the yield of enzymatic hydrolysis
of POME.
Production of Bioflocculant in a 5‑L Bioreactor
The strain seed was prepared as described previously
[24]. The fermentation medium (pH 7.0) composed of
(per liter): 200 mL POME hydrolysate, 5 g ­K2HPO4, 2 g
­KH2PO4, 0.5 g urea, 0.2 g M ­ gSO4·7H2O, 0.2 g (­ NH4)2SO4,
0.5 g yeast extract. All the fermentation experiments were
performed in a 5-L bioreactor (Minifors™, Infors AG)
with 3.0 L working volume. The bioreactor was equipped
with control modules for pH, temperature, agitation, air
flow and dissolved oxygen (DO). Pre-cultures were pre-
pared by transferring a single fresh colony into 10 mL
nutrient broth (NB) and shaken at 150 rpm overnight at
37 °C. Then, the culture was used to inoculate 300 mL
NB in a 1-L baffled flask held at 30 °C and 150 rpm for
24 h and the resulting pre-culture was added to 2.1 L of
growth medium. An antifoaming agent (J673A Struktol,
Germany) at 5% (v/v) was used to control foaming. The
temperature was maintained at 37 °C by a cooling element
connected to a circulating chiller held at 5 °C. Throughout
the fermentation, pH was maintained at 7.0 by automati-
cally adding 5 M ­H2SO4 and 30% (w/v) ­NH4OH, while the
rest—agitation within the range of 200–600 rpm, aeration
at 1 L/min, and DO above 20% by cascading the agita-
tion. IRIS (Infors AG) software was employed to moni-
tor and record all available fermentation parameters. The
samples were collected at predetermined time intervals to
determine the bioflocculant production yield and cell dry
weight (CDW). The culture broth was harvested by cen-
trifugation at 4000 rpm for 15 min and the bioflocculant
was purified using cold ethanol precipitation [24]. The
precipitate was dried to obtain bioflocculant (designated
as BM-8) and used for analysis.
Flocculating Properties of BM‑8
A kaolin clay solution was used as the test material to deter-
mine the flocculating properties of BM-8. Briefly, 5 g of
kaolin clay was suspended in 1 L of distilled water to make
a solution with suspension concentration of 5 g/L. 100 mL
of kaolin suspension, 3 mL of 1% (w/v) ­CaCl2 and 3 mL
of culture supernatant were added into a 500-mL flask.
The mixture was agitated vigorously for 60 s, then poured
into a 100-mL measuring cylinder and allowed to settle for
5 min at room temperature. The optical density (OD) of the
clarifying supernatant was measured at 550 nm using a UV
spectrophotometer (Genesys, Thermo Scientific, USA). The
resulting flocculating activity of BM-8 was determined as
in Eq. (2).
13
Waste and Biomass Valorization
(B − A)
Flocculating activity = × 100%
B (2)
where A and B are OD of the sample and the control super-
natant measured at 550 nm, respectively.
The effect of pH of Kaolin clay suspension on flocculat-
ing activity of BM-8 was determined by adjusting the pH
from 2 to 10 using ­H2SO4 or NaOH, before adding BM-8
and ­CaCl2 as described previously. Beside ­CaCl2, other cat-
ion sources i.e. NaCl, M­ gCl2, ­AlCl3, ­CaCl2, ­MnCl2, ­FeCl2,
­FeCl3, ­CoCl2, ­NiCl2, ­CuCl2 and ­ZnCl2 were each added to
assess if a cation was indeed required by BM-8 to trigger
flocculation.
Characterization of the Novel BM‑8
The molecular composition of BM-8 i.e. carbohydrate was
determined in accordance to the phenol–sulphuric acid
method using glucose as standard [28]. Its total protein and
DNA contents were analyzed using a digestor/auto distil-
lation unit (KjelROC, OPSIS LIQUIDLINE, Sweden) and
a NanoDrop spectrophotometer (ND-1000) equipped with
NanoDrop 3.0.1 software (Coleman Technologies Inc),
respectively.
The functional groups of BM-8 was analysed using a Fou-
rier transform infrared (FTIR) spectrometer (Perkin-Elmer
System, USA). The resulting spectra were recorded over a
wavelength range of 650–4000 cm−1 under ambient condi-
tions. The thermogravimetric analysis (TGA) of BM-8 by
a thermogravimetric analyser (Perkin-Elmer System, USA)
was carried out at a temperature range of 20–900 °C with a
heating rate of 20 °C/min under a constant flow of nitrogen
gas. The elemental content of BM-8 was analysed using an
elemental analyser (CHN628S, LECO, USA).
The thermo-stability of BM-8 was determined by incubat-
ing the bioflocculant solutions for 30 min at different incuba-
tion temperatures (10–100 °C), and its residual flocculating
activity measured.
Precipitation of Microalgae by BM‑8
A microalgae species—C. vulgaris UMACC 283 was
donated by University of Malaya, Malaysia. The micro-
algae was cultivated on agar slant and kept in a culture
chamber (25 ± 1 °C and irradiance of 8 µmol photon/m2/s).
The single colony was inoculated in Bold’s Basal Medium,
followed by flask cultivation for 12 days. The flasks were
incubated in a controlled environment at 25 ± 1 °C, illumi-
nated with cool white fluorescent lamps (40 µmol photon/
m−2/s) on a 12:12-h light–dark cycle and supplied with
100 mL/min filtered ambient air [29]. Subsequently, the
flask culture was transferred to an outdoor enclosed 200-L
photobioreactor (PBR) i.e. high rate alga pond and grown
Waste and Biomass Valorization
at ambient conditions for 12 days using 5% (v/v) POME
with 10% (v/v) inoculum [30]. For sampling purpose, a
100-mL C. vulgaris culture was aliquoted from the PBR
into a 250-mL beaker.
The collected culture was dosed with BM-8 at various
concentrations from 50 to 1000 mg/L followed by vigorous
stirring at 150 rpm for 2 min until completely dissolved.
A gentle agitation mode at 20 rpm was then applied for
2 min to facilitate the formation of flocs. The suspension
was allowed to rest for another 15 min. Additionally, four
types of chemical flocculant—solid poly aluminium chlo-
ride (PAC)—were tested accordingly for comparison: (1)
high-purity PAC (aluminium hydroxide + synthetic hydro-
chloric); (2) PAC-S (aluminium hydroxide + synthetic
hydrochloric + aluminium sulphate); (3) PAC-V (alumin-
ium hydroxide + synthetic hydrochloric + aluminium cal-
cium) and (4) PAC-V Industrial class (bauxite + industrial
hydrochloric).
The algal biomass recovery efficiency was calculated
according to Eq. (3).
( )
A750 t0 − A750 (t)
Biomass recovery (%) = × 100 (3)
A750 (t0 )
where the culture absorbance (A) before the clarified zone
(t0) and after (t) was measured at 750 nm.
Results and Discussion
The raw POME has about 95–96% water, 0.34% oil and
2.6% total solid (Table 2). It is acidic (pH 4.27 ± 0.1) due
to the presence of complex organic acids. The high BOD
and COD of POME in the range of 15,800–19,300 mg/L
and 95,313–96,250 mg/L, respectively reveals that it can be
favourably hydrolyzed into simple or reducing sugars to aid
microbial fermentation process. In this study, the enzymatic
hydrolysis of POME was thus optimized using response sur-
face methodology (RSM) to find the optimum conditions
required to produce reducing sugars.
Table 2  Characteristics of raw Parameters
and hydrolysed POME
Biochemical oxygen demand (BOD)
Chemical oxygen demand (COD)
Total solid (TS)
Oil and grease (O&G)
Volatile fatty acid (VFA)
pH
Temperature
Moisture content
Optimization via Response Surface Methodology
(RSM)
Through PBD screening, some of the process variables such
as pH and temperature which had been previously optimized
(pH 5.0 and T: 50 °C) [24] were eliminated. The resulted
experimental sugar yield (RSC) ranged 15.92–38.44 g/L
(Table 1). In Table 1, the investigated ranges for agitation
(X1), enzyme dosage (X2), incubation time (X3), substrate
concentration (X4) and Triton X-100 (X5) were 100–200 rpm,
0.5–1.0% (v/v), 18–24 h, 50–100% (v/v) and 0.05–1.00%
(v/v), respectively. The decreasing order of the standard-
ized effect and percentage contribution by the tested vari-
ables was: substrate concentration > agitation > enzyme
dosage > incubation time > Triton X-100, thus devoting
that substrate concentration was the most significant vari-
able towards the response (RSC), followed by agitation and
enzyme dosage. The incubation time was insignificant, prob-
ably due to the employed narrow range of reaction interval
(6 h); in fact it would cause a decreased sugar yield as was
indicated by the negative standardized effect (Table 1). Thus,
a 18-h period was used for subsequent experiment. Similarly,
the Triton X-100 concentration was found far too insignifi-
cant; hence the lowest concentration of 0.05% (v/v) was
selected. The PBD screening revealed that substrate con-
centration, agitation and enzyme dosage would give greater
influence on enzymatic hydrolysis of POME.
The response (RSC) from POME hydrolysis, as a func-
tion of agitation (X1), enzyme dosage (X2) and substrate
concentration (X3) was evaluated based on CCD. The pro-
posed quadratic model by Design-Expert 9.0.5.1. showed
low standard deviation (0.99), high R2 value (0.9877) and
insignificant lack-of-fit p-value (0.0785). The design and
results of CCD are shown in Table 3. The sugar concentra-
tions at the centre points (Run no: 2, 3, 4, 5, 6 and 18) were
close to each other (standard deviation = 0.64) indicating the
reproducibility of the experiments. The interactive relation-
ship between two variables on the sugar yield was visualized
in three dimensional (3D) response surface plots while the
other variable was kept at level 0 (Fig. 1a–c).
Unit Raw POME Hydrolysed POME
mg/L 17,550 ± 2475 59,483 ± 1200
mg/L 95,912 ± 520 72,727 ± 6389
mg/L 26,422 ± 139 78,956 ± 4337
mg/L 3400 ± 141 9033 ± 368
mg/L 3843 ± 89 1287 ± 120
4.27 ± 0.1 4.47
°C 80–90 50
% 95–96 95.8–99.1
13
 Waste and Biomass Valorization

Table 3  CCD matrix showing Run Variable Sugar concentration (g/L)

the independent variables and
the experimental response of Agitation, X1 (rpm) Enzyme dosage, Substrate concentra- Experimental Predicted
reducing sugar yield X2 (%, v/v) tion, X3 (%, v/v)

1 1 (200) 1 (1.00) − 1 (50) 19.77 20.31

2 0 (150) 0 (0.75) 0 (75) 26.40 27.04
3 0 (150) 0 (0.75) 0 (75) 27.53 27.04
4 0 (150) 0 (0.75) 0 (75) 27.03 27.04
5 0 (150) 0 (0.75) 0 (75) 26.82 27.04
6 0 (150) 0 (0.75) 0 (75) 27.69 27.04
7 1 (200) 1 (1.00) 1 (100) 35.65 34.69
8 0 (150) − α (0.25) 0 (75) 23.69 24.68
9 1 (200) − 1 (0.50) − 1 (50) 17.20 16.48
10 α (250) 0 (0.75) 0 (75) 22.74 23.45
11 − α (50) 0 (0.75) 0 (75) 19.46 19.45
12 0 (150) 0 (0.75) α (125) 32.19 33.42
13 − 1 (100) 1 (1.00) − 1 (50) 15.13 15.42
14 − 1 (100) 1 (1.00) 1 (75) 28.87 28.89
15 1 (200) −α (0.25) 1 (75) 31.62 28.25
16 0 (150) α (1.25) 0 (75) 28.54 28.24
17 − 1 (100) − 1 (0.50) − 1 (50) 18.04 18.30
18 0 (150) 0 (0.75) 0 (75) 26.04 27.04
19 − 1 (100) − 1 (0.50) 1 (75) 29.47 29.16
20 0 (150) 0 (0.75) − α (25) 8.72 8.18

Fig. 1  Three dimensional response surface plots showing the com- and substrate concentration, enzyme dosage = 0.75% (v/v); c enzyme
bined interactive effects of variables on POME hydrolysis: a agitation dosage and substrate concentration, agitation = 150 rpm
and enzyme dosage, substrate concentration = 75% (v/v); b agitation

The statistical significance of the developed model was
evaluated by analysis of variance (ANOVA) (Table 4). The
adequacy of the model generated was indicated by p-value
of < 0.0001 and R2 of 0.9877, implying that 98.77% of sam-
ple variation for sugar production was attributed to the 3
independent variables. The p-values of X1 (agitation), X2
(enzyme dosage), X3 (substrate concentration), X1X2, X12 and
X32 were all lower than 0.05 indicating that they were the
significant factors influencing the sugar yield from POME.
The quadratic effects of agitation (X12) and substrate concen-
tration (X32) were found dominant with high F-value (49.98
vs. 62.18) and low p-value (< 0.0001), when compared to
enzyme dosage (X22; F = 0.53, p = 0.4832) (Table 4). X1X2
was also significant (p = 0.0007) in demonstrating the inter-
action between agitation and enzyme dosage unlike X1X3 and
X2X3 with poor interaction. This interaction implies that the
faster the agitation rate, the more dispersion the enzyme is
towards the substrate, thus more reducing sugars is released.
The insignificant lack of fit (p = 0.0785) further validated
the model terms (X1, X2, X3, X1X2, X12, X32) used in this
response model.

13
Waste and Biomass Valorization

Table 4  Analysis of variance Source Sum of squares df Mean square F-value p-value

(ANOVA) of response surface
model for reducing sugar yield Model 786.83 9 87.43 88.96 < 0.0001*
X1—agitation 15.96 1 15.96 16.24 0.0024*
X2—enzyme dosage 12.67 1 12.67 12.90 0.0049*
X3—substrate concentration 637.06 1 637.06 648.25 < 0.0001*
X1 X2 22.51 1 22.51 22.91 0.0007*
X1 X3 0.41 1 0.41 0.42 0.5309
X2 X3 3.41 1 3.41 3.47 0.0923
X12 49.12 1 49.12 49.98 < 0.0001*
X22 0.52 1 0.52 0.53 0.4832
X32 61.11 1 61.11 62.18 < 0.0001*
Residual 9.83 10 0.98
Lack of fit 7.85 5 1.57 3.96 0.0785
Pure error 1.98 5 0.40
Cor total 796.66 19

R 2 = 0.9877; adjusted R2 = 0.9766

*
 Significant

results. The RSM employed was able to optimize the enzy-

matic hydrolysis of POME to produce 17.3% more sugar
compared to the conventional one-factor-at-a-time method
previously studied [24].

Production of BM‑8

The released sugars from the hydrolyzed POME were sub-

Fig. 2  Time course of BM-8 production by B. marisflavi NA8 in
hydrolyzed POME
Equation (4) represented the relationship between the
significant variables and the sugar yield from enzymatic
hydrolysis of POME in the resulted factors.
Yi = −2.44534 + 0.073402 X1 − 20.94455 X2 + 0.52095 X3
+ 0.13420 X1 X2 − 5.59091 × 10−4 X1 2 − 2.49436 × 10−3 X3 2
(4)
While positive coefficient improves the response vector,
the negative value suggests inverse relationship. The pre-
dicted sugar yield based on Eq. 4 are very close to the exper-
imental results (Table 3). The model performed optimum
sugar production occurred at 200 rpm agitation, 1% (v/v)
enzyme dosage and 100% (v/v) substrate concentration. The
experimental yield of 35.65 g/L sugar was in good agree-
ment with the RSM model predicted value of 34.69 g/L.
Validation of the model was done by conducting three con-
firmation experimental runs at selected optimal level. One
sample t test with p-value of 0.08 (> 0.05) confirmed that the
model was valid and applicable to predict the experimental
sequently fed into a 5-L bioreactor containing B. marisflavi
NA8, and fermentation was performed using the previously
optimised conditions: pH 7.0, 37 °C and without N supple-
ment [24] to ensure cell growth and bioflocculant formation.
The POME was enriched with proteins sufficient for bacte-
rial growth. The DO set at above 20% provided enough oxy-
gen for B. marisflavi to metabolize the POME. This coupled
with appropriate agitation rate (200–600 rpm) maximized
the oxygen concentration to ensure the cells receiving an
adequate supply of air during their growth.
The cell growth correlates with the DO saturation
curve (Fig. 2). Initially for the first 6 h, the NA8 became
acclimated to the new environment i.e. POME medium. Its
growth accelerated exponentially within the next 9-h fer-
mentation period, due to the excess available carbon sources.
The BM-8 production reached its maximum of 9.72 g/L at
the 24 h with a productivity of 0.41 g/L/h. The DO level
above 20% was maintained for about 50 h (from 10 to 60 h
fermentation time) to support the growth of NA8 throughout
and for another day before decreasing drastically at 72 h;
probably due to denatured activities caused by accumu-
lation of toxic metabolites [31]. This was followed by an
increase in the DO level at 60 h indicating the depleted car-
bon source for growth; hence the beginning of cell death
phase. Therefore, it was best to harvest BM-8 at the 24 h

13

with a maximum CDW of 8.4 g/L. The ability of BM-8 to
reach maximum yield and productivity at the early stage of
fermentation would be economical for commercial produc-
tion in the future.
Physico‑chemical Properties of BM‑8
Biochemical Composition
A bioflocculant yield of 9.72 g BM-8 was precipitated from
a litre of 24-h culture, using cold ethanol. On the contrary,
the use of acetone hardly gave any precipitation. As such,
a polysaccharide-based flocculant was indeed isolated [32].
The biochemical analysis further confirmed that BM-8
exhibited 74% polysaccharides and 25% protein. Carbohy-
drate is functionally rich with many active sites that play
a major role in flocculation due to an increased polymeric
electrostatic interactions [33, 34], and aggregating binding
strength via hydrophobic interactions and polyvalent cations
bridging leading to an increased stability of the resulting
biopolymer network [35]. The major component in BM-8
implies that it has potential in flocculation. The minor com-
ponent of BM-8 was deoxyribonucleic acid (DNA) (1%).
This low level is likely due to the presence of a few dead/
lysed cells, or active generation of extracellular DNA [36].
Functional Group, Weight Degradation and Elemental
Composition
Flocculating activity of a microbial bioflocculant is depend-
ent on its chemical structure. The purified BM-8 had several
functional groups responsible for flocculation as shown in
Fig. 3a. A broad stretching peak at 3264 cm−1 in the FTIR
spectrum suggested the presence of hydroxyl and amine
groups, probably attributed to the –OH or –NH stretch-
ing modes present in the sugar ring [37]. Both the alkyl
C–H stretching vibration at 2852 cm−1, and C–H asym-
metrical stretching vibration at 2921  cm−1 were typical
of carbohydrate derivatives. The peak at 1744 cm−1 cor-
responded to asymmetric C=O stretching of a carboxylate
anion, while that at 1416 cm−1 due to symmetric stretch-
ing of the –COO– group. The broad absorption bands at
1000–1100 cm−1 were characteristics of C–O–C and C–O
of carbohydrate present. The polar functional groups in
BM-8 i.e. hydroxyl, carboxyl and probably amine, were the
major adsorptive forces responsible for flocculation due to
their high binding capacities [38]. Our findings revealed that
majorly the present carboxyl groups served as negative bind-
ing sites for cationic substances allowing for an improved
flocculation similar to that observed by Sekelwa et al. [22].
The TGA degradation profile of BM-8 showed an initial
weight loss between 50 and 125 °C due to evaporation of
water during degradation of the hydroxyl/carboxyl groups
13
Waste and Biomass Valorization
present in the polysaccharide [22]. The decline in the weight
above this temperature was attributed to thermal degradation
[39]. Figure 3b showed 20% mass reduction at a degrada-
tion temperature of 270 °C. The decomposition eventually
reached 50% mass loss at 500 °C.
The elemental weight fractions of the purified BM-8 were
29.6 ± 2.9% C, 6.4 ± 0.58% H, 4.5 ± 0.25% N, 0.74 ± 0.04%
S and 58.76% O (calculated by difference). The high N and
O contents revealed that possibly many N- and O-containing
functional groups are available offering electron lone pairs
as preferred coordination sites for flocculation [40].
Thermal Stability and Flocculating Property
Industrial applications of bioflocculant often involve
relatively harsh conditions. In this regards, the flocculat-
ing activity of BM-8 over a wide range of temperature
(10–100 °C) was assessed at a fixed 30-min time. BM-8 was
found thermally stable as ~ 80% of its flocculating activity
was retained (Fig. 4a). The thermal stability of biological
products depends on their activity ingredients. Generally,
polysaccharide-based bioflocculants are thermo-stable,
while those made of protein are heat-sensitive [38] as pro-
tein is easily denatured by external stress such as heat. In this
study, the excellent stability exhibited by BM-8 indicated
that its main backbone was polysaccharide-based. BM-8 is
thus able to tolerate high temperature when applied indus-
trially at drastic conditions. Other bioflocculants deriving
from Bacillus sp. i.e. Bacillus mojavensis [41], Bacillus
licheniformis [42] and Bacillus velezensis [43] were simi-
larly reported.
Adjusting the pH of the kaolin clay suspension from
­ + con-
acidic to alkaline showed recalcitrance effects to the H
centration [44]. BM-8 maintained high flocculating activity
(> 70%) at a wide pH range assessed (2–10); the highest
being 85% at pH 6 (Fig. 4b). Again, BM-8 is highly desir-
able for industrial applications. Other studies showing opti-
mum flocculating activity were at pH 6 via a mixed culture
of Staphylococcus and Pseudomonas [45], and at pH 7 by
bioflocculants from B. subtilis, B. mojavensis, B. licheni-
formis and B. velezensis [3, 41–43].
Various cations (mono, di and trivalent) were tested and
then assayed for flocculating activity of BM-8. Figure 4c
shows that F ­ e3+ performed the most in stimulating the floc-
culating activity reaching ~ 90%, followed by M ­ g2+, ­Na+,
2+ 2+
­Ca and ­Cu . This indeed showed that the tested floc-
culating activity was cation-dependent. All of the cations
stimulated the activity to a level above that of control i.e.
60%. Unlike other previously produced bioflocculants by
B. mojavensis [41], B. velezensis [43] and Pseudomonas
aeruginosa [46] having been inhibited by A ­ l3+ and F ­ e3+,
the flocculating activity of BM-8 was not at all affected.
Our findings further corresponded with Okeiyeto et al.
Waste and Biomass Valorization
Fig. 3  Physico-chemical characterization of BM-8: a FTIR spectra; b TGA thermogram
[47] and Wang et al. [48] reaffirming the necessity of add-
ing cations to induce flocculation amongst the microbial
flocculants. In fact, cations is often used as coagulation aid
in achieving high flocculating activity by neutralizing the
negatively charged functional groups on the bioflocculant
and suspending particles thereby increasing the adsorp-
tion of bioflocculant to the suspended particles [49]. In
our study, the practicality of adding cations into BM-8
for specific flocculation activity needs to be investigated.
Microalgae Harvesting by BM‑8
Chlorella vulgaris UMACC 283 has potential as a bio-
diesel feedstock [30]. Flocculants show potential as a
13
 Waste and Biomass Valorization

Fig. 5  Recovery efficiency (%) of BM-8 and other flocculants

[PAC-HP (high-purity PAC; aluminium hydroxide  + 
synthetic
hydrochloric), PAC-S (aluminium hydroxide + synthetic hydrochlo-
ric + aluminium sulphate), PAC-V (aluminium hydroxide + synthetic
hydrochloric + aluminium calcium), PAC-V Industrial class (baux-
ite + industrial hydrochloric)] in C. vulgaris harvesting

Fig. 4  The effect of: a temperature; b pH; c cations on flocculating

activity of BM-8

low-cost agent to aggregate microalgae. The newly char-

acterized BM-8 (this study) shows potential too as a floc- Fig. 6  BM-8’s flocculating efficiency on C. vulgaris at 0.1  g/L and
examination under microscopic view at × 100 magnification: a before
culating agent. In this regard, the feasibility of flocculat- flocculation; b after 30 min flocculation
ing the C. vulgaris cells using BM-8 was investigated.
Interestingly, BM-8 was found more efficient than the
chemical PACs in microalgae harvesting (Fig. 5). It was
able to precipitate about 60% C. vulgaris biomass at lower
dose (100 mg/L), while > 500 mg/L of PACs was required
to achieve similar recovery efficiency over 15 min. Using
this, a dilute suspension of 0.1 g/L dry matter of C. vul-
garis could be concentrated to a 5-g/L biomass slurry, i.e.
a 50 × concentration achieved (Fig. 6). Due to BM-8’s
unique compositions, we suggested that an electrostat-
ically-charged patching could be the mechanism behind
the flocculation of (BM-8)—(C. vulgaris) network, as
they were seemingly more connected locally (Fig. 6b).
BM-8 offers a net positive charge that may quickly and Fig. 7  Proposed mechanism of BM-8’s flocculation by patching

13
Waste and Biomass Valorization

Table 5  Comparison of chemicals flocculants and bioflocculant used for microalgae harvesting

Flocculants (dosage) Classification Microalgae (cell density) FE (setting time) References Features Cost (USD/kg)

Al2(SO4)3 (0.1 g/L) Inorganic Scenedesmus sp. > 90% (60 min) [51] Risk of secondary pol- 5.70
(0.23 g/L) lution
Fe2(SO4)3 (1 g/L) Inorganic Chlorella minutissima > 90% (60 min) [52] High dosage, cell dam- 10.00
age
Polyaluminium chloride Inorganic Nannochloropsis 70–80% (30 min) [53] Low cost, risk of second- 0.30
(PAC) (20–40 mg/L) gaditana (132 mg/L) ary pollution
Aluminium hydroxide Inorganic C. vulgaris (0.1 g/L) 60% (15 min) This study 0.50
(500 mg/L)
Polyacrylamide (PAM) Inorganic Scenedesmus sp. 60% (10 min) [51] Risk of toxic acrylamide 2.00
(50 mg/L) (0.54 g/L)
Chitosan (10 mg/L) Organic Chlorella sorokiniana 99% (30 min) [54] High cost of chitosan 20.00
(2 g/L)
BM-8 (100 mg/L) Organic C. vulgaris (0.1 g/L) 60% (15 min) This study Medium cost 5.14
90% (30 min)

FE flocculation efficiency

completely bind the negatively-charged C. vulgaris cells
to create a patched-network with positive surface charges
accessible to more microalgae cells and this attractive
interaction resulted in cells flocculation (Fig. 7) [50].
Comparing the efficacy and cost of BM-8 to those
of chemical flocculants (inorganic) and organic floccu-
lants (Table 5), we found that the flocculation times for
100 mg/L of BM-8 to achieve ~ 60% and 90% precipita-
tion of C. vulgaris i.e. 15 and 30 min, respectively were
much lower than in several other studies where > 90% of
Scenedesmus sp. and C. minutissima biomass were recov-
ered using 0.1 g/L and 1 g/L of sulphate salts, respectively
[51, 52]. The one-step solvent extraction of BM-8 using
ethanol was relatively inexpensive as the solvent can
be recycled. The estimated processing cost of BM-8 in
this study was USD 5.14/kg, which appeared much more
attractive compared to sulphate salts (inorganic floccu-
lant) and chitosan (organic flocculant). Hence, BM-8 pro-
duced from an abundantly available wastewater source—
POME—at zero feedstock cost is more advantageous (in
terms of cost and environmental benefits) compared to
harvesting methods using the chemical flocculants and
some of the bioflocculants studied. It can potentially be
an alternative harvesting method in algal technology and
for hazardous chemical flocculants substitution.
Conclusion
Enzymatic hydrolysis of POME was optimized in the present
study by RSM with PBD and CCD. The hydrolyzed POME
containing an optimal level of reducing sugars served as a
carbon source in producing 1.5-fold higher yield of BM-8
using B. marisflavi NA8, under a controlled environment
(bioreactor). The results show that 48.6 kg of BM-8 can
be derived from 1 tonne of POME. BM-8’s biochemical
and molecular characteristics reveal its potential flocculat-
ing capability and was extended to microalgae harvesting.
Indeed, it showed higher efficiency in precipitating microal-
gae than the conventional chemical flocculants.
Acknowledgements  The authors thank the Malaysian Palm Oil Board
(MPOB) for permission to publish the findings. Thanks are also due to
the Malaysia Genome Institute (MGI) for providing the enzyme under
Ministry of Agriculture No. TF0310F086, and Shanxi Tianli Jinrun
Industrial Co. Ltd. for the chemical flocculants provided. The technical
assistance provided by the interns and the staff of the Engineering and
Processing Research Division of MPOB is also deeply appreciated.
Compliance with Ethical Standards 
Conflict of interest  The authors declare that they have no conflict of
interest.
References
1. Kushairi, A., Singh, R., Ong-Abdullah, M.: The oil palm indus-
try in Malaysia: thriving with transformative technologies. J. Oil
Palm Res. 29(4), 431–439 (2017)
2. More, T.T., Yadav, J.S.S., Yan, S., Tyagi, R.D., Surampalli,
R.Y.: Extracellular polymeric substances of bacteria and their
potential environmental applications. J. Environ. Manag. 144,
1–25 (2014)
3. Chaisorn, W., Prasertsan, P., O-Thong, S., Methacanon, P.: Pro-
duction and characterization of biopolymer as bioflocculant from
thermostable Bacillus subtilis WD161 in palm oil mill effluent.
Int. J. Hydrog. Energy 41, 21657–21664 (2016)
4. Czemierska, M., Szczes, A., Holysz, L., Wiater, A., Jarosz-
Wilkolazka, A.: Characterisation of exopolymer R-202 isolated

13

from Rhodococcus rhodochrous and its flocculating properties.
Eur. Polym. J. 88, 21–33 (2017)
5. Rani, R.P., Anandharaj, M., Sabhapathy, P., Ravindran, A.D.:
Physiochemical and biological characterization of novel exopol-
ysaccharide produced by Bacillus tequilensis FR9 isolated from
chiken. Int. J. Biol. Macromol. 96, 1–10 (2017)
6. Buthelezi, S.P., Olaniran, A.O., Pillay, B.: Turbidity and microbial
load removal from river water using bioflocculants from indig-
enous bacteria isolated form wastewater in South Africa. Afr. J.
Biotechnol. 8, 3261–3266 (2009)
7. Gong, W.X., Wang, S.G., Sun, X.F., Liu, X.W., Yue, Q.Y., Gao,
B.Y.: Bioflocculant production by culture of Serratia ficaria and
its application in wastewater treatment. Bioresour. Technol. 99,
4668–4674 (2008)
8. Guo, J., Chen, C.: Removal of arsenite by a microbial biofloccu-
lant produced from swine wastewater. Chemosphere 181, 759–766
(2017)
9. Sajayan, A., Kiran, G.S., Priyadhashini, S., Poulose, N., Selvin,
J.: Revealing the ability of a novel polysaccharide bioflocculant
in bioremediation of heavy metals sensed in a Vibrio biolumines-
cence reporter assay. Environ. Pollut. 228, 118–127 (2017)
10. Zhao, H., Zhong, C., Chen, H., Yao, J., Tan, L., Zhang, Y.,
Zhou, J.: Production of bioflocculants prepared from formalde-
hyde wastewater for the potential removal of arsenic. J. Environ.
Manag. 172, 71–76 (2016)
11. Zhang, Y., Wang, F., Yang, X., Gu, C., Kengara, F., Hong, Q., Lv,
Z., Jiang, X.: Extracellular polymeric substances enhanced mass
transfer of polycyclic aromatic hydrocarbons in the two-liquid-
phase system for biodegradation. Appl. Microbiol. Biotechnol.
90, 1063–1071 (2011)
12. Li, R., Ning, X., Sun, J., Wang, Y., Liang, J., Lin, M., Zhang,
Y.: Decolorization and biodegradation of the Congo red by Aci-
netobacter baumannii YNWH 226 and its polymer production’s
flocculation and dewatering potential. Bioresour. Technol. 194,
233–239 (2015)
13. Sathiyanarayanan, G., Kiran, G.S., Selvin, J.: Synthesis of sil-
ver nanoparticles by polysaccharide bioflocculant produced from
marine Bacillus subtilis MSBN17. Colloids Surf. B 102, 13–20
(2013)
14. Drakou, E.M., Amorim, C.L., Castro, P.M.L., Panagiotou, F.,
Vyrides, I.: Wastewater valorization by pure bacterial cultures
to extracellular polymeric substances (EPS) with high emulsify-
ing potential and flocculation activities. Waste Biomass Valoriz.
(2017). https​://doi.org/10.1007/s1264​9-017-0016-9
15. Wan, C., Zhao, X.Q., Guo, S.L., Alam, M.A., Bai, F.W.: Biofloc-
culant production from Solibacillus silvestris W01 and its applica-
tion in cost-effective harvest of marine microalga Nannochloro-
psis oceanica by flocculation. Bioresour. Technol. 135, 207–212
(2013)
16. Liu, C., Wang, K., Jiang, J.H., Liu, W.J., Wang, J.Y.: A novel
bioflocculant produced by a salt-tolerant, alkaliphilic and biofilm-
forming strain Bacillus agaradhaerens C9 and its application in
harvesting Chlorella minutissima UTEX2341. Biochem. Eng. J.
93, 166–172 (2015)
17. Li, Y., Xu, Y., Liu, L., Jiang, X., Zhang, K., Zheng, T., Wang,
H.: First evidence of bioflocculant from Shinella albus with floc-
culation on harvesting of Chlorella vulgaris biomass. Bioresour.
Technol. 218, 807–815 (2016)
18. Li, Y., Xu, Y., Liu, L., Li, P., Yan, Y., Chen, T., Zheng, T., Wang,
H.: Flocculation mechanism of Aspergillus niger on harvesting of
Chlorella vulgaris biomass. Algal Res. 25, 402–412 (2017)
19. Li, Y., Xu, Y., Zheng, T., Wang, H.: Flocculation mechanism of
the actinomycete Streptomyces sp. hsn06 on Chlorella vulgaris.
Bioresour. Technol. 239, 137–143 (2017)
20. Pu, S., Ma, H., Deng, D., Xue, S., Zhu, R., Zhou, Y., Xiong, X.:
Isolation, identification, and characterization of an Aspergillus
13
Waste and Biomass Valorization
niger bioflocculant-producing strain using potato starch wastewa-
ter as nitrile and its application. PLoS ONE 13(1), 1–17 (2018)
21. Okeiyeto, K., Nwodo, U.U., Okoli, S.A., Mabinya, L.V., Okoh,
A.I.: Implications for public health demands alternative to inor-
ganic and synthetic flocculants: bioflocculants as important can-
didates. Microbiol. Open 5(2), 177–211 (2016)
22. Sekelwa, C., Anthony, U.M., Mabinya, L.V., Anthony, O.I.: Char-
acterization of a thermostable polysaccharide bioflocculant pro-
duced by Virgibacillus species isolated from Algao bay. Afr. J.
Microbiol. Res. 7(23), 2925–2938 (2013)
23. Nurul Adela, B., Nasrin, A.B., Loh, S.K., Madihah, A.Z.: Isolation
and identification of novel bioflocculant-producing bacteria from
palm oil mill effluent. J. Pure Appl. Microbiol. 9, 1–12 (2015)
24. Nurul-Adela, B., Nasrin, A.B., Loh, S.K.: Palm oil mill effluent
as a low-cost substrate for bioflocculant production by Bacillus
marisflavi NA8. Bioresour. Bioprocess. 3(20), 1–8 (2016)
25. APHA: Standard Methods for the Examination of Water and
Wastewater, 21st  edn. American Public Health Association
(APHA), Washington, DC (2005)
26. Loh, S.K., Lai, M.E., Ngatiman, M., Lim, W.S., Choo, Y.M.,
Zhang, Z., Salimon, J.: Zero discharge treatment technology of
palm oil mill effluent. J. Oil Palm Res 25(3), 273–281 (2013)
27. Miller, G.L.: Use of dinitrosalicylic acid reagent for determination
of reducing sugars. Anal. Chem. 13, 420–428 (1959)
28. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith,
F.: Colorimetric method for determination of sugars and related
substrates. Anal. Chem. 28(3), 350–356 (1956)
29. Vello, V., Phang, S.M., Chu, W.L., Nazia, A.M., Lim, P.E., Loh,
S.K.: Lipid productivity and fatty acid composition-guided selec-
tion of Chlorella strains isolated from Malaysia for biodiesel pro-
duction. J. Appl. Phycol. 26, 1399–1413 (2014)
30. Idris, N.A., Loh, S.K., Lau, H.L.N., Mustafa, E.M., Vello, V., Tan,
C.Y., Phang, S.M.: Cultivation of microalgae in medium contain-
ing palm oil mill effluent and its conversion into biofuel. J. Oil
Palm Res. 29(2), 291–299 (2017)
31. Zheng, Y., Ye, Z.L., Fang, X.L., Li, Y.H., Cai, W.M.: Production
and characteristics of a bioflocculant produced by Bacillus sp.
F19. Bioresour. Technol. 99, 7686–7691 (2008)
32. Gao, Q., Zhu, X.H., Mu, J., Zhang, Y., Dong, X.W.: Using Rudi-
tapes philippinarum conglutination mud to produce bioflocculant
and its applications in wastewater treatment. Bioresour. Technol.
100, 4996–5001 (2009)
33. Badireddy, A.R., Chellam, S., Gassman, P.L., Engelhard, M.H.,
Lea, A.S., Rosso, K.M.: Role of extracellular polymeric substances
in bioflocculation of activated sludge microorganisms under glu-
cose-controlled conditions. Water Res. 44, 4505–4516 (2010)
34. Aqma, W.S., Quilty, B.: Influences of extracellular polymeric
substances (EPS) for autoaggregation of Pseudomonas putida
CP1 during growth on mono-chlorophenol. Malays. J. Microbiol.
11(13), 246–253 (2015)
35. Jorand, F., Boue-Bigne, F., Block, J.C., Urbain, V.: Hydropho-
bic/hydrophilic properties of activated sludge exopolymeric sub-
stances. Water Sci. Technol. 37, 307–315 (1998)
36. Suzuki, H., Daimon, M., Awano, T., Umekage, S., Tanaka, T.,
Kikuchi, Y.: Characterization of extracellular DNA production
and flocculation of the marine photosynthetic bacterium Rhodo-
vulum sulfidophilum. Appl. Microbiol. Biotechnol. 84, 349–356
(2009)
37. Xiong, Y.Y., Wang, Y.P., Yu, Y., Li, Q.B., Wang, H.T., Chen,
R.H., He, N.: Production and characterization of a novel biofloc-
culant from Bacillus licheniformis. Appl. Environ. Microbiol. 76,
2778–2782 (2010)
38. Guo, J., Yu, J., Xin, X., Zou, C., Cheng, Q., Yang, H., Nengzi, L.:
Characterisation and flocculation mechanism of a bioflocculant
from hydrolyzate of rice stover. Bioresour. Technol. 177, 393–397
(2015)
Waste and Biomass Valorization
39. Czemierska, M., Szczes, A., Pawlik, A., Wiater, A., Jarosz-
Wilkolazka, A.: Production and characterisation of exopolymer
from Rhodococcus opacus. Biochem. Eng. J. 112, 143–152 (2016)
40. Li, Z., Zhong, S., Lei, H.Y., Chen, R.W., Yu, Q., Li, H.L.: Pro-
duction of a novel bioflocculant by Bacillus licheniformis X14
and its application to low temperature drinking water treatment.
Bioresour. Technol. 100, 3650–3656 (2009)
41. Elkady, M., Farag, S., Zaki, S., Abd-El-Haleem, D.: Bacillus
mojavensis strain 32A, a bioflocculant-producing bacterium iso-
lated from an Egyptian salt production pond. Bioresour. Technol.
102, 8143–8151 (2011)
42. Ji, B., Zhang, X.Y., Li, Z., Xie, H.Q., Xiao, X.M., Fan, G.J.:
Flocculation properties of a bioflocculant produced by Bacillus
licheniformis.. Water Sci. Technol. 62, 1907–1913 (2010)
43. Zaki, S.A., Elkady, M.F., Fareg, S., Abdel-Haleem, D.: Charac-
terization and flocculation properties of a carbohydrate biofloc-
culant from a newly isolated Bacillus velezensis 40B. J. Environ.
Biol. 34, 51–58 (2013)
44. Luvuyo, N., Nwodo, U.U., Mabinya, L.V., Okoh, A.I.: Studies on
bioflocculant production by a mixed culture of Methylobacterium
sp. Obi and Actinobacterium sp. Mayor. BMC Biotechnol. 13, 62
(2013)
45. Zhang, Z.Q., Lin, B., Xia, S.Q., Wang, X.J., Yang, A.M.: Produc-
tion and application of a novel bioflocculant by multiple-micro-
organism consortia using brewery wastewater as carbon source.
J. Environ. Sci. 19, 667–673 (2007)
46. Gomma, E.Z.: Production and characteristics of a heavy metal
removing bioflocculant produced by Pseudomonas aeruginosa.
Pol. J. Microbiol. 4, 281–524 (2012)
47. Okeiyeto, K., Nwodo, U.U., Mabinya, L.V., Okili, A.S., Okoh,
A.I.: Characterisation of a bioflocculant (MBF-UFH) produced by
Bacillus sp. AMREG7. Int. J. Mol. Sci. 16, 12986–13003 (2015)
48. Wang, L., Ma, F., Lee, D.J., Wang, A., Ren, N.: Bioflocculants
from hydrolysates of corn stover using isolated strain Ochrobac-
tium ciceri W2. Bioresour. Technol. 145, 259–263 (2013)
49. Mabinya, L.V., Cosa, S., Mkhwetshana, N., Okoh, A.I.: Halo-
monas sp. OKOH- a marine bacterium isolated from the bottom
sediment od Algoa bay—produces a polysaccharide bioflocculant:
partial characterization and biochemical analysis of its properties.
Molecules 16, 4358–4370 (2011)
50. Salim, S., Vermue, M.H., Wijffels, R.H.: Ratio between autofloc-
culating and target microalgae affects the energy-efficient harvest-
ing by bio-flocculation. Bioresour. Technol. 118, 49–55 (2012)
51. Chen, L., Wang, C., Wang, W., Wei, J.: Optimal conditions of
different flocculation methods for harvesting Scenedesmus sp. cul-
tivated in an open-pond system. Bioresour. Technol. 133, 9–15
(2013)
52. Papazi, A., Makridis, P., Divanach, P.: Harvesting Chlorella
minutissima using cell coagulants. J. Appl. Phycol. 22, 349–355
(2010)
53. Sirin, S., Clavero, E., Salvadó, J.: Potential pre-concentration
methods for Nannochloropsis gaditana and a comparative study
of pre-concentrated sample properties. Bioresour. Technol. 132,
293–304 (2013)
54. Xu, Y., Purton, S., Baganz, F.: Chitosan flocculation to aid the
harvesting of the microalga Chlorella sorokiniana. Bioresour.
Technol. 129, 296–301 (2013)
13