Bioresource Technology 288 (2019) 121505

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Bioconversion of kitchen wastes into bioflocculant and its pilot-scale T

application in treating iron mineral processing wastewater
Weijie Liua,1, Zhen Donga,1, Di Suna, Ying Chena, Shiwei Wangb, Jingrong Zhua, Cong Liua,
⁎

a
School of Life Science, Jiangsu Normal University, Xuzhou 221116, Jiangsu Province, China
b
Key Laboratory of Resources Biology and Biotechnology in Western China, Ministry of Education, College of Life Science, Northwest University, Xian 710069, Shanxi
Province, China

ARTICLE INFO ABSTRACT

Keywords: In this study, the feasibility of converting kitchen waste into bioflocculant using Bacillus agaradhaerens C9 was
Kitchen waste analyzed. The result showed that strain C9 could secrete various degrading enzymes, including amylase, pro-
Bioflocculant tease, lipase, cellulase, xylanase and pectinase, promoting the hydrolysis of kitchen waste. Strong alkaline fer-
Mineral processing wastewater mentation condition was able to induce the bioflocculant production, and inhibit the growth of contaminated
Bacillus agaradhaerens
bacteria, which avoids the sterilization process of kitchen waste. The optimum fermentation condition for en-
zymatic hydrolysis and bioflocculant production was 40 g/L kitchen waste, 37 °C, pH 9.5, and the highest bio-
flocculant yield of 6.92 g/L was achieved. Furthermore, bioflocculant was applied to treat pilot-scale (30 L) of
mineral processing wastewater for the first time, and the removal rate of 92.35% was observed when 9 mg/L
bioflocculant was added into wastewater. Therefore, this study could promote the resource utilization of kitchen
waste and recycling of mineral processing wastewater.

1. Introduction
Kitchen waste is usually generated at the customer level, such as
restaurants, households and canteens of school and factory (Jiang et al.,
2018; Karthikeyan et al., 2018). In china, over 30 million tons of
kitchen wastes are produced per year, which cause serious environ-
mental pollution and resource waste if they are discharged without
proper treatment (Li et al., 2016; Wang et al., 2016). Kitchen waste is a
biodegradable organic substance (Ye et al., 2013), which mainly con-
tains starches, polysaccharides, proteins, celluloses, pectins and in-
organic salts. These substances can be used as adequate nutrients to
support the growth of microorganisms, thereby producing value-added
microbial metabolites (Li et al., 2017; Ntaikou et al., 2018). In recent
researches, kitchen wastes were used as substrate to produce bio-
surfactant (Chen et al., 2018), xanthan (Li et al., 2017), short-chain
fatty acid (Chen et al., 2013), lactic acid (Ohkouchi and Inoue, 2006),
cellulase (Bansal et al., 2012), glucoamylase (Wang et al., 2008), or
produce volatile fatty acids (Wang et al., 2014), methane (Campuzano
and González-Martínez, 2016), ethanol (Hafid et al., 2017; Ntaikou
et al., 2018) and hydrogen (Karthikeyan et al., 2018; Srivastava et al.,
2017) through anaerobic digestion. However, the amount of kitchen
waste available far exceeds the requirement for resource utilization.
Therefore, it is urgent to develop new ways to achieve the resource
utilization of kitchen waste.
The mining and ore washing processes discharged a huge amount of
mineral processing wastewater, which accounts for 10% of the total
industrial wastewater in the world (Park et al., 2016). The mineral
processing wastewater poses a serious threat to the environment due to
its high concentration of solid suspended particles and various heavy
metal ions, such as Cd, Zn, Pb and Cu (Benavente et al., 2011; Hu et al.,
2014). The released heavy metal ions seriously inhibit the growth of
crops in the surrounding environment, and enter the human body along
with the food chain, threating the human health. Furthermore, most
mining areas lack water resource (Iakovleva and Sillanpaa, 2013; Park
et al., 2016), the recycling of mineral mining wastewater is necessary
for environmental protection and water saving.
Flocculation is an economic and effective strategy to treat the mi-
neral mining wastewater (Chekli et al., 2015; Park et al., 2016). Bio-
flocculant are environment-friendly macromolecular polymers pro-
duced by microorganisms (Pathak et al., 2017; Liu et al., 2010).
Compared with traditional inorganic and organic synthetic flocculants,
bioflocculant exhibits the advantages of high flocculation efficiency,

Corresponding author at: School of Life Science, Jiangsu Normal University, No. 101, Shanghai Road, Tongshan District, Xuzhou 221116, Jiangsu Province,
⁎

China.
E-mail address: liucong0426@126.com (C. Liu).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.biortech.2019.121505
Received 3 April 2019; Received in revised form 14 May 2019; Accepted 17 May 2019
Available online 18 May 2019
0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
W. Liu, et al.
non-toxicity, harmlessness and biodegradability (Aljuboori et al.,
2015). More importantly, chemical flocculants are sensitive to the pH
change of wastewater (Liu et al., 2016). Thus, bioflocculant is an ideal
choice for the treatment of mineral mining wastewater with large
fluctuation of pH value. In previous studies, bioflocculant has been
widely applied in harvest of microalgae (Liu et al., 2015b;
Ndikubwimana et al., 2016), dewatering of activated sludge (Guo et al.,
2017) and the treatment of wastewater, such as decolorization of dying
wastewater (Liu et al., 2009), adsorption of heavy metal wastewater
(Fan et al., 2019) and the settlement of solid suspended particles in ash-
flushing wastewater (Liu et al., 2016). However, bioflocculant only
occupy a lower market share, mainly due to its high production cost
caused by fermentation medium (Cao et al., 2015; Guo et al., 2015).
Over the past decade, efforts have been made to cheaply produce bio-
flocculant and expand its application fields. Various wastewaters with
high concentration of organic matter were used as alternative carbon
sources of fermentation medium, such as potato starch processing
wastewater (Pu et al., 2014), dairy wastewater (Wang et al., 2007), and
palm oil processing wastewater (Aljuboori et al., 2014). Agricultural
wastes (Liu et al., 2017; Liu et al., 2015b) and their thermo-acid hy-
drolysates (Wang et al., 2013) have also been used as inexpensive
carbon sources for bioflocculant production. However, in addition to
these alternative carbon sources, nitrogen sources and inorganic salts
are required to be supplemented to support the microbial fermentation.
Kitchen wastes are rich in carbon source, nitrogen source and inorganic
salts (Jiang et al., 2018), can be used as a complete fermentation
medium for the production of bioflocculant.
B. agaradhaerens C9 has been reported as a high-yield bioflocculant-
producing strain, and the strong alkaline condition (adding Na2CO3 to
adjust pH) can induce strain C9 to produce high-yield of bioflocculant
(Liu et al., 2015a). This study found that strain C9 was able to secrete
various degrading enzymes, including amylase, cellulase, xylanase,
protease, pectinase and lipase. So the feasibility of converting kitchen
waste into bioflocculant using strain C9 was analyzed. The results
showed that alkaline condition not only induces the production of
bioflocculant, but also realizes the pretreatment of kitchen waste. The
fermentation process integrates four processes: pretreatment of kitchen
waste, enzyme production, enzymatic hydrolysis of kitchen waste and
bioflocculant production, thereby improving the bioconversion effi-
ciency of kitchen waste into bioflocculant. Moreover, the sterilization of
kitchen waste can be avoided due to the strong alkaline fermentation
condition can effectively inhibit the growth of contaminated bacteria in
kitchen waste. In addition, bioflocculant was applied for the first time
in the treatment of mineral processing wastewater. Thus, this study
could promote the resource utilization of kitchen waste and the re-
cycling of mineral processing wastewater.
2. Materials and methods
2.1. Strain and culture conditions
The bioflocculant-producing strain B. agaradhaerens C9 was isolated
in a previous study (Liu et al., 2015a) and preserved in the China
General Microbiological Culture Collection Center (CGMCC, Beijing,
China) with a deposition number of CGMCC13057. B. agaradhaerens C9
was mixed with 25% glycerol and stored in a −80 °C freezer. The strain
C9 was inoculated into 100 mL flask with 25 mL seed medium which
contained 10 g/L glucose, 10 g/L yeast extract, 1.3 g/L K2HPO4, 0.2 g/L
MgSO4·7H2O, and supplemented with 10 g/L Na2CO3. After 12 h in-
cubation at 180 rpm and 37 °C, the seed culture was obtained and used
for further experiments. To verify the activity of degradation enzymes
secreted by B. agaradhaerens C9, 10 μL of seed culture was dropped onto
various test agar plates which contained 2 g/L yeast extract, 3 g/L
peptone, 1.2 g/L K2HPO4, 0.2 g/L MgSO4·7H2O, 15 g/L agar, 10 g/L
Na2CO3 (added before pouring plate), and supplemented with 10 g/L of
CMC-Na for cellulase assay, or 10 g/L xylan for xylanase assay, or 10 g/
2
Bioresource Technology 288 (2019) 121505
L starch for amylase assay, or 10 g/L pectin for pectinase assay, or 10 g/
L skimmed milk powder for protease assay, or supplemented with 1%
soybean oil and 1 mg/L neutral red for lipase assay. After incubation at
37 °C for 48 h, the cellulase and xylanase were analyzed by observing
hydrolytic zones on the plates by staining with 0.3% Congo red solution
for 30 min and destaining with 0.9% NaCl for 30 min; the clear zones on
the plates for amylase and pectinase test were observed after flooding
the plates with iodine solution (dissolved 1 g iodine and 2 g potassium
iodide into 300 mL deionized water) for 5 min and washing with
deionized water for 1 min; the protease and lipase were verified by
directly detecting hydrolytic zones on the plates and red spot of the
colony, respectively.
2.2. Enzyme activity assay
The obtained seed culture was inoculated into the enzyme activity
analysis medium which contained 10 g/L glucose, 10 g/L yeast extract,
1.3 g/L K2HPO4, 0.2 g/L MgSO4·7H2O, and 10 g/L Na2CO3. After cul-
ture at 37 °C for 24 h, the fermentation broth was collected and cen-
trifuged at 4 °C, 12000 rpm for 10 min to remove the cells. The enzyme
activities of amylase, protease, pectinase, cellulase, xylanase and lipase
in the supernatant were measured at different pH values (5.0–11.0 with
pH intervals of 0.5) and different temperatures (30, 37, 40, 50, 60, 70,
80, 90 and 100 °C) to determine the optimal pH and temperature range
of enzymes. The substrates used for the assay of amylase, protease,
pectinase, cellulase, xylanase and lipase were dissolved in 0.25 M
phosphate buffer (pH 5.0–8.0) and 0.25 M NaHCO3-Na2CO3 buffer (pH
8.0–11.0).
2.2.1. Determination of amylase, cellulase, xylanase and pectinase
1% (w/v) of soluble starch (Sinopharm), sodium carboxymethyl
cellulose (Sinopharm), xylan (Sigma) and pectin (Solarbio) dissolved
into phosphate buffer and NaHCO3-Na2CO3 buffer were respectively
used as substrates for amylase, cellulase, xylanase and pectinase assay.
The reaction systems contained 150 μL of substrate solution and 50 μL
of broth supernatant were reacted for 30 min, followed by the addition
of 100 μL of 1 M NaOH and 150 μL dinitrosalicylic acid (DNS) to stop
the enzyme reaction. After boiling for 5 min, 550 μL of deionized water
was added into the mixture, and the OD540 was measured using a
spectrophotometer (Unic-7200, China). The enzyme solution in-
activated by autoclaved sterilization at 121 °C for 20 min was set as a
control. One unit of enzyme activity was defined as the amount of
enzyme that required to release 1 μM reducing sugar (for amylase,
cellulase and xylanase) or produce 1 μM galacturonic acid (for pecti-
nase) per minute under the assay conditions.
2.2.2. Protease assay
For determination of protease, 300 μL of 0.8% casein solution and
100 μL of the broth supernatant were mixed well and incubated at 37 °C
for 30 min. Then, 400 μL 0.4 M trichloroacetic acid (TCA) was added to
stop the enzymatic reaction. After centrifugation at 12000 rpm for
5 min, 400 μL of the supernatant, 500 μL of 0.4 M sodium carbonate and
100 μL of Folin-Phenol were mixed and incubated at 40 °C for 20 min.
The absorbance was determined at 680 nm using a Unic-7200 spec-
trophotometer. The enzyme solution inactivated by the TCA treatment
was used as a control. One unit of protease activity was defined as the
amount of enzyme that produced 1 μM tyrosine per minute.
2.2.3. Lipase assay
p-Nitrophenol palmitate (p-NPP, Aladdin) dissolved in isopropanol
was used as a substrate for lipase assay. The reaction system consisted
of 100 μL of 0.1 mM p-NPP solution, 100 μL of the broth supernatant
and 800 μL of buffer solution was reacted at 37 °C for 30 min, followed
by boiling 5 min to stop the reaction. After adding 250 μL of 0.1 M
Na2CO3, the mixture was centrifuged at 12000 rpm for 5 min. Then the
OD410 was measured with a Unic-7200 spectrophotometer. The enzyme
W. Liu, et al.
solution inactivated by autoclaved sterilization at 121 °C for 20 min was
set as a control. One unit of lipase activity was defined as the amount of
enzyme that released1 µM p-nitrophenol (p-NP) per minute.
2.3. Production and extraction of bioflocculant from kitchen wastes
The kitchen wastes were sampled from five school dining rooms and
a restaurant of Xuzhou city in Jiangsu Province. The compositions of
kitchen wastes were determined by Qingdao Hengxin Testing
Technique Service Company according to the national food safety
standards of china. After crushing using a homogenizer (PY-790,
HanJiaOurs, Zhongshan, China), six kitchen waste samples were ad-
justed to a concentration of 40 g/L (dry weight concentration) using
deionized water, and then sterilized at 115 °C for 30 min. The 100-mL
flasks which contained 25 mL of 40 g/L kitchen waste were inoculated
with 0.25 mL seed culture of B. agaradhaerens C9 and supplemented
with 10 g/L Na2CO3 before the fermentation. After incubation at
180 rpm and 37 °C for 12, 24, 36 and 48 h, the flocculating activity and
bioflocculant yield were determined. The flocculating activity assay
was performed according to a previous study (Liu et al., 2015a). To
extract the bioflocculant, the fermentation broths of six kitchen wastes
were centrifuged at 12,000 rpm, 4 °C for 10 min to remove the cells and
the residues of kitchen waste. The bioflocculant was precipitated from
the obtained supernatant by adding two volumes of pre-cooled ethanol,
and collected by centrifugation at 10000 rpm, 4 °C for 5 min, followed
by washing twice with 75% ethanol and lyophilized to obtain bio-
flocculant product (named as BF-KW, means Bioflocculant from Kitchen
Waste).
In order to analyze whether bioflocculant can be produced using
unsterilized kitchen waste, 40 g/L kitchen waste without autoclaving
treatment was directly used as fermentation medium, which floccu-
lating activity and bioflocculant yield were compared with those of the
sterilized kitchen waste.
2.4. Growth curve of B. agaradhaerens C9 in kitchen waste medium
The seed culture of B. agaradhaerens C9 was inoculated into 100-mL
flasks with 25 mL 40 g/L kitchen waste fermentation medium. Three
bottles were taken out from shaker at different time intervals, and the
cell growth was monitored by colony-counting method; the flocculating
activity of fermentation broth was determined according to a previous
study (Liu et al., 2015a); the bioflocculant yield was measured by
ethanol precipitation method; and the activity of amylase, protease,
pectinase, cellulase, xylanase and lipase in the fermentation medium
was analyzed according to the methods mentioned above.
2.5. The application of BF-KW in iron mineral processing wastewater
The iron mineral processing wastewater was sampled from iron ore
processing plant of Xuzhou in Jiangsu province, China, which con-
taining 22.86 g/L solid suspended particles and pH 7.96. In order to
analyze the application of bioflocculant BF-KW in wastewater treat-
ment, BF-KW was added into 60 mL and 30 L of iron mineral processing
wastewater to different concentrations. Then the solution was stirred
with a rapid mixing for 10 min, followed by a slow mixing for 2 min.
After settlement for 10 min, the absorbance at 550 nm of the super-
natant was measured by a spectrophotometer (Unic-7200). The floc-
culating activity was calculated according to the following equation:
FR = [X − Y]/X × 100%, where FR is the flocculating rate of iron
mineral processing wastewater; Y represents the turbidity (OD550) of
the supernatant of the flocculated wastewater and X represents the
turbidity of the control without addition of bioflocculant. To test the
effect of metal ions on the flocculating activity of iron mineral pro-
cessing wastewater, 1 mL 0.5% metal ions solution was added into
60 mL wastewater with 9 mg/L BF-KW, and then the flocculating ac-
tivity was determined.
3
Bioresource Technology 288 (2019) 121505
3. Results and discussion
3.1. The fermentation medium and enzyme production characteristics of B.
agaradhaerens C9
Kitchen wastes are rich in nutrients, mainly containing starch,
protein and lipid, in addition to a small amount of cellulosic materials,
pectin and salts (Lau et al., 2014), thus it shows a good potential to be
converted into other value-added products by microbial fermentation,
such as biosurfactant (Chen et al., 2018), xanthan (Li et al., 2017),
lactic acid (Ohkouchi and Inoue, 2006). However, there is no report on
the production of bioflocculant using kitchen wastes as fermentation
medium. In a previous study, B. agaradhaerens C9 has been reported as
a bioflocculant-producing strain. In order to verify that whether B.
agaradhaerens C9 could convert kitchen wastes into bioflocculant, the
extracellular degradation enzymes secreted by B. agaradhaerens C9
were analyzed. The clear hydrolyzed circle presented separately on the
solid agar medium containing starch, skim milk, pectin, cellulose and
xylan, indicating that B. agaradhaerens C9 could produce amylase,
protease, pectinase, cellulase and xylanase. In addition, the appearance
of red colony on the solid medium containing soybean oil indicated that
B. agaradhaerens C9 could produce lipase. Therefore, B. agaradhaerens
C9 showed a good potential to produce the bioflocculant by using en-
zymatic hydrolysates of kitchen wastes as nutrition.
3.2. The optimum pH and temperature of enzymes from strain C9 and
stability assay
In order to efficiently produce bioflocculant from kitchen wastes, it
is very important to determine an optimal condition in which most
degrading enzymes secreted by B. agaradhaerens C9 can exhibit rela-
tively high enzyme activity. Thus, the optimum pH range and tem-
perature range of different enzymes were analyzed. As shown in
Fig. 1A, the optimum pH ranges for amylase, protease, pectinase, cel-
lulase, xylanase and lipase, were 8.5–10.0, 9.5–10.5, 9.5, 5.0–5.5,
7.5–9.0, 8.0–9.5, respectively. Considering the high content of starch,
protein and lipid in kitchen waste, the pH value of 9.5 was selected as
fermentation condition, in which amylase, protease, pectinase, xylanase
and lipase can achieve relatively high enzyme activities. More im-
portant, the production of bioflocculant can be induced by strong al-
kaline condition (pH 9.5–10.0 with Na2CO3 as buffer) (Liu et al.,
2015a). Although the optimum pH range for cellulase from B. agar-
adhaerens C9 is weak acidic condition, more than 70% of its maximum
enzyme activity was observed around pH 9.5. This result is consistent
with a previous report (Liu et al., 2017). Furthermore, the pH stabilities
of different degrading enzymes were analyzed (Fig. 2A). Amylase, cel-
lulase and lipase maintained stable enzyme activity after incubation at
pH 9.0–9.5 for 30 min. Although protease, pectinase and xylanase
showed instability in the alkaline condition, more than 50% enzyme
activities were observed after the treatment at pH 9.0–9.5 for 30 min.
The effects of temperature on the activity of different enzymes were
shown in Fig. 1B. It can be seen that the optimum temperature of
amylase, protease, cellulase and xylanase from B. agaradhaerens C9 was
60 °C, while the highest activity of pectinase and lipase was observed at
40 and 37 °C, respectively. The temperature stabilities of different en-
zymes were also analyzed. As shown in Fig. 2B, amylase, protease,
cellulase and lipase were unstable at 60 °C; and all the test enzymes
showed relatively high stability at 37 °C, which is beneficial to the ac-
cumulation of enzyme activity. Moreover, high temperature inhibited
the flocculating activity and production of bioflocculant, and the
highest flocculating activity and yield were achieved at 37 °C. Com-
prehensively considering the energy consumption and bioflocculant
production, the temperature of 37 °C was selected as fermentation
temperature.
W. Liu, et al.
Fig. 1. The optimum pH (A) and temperature (B) of different enzymes were
determined. The substrates used for the assay of amylase, protease, pectinase,
cellulase, xylanase and lipase were dissolved in 0.25 M phosphate buffer (pH
5.0–8.0) or 0.25 M NaHCO3-Na2CO3 buffer (pH 8.0–11.0).
Fig. 2. The pH stability (A) and temperature stability (B) of different enzymes
produced from B. agaradhaerens C9. The enzyme activities were determined
after incubation at different pH values or temperatures for 30 min.
4
Bioresource Technology 288 (2019) 121505
3.3. Applicability of B. agaradhaerens C9 in converting kitchen wastes into
bioflocculant
The composition of kitchen waste varies significantly along with its
different sources. In order to analyze the universal applicability of B.
agaradhaerens C9 for converting different kitchen wastes into bio-
flocculant, six kitchen waste samples were collected from different
canteens and a restaurant of Jiangsu Normal University and Kewen
college. The composition analysis found that six kitchen waste samples
contained 13.1–14.1% total solid for wet basis; and contained
33.1–42.8% starch, 1.8–5.3% protein, and 0.4–0.9% lipid for dry basis,
suggesting that six sampled kitchen wastes could support the cell
growth and bioflocculant production. As shown in Fig. 3A, all the fer-
mentation broth using six selected kitchen wastes as nutrition showed
more than 90% of flocculating activity. The highest flocculating activity
of 96.27% to kaolin solution was observed when the restaurant kitchen
waste was used as medium after 24 h fermentation. As shown in Fig. 3B,
the yield of bioflocculant increased gradually with the extension of the
culture time within 48 h, and 1.51–3.38 g/L bioflocculant was
achieved, indicating that B. agaradhaerens C9 was able to utilize various
kitchen wastes from different sources to produce bioflocculant. Con-
sidering that the highest flocculating activity and relatively high bio-
flocculant yield, the kitchen wastes sampled from the restaurant was
used in following experiments.
Furthermore, the effects of kitchen waste concentrations on the
production of bioflocculant were investigated. As shown in Fig. 3C, the
yield of bioflocculant was enhanced with the increase of kitchen waste
concentration. The highest flocculating activity and the highest yield
were achieved when 40 g/L restaurant kitchen waste was used as fer-
mentation medium. Further increase of kitchen waste concentration
inhibited the production of bioflocculant, which may be due to the
reduction of the efficiency of oxygen supply and the extraction effi-
ciency of bioflocculant.
3.4. Growth curve of B. agaradhaerens C9 in restaurant kitchen waste
The time curves of cell growth, six enzyme activities and bio-
flocculant production were monitored when 40 g/L restaurant kitchen
waste was used as fermentation medium (Fig. 4). As shown in Fig. 4A,
the cell growth entered exponential phase from 6 h; the flocculating
activity and bioflocculant yield increased sharply and showed a good
correlation with the cell growth in exponential phase. The cell growth
declined after 24 h culture; however, the bioflocculant yield further
increased to 6.92 g/L of 48 h. Then the yield decreased gradually,
which may be due to the hydrolysis effect of degradation enzymes re-
leased during the cell lysis. Therefore, the fermentation time of 48 h
was selected to extract the bioflocculant product. As shown in Fig. 4B,
in first 9 h, most tested enzymes decreased slightly, which may be
caused by instability of enzyme, and then all the enzyme activities in-
creased obviously to a high level with the cell growth, thereby pro-
moting the conversion of kitchen wastes into bioflocculant. Further-
more, the maximum enzyme activities measured in fermentation
medium with kitchen wastes as nutrition were higher than those in
enzyme activity analysis medium, indicating that the kitchen waste
could induce the secretion of degradation enzymes.
3.5. Production of bioflocculant from the unsterilized kitchen waste
Sterilization process of kitchen waste increases the production cost
of bioflocculant and the denaturation of nutrients in the kitchen waste
caused by high sterilization temperature inhibits the production of
bioflocculant. B. agaradhaerens C9 used in this study is an alkali-re-
sistant strain and the alkaline condition can induce the secretion of
bioflocculant (Liu et al., 2015a). The strong alkaline condition (pH 9.5)
can inhibit the growth of natural bacteria in the kitchen waste and
promote B. agaradhaerens C9 to become a dominant strain. The result
W. Liu, et al.
Fig. 3. The flocculating activities (A) and yields (B) of bioflocculant produced from six different kitchen wastes. And the effects of concentrations of restaurant
kitchen waste on the flocculating activity and yield of BF-KW (C).
showed that the flocculating activity of 95.57% to kaolin clay solution
was achieved when the unsterilized kitchen waste was used as nutri-
tion, suggesting that B. agaradhaerens C9 could produce bioflocculant
through directly utilizing unsterilized kitchen waste, and thereby re-
ducing the equipment input and production cost.
5
Bioresource Technology 288 (2019) 121505
3.6. The application of BF-KW in treating iron mineral processing
wastewater
Iron mineral processing wastewater is generally produced in the
mining and ore washing processes. The main pollutants in mineral
processing wastewater are solid suspended particles, such as mineral
W. Liu, et al.
Fig. 4. Time curves of flocculating activity, yield (A) and different enzyme activities (B) produced from B. agaradhaerens C9 during cell growth in fermentation
medium containing 40 g/L kitchen wastes and supplemented with 10 g/L Na2CO3.
clay particles. Its direct discharge leads to the contamination of the
surrounding environment. Therefore, the removal of solid suspended
particles is necessary for reducing environmental pollution and pro-
moting the recycling of iron mineral processing wastewater. Thus, the
flocculating rate of solid suspended particles in iron mineral processing
wastewater was mainly determined. In 60 mL of wastewater system, the
flocculating activity enhanced with the increase of BF-KW concentra-
tion, and the highest flocculating activity was achieved when 9 mg/L
BF-KW was added into iron mineral processing wastewater. Then fur-
ther increase of BF-KW concentration led to decreased removal effi-
ciency. This phenomenon can be explained by the insufficient bridging
effect when BF-KW concentration was lower than 9 mg/L; when BF-KW
concentration more than 9 mg/L, the decreased flocculating efficiency
is caused by the repulsion effect between solid suspended particles with
excessive negative charged bioflocculant. Furthermore, divalent metal
ions, Ca2+, Zn2+ and Mg2+, were found to be able to improve the
flocculating efficiency of BF-KW, also suggesting that bridging phe-
nomenon mediated by divalent metal ion plays a key role in the floc-
culating process. In addition, 30 L of mineral processing wastewater
was flocculated using 9 mg/L BF-KW. The flocculating rate of 92.35%
was achieved for 30 L wastewater without adding additional metal ions,
and the pH of wastewater was slightly changed to 7.72. Thus, this study
demonstrated a good potential of BF-KW in the treatment and recycling
of iron mineral processing wastewater.
4. Conclusions
B. agaradhaerens C9 can secrete various degrading enzymes, thereby
converting kitchen wastes into bioflocculant BF-KW. The strong
6
Bioresource Technology 288 (2019) 121505
alkaline fermentation condition can inhibit the growth of contamina-
tion bacteria, thereby promoting the production of bioflocculant using
unsterilized kitchen waste. The highest yield of 6.92 g/L bioflocculant
was obtained using 40 g/L kitchen waste as nutrition medium at 37 °C
and pH 9.5. Furthermore, BF-KW exhibited a good flocculating effi-
ciency of 92.35% to 30 L iron mineral processing wastewater. This
study reported for the first time that bioflocculant was produced from
kitchen wastes and effectively applied in the recycling of iron mineral
processing wastewater.
Declaration of Competing Interest
The authors declare no competing interest.
Acknowledgments
This study is supported by the National Natural Science Foundation
of China (31300054, 31800020), Six Talent Peaks Project of Jiangsu
Province (JNHB-103), Qing Lan Project of Jiangsu Province of China,
Natural Science Foundation of Jiangsu Province of China
(BK20171163, BK20181009), Agriculture Research System of China
(CARS-10-B03) and Priority Academic Program Development of
Jiangsu Higher Education Institutions (PAPD).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.biortech.2019.121505.
W. Liu, et al.
References
Aljuboori, A.H.R., Idris, A., Al-joubory, H.H.R., Uemura, Y., Ibn Abubakar, B.S.U., 2015.
Flocculation behavior and mechanism of bioflocculant produced by Aspergillus flavus.
J. Environ. Manage. 150, 466–471.
Aljuboori, A.H.R., Uemura, Y., Osman, N.B., Yusup, S., 2014. Production of a bio-
flocculant from Aspergillus niger using palm oil mill effluent as carbon source.
Bioresour. Technol. 171, 66–70.
Bansal, N., Tewari, R., Soni, R., Soni, S.K., 2012. Production of cellulases from Aspergillus
niger NS-2 in solid state fermentation on agricultural and kitchen waste residues.
Waste Manage. 32, 1341–1346.
Benavente, M., Moreno, L., Martinez, J., 2011. Sorption of heavy metals from gold mining
wastewater using chitosan. J. Taiwan Inst. Chem. Eng. 42, 976–988.
Campuzano, R., González-Martínez, S., 2016. Characteristics of the organic fraction of
municipal solid waste and methane production: a review. Waste Manage. 54, 3–12.
Cao, G., Zhang, Y., Chen, L., Liu, J., Mao, K., Li, K., Zhou, J., 2015. Production of a
bioflocculant from methanol wastewater and its application in arsenite removal.
Chemosphere 141, 274–281.
Chekli, L., Galloux, J., Zhao, Y.X., Gao, B.Y., Shon, H.K., 2015. Coagulation performance
and floc characteristics of polytitanium tetrachloride and titanium tetrachloride
compared with ferric chloride for coal mining wastewater treatment. Sep. Purif.
Technol. 152, 94–100.
Chen, C., Sun, N., Li, D., Long, S., Tang, X., Xiao, G., Wang, L., 2018. Optimization and
characterization of biosurfactant production from kitchen waste oil using
Pseudomonas aeruginosa. Environ. Sci. Pollut. Res. Int. 25, 14934–14943.
Chen, Y., Luo, J., Yan, Y., Feng, L., 2013. Enhanced production of short-chain fatty acid by
co-fermentation of waste activated sludge and kitchen waste under alkaline condi-
tions and its application to microbial fuel cells. Appl. Energy 102, 1197–1204.
Fan, H.C., Yu, J., Chen, R.P., Yu, L., 2019. Preparation of a bioflocculant by using acet-
onitrile as sole nitrogen source and its application in heavy metals removal. J.
Hazard. Mater. 363, 242–247.
Guo, J., Du, J., Chen, P., Tan, X., Huang, X., Gan, P., Fu, L., 2017. Enhanced efficiencies of
sludge dewatering and domestic wastewater treatment by using the bioflocculant
from rice stover. Water Environ. J. 31, 120–126.
Guo, J., Lau, A.K., Zhang, Y., Zhao, J., 2015. Characterization and flocculation me-
chanism of a bioflocculant from potato starch wastewater. Appl. Microbiol.
Biotechnol. 99, 5855–5861.
Hafid, H.S., Rahman, N.A.A., Shah, U.K.M., Baharuddin, A.S., Ariff, A.B., 2017. Feasibility
of using kitchen waste as future substrate for bioethanol production: a review.
Renew. Sustain. Energy Rev. 74, 671–686.
Hu, X.F., Jiang, Y., Shu, Y., Hu, X., Liu, L., Luo, F., 2014. Effects of mining wastewater
discharges on heavy metal pollution and soil enzyme activity of the paddy fields. J.
Geochem. Explor. 147, 139–150.
Iakovleva, E., Sillanpaa, M., 2013. The use of low-cost adsorbents for wastewater pur-
ification in mining industries. Environ. Sci. Pollut. Res. 20, 7878–7899.
Jiang, J., Li, L., Cui, M., Zhang, F., Liu, Y., Liu, Y., Long, J., Guo, Y., 2018. Anaerobic
digestion of kitchen waste: the effects of source, concentration, and temperature.
Biochem. Eng. J. 135, 91–97.
Karthikeyan, O.P., Trably, E., Mehariya, S., Bernet, N., Wong, J.W.C., Carrere, H., 2018.
Pretreatment of food waste for methane and hydrogen recovery: a review. Bioresour.
Technol. 249, 1025–1039.
Lau, K.Y., Pleissner, D., Lin, C.S.K., 2014. Recycling of food waste as nutrients in Chlorella
vulgaris cultivation. Bioresour. Technol. 170, 144–151.
Li, P., Zeng, Y., Xie, Y., Li, X., Kang, Y., Wang, Y., Xie, T., Zhang, Y., 2017. Effect of
pretreatment on the enzymatic hydrolysis of kitchen waste for xanthan production.
Bioresour. Technol. 223, 84–90.
Li, Y., Jin, Y., Li, J., Li, H., Yu, Z., 2016. Effects of thermal pretreatment on the bio-
methane yield and hydrolysis rate of kitchen waste. Appl. Energy 172, 47–58.
Liu, C., Hao, Y., Jiang, J., Liu, W., 2017. Valorization of untreated rice bran towards
Bioresource Technology 288 (2019) 121505
bioflocculant using a lignocellulose-degrading strain and its use in microalgal bio-
mass harvest. Biotechnol. Biofuels 10, 90.
Liu, C., Wang, K., Jiang, J.H., Liu, W.J., Wang, J.Y., 2015a. A novel bioflocculant pro-
duced 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.
Liu, W., Hao, Y., Jiang, J., Zhu, A., Zhu, J., Dong, Z., 2016. Production of a bioflocculant
from Pseudomonas veronii L918 using the hydrolyzate of peanut hull and its appli-
cation in the treatment of ash-flushing wastewater generated from coal fired power
plant. Bioresour. Technol. 218, 318–325.
Liu, W., Zhao, C., Jiang, J., Lu, Q., Hao, Y., Wang, L., Liu, C., 2015b. Bioflocculant pro-
duction from untreated corn stover using Cellulosimicrobium cellulans L804 isolate and
its application to harvesting microalgae. Biotechnol. Biofuels 8, 170.
Liu, W.J., Wang, K., Li, B.Z., Yuan, H.L., Yang, J.S., 2010. Production and characterization
of an intracellular bioflocculant by Chryseobacterium daeguense W6 cultured in low
nutrition medium. Bioresour. Technol. 101, 1044–1048.
Liu, W.J., Yuan, H.L., Yang, J.S., Li, B.Z., 2009. Characterization of bioflocculants from
biologically aerated filter backwashed sludge and its application in dying wastewater
treatment. Bioresour. Technol. 100, 2629–2632.
Ndikubwimana, T., Zeng, X., Murwanashyaka, T., Manirafasha, E., He, N., Shao, W., Lu,
Y., 2016. Harvesting of freshwater microalgae with microbial bioflocculant: a pilot-
scale study. Biotechnol. Biofuels 9, 47.
Ntaikou, I., Menis, N., Alexandropoulou, M., Antonopoulou, G., Lyberatos, G., 2018.
Valorization of kitchen biowaste for ethanol production via simultaneous sacchar-
ification and fermentation using co-cultures of the yeasts Saccharomyces cerevisiae and
Pichia stipitis. Bioresour. Technol. 263, 75–83.
Ohkouchi, Y., Inoue, Y., 2006. Direct production of L (+)-lactic acid from starch and food
wastes using Lactobacillus manihotivorans LMG18011. Bioresour. Technol. 97,
1554–1562.
Park, J.H., Oh, C., Han, Y.S., Ji, S.W., 2016. Optimizing the addition of flocculants for
recycling mineral-processing wastewater. Geosyst. Eng. 19, 83–88.
Pathak, M., Sarma, H.K., Bhattacharyya, K.G., Subudhi, S., Bisht, V., Lal, B., Devi, A.,
2017. Characterization of a novel polymeric bioflocculant produced from bacterial
utilization of n-hexadecane and its application in removal of heavy metals. Front.
Microbiol. 8, 170.
Pu, S.Y., Qin, L.L., Che, J.P., Zhang, B.R., Xu, M., 2014. Preparation and application of a
novel bioflocculant by two strains of Rhizopus sp. using potato starch wastewater as
nutrilite. Bioresour. Technol. 162, 184–191.
Srivastava, S., Kumar, A., Pandey, A., Pandey, A., 2017. Intensification of hydrogen
production by B. licheniformis using kitchen waste as substrate. Int. J. Hydrogen
Energy 42, 21659–21666.
Wang, K., Yin, J., Shen, D.S., Li, N., 2014. Anaerobic digestion of food waste for volatile
fatty acids (VFAs) production with different types of inoculum: effect of pH.
Bioresour. Technol. 161, 395–401.
Wang, L., Ma, F., Lee, D.J., Wang, A., Ren, N., 2013. Bioflocculants from hydrolysates of
corn stover using isolated strain Ochrobactium ciceri W2. Bioresour. Technol. 145,
259–263.
Wang, Q.H., Wang, X.Q., Wang, X.M., Ma, H.Z., 2008. Glucoamylase production from
food waste by Aspergillus niger under submerged fermentation. Process Biochem. 43,
280–286.
Wang, S.G., Gong, W.X., Liu, X.W., Tian, L., Yue, Q.Y., Gao, B.Y., 2007. Production of a
novel bioflocculant by culture of Klebsiella mobilis using dairy wastewater. Biochem.
Eng. J. 36, 81–86.
Wang, Y., Zang, B., Li, G., Liu, Y., 2016. Evaluation the anaerobic hydrolysis acidification
stage of kitchen waste by pH regulation. Waste Manage. 53, 62–67.
Ye, J.Q., Li, D., Sun, Y.M., Wang, G.H., Yuan, Z.H., Zhen, F., Wang, Y., 2013. Improved
biogas production from rice straw by co-digestion with kitchen waste and pig
manure. Waste Manage. 33, 2653–2658.