Journal of Cleaner Production 323 (2021) 129195

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Journal of Cleaner Production

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

Biowaste-based biodegradable flocculants for clean and sustainable tailings

management in industrial mining and mineral processing
Yeling Zhu a, 1, Michael Chae a, Jie Wang a, Birendra Adhikari a, Paolo Mussone b, David
C. Bressler a, *
a
Department of Agricultural, Food & Nutritional Science, University of Alberta, 116 St. and 85 Ave., NW, Edmonton, Alberta, T6G 2P5, Canada
b
Applied BioNanotechnology Industrial Research Chair, Industry Solutions, Northern Alberta Institute of Technology, 10210 Princess Elizabeth Ave., NW, Edmonton,
Alberta, T5G 0Y2, Canada

A R T I C L E I N F O A B S T R A C T

Handling editor: Cecilia Maria Villas Bôas de Massive fluid tailings discharged from industrial mining and mineral processing operations pose numerous
Almeida economic and environmental challenges. To remove water and condense tailings into sediment, we developed a
novel bioflocculant from specified risk materials, a particular type of waste biomass from the animal rendering
Keywords: industry. By properly controlling key synthesis conditions, the bioflocculant successfully met industrial re­
Wastewater treatment
quirements for fluid tailings treatment, including enhanced short-term tailings settling and improved ultimate
Clean mining
water removal extent. Settling tests using real industrial fluid tailings showed that the synthesized bioflocculant
Clean mineral processing
Biodegradable enabled an initial settling rate 4 magnitudes higher than the control case and achieved 93.2% the dewatering
Specified risk materials efficiency of a commercial flocculant (hydrolyzed polyacrylamide; HPAM). In contrast to HPAM, our bio­
Land reclamation flocculant can achieve tailings settling without gypsum, a common processing aid that can hinder downstream
industrial operations. More importantly, growth tests using two common microorganisms (Saccharomyces cer­
evisiae and Escherichia coli) suggested that the bioflocculant can be easily degraded, indicating reduced negative
environmental impacts after its use. Taken together, this study demonstrates the great potential of implementing
a biodegradable bioflocculant from proteinaceous wastes for clean and sustainable management of mining and
mineral processing fluid tailings.

1. Introduction removal/recycling for clean and sustainable management of these

Industrial mining and mineral processing operations are generating
huge quantities of fluid tailings (Northey et al., 2016; Yan et al., 2019).
Due to the lack of efficient and economically-viable dewatering tech­
niques, billions of tonnes of fluid tailings are currently impounded in
tailings ponds worldwide and subject to time-consuming natural settling
(C. Wang et al., 2014; Y. Zhu et al., 2020). This practice not only makes
the entrapped water inaccessible, but also brings numerous concerns
about the economy and environment, such as the seepage of fluid tail­
ings to surface/ground water (Palmer et al., 2010) and the leakage of
toxic organics, e.g., polycyclic aromatic hydrocarbons and synthetic
surfactants (Gosselin et al., 2010; Redman et al., 2018). Therefore, green
and inexpensive dewatering techniques are urgently demanded to
simultaneously reduce the mobility of fluid tailings and achieve water
intractable tailings (Asr et al., 2019).
Aggregating suspended fine solids through flocculation has been
widely recognized as an effective strategy to densify fluid tailings
(Gosselin et al., 2010; Qi et al., 2018). A flocculant is able to bridge
multiple solid particles along its molecular chains. Such bridging leads
to the generation of large solid clusters with greater settling speed
(Gregory and Barany, 2011), which accelerates the separation of fluid
tailings into clear water that can be recycled and solid sediment that can
be stacked in post-mining areas. Unfortunately, since most commercial
flocculants, such as polyacrylamide (Alamgir et al., 2012), are derived
from petrochemicals (Vajihinejad et al., 2019), implementing such
synthetic-polymer flocculants intensifies the consumption of
non-renewable petroleum resources. In addition, certain
synthetic-polymer flocculants contain unreacted monomers that are

* Corresponding author.
E-mail addresses: yeling2@ualberta.ca (Y. Zhu), mchae@ualberta.ca (M. Chae), jie13@ualberta.ca (J. Wang), badhikar@ualberta.ca (B. Adhikari), pmussone@
nait.ca (P. Mussone), dbressle@ualberta.ca (D.C. Bressler).
1
Present Address: Department of Wood Science, University of British Columbia, 2900-2424 Main Mall, Vancouver, BC, Canada V6T 1Z4.

https://doi.org/10.1016/j.jclepro.2021.129195
Received 11 May 2021; Received in revised form 30 August 2021; Accepted 26 September 2021
Available online 27 September 2021
0959-6526/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Y. Zhu et al.
toxic (e.g., carcinogenic acrylamide monomers in commercial poly­
acrylamide products (Guezennec et al., 2015)), which could bring
long-term hazards to the environment.
A promising technological alternative toward clean and sustainable
industrial tailings management can be achieved by using flocculants
derived from biomass sources such as cellulose (Kemppainen et al.,
2016), proteins (Piazza et al., 2011), and alginate (Maruyama et al.,
2020), due to their renewability, availability, and inherent biodegrad­
ability (Vajihinejad et al., 2019). In particular, to create greater eco­
nomic and ecological benefits, specified risk materials (SRM), a type of
waste biomass produced in massive quantities by the animal rendering
industry and disposed of at a high cost, have been investigated as
feedstock for the production of bioflocculants. The feasibility of using
SRM-derived polymers as a bioflocculant has been demonstrated in
previous proof-of-concept studies, which demonstrated that unmodified
SRM-derived peptides could flocculate both synthetic fluid tailings as
well as industrial tailings (Y. Zhu, Gong, et al., 2021) and that cross­
linking of SRM-derived peptides with polyamidoamine epichlorohydrin
(PAE) led to better flocculation of synthetic fluid tailings (Y. Zhu, Chae,
et al., 2021).
In this study, three knowledge gaps will be bridged to push forward
the technology development for scale-up. Firstly, how synthesis factors
impact the product’s performance will be investigated to facilitate
production of bioflocculants that meet different tailings settling goals.
For instance, a bioflocculant that enables greater ultimate dewatering
efficiency is of great value for mining operations that are heavily
dependent on the amount of water that can be recycled, e.g., mining oil
sands industry; alternatively, a bioflocculant that facilitates rapid tail­
ings settling in a short time is important for facilities with limited tail­
ings processing capability. Secondly, based on the successful application
of peptides-PAE for the flocculation of synthetic tailings (Y. Zhu, Chae,
et al., 2021), this bioflocculant will be employed for tailings treatment of
industrial streams to validate its effectiveness, as it may behave differ­
ently than observed in synthetic process water. Thirdly, although the
SRM-derived peptides coming from cattle are biodegradable, it is
necessary to study whether the peptides-PAE bioflocculant can still
retain this characteristic.
Hence, this study focused on developing an SRM-derived bio­
flocculant through adjusting key synthesis factors, investigating its
performance in treating industrial fluid tailings, and studying the po­
tential of biodegradability. The Taguchi method (Antony and Kaye,
2000) was implemented to screen for the best condition tested with
reduced experimental trials, and two characteristic functions were used
to evaluate the settling of synthetic fluid tailings. Settling of industrial
tailings stream was carried out using the developed bioflocculant and
compared with a commercial synthetic flocculant. The biodegradability
of the bioflocculant was examined through degradation tests, quantified
by microbial growth experiments conducted using two common
microorganisms.
2. Experimental
2.1. Materials and chemicals
Rendered SRM, provided by a large rendering company in western
Canada, were handled following the Canadian Food Inspection Agency
(CFIA) recommended protocol for safe handling and disinfection
(Mekonnen et al., 2013). PAE resin with the chemical formula HO
(C13H24N3O3Cl)nH and molecular weight of the repeating unit of 305.8
g/mol, was purchased in the form of a 12.5 wt% aqueous solution from
Solenis, DE, USA, and stored at 4 ◦ C. According to the manufacturer’s
information, the resin has a molecular weight in the range of 750,
000–850,000 g/mol. PolyGloss-90 kaolinite was purchased from KaMin
Performance Minerals, GA, USA. CaCl2 (98%), KCl (99%), NaCl (99%),
Na2CO3 (99%), CH3OH (99.9%), hexane (99.9%), MP Biomedicals™
tryptone, BD Bacto™ tryptic soy broth (TSB; 1-liter medium contains
2
Journal of Cleaner Production 323 (2021) 129195
2.5 g glucose, 17 g pancreatic digest of casein, 3 g papaic digest of
soybean, 5 g sodium chloride, and 2.5 g dipotassium phosphate), Fish­
erbrand™ agar, and Whatman filter papers (diameter: 11 cm; pore size:
20–25 μm) were purchased from Fisher Scientific, PA, USA. According to
data obtained using a CHNS/O Analyser Series II (PerkinElmer Inc., MA,
USA), the tryptone contains a nitrogen content of 13.5 ± 0.1%.
MgCl2∙6H2O (99%) was purchased from EMD Millipore, MA, USA.
Calcium sulfate dihydrate (gypsum, 98%) was purchased from Acros
Organics, NJ, USA. Flopam A3338™, an anionic hydrolyzed poly­
acrylamide (HPAM) was purchased from SNF Canada Ltd., AB, Canada.
Industrial oil sands mature fine tailings (MFTs) were purchased from
InnoTech Alberta, AB, Canada, and were stored in a 4 ◦ C fridge.
2.2. Hydrolysis of SRM and recovery of peptides
Rendered SRM was treated using a CFIA approved thermal hydro­
lysis protocol for disinfection and peptide production; detailed proced­
ures were described elsewhere (Mekonnen et al., 2013). The protein
fragments (referred to as “peptides” hereafter) after hydrolysis were
recovered following a standard protocol detailed previously (Adhikari
et al., 2016) and stored in a 4 ◦ C fridge before use. The peptides contain a
nitrogen content of 15.3 ± 0.1% (C. Zhu, 2019).
2.3. Synthesis of peptides-PAE bioflocculant
For the synthesis of peptides-PAE materials, the reactants’ concen­
tration (on dry basis) in suspension, PAE% (with respect to the total
amount of peptides and PAE), reaction temperature, and reaction time
were varied at three levels using the Taguchi method as given in Table 1.
The selection of these factors, as well as their variance levels, were
determined based on previous studies (Adhikari et al., 2016, 2019) and
preliminary experimental trials. Particularly, the reactants’ concentra­
tion (Factor 1) was found to affect the gelation behaviors of product,
which is important to the structure of the final product; PAE% (Factor 2)
dominated the crosslinking behavior between SRM peptides and PAE;
reaction temperature and time (Factors 3 and 4) were critical in deter­
mining the extent of condensation between the two reactants. Synthesis
of the materials were then carried out systematically according to the
experimental design that was generated based on the Taguchi-L9
method, as detailed in Table 2. Specifically, peptides were diluted with
Milli-Q water to reach certain reactants’ concentrations (peptides and
PAE combined; Table 2). To maintain a thermostatic reaction, a coiled
condenser and an oil bath with a feed-back thermocouple were
deployed. After the specified temperature was reached, a predetermined
amount of PAE resin (Table 2) was added drop by drop. Mixing using a
magnetic bar at a speed of 300 rpm was implemented throughout the
experiment. After the reaction, the product was transferred to a 50 mL
centrifuge tube and cooled down in a − 20 ◦ C freezer. The concentration
of solid materials in the final products was quantified by drying 2.00 ±
0.05 g of vortexed suspension at 80 ◦ C until the weight became stable
over time, based on which all the products were diluted with Milli-Q
water to an identical concentration of 1.43 wt% as working suspensions.
2.4. Tailings settling
Bench-scale cylinder settling tests were performed to evaluate the
flocculation behaviors of the synthesized peptides-PAE materials
Table 1
Selected synthesis factors and their variation levels.
Synthesis factor Level 1 Level 2 Level 3
Reactants’ concentration (wt%) 8 10 12
PAE% (wt%; dry basis) 11.4 22.8 34.2
Temperature (◦ C) 60 75 90
Reaction time (h) 0.5 2 20
Y. Zhu et al. Journal of Cleaner Production 323 (2021) 129195

Table 2 synthesis factor was predicted based on the S/N analyses of the char­
Taguchi-L9 orthogonal array of experimental design. acteristic functions. New peptides-PAE materials were prepared
Experimental Reactants’ PAE Temperature Reaction accordingly and, to validate the Taguchi method, their performance on
group concentration proportion (◦ C) time (h) actual tailings settling was compared with the theoretical values. The
(wt%) (wt%; dry supernatant at the end of 72 h was collected and homogenized prior to
basis)
turbidity measurement using an Orion™ AQ4500 turbidimeter (Thermo
E1 8 11.4 60 0.5 Fisher Scientific, MA, USA).
E2 8 22.8 75 2 Industrial tailings, prepared by diluting the original MFTs with
E3 8 34.2 90 20
E4 10 11.4 75 20
synthetic process water at a 1:2 mass ratio to achieve a solid content of
E5 10 22.8 90 0.5 8.9 wt%, were tested for tailings settling. The select bioflocculant and its
E6 10 34.2 60 2 two reactants (SRM peptides and PAE) were tested at a dosage of 10 kg/
E7 12 11.4 90 2 tonne tailings solid equivalent, similar to the case of synthetic kaolinite
E8 12 22.8 60 20
tailings. HPAM was tested in parallel at the optimum dosage (i.e., 0.14
E9 12 34.2 75 0.5
kg/tonne) identified by Kaminsky and Gong (Kaminsky and Gong,
2018).
(Gumfekar and Soares, 2018; Vajihinejad et al., 2019). The synthetic
process water contained 28.3 ppm KCl, 545 ppm NaCl, 39.3 ppm MgCl2, 2.5. Biodegradation measurements
41.5 ppm CaCl2, 895 ppm NaHCO3, and 443 ppm Na2SO4, with pH
adjusted to 8.4 using 5 mol/L NaOH; detailed preparation of the syn­ Two microorganisms, Superstart™ Saccharomyces cerevisiae (Lalle­
thetic process water is described elsewhere by Y. Zhu, Chae, et al. mand Inc., WI, USA) and Invitrogen™ Escherichia coli (DH5α) (in the
(2021). Synthetic process water carrying 4 wt% kaolinite clays was used form of glycerol stock, Thermo Fisher Scientific, CA, USA), were selected
as model fluid tailings. Tailings settling was carried out by mixing 243 as the yeast and bacterial strain, respectively, for biodegradation tests. A
mL of synthetic kaolinite tailings with 7 mL flocculant working sus­ yeast glycerol stock was prepared according to J. Wang et al. (2020).
pensions (carrying flocculant at a dosage of 10 kg/tonne tailings solid in Both glycerol stocks were stored at − 80 ◦ C before use.
equivalent), after which the normalized mudline height, ̂ h (equals to the All test agar plates contained the following chemicals (final amount
actual mudline height divided by that of the whole tailings), was in 1 L of medium): 8 g glucose, 15 g agar, 6.78 g Na2HPO4, 3 g KH2PO4,
recorded at various timepoints over a period of 72 h. To obtain char­ 0.5 g NaCl, 0.24 g MgSO4, 11 mg CaCl2, 2.4 mg FeCl3⋅6H2O, 100 μg
acteristic functions, the normalized mudline height was fitted in CuSO4⋅5H2O, 50 μg MnSO4⋅5H2O, 50 μg ZnSO4⋅7H2O, 10 μg CoCl2, 7 μg
response to time using OriginPro 2017 (non-linear fitting) with a hy­ H3BO3, and 10 μg NaMoO4. The above medium was autoclaved and
perbolic equation (Equation (1)) as the fitting equation (Y. Zhu et al., mixed with various nitrogen-bearing chemicals (final concentration): (a)
2018). none (negative control); (b) 2 g/L tryptone (positive control); (c) 1.76 g/
( ) L SRM-derived peptides (contained the same nitrogen amount as (b));
αt
̂
h= + 1 × 100% (1) and (d) 2.28 g/L peptides-PAE materials (contained the same peptides
β+t
amount as (b)). To prepare agar plates, the mixture was poured into
Fisherbrand™ petri dishes (100 mm × 15 mm in size, Fisher Scientific,
where, α (%) and β (min) are two fitting parameters. To identify a bio­
CA, USA) in a biosafety cabinet.
flocculant with enhanced tailings settling performance, two character­
For cultivation, microorganisms were first streaked onto TSB agar
istic functions were used to evaluate the settling behaviors of synthetic
plates (containing 30 g/L TSB and 15 g/L agar) and incubated for 48 h,
kaolinite tailings, i.e., v0 (normalized initial settling rate) and hULT (ul­
at 30 ◦ C for S. cerevisiae or 37 ◦ C for E. coli. A single colony isolate was
timate sediment height). These two characteristic functions are associ­
then selected and transferred to a 250 mL shaking flask containing 50
ated with the short-term tailings settling and the ultimate water removal
mL TSB medium (30 g/L TSB) and incubated in shakers (New Bruns­
extent, respectively, which are two important considerations for in­
wick™ Innova 44, Eppendorf Canada, ON, Canada) for 36 h at 200 rpm,
dustrial operators. These two characteristic functions can be calculated
30 ◦ C for S. cerevisiae (37 ◦ C for E. coli). The broth was then subject to
following Equations (2) and (3) (Y. Zhu et al., 2018).
serial dilution using sterile phosphate buffer (pre-autoclaved and con­
( )
dh α taining 6.78 g/L Na2HPO4, 3 g/L KH2PO4, and 0.5 g/L NaCl; pH:
v0 = − =− (2)
dt t=0 β 6.9–7.0) to prepare inoculum to make the final colony count falling
between 30 and 200 per plate. After this, 100 μL of the diluted culture (i.
hULT = lim h = 1 + α (3) e., 30–200 CFU) was applied to plates using a Fisherbrand™ L-shape
t→+∞
spreader (Fisher Scientific, PA, USA). The agar plates were then wrapped
In other words, v0 and hULT refer to the normalized settling rate at t = with Parafilm™ and incubated at 30 ◦ C for S. cerevisiae (37 ◦ C for E. coli).
0 and the thickness of sediment at t→ + ∞, respectively. The signal-to- Cell counts were performed for each plate every 24 h until 96 h and were
noise (S/N) was employed to indicate the extent of changes in the two carried out using 5 replicates. Both the TSB medium and phosphate
characteristic functions due to variations of the selected synthesis fac­ buffer were sterilized before use following J. Wang et al. (2017).
tors with respect to errors, as given in Equation (4) (Antony and Kaye,
2000).
2.6. Statistical analysis
S / N = − 10 log(MSD) (4)
All experiments were conducted using at least 3 replicates and results
∑n
(1/y2i ) were presented in the form of “mean value ± standard deviation”. The
where, MSD (mean square deviation) equals to i=1
n for the Larger-
∑n 2 statistical analyses of the data were carried out using Minitab (version
y
the-Better case (i.e., v0 ) and i=1
n
i
for the Smaller-the-Better case (i.e., 15), a statistical software package. Single factor Analysis of Variance
hULT ); n refers to number of observations; yi refers to the value of (ANOVA) was applied to the data populations examined to identify
characteristic function in each experimental trial. The best level for each significant differences among mean values, based on the Least Signifi­
cant Difference (LSD) criteria at a 95% confidence level.

3
Y. Zhu et al.
3. Results and discussion
3.1. Formulation optimization using synthetic kaolinite tailings
3.1.1. Taguchi method-based optimization
SRM feedstock was processed using a CFIA-approved thermal hy­
drolysis protocol to destroy any potential prions and to obtain peptides
for bioflocculant development. Syntheses of peptides-PAE materials
were carried out based on Taguchi-L9 design (Table 2) and were then
used for settling synthetic kaolinite tailings. According to the fitting
results of using the formulated peptides-PAE materials to settle the
synthetic kaolinite tailings (Tables S1 and S2) (Y. Zhu et al., 2018), the
results of v0 and hULT were obtained and presented in Fig. 1 as functions
of each individual formulation. Except for the blank reference where
tailings settling barely took place (Tables S1 and S2), a high coefficient
of determination (r2 > 0.992) was obtained from the fittings of all the
other groups, suggesting that the settling kinetics were well described by
the two characteristic functions. As shown in Fig. 1, all formulated
bioflocculants gave much greater v0 than the blank reference, which
recorded a v0 of (8.1 ± 1.3) × 10− 3%/min, suggesting that the
peptides-PAE materials triggered a rapid phase separation at the early
settling stages. Meanwhile, all hULT values observed in these experiments
were much smaller than the blank reference (94.1 ± 0.2%), indicating
that peptides-PAE materials facilitated a more complete water recovery.
Both trends suggested the advantages of using peptides-PAE bio­
flocculants for settling clay-enriched tailings, which was in line with a
previous study (Y. Zhu, Chae, et al., 2021). More importantly, a dramatic
difference in characteristic function values was observed among
different individual formulations (Fig. 1). For instance, E8 gave a v0
(76.8 ± 11.1%/min) that was ~2.5 folds of the E1 group (29.6 ±
1.2%/min); E3 indicated a much denser sediment (hULT of 11.3 ± 0.1%)
with the volume being only 61.7% of the E6 group (18.3 ± 0.1%). Such
variations demonstrated that the factors selected in this study do have a
significant effect on the desired tailings settling properties.
3.1.2. Analysis of the S/N ratios and the effect of select synthesis factors
S/N ratio represents the sensitivity of the select characteristic func­
tion to the noise factors, which reflects both the average and variation
trend of the quality characteristics in the Taguchi method (Antony and
Kaye, 2000). The obtained S/N ratios are presented in the form of main
effect plots in Fig. 2, to identify the best level of each synthesis factor for
bioflocculant development (Table 2). The variation of S/N ratio in
response to each synthesis factor was ranked in the order of relative
importance, with a complete dataset given in Table S3 and results
denoted in Fig. 2. Note that a greater v0 and a smaller hULT were
preferred in this analysis; hence, S/N ratios of v0 and hULT were studied
Fig. 1. v0 (panel a) and hULT (panel b), derived from the settling of synthetic kaolinite tailings using peptides-PAE materials (Table 2). As the benchmark, tailings
settling without peptides-PAE materials gives v0 of (8.1 ± 1.3) × 10− 3%/min and hULT of 94.1 ± 0.2%. The synthesis factors used to generate each of the experimental
groups can be found in Table 2.
4
Journal of Cleaner Production 323 (2021) 129195
in larger-the-better and smaller-the-better modes of Taguchi method,
respectively (Antony and Kaye, 2000).
Effect of reactants’ concentration. The reactants’ concentration
(on a dry basis) was the second major influential factor (ranked 2) for v0
(Fig. 2a) and was the third influential factor (ranked 3) for hULT (Fig. 2b).
Both v0 and hULT appeared to be positively correlated with the reactants’
concentration in the test range (Fig. 2). Such a trend is likely associated
with the density of crosslinking nodes in the resultant bioflocculant.
According to previous literature (Adhikari et al., 2016), the synthesis of
peptides-PAE materials depends on the reactions between the azetidi­
nium group of PAE resin (molecular weight in the range of 750–850 kDa
(Y. Zhu, Chae, et al., 2021)) and the functional groups (-NH2, –COOH,
etc.) of the peptide molecules (molecular weights mainly in the range of
10–50 kDa (Y. Zhu, Chae, et al., 2021)). Thus, with a greater reactants’
concentration, crosslinking nodes between PAE and peptides could be
densified, which increased the molecular weight of the resultant bio­
flocculant. According to Vajihinejad et al. (2019), a flocculant with a
large molecular weight is beneficial for aggregating the fine particles in
suspensions, leading to the formation of larger aggregates with a greater
settling rate, which agreed with the greater v0 at higher concentration in
Fig. 2a. However, a high concentration of reactants also increased the
system viscosity. This could hinder the movement of molecular chains
and reduce the crosslinking extent, which might account for the pla­
teaued v0 at a reactant concentration greater than 10%. A similar
observation was reported by Adhikari et al. where a highly viscous
binder was produced at a reactant concentration of 20% (Adhikari et al.,
2019). On the other hand, larger aggregates could trap more water
(intra-aggregate water), which is more difficult to drain compared to the
water between aggregates (inter-aggregate water) and has been recog­
nized as the major contributor for the swelling of sediment solids
(Costine et al., 2019; Grabsch et al., 2013). Such a difference could ac­
count for the lower compactness of the final sediment (a higher hULT ) at
higher reactants’ concentration in Fig. 2b.
Effect of PAE proportion. The PAE proportion with respect to the
total reactants (dry basis) was the most influential factor (ranked 1) for
v0 (Fig. 2a). The main effect plots showed that the S/N ratio of v0
exhibited a sharp increase as the PAE proportion increased from 11.4%
to 22.8%, and a sharp decrease with PAE proportion reaching 34.2%.
This observation may be explained by the effect of PAE proportion, or
specifically, the ratio between the reactive functional groups of PAE and
peptides, on the reaction types and hence the product. According to
Adhikari et al. (2016), the reactive functional group (azetidinium group)
in PAE was present at 3.27 mmol/g; in contrast, the occurrence of major
reactive functional groups in peptides were reported to be: carboxylic
acid groups – 1.61 mmol/g, and primary amine – 0.53 mmol/g (C. Zhu,
2019). At a low PAE proportion of 11.4%, the azetidinium groups of PAE
Y. Zhu et al.
Fig. 2. Main effect plot of the key synthesis factors for v0 (panel a) and hULT (panel b). The four synthesis factors, including reactants’ concentration, PAE proportion
with respect to total reactants in weight, reaction temperature, and reaction time, are listed at the horizontal axis, each presented at three variation levels.
(0.37 mmol/g with respect to overall reactants) were more likely to
react with the relatively small peptide molecules that carried abundant
functional groups (1.89 mmol/g of –COOH and –NH2 combined). With
increasing PAE proportion, more azetidinium groups were introduced to
the reactions, where both intramolecular and intermolecular
homo-crosslinking of PAE was more likely to take place (Adhikari et al.,
2016). For example, at a higher PAE proportion of 22.8%, the intensity
of azetidinium groups (0.75 mmol/g) in reactants was closer to the 1.65
mmol/g of –COOH and –NH2 combined of peptides, which increased the
probability of homo-crosslinking of PAE. In particular, the intermolec­
ular homo-crosslinking of PAE dramatically improved the molecular
weight of the resultant peptides-PAE materials, which incurred faster
flocculation of the suspended clay particles as suggested by the sub­
stantially greater v0 at a PAE proportion of 22.8% in Fig. 2a. However,
the intensified homo-crosslinking of PAE not only elevated the molec­
ular weight of the product but also led to the formation of rigid,
three-dimensional matrix of polymers interlinked via covalent bonds.
This effect could become dominating at high initial PAE proportion
(34.2%), which could hamper the relaxation of the bioflocculant’s chain
in an aqueous solution. Hence, the decrease of v0 at a PAE proportion of
34.2% in Fig. 2a can be attributed to the reduced flexibility of the bio­
flocculant’s polymeric chains and restrained ability to bridge the sus­
pended clay particles in tailings. An optimum PAE proportion of ~23%
was also reported by Adhikari et al. in developing peptide-PAE adhe­
sives (Adhikari et al., 2019).
Similarly, the PAE proportion was the most influential factor (ranked
1) for hULT (Fig. 2b). Specifically, the S/N ratio of hULT versus PAE
proportion followed a similar trend to the v0 case (Fig. 2a), i.e., it
increased as the PAE proportion increased from 11.4% to 22.8% and
then decreased with PAE proportion up to 34.2%. Such variation may
have resulted from the dewatering of intra- and inter-aggregate water in
sediment as well, as the bioflocculant with greater molecular weight and
more flexible polymeric chain incurred faster aggregation and settling of
suspended solid particles (greater v0 ), and it simultaneously led to a
greater content of intra-aggregate water that resulted in less compact
sediment (greater hULT ).
Effect of reaction temperature and time. As shown in Fig. 2a, re­
action temperature and time were ranked fourth and third, respectively,
with regard to their influence on v0 . Interestingly, their effects on v0
exhibited a high similarity in trend; though the overall variation was
marginal, the S/N of v0 was found to increase and then decrease with
increasing reaction temperature in the range of 60–90 ◦ C, while a similar
trend was observed with regard to reaction time in the range of 0.5–20 h.
As demonstrated by Adhikari et al., 2016, 2019, reaction temperature
and time affected the shear strength of peptides-PAE solution in a binder
application, which was associated with the extent of chemical reactions
between the two multi-functional reactants. The increase of tailing
settling rate (Fig. 2a) with temperature increasing to 75 ◦ C or reaction
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Journal of Cleaner Production 323 (2021) 129195
time reaching 2 h can be attributed to the formation of a larger bio­
flocculant. In contrast, at an even higher temperature of 90 ◦ C or a
sufficiently long reaction time of 20 h, a substantial amount of the
functional groups in peptides (-COOH, –NH2, etc.) would be consumed
in the reaction. This would increase the hydrophobicity of the product
and reduce molecular stretching in aqueous environments, which could
lead to a lower settling rate (lower v0 in Fig. 2a). The increased hydro­
phobicity of the bioflocculant also explained the decreasing S/N ratio of
hULT with increasing reaction temperature or time (Fig. 2b), as its
reduced affinity with water could facilitate drainage of water from
sediment, leading to sediment with a greater compactness (lower hULT ).
Taken together, bioflocculants prepared at 75 ◦ C and 2 h facilitated
crosslinking levels that benefited rapid aggregation of tailing solids,
while the one prepared in a more severe condition (90 ◦ C and 20 h)
allowed the ultimate release of more water.
Interaction of factors. Results of the Taguchi method provided in­
formation about the interaction between the factors examined. A matrix
of interaction plot for each pair of the four key synthesis factors (6 two-
factor interactions in total) on the means of v0 and hULT are provided in
Figs. S1 and S2, respectively. Since neither the curves in Fig. S1 nor in S2
shows any parallel pattern, a significant degree of interaction was pre­
sent between the factors within the test range.
3.1.3. Validation of the optimized formulation
Based on analyzing the effects of the synthesis factors on the S/N
ratio of v0 (Fig. 2a) and hULT (Fig. 2b), the corresponding variation level
of each synthesis factor was predicted for the theoretically largest v0
(Case 1) and smallest hULT (Case 2). To achieve the predicted largest v0 ,
the bioflocculant needed to be formulated at a reactants concentration of
12 wt%, a PAE proportion of 22.8 wt%, a reaction temperature of 75 ◦ C,
and a reaction time of 2 h; in contrast, the bioflocculant corresponding
to the theoretically smallest hULT needed to be formulated at a reactants
concentration of 8 wt%, a PAE proportion of 11.4 wt%, a reaction
temperature of 90 ◦ C, and a reaction time of 20 h. Tailings settling ex­
periments using these select conditions were conducted, with their
corresponding characteristic function values presented in Fig. 3;
meanwhile, the predicted characteristic function values of the optimized
formulations were calculated using Minitab software and are also pre­
sented in Fig. 3. It was observed that the experimental values were in
good agreement with those predicted; the v0 presented in Fig. 3a was the
highest compared to all the groups in Fig. 1a, and the hULT given in
Fig. 3b was much lower than any of the 9 tests in Fig. 1b. This validated
the Taguchi method-based formulation optimization.
3.2. Treatment of industrial fluid tailings
Mining oil sands mature fine tailings (MFTs) were studied as the
target stream in this study to characterize the performance of peptides-
Y. Zhu et al.
Fig. 3. Comparison of the experimental and predicted characteristic function
values of the optimized formulations. Panel a and b refers to the v0 in Case 1
(largest v0 ) and the hULT in Case 2 (smallest hULT ), respectively.
PAE bioflocculant in settling industrial fluid tailings using bench-scale
cylinder settling test (Gumfekar and Soares, 2018; Vajihinejad et al.,
2019). As the developed bioflocculant is expected to be applied at the
stage of thickener prior to the tailings’ discharge at the tailings pond
(Masliyah et al., 2010), MFTs pre-diluted with process water at a 1:2
mass ratio with a solid content of 8.9 wt% was used as the sample stream
to better represent the raw tailings at this stage (Gosselin et al., 2010).
The industrial mining oil sands operation has been challenged by limited
freshwater intake during the wintertime and is heavily dependent on the
amount of water that can be recovered (Masliyah et al., 2010). There­
fore, a complete water removal (i.e., a small hULT ) is more important in
this case compared to a fast tailing settling (i.e., a large v0 ). In this study,
the developed bioflocculant associated with the smallest hULT for syn­
thetic kaolinite tailings was evaluated in settling industrial fluid tailings.
A preliminary examination of the dosage effects showed that a complete
solid sedimentation can be achieved at a threshold dosage of 10
kg/tonne (Fig. S3), which was further investigated for comparison with
the industrial practice. As given in Fig. 4a, the settling of the industrial
fluid tailings was substantially improved with the use of the
SRM-derived bioflocculant, which agrees with the observations for
synthetic kaolinite tailings. In particular, the use of the bioflocculant
Fig. 4. Settling of prediluted mature fine tailings (MFTs) from the mining oil sands industry using the select peptides-PAE bioflocculant at a dosage of 10 kg/tonne
tailings solids in equivalent (panel a) and the corresponding v0 (panel b) and hULT (panel c). The inlet of panel a shows the settling behaviors within the initial
120 min.
6
Journal of Cleaner Production 323 (2021) 129195
substantially increased v0 to a level (~101%/min) comparable to the
industrially applied HPAM flocculant (with gypsum) (Y. Zhu, Chae,
et al., 2021) and was 4 magnitudes faster than the blank reference
(~10− 3%/min; Fig. 4b). Meanwhile, hULT was found to be 48.1 ± 0.6%
through implementing the bioflocculant, which indicates a dewatering
efficiency as high as 93.2% of the HPAM case (Fig. 4c). More impor­
tantly, unlike HPAM, the bioflocculant enabled a satisfactory tailings
settling performance without the use of gypsum coagulant, which agrees
with a previous publication (Y. Zhu, Chae, et al., 2021). Hence, the
SRM-derived bioflocculant opens up the potential of a gypsum-free
approach to settle industrial fluid tailings, which is critical for in­
dustries where gypsum use is unfavored, for instance, the mining oil
sands industry (Kasongo et al., 2000; Liu et al., 2002).
On the other hand, the use of either SRM peptides or PAE alone was
unable to achieve an industrial fluid tailing dewatering as fast (reduced
v0 in Fig. 4b) and complete (greater hULT in Fig. 4c) as that of the
peptides-PAE bioflocculant. Such observations agree with previous
literature for synthetic kaolinite tailings (Y. Zhu, Chae, et al., 2021).
Taken together, the peptides-PAE bioflocculant demonstrated improved
dewatering capability in treating complicated industrial fluid tailings.
Future studies will assess the performance of the peptides-PAE bio­
flocculant at larger scale in order to validate the findings of our
bench-scale work.
3.3. Biodegradation
Since SRM-derived peptides are hydrolyzed proteins from biological
sources, one of their major advantages is the potential of biodegradation
and low toxicity. This makes peptides and their derivatives of particular
interest in terms of reduced environmental impacts compared to HPAM,
a commercial flocculant that is not easily broken down in nature (Sun
et al., 2017). As peptides and peptides-PAE materials were soluble in
water and impractical to be quantified after tailings treatment, their
biodegradation behaviors were quantitatively investigated using a col­
ony count method. Due to the high nitrogen content in SRM-derived
peptides (i.e., 15.3 ± 0.1% C. Zhu (2019)), the biodegradation of the
peptides-PAE bioflocculant was tested by evaluating its potential to
serve as the sole nitrogen source for the growth of two common mi­
croorganisms, S. cerevisiae (yeast) and E. coli (bacteria), on agar plates.
For comparison, agar plates containing all essential nutrients except
nitrogen were used as the negative controls, while the same plates
supplemented with tryptone, a commonly used organic nitrogen source
in microbiology, were used as the positive controls. Furthermore, during
inoculum preparation, broth containing live cells was diluted using
Y. Zhu et al.
phosphate buffer (at least 105 folds), to minimize nitrogen levels in the
inoculum.
The results of the colony count experiments are summarized in
Fig. 5a (S. cerevisiae) and 5b (E. coli). In both cases, no colonies were
observed throughout the 4-day cultivation on the negative control
plates, which contained no nitrogen source (Fig. 5a and b). Conversely,
visible microbial colonies were observed on the positive control plates
within the first 24 h (Fig. 5), suggesting that addition of tryptone to the
media would facilitate cell growth.
SRM-derived peptides were also analyzed as a potential nitrogen
source. Compared to the positive controls, yeast growth on the peptides
plates (Fig. 5a) exhibited slightly lower colony counts before 48 h, but
reached similar levels by 72 h; meanwhile, no statistical difference in
bacterial colony counts was observed relative to the positive control
plates throughout the entire experiment (Fig. 5b). Taken together, these
data suggested that nitrogen present in the SRM-derived peptides is
easily accessible to microbes. Since the only source of nitrogen in these
plates was from the SRM-derived peptides, these results suggest that the
nitrogen must be easily accessible to microbes, clearly indicating that
the bioflocculant is biodegradable.
The peptides-PAE plates (Fig. 5) were found to support the growth of
both microorganisms as well, albeit at lower levels. Interestingly, no
yeast colonies were observed on the peptides-PAE plates before 24 h,
with the quantity of visible colonies gradually increasing until they
reached levels similar to the positive control plates by 72 h (Fig. 5a). On
the other hand, the number of bacterial colonies plateaued by 24 h,
reaching values that were ~1/3 that of the positive controls (Fig. 5b).
One possible explanation is that E. coli is unable to break down certain
bonds within the peptide-PAE materials, resulting in an incomplete
usage of the materials. Nevertheless, the data in Fig. 5 demonstrated that
the peptides-PAE materials can still serve as a nitrogen source for the test
microorganisms, indicating that the bioflocculant is biodegradable.
It should also be noted that common laboratory yeast and bacterial
strains were used in these studies; mixed populations of environmental
strains would likely be much more adept at breaking down the peptides-
PAE materials (Naranjo et al., 2020). Regarding actual industrial fluid
tailings, diverse microorganisms from the environment can be easily
introduced to the fluid tailings through the open-pit mining operations,
open-air tailings ponds, and the dykes impounding the tailing ponds
(Foght et al., 2017). Therefore, if applied for industrial fluid tailings
dewatering, this peptide-PAE bioflocculant has great potential to be
biodegraded by environmental strains. Future work will investigate the
effect of the bioflocculant on microorganism activities in the actual
tailings, as well as evaluate the impact on the landscape reclamation of
the tailings pond areas. In addition, although it is most desirable to
achieve both rapid settling and minimum sediment height simulta­
neously in tailings treatment, the conditions for largest v0 do not
necessarily match those for smallest hULT in this study. In this case, a
Fig. 5. Cell counts of (a) S. cerevisiae and (b) E. coli, on agar plates containing various media. Data points sharing the same letter within the same time groups
represent no statistical difference at a confidence interval of 95%.
7
Journal of Cleaner Production 323 (2021) 129195
ternary bioflocculant with the inclusion of a thermo-responsive agent
(with a lower critical soluble temperature, or LCST) as the third reactant
may be of interest (Vajihinejad et al., 2019). The bioflocculant with the
thermo-responsive segment becomes more relaxed and flexible at a
higher operation temperature (T > LCST), which results in rapid solid
particles aggregation; as temperature ramps down during tailing settling
(T < LCST), the bioflocculant tends to shrink and become less soluble in
water, which facilitates the drainage of intra-aggregate water and leads
to a sediment with greater compactness. Hence, the ternary bio­
flocculant has a chance to achieve optimum v0 and hULT simultaneously
and will be investigated in future work.
4. Conclusions
This is the first study demonstrating the development of peptide-PAE
bioflocculant for real industrial fluid tailings treatment. By properly
controlling key synthesis factors, bioflocculants with different perfor­
mance can be synthesized to meet particular industrial purposes, which
can be enhanced short-term tailings settling or improved ultimate water
removal. Compared to the commercial HPAM flocculant, the developed
SRM-derived bioflocculant not only achieved a comparably fast and
complete densification (93.2% the dewatering efficiency of the HPAM
case) of real industrial fluid tailings, but also allowed tailings settling in
a gypsum-free condition. In addition, the bioflocculant can be degraded
and used as a nitrogen source by two common microorganisms. Overall,
bioflocculants derived from negative-value SRM feedstocks shows a
great potential for clean and sustainable management of industrial fluid
tailings, as well as certain biodegradability that minimizes environ­
mental impacts.
CRediT authorship contribution statement
Yeling Zhu: Conceptualization, Methodology, Validation, Formal
analysis, Investigation, Data curation, Writing – original draft, Writing –
review & editing, Visualization. Michael Chae: Conceptualization,
Methodology, Validation, Resources, Writing – review & editing, Su­
pervision, Project administration, Funding acquisition. Jie Wang:
Conceptualization, Methodology, Validation, Formal analysis, Writing –
original draft, Writing – review & editing. Birendra Adhikari: Meth­
odology, Writing – review & editing. Paolo Mussone: Conceptualiza­
tion, Resources, Validation, Writing – review & editing, Supervision,
Funding acquisition. David C. Bressler: Conceptualization, Methodol­
ogy, Validation, Resources, Writing – review & editing, Supervision,
Project administration, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
Y. Zhu et al. Journal of Cleaner Production 323 (2021) 129195

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