Journal of Environmental Management 253 (2020) 109706

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Journal of Environmental Management

journal homepage: http://www.elsevier.com/locate/jenvman

Research article

Bioremediation of copper by active cells of Pseudomonas stutzeri LA3

isolated from an abandoned copper mine soil
Thenmozhi Murugaian Palanivel, Nallusamy Sivakumar, Aliya Al-Ansari, Reginald Victor *
Department of Biology, Sultan Qaboos University, P.O. Box 36, 123 Al-Khoud, Muscat, Oman

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

Keywords: Copper bioremoval efficiency and bioaccumulation capacity of Pseudomonas stutzeri LA3 isolated from copper
Copper contaminated soil were investigated. P. stutzeri LA3 removed about 50% of Cu (II) at 50 mg l 1 of concentration
Pseudomonas stutzeri LA3 and accumulated a maximum of 1.62 mg of Cu g 1 biomass dry weight. Bioremediation by P. stutzeri LA3
Bioremediation
partially depended on the production of extracellular polymeric substances, composed of proteins and carbo-
Extracellular polymeric substances
hydrates. Cell surface alterations were observed on the Cu (II) treated biomass through a scanning electron
Scanning electron microscope
FTIR analysis microscope (SEM). Energy dispersive spectrometer (EDX) investigation of Cu (II) treated biomass showed clear
signals of Cu, confirming the presence of copper ions on the cell surface. Fourier transform infrared spectroscopy
(FTIR) showed the contribution of functional groups such as hydroxyl (–OH), carboxyl (–COOH), amide and
amine (–NH2) in the remediation process. Based on the results, the isolated strain P. stutzeri LA3 could serve as a
potential candidate for copper due to its significant copper removal effeciency.

1. Introduction complete restoration (Siddiquee et al., 2015; Ayangbenro and Babalola,

2017).
Heavy metals are termed as metals and metalloids with the atomic One of the processes of bioremediation is the use of microbes to
density more than 4000 kg m 3 or five times greater than that of water remove environmental contaminants. Despite the opinion of some sci-
(Hookoom and Puchooa, 2013). Heavy metals discharged from several entists who think bioremediation of heavy metals is not useful due to
industrial activities, for instance mining, smelting and ore processing their lethality in high concentrations (Rizwan et al., 2014), it has some
processes easily generate pollution and cause adverse impacts on living strong merits. However, integrating nanoparticles with the microbial
organisms and environment (Tunali et al., 2006). cells can enhance the clean-up of polluted environment because they
Even at minimum concentrations, toxic metals could be the source have strong reactive sites which in contact with contaminants may
for several diseases and negative effects on health and so, heavy metals facilitate immobilization or detoxification (Tratnyek and Johnson,
present in concentrations above standard limits should be removed from 2006; Jacob et al., 2018).
the environment (Aryal and Liakopoulou-Kyriakides, 2015). Physico- Microbial bioremediation can be carried out in situ with a low
chemical and biological methods could be used for remedying the metal negative impact on the environment (Farhadian et al., 2008; Guo et al.,
polluted sites. Another recent advance is to use environmental nano- 2010). It does not create secondary toxic compounds and is inexpensive
technology for removing heavy metals (Rizwan et al., 2014; Street et al., when compared to chemical processes (Kadukova and Vircikova, 2005;
2014; Rawtani et al., 2018). Surface modified halloysite nanotubes Ayangbenro and Babalola, 2017). Many microbes effectively act as
(HNT) have been used to remove dyes and heavy metals such as Cu, Pb, adsorbing agents in the removal process of heavy metals (Borah and
Fe, Pd, Cr, Zn, Co, Ag, and Bi (Yu et al., 2016) and several HNT-based Yadav, 2014; Iram et al., 2015). They could also accumulate heavy
nanocomposites have also been developed for the same purpose (Thar- metals through absorption. Adsorption is a surface phenomenon, while
mavaram et al., 2018). The physicochemical processes such as reverse absorption involves the entire volume of material (Jin et al., 2018).
osmosis, ion-exchange resins, filtration, evaporation and chemical pre- Adsorption comprises complexation of heavy metal on the cell surface,
cipitation are very expensive and inefficient. They also produce sec- from where it could be absorbed into the cell (Danis et al., 2008).
ondary toxic compounds, which should be neutralized to achieve According to Malik (2004), the uptake of toxic heavy metals by

* Corresponding author. Department of Biology, College of Science, P.O. Box 36, PC 123, Sultan Qaboos University, Al Khoud, Oman.
E-mail address: [email protected] (R. Victor).

https://doi.org/10.1016/j.jenvman.2019.109706
Received 29 June 2019; Received in revised form 9 October 2019; Accepted 12 October 2019
Available online 18 October 2019
0301-4797/© 2019 Elsevier Ltd. All rights reserved.
T.M. Palanivel et al. Journal of Environmental Management 253 (2020) 109706

bacterial cells and their transport into the cell by the metabolic cycle is followed by 25 cycles of 94 � C for 30 s, 50 � C for 30 s, 72 � C for 4 min and
defined as bioaccumulation. It involves various mechanisms like metal the final extension was performed with 72 � C for 10 min. The amplified
binding on inter- and intracellular compounds, precipitation and PCR products stained with ethidium bromide in 2% agarose gels were
methylation. The components of bacterial cell wall and different pro- examined by electrophoresis. The gels were checked by gel photo
teins in the cell wall are responsible for the binding of metals (Aryal and GS-800 system (Ingenius, Rodermark). PCR products were purified
Liakopoulou-Kyriakides, 2015). Bacteria also have numerous functional using QIA PCR purification kit. PCR products were sequenced with Big
groups such as –COOH, –SH, and –OH on their surfaces that help in the Dye Terminator kit, consisted of Big dye sequencing mix, 5X sequence
metals adsorption (Gadd, 2009). buffer, PCR product, PCR water, forward, reverse and internal primers
In general, bacteria respond to harsh environments like heavy metal 3 pmol/μL each. Sequencing purification was done using the BigDye
contaminated sites by discharging extracellular polymeric substances XTerminator purification kit and finally, sequencing was performed by
(EPS) from the cell surface (Colica et al., 2014). In general EPS exhibit a Genetic analyzer 3130 xl. Bio edit software was used to edit the se-
high affinity for copper (Fang et al., 2011; Cornu et al., 2017). The quences and was compared against the 16S rRNA collection of sequences
prospective role of EPS in the removal of heavy metals from the envi- in the GenBank database with the BLASTn program. A phylogenetic tree
ronment is because of their contribution in flocculation and ability to was constructed with MEGA7 software by UPGMA method.
bind metal ions from solutions (Pal and Paul, 2008). EPS matrix exhibits
adsorption and biodegradability owing to its special components such as 2.2. Copper removal efficiency
carbohydrates, proteins, humic substances and nucleic acids. It plays a
critical role in biofilm formation, mass transfer of heavy metals via The copper removal efficiency was investigated in 250 ml Erlen-
biofilm and adsorption through biofilm; it also offers extensive struc- meyer flasks containing 100 ml LB broth with various concentrations of
tural support to the biofilm (More et al., 2014). Cu (II) (50, 100, 150, 200 and 400 mg l 1). The flasks were inoculated
Copper is a trace metal and its average concentration in soil ranges with overnight culture and incubated at 32 � C for 96 h at 160 rpm. Cell
between 13 and 24 mg kg 1 (Kabata-Pendias and Pendias, 2001). The growth and the residual copper concentrations were analyzed from the
topsoil (0–30 cm) of the copper-mined tailing areas contained copper in aliquots (5 mL) collected at regular intervals (24 h). Growth was moni-
several thousand mg kg 1 of soil (Shutcha et al., 2015; Cornu et al., tored by measuring the optical density using a UV–Vis spectrophotom-
2017). In general, anthropogenic copper is more mobile as it is more eter at 600 nm (Thermo Scientific) and Cu concentrations were
bioavailable to the living organisms than pedogenic copper (Duplay measured using an inductively coupled plasma optical spectrometer
et al., 2014). Copper can directly enter into the food cycle from plants (ICP-OES) (PerkinElmer Inc. OPTIMA-7300DV, USA). Control experi-
and tend to accumulate in the human body where, the excess copper ments were also conducted in parallel without the addition of bacterial
causes various health problems including chronic anemia, liver cells. All the experiments were performed in triplicate. Percentage (%)
cirrhosis, brain and kidney damage (Dixit et al., 2015). removal of Cu (II) was calculated.
The present study aimed to examine the Cu (II) removal and bio-
accumulation capability of Pseudomonas stutzeri LA3, isolated from 2.3. Bioaccumulation of copper
Lasail abandoned copper mine soil in Oman. Further, the components of
extracellular polymeric substances (EPS) produced by P. stutzeri LA3 The Cu bioaccumulation ability of the isolate, P. stutzeri LA3 was
were analyzed. The interactions between Cu (II) ions and functional investigated by the modified method of Wong and So (1993). Cu content
groups on the cell wall surface of its biomass was evaluated using FTIR, in the solution was analyzed by ICP-OES. The bacteria grown without Cu
SEM and EDX analyses. At present, there are about 150 abandoned were used as a blank.
copper mine sites in Oman and this is the first study using microbes to
remediate the contaminated copper mine soil. The strain P. stutzeri LA3, 2.4. Biochemical analysis of the EPS
isolated from the abandoned copper mine soil is indigenous, adopted to
the arid and contaminated environment. It can be cultivated easily EPS produced by the isolate was extracted by a modified method
under in situ conditions. described by Adav and Lee (2008). Carbohydrate content of the
extracted EPS was measured by the phenol-sulfuric acid method, glucose
2. Materials and methods was used as a standard (Dubois et al., 1956) and the protein content was
estimated by the Bradford method with bovine albumin serum as a
2.1. Isolation and identification of copper-resistant bacteria standard (Bradford, 1976).

For isolation of potential copper resistant bacteria, 1 g of soil sample 2.5. Scanning electron microscope (SEM) and energy dispersive X-ray
was added to 100 ml of Luria- Bertani (LB) broth containing 200 mg l 1 spectroscopy (EDX)
of Cu (II) and the flasks were incubated at 32 � C for 48 h at 160 rpm for
enrichment. Further, the enriched broth was serially diluted, and plated A SEM (JEOL JSM-77600F; 20 keV, 76 A, working distance 8 mm,
on LB plates comprising 200 mg l 1 Cu (II), and incubated at 32 � C for probe current 8 A) was used to identify the effect of Cu (II) on the surface
48 h. The colonies on the plates were purified and stored on fresh agar topology and morphology followed by the EDX analysis for the
slants for further study. elemental composition of the isolates before and after the accumulation
The potential strain was identified using 16S rRNA gene sequencing. process (Tunali et al., 2006). For SEM analysis, the cells (Cu (II) treated
The genomic DNA was extracted using Power soil DNA extraction kit and untreated) were washed twice with distilled water, smeared on the
from the fresh cultures using the manufacturer’s instructions. The aluminium sample holder and left for complete drying of bacterial cells.
extracted DNA quantity and quality were examined on 1% agarose gel The dried cells were coated with carbon and examined under SEM
and NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific coupled with energy dispersive X-ray analysis. EDX is one of the
Inc.). The PCR mixture (25 μl) contains 12.5 μl of Master Mix, 1 μl of appropriate tools to assess the elemental and chemical characteristics of
10 pmol 16S rRNA bacterial forward primer 27f (50 -GAGTTTGAT- the adsorbent (Tunali et al., 2006).
CACTGGCTCAG-30 ), reverse primer 1492r (50 - TACGGCTACCTTGT-
TACGACTT-30 ) (Guo et al., 2010), PCR water 3.5 μl and 7 μl of extracted 2.6. Fourier transform infrared spectroscopy (FTIR) analysis
DNA. PCR was performed in a thermal cycler (Eppendorf) with the
following program: five cycles of 94 � C for 30 s, 60 � C 30 s, 72 � C for FTIR was used to distinguish the molecular structural confirmation
4 min and five cycles of 94 � C for 30 s, 55 � C for 30 s and 72 � C for 4 min and fluctuations of functional groups before and after Cu (II)

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T.M. Palanivel et al. Journal of Environmental Management 253 (2020) 109706

Fig. 2. Copper removal effeciency of P. stutzeri LA3 at various concentrations of

Cu (II) (50, 100, 150, 200, 400 mg l 1) and at different times (0, 24,48,
Fig. 1. Growth of P. stutzeri LA3 as biomass (OD) at various concentrations of 72, 96 h).
Cu (II) (0, 50, 100, 150, 200 and 400 mg l 1) and at different times (0, 24, 48,
72, 96 h).
150, 200 and 400 mg l 1 respectively (Fig. 2). The results revealed that
the higher removal efficiency was achieved at 50 mg l 1 of Cu (II) con-
bioaccumulation. The biomass was dried, mixed with a KBr matrix
centration. A two-way ANOVA exhibited that a significant difference in
(Merck) at a ratio of 1 : 100 to prepare pellets. The band was recorded in
the removal of copper (primary variable) by P. stutzeri LA3 among Cu (II)
the range of 400–4000 cm 1 with a resolution of 4 cm 1 (Perkin–Elmer
concentrations of 50, 100, 150, 200 and 400 mg l 1 (F ¼ 295.379;
Spectrum RX I FTIR). The band of background was plotted by scanning
P < 0.0001). Post hoc comparisons using Tukey HSD showed no signifi-
the pure KBr using the same scale on the transmittance axis (Pugaz-
cant difference between 150 and 200 mg l 1 of Cu (II) and the removal at
hendhi et al., 2018).
50, 100 and 400 mg l 1 concentrations were significantly different from
each other (P < 0.0001). The incubation time (secondary variable) also
2.7. Statistical analysis significantly influenced the removal rate of copper (F ¼ 1991.807;
P < 0.0001). Also, the removal efficiency of copper is negatively corre-
All statistical procedures were performed using Microsoft Office lated to the initial Cu (II) concentration. Likewise, Aryal and
Excel 2016 and the Statistical Package for the Social Sciences (SPSS) Liakopoulou-Kyriakides (2015) suggested that at low concentrations,
Version 21. the metal ions existing in the solution could augment the interaction
among the binding sites of bacteria and metal ions resulting in increased
3. Results and discussion sorption effectiveness. Therefore, it seems that high concentaration in-
hibits the growth rate of bacteria and reduce the metabolic rate by
3.1. Isolation and characterization of copper resistant bacterium decreasing the number of active cells and potential binding sites. The
increasing concentration of heavy metals reduced the removal efficiency
The copper resistant bacterium isolated from Lasail abandoned of bacterium due to the toxicity of metals, which caused considerable
copper mine soil was identified through 16S rRNA gene sequencing. changes in biochemical and physiological properties such as disturbed
Based on the sequence analysis, the strain was identified as P. stutzeri cellular activities, reduced biomass and denaturation of bacterial en-
LA3, non-spore forming, an aerobic, gram negative rod-shaped bacte- zymes (Oves et al., 2016).
rium (GenBank accession number: MH298766). P. stutzeri is distributed Cu (II) removal of 29, 27, 18, 13 and 4% were achieved at 24 h with
widely in the environment. It is identified as a useful bacterium with initial Cu (II) concentration of 50, 100, 150, 200 and 400 mg l 1
high degradation capabilities for harmful chemical compounds and is respectively. The results confirmed that the removal efficacy was
therefore useful for bioremediation (Cho et al., 2001; Shimada et al., comparatively higher at the log phase of the bacterial growth due to the
2012., Deb et al., 2013; Halder and Basu, 2016). The phylogenetic tree significant increase in growth at 24 h. Anand et al. (2006) reported that
was constructed using the UPGMA method (Supplementary Fig. 1). a good percentage accumulation of Cu (II) was observed with Tricho-
Evolutionary analyses were done in MEGA7 (Kumar et al., 2016). derma viride in lag and early log phase. However, in this study the
removal was maximum at the stationary phase due to the further growth
3.2. Bioremoval of copper by P. stutzeri LA3 of bacteria after the log phase. Malik (2004) suggested that metaboli-
cally active cells arising from the exponential growth phase possibly
The results of the growth studies revealed that the bacterial growth comprise of very active enzymes that are contributing in the process of
increased at 24 h, continued to increase at a lesser rate at 48 and 72 h complexing and binding metal ions from the solution. Rajeshkumar and
and decreased at 96 h (Fig. 1). A two-way ANOVA showed that a sig- Kartic (2011) concluded that the removal of copper was high at sta-
nificant difference in the growth of P. stutzeri LA3 (primary variable) tionary phase though, the number of bacterial cells remains constant but
between control, and Cu (II) concentrations of 50, 100, 150, 200 and metabolically active. The removal of Cu increased with time due to the
400 mg l 1 (F ¼ 147.045; P < 0.0001). Post hoc comparisons by Tukey increase in the number of growing cells providing new binding sites for
HSD showed no significant differences among 50, 100 and 150 mg l 1 of Cu (Pugazhendhi et al., 2018). Manohari and Yogalakshmi (2016) also
Cu (II). As expected, the incubation time (secondary variable) also found that the increase in bacterial biomass leads to increase in binding
significantly influenced the growth rate (F ¼ 2077.456; P < 0.0001). sites for the metal uptake with respect to the increase in time.
Thus, the results of the study showed that the copper concentration
exhibited a significant effect on the growth of the bacterium. 3.3. Bioaccumulation of copper by P. stutzeri LA3
The results of bioremoval study revealed that the Cu removal effe-
ciency of the isolate, P. stutzeri LA3 was about 50, 41, 32, 25 and 9% The copper bioaccumulation study revealed that the isolate,
from the solution at 96 h when the initial concentration was 50, 100, P. stutzeri LA3 exhibited significant accumulation of copper after 96 h.

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T.M. Palanivel et al. Journal of Environmental Management 253 (2020) 109706

(Choudhury and Srivastava, 2002).

3.4. EPS composition and production by P. stutzeri LA3

Polysaccharides and protein are the main biochemical constituents

of the EPS, with a proportion of more than 95% (Zhang et al., 2015).
Table 1 illustrates the EPS composition and production. The results
showed that EPS production was significantly high when Cu (II) was
added in the growth medium. However, the composition and production
varied in different Cu (II) concentrations (Table 1). The means of car-
bohydrate and protein contents in the control were 41.54 and
93.58 mg l 1 at a ratio of 1.00 : 2.25 wheras, the ratios were 1.00 : 1.50;
Fig. 3. Bioaccumulation of Cu (II) by P. stutzeri LA3 at various concentrations. 1.00 : 2.89; and 1.00 : 1.07 at 100, 200 and 400 mg l 1 Cu (II) respec-
The letters a, b, c, d and e show the results of Tukey test. Bars with the different tively. The above findings indicate that there was more EPS production
letters are significantly different at P < 0.05. Error bars represent the standard till the Cu (II) concentration was 200 mg l 1 ;later, the production was
errors of the means (n ¼ 3). decreased due to the addition of more Cu (II); i.e., 400 mg l 1. The dif-
ferences in the ratios of EPS observed in microorganisms could
The accumulated mean values of Cu (II) were 0.54, 1.06, 1.23 and encourage metallic trace element resistance (Ozturk et al., 2010).
1.62 mg g 1 biomass at 50, 100, 150 and 200 mg l 1 of Cu (II), respec- Generally, microorganisms could respond by excreting EPS to ease the
tively (Fig. 3). The results suggested that the accumulation increased effects produced by a sudden exposure to Cu. The EPS are able to
with increasing Cu (II) concentration from 50 to 200 mg l 1 and establish a shield to safeguard the cells from the negative impact of the
considerably reduced (1.34 mg g 1) at 400 mg l 1. The ANOVA test increased Cu (II) concentrations by chelating with Cu (II) (Wang et al.,
confirmed that a significant difference in the accumulation of Cu with 2015).
different concentrations of Cu (II) (F ¼ 142652.68; P < 0.0001). The Chi-square results showed that all concentrations were significantly
maximum accumulation of 1.62 mg g 1 copper was accomplished at the different when compared with the control at P < 0.05. Highest protein
concenttration of 200 mg l 1. Li and Ramakrishna (2011) reported that a and carbohydrate production was reported at 200 mg l 1 Cu (II). The
Pseudomonas strain accumulated a maximum Cu of 0.838 mg g 1 of compositional analysis of EPS showed that protein is the major con-
biomass dry weight at 3 mM Cu. Wong and So (1993) found that the stitutent followed by carbohydrate. Hence, these results demonstrated
Pseudomonas putida accumulated the highest Cu (II) of 65 mg g 1 of dry that in the present study, copper could be more favorably adsorbed
biomass. The results from this study indicated that this strain of extracelluarly by proteins than the carbohydrates. Sheng et al. (2005)
P. stutzeri LA3 also removed Cu through the intracellular seques- stated that the protein present in the EPS played a major role in the Cu
tration/precipitation of metal in addition to adsorption. Gekeler et al. (II) binding. It increased instantaneously while removing the metal ions
(1988) reported that the diffused metals into the cells bind to the che- whereas, the carbohydrate sustained unchanged. The EPS production by
latins or intracellular proteins before getting accumulated to other pure cultures of bacteria derived from the activated sludge mostly
intracellular sites and vacuoles. Furthermore, there are fewer chances consists of proteins and fewer amounts of carbohydrates (Guibaud et al.,
for metals to be returned back to the environment as the processes are 2005). As in this study, Pal and Paul (2008) demonstrated that another
irreversible. Similarly, P. putida strain was able to accumulate metals bacterium, Rhodopseudomonas acidophila produced higher amount of
from the growth medium as well as from Cu mine effluents and tailings protein than carbohydrate when grown with toxic Cu (II), Cd (II) and Cr

Table 1
Composition and production of EPS by P. stutzeri LA3 at different Cu (II) concentrations; Values represented mean � SD; different letter(s) following values within the
columns are significantly different at P � 0.05.
1 1 1
Cu (II) concentration (mg l ) Carbohydrates (mg l ) Protein (mg l ) Ratio Chi-square value Significance
a a
Control 41.54 � 2.24 93.58 � 3.43 1.00:2.25 – –
100 94.09 � 3.46b 141.15 � 2.64c 1.00:1.50 90.66 p < 0.05
200 97.76 � 2.61b 273.60 � 3.31d 1.00:2.79 422.39 p < 0.05
400 94.46 � 4.17b 101.23 � 1.44b 1.00:1.07 68.04 p < 0.05

Fig. 4. Scanning electron microscope images of P. stutzeri LA3 (a) before Cu (II) treated (control) and (b) after Cu (II) treated cells.

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T.M. Palanivel et al. Journal of Environmental Management 253 (2020) 109706

Fig. 5. FTIR spectrum of P. stutzeri LA3 (a) before Cu (II) treated (control) (b) after Cu (II) treated.

(VI). Vijayaraghavan and Yun (2008) reported that the constitutents of these bacteria are thin. A recent study reported that EPS produced by a
gram negative bacteria such as peptidoglycan, phospholipids, and li- strain of Stenotrophomonas maltophilia exhibited 69.1% and 43% of Cu
popolysaccharides are mainly accountable for the anionic nature and and chromium ion removal respectively at the initial concentration of
binding ability of the metal to the cell wall, although the cell walls of 25 mg l 1 (Gupta and Diwan, 2017). Thus the production of EPS seems

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T.M. Palanivel et al. Journal of Environmental Management 253 (2020) 109706

to be an intrinsic self defense mechanism against exposure to high and FTIR results indicated the bioremediation ability of P. stutzeri LA3.
concentrations of heavy metals. The studied strain of P. stutzeri LA3 is indigenous, adapted to the arid
environment and thus, can easily be cultivated under field conditions.
3.5. SEM and EDX analysis However, field trials are necessary to consider its efficacy and cost-
effectiveness in the natural environment.
The surface morphology of the isolate, P. stutzeri LA3 treated and
untreated with Cu (II) was investigated using scanning electron micro- Declaration
scopy (Fig. 4a and b). There was a significant morphological difference
between Cu (II) treated and untreated cells. The untreated cells were The authors express that they do not have any conflict of interest.
very clear and discrete in appearance, while the treated cells were
clumped and shiny because the cell surface was covered by Cu (II) Acknowledgements
particles in addition to the production of EPS. Several other studies re-
ported that the metal treated bacterial cell surface was altered and We greatly acknowledge the Department of Biology, College of Sci-
impaired by the heavy metal ion accumulation (Colak et al., 2013; Aryal ence, Sultan Qaboos University for partially supporting this project.
and Liakopoulou-Kyriakides, 2015). The heavy metal remediation by
microbes dictates the alteration of cell surface and clustering of cells to
Appendix A. Supplementary data
alleviate the toxic effects (Jacob et al., 2018).
The adsorption of Cu (II) ions on to P. stutzeri LA3 was analyzed by
Supplementary data to this article can be found online at https://doi.
EDX. Supplementary Fig. 2a and b showed the typical EDX pattern for
org/10.1016/j.jenvman.2019.109706.
Cu (II) untreated and treated, P. stutzeri LA3 respectively. The EDX
pattern for untreated biomass did not exhibit the indication for Cu
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