Environmental Technology & Innovation 18 (2020) 100714

Contents lists available at ScienceDirect

Environmental Technology & Innovation

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

Development and performance evaluation of native microbial

consortium for multi metal removal in lab scale aerobic and
anaerobic bioreactor
∗
Deepak Gola a,c , Pushap Chawla a , Anushree Malik a , , Shaikh
Ziauddin Ahammad b
a
Applied Microbiology Lab, Centre for Rural Development and Technology, India
b
Department of Biochemical Engineering & Biotechnology, Indian Institute of Technology, Hauz Khas, Delhi, India
c
Noida Institute of Engineering and Technology, Greater Noida, Uttar Pradesh, India

article info a b s t r a c t

Article history: Shortage of freshwater force farmers to use untreated wastewater for irrigation which
Received 31 December 2019 leads to the accumulation of contaminants like heavy metals in the food chain and
Received in revised form 28 February 2020 deteriorate the quality of produce and soil. Present study focused on bioremediation
Accepted 29 February 2020
of multiple heavy metals (Cd, Cu, Cr, Zn. Pb and Ni) from wastewater used for irrigation
Available online 4 March 2020
of food crops using four continuous Stirred Tank Reactor (CSTR), two aerobic and two
Keywords: anaerobic. One set of bioreactor (aerobic and anaerobic) was fed with actual wastewater,
Heavy metal showed heavy metal removal up to 30%–100%. Whereas other set fed with spiked with
Bioreactor metal ion concentration showed removal up to 70%–100%, indicating the improvement
CSTR in removal efficiency due to acclimatization of microbial consortia at higher metal
Microbial consortium
concentration. Present study clearly shows the ability of native microbial consortia in
removing even higher concentrations of heavy metals from actual wastewater under
ambient conditions. The present bioreactors configuration can be scale up for the on-
site wastewater treatment with further optimization of the removal process. It was
recommended that after using the present treatment process, treated water is safe for
irrational purposes.
© 2020 Published by Elsevier B.V.

1. Introduction

Industrialization has led to the contamination of water bodies with toxic contaminants like heavy metals (Beni and
Esmaeili, 2020; Liu et al., 2020; Verma and Balomajumder, 2019). It was observed that these water bodies are directly
receiving metal laden effluents from the industries like paint, metal, batteries, without any prior treatment (Gupta et al.,
2019; Khan et al., 2019). As a regular practice, this contaminated water is being used for irrigational purposes by the
farmers due to the ease of availability (Gola et al., 2019). Due to such activities heavy metals enter into the food products
(crops, vegetables and fruits) which are later on consumed by the human beings (Gümüş and Gümüş, 2019). Consumption
of heavy metals along with food can cause multiple disorder and diseases as heavy metals are mutagenic and carcinogenic
in nature (Sodhi et al., 2019; Ubuoh et al., 2019). Hence methods for removal of these metals from water bodies prior to
irrigation is a prime necessity.

∗ Corresponding author.
E-mail addresses: deepakgola@gmail.com (D. Gola), pushapchawla@gmail.com (P. Chawla), anushree@rdat.iitd.ac.in (A. Malik),
zia.iitd@gmail.com (S.Z. Ahammad).

https://doi.org/10.1016/j.eti.2020.100714
2352-1864/© 2020 Published by Elsevier B.V.
2 D. Gola, P. Chawla, A. Malik et al. / Environmental Technology & Innovation 18 (2020) 100714

Studies related to the treatment of the synthetic wastewater using isolated biological identity or microbial consortium
under well optimized lab conditions, with high removal efficiency are widely available (Ali et al., 2015; Gola et al., 2016a;
Ravikumar et al., 2018; Kalpana et al., 2018). However, efficiency of these isolated microbial species or microbial consortia
might deplete drastically with actual wastewater (Gola et al., 2016b; Malik, 2004; Mishra and Malik, 2012). In addition to
this, more problems are associated with the application of isolated species for the treatment of wastewater as washout
condition may prevail due to the dominance of micro-organism present in wastewater itself (Gola et al., 2016b; Jackson
et al., 2009; Mishra and Malik, 2012; Mishra et al., 2018). Considering all the above situations, present study was focused
upon developing two microbial consortia i.e. aerobic and anaerobic. Further, developed microbial consortium was tested
for its heavy metal and COD (chemical oxygen demand) removal capacity in novel lab scale bioreactor system (aerobic
and anaerobic).

2. Material and methods

2.1. Wastewater collection

Water samples were collected from a depth of 15–30 cm in duplicates in 5 l sampling bottles (polypropylene, Borosil)
from the selected location of Loha mandi drain. Bottles were cleaned (using diluted acid) and rinsed with tap water before
sampling. Wastewater was filtered using cotton cloth to remove large suspended particles/debris and refrigerated at 4 ◦ C
prior to processing and analysis.

2.2. Development of microbial consortium (aerobic and anaerobic) from wastewater under multi metal stress

To develop multi metal resistance aerobic microbial consortium, 1 mL of the wastewater from drain (Loha Mandi,
located in Delhi, India) was inoculated in 100 mL flask containing nutrient broth (composition) with 6 mg L−1 multi
metal mix (1 mg L−1 of each heavy metal). The flask was incubated at 30 ◦ C with 150 rpm for 48 h. To enhance the heavy
metal removal ability of the microbial consortium at higher concentrations, multi metal ions concentration in the nutrient
broth was increased sequentially from 6 mg L−1 to 12 mg L−1 , 18 mg L−1 and 30 mg L−1 with above mentioned growth
conditions. The acclimatization process was carried out until the microbial consortium showed the maximum growth as
observed in the control medium without metal. The developed microbial consortium was used as inoculum for bioreactor.
Further, to developed anaerobic culture, similar protocol was followed as described above along with gas sparging [CO2
(70%):N2 (30%)] for 10 min.

2.3. Experimental set-up

The schematic diagram of aerobic and anaerobic bioreactors are presented in Fig. 1(X and Y).

2.3.1. Bioreactors
Four parallel CSTR (Continuous Stirred Tank Reactor) bioreactors (R1, R2, R3 and R4) of 4L working capacity (Total
volume = 5 L) were fabricated using acrylic cylinder (Length: 13 cm, inner diameter: 13 cm and outer diameter:
15 cm). CSTR type bioreactor was selected for experiments because in CSTR bioreactors concentration of nutrients and
contaminants remains uniform throughout the bioreactor and it helps in maintaining the consortium in a constant average
growth state. Bioreactor R1 and R2 were operated with aerobic conditions while bioreactors R3 and R4 were operated with
anaerobic conditions. All the bioreactors were kept at 30 ◦ C, during batch and continuous mode. Mixing was achieved
by magnetic stirrer (REMI 2 ML) at the speed of 400 rpm in aerobic bioreactor. For anaerobic bioreactors, mixing was
achieved via impeller attached to a shaft rotating at approximately 400 rpm. To maintain the aerobic condition in the
bioreactor diffused air was provided in the cylinder with the help of an air pump and diffuser (located at the bottom of
the cylinder), whereas for anaerobic conditions sparging was done for 10 min at regular interval of 24 h.

2.3.2. Feeding tank

Wastewater collected from the Loha mandi drain was used to feed all the bioreactor systems. Wastewater from feeding
tank (Length: 13 cm, inner diameter: 13 cm and outer diameter: 15 cm) was fed into the bioreactor from top by a
peristaltic pump (Designinnova, Acuflo D1X-01) with a volumetric flow rate of 2.8 mL min−1 . Magnetic stirrers (REMI
2 ML) were used to maintain the homogenous condition in the feeding tank.

2.4. Wastewater composition for bioreactor

Rivers and drains contain low concentrations of multiple heavy metals because of the mixing of contaminated industrial
wastewaters (Gola et al., 2016b). Similarly, low level of heavy metals was detected in the Loha mandi drain wastewater
 D. Gola, P. Chawla, A. Malik et al. / Environmental Technology & Innovation 18 (2020) 100714 3

Fig. 1. (X-above) Schematic diagram of aerobic bioreactor (R1 and R2) and (Y-above) Schematic diagram of anaerobic bioreactor (R3 and R4). *R3
was fed with actual wastewater and R4 was fed with multi metal spiked wastewater with 30 mg L−1 hexa metal mixture (5 mg L−1 each of Cr6+ ,
Cd2+ , Pb2+ , Ni2+ , Zn2+ and Cu2+ ). *R1 was fed with actual wastewater and R2 was fed with multi metal spiked wastewater with 30 mg L−1 hexa
metal mixture (5 mg L−1 each of Cr6+ , Cd2+ , Pb2+ , Ni2+ , Zn2+ and Cu2+ ).

which is used for irrigation purposes (Gola, 2018). In preliminary, it was observed that microbial consortia was unable to
grow in wastewater without any supplements. Hence, yeast extract (1 g L−1 ) was added as substrate at an interval of 5
day in wastewater to support the growth of microbial community in bioreactor system during batch operation. Thereafter,
wastewater supplemented with 1 g L−1 yeast extract was fed in continuous mode. Bioreactor R1 and R3 were fed with
only wastewater supplemented with yeast extract whereas bioreactor R2 and R4 were fed with wastewater supplemented
with yeast extract and spiked with multi heavy metals of total concentration of 30 mg L−1 (5 mg L−1 of each Cu2+ , Cr6+ ,
Cd2+ , Zn2+ , Pb2+ and Ni2+ ). Concentration of multi metal ion was fixed to 30 mg L−1 because it was the maximum possible
concentration expected in rivers and drains (across India) as per the literature survey (Bhattacharya et al., 2015; Bhardwaj
et al., 2017; Rahman and Singh, 2018).

2.5. Experimental procedure

All bioreactor systems were first inoculated with acclimatized and enriched microbial consortium (10% v/v). Bioreactor,
R1 and R3 were operated for 35 days, whereas bioreactor R2 and R4 were operated for 40 days, in two different phases.
In first phase, all bioreactors were incubated for 20 days at batch operating condition using wastewater (supplemented
with yeast extract 1 g L−1 ). After pre-incubation period of 20 days in batch mode, in phase two bioreactor R1 and R3,
were then fed continuously with drain (Loha mandi) wastewater amended with yeast extract (1 g L−1 ) for next 15 days.
On the other hand, bioreactor R2 and R4 were operated for 20 days in continuous mode in second phase with drain (Loha
mandi) wastewater spiked with multi metal mixture (30 mg L−1 ). The hydraulic retention time (HRT) was kept 24 h during
second phase for all the bioreactors. All the bioreactor systems, were kept at 30 ◦ C and mixing was done using magnetic
stirrer (REMI 2 ML) shaft at 400 rpm. Samples were withdrawn at an interval of 24 h from inlet and outlet in order to
estimate the concentration of heavy metal ions and COD.

2.6. Analytical techniques

2.6.1. Residual heavy metal concentration

Samples (5 ml) from the above experiments were drawn at an interval of 24 h and centrifuged at 10 000 rpm for 10
min. The supernatant broth was digested in microwave digester (at 160 ± 4 ◦ C for 10 min followed by at 165 ◦ C to 170 ◦ C
for 10 min for reaction (Anton Paar Multiwave Pro) and analysed for metal ions via MPAES (4200, Agilent)
4 D. Gola, P. Chawla, A. Malik et al. / Environmental Technology & Innovation 18 (2020) 100714

Table 1
Inlet and outlet heavy metal concentration in bioreactor R1 (aerobic) and R3 (anaerobic) after treatment.
Bioreactor Heavy metal concentration (mg L−1 )
Pb2+ Zn2+ Cd2+ Cr6+ Cu2+ Ni2+
R1 (inlet) 0.234 ± 0.04 0.5340 ± 0.004 00 ± 0.000 0.2530 ± 0.002 0.1340 ± 0.002 0.1430 ± 0.002
R1 (Batch) 0 ± 0.000 0.020 ± 0.001 00 ± 0.000 0.0190 ± 0.001 0.0480 ± 0.001 00 ± 0.000
R1 (Continuous) 0.14 ± 0.01 0.0210 ± 0.001 00 ± 0.000 0.1460 ± 0.002 0.0810 ± 0.001 0.0780 ± 0.002
R3 (inlet) 0.098 ± 0.02 0.5660 ± 0.002 00 ± 0.000 0.0940 ± 0.004 0.6260 ± 0.002 0.3080 ± 0.004
R3 (Batch) 00 ± 0.000 0 00 ± 0.000 00 ± 0.000 00 ± 0.000 00 ± 0.000
R3 (Continuous) 00 ± 0.000 0.0050 ± 0.000 00 ± 0.000 00 ± 0.000 0.01870 ± 0.002 0.0910 ± 0.001
FAO (limitsa ) 5 2 0.01 0.1 0.2 0.2
a
The concentration of heavy metals which lies above permissible limits (FAO) are indicated with bold numeric values.

2.6.2. COD (chemical oxygen demand) estimation

For COD estimation, collected samples were centrifuged at 8000 g for 10 min in order to separate the suspended matter,
if any. Sample COD was estimated by oxidizing the known volume of sample via potassium dichromate (HACH method
8000; detection limit 0–1500 mg L−1 ) resulting in the change of chromium from hexavalent state to trivalent state. These
samples were digested for two hours at 150 ◦ C in digester (Hach digital reactor DRB200). The Cr6+ ion absorbs strongly at
400 nm wavelength and Cr3+ ion absorbs strongly at 600 nm wavelength, the dichromate showed no absorbance at any
wavelength. After digestion, the samples were centrifuged to remove insoluble suspended matter. The blank was prepared
using the distilled water and was subjected to the same conditions as mentioned above.

3. Results and discussion

3.1. Heavy metal removal

3.1.1. First phase (batch mode)-R1 and R3 [R1 (aerobic bioreactor) and R3 (anaerobic bioreactor) fed with actual drain
wastewater]
The bioreactor systems (R1 and R3) were operated in batch mode to stabilize the native microbial community. In the
wastewater collected during July and Aug-2015 and fed to reactor R1, average concentration of Zn2+ was highest (0.53
mg L−1 ) followed by Cr6+ and Pb2+ (0.25 mg L− -0.23 mg L−1 ), Cu2+ and Ni2+ (0.13 mg L−1 and 0.14 mg L−1 ) and Cd2+
(0 mg L−1 ). On the other hand, in wastewater collected during April-May-2017 and fed to anaerobic bioreactor (R1), the
average of Pb2+ was the highest (0.98 mg L−1 ) followed by Cu2+ and Zn2+ (0.62 mg L−1 -0.56 mg L−1 ), and Cd2+ (0 mg
L−1 ) (Table 1). The difference in heavy metal concentrations in the wastewater was due to seasonal variation in which
the wastewater was collected for bioreactors. The residual heavy metal concentration after first phase i.e. batch mode
for both the bioreactors (inoculated with aerobic and anaerobic microbial community) are shown in Figs. 2(A) and 3(A),
respectively.
Fig. 2(A) illustrates the metal removal profile in aerobic bioreactor (R1) during batch operation. Although the
concentration of Zn2+ was highest in collected wastewater but its concentration drops rapidly to almost zero from 0.53
mg L−1 within 8 days of operation and thereafter fluctuate in between 0.027–0 mg L−1 . The removal pattern of Ni2+ and
Cr6+ seem to be similar and shows the constant drop till 20th day. There was no removal of Cu2+ until first 9 days of
operation but a constant drop was observed after 9 day of treatment from 0.13 mg L−1 to 0.04 mg L−1 . In addition to this,
concentrations of Cr6+ dropped below the permissible limits (FAO guidelines), on 8th day.
Fig. 3(A) illustrates the metal removal profile in anaerobic bioreactor (R3) during batch mode. It was detected that
concentration of Cu2+ was highest in wastewater and drop to zero within 4 days of operation. On the other hand, the
removal profile of Zn2+ and Ni2+ indicate the slow removal of these metals as compared to Cu+2 and drop to zero after
7 and 9 days of operation, respectively.
Total concentration of metal ions was high in wastewater fed to reactor R3 (1.69 mg L−1 ) as compared to wastewater
fed to reactor R1 (1.29 mg L−1 ). It was observed that removal efficiency of bioreactor R3 was higher as compared to
bioreactor R1 showing almost complete removal for all the heavy metals (Cr6+ , Cu2+ , Cd2+ , Zn2+ , Pb2+ and Ni2+ ) (Fig. 3).
On the other hand, removal efficacy of bioreactor R1 was highest for Pb and Ni (100%) followed by Zn2+ and Cr6+ (94%–
92%) and least removal was observed for Cu2+ (64%). The final residual metal concentration treated water after batch
mode is illustrated in Table 1. It was observed that concentrations of Cr6+ was above the FAO permissible limit in the
inlet wastewater for bioreactor R1. On the other hand, concentrations of Cu2+ and Ni2+ were on above permissible limit
in the inlet wastewater for R3 and drop to permissible level after batch operation.

3.1.2. Second phase (continuous mode)-R1 and R3 [R1 (aerobic bioreactor) and R3 (anaerobic bioreactor) fed with actual drain
wastewater]
During second phase of operation, bioreactor R1 and R3 were continuously fed with Loha mandi drain wastewater for
next 15 days and removal efficiency of both bioreactors were examined in term of heavy metal removal as depicted in
Figs. 2(B) and 3(B).
 D. Gola, P. Chawla, A. Malik et al. / Environmental Technology & Innovation 18 (2020) 100714 5

Fig. 2. (A) Outlet heavy metal concentration in bioreactor R1 (aerobic) during continuous operation and (B) Outlet heavy metal concentration in
bioreactor R1 (aerobic) during batch operation.

Fig. 2(B) illustrates the metal removal profile of bioreactor R1 (aerobic) during continuous mode. Removal profile of
Zn2+ in continuous mode indicates that concentration of Zn2+ increased slightly in the outlet during first 10 days of
operation and decreased to zero thereafter. It was observed that, after 11 days of continuous operation, no further drop
in outlet concentration of Cu2+ and Ni2+ was observed, whereas the concentration of Cr6+ and Pb2+ kept fluctuating
throughout the continuous operation.
Fig. 3(B) illustrates the removal of heavy metal ions from wastewater in anaerobic condition (R3) during continuous
mode. Concentration of Ni2+ drop constantly till 30th day in outlet water and no further reduction in outlet concentration
was observed thereafter. Overall, it was observed that the concentration of other metal ion such as Cu2+ , Cr6+ and Zn2+
in outlet become constant after 10 days of treatment process.
In bioreactor R1 (aerobic), maximum removal was observed for Zn2+ (96%) whereas the removal efficiency of other
metal ions (Pb2+ , Cr6+ , Cu2+ and Ni2+ ) was significantly lower (30%–40%). On the other hand, removal efficiency of
bioreactor R3 was on substantially higher as compared to bioreactor R1, in second phase also. In bioreactor R3, almost
complete removal was observed for Pb2+ , Zn2+ , Cr6+ and Cu2+ while minimum removal was observed for Ni2+ (70%).
Initial and final concentration of each metal ion obtained after batch and continuous mode operations is also illustrated
6 D. Gola, P. Chawla, A. Malik et al. / Environmental Technology & Innovation 18 (2020) 100714

Fig. 3. (A) Outlet heavy metal concentration in bioreactor R3 (anaerobic) during batch operation and (B) Outlet heavy metal concentration in
bioreactor R3 (anaerobic) continuous operation.

in Table 1. Above results shows that a mixture of metals were present in the Loha mandi drain wastewater and Cr6+ ,
Cu2+ and Ni2+ were recorded above FAO permissible limits. The concentrations of all the heavy metals were below the
permissible limit in the treated water in both bioreactors (R1 and R3), except for Cr6+ for in bioreactor R1 (continuous
mode). Hence, the treated wastewater was observed to be safe for irrigational usage as per the FAO guidelines (Codex
Alimentarious Commission, 1984). While handling the ambient concentration found in Loha mandi drain during Apr–Aug
2015. However, since the metal concentration fluctuate depending upon the seasonal variation and industrial activities,
it is desirable to test the bioreactor performance under shock loading with higher metal concentrations. For this, in next
study, similar bioreactors were run simultaneously, in which the fed wastewater was amended with the multi metal mix
(30 mg L−1 ).

3.1.3. First phase (batch mode)-R2 and R4 [R2 (aerobic bioreactor) and R4 (anaerobic bioreactor) fed with actual drain
wastewater amended with 30 mg L−1 multi metal mix]
To study the metal removal ability of developed native microbial consortium at higher concentrations, wastewater was
amended with metal ion solution (30 mg L−1 multi metal mixture containing 5 mg L−1 each of Cr6+ , Cd2+ , Pb2+ , Ni2+ ,
 D. Gola, P. Chawla, A. Malik et al. / Environmental Technology & Innovation 18 (2020) 100714 7

Fig. 4. (A) Outlet heavy metal concentration in bioreactor R2 (aerobic) fed with heavy metal spiked (30 mg L−1 ) wastewater during operation and
(B) Outlet heavy metal concentration in bioreactor R2 (aerobic) fed with heavy metal spiked (30 mg L−1 ) wastewater during continuous operation.
*[5 mg L−1 each of Cr6+ , Cd2+ , Pb2+ , Ni2+ , Zn2+ and Cu2+ ].

Zn2+ and Cu2+ ). The concentration of heavy metals in wastewater varies with the seasons as well as the quality of the
industrial effluents merging into the drain at that particular site, hence heavy metal removal efficiency was investigated
at higher metal ion concentrations. The metal spiked (30 mg L−1 ) wastewater was fed in bioreactor R2 (aerobic) and R4
(anaerobic). After spiking, initial concentration of heavy metals (Pb2+ , Zn2+ , Cd2+ , Cr6+ , Cu2+ and Ni2+ ) in bioreactor R2
ranged from 5.21 mg L−1 to 4.62 mg L−1 , whereas for bioreactor R4 ranged from 5.72 mg L−1 to 4.46 mg L−1 . The results
pertaining to the residual metal ion concentration after 20 days of treatment in both the systems are shown Figs. 4(A)
and 5(A).
Fig. 4(A) illustrates removal profile of heavy metal in wastewater amended with 30 mg L−1 multi metal mix in aerobic
bioreactor (R2) during batch operation. As compared to other heavy metal ions in wastewater, removal profile of Zn2+
shows sharp decrease in outlet concentration from 4.62 mg L−1 to 1.92 mg L−1 within 3 days of operation and thereafter
drop slowly. Removal profile of other heavy metal (Cd2+ , Cu2+ , Cr6+ and Pb2+ ) showed constant reduction in treated
wastewater, except for Ni2+ . Concentration of Ni2+ drop till 9th day and no further drop in outlet concentration was
detected.
8 D. Gola, P. Chawla, A. Malik et al. / Environmental Technology & Innovation 18 (2020) 100714

Fig. 5. (A) Outlet heavy metal concentration in bioreactor R4 (anaerobic) fed with heavy metal spiked (30 mg L−1 ) wastewater during batch
operation and (B) Outlet heavy metal concentration in bioreactor R4 (anaerobic) fed with heavy metal spiked (30 mg L−1 ) wastewater during
continuous operation. *[5 mg L−1 each of Cr6+ , Cd2+ , Pb2+ , Ni2+ , Zn2+ and Cu2+ ].

Fig. 5(A) illustrate removal profile of heavy metal in wastewater amended with 30 mg L−1 multi metal mix in anaerobic
bioreactor (R4) during batch operation. It was observed that concentration of Cd2+ and Zn2+ in bioreactor drop to 1.77
mg L−1 and 2.11 mg L−1 after 3 days of operation, respectively. There was a continuous decline in concentration of Cu2+
and Cr6+ and become zero after 11 days of operation. Whereas removal profile of Pb2+ indicated that removal of Pb2+
was delayed as compared to other heavy metal ion and drop to almost zero only after 17 days of operation.
After operating the bioreactor for 20 days in batch mode, the concentrations of Pb2+ , Zn2+ , Cd2+ , Cr6+ , Cu2+ and Ni2+
ranged from 0–2.70 mg L−1 in bioreactor R2 (aerobic). It was observed that concentrations of Cr6+ , Cu2+ , Ni2+ and Cd2+
was above the permissible limits in the outlet effluent, whereas the concentration of Pb2+ and Zn2+ was below permissible
limit in treated water, in bioreactor R2. On the other hand, the concentrations of Pb2+ , Zn2+ , Cd2+ , Cr6+ , Cu2+ and Ni2+
ranged from 0–1.14 mg L−1 in bioreactor R4 (anaerobic). The outlet concentration of all the heavy metal ions were below
the permissible limit (except for Ni2+ ). On comparing the removal efficiency of both the bioreactor it was observed that
efficiency of the bioreactor R4 (77%–100%) was higher as compared to bioreactor R2 (53%–100%).
 D. Gola, P. Chawla, A. Malik et al. / Environmental Technology & Innovation 18 (2020) 100714 9

Table 2
Inlet and outlet heavy metal concentration in bioreactor R2 and R4 after treatment.
Reactor Heavy metal concentration (mg L−1 )
Pb2+ Zn2+ Cd2+ Cr6+ Cu2+ Ni2+
R2 (inlet) 5.02 ± 0.002 5.32 ± 0.030 4.80 ± 0.012 4.98 ± 0.014 4.50 ± 0.002 4.67 ± 0.016
R2 (Batch) 0 ± 0.000 0.41 ± 0.010 0.28 ± 0.002 2.34 ± 0.007 0.99 ± 0.002 2.70 ± 0.009
R2 (Continuous) 1.5 ± 0.001 0.37 ± 0.008 0 ± 0.000 3.82 ± 0.012 0.57 ± 0.001 3.15 ± 0.008
R3 (inlet) 4.88 ± 0.021 5.12 ± 0.012 4.87 ± 0.007 5.01 ± 0.021 5.18 ± 0.009 5.05 ± 0.011
R4 (Batch) 0 ± 0.000 0.21 ± 0.001 0 ± 0.000 0 ± 0.000 0 ± 0.000 1.14 ± 0.002
R4 (Continuous) 1.76 ± 0.002 0.10 ± 0.002 1.4 ± 0.005 1.14 ± 0.009 0.08 ± 0.001 0.42 ± 0.004
FAO (limits) 5 2 0.01 0.1 0.2 0.2

The concentration of heavy metals which lies above permissible limits (FAO) are indicated with bold numeric values.

Table 3
Comparison of present study with recently developed various biological methods for heavy metal removal from wastewater.
Reactor type and Micro- HRT, temperature, Metal Removal (%) Reference
volume Organism wastewater
HRBT-Horizontal Microbial Sterile condition with Cr, Mn and Co (125 mg L−1 each) 69–100 Zeiner et al.
Rotating Tubular consortia 26 ◦ C (2012)
Bioreactor (15 L)
Small Scale Bioreactor SRB Synthetic wastewater Cr(V1) (50 mg L−1 ) 96 Singh et al.
(0.5 L) 2011
Anaerobic SCST (2 L) SRB 20 days Cu, Zn, Ni and Cr (30–150 mg L−1 ) 94–100 Kieu et al.
(2011)
HRBT Microbial Sterile condition and Fe, Cu, Ni and Zn 38.1–95.5 Zeiner et al.
consortia synthetic wastewater (2010)
Fixed Bed Reactor SRB Synthetic wastewater Cd 0.1 mM, Cr(VI) 0.1 mM, Cu 100 Viggi et al.
(6.65 L) 0.1 mM, (2010)
Zn 0.1 mM and As(V) 27 mM
Anaerobic Bioreactor SRB HRT 13 h with 25 ◦ C Cd (2 mg L−1 ) and Zn(50–70 mg L−1 ) >99 Gonçalves et al.
(13 L) (2007)
Aerobic Bioreactor Microbial HRT 24 h, ambient Pb (0.23 mg L−1 ), Zn (0.53 mg L−1 ), 30–96 Present study
(4 L)-R1 consortia temperature and actual Cd (0 mg L−1 ), Cr (0.25 mg L−1 ),
wastewater Cu (0.13 mg L−1 ), Ni (0.14 mg L−1 )
Aerobic Bioreactor Microbial HRT 24 h, ambient Pb ( 5.02 mg L−1 ), Zn (5.32 mg L−1 ), 23–100 Present study
(4 L)-R2 consortia temperature and actual Cd (4.80 mg L−1 ), Cr (4.98 mg L−1 ),
wastewater spiked with Cu (4.50 mg L−1 ), Ni (4.67 mg L−1 )
multi metal ion
Anaerobic Bioreactor Microbial HRT 24 h, ambient Pb (0.98 mg L−1 ), Zn (0.56 mg L−1 ), 70–100 Present study
(4 L)-R3 consortia temperature and actual Cd (0 mg L−1 ), Cr (0.09 mg L−1 ),
wastewater Cu (0.62 mg L−1 ), Ni (0.30 mg L−1 )
Anaerobic Bioreactor Microbial HRT 24 h, ambient Pb (4.88 mg L−1 ), Zn (5.12 mg L−1 ), 72–98 Present study
(4 L)-R4 consortia temperature and actual Cd (4.87 mg L−1 ), Cr (5.01 mg L−1 ),
wastewater spiked with Cu (5.10 mg L−1 ), Ni (5.05 mg L−1 )
multi metal ion

3.1.4. Second phase (continuous mode)-R2 and R4 [R2 (aerobic bioreactor) and R4 (anaerobic bioreactor) fed with actual drain
wastewater amended with 30 mg L−1 multi metal mix]
In this phase, bioreactors R2 and R4 were run in continuous mode for next 20 days. The average inlet concentrations
of heavy metals (Pb2+ , Zn2+ , Cd2+ , Cr6+ , Cu2+ and Ni2+ ) in wastewater to be fed in bioreactor R2 (aerobic) were ranged
from 5.32 mg L−1 to 4.50 mg L−1 . On the other hand, for bioreactor R4 (anaerobic) average inlet concentration of heavy
metals were ranged from 5.18 mg L−1 to 5.05 mg L−1 . The results pertaining to the residual metal ion concentration in
both the systems are shown in Figs. 4(B) and 5(B).
Fig. 4(B) illustrates removal profile of heavy metal in wastewater amended with 30 mg L−1 multi metal mix in aerobic
bioreactor (R3) during continuous operation. It was observed that, similar to batch operation concentration of Ni2+ and
Cr6+ remain high in outlet during continuous operation. On the other hand, outlet concentration of Zn2+ and Cu2+ drop to
1.2 4 mg L−1 and 2.03 mg L−1 during 2 days of operation, respectively, and thereafter their concentration in outlet remain
almost same. Removal profile of Cd2+ shows constant decrease in outlet concentration and drop to zero after 9 days of
continuous operation. Fig. 5(B) illustrates removal profile of heavy metals in wastewater amended with 30 mg L−1 multi
metal mix in anaerobic bioreactor (R4) during continuous operation. It was observed that the outlet concentration of Cd2+
drop persistently from 4.35 mg L−1 to 1.40 mg L−1 during continuous operation. On the other hand, the concentration
of Pb+2 in the outlet drop substantially to 2.76 mg L−1 within 5 days of operation and thereafter concentration declined
10 D. Gola, P. Chawla, A. Malik et al. / Environmental Technology & Innovation 18 (2020) 100714

slowly to 1.76 mg L−1 in the next 15 days of operation. Removal of Cr6+ seems to be delayed and no major drop in the
outlet concentration of Cr6+ was observed in first 11 days, but thereafter dropped consistently to 1.42 mg L−1 . Table 2
illustrate the final concentrations of metal ion after batch and continuous operation in reactor R2 and R4.
After operating the bioreactor for 20 days in continuous mode, concentrations of metal ion in outlet ranged from 3.82
to 0 mg L−1 in bioreactor R2 (aerobic). The outlet concentration of Pb2+ , Zn2+ and Cd2+ were below the permissible limits,
while the concentration of Cr6+ , Cu2+ and Ni2+ remain high as compared to FAO permissible limits. On the other hand,
concentration of metal ions in outlet stream for anaerobic bioreactor (R4) ranged from 1.76 to 0.08 mg L−1 . The outlet
concentration of Pb2+ and Zn2+ were below the permissible, while the outlet concentration of Cd2+ , Cr2+ , Cu2+ and Ni2+
lies above the permissible limits. Overall, the removal efficiency of anaerobic bioreactor (R4) was higher as compared to
the aerobic bioreactor (R2).
As compared to batch mode, the outlet concentration of Zn2+ , Cd2+ and Cu2+ decreases in continuous mode of R2, while
the outlet concentration of Ni2+ , Pb2+ and Cr6+ were increased. In case of R4, drop in outlet concentration of Zn2+ and Ni2+
were observed in continuous mode as compared to batch mode, while the concentration of Pb2+ , Cr6+ , Cd2+ , and Cu2+
were increased. After 20 days of operation (R2), it was observed that maximum removal was obtained for Cd2+ (100%),
followed by Zn2+ (93%), Cu2+ (87%) and Pb2+ (70%). Whereas, the removal efficiency for Ni2+ (32%) and Cr6+ (23%) was
lower as compared to other metal ions. It was observed that, maximum removal (90%–98%) was observed for Cu2+ , Ni2+
and Zn2+ followed by Cd2+ (72%), Cr6+ (77%) and least removal was obtained for Pb2+ (64%), in anaerobic bioreactor (R4).
High removal efficiency in anaerobic bioreactor might be due to the precipitation of metal during the sludge formation
by anaerobic microbial community present in the bioreactor (Hawari and Mulligan, 2007; Kieu et al., 2011; Kijjanapanich
et al., 2012; Kiran et al., 2015). Anaerobic bacteria utilize the inorganic ions and get precipitated with metal during sludge
formation. Many investigator, employs anaerobic microbial consortia to remediate heavy metal ions from the wastewater.
Kieu et al. (2011) observed 94%–100% removal of various heavy metals like Cu2+ , Zn2+ , Cr6v and Ni2+ in anaerobic semi
continuous stirred tank reactor using a consortium of sulphate-reducing bacteria with HRT of 20 days, reducing HRT to
12 days decreased the overall metal removal efficiency by 30%–50%. In another study, Dvorak et al. (1992) observed up to
95% heavy metal removal from underground heavy metal contaminated water of coal mine (Cu2+ , Ni2+ and Zn2+ ) using
SRB consortium in anaerobic bioreactor system, but require limestone to maintain the alkaline pH inside the bioreactor.
Investigator also evaluated the performance of aerobic bioreactor such as RBC (rotating biological contactor) and HRTB
(horizontal rotating tubular bioreactor) in term of metal removal. Zeiner et al. (2010) obtained up to 38.1%–95.5% removal
of Fe3+ , Cr6+ , Ni2+ and Zn2+ from the wastewater using bioreactor system. In another study by same author, Zeiner et al.
(2012) obtained over 93%, 100% and 69% removal of Mn+2 , Cr+2 and Co+2 , respectively, in horizontal rotating tubular
bioreactor (HRTB), using synthetic wastewater and sterilized conditions. Costley and Wallis (2000) observed maximum
removal for Cu2+ (85%) as compared to Zn2+ (57%) and Cd2+ (34%) in rotating biological contractor (under aerobic
conditions) with HRT of 24 h and at a specific temperature of 26 ◦ C . In another study, Diels et al. (2003) observed up to
100% removal for Cu2+ and Zn2+ in airlift bioreactor, whereas less removal was seen in case of Ni2+ and Co2+ . Similarly,
Jackson et al. (2009) evaluated the performance of various bioreactors (two laboratory-scales and one on-site) for Al3+ ,
Ni2+ , Fe3+ , Mn2+ and Zn2+ removal having microbial consortium (Pseudomonas sp., Sphingomonas sp., and Bacillus sp.)
and obtained significant removal up to 44%–97% for Al3+ , Ni2+ , Fe3+ , Mn2+ and Zn2+ . From the available literature it can
be seen that, the removal trend depends not only on the concentration of heavy metal ions in the solution but also the
micro-organism used for remediation process. Heavy metal removal efficiency is dominated by the metabolic activity of
microorganisms. Azizi et al. (2016) studied the effect of initial multi metal ion (Zn2+ , Cu2+ , Cd2+ and Ni2+ ) concentration
(8–40 mg L−1 ) on removal efficiency of packed bed biofilm reactor. Metal removal efficacy first increased from 8 mg L−1
to 20 mg L−1 and then decreases with the increase in concentration, which was attributed to the decreased in metabolic
activity of micro-organism. Toxic effect of heavy metal at higher concentration can cause negative influence on structure
and metabolism of microorganisms present in the bioreactor, thus reducing their metal removal efficiency. Stasinakis
and Thomaidis (2010) also reported that higher metal ion concentration in wastewater inhibit microbial respiration, thus
causing a change in the microbial community in term of activity and adversely affecting the treatment process. From
available literature it can be seen that bioreactors either require high HRT or specific temperature for high metal removal.
Moreover, most of the studies are conducted with synthetic wastewater and lack performance evaluation with actual
wastewater. The present study was performed with actual wastewater and with a short HRT of 24 days without demanding
specific temperature requirement. Hence bioreactor performance mimic actual treatment process without demanding any
specific conditions. Table 3 illustrates comparison of present study with recently developed various biological methods
for heavy metal removal from wastewater
Moreover, it was observed that reactor R2 and R4 are able to remove high concentration of wastewater in an efficient
manner, with up to 23%–98%, in continuous mode. Concentration of heavy metals are high in actual effluent generated by
electroplating, paint, and battery industries, for such type of wastewater bioreactor R2 and R4 can be employed for the
treatment process. However, the concentration of heavy metal ions in Loha mandi drain was on lower side as compared to
the concentration studied in bioreactor R2 and R4. When actual wastewater was used in the bioreactor R1 (aerobic) and R3
(anaerobic) up to 30%–100% removal efficiency was observed. Higher removal efficacy at lower metal ion concentration
might be due to the decrease in toxicity caused by metal ion. The above study concluded that configured bioreactors
system are very much efficient in handling the multiple metal ion containing (at low or higher concentration) wastewater
with good metal removal efficiency ranging from 23%–100%.
 D. Gola, P. Chawla, A. Malik et al. / Environmental Technology & Innovation 18 (2020) 100714 11

Fig. 6. Average COD value with removal percentage in aerobic bioreactors (R1 and R2) and anaerobic bioreactor (R3 and R4) in batch mode. (*R1
was fed with actual wastewater and R2 was fed with multi metal spiked wastewater). (*R3 was fed with actual wastewater and R4 was fed with
multi metal spiked wastewater).

3.2. COD removal

Application of microorganisms for the removal of organic/inorganic matter from the wastewater has been used widely
in different wastewater treatment processes. Heavy metals at high concentrations are toxic to microorganisms present in
wastewater, which may affect the metabolic functioning of the microorganisms and subsequently the treatment process.
Performance of all the bioreactors in terms of COD removal efficiency was evaluated in treating actual wastewater as well
as wastewater spiked with heavy metals, both in aerobic and anaerobic conditions. During characterization of wastewater
it was observed that average COD of Loha mandi drain was around 297 mg L−1 . Further, it was noted that developed
microbial consortium was unable to grow without addition of an external substrate in the wastewater.
Hence, yeast extract (1 g L−1 ) was added as substrate at an interval of 5 days (0, 5, 10, 15 and 20 day) in wastewater
to support the growth of microbial community in bioreactor system during batch operation. Thereafter, wastewater
supplemented with 1 g L−1 yeast extract was fed in continuous mode. Overall, average COD removal (%) in bioreactor
R1, R2, R3 and R4 (Fig. 6) were 71%, 68%, 76% and 72%, respectively. Bioreactor R1 (aerobic) and R3 (anaerobic) without
any metal spiking showed higher COD removal as compared to metal spiked bioreactors R2 (aerobic) and R4 (anaerobic).
This clearly indicate the toxic effect of heavy metal on treatment efficiency.
On the other hand, average COD removal (%) in bioreactor R1, R2, R3 and R4 were 84%, 66%, 87% and 74%, respectively,
when the reactors were operated in continuous mode. As compared to the batch mode, COD removal efficiency of all the
bioreactors increased in continuous mode (except for R2-aerobic where slight decrease from 68% to 66% was observed).
Metal spiking in the wastewater inhibited the COD removal efficacy of the bioreactor as shown in Figs. 7 and 8. The
possible cause of lower COD reduction at higher concentration of heavy metal in wastewater might be due to the increase
in toxicity which influence the overall performance of micro-organisms present in the bioreactor system. It can be noted
that anaerobic reactors displayed better metal removal efficiency due to which toxicity would have been alleviated leading
to higher COD removal in anaerobic reactor. This difference was more clearly observed while treating metal spiked
wastewater (Fig. 8). Beyond 28 days in anaerobic bioreactor (R4), the concentration of certain metals (Ni2+ , Zn2+ and
Cu2+ ) dropped and hence the COD removal (%) in R4 stand significantly higher than aerobic bioreactor (R2), where the
concentration of all metals including Pb2+ , Cu2+ , Cr6+ and Zn2+ were still higher. Earlier, Oviedo et al. (2002)s heavy metal
(Zn2+ , Cu2+ and Cd2+ ) toxicity decreased COD removal up to 30%–40% in aerated lab scale bioreactor. It was observed
that drop in COD removal was largely depend on the different metal ions present in the wastewater.
The COD removal was inhibited significantly in presence of Cd2+ even at low concentration (0.3 mg L−1 ), whereas
exposure of Zn2+ at 3 mg L−1 did not any effect on COD removal efficiency. Similar decrease in COD removal efficiency in
presence of multi metal ions (Cu2+ , Cd2+ , Zn2+ , Ni2+ and Pb2+ ) was also observed with three different lab scale bioreactor
configurations (Kiran et al., 2015). A sharp fall in COD removal from 98% to 41% was observed in a sequential biological
12 D. Gola, P. Chawla, A. Malik et al. / Environmental Technology & Innovation 18 (2020) 100714

Fig. 7. Average COD value with removal percentage in bioreactor [R1 (aerobic) and R3 (anaerobic)] in continuous mode with actual wastewater.
(*R1 and R3 was fed with actual wastewater).

Fig. 8. Average COD value with removal percentage in bioreactor [R2 (aerobic) and R4 (anaerobic)] in continuous mode with multi metal spiked
wastewater. (*R2 and R4 was fed with multi metal spiked wastewater).

reactor when the concentration of Cd2+ increased above 14 mg L−1 (Santos et al., 2004). Taşeli et al. (2008) concluded that
Cr6+ caused more serve reduction in COD removal (27%) as compared to Ni+2 (22%), in a rotating biological contractor.
Azizi et al. (2016) also observed similar reduction in COD removal efficiency in bioreactor system (modified pack bed
reactor) with the increase in the heavy metal concentrations from 20 mg L−1 to 40 mg L−1 , using microbial consortium
in bed biofilm bioreactor.
 D. Gola, P. Chawla, A. Malik et al. / Environmental Technology & Innovation 18 (2020) 100714 13

Sujarittanonta and Sherrard (1981) reported that microbial community was adversely affected by the presence of
Ni2+ and Cr6+ at the concentration of 10 mg L−1 and hence causing significant drop in the COD removal rate. Therefore,
difference in COD removal efficiency for aerobic and anaerobic bioreactor may also be attributed to difference in microbial
community present in the bioreactor (Hartmann et al., 2013). Hence based on the results from the above experiments
and available literature, it can be concluded that COD removal depends on many factors such as microbial community in
the bioreactor, type of bioreactor, wastewater composition, metal ion and its concentration and operating conditions of
the bioreactor.

4. Conclusion

The experimental setup used above mimics the wastewater treatment process as actual wastewater was used during
operations. With the simplified design of bioreactor systems and its treatment efficacy for multi metal removal, it is
perfectly compatible even with heavy metal concentration fluctuations in wastewater. In addition to this, the usage of
native microbial consortium for bioremediation process can reduce the acclimatization period and can result in higher
removal efficacy. The above data indicates that two alternative approach can be used to treat same wastewater. It was
observed that metal removal efficiency of anaerobic native microbial consortium was higher as compared to aerobic
one. However heavy metal removal efficiency might reduce in actual field conditions. Hence the developed microbial
consortium was further tested in on-site bioreactors of different scales under field conditions.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper.

Acknowledgements

Authors gratefully acknowledge Mr. Sabal Singal and Vinod Kumar of Applied Microbiology Lab, Centre for Rural
Development and Technology, IIT Delhi.

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