Journal of Water Process Engineering 42 (2021) 102123

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Journal of Water Process Engineering

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Bioremediation of copper polluted wastewater-like nutrient media and

simultaneous synthesis of stable copper nanoparticles by a viable green alga
Urška Žvab a, Danijel Stojković Kukulin a, Mattia Fanetti a, Matjaz Valant a, b, *
a
University of Nova Gorica, Materials Research Laboratory, 5270, Ajdovščina, Slovenia
b
Institute of Fundamental and Frontier Sciences, University of Electronic Science and Technology of China, Chengdu, 610054, China

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

Keywords: Environmentally benign, algae-mediated biosynthesis of valuable copper-based materials in wastewater, com­
Chlamydomonasreinhardtii bined with cupric ion (Cu2+) bioremediation, has the potential to reduce the cost of wastewater treatment and
Viable microalga generate high quality biosolids. This study investigated the ability of a wild-type strain of Chlamydomonas
Simulated municipal wastewater
reinhardtii to bioremediate free Cu2+ in wastewater-like nutrient media during the biosynthesis of copper
Cupric ion bioremediation
Hydrogen photoproduction
nanoparticles (Cu NPs). The color of supernatants from treated Cu-polluted media provided a first indication of
Copper nanoparticle biosynthesis Cu-based NPs formation in the aqueous phase. Analysis by fluorescein diacetate hydrolysis, observations of cell
morphology, and algal regrowth experiments after treatment showed that algal viability was crucial for efficient
Cu2+ reduction to Cu NPs. UV–vis absorption spectroscopy demonstrated that sulfur-free medium, which enables
sustained hydrogen photoproduction in alga, was not the most efficient in NPs formation. Dark-field scanning
transmission electron microscopy (STEM) images overlapped with Cu signal map from energy dispersive X-ray
spectrometry (EDS) and high-resolution transmission electron microscopy confirmed the presence of poly­
disperse, spherical, and well-dispersed sub-10 nm Cu NPs crystallites exclusively in algae-treated heavily Cu-
polluted media (10 mg L− 1). STEM and EDS also demonstrated the affinity of Cu NPs for carbon-rich
(organic) objects. Overall, this study demonstrates the feasibility of Cu2+ bioremediation from the
wastewater-like complex nutrient media and the simultaneous biosynthesis of Cu NPs by viable green microalga.

1. Introduction electrochemical and physicochemical methods typically employed.

Copper (Cu) is a frequently encountered metal contaminant in
municipal wastewater. It has adverse environmental effects on receiving
water bodies and reduces the quality of the biosolids below the standard
required for their use as fertilizer or safe disposal. Exceedances often
relate to various industrial sources and the use of Cu-containing pesti­
cides in conventional and organic agriculture [1]. While a wide range of
metabolic processes in both prokaryotes and eukaryotes requires Cu,
increased concentration can have detrimental effects on organisms. For
example, contaminated coastal seawater has been found to exhibit
decreased biodiversity [2]. Elevated Cu levels in the environment can
cause co-selection for antibiotic resistance [3] and poses an additional
threat to coral health in warming oceans [4]. Free or weakly complexed
copper can also be harmful to humans [5]. In general, free cupric ion
(Cu2+) is evidently very toxic Cu-chemical species [6,7].
Removal of trace metals (in the 10− 100 mg L− 1 concentration
range) from wastewater is costly and challenging to achieve with the
Drawbacks to the conventional approaches of metal-polluted waste­
water treatment include demanding chemical and energy requirements,
and the formation of hazardous biosolids or other waste byproducts that
require specific disposal [8–12]. An attractive alternative to these ap­
proaches is offered by the biosynthesis of zero-valent metals, alloys, or
inorganic-organic composites, which take place together with the
bioremediation process. This promising biotechnological approach has
the potential to remove trace concentration of metal ions, improving the
quality of biosolids while producing no toxic waste, and reducing the
cost of wastewater treatment.
The production of various nanosized particles by prokaryotic and
eukaryotic microorganisms has been reported through both intracellular
or extracellular routes. A wide variety of organisms also form organic/
inorganic composites with an ordered structure using biopolymers, such
as proteins and microbe cells. Different physical and chemical condi­
tions, exposure time, microorganism species and strains, and biomin­
eralization scaffold proteins can control the rate of particle formation,

* Corresponding author at: University of Nova Gorica, Materials Research Laboratory, 5270, Ajdovščina, Slovenia.
E-mail address: matjaz.valant@ung.si (M. Valant).

https://doi.org/10.1016/j.jwpe.2021.102123
Received 19 February 2021; Received in revised form 30 April 2021; Accepted 30 April 2021
Available online 8 May 2021
2214-7144/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
U. Žvab et al.
size, and morphology of bio-produced nanoparticles (NPs) [13]. From
the perspective of NP production, biological synthesis alone has many
advantages regarding cost, speed, simplicity, and scalability. It is also
environment friendly as it does not require organic solvents or any other
hazardous chemicals and has low energy demands [14,15]. Just as the
recovery of precious metals has been proposed to reduce the cost of
wastewater treatment [10], the recovery of many other biominerals may
be of significant value [15–17].
The importance of algae in wastewater treatment based on nutrient
assimilation and valuable biomass formation is well-documented. The
biosynthesis of silver, palladium, silver chloride, gold, zinc oxide and,
copper NPs (among others), by algae have been reported [18,19]. Bar­
wal et al. (2011) described the formation of silver NPs (SNP) by cellular
proteins of C. reinhardtii. Most proteins associated with biosynthesis and
stabilization of SNP were part of cellular oxido-reductive processes [20].
However, various functional groups of algal phytochemicals, such as
polysaccharides, alkaloids, polyphenols, and, proteins can serve as
effective metal-reducing, capping, and stabilizing agent in the biosyn­
thesis of metallic NPs [18,21,22].
Posewitz et al., 2009 reported that numerous species of Chlamydo­
monas produce hydrogen, which can contribute to the reduction of metal
pollutants. Standard electrode potentials (E◦ ) at 25 ◦ C and 101.325 kPa
(1 atm) predict the ability of H2 to reduce cuprous ion (Cu+) and Cu2+ to
zero-valent Cu (as well as two other major metal contaminants, Hg2+
and Cr6+ to their less toxic forms Hg◦ and Cr3+) [23]. Therefore,
H2-producing algae are expected to facilitate the reduction of Cu2+ and
the formation of metallic Cu. A powerful tool for sustained H2 produc­
tion in C. reinhardtii was observed to be sulfur deprivation [24,25,31].
No published study yet has investigating bioremediation of the
common metal pollutant, Cu2+ coupled with the biosynthesis of valu­
able Cu-based NPs in the wastewater-like, complex, nutrient media by
ubiquitous green microalga.
The main objective of this research was to study the ability of the
wild-type (wt) strain of green alga Chlamydomonas reinhardtii
(C. reinhardtii) for the bioremediation of heavily Cu-polluted municipal
wastewater and simultaneous production of valuable Cu NPs.
Bioremediation and biosynthesis were carried out in aerobic and
anoxic conditions in a complete tris-acetate-phosphate (TAP) medium
that simulated raw municipal wastewater (Table S1) and sulfur-free TAP
(TAP-S). The wastewater’s characteristics listed in Table S1 were
collected from reports of two local wastewater treatment plants
(WWTP), from the Food and Agriculture Organization (FAO) [26], and
scientific publications [27–29] Successful Cu2+ bioremediation and
Cu-based NPs biosynthesis was initially assessed based on media color
change and UV–vis analysis of their supernatant. Total Cu concentration
(CuT) and free Cu2+ concentration (CuF) was then determined by flame
atomic absorption spectroscopy (AAS) and with a cupric ion selective
electrode (ISE), respectively. This was analyzed alongside pH and algal
metabolic activity. Reliable determination of algal viability during
treatment enabled the fluorescein diacetate (FDA) method, cell
morphology observations, and algal regrowth. Several control experi­
ments were conducted to rigorously assess Cu2+ bioremediation and Cu
NPs biosynthesis by algae, including the use of a light-sensitive rubisco
(rbc) strain of algae, the wt strain of algae under dark conditions, and
ultrasonicated algal culture followed by Cu exposure. Dark-field scan­
ning transmission electron microscopy (STEM), high-resolution trans­
mission electron microscopy (HRTEM), and energy dispersive X-ray
spectroscopy (EDS) were used for direct observation of NPs.
2. Materials and methods
2.1. Preparation of media and Cu2+ solutions
TAP medium was prepared according to guidelines from the Chla­
mydomonas Resource Center (CRC, at the University of Minnesota, USA)
[30]. To create TAP-S medium, 5 g of MgSO4 ∙ 7H2O and Hunter’s trace
2
Journal of Water Process Engineering 42 (2021) 102123
elements (HTE), were replaced by 4.12 g of MgCl2∙ 6H2O and sulfur-free
Hunter’s trace elements (HTE-S), respectively. HTE and HTE-S were
purchased from CRC. A stock solution of Cu2+ (10 g L− 1 was prepared by
dissolving CuCl2∙ 2H2O (Fischer Scientific, USA) in ddH2O and stored at
5− 7 ◦ C. TAP and TAP-S media were polluted with a filter-sterilized Cu2+
solution of 1 g L− 1 in ddH2O. Standard Cu2+ solutions of 1, 2, 5, and
10 mg L− 1 were prepared in TAP media for use in AAS and free Cu2+
analysis.
2.2. Growth and maintenance of the algae
C. reinhardtii strain C-5075 (mt+), sequence-verified clone of the
wild-type (wt) strain CC-125 and control light-sensitive rubisco (rbc)
mutant CC-4731 (mt-) (CRC, USA) were grown photoheterotrophically
in TAP medium, pH 7.0, under continuous cool-white fluorescence
illumination. Dim light conditions were created by entirely encasing the
light-sensitive rbc culture with aluminium foil. Algal cultures were
maintained in a solid medium under a low photon flux of 5− 10 μmol
m− 2 s-1 or in dim light for up to 6 months after being received from CRC.
Liquid cultures were grown in Erlenmeyer flasks at a photon flux density
of 30 μmol m-2 s-1 or in dim light, in an orbital shaker at 100 rpm and
26− 28 ◦ C. For new algal culture, a loopful of algae from solid TAP
medium was transferred into 50 mL of liquid TAP media and grown to
the mid-exponential phase. A 10 mL aliquot of cell suspension was used
to prepare 150 mL of culture then grown to the late exponential phase.
Small, oval, and motile cells with a cell density of 3− 6 × 106 cells mL-1
were harvested by centrifugation at 1000x g for 5 min at 23 ◦ C twice,
and resuspended in TAP or TAP-S medium. Algal intactness and motility
were preserved during the described procedures based on the proced­
ures reported by Melis et al., (2000) and Pinto et al. (2013). Algal
samples were inspected by optical microscope at 100x and 400x
magnification for their motility, size, shape, and contaminations. Cells
stained with Lugol’s iodine solution (10 μL per 1 mL of algal cultures)
were counted with a hemocytometer.
2.3. Algal treatment of Cu-polluted media
Algal cultures were placed into 250 mL borosilicate reagent bottles
covered with an H2-tight modular lid without or with an outlet for gases.
Borosilicate reagent bottles and stirring bars were washed in 20 % HNO3
(technical grade) overnight between experiments. Lids were made of
multiple distributors for bottles D612− 08, screw joints D598− 06 and
D593− 06, an airtight tube S1815− 04, and gaskets H973− 14 for sam­
pling (Bohlender, Germany). For the aerobic TAP-S culture, the reagent
bottle was covered with a cotton plug. Aerobic TAP sample was prepared
with the modular lid which enabled accurate sampling of gases that had
evolved during treatment. Anaerobic conditions were not created as
photosynthesis was not inhibited in the setup. Cultures were incubated
at 25 ◦ C, with minimal stirring, at continuous low-moderate illumina­
tion of 40 μmol m− 2 s-1 (measured inside bottles). The setup is shown in
Fig. S1. To estimate the amount of H2 produced by the algal culture,
2 mL of gas was drawn from the headspace of the culture and analyzed
by mass spectrometry (ThermoStar™, Pfeiffer Vacuum, Germany). Algal
cultures were pre-incubated for 3.5 days, by which time a significant
amount of H2 accumulated in the headspace of anaerobic TAP-S cul­
tures. Other samples were treated in the same way to provide compa­
rable experimental conditions. After the pre-incubation period, Cu2+
was injected into the media to the final concentration of 10 mg L-1.
Samples were collected at day 0 (2 h) and during 1, 2, and 3 days after
metal addition. Algae were sonicated with an ultrasonic homogenizer
(Labsonic M, Sartorius Group, Germany) on ice, repeatedly in 20 s in­
tervals until the absorbance of about 1 AU at 260 nm was measured.
After sonication, samples with impaired algal cells were incubated with
Cu2+ for 1 h. UV–vis absorption spectra were then recorded for uncon­
taminated and Cu-spiked supernatants. The experiments were repeated
two to eight times. The control sonication experiments were not
U. Žvab et al.
repeated.
2.4. Analysis of supernatants
Supernatants with biosynthesized Cu NPs were separated from algae
by centrifugation at 11,000x g for 10 min at room temperature. Imme­
diately after centrifugation, formation of Cu-based NPs was monitored
on a Lambda 35 UV–vis spectrometer (PerkinElmer, USA) between
200–700 nm for each sample. Up to one week after treatment CuT was
determined with AAS (SpectrAA 10 instrument, Varian, USA). Before
analysis, samples were digested with HNO3 (analytical grade) with the
final concentration of 1%. CuF was measured with an ISE by using a
direct calibration procedure. A cupric half-cell electrode was used in
combination with the double junction reference electrode (Thermo
Fischer Scientific, USA). Measurement of pH was performed alongside
measurement of CuF. Microscopy characterization of Cu NPs was done
by transmission electron microscope (TEM) JEM-2100f (JEOL, Japan)
operated at 200 kV. Images were obtained using a charge-couple device
(CCD) camera. The microscope is equipped with a Scanning Trans­
mission Electron Microscope (STEM) unit (operated with an annular
dark-field detector) and an Energy Dispersive X-ray Spectrometer (EDS)
X-Max 80 T (Oxford Instruments, UK), which enables spatially resolved
elemental analysis. TEM samples were prepared by dipping a 400 mesh
nickel grid (coated by amorphous C lacey supporting film) into super­
natant and air drying. Several samples were also analyzed after being
aged on the TEM grid. Between analyses, supernatants were stored at
5− 7 ◦ C.
2.5. Analysis of algae
Algal morphology, enzymatic capacity for fluorescein diacetate
(FDA) cleavage and cell capability for the retention of the fluorescent
product were analyzed immediately after sampling using an Axio
Observer.Z1 epifluorescence microscope (Zeiss, Germany). Algal capa­
bility to recover after Cu2+ bioremediation was evaluated by regrowing
cells in a transparent 96-well microtiter plate containing TAP medium.
Cell size, morphology, and motility were examined in a bright-field.
Selected samples were analyzed by differential interference contrast
(DIC) microscopy. Enzymatic activity and cell membrane integrity was
assessed by green fluorescence of cells stained by FDA (Sigma, USA)
using a 38 HE GFP filter set (for green fluorescent protein). The FDA
probe was prepared and handled as recommended by Jochem (1999),
with some modifications. Briefly, stock solution of FDA of 5 mg L− 1 was
made in acetone and stored at -20 ◦ C. The working solution was a 50-
fold dilution of stock solution in ddH2O. FDA (3 μL) was added
to100 μL of algal culture and incubated at RT in the dark for 20 min.
Cells were then harvested by centrifugation at 1000x g for 5 min at RT,
resuspended in 20 μL of filter-sterilized 0.9 % NaCl and observed under
the microscope. For regrowth experiment algae were harvested by
centrifugation at 1000x g for 5 min at 23 ◦ C and resuspended in TAP
medium. Equal volumes of algal cultures and TAP were combined in
microplate wells. The microtiter plate was covered with a transparent lid
and wrapped with parafilm to prevent evaporation before being placed
under a continuous illumination of 30 μmol m-2 s-1 for a week in an
orbital shaker at 100 rpm at 26− 28 ◦ C. Optical microscopy indicated
that dark green cultures were the densest and contained motile cells.
2.6. Statistical analysis
Mean values and standard deviations (SD) were calculated,
normality test was performed and outliers were identified with Dixon’s
Q test. To test for the homogeneity of variances, Leven’s test was
applied. One-way analysis of variance (ANOVA) and post-hoc Tukey test
determined significantly different mean absorption values; at around
250 nm or exactly at 250 nm for samples without peak (Absorbance)
and to determine the significantly different mean pH values.
3
Journal of Water Process Engineering 42 (2021) 102123
Significantly different mean CuT and mean CuF values were determined
with Welch’s test as the homogeneity assumption was violated for these
two variables [34]. The selected confidence level was 95 %. Multivariate
plots were applied for graphical presentation of the inter-relations
among Absorbance, pH, CuT, CuF, H2, and algal viability.
3. Results and discussion
3.1. Optical properties of algae-treated Cu-polluted media supernatants
The color of anaerobic TAP-S culture with abundant H2 photopro­
duction and the addition of 10 mg L− 1 Cu2+ (TAP-S an H2 wt 10Cu;
Fig. S2A) turned green during treatment. Algae in aerobic Cu-polluted
TAP-S medium (TAP-S a wt 10Cu) and TAP medium (TAP a wt 10Cu)
formed brown supernatants (Fig. 1). A control sample of anaerobic TAP-
S culture that photoproduced H2 and contained a small amount of Cu2+
(0.4 mg L− 1 for optimal algal growth; TAP-S an H2 0.4Cu wt) was only
lightly colored at the end of the experiment. Other Cu-polluted control
samples remained colorless, including anaerobic TAP-S media i) without
algae (TAP-S no algae 10Cu), ii) with algae kept in dark conditions (TAP-
S an lowH2 dark wt 10Cu), and iii) with rbc mutant (TAP-S an lowH2 rbc
10Cu).
The observed color change of media upon treatment are in agree­
ment with previously reported green syntheses of Cu-based NPs [21,
35–39]. Tatsuma (2013) discussed that metal clusters with a diameter of
≤ 2 nm absorb light based on electron transition from occupied to un­
occupied orbitals while larger NPs up to several hundred nm absorb and
scatter light by localized surface plasmon resonance (SPR). Optical
properties, including resonance wavelength and absorption intensity,
depend on particle size, shape, orientation, spacing, and dielectric en­
vironments [40]. The colorless supernatants formed in control samples
suggest the absence of Cu2+ reduction and NPs synthesis.
While color change is a widely used indicator of Cu-based NP
biosynthesis, insight can be gained from the light absorbance of samples
by UV–vis absorption spectroscopy, as has been used in previous studies
that characterized semiconductor NPs [41]. Absorption peaks at
246− 258 nm were observed for the samples shown in Fig. 2A-D, and for
the control sample, TAP-S an lowH2 dark wt 10Cu (Fig. S3A), charac­
teristic of Cu and CuO NPs due to dipole oscillation [37,42–44].
Absorbance peak intensity for different samples and treatment times can
be seen in Fig. 2F. Smaller features were observed between 500− 300 nm
for the three primary samples under study (Fig. 2A–C) possibly related to
Cu2O NPs [45], CuO NPs [37,46], or sub-10 nm Cu NPs with thin CuO
shell [21,47]. Therefore, in accordance with observed color change,
UV–vis absorption spectra confirmed the biosynthesis of metallic Cu or
CuO NPs, and provided some evidence for the existence of Cu2O NPs.
The SPR peak at ~570 nm, characteristic of Cu NPs, was not seen for any
of the samples. The absence of this peak has been rationalised previously
due to the formation of CuO or CuCl2 layers around NPs and the small
size of Cu NPs in the sample [36,39,43,47]. This is supported by the lack
of an intense absorbance observed at 570 nm, known to be an SPR
feature (Creighton and Eadon 1991), as a possible result of low con­
centration and polydisperse Cu NPs (given that the green and brown
colors of these samples correspond to the absorption at 570 nm for
metallic Cu NPs). A small broad UV peak characteristic of Cu and CuO
NPs appeared for control samples TAP-S an H2 0.4Cu wt (Fig. 2D), and
Fig. 1. Colored supernatants of algae-treated Cu-polluted media. Green su­
pernatant from TAP-S an H2 wt 10Cu (A) and brown supernatants from TAP-S a
wt 10Cu (B) and TAP a wt 10Cu (C).
U. Žvab et al. Journal of Water Process Engineering 42 (2021) 102123

Fig. 2. UV–vis absorption spectra (A-E) and absorption intensities at peak wavelengths (λ) (F) for algae-treated media supernatants. Spectra were recorded at several
time points during bioremediation: at day 0 (2 h) and day 1-3. Absorption spectra of supernatants are shown for Cu2+-polluted (10 mg L− 1) samples: TAP-S an H2 wt
10Cu (A), TAP-S a wt 10Cu (B), and TAP a wt 10Cu (C); from the non-Cu2+ polluted sample TAP-S an H2 0.4Cu wt (D) and sonicated algal culture, polluted or non-
Cu2+ polluted after sonication (E). (F) shows mean peak intensities with error bars representing SD. Bars (mean values) with no common letters are significantly
different (p ≤ 0.05); for instance, first and second means are not significantly different while first and seventh means are significantly different.

TAP-S an lowH2 dark wt 10Cu (Fig. S3A) likely due to the small amount of the aqueous solutions of Cu NPs remained stable at least up to 1 month at
copper in the former and the suboptimal dark conditions for photosyn­ 5− 7 ◦ C. That aqueous phase stability of biosynthesized Cu NPs was
thetic algal cells in the latter (decreasing metabolic activity and NPs observed before. It was attributed to the strongly-capped sulfur-con­
formation). Characteristic UV peak did not form for control samples taining amino acids (cysteine/methionine) [36,49]. Other biomolecules
TAP-S an lowH2 rbc 10Cu (Fig. S3B) and TAP-S an no alga 10Cu might be involved in formation of stable solution of NPs according to
(Fig. S3C). The light-sensitive rbc culture was not capable of NP syn­ Arya et al. [21].
thesis following extensive destruction during irradiation (Fig. S3B).
Irradiation alone is not capable of NPs synthesis either (Fig. S3C).
3.2. CuT, CuF and pH in supernatants of algae-treated Cu-polluted media
Slightly elevated levels of H2 in TAP-S an lowH2 dark wt 10Cu and TAP-S
an lowH2 rbc 10Cu samples (Fig. S2B,C) did not improve NPs formation
If absorption peaks correspond to Cu or CuO NPs, supernatants
according to UV–vis spectra, in agreement with observed color change
should be depleted of free Cu2+ and Cu must remain in media after
during treatment. The supernatant of sonicated algae spiked with Cu2+
treatment. Therefore, analysis of CuT and CuF in supernatants was per­
after sonication exhibited increased absorbance at ~250 nm without the
formed. CuF in control TAP-S samples indicated the extent of Cu2+
defined peak shape associated with Cu and CuO NPs (Fig. 2E; black
complexation with algal biomolecules and the influence of algal meta­
curve). That absorbance was ascribed to soluble complexes of Cu2+ with
bolic activity on bioremediation and biosynthesis. Concomitant pH
cellular components of impaired algal cells [48] as supernatants of
measurements were taken to assess influence on Cu speciation [50], and
sonicated algal cells without addition of Cu2+ displayed less absorption
as an indication of algal metabolic activity.
in the 200− 300 nm range (Fig. 2E; red curve). Similar absorption
AAS measurements of supernatant CuT at the end of the treatment
spectra shape in the 250− 300 nm range was observed of supernatants
showed that most of the initial Cu remained in the media after algae
for 2 h polluted anaerobic and aerobic TAP-S cultures (Fig. 2A,B; Day
were removed (Fig. 3A). Up to ~20 % of Cu was removed with algae
0 spectra), and Cu2+-polluted sonicated algal culture. The observation
from the primary samples (TAP-S an H2 wt 10Cu, TAP-S a wt 10Cu, and
indicated formation of soluble complexes of Cu2+ with algal bio­
TAP a wt 10Cu). About 30 % of Cu was removed from the TAP-S an lowH2
molecules at the beginning of Cu2+ bioremediation and slower initiation
dark wt 10Cu control sample. A decrease of ~5% of CuT was observed for
of biosynthesis of Cu NPs. Delayed nucleation in Cu NPs synthesis
the TAP-S no algae 10Cu control sample, after being illuminated for 7
following the formation of soluble complexes of Cu2+ with biomolecules
days, providing evidence of algae-independent sedimentation of Cu in
can be the rationale for observed pattern in absorbance increase for
irradiated media. Only a small decrease in CuT was observed for the
TAP-S an H2 0.4Cu wt (Fig. 2D) and TAP-S an lowH2 dark wt 10Cu
TAP-S an lowH2 rbc 10Cu control sample. Algae from aerobic TAP-S
(Fig. S3A) supernatants. In contrast, for TAP a wt 10Cu supernatant
supernatant were brown, providing another indication that some NPs
immediately after Cu2+ addition UV peak appeared indicating the
were inside or attached to algal cells, removed from media together with
presence of more or more active biomolecules in this culture.
algae. Therefore, analysis of CuT after treatment in the three primary
Sulfur-depletion decreased the rate of NPs formation in TAP-S medium
samples under study and the control samples (with Cu2+ treatment in
and low concentration of Cu2+ additionally in non-polluted control
dark conditions and with irradiated light-sensitive rbc strain of alga)
culture.
suggested the involvement of algal cell with intact cell structure in Cu2+
UV–vis spectra recorded after one month of samples storage at
adsorption, reduction, and Cu NPs synthesis. Suggested occurrence of
5− 7 ◦ C indicated just minor changes in absorption (Fig. S4A). Therefore,
Cu2+ interactions with algae in processes of bioremediation and

4
U. Žvab et al. Journal of Water Process Engineering 42 (2021) 102123

Fig. 3. Indicators of Cu2+ bioremediation and biosynthesis of Cu-based NPs: pH, CuT, and CuF obtained in supernatants of algae-treated Cu-polluted (10 mg L− 1)
samples (A) and scatter plots showing inter-relation among analyzed variables (B), where rows of the matrix have the variable that appears in that row on the y-axis.
The columns of the matrix have the variable that appears in that column on the x-axis. The analyzed supernatants were from the primary samples: TAP-S an H2 wt
10Cu, TAP-S a wt 10Cu and TAP a wt 10Cu, and control samples: TAP-S an lowH2 dark wt 10Cu, TAP-S an lowH2 rbc 10Cu, TAP-S no algae 10Cu, TAP-S an H2 0.4Cu
wt, and a standard solution of Cu2+ in TAP-S. Mean pH, CuT and CuF statistically compared values. Bars (mean values) with no common letter are significantly
different (p ≤ 0.05); for instance, first and fourth means are significantly different while second and fifth means are not significantly different. Error bars repre­
sent SD.

biosynthesis is supported by the brown appearance of algal biomass in
brown aerobic TAP-S medium. Reduction and cell surface-adsorption of
metal ion (Au3+) has already been demonstrated for pristine unicellular
green alga [51]. However, acidic conditions (pH 3–5) are most favorable
for metal ion sorption [8], which was not observed in any of the samples
(Fig. 3A).
Cupric ISE measurements showed that in the TAP-S no algae 10Cu
control sample, most of the Cu was in the form of free Cu2+. About
4 mg L− 1 of free Cu2+ remained in the media of the control samples
(TAP-S an lowH2 dark wt 10Cu and TAP-S an lowH2 rbc 10Cu) after 3 days
of treatment (Fig. 3A). Conversely, only small amount (~1 mg L− 1) of
free Cu2+ was left in the primary samples (TAP-S an H2 wt 10Cu, TAP-S a
wt 10Cu, and TAP a wt 10Cu). Cupric ISE therefore demonstrated the
removal of toxic free Cu2+ from polluted media by metabolically active
algae. ISE also showed the absence of Cu2+ complexation and reduction
in TAP-S no algae 10Cu control sample. Cell constituents released from
light-damaged light-sensitive rbc cells did not adequately bind Cu2+. In
addition, low algal metabolic activity in a suboptimal dark condition
may have given rise to reduced complexation and transformation of free
Cu2+, inferred already from optical analysis.
Measurements of CuF are consistent with the Pourbaix diagram
(potential-pH diagram) of Cu [50]. Chemical equilibrium should move
from CuF towards precipitates of cupric oxide (CuO), cuprous oxide
(Cu2O), or metallic Cu in the primary samples (TAP-S an H2 wt 10Cu,
TAP-S a wt 10C and TAP a wt 10Cu) at the measured pH values (Fig. 3A).
Graphical presentation of inter-relations between variables showed a
strong positive correlation between absorbance and pH and mostly
confirms a negative correlation between pH and CuF in different samples
(Fig. 3B). The largest pH value (~8) were observed for aerobic media.
Biosynthesis of more stable and smaller Cu NPs at higher pH has already
been demonstrated (Podstawczyk et al., 2019). At larger pH, negative
charge and the reducing power of various functional groups of phyto­
chemicals increases [18]. For both Cu-polluted and non-polluted media
where significant H2 production was measured (TAP-S an H2 wt 10Cu
and TAP-S an H2 0.4Cu wt) the final pH was around 7.7. Therefore, an
increase in the pH was probably a consequence of normal and preserved
cell metabolism and was not influenced by exposure to a high level of
Cu. The pH of the TAP-S no algae 10Cu control sample did not change
substantially from an initial value of 7.0 during treatment. The same was
observed for control samples (TAP-S an lowH2 dark wt 10Cu and TAP-S
an lowH2 rbc 10Cu) indicating diminished algal metabolism and
viability. Viability (treated as a dichotomous variable based on
semi-quantitative evaluation by fluorescence microscopy and auxiliary
indicators: pH and algal morphology) was positively correlated with pH
and negatively with CuF as can be seen in the scatterplots presented in
Fig. 3B. This implies that algal viability is required to remove CuF effi­
ciently from the media. Cellular activity of viable alga can act through
changes in media pH, providing biomolecules that act as complexing
agents, reductants, capping and stabilizing agents. Algal cell surface,
vacuoles, and vesicles also provide local environments with distinct pH,
ion concentration, shape, size, enzyme catalytic activity, chemical and
physical properties of other biomolecules, which might influence the
formation and stability of the particles [18,52,53].
A small percentage (5%) of CuT was removed in aged anaerobic TAP-
S an H2 wt 10Cu supernatant during storage. In contrast, aged aerobic
TAP and TAP-S showed stable CuT values. However, there were no
statistically significant differences in CuT, CuF, or pH values between Cu-
polluted fresh and aged primary samples (TAP-S an H2 wt 10Cu, TAP-S a
wt 10Cu, and TAP a wt 10Cu) (Fig. S4B). Samples showed stable pH, CuT,
and CuF values on aging and were not found to be inter-related with
photoproduced H2 (Fig. 3B), conter to our predictions.
3.3. Direct analysis of Cu NPs by TEM
TEM studies were conducted to provide proof of the biosynthesis of
stable sub-10 nm phase-pure metallic Cu NPs in the three primary
samples. TEM and EDS analysis were performed on supernatants that
had indicated presence of Cu (CuO) NPs based on optical, CuT, CuF, and
pH analysis. Supernatants from analyzed TAP-S an H2 wt 10Cu, TAP-S a
wt 10Cu, and TAP a wt 10Cu were found to contain NPs with similar
morphology and size, which suggest similar mechanism of NPs biosyn­
thesis in all three samples. The dark-field STEM (Fig. 4A) overlapped
with the Cu signal map from EDS (Fig. 4B) and high-resolution trans­
mission electron microscopy (HRTEM) (Fig. 4C,D) indicated the pres­
ence of polydisperse, spherical, and well-dispersed sub-10 nm Cu NPs.
The crystallinity of the Cu NPs in all samples was confirmed by observed
lattice fringes in 5− 6 nm NPs at high resolution. Lattice d-spacing values
of 0.21 nm and 0.18 nm that appeared in different particles (Fig. 4C,D)
were ascribed to (111) and (200) crystal planes of metallic Cu. The
planes (111) for CuO and Cu2O crystallites with lattice d-spacing values
of 0.23 nm and 0.25 nm were not found in any of the NPs analyzed.
Crystals containing sulfur, chlorine, or phosphorus in addition to copper

5
U. Žvab et al. Journal of Water Process Engineering 42 (2021) 102123

Fig. 4. TEM analysis demonstrating biosynthesis of Cu NPs in Cu-polluted TAP-S an H2 wt 10Cu culture. Dark-field STEM image of sub-10 nm Cu NPs (A) and STEM
image overlapped with Cu signal map from EDS that confirmed the presence of Cu in NPs (B) are shown. The HRTEM images of sub-10 nm Cu NPs revealed particles
with lattice d-spacings of 0.21 nm and 0.18 nm that corresponding to (111) and (200) crystal planes of Cu.

were not found. TEM analysis of TAP-S a wt 10Cu supernatant aged in
the ambient atmosphere on the TEM grid for several weeks still
demonstrated the presence of pure Cu NPs (Fig. S5).
Broader size distribution suggest the formation of NPs through
multiple nucleation events [17]. Organic ligands with high affinity for
metal surfaces might protect sub-10 nm Cu NPs from further growth and
oxidation during nucleation and growth processes [17,36,41,43,54,55].
Indeed, STEM and EDS analysis demonstrated the co-localization of Cu
NPs with large C-rich (organic) objects (< 1 μm) on the TEM grid sup­
porting film in all three samples. One such objects is presented in Fig. 5.
Surface organic ligands can have both beneficial or detrimental effects
on the properties of bare NPs [41,56].
3.4. Algal viability
During the treatments, algal viability was evaluated by following
changes in cell morphology and FDA enzymatic cleavage and intracel­
lular retention of the fluorescent product. The algae from TAP-S an H2
wt 10Cu and TAP-S a wt 10Cu samples lost their swimming ability and
surface smoothness (Figs. 6A,B and S6c fluorescence of fluorescein also
decreased significantly for 3 day Cu-exposed algae (Fig. 6A,B). On the
contrary, at least part of the algae of TAP a wt 10Cu culture were still
viable at the end of the 3 day treatment. Flagella on the bright-field
micrograph are evidence of the motility of a healthy part of the cul­
ture. Algae did not appear distended in the bright-field and DIC. TAP
medium contains sulfur that prevents Cu hyperaccumulation inside
cells. Still, CuT analysis showed that the total amount of Cu inside or

Fig. 5. The STEM image (A) and EDS spectra (B) indicate an organic object with a high affinity for Cu NPs. The presented organic object was found in the supernatant
from TAP-S an H2 wt 10Cu culture.

6
U. Žvab et al.
attached to algal cells after 3 days of treatment was similar for all tree
primary cultures in both TAP and TAP-S media. Fast development of
strong brown color and intense absorption peak, characteristic of Cu
NPs, in TAP a wt 10Cu sample also suggest quick Cu2+ reduction and
formation of Cu NPs. Strong green fluorescence, which appeared for a
large part of the culture, is yet another evidence of the algal viability
(Figs. 6C and S6). However, viability decreased gradually during the 3-
day bioremediation of TAP-S an H2 wt 10Cu and TAP-S a wt 10Cu
samples. Decrease in algal viability coincided with an increase in
absorbance for a peak characteristic of Cu NPs in UV–vis spectra
(Figs. 2A,B and 6 F). Algae in the TAP-S an lowH2 dark wt 10Cu control
sample were small, lost their motility and had diminished viability, as
demonstrated by faint green fluorescence (Fig. 6D), in agreement with
previous analysis of absorbance, pH, and CuF. The smooth appearance of
cells suggested limited intracellular accumulation of Cu. In contrast, a
larger amount of Cu was attached with algae (CuT analysis). That indi­
cated that metal adsorption on the cell surface is a passive process.
Complete absence of green fluorescence was observed for the light-
sensitive rbc strain of C. reinhardtii. The cells in rbc culture appeared
severely damaged (Fig. 6E). Therefore, algal viability was confirmed to
be vital for the efficient transformation of excess Cu2+ into Cu NPs under
the conditions tested. The detailed underlying mechanism of Cu NPs
synthesis in C. reinhardtii remains to be distinguished.
After treatment of Cu polluted media for different periods, algal
regrowth was examined by placing algae in fresh nutrient media, i.e.,
wastewater-like TAP medium, in small wells of a sterile transparent
microplate for a week. Algal samples were collected from the reactors at
2 h (Day 0) and 1, 2, and 3 days after Cu2+ addition. The algae collected
from the TAP a wt 10Cu sample regrew into viable dense algal cultures
with highly motile cells that appeared as dark green cultures in the
microplate wells (Fig. 7; first line). Algae from three TAP-S an H2 wt
10Cu samples had small regrowth after exposure to Cu for 1, 2, and 3
days. A profuse culture recovery was observed only for 2 h of exposure
7
Journal of Water Process Engineering 42 (2021) 102123
Fig. 6. Algal viability as observed in bright-
field and fluorescence (inserts) micrographs of
algae. Cultures of algae were used for biore­
mediation of Cu-polluted media for 3 days (A-
E). Using the FDA method, a gradual decrease
of algal viability during 3 days of treatment is
demonstrated (F). Algal morphology and enzy­
matic capacity for FDA cleavage to green fluo­
rescein and its intracellular retention were
evaluated for algae from TAP-S an H2 wt 10Cu
(A), TAP-S a wt 10Cu (B), TAP a wt 10Cu (C),
TAP-S an lowH2 dark wt 10Cu (D), and TAP-S
an lowH2 rbc 10Cu cultures; insert demon­
strates severe damages in rbc culture (E). Two
sequences of images show decreasing algal
viability with the time of treatment for TAP-S
an H2 wt 10Cu culture (upper) and TAP-S a
wt 10Cu culture (lower) (F).
Fig. 7. Algal cultures regrown in microplate with fresh medium following
treatment of Cu-polluted media. Algae were collected from media at different
treatment times: at days 0 (2 h), 1, 2, and 3. The samples analyzed were TAP-S
an H2 wt 10Cu (samples _1, _2, and _3), TAP-S a wt 10Cu, TAP a wt 10Cu, TAP-S
an lowH2 dark wt 10Cu, and TAP-S an lowH2 rbc 10Cu. (After the first and the
second day post-Cu injection into the TAP medium, low pressure in a glass
reactor did not permit collecting samples; see the empty wells in the first line of
the microplate).
to Cu (Fig. 7; second, fifth, and seventh lines). A combination of sulfate
deprivation, anoxia (required for H2 production), and Cu exposure
combined with irradiation impaired algae considerably. The observed
impairments manifested in reduced Cu NPs synthesis as suggested by
UV–vis spectra (Fig. 2A). In contrast, significant algal recovery was
observed from the TAP-S a wt 10Cu culture. Regardless of the Cu
treatment length, the algae recovered into dark green cultures (Fig. 7;
fourth line).
The algae from TAP-S an lowH2 dark wt 10Cu culture showed limited
viability by the fluorescent FDA method and preserved cell integrity in
the bright-field (Fig. 6D). This algal culture recovered better than the H2
producing culture. Algal regrowth was successful for all Cu-pollution
U. Žvab et al.
exposure times (Fig. 7; third line). The anaerobic TAP-S culture of the
light-sensitive algal strain rbc was destroyed after the Cu treatment
under moderate light conditions as indicated by bright-field micro­
graphs (Fig. 6E). However, the rbc cells successfully recovered in the
microplate wells under the low-moderate light conditions in fresh
nutrient media (Fig. 7; sixth line). Recovery was slower than for the wt
algae from the aerobic TAP a wt 10Cu or TAP-S a wt 10Cu samples, but
faster than for the wt algae from the TAP-S an H2 wt 10Cu samples.
Regardless of the relatively high Cu-pollution previously observed to be
toxic for the wt C. reinhardtii [60], the algal culture was still able to
recover once Cu exposure had ended. The regrowth method provided
additional evidence for the importance of algal viability for efficient Cu
NPs synthesis. Furthermore, the regrowth method has demonstrated
that after Cu-bioremediation and Cu NPs recovery from temporally
high-Cu industrial discharge, algae may continue to be used in waste­
water treatment.
4. Conclusions
Treatment of Cu polluted wastewater-like complex nutrient media
with C. reinhardtii in aerobic and anoxic conditions with photoproduced
H2 formed brown and green media supernatants. Cupric ISE demon­
strated the removal of toxic free Cu2+ from polluted media by algal
treatment. AAS measurements revealed Cu’s persistence in media after
treatment, while the UV–vis spectra provided evidence for the formation
of Cu-based NPs. Experiments conducted with the impaired light-
sensitive rbc strain of C. reinhardtii and with sonicated algal cells,
disprove only the formation of soluble Cu complexes with cellular
components. Together with experiments conducted in dark conditions,
these experiments also suggest that viable algae are required for Cu NPs
biosynthesis. Algal viability assessment by FDA method, cell
morphology observations, and post-treatment regrowth of algae
confirmed efficient biosynthesis of Cu NPs by metabolically more active
and less impaired algal cultures. TEM observations of supernatants
proved the existence of stable sub-10 nm phase-pure metallic Cu NPs in
different samples. Biosynthesized Cu NPs were co-localized with organic
objects in all three samples analyzed by TEM. Organic ligands appear to
be responsible for protecting Cu NPs from further growth, agglomera­
tion, and oxidation between and after biosynthesis. The observed af­
finity of biomineralized Cu NPs for organic objects promises Cu NPs
recovery from complex media by tailored organic adsorbent in the next
step.
Funding information
This work was supported by the European Regional Development
Fund and Ministry of Education, Science, and Sport of the Republic of
Slovenia (Raziskovalci-2.0-UNG-529037); and Slovenian Research
Agency (research core funding No. P2− 0412).
Declaration of Competing Interest
The authors declare that they have no conflict of interest.
Acknowledgments
We appreciate the knowledge provided and data shared by the
members of the wastewater treatment section of Komunalno stano­
vanjsko podjetje d.o.o. Ajdovščina, Slovenia. We thank Philipp Mäckle
for his help with the set-up of fluorescence microscopy analysis. We are
very grateful to the Wine Research Center (UNG) for sharing their fa­
cilities for the microbiological research work and Laboratory for Envi­
ronmental and Life Sciences (UNG) for cupric ISE and allowance for AAS
instrument use. We thank Petra Makorič (Laboratory for Environmental
and Life Sciences, UNG) for assistance with the AAS measurements. We
also thank dr. Iain Robert White, dr. Vidya Kunnathully and Michael
8
Journal of Water Process Engineering 42 (2021) 102123
Saglimbene for their assistance with the final text editing. The authors
gratefully acknowledge the funding of this work by the European
Regional Development Fund and the Ministry of Education, Science, and
Sport of the Republic of Slovenia (Raziskovalci-2.0-UNG-529037) and
the Slovenian Research Agency (research core funding No. P2-0412).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jwpe.2021.102123.
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