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Bioremediation of noxious metals from e-waste printed circuit boards by

Frankia

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DOI: 10.1016/j.micres.2021.126707

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Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Bioremediation of noxious metals from e-waste printed circuit boards

by Frankia
Marappa Narayanasamy, Dharumadurai Dhanasekaran *, Nooruddin Thajuddin
Department of Microbiology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu, 620 024, India

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

Keywords: The environmental noxious e-waste was collected and physicochemical characterized by Scanning electron
E-waste microscopy (SEM) along with energy dispersive X-ray spectroscopy (EDX), Atomic absorption spectrometry
Toxic heavy metals (AAS), and X-ray diffraction analysis (XRD) exploration to understand the presence of toxic metals like Hg, Cd,
Frankia
Pd, Si, Ru. Therefore, the finding provides vital knowledge about the impact of toxic metals from e-waste printed
Two step bioremediation
circuit boards as contaminants in the environment and its impact on humans. The Frankia sp. DDNSF-03 and
Frankia casuarinae DDNSF-04 were isolated and identified, further utilized for removal of e-waste toxic metals by
one and two steps bioremediation experiments executed with various e-waste concentrations. The two-step
bioremediation experiment is efficient in the expression of toxic metals that were removed at a lesser concen­
tration of e-waste. Consequently, the presence of organic acids in the Frankia primary metabolites was confirmed
by FT-IR analysis besides decreasing the pH level in the Frankia growth medium. The positive control Frankia and
negative control e-waste were maintained throughout the bioremediation experiments. The initial Hg 4.3, Cd 8.3,
Pd 4.6 (ppm) in the e-waste and final treated with Frankia sp. DDNSF-03 Hg 0.09, Cd 5.09, Pb 0.49 (ppm), and
Frankia casuarinae DDNSF-04 Hg 2.15, Cd 5.6, Pb 2.82 (ppm) concentration of toxic metals was quantified by
AAS spectrum analysis. The toxic metals mercury and lead were significantly mineralized by Frankia sp. when
compare the Frankia casuarinae. The above finding was confirmed the manifestation of morphological changes by
an accumulation of e-waste in Frankia hyphae using SEM analysis and obtain the qualitative of toxic metals
parallel peaks in EDX analysis.

1. Introduction
Nowadays e-waste is one of the emerging problems in environmental
and human health, due to the large modern electronic uses and short
lifespan. The overuses of innovative electronic product and short life­
span it generated numerous amount e-waste (Narayanasamy et al.,
2017, 2018, 2020a,b,c,d). According to the United Nations Environment
Programme (UNEP) report in 2017 worldwide e-waste generation is
44.7 million metric tonnes (Mt) and it visualizes by the year 2021,
e-waste generation increased 200–500 % from 2017 in developing and
underdeveloped countries such as India, South Africa, and China. The
total e-waste accumulation in worldwide only 15–20 % was recycled or
reused and 70–80 % is discarded directly in the environment. Vats and
Singh (2014) were described the dispose of cell phones will be 9–22
times higher in 2009 condition particularly in China and India. The
e-waste printed circuit boards (PCBs) consist of 1000 different materials
under harmful and harmless groups like Lead 1.03 %, Copper 20.01 %,
Tin 2.05 %, Aluminium 7.03 %, and Nickel 5.76 % (Leung et al. (2006),
Sohaili et al.2012; Willner (2012), Zhang et al.2015; Yuan et al. (2018).
Liu et al. (2017) described industrial wastes and their environmental
issues. During the production of industrial wastes, some toxic elements
are leached into the ambiance and create severe ecological issues. In­
dustrials are mainly produced some different verities of wastes such as
fly ash, e-scraps, lithium batteries, petroleum catalysts, sewage sludge,
etc. E-waste PCBs encloses high level of noxious materials that have
critical impacts on ecology and human being if not carry out perfectly or
improper recycling (Pinto, 2008). The major issue of e-wastes contam­
inated in soil, groundwater and atmosphere due to their occurrence of
toxins, heavy metals, dioxins and furans are responsible for the high
level of pollutions (Lee et al. (2007), Yoo et al.2009; Yamane et al.
(2011), Kumar et al.2013.
E-waste PCBs recovery and recycling is essential because of their
noxious pollution effect and demand for precious electronic raw re­
sources (Narayanasamy et al., 2017, 2018 and 2020a,b,c,d, Guo et al.,

* Corresponding author.
E-mail addresses: sam16@bdu.ac.in (M. Narayanasamy), ddhanasekaran@bdu.ac.in (D. Dhanasekaran).

https://doi.org/10.1016/j.micres.2021.126707
Received 28 October 2020; Received in revised form 20 December 2020; Accepted 9 January 2021
Available online 19 January 2021
0944-5013/© 2021 Published by Elsevier GmbH.
M. Narayanasamy et al.
2009). Generally the conventional hydrometallurgical and pyrometal­
lurgical approaches are normally used to recycling and recovery of
metals from e-waste (Xu et al. (2010), Pant et al.2012; Achal et al.
(2012). The traditional methods of e-waste recycling polluted the total
environment via expulsion of dioxins and furans and also high energy
expenditure process for the recycling of e-waste (Mack et al. (2007);
Borthakur and Sinha (2013); Khaliq et al. (2014), Liu et al.2017; Hu
et al. (2020).
In a vision of the ecological and financial profit, increasing aware­
ness has been selected to the advance procedure to the recovery of
metals from e-waste PCBs.
Recently introduced promising bio-based approach for microbial
cells interact to extort and revival of metals from e-waste. The bio-based
progress is most inexpensive and alternative to other previous expensive
techniques to extract expensive metals elements and recycling the
noxious components from the e-waste PCBs. This process demand low
energy and a reduced amount of e-waste production outcome (Nar­
ayanasamy et al., 2020a,b,c,d, Kavitha (2014), Khaliq et al.2014; Liu
et al. (2017). Ilyas and Lee (2014) and Das (2010) discuss the biological
process and there dynamic metabolism of microorganisms. The organ­
isms are conveying into biomass inside the cellular arrangement
concerted there, course of action demand vital metabolism is called
absorption or bioaccumulation. During this process, some toxic e-waste
metals are accumulated like lead, cadmium, uranium, and chromium.
Generally, the effective organisms are used for bioabsorption such as
Micrococcus luteus, Aspergillus niger, Bacillus spp. etc. (Narayanasamy
et al., 2017; Borthakur and Sinha, 2013). Andres and Gerente (2011),
Tanaka and Watanabe2015 are clarified about the biotransformation
process of heavy metals from e-waste. Generally, this process occurs via
biochemical reaction of oxidation and reduction of chemical exchange.
Biotransformation occurs in 2 ways directly enzymatic heavy metals
reduction and cell indirect e-waste metals reduction. During the process
in which noxious metal ions join between ions or ligands formed since
the microorganisms to appearance precipitation. The mineralization of
toxic metals from e-waste PCBs is very difficult and major challenges in
the worldwide. However, the e-waste bioremediation potential of native
isolate Actinobacteria Frankia from Tamil Nadu, India is unexplored.
Hence, the present research was undertaken to perform mineralization
of toxic metals from e-waste PCBs using native isolates of Frankia sp.
DDNSF-03 and Frankia casuarinae DDNSF-04 under one-step and
two-step bioremoval experiments.
2. Materials and methods
2.1. E-waste collection and process
The electronic waste in the form of printed circuit boards (PCBs) was
collected together from marine polluted environment located in Pudu­
pattinam, Muthupettai (Latitude: N 10◦ 47’ 13”, Longitude: E 79◦ 8’
16”), Tamil Nadu, India. Collected e-waste PCBs were crushed by we
previously published novel and economic method of Narayanasamy
et al. (2018, 2020a,b,c,d). The crushed fine particles were used under
one-step and two-step bioremediation experiments.
2.2. Actinobacteria Frankia strains
The Frankia was isolated from root nodules of Casuarina cunning­
hamiana plant from an estuarine region of Casuarina forest of sandy soil
in Pudupattinam, Muthupettai (Latitude: N 10◦ 47’ 13”, Longitude: E
79◦ 8’ 16”). Saline region of sandy soil in Dhanushkodi, Rameswaram
(Latitude: N 34◦ 9’ 9", Longitude: E 77◦ 34’ 37") Tamil Nadu, India. The
identified Frankia sp. DDNSF-03 and Frankia casuarinae DDNSF-04 were
deposited in NCBI database with the accession numbers MH797560 and
MH797561. The Frankia were maintained in standard DPM medium
(Defined propionate minimal medium) and used for bioremediation
experiments. DPM medium contains in 1000 mL composition: K2HPO4 4
2
Microbiological Research 245 (2021) 126707
g, KH2PO4 7 g, MgSO4 0.1 g, CaCl2 0.07 g, Fe Na EDTA 1 mL, NH4Cl 0.5
g, Pyruvate 1 g, Propionate 1 g, Microelements 1 mL, Biotin 1 mL,
Pyridoxamine dihydrochloride 0.1 g, NaCl 1 g (Gtari et al., 2013) and
(Narayanasamy et al., 2020a,b,c,d).
2.3. Acid digestion of e-waste
2.3.1. Hot plate aqua-regia digestion
The crushed e-waste was acid digested with slight modification of
Ehi-Eromosele et al. (2012) method. 0.5 g of e-waste crushed fine par­
ticles was adding to a pointed container and 15 mL of newly prepared (5
mL HNO3 + 10 mL HCl ratio 1:2) was added. The mixture allowed
heating continually for 2–4 h on a hot plate and the mixture of intensity
was maintained the same condition. After heating treatment allowed to
chill completely and filtered fine suspension from a mixture of e-waste
treatment. The filtrate suspension was concentrated in 50 mL of
deionized distil water. The diluted suspensions were consequently
explored via atomic absorption spectroscopy (SHIMADZU- AAS, Japan)
(Narayanasamy et al., 2018, 2020a,b,c,d).
2.4. Characterization of e-waste
2.4.1. Analysis of e-waste PCBs by SEM with EDX
Investigation of e-waste PCBs through scanning electron microscope
beside through energy-dispersive X-ray spectroscopy followed by
methods of Bajestani et al. (2014) and Narayanasamy et al. (2018,
2020a,b,c,d). Scanning electron microscope JEOL JSM-5600LV exposes
and understanding the morphological arrangements in e-waste PCBs
sample. The e-waste PCBs samples desiccated fine particles were laden
resting on to copper stubs with help of carbon tape and covered with
10− 1 m gold atoms with an auto-fine coater over vacuity on 40 mA in
favor of 60 s as well as analyzed the morphology of e-waste. The
energy-dispersive X-ray spectroscopy (VEGA3 TESCAN Bruker, USA)
were clarified and understanding the heavy metals element composition
their occurrence in the e-waste PCBs sample.
2.5. X-ray diffraction (XRD)
Exploration of e-waste PCBs heavy metal elements intensity peaks
through X-ray diffraction (Rigaku Ultima all, Japan) investigation be
there followed by the method of Chatterjee and Kumar (2009) and
Zhang et al. (2012). The e-waste PCBs samples were dried and fine
particles were allowed to the internal center of the surrounding sub­
stance in the matrix, furthermore, the reach your peak range be explored
in favor of every metal beginning 0 to 380 2Ø toward identifying the
metal elements found on top of the peak vary. The specific peaks go on
documented resting from the source of Joint Committee Powder
Diffraction Standards (JCPDS) statistics file.
2.6. Bioremediation experiments
Bioremediation experiments were performed under the one-step and
two-step bioremediation the process followed by the method of Nar­
ayanasamy et al. (2018, 2020a,b,c,d). One-step and two-step bioreme­
diation were performed with Frankia sp. DDNSF-03 and Frankia
casuarinae DDNSF-04 along with different percentage of e-waste 0.2,
0.4, 0.6, 0.8, 1.0 % in standard DPM medium (Defined Propionate
Minimal medium). In one-step bioremediation the Frankia and e-waste
pulp density was allowed in DPM growth medium at the same time of
the experiment. In two-steps initially the Frankia the culture was alone
to grown in standard DPM medium for 3–5 days. After incubation to add
e-waste pulp density same as one-step process furthermore incubated on
28 ◦ C for 30 days.
Therefore positive control (medium beside with culture) and nega­
tive control (medium along with e-waste) were maintained during the
bioremediation experiments for confirmation of pH variation.
M. Narayanasamy et al.
Fig. 1. Process of two-step bioremediation with different concentration (0.2, 0.4, 0.6, 0.8 and 1.0 %) of e-waste by Frankia. NC-Negative control (e-waste along with
medium without culture), PC-Positive control (medium with Frankia culture without e-waste).
2.7. Determination of pH and biomass
Investigation of pH and biomass variation during one-step and two-
step bioremediation experiments were carried out by slight modification
methods of Lamma and Swamy (2015), Shah et al.2015 and Nar­
ayanasamy et al. (2018, 2020a,b,c,d). 5− 10 mL of bio removal residue
solution was obtained during the bioremediation experiments for
frequently at 5-day intermissions. The pH variations were observed via
digital pH measuring device (Analab Scientific Instruments Pvt. Ltd,
India). The bio removal residue culture broth was obtained besides
filtered the biomass and calculate the wet and dried biomass weight with
the help of a weight measuring device.
2.8. Analysis of bioremoval residue by AAS
Exploration of noxious heavy metals elements via atomic adsorption
spectroscopy (SHIMADZU- AAS, Japan) from bio removal residues of
bioremediation experiments followed by the method of Narayanasamy
et al. (2018, 2020a,b,c,d). After bioremediation treatment, the bio
removal residues were acquired and acid digested through hot plate
aqua-regia digestion method already discussed above. The concentrated
the fine suspension was used for investigation of noxious heavy metals
elements (ppm) from bio removal residues.
Fig. 2. Mechanism of bioremediation (a). Bioaccumulation of e-waste PCBs mechanism by Frankia (b). Bioremediation of e-waste by phosphatase exoemzymes
of Frankia.
3
Microbiological Research 245 (2021) 126707
2.9. SEM with EDX spectra of bioremoval residue
Investigation of bioremoval residue by the use of scanning electron
microscope via the side with energy-dispersive X-ray spectroscopy
(VEGA3 TESCAN Bruker, USA) followed by methods of Narayanasamy
et al. (2020a,b,c,d), Bajestani et al. (2014), Rastegar et al.2015. The
scanning electron microscope was revealing the Frankia morphological
variations during the bioremediation experiment and understanding the
e-waste accumulation on the bacterial hyphae. After bioremediation
treatments, the bio removal residue was collected and dried and the
desiccated fine residue was set by way of 3% (v/v) glutaraldehyde
within distill water on 4◦ c as well as dehydrated in excess of ethanol
gradient and analyzed the morphology of bio removal residue. The
energy-dispersive X-ray spectroscopy was elucidating the heavy metals
element composition and understanding their happening in the removal
residue sample.
2.10. XRD spectrum of bioremoval residue
Detection of heavy metal elements from two-step bioremediation
experiment of bio removal residue based on their intensity of peaks
range via X-ray diffraction (Rigaku Ultima all, Japan) analysis followed
by methods of Tanaka and Watanabe (2015) and Rastegar et al. (2015).
After bio removal treatments, the residue was collected and dried with
the help of lyophilization. The desiccated fine residue was obtained and
M. Narayanasamy et al.
Fig. 3. pH and biomass profile of e-waste bioremediation using Frankia sp. DDNSF-03. and F. causarinae DDNSF-04 (a). One step bioremediation and (b). Two step
bioremediation (c). Positive and negative control both steps.
allowed to the internal central part of surrounding substance in the
matrix and explore in favor of heavy metal component based on the
intensity of peaks size. The identified intensity peaks were used Joint
Committee Powder Diffraction Standards (JCPDS).
2.11. FT-IR spectra of bio removal residue
Exploration of organic acid dynamic functional groups from bio
removal residue via Fourier transforms infrared spectroscopy (FT-IR)
(PerkinElmer, Spectrum TWO, Llantrisant, UK) was executed by minor
alteration of Bullen et al. (2008), Sethurajan et al.2012 and Nar­
ayanasamy et al. (2018, 2020a,b,c,d) methods. Following the two-step,
bioremoval treatments of residue were obtained furthermore filtered
throughout Whatman No. 1 sifts term paper for 2–3 times to eliminate
solid particles. Therefore the superior suspension was used for revealing
the organic acids functional groups occurrence in the removal residue.
3. Result and discussion
3.1. Mechanism of bioremediation
The mechanism of bioremediation is the conversion of materials into
a new-fangled composite substance throughout biochemical and meta­
bolic response of bacteria. Frankia sp. DDNSF-03 and Frankia casuarina
DDNSF-04 have produced a combination of secondary metabolites of
organic acids and enzymes are complicated with toxic heavy metals
from e-waste designed for bioremediation (Fig. 1). The growth system of
one-step bioremediation experiment emergence less turbidity formed
with 0.2, 0.4, 0.6, 0.8, and 1.0 % of e-waste fine particles (Fig. 2). The
particular growth rapidity was reduced at the same time as e-waste
concentration was increased suitable in the direction of the inhibition
consequence toward noxious e-waste PCBs.
4
Microbiological Research 245 (2021) 126707
Narayanasamy et al. (2018, 2020a,b,c,d), Yang et al. (2009a,b) and
Rastegar et al. (2015) previous researchers are reported the turbidity
formation or precipitation velocity is affected appropriate toward high
e-waste flesh concentration during one-step bioleaching process.
The growth system of two-step biodegradation experiment appear­
ance cluster turbidity fashioned with the same concentration of e-waste
as discuss above one-step experiment. In the two-step process to form
rapid metal precipitation affects along with bacteria. The pH was highly
decreased within two-step experiment as the organic acids are previ­
ously there inside the medium earlier toward the adding e-waste PCBs.
Consequently, the biomass was highly increased in low flesh solidity of
e-waste concentration. As a result, secondary metabolites were highly
secreted due to the pH and biomass effect on two-step biodegradation
experiment. Lamma and Swamy (2015) and Narayanasamy et al. (2018)
researchers have described as the two-step bioleaching process. During
the extraction of the metals in the two-step, there is no more toxic effect
when viewing of the one-step metal extraction process due to the high
level of secondary metabolites and organic acid production.
Yang et al. (2009a,b); Tanaka and Watanabe (2015) researchers are
communicated about the enzymatic reaction of toxic metals biodegra­
dation. The enzymatic reaction occurs in two way direct and indirect
reaction of metals reduction. The indirect reaction of secondary me­
tabolites involved a high level of toxic metals removal when compared
to the direct method. Gtari et al. (2013) was suggested about the sym­
biotic role of Actinobacteria and their importance of Actinorhizal plants.
Actinobacteria Frankia capable to bioremoval the toxic heavy metals
from e-waste which polluted the marine and other agricultural lands.
3.2. pH and biomass profiles during bioremediation
The manifestation of pH and biomass variations during the biore­
mediation process implicated a vital role in the elimination of noxious
M. Narayanasamy et al.
Fig. 4. AAS bioremoval of toxic metals from e-waste by Frankia untreated e-
waste, treated with Frankia sp. DDNSF-03 and treated with F. causarinae
DDNSF-04.
metals elements from e-waste PCBs. In the one-step bioremediation, the
pH and biomass similarity was remarkably slow at a particular con­
centration of e-waste PCBs. During the one-step bioremediation process,
the e-waste pulp density was prevented the growth expansion of Frankia
due to their toxic effect on immature bacteria. Therefore the pH was
reduced due to the initiate organic acids creation on day by day inter­
mission based on the biomass enhancement of Frankia and fewer con­
centrations of e-waste PCBs (Fig. 3).
Narayanasamy et al. (2018, 2020a,b,c,d) was described the bio­
leaching of e-waste, during the progression of one-step bioleaching the
fungal originate growth was slow down proper toward the noxious a
consequence of e-waste high resistance. In two-step bio-removal process
the pH and biomass affinity was tremendously high variation at a
particular combination of e-waste PCBs. Accordingly the two-step
bio-removal progression the bacteria was tolerant of the e-waste pulp
density due to their high intensity of biomass and secondary metabolites
of organic acids. Based on the e-waste concentration the level of toxic
heavy metal elimination course of action was varied when estimate in
one-step removal process two-step was immensely removed the noxious
metals (Fig. 3). In the positive control pH and biomass were gradually
increased at a high level for day by day intermission when appraising to
bio-removal treatments. The major explanations for pH and biomass
Fig. 5. SEM with EDX spectrum of e-waste printed circuit board. (A). SEM Photographs of e-waste different size of view (5, 10, 20, 50 μm) (B). EDX spectrum view of
toxic metals from e-waste.
5
Microbiological Research 245 (2021) 126707
expansion in positive control their secretion of primary metabolites
fabrication and there are no e-waste toxic effects. In the negative con­
trol, there is no pH variations because the bacteria are only responsible
for pH changes due to their production of organic acids it’s not involved
for e-waste particles.
Saravanan et al. (2008), Shah et al.2015 and Narayanasamy et al.
(2018, 2020a,b,c,d) researchers are expressing about the pH and
biomass variations during the bio removal and bioleaching progress. The
comparable conclusion was justified all the investigators in two-step bio
removal treatments than the other system of action. Therefore the key
justification for two-step bio removal treatment was greatest due to their
secretion of primary metabolites fabrication before e-waste action.
3.3. AAS spectra of e-waste PCBs and bioremoval residue
Atomic absorption spectroscopy was exposing the noxious heavy
metals elements occurrence in the crude e-waste PCBs and bio removal
treatments of heavy metals variations.
The raw e-waste PCBs proved the heavy metals concentration being
there before the removal treatments and it resembling Hg 4.31, Cd 8.30,
and Pb 4.60 (ppm) in the e-waste PCBs (Fig. 4). In one-step bio removal
treatment, the heavy metals similarity for removal is exceptionally low
and it’s paralleled to unprocessed. In a two-step, bio removal experi­
mentation showed the excessive level of heavy metals variation resting
on both Frankia sp. DDNSF-03 and Frankia casuarinae DDNSF-04
treatments.
Treated with DDNSF-03 provide evidence for toxic heavy metals
elimination during the bio removal course of action via AAS represents
resembling Hg 0.09, Cd 5.09 and Pb 0.49 (ppm). Treated with DDNSF-04
demonstrate the variation of the heavy metal during bio removal pro­
gressions like Hg 2.15, Cd 5.61 and Pb 2.82 (ppm). The toxic metals like
mercury and lead are successfully bioremediation through isolate
DDNSF-03 resting on two-step bio removal course of action as appraise
to isolate DDNSF-04 and also other toxic elements (Fig. 4). As a result,
were key justified in favor of two-step bio removal experiments showed
a high level of metal bioremediation due to their prominent amount of
secondary metabolites and organic acid fabrication prior to the e-waste
treatment.
Ehi-Eromosele et al. (2012), Narayanasamy et al.2020 and Shah et al.
(2015) researchers are giving the justification about the bioremediation
of noxious heavy metals from different resource through microbial
M. Narayanasamy et al.
Fig. 6. EDX analysis of toxic metals from both control and bioremoval residues
by Frankia EDX results views untreated e-waste and treated with Frankia sp.
DDNSF-03 and treated with F. causarinae DDNSF-04.
achievements. Throughout two-step bio removal course of action the
potable water polluted toxic heavy metals resembling Cr, Cu, Ni, Pb, As,
B, Mo, Ni, Cd, Co, Pb, Zn, Hg, and sewer successfully bioremediation. As
a result of the use of various forceful microorganisms such as Aspergillus
niger, Bacillus megaterium, Bacillus circulans, Micrococcus luteus.
3.4. SEM with EDX spectrum of e-waste PCBs and bioremoval residue
The scanning electron microscope has interpreted the shape, size,
and structure of e-waste PCBs fine particles prior to bio removal treat­
ments. Therefore it demonstrates e-waste fine elements are soft rod-like
and multilateral shaped particles among irregular crystals were attached
to the surfaces (Fig. 5). Energy-dispersive X-ray spectroscopy represents
the silicon and carbon fragments of e-waste PCBs.
As a result proves the composition of the heavy metals did occur­
rences in the crude e-waste resemble Si 370, Hg 180, Ru 70, Cd 40, and
Pd 50 (mg/g) (Fig. 6). In the two-step bio removal treatments both
Frankia sp. DDNSF-03 and Frankia casuarinae DDNSF-04 were signifi­
cantly representations of their ability to toxic heavy metals bioremedi­
ation course of action. SEM to be evidence for the view of solid e-waste
particles were accumulated on the outer surface of Actinobacteria hy­
phae (Figs. 7 and 8). EDX significance was inveterate the heavy metals
Fig. 7. SEM with EDX spectrum of bioremoval residues by Frankia sp. DDNSF-03. (A). SEM Photographs different views of bioremoval residues (5, 10, 20, 50 μm) (B).
EDX spectrum view of toxic metals bioremoval residues.
6
Microbiological Research 245 (2021) 126707
elements variations during the bio removal process and prior to treat­
ment metals quantity. Treated with isolate DDNSF-03 were expose the
evidence for noxious heavy metals exclusion during the removal pro­
gression of achievement by the use of EDX convey similar to Si 330, Hg
140, Ru 50, Cd 0.00, and Pd 10 (mg/g). Consequently treated with
isolate DDNSF-04 be evidence for the toxic heavy metals elimination
during bio removal process achievements are look like Si 320, Hg 120,
Ru 40, Cd 0.00, and Pd 0.00 (mg/g) (Fig. 6). The toxic metal cadmium
was competently bioremediation as a result of both strains of Frankia.
Therefore the heavy metal lead was moreover absolutely bioreme­
diation through isolate DDNSF-03 when compared to other toxic ele­
ments. In view of that frequently justified in two-step bio removal
activity was effectively bioremediation. Due to their mechanism for
adsorption of sulfur oxidized precipitation among e-waste particles on
the surface of bacteria and it ionized the toxic metals. Das (2010) and
Mack et al. (2007) researchers were explained about the mechanism of
biosorption among the toxic heavy metals from a different resource. The
toxic heavy metals from aqueous solutions they observed the high in­
tensity of biosorption along with different microbes such as bacteria,
fungi, and algae. They mostly clarify the mechanism for the positively
stimulating amine groups toward magnetize metal ions. Tanaka and
Watanabe (2015) were described metal ions exchanges during the bio­
logical process Pt chlorine ions and Pt42- amine groups variations on
bacterial cells during bio removal reactions. Rastegar et al. (2015) were
also being evidence about biosorptions of toxic heavy metals from sol­
uble contaminants source along with bacteria. As a result, proved the
chemical reaction of iron exchanges during the removal mechanism for
ionized the toxic heavy metals.
3.5. XRD spectrum of the e-waste PCBs and bioremoval residue
X-ray diffraction it revels both anode and cathode catalysts structure
of diffraction peaks and also identified crystallinity of polymers.
Therefore it exposes the microstructural diffraction peaks of toxic heavy
metals from crude e-waste prior to bio removal treatments.
As a result understanding via XRD exploration revealed major peaks
at 2θ values of noxious heavy metals such as (25.3), (79.5), (22.8),
(26.8), and (30.1). In view of that 2θ peaks values corresponding to the
intensity values (1400), (700), (1300), (1200), and (1700). Conse­
quently above finding intensity values was demonstrate the occurrence
of toxic heavy metals like Si, Hg, Ru, Cd, and Pb in crude e-waste PCBs
M. Narayanasamy et al.
Fig. 8. SEM with EDX spectrum of bioremoval residues by F. causarinae DDNSF-04. (A). SEM Photographs different views of bioremoval residues (5, 10, 20, 50 μm)
(B). EDX spectrum view of toxic metals bioremoval residues.
Fig. 9. XRD spectrum of the e-waste printed circuit boards. XRD views toxic
metals 2-theta intensity picks.
(Fig. 9).
In the two-step bio removal treatments both Frankia sp. DDNSF-03
and Frankia casuarinae DDNSF-04 were extensively showed their po­
tential to noxious heavy metals elimination achievements. XRD to be
support intended for the observation of toxic heavy metals microstruc­
tural diffraction peaks emission on bio removal reduces. Therefore the
XRD expose diffraction as of 0–380 2θ reach your peak value based on
top of the peak range toward recognized the metal elements. Conse­
quently, above the finding, similar diffraction peaks were observed in
two-step bio removal treatments along with both Frankia. The occur­
rence of toxic heavy metals like Si, Hg, Ru, Cd, and Pd in bio removal
reduce is evidence for metals deletion (Fig. 10). Gtari et al. (2013) and
Rastegar et al. (2015) researchers are discussed about toxic metals risk
in environment and microbes importance for bioremediation. They
provide and make clear about the bioremediation of toxic elements
which polluted the environment. Thus they described the importance of
Frankia for their capacity to bind and mineralized the toxic metals
particularly which contaminated the land.
7
Microbiological Research 245 (2021) 126707
3.6. FT-IR spectra of two-step bioremoval residue
Fourier transforms infrared spectroscopy it revels dynamic func­
tional groups of organic acid produced via bacteria during bio removal
course of action. In a two-step, bio removal residue exposes a more
active functional group of transmittance bands from both Frankia which
give evidence for bio removal of toxic metals. Treated through isolate
DDNSF-03 were showed a range of transmittance bands on 1017, 515,
602, 482.72 cm-1 which be correlated toward C–O, CCN, RCO− OH,
CC–––, correspondingly.
Therefore the occurrence of active functional groups represents the
proof for noxious heavy metals elimination throughout the bio removal
evolution. Treated in the course of isolate, DDNSF-04 demonstrated
some similar transmittance bands on 668, 1637, 3271, 664, 1063, 2123,
1637 cm-1 which be interconnected toward C–H–C–H, OH– as well as
RCO− OH active functional groups correspondingly (Fig. 11). As a result,
recognized for the occurrence of various organic acids resembles citric
acid as well as oxalic acids. Therefore the findings were justified to a
possible explanation for decreased pH during the bio removal course of
action. Bullen et al. (2008) and Grube et al., (2008) researches were
express about the active functional groups of organic acids secretion
throughout the bioleaching of voluble metals.
Therefore they finding transmittance bands of functional groups are
parallel to our current consequences and its evidence for the biological
process of heavy metals exclusion. Sethurajan et al. (2012); Willner
(2012) and Narayanasamy et al. (2018, 2020a,b,c,d) researchers are
specified designed for information about two-step bioleaching. They are
finding very similar transmittance bands of functional groups during the
bioleaching of e-waste elements. As a result, given a confirmation for the
above findings, functional groups are involved for bio removal of toxic
heavy metals from e-waste.
4. Conclusions
The present research was concluded with the intention of native
isolates of Frankia sp. DDNSF-03 and Frankia casuarinae DDNSF-04 is an
approaching biological mediator in bioremediation of noxious heavy
metal elements from e-waste. The findings were further investigating in
M. Narayanasamy et al. Microbiological Research 245 (2021) 126707

Fig. 10. XRD spectrum of bioremoval residues using Frankia. XRD views toxic metals 2-theta intensity picks of bioremoval residues. (A). Frankia sp. DDNSF-03. (B).
F. causarinae DDNSF-04.

Fig. 11. FT-IR spectra of bioremoval residues using Frankia FT-IR spectra views functional groups of bioremoval residues. (A). Frankia sp. DDNSF-03. (B).
F. causarinae DDNSF-04.

large scale optimization of bioremoval conditions, mass production as
well as quantify the bioremoval noxious metals. Therefore the findings
recommended as bioremediation is a successful method in favor of
recycling e-waste toxic heavy metals from marine polluted environment
both economic and ecofriendly approach.
Funding information
This work was supported by Research Foundation of Bharathidasan
University (URF) (02492/URF/K7/2016).
University for the fellowship (URF) (02492/URF/K7/2016. DST-FIST
(Department of Science and Technology-Fund for improvement of S&T
infrastructure), New Delhi, is express gratitude for the instrumentation
facility (DST Sanction Order No.: SR/FIST/LSI-013/ 2012/
Dt.13.08.2012). We acknowledge the financial assistance of the
Department of Science and Technology, Government of India, under
DST-Promotion of University Research and Scientific Excellence
(PURSE) scheme-Phase II, Rashtriya Uchchatar Shiksha Abhiyan
(RUSA)-2.O. Gandhigram Rural University, Gandhigram, Dindigul dis­
trict, Tamil Nadu 624302, India, is express thanks for the providing
SEM-EDX instrumentation facility.

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