Science of the Total Environment 715 (2020) 136885

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Science of the Total Environment

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Bioconversion of citrus peel wastes into bioflocculants and their

application in the removal of microcystins
Xiaoli Qi a,b,1, Yongliang Zheng c,1, Ningjia Tang b, Jiangang Zhou b,d,⁎, Su Sun e
a
College of Life Sciences, Jiamusi University, Jiamusi 154007, China
b
School of Environmental Engineering, Wuhan Textile University, Wuhan 430073, China
c
Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resources Comprehensive Utilization, Huanggang Normal University, Huanggang 438000, China
d
Engineering Research Centre for Clean Production of Textile Dyeing and Printing, Ministry of Education, Wuhan Textile University, Wuhan 430073, China
e
School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• A novel bioflocculant-producing bacte-

rium was isolated from orange tin pro-
duction wastewater.
• Citrus peel wastes (CPW) were utilised
to prepare enzymes and bioflocculants.
• Molecular distillation was used to re-
move antimicrobial limonene.
• The bacterium can secrete cellulase,
hemicellulase, pectinase, protease, and
ligninase.
• The bioflocculant was used to eliminate
microcystins for the first time.

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

Article history: In this study, the mechanism for converting citrus peel wastes (CPW) into bioflocculants using Alcaligenes faecalis
Received 21 December 2019 subsp. phenolicus ZY-16 was analysed. The results demonstrated that the ZY-16 strain could produce various
Received in revised form 21 January 2020 lignocellulolytic enzymes, containing cellulase, hemicellulase, pectinase, protease, and ligninase, enhancing the
Accepted 21 January 2020
hydrolysis of citrus peel wastes. Molecular distillation removes antimicrobial limonene, which could inhibit
Available online 23 January 2020
bioflocculant production. The optimal fermentation conditions with the highest bioflocculant yield (3.49 g/L)
Editor: Yifeng Zhang were 38.79 g/L of CPW, 35.54 °C, and pH 4.48. Furthermore, the bioflocculant was used to eliminate microcystins
for the first time, and the highest removal efficiency (90.05%) was achieved at a pH of 3.0, after 800 mg/L of
Keywords: bioflocculant was added into the microcystins solution (10 mg/L) for 60 min. Therefore, this paper demonstrated
Bioflocculant that CPW could be a cost-effective feedstock for the production of bioflocculants, which have potential applica-
Citrus peel wastes tion in microcystin removal.
Alcaligenes faecalis subsp. phenolicus © 2020 Elsevier B.V. All rights reserved.
Microcystins

⁎ Corresponding author at: School of Environmental Engineering, Wuhan Textile University, Wuhan 430073, China.
E-mail address: wallice24@hotmail.com (J. Zhou).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.scitotenv.2020.136885
0048-9697/© 2020 Elsevier B.V. All rights reserved.
2 X. Qi et al. / Science of the Total Environment 715 (2020) 136885
1. Introduction
Harmful cyanobacterial blooms (HCB) have become a worldwide
environmental issue and are prevalent with an increasing magnitude,
duration, and frequency (Weber et al., 2019). Microcystins (MCs) are
known to be potent heptapeptide hepatotoxins released by HCB,
among which microcystin-LR (MCLR) is the most prominent and toxic
in nature (Su et al., 2019). The World Health Organisation (WHO) sug-
gests a safe critical level of MCLR equivalents to be ≤1 ng/mL in drinking
water for adults and ≤0.3 ng/mL for children (Torbick et al., 2018). MCLR
is recalcitrant to inactivation and degradation by traditional water treat-
ment including ozonation, filtration, and chlorination (Turner et al.,
2018). The resistance of MCLR to typical environmental stressors has
forced scientists to create new technologies to eliminate it.
Flocculation technology is found to be an effective method for deal-
ing with various contaminants. Currently, chemical flocculants, such as
polyacrylamide and aluminium sulphate, are widely employed for agro-
industrial wastewater treatment (Muthulakshmi et al., 2019). However,
inorganic and organic synthetic flocculants are detrimental to human
health and the environment (Bisht and Lal, 2019). Generally speaking,
bioflocculants are extracellular macromolecule polymers, such as glyco-
protein, polysaccharide, and proteoglycan, secreted by microorganisms
(Abu Tawila et al., 2019). Thus, the flocculants of microbiological origin
are advantageous over chemical flocculants due to their safety, biocom-
patibility, renewability, and biodegradable properties (Zhong et al.,
2016). Thus far, they have been commercialised for use in the fermenta-
tion, pulp, paper, and textile industries (Maliehe et al., 2019). Among
these biopolymers, proteoglycan-based bioflocculants have attracted
great attention in recent years, owing to their high-performance when
removing toxic wastes and pollutants. However, the high cost of the cul-
ture medium was the major hindrance concerning the economic pro-
duction of bioflocculants. To lower the production expense,
agricultural and industrial wastes, such as kitchen wastes (Liu et al.,
2019), rice bran (Liu et al., 2017), cactus juice (Sellami et al., 2014),
corn stover (Guo et al., 2017; Liu et al., 2015a), palm oil mill effluent
(Bukhari et al., n.d.), and peanut hull (Liu et al., 2016), have been used
as fermentation media for bioflocculant production. Although most of
these agro-industrial by-products contain a high organic matter for bac-
terial growth, their unbalanced nutrition is likely to inhibit the biosyn-
thesis of polymers (Mohammed and Dagang, 2019). Therefore, a new
low-cost substrate should be investigated.
In 2018, the total amount of citrus in China was 41.381 million t/
year, with citrus peel waste (CPW) making up to 50% of the total fruit's
weight (Sharma et al., 2019). CPW includes large amounts of cellulose,
hemicellulose, pectin, lignin, monosaccharide, and oligosaccharides,
they perish easily, and even increase soil pollution (Fukada et al.,
2014). Stricter environmental legislation for citrus industries in China
has compelled these producers to develop an environmentally benign
solution for the valorisation of citrus waste. Some attempts have been
made to utilise CPW, such as cattle feed, composting, and dump storage
(Zema et al., 2018). However, waste valorisation through the aforemen-
tioned projects can give rise to serious environmental and economic is-
sues, because of the uncontrolled fermentation and high disposal costs
(Solidum, 2013). Thus, using CPW as a cheap cultivation medium for
biopolymer production is favourable for environmental protection and
cost reduction. To our knowledge, there have been almost no studies
utilising CPW as a promising feedstock for bioflocculant production.
Here, a new bacterium, Alcaligenes faecalis subsp. phenolicus ZY-16,
which secretes pectinase, ligninase, protease, hemicellulase, and cellu-
lase, showed simultaneous production of bioflocculants from CPW.
In this study, we firstly reported a novel lignocellulase-producing
bacterium ZY-16, with the potential ability to directly convert CPW to
bioflocculants, and demonstrated that limonene in the medium is an in-
hibitor of this process. The biodegradability of CPW was determined and
bioflocculants' production of ZY-16 was evaluated by optimizing its fer-
mentation conditions.
As hydrodistillation was carried out at high temperature which may
cause degradation of thermolabile nutrients (Lemes et al., 2018), we
used a molecular distillation method to remove antimicrobial limonene
and increase bioflocculants yield. The whole fermentation process con-
solidates three parts: pre-treatment of CPW, enzyme secretion, and
bioflocculant production, thereby enhancing the bioconversion rate of
CPW into bioflocculants. The most convenient method for the removal
of MCLR is to use M. oleifera Lam seed extracts, which acts as natural
flocculants and can terminate hydrophobic organic pollutants from
water resources (Yasmin et al., 2019). In the present study, the micro-
bial flocculant was applied for the first time in the effective removal of
microcystins from wastewaters. Thus, this study promotes the
valorisation of CPW and can be applied to remove microcystins from
aqueous solutions.
2. Materials and methods
2.1. Raw materials
Citrus peel waste was collected from a local Walmart market
(Wuhan, China) and kept in a freezer until further use. CPW was shred-
ded into particles of b1 mm in diameter. The contents of hemicellulose
and cellulose were calculated according to the contents of pentose and
hexose, respectively. The monosaccharide and limonene content were
determined according to the GC–MS method (Barreto et al., 2017).
The lignin content was determined using a UV-spectrophotometer
(METASH, UV 5100B, Shanghai) at the wavelength of 205 nm. The pro-
tein content of the sample was measured by the Bradford method (Chen
et al., 2016). The contents of the dried CPW have been estimated as fol-
lows: 22.30% cellulose, 11.32% hemicellulose, 2.99% lignin, 7.04% glu-
cose, 11.06% fructose, 2.03% sucrose, 26.54% pectin, 5.02% protein, and
4.02% limonene. The standard MCLR was purchased from Sigma-
Aldrich (Beijing, China).
2.2. Screening and identification of bioflocculant-producing strains
CPW-degrading strains were isolated from the activated sludge of
orange tin production wastewater obtained from the YuGuo Company
in Hubei, China. When culturing the strains in the screening medium
(1.0 g/L of K2HPO4·3H20, 2.0 g/L of KH2PO4, and 0.1 g/L of
MgSO4·7H20) containing 3.5% CPW, some culture supernatants became
more transparent after 60 h of incubation at 30 °C. Then, the flocculating
activity of each bacterial broth was evaluated. Finally, a CPW-degrading
bacterium (ZY-16), which showed the maximum flocculating efficiency,
was selected for next experiments. To identify this selected isolate, its
genome was extracted using a Gene Jet Purification Kit (Invitrogen).
The 16S rDNA gene fragment was amplified according to our previous
works (Zhong et al., 2016). Then, the PCR amplicons were purified
and sequenced by Shengong Biotech Co., Ltd. (Shanghai, China). The
16S rDNA sequence of strain ZY-16 was deposited in the NCBI data li-
brary (Sik Nam et al., 1996).
2.3. Removal of limonene by molecular distillation
The limonene in the CPW was eliminated by molecular distillation at
various wiper rolling speeds. After 500 mL of the prepared CPW broth
was put into the distillation flask, the wiper rolling speed was adjusted
to different values (60, 90, 120, 150, or 180 r/min). Then, the limonene
in the CPW was evaporated by a wipe-film molecular distillation (MD)
apparatus (Pope Scientific, Inc., USA). Distillation experiments were
performed at 50 °C and 50 Pa. After cooling, purified water was added
to the flask to restore the original volume. The original and residual lim-
onene contents of the CPW both were measured by an Agilent 6890
model gas chromatograph (GC), and the limonene removal efficiency
 X. Qi et al. / Science of the Total Environment 715 (2020) 136885
was then calculated using Eq. (1):
Limonene removal rateð%Þ
ðoriginal limonene content−residual limonene contentÞ  100
¼
original limonene content
ð1Þ
2.4. Evaluation of CPW as an alternative medium for bioflocculant
production
To evaluate the effects of CPW on the bioflocculant production by
ZY-16, two culture broths were tested. In the first culture broth, CPW
(40 g/L) was used as a nutrition source, while in the second culture
broth, glucose (40 g/L) was used as a carbon source. For the latter
(seed fermentation medium), peptone (5 g/L), yeast extract (5 g/L),
NaNO3 (1 g/L), K2HPO4 (1.5 g/L), MgSO4 (0.2 g/L), and KH2PO4
(1.5 g/L) were added. The fermentation conditions were: inoculum
size 5% (106 CFU/mL), initial pH 5.0, cultural temperature 30 °C, and in-
cubation at 160 rpm for 96 h. The bioflocculant production, cell prolifer-
ation, substrate utilization, and pH of the remaining broth were
determined along with the whole fermentation process. A batch of fer-
mentation experiments, in which ZY-16 was grown on different con-
centrations of the CPW medium and seed fermentation medium, were
performed.
2.5. Production and extraction of bioflocculant from CPW
There are many constraints that can affect bioflocculant production
by ZY-16. In this study, the effects of three numerical factors, including
the CPW concentration (%), pH, and incubation temperature (°C),
were evaluated using a central composite design (CCD) (Table 1).
After data collection, ANOVA was used to test the significance of the pa-
rameters and their complex interactive effects. Then, numerical optimi-
sation was used to maximise the bioflocculant production. Minitab17
software was used for the statistical analysis. Based on these results, a
confirmation assay was also applied to verify the optimum conditions.
Bioflocculant extraction was performed as previously described (Li
et al., 2013).
2.6. Enzyme activity assay
Samples collected at 12-h intervals over five days were taken for an
enzymatic activity assay. The activities of pectinase, hemicellulase, and
cellulase were estimated by determining the concentrations of reducing
sugars liberated from pectin, xylan, and carboxymethyl cellulose, re-
spectively, according to a previously reported method (Liu et al.,
2015a). The ligninase (laccase) activity in the fermentation samples
was assessed using syringaldazine as a reactant (Nypelo et al., 2018).
The reaction mixture, containing 1.0 mL of cell-free supernatant,
1.0 mL of 0.05 M citrate–phosphate buffer (pH 4.5), and 1.0 mL of
1.0 mM syringaldazine, was incubated at 35 °C for 20 min. The optical
density was read at 525 nm for the different parameters. Casein was
used as a substrate for the protease assay. The reaction system,
consisting of 0.3 mL of 0.8% casein and 0.2 mL of the broth supernatant,
was reacted at 35 °C for 60 min, followed by boiling for 10 min to
Table 1
Independent variables for the MBF-16 production.
Factors Coded levels
−1.682 −1.000 0 1.000
CPW concentration (g/L) 23.18 30.00 40.00 50.00
Initial pH 3.30 4.00 5.00 6.00
Temperature (°C) 26.60 30.00 35.00 40.00
3
inactivate the enzyme. After adding 0.5 mL of 0.5 M Na2CO3 and
0.15 mL of Folin-Phenol, the solution was incubated at 35 °C for
30 min and centrifuged at 12000 rpm for 10 min. Then, the OD680 was
measured with a UV 5100B spectrophotometer.
2.7. Chemical analysis of MBF-16
The carbohydrate component of MBF-16 was elucidated using the
phenol–sulfuric acid assay (Taokaew et al., 2015). The protein content
of MBF-16 was determined using the Bradford method (Tang et al.,
2014a). After which, the sample was hydrolysed with 5 M
trifluoroacetic acid at 120 °C for 2 h. Neutral sugar, uronic acids and
amino sugar were quantified by the anthrone reaction, the carbazole-
sulfate reaction and the Elson-Morgan reaction, respectively, using the
procedures of Chaplin and Kennedy (Peng et al., 2014). Size exclusion
chromatography (SEC), using a Hitachi L-6200 system controller, was
used to evaluate the molecular weight (MW) of MBF-16 (Pathak et al.,
2017). Elemental analysis was employed using an atomic absorption
spectrophotometer (Model 2400, USA). The functional groups of MBF-
16 were analysed with a Fourier-transform infrared spectrometer
(FTIR-6100, Japan).
2.8. MCLR removal assay
To further analyse the feasibility of MCLR removal in practical appli-
cations, batch flocculation experiments were performed in 100 mL of
MCLR solution with various initial concentrations (1–100 mg/L) for dif-
ferent times (20–200 min). Different doses of MBF-16 were added to
the MCLR solution and the pH was adjusted to the required value with
diluted HCl and NaOH. After the remaining MCLR concentrations in
the solution were determined by Reverse-phase High-performance Liq-
uid Chromatography (RHPLC), using a photodiode array detection sys-
tem, the percentage removal efficiency of MCLR was expressed
according to the following Eq. (2):


C i −C f  100
%removal of MCLR ¼ ð2Þ
Ci
where Ci is the initial MCLR concentration and Cf is the final concentra-
tion of MCLR in the bulk solution.
3. Results and discussion
3.1. Screening and characterization of bioflocculant-producing ZY-16 strain
In total, twenty one isolates were collected from the screening me-
dium. Three of these isolates showed high flocculating activity. Isolate
ZY-16, which showed a higher flocculating efficiency of 95.35% within
all the results, was selected for further studies. According to the degra-
dation enzyme ability test, the ZY-16 strain produces pectinase,
ligninase, protease, cellulase, and hemicellulase. The novel Gram-
negative strain is an actively motile, coccobacillary, peritrichously flag-
ellated, non-spore forming bacterium. Growth occurs at 20, 25, 30, 35,
and 42 °C, with optimal growth occurring at 35 °C. Pectin, hemicellulose,
lignin, and cellulose are depolymerised under aerobic conditions. Lig-
nin, dextrin, glycogen, phenol, L-lactate, 3-hydroxybutyrate, and etha-
nol were utilized.
The 16S rDNA sequence of isolated ZY16 was determined and sub-
mitted in the GenBank nucleotide database (accession number:
MN339607). The results of the physiological and biochemical proper-
1.682
ties, the morphological characteristics, and the 16SrDNA sequence
56.82 BLAST of the strain showed that isolated ZY-16 was identified as
6.70 Alcaligenes faecalis subsp. phenolicus. The extracellular bioflocculant se-
43.40
creted by this isolate was named MBF-16.
4 X. Qi et al. / Science of the Total Environment 715 (2020) 136885
3.2. Effects of limonene on MBF-16 production and limonene removal from
CPW
To study the effects of limonene on MBF-16 production, limonene
was dropped exogenously into the seed fermentation medium at differ-
ent dosages and the MBF-16 yields were compared. Fig. 1 shows that the
MBF-16 production was inhibited by the increasing limonene content in
the seed fermentation medium. Particularly, when the limonene con-
tent increased from 0 to 0.5 g/L, the MBF-16 yield was moderated
slightly from 1.81 to 1.73 g/L. However, the MBF-16 yield shows a
sharp drop (1.08 g/L) when the limonene concentration reached
1.0 g/L. After the final concentration of limonene exceeded 1.5 g/L, the
MBF-16 production was only 0.67 g/L, which was almost one-third of
that observed in the limonene-free seed fermentation medium. Limo-
nene toxicity to microorganisms has been investigated (Gu et al.,
2019). In this study, limonene significantly inhibited MBF-16 produc-
tion at high concentrations. Thus, limonene should be eliminated
when using CPW as a feedstock to improve MBF-16 yield. So far, two
conventional methods were reported to remove limonene from the
CPW, one is assimilated by microorganisms and the other one is molec-
ular distillation (Rossi et al., 2011). The former mainly focuses on catab-
olism by the microbe, such as Sphingobium sp., Pseudomonas fluorescens,
and Fusarium oxysporum (Yan et al., 2019; Chen et al., 2018; Cheng et al.,
2019). Nevertheless, the nutrition in the CPW could also be consumed
by these microbes during the metabolism process, which may affect
the MBF-16 production in this study. Thus, to remove the limonene
from the CPW, molecular distillation was performed at different wiper
rolling speeds. The limonene removal rate, total residual carbohydrates,
and MBF-16 production were obtained (Fig. 2). The results show that
the limonene removal rate increased with the rise in wiper rolling
speeds. The removal rate of limonene was 74.4% at a wiper rolling
speed of 90.0 r/min, 86.9% at a wiper rolling speed of 120.0 r/min, and
86.5% at a wiper rolling speed of 150.0 r/min. This implies that the lim-
onene removal rate increases when the liquid film in the internal sur-
face of the evaporator continuously becomes more and more uniform
with the increase in wiper rolling speed. However, such effect can be
out of consideration when the wiper rolling speed is N120.0 r/min.
Fig. 2 also showed that a wiper rolling speed of 120.0 r/min was desir-
able in the experiments.
After removing the limonene, pure water was added back, and the
carbohydrate content and MBF-16 production were studied. The
highest content of residual carbohydrate (21.53 g/L) was observed at a
wiper rolling speed of 30.0 r/min, with residual carbohydrate levels of
21.33 g/L at a wiper rolling speed of 60.0 r/min, and 21.35 mg/mL at a
wiper rolling speed of 90.0 r/min. The lowest residual carbohydrate con-
tent (21.2 g/L) occurred at a wiper rolling speed of 180.0 r/min. Fig. 2 in-
dicates that the carbohydrate content was unchanged when limonene
was removed at different wiper rolling speeds. Overall, the results
Fig. 1. Effects of limonene on the MBF-16 yield in the seed fermentation medium (n = 3).
Fig. 2. Limonene removal efficiency, MBF-16 production of CPWs, and residual
carbohydrate at different wiper rolling speeds (n = 3).
suggest that molecular distillation was an efficient approach to remove
limonene while preserving the sugar content. The MBF-16 production
increased steadily with the increase in limonene removal, while the car-
bohydrate content remained unchanged in the CPW. After removing
86.9% of the limonene in the CPW at a wiper rolling speed of 120.0 r/
min, the highest MBF-16 production (3.04 g/L) was achieved. Compared
with that of the original CPW (1.06 g/L), the MBF-16 production
(3.04 g/L) increased ~186.8% after removing the limonene. A similar
MBF-16 yield was obtained at a wiper rolling speed of 150.0 r/min,
which was also attributed to the high limonene removal efficiency.
This result was in line with that observed with the seed fermentation
medium, in that the MBF-16 production was significantly suppressed
when the limonene concentration was higher than 0.5 g/L (Fig. 1).
3.3. Evaluation of CPW as an alternative medium for MBF-16 production
In this study, CPW was directly supplied to Alcaligenes faecalis subsp.
phenolicus ZY-16 as nutrients to produce MBF-16 without any extra
feedstock added to generate manufacturing cost savings. The effects of
CPW strength (with various CPW concentrations) on the MBF-16 yield
were studied. As shown in Fig. 3, the yield and flocculating efficiency
of MBF-16 were improved with the increase of the CPW concentration.
The highest flocculating efficiency and yield were obtained when 35 g/L
of CPW was used as a broth medium. A further increase in the CPW con-
centration suppressed the production of MBF-16, which might be attrib-
uted to the reduction of the oxygen supply, which aligned with the
reports from previous studies (Ahmed et al., 2016).
3.4. Optimal pH and temperature of degradation enzymes secreted by strain
ZY16 and stability assay
To effectively convert CPW into bioflocculants, it was indispensable
to confirm the optimal conditions in which the majority of catabolic en-
zymes could show relatively high activities. Thus, the optimal pH range
and temperature of various enzymes were studied. As indicated in
Fig. 4A, the optimum pH for ligninase, pectinase, protease, cellulase,
and hemicellulase was 4.5, 5.0, 5.5, 4.0, and 4.0, respectively. Further-
more, the biosynthesis of flocculants could be induced by acidic condi-
tions. In addition, the pH stabilities of these enzymes were analysed.
Pectinase, cellulase, and hemicellulase activities remained unchanged
after incubation at pH 4.5 for 30 min (Fig. 5A). Although ligninase and
protease showed instability in the acidic conditions, N60% of enzyme ac-
tivities were retained after the treatment at pH 4.5 for 30 min.
The effects of temperature on the enzyme activities were illustrated
in Fig. 4B. The optimal temperature for cellulase and hemicellulase from
strain ZY-16 was 60 °C, while the best activity of protease, pectinase,
and ligninase was obtained at 50, 40, and 40 °C, respectively. The
 X. Qi et al. / Science of the Total Environment 715 (2020) 136885
Fig. 3. Effects of the concentrations of CPW on the flocculating activity and yield of MBF-16
(n = 3).
thermal stabilities of these enzymes were also determined. As shown in
Fig. 5B, cellulose, hemicellulose, protease, pectinase, and ligninase were
unstable at 60 °C, and all the test enzymes exhibited the best stability at
35 °C. Moreover, high temperatures suppressed the flocculating effi-
ciency and synthesis of bioflocculants, and the maximum flocculating
activity and yield were obtained at 35 °C. Considering the energy expen-
diture and bioflocculant production, 35 °C was selected as the culture
temperature.
3.5. Growth curve of Alcaligenes faecalis subsp. phenolicus ZY-16 in CPW
The time profiles for cell growth, five enzyme activities, pH, and
MBF-16 production were studied when ZY-16 was cultured in the
3.5% CPW medium. Fig. 6A shows that the pH increased from 4.5 to
6.5 in the first 24 h, and then gradually rose to 7.5. This increased pH
may be caused by the consumption of various organic acids as sub-
strates. Some scientists have reported that bioflocculant excretion is
parallel to cell proliferation. For example, profiles of the bioflocculant
synthesis of Pseudomonas veronii L918 (Liu et al., 2016) and Bacillus
agaradhaerens C9 (Liu et al., 2015b) showed good relationships with
the cell proliferation curve, and achieved its maximum flocculating
yield during the stationary phase. In this study, Fig. 6B shows that the
cell proliferation increased sharply from 24 to 36 h and the production
of MBF-16 was positively correlated with the cell multiplication during
the exponential growth stage. The cell reproduction declined after 48 h
of culture, however, the MBF-16 yield rose to 3.22 g/L at 60 h. Therefore,
the reduction in the flocculating efficiency and yield of MBF-16 may be
due to the release of degradation enzymes during cell death. Conse-
quently, the fermentation time of 60 h was selected to harvest the
MBF-16 product. As shown in Fig. 6C–D, during the first 12 h most
Fig. 4. Optimum pH (A) and temperature (B) of different enzymes secreted from Alcaligenes faecalis subsp. phenolicus ZY-16.
5
tested enzyme activities increased slightly, which may be due to the
low content of the ZY-16 strain, and then all the enzyme activities obvi-
ously increased, thereby accelerating the conversion of CPW into MBF-
16.
The concentrations of lignin, carbohydrate, and pectin in the CPW
after strain cultivation were 8.6, 26.9, and 10.7 mg/L, with a removal ef-
ficiency of 65.4%, 71.7%, and 75.8%, respectively. The presence of cellu-
lase, pectinase, hemicellulase, and ligninase in the medium may lead
to the decrease in the lignocellulose content of the broth when inocu-
lated with ZY-16. All these enzymes are critical for their pectin and
lignocellulose-degrading capabilities (Guo et al., 2018). Additionally,
MBF-16 production costs largely reduced when the ZY-16 strain was
cultivated in CPW, while the flocculating activity did not decline. Thus,
this method can minimise MBF-16 manufacturing costs.
3.6. Optimisation of CPW culture conditions
For the optimum cultural conditions of CPW, the central composite
design was carried out to determine the optimum levels of the three sig-
nificant factors that caused maximum MBF-16 production. The second
order polynomial equation was expressed as follows:
YMBF−16 production ¼ 3:51 þ 0:14  A−0:014  B−0:031  C
þ 0:151  A  B þ 0:13  A  C þ 0:057  B
 C−0:747  A2 −0:433  B2 −0:321  C2 ð3Þ
where the variable parameters took their coded values, representing the
CPW concentration (A), temperature (B), and pH (C). The fit was
checked by the coefficients of determination R2, which was calculated
to be 0.9121, highlighting that 91.21% of the variation in the response
could be interpreted by Eq. (3). The quadratic regression results demon-
strate that the model was actually significant due to a high F-value
(85.76) and low probability value (Pmodel b 0.01). From the regression
model analysis, the optimal production conditions for MBF-16 were as
follows: CPW concentration of 38.79 g/L, initial pH of 4.48, and fermen-
tation temperature of 35.54 °C. Under these optimal conditions, an ac-
tual yield of 3.49 g/L was achieved, which was very close to the
predicted maximum values (3.58 g/L). This value (3.49 g/L) is a 2-fold
increase in the MBF-16 yield, when compared to the results (1.81 g/L)
obtained from the seed fermentation medium.
3.7. Characteristics of MBF-16
Purified MBF-16 is a heteroglycan, mainly composed of 90.9% poly-
saccharide and 7.1% protein. Further chemical analysis of the MBF-16
showed that the relative mass proportions of neutral sugar, amino
sugar, and uronic acid were 20.11%, 0.88%, and 30.23%, respectively.
6 X. Qi et al. / Science of the Total Environment 715 (2020) 136885

Fig. 5. pH stability (A) and temperature stability (B) of different enzymes produced from Alcaligenes faecalis subsp. phenolicus ZY-16 (n = 3).

The average molecular weight of MBF-16 determined by SEC was
9.2498 × 105 Da. Elemental analysis of MBF-16 revealed that the relative
mass proportions of C, O, H, N, and S were 39.61, 33.42, 7.43, 7.22, and
0.92 (w/w), respectively.
3.8. Application of MBF-16 for MCLR removal
The effects of MBF-16 dosage on the flocculation efficiency of MCLR
at pH 3.0 were studied. The highest MCLR flocculation activity was
achieved with 800 mg/L of MBF-16, with increased MBF-16 doses lead-
ing to an unchanged flocculation efficiency (Fig. 7A). The enhanced
MCLR removal capacity could be due to an abundant MBF-16 dose.
However, increased MCLR removal will never occur when all positive
MBF-16 molecules are occupied by MCLR molecules. Additionally,
MCLR solutions with different initial concentrations (1–100 mg/L)
were studied to observe the flocculation performance of MBF-16. The
effects of the initial concentration of MCLR on the flocculation efficiency
are illustrated in Fig. 7B. The flocculation efficiency gradually increased
to 90.05%, with the increase of the initial concentration of MCLR, and an
equilibrium could be achieved. At a low concentration, the ratio of avail-
able flocculation sites to the total MCLR was high and all MCLR mole-
cules could be absorbed to the active sites of MBF-16. Whereas, at a
high concentration, the ratio was reduced and consequently the floccu-
lation ability of MBF-16 was inversely correlated with the initial concen-
tration. The maximum flocculating activity of 90.05% was achieved with
a 10 mg/L MCLR solution at pH 3.0.

Fig. 6. Time curves of the pH and flocculating activity (A), cell quantity and MBF-16 production (B), cellulase and hemicellulase (C), and ligninase, pectinase, and protease (D) during cell
growth in the optimised fermentation medium (n = 3).
 X. Qi et al. / Science of the Total Environment 715 (2020) 136885 7

Fig. 7. Effects of MBF-16 dosage, initial concentration of MCLR, pH, and time on the MCLR removal efficiency (A, 10 mg/L of MCLR, 1 h, and pH 3.0; B, 800 mg/L of MBF-16, 1 h, and pH 3.0; C,
800 mg/L of MBF-16, 10 mg/L of MCLR, and 1 h; and D, 800 mg/L of MBF-16, pH 3.0, and 10 mg/L of MCLR) (n = 3).

The flocculation performance of MBF-16 was investigated to study increasing coverage, and competition between flocculated MCLR and
the effects of pH with 10 mg/L of MCLR for 120 min. Turner et al. dem- free MCLR took place. Thus, with the elapsing time, a stable flocculation
onstrated that the MCLR molecules remain neutral within a narrow pH rate was obtained, which led to an equilibrium.
range of 2.09–2.19 (Turner et al., 2018). Better flocculation performance This study demonstrates the potential of microbiological flocculants,
is expected at pH values where both MBF-16 and MCLR carry opposing especially MBF-16 secreted by Alcaligenes faecalis subsp. phenolicus, to
electrical charges due to the ion pairing effect. In our experiments, the scavenge MCLR from aqueous solutions. MCLR is a typical example of
maximum percentage removal of MCLR was achieved at pH 3.0. As the family of microcystins and, with its evident affinity for MBF-16, it
shown in Fig. 7C, the flocculation efficiency gradually decreased when is possible that other similar compounds of this family of toxins will
the pH value increased from 3.0 to 9.0, because the hydrogen ions show similar behaviours.
could make MBF-16's functional groups, such as –NH2 protonated, and
enhanced the MCLR flocculation capacity. Whereas, when there was a 3.9. Comparison of the performance of MBF-16 versus the other conven-
pH below 3.0 there was a rapid drop in the flocculation performance. tional flocculants
The high efficiency of MBF-16 at pH 3.0 may be due to the positively
charged MBF-16 surface and negatively charged MCLR anions. A similar Bioflocculants are macromolecular biopolymers with abundant
result of good flocculence in a highly acidic environment was also re- functional groups that can bind to various environmental contaminants.
ported for the flocculation of MCLR using Moringa oleifera Lam. seeds These characteristics give the bioflocculants the ability to aggregate
(Yasmin et al., 2019). MCLR removal with MBF-16 was 47.08% at
pH 9.0, which was almost 50% less at pH 3.0. In basic conditions, hy-
Table 2
droxyl ions decreased the flocculence of MCLR due to fierce competition
Removal efficiencies of MCLR with different flocculants (%).
among MCLR anions and hydroxyl ions.
The flocculation capacity of MBF-16 onto MCLR was investigated for Flocculants Optimal Optimal Optimal Optimal MCLR Maximum
pH dosage reaction concentration removal of
the reaction time, varying from 0 to 200 min. Fig. 7D shows that the floc-
(mg/L) time (mg/L) MCLR (%)
culation capacity of MBF-16 onto MCLR enhanced from 0 to 90.05% (min)
when the reaction time increased from 0 to 60 min. However, the floc-
MBF-16a 3 80 60 10 90.5
culation efficiency maintained constant eventually. These results sug- MBF-16b 3 50 60 10 92.0
gest that a flocculation equilibrium could be achieved within 60 min. PAM 7 300 30 100 59.5
The high rate of MCLR uptake at the beginning was due to the initial PAC 8 200 30 100 30.8
availability of the large accessible surface area of MBF-16 to MCLR. Af- a
MBF-16 produced from CPW.
terwards, the bare MBF-16 surface decreased rapidly with the b
MBF-16 produced from the seed fermentation medium.
8 X. Qi et al. / Science of the Total Environment 715 (2020) 136885
Table 3
The comparisons of different bioflocculants.
Microorganism Dosage used for kaolin removal (mg/L)
Stenotrophomonas maltophilia 10
Bacillus licheniformis 5.8
Paenibacillus mucilaginosus 4 97%
Bacillus megaterium TF10 30.2
Bacillus mojavensis 32A 10
Alcaligenes faecalis subsp. phenolicus ZY-16 2 95.4%
suspended and dissolved particles. To determine the potential of MBF-
16, it was compared to other conventional chemical flocculants: non-
ionic polyacrylamide (PAM) and polyaluminum chloride (PAC), at
their optimal conditions for the removal of MCLR. Table 2 illustrates
that MBF-16 performed much better than PAM and PAC, especially for
a low concentration of MCLR. Therefore, it is a perfect new flocculant
to remove MCLR. While the detailed chemical mechanisms through
which MBF-16 interacts with MCLR are not understood, many primary
studies have been carried out for developing a simple and useful
method for the removal of MCLR in water. Further research on the
exact mechanisms for the interaction of MCLR with MBF-16 may facili-
tate the engineering of reactive radicals to generate potential new poly-
saccharide variants.
Table 3 provides the comparison of exopolysaccharide-based
bioflocculants yields by different strains. The flocculation efficiency of
MBF-16 is higher than the other reported bioflocculants. For instance,
2.0 mg/L MBF-16 could obtain 95.4% flocculation efficiency, adversely,
30.2 mg/L Bacillus megaterium TF10 gained 95.0% flocculation efficiency.
4. Conclusions
Alcaligenes faecalis subsp. phenolicus ZY-16 can secrete various
biomass- degrading enzymes, thereby converting CPW into MBF-16.
The limonene in CPW was demonstrated to inhibit MBF-16 production
and could be removed by molecular distillation at a wiper rolling speed
of 120 r/min. Under these conditions, the limonene removal was 86.9%,
increasing the MBF-16 yield from 1.06 to 3.04 g/L. Using central com-
posite design optimisation, the MBF-16 yield could be further promoted
to 3.49 g/L by adjusting the initial pH to 4.48, CPW concentration to
38.79 g/L, and fermentation temperature to 35.54 °C. Furthermore,
MBF-16 showed higher efficiency in precipitating MCLR than the con-
ventional chemical flocculants.
This research reported for the first time that MBF-16 was produced
from CPW and effectively applied in the removal of MCLR from water.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgement
This work was supported by the National Natural Science Founda-
tion of China (31700110) and Foundation for Innovative Research
Group of Hubei Educational Office (T201619).
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