Ecotoxicology and Environmental Safety 172 (2019) 281–289

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Ecotoxicology and Environmental Safety

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

In situ bioremediation of hexavalent chromium in presence of iron by dried T

sludge bacteria exposed to high chromium concentration
Neetu Bansal , Johan J. Coetzee, Evans M.N. Chirwa
⁎

Department of Chemical Engineering, University of Pretoria, Pretoria 0002, South Africa

ARTICLE INFO ABSTRACT

Keywords: Stability of chromium in the ferrochrome slag dumps and leachate are affected by pH, redox potential and the
In situ bioremediation presence of other metallic species in the slag. It is desirable to keep chromium in slag dumps in the trivalent [Cr
Chromium stabilisation (III)] state because trivalent chromium is 1000 times less toxic to living organisms than the hexavalent form [Cr
Biocatalysis (VI)]. Due to the low toxicity and low mobility of Cr(III), it is recommended to convert Cr(VI) to Cr(III) wherever
Ferrochrome slag
possible to protect the health of living organisms. In this study, the role of Cr(VI) reducing organisms for sta-
Chromium reducing bacteria
Inhibition kinetics
bilising chromium in slag dumps was evaluated in the presence of iron [oxidation states Fe(II) and Fe(III)]. The
study showed that stabilisation of chromium species in the trivalent state was most favourable under aerated
conditions. Up to 100 mg/L Cr(VI) was reduced in less than 24 h by cultures grown under aerobic conditions in
the presence of Fe(III). A much shorter time (6 h) was required to reduce the same amount of Cr(VI) in the
presence of Fe(II). When oxygen was completely excluded, it was only possible to reduce 20 mg/L in about 48 h
which was much slower than the removal of 100 mg/L in less than 24 h under aerated conditions. Fe(II) con-
tributed directly to catalytic reduction of Cr(VI) reduction whereas Fe(III) was beneficial to Cr(VI) reduction up
to an initial Cr(VI) concentration of 75 mg/L. Evaluation of Cr(VI) reduction kinetics showed that Cr(VI) re-
duction under aerobic conditions followed the non-competitively inhibited mixed-order reaction. Cr(VI) re-
duction in sealed reactor vessels, under anaerobic conditions, followed a modified non-competitive inhibition
reaction model. The results indicate that chromium stabilisation in ferrochrome slag dumps would require
maintenance of a fully aerated dump supplemented by a culture of Cr(VI) reducing organisms.

1. Introduction
Chromium in the environment exists in a range of oxidation states
ranging from Cr(-II) to Cr(+VI). However, the most prevalent forms in
the environment are chromium oxide [Cr(III)2O3] found in rocks and
chromite [FeCr(III)2O4] which is a significant component of geological
chrome ore seams. Chromite ore contains up to 80% chromium mainly
in the trivalent state (Dhal et al., 2013; Erdem et al., 2005). On the
other hand, chromium emanating from chromium refining is dis-
charged mainly as slag, sludge and/or dust slurry containing high levels
of chromium in the hexavalent form [Cr(VI)] (van Staden et al., 2014).
High temperatures in kilns and chemicals used during refining convert
Cr(III) from the rocks to Cr(VI), which consequently produces waste
product streams with high levels of Cr(VI). The slag portion of the waste
makes up the largest volume and is typically discharged into slag dumps
(slag heaps) sometimes together with rock residue tailings (Panda et al.,
2013). Leachate from the long–term storage of waste and slag pose a
perpetual risk to the higher order organisms living in receiving waters
(Satarupa and Paul, 2013). The release of Cr(VI) into the surrounding
environment can lead to bioaccumulation in humans and mammals,
while also causing major health issues (Coetzee et al., 2018; Guertin
et al., 2016). This especially problematic in situations where the lea-
chate water enters agricultural supply water. Under these conditions,
there is heightened risk of contamination of food products and bioac-
cumulation in fish and livestock products (Chirasha and Shoko, 2010).
Chromium, in its trivalent state, readily precipitates as chromium
hydroxide, Cr(OH)3(s), at near neutral to alkaline pH conditions
(pH > 6). The tendency of Cr(III) to precipitate makes Cr(III) less mo-
bile in the water/leachate which makes it much easier to manage its
ecological impacts as against Cr(VI) (Dogan et al., 2011; Madhavi et al.,
2013). Furthermore, the Cr(VI) is highly mobile in water and is known
to be carcinogenic in mammalian cells (Federal Register, 2004;
Zhitkovich, 2011).
Chromium in leachate and dust slurry is usually treated using con-
ventional physical-chemical processes by applying chemical reducing
agents to reduce Cr(VI) to Cr(III) followed by precipitation of Cr(III) as

⁎
Corresponding author.
E-mail address: neetu.bansal@uqconnect.edu.au (N. Bansal).

https://doi.org/10.1016/j.ecoenv.2019.01.094
Received 8 June 2018; Received in revised form 25 January 2019; Accepted 28 January 2019
Available online 01 February 2019
0147-6513/ © 2019 Elsevier Inc. All rights reserved.
N. Bansal et al.
a chromium hydroxide at a higher pH (< 8.0). This process leaves
behind a toxic chemical sludge that is even more costly to treat (Fiúza
et al., 2010; Hawley et al., 2004; Stasinakis et al., 2003). Alternatively,
a more passive biological process could be used to facilitate conversion
of the Cr(VI) to ensure long-term protection of the environment. The
viability of in situ biotransformation of Cr(VI) to Cr(III) has been de-
monstrated in both laboratory scale and pilot scale systems for effective
removal of Cr(VI) from water but has not been applied at full scale
(Bharagava and Mishra, 2018; Francisco et al., 2002; Jobby et al., 2018;
Liu et al., 2018).
In this study, the reduction of toxic hexavalent chromium by natural
occurring bacteria in the presence of Fe(II) and Fe(III), a common co-
pollutant that exists in most chrome refinery waste and residue from ore
extraction, is evaluated. The hypothesis is tested, if the ferrous/ferric
redox couple may act as a cyclic catalyst for Cr(VI) reduction to the
trivalent state, and as a stabilizer of chromium in the less toxic trivalent
state. A Cr(VI) reducing culture isolated from Cr(VI) contaminated
waste water plant sludge was used as the biocatalyst to achieve the
stabilisation of chromium as Cr(III) in aqueous solution.
2. Materials and methods
2.1. Chemical reagents
Analytical grade reagents with high purity level (99% and above)
were purchased from Sigma-Aldrich Pty Ltd (Industrial Park, Jet Park,
Johannesburg, South Africa) used in the experiments without further
purification. Standard solutions of chromium were prepared daily by
diluting 1 g/L of chromium stock solution. 1,5-diphenylcarbazide (DPC)
solution was prepared by dissolving specific amount of DPC in HPLC
grade acetone and stored in a brown bottle covered with aluminium
foil. 0.85% NaCl was prepared in distilled water and sterilized by au-
toclaving at 125 °C and 30 psi for 15 min. Iron species were added as
FeCl3·6H2O for Fe(III) and Mohr's salt for Fe(II). Mineral Salt Media
(MSM) was prepared by dissolving: 10 mM NH4Cl, 30 mM Na2HPO4,
0.8 mM Na2SO4, 0.2 mM MgSO4, 50 μM CaCl2, 25 μM FeSO4, 0.1 μM
ZnCl2, 0.2 μM CuCl2, 0.1 μM NaBr, 0.05 μM Na2MoO2, 0.1 μM MnCl2,
0.1 μM KI, 0.2 μM H3BO3, 0.1 μM CoCl2, and 0.1 μM NiCl2 into 1 L of
distilled water. The MSM solution was sterilized at 125 °C for 15 min
and 1 g/L of D-glucose was added to act as carbon source for the bac-
teria.
Luria–Bertani (LB) broth and Luria-Bertani (LB) agar media were
prepared by dissolving (in 1 L distilled water), 25 g and 35 g of the
respective commercial media powder purchased from Sigma–Aldrich
(Johannesburg, South Africa). Solid agar media was allowed to cool
down to 30 °C after sterilisation at 121 °C for 15 min before introducing
samples for streaking and/or colony development.
2.2. Bacterial culture
2.2.1. Sources of chromium reducing bacteria
The mixed culture of bacterial culture was collected from the sand
drying beds and dried sludge from the belt filter press at the Brits
Wastewater Treatment Works in Brits (North West Province, South
Africa). The samples were stored in sterile containers at 4 °C. The source
of organisms was chosen because the Wastewater Treatment Works had
received periodic flows of accidental discharges from an abandoned
Ferrochrome processing foundry, which discharged varying loadings of
sodium dichromate into the municipal system.
2.2.2. Culture isolation
Initial cultures were grown in Luria-Bertani (LB) broth by in-
oculating the solution with a grain (0.2 g) of collected sludge sample.
The inoculated broth was also spiked with 50 mg/L Cr(VI) and in-
cubated for 24 h at 30 ± 2 °C in a lateral shaker (Labotec, Gauteng,
South-Africa). Aerobic cultures were grown in 500 mL Erlenmeyer
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Ecotoxicology and Environmental Safety 172 (2019) 281–289
flasks containing 200 mL LB broth and covered with cotton wool plugs.
Anaerobic cultures were grown in 100 mL serum bottles that were
purged with pure nitrogen gas (99% pure grade) and sealed with sili-
cone rubber stoppers and aluminium caps.
Pure cultures were grown by transferring 1 mL of a serial diluted
sample onto a petri dish containing LB Agar. The cell was then in-
cubated at 30 °C to develop separate identifiable colonies. The in-
dividual colonies were transferred using a sterile wired loop and the
process was repeated three times until there were no more separate
identifiable colonies.
2.2.3. Culture characterization
Individual colonies developed were characterized by using phylo-
genetic characterization of the cells from the 7th to the 10th tube in the
serial dilution. Genomic DNA was extracted from the pure cultures
using a DNeasy tissue kit (QIAGEN Ltd, West Sussex, UK) as per man-
ufacturer's instructions. The 16S rRNA genes of isolates amplified by
reverse transcriptase-polymerase chain reaction (RT-PCR) using pri-
mers pA and pH1 (Primer pA corresponds to position 8–27; Primer pH
to position 1541–1522 of the 16S gene) (Coenye et al., 1999). An in-
ternal primer pD was used for sequencing (corresponding to position
519–536 of the 16S gene). Species identification was conducted by
BLAST search of the NCBI database. Phylogenetic tree diagrams were
then constructed using the neighbour-joining method (Tamura et al.,
2013). Confidence in the tree topology was determined by the bootstrap
analysis based on 100 re-samplings (Felsenstein, 1985).
2.3. Ferrochrome slag samples
Ferrochrome slag samples for evaluation of chromium and iron
species content in tailings was collected from waste dumps located at
separate open and closed furnace ferrochrome smelters located in the
North West Province (South Africa). Only samples from mixed waste
tailing dumps were used during this study.
2.4. Batch studies
2.4.1. Aerobic reduction experiments
Aerobic cultures were grown in 1 L Erlenmeyer flasks containing
400 mL LB Broth spiked with 50 mg/L Cr(VI) for 24 h. Cells were har-
vested by centrifuging at centrifugal force of 3220×g for 10 min at 5 °C.
The supernatant was decanted and the remaining pellet was washed
twice in a sterile saline solution (0.85% NaCl) while centrifuging.
Aerobic Cr(VI) reduction experiments were conducted in 250 mL
Erlenmeyer flasks containing 20, 50, 100, 200, and 400 mg/L Cr(VI).
An initial 1 mL sample was taken to measure initial concentration of Cr
(VI) before the cells were inoculated. The flasks were covered with
cotton wool plugs to prevent contamination, while still allowing oxygen
transfer. The batches was incubated at 30 ± 2 °C with continuous
shaking on a lateral shaker (Labotec, Gauteng, South Africa). All ex-
periments were conducted in duplicate and performed using cells har-
vested during the log-growth phase (16–24 h incubation). 1 mL with-
drawn at regular intervals were stalled for Cr(VI) and total chromium
determination over time. The effect of Fe(II) and Fe(III) on the reduc-
tion of Cr(VI) was evaluated at the optimum initial Cr(VI) concentration
of 50 mg/L using added Fe(II) and Fe(III) concentrations at 5, 20, 50,
75, and 100 mg/L.
2.4.2. Anaerobic reduction experiments
Anaerobic cultures were grown in 100 mL serum bottle containing
100 mL LB Broth (purged with 99% pure N2 gas for 15 min) spiked with
50 mg/L Cr(VI) for 24 h. 5 mL of the cells were then transferred anae-
robically to 500 mL serum bottles containing 400 mL LB broth and also
grown for 24 h. Cells were harvested by centrifugation at 3220×g for
10 min at 5 °C. The supernatant was decanted and the remaining pellet
was washed twice in a sterile saline solution (0.85% NaCl) after each
N. Bansal et al.
centrifugation cycle. Anaerobic Cr(VI) reduction experiments were
conducted in 100 mL serum bottles containing 20, 50, 100 mg/L Cr(VI)
in absence of iron and 5, 20, 50, 75, 100 mg/L Cr(VI) in presence of Fe
(II) or Fe(III). An initial 1 mL sample was taken to measure initial
concentration of Cr(VI) before the cells were inoculated. The serum
bottles containing the suspended cells were then purged with 99% pure
N2 gas and sealed with a silicon stopper and aluminium cap and then
incubated at 30 ± 2 °C with continuous shaking on a lateral shaker
(Labotec, Gauteng, South Africa). Cells were harvested during the log-
growth phase to ensure maximum viability of the cells used in the ex-
periment The effect of Fe(II) and Fe(III) on the reduction of Cr(VI) was
performed like aerobic batches except in anaerobic conditions.
2.4.3. Abiotic experiments
Abiotic experiments were conducted by heat killing grown cells
before inoculation to determine the abiotic reduction of Cr(VI).
Cultures were harvested by centrifuging for 10 min at centrifugal force
of 3220×g, and then washed twice in a sterile saline solution (0.85%
NaCl). The remaining pellet was used for the reduction experiments.
The supernatant from the biomass analysis was used for pH measure-
ments of the experimental batches.
2.5. Analytical methods
2.5.1. Cr(VI) analysis
Aliquote samples were centrifuged in 2 mL Eppendorf tubes for
10 min at 3220×g and 5 °C in a Minispin® Microcentrifuge (Eppendorf,
Hamburg, Germany) to remove suspended cells. The supernatant was
used for Cr(VI) concentration analysis. Cr(VI) concentration in the su-
pernatant was measured using a UV/Vis Spectrophotometer (WPA,
Light Wave II, and Labotech, South Africa) after reacting acidified
samples with 0.2 mL 1,5 DPC to yield a bright yellow colour. The so-
lution was scanned at the wavelength γ = 540 nm across a 10 mm light
path (Eaton et al., 2005). Fe(II) and Fe(III) is known to cause inter-
ference to the spectrophotometric measurement of Cr(VI) using the
reactive dyes (Mahesvari and Balasabramanian, 1996). The interference
was eliminated by acidifying the samples by adding 1.0 mL of 1 N sul-
furic acid to the 10 mL flasks before dilution of the sample to the 10 mL
mark. A similar method was used (Mahesvari and Balasabramanian,
1996) to eliminate Fe(II) and Fe(III) interference in a spectro-
photometric method based on ion-pair formation with the cationic
dye–rhodamine 6G. The elimination of the interference was verified by
preparation of standard curves of both species in medium without Cr
(VI) which showed no linear correlation between Fe(II), Fe(III) and
absorbance after the above treatment.
2.5.2. Total chromium analysis
Total chromium was measured at a wavelength of 359.9 nm using a
Varian AA–1275 Series Flame Atomic Adsorption Spectrophotometer
(AAS) (Varian, Palo Alto, CA) equipped with a 3 mA chromium hollow
cathode lamp. Before analysis, samples were diluted 5–50 time with
distilled water to bring the concentration of samples in calibration
range of the instrument. The lower Instrument Detection Limit (IDL) of
the AAS is 0.1 mg/L. Cr(III) was determined as the difference between
total chromium measured by AAS and Cr(VI) concentration measured
by UV/Vis spectrophotometer using the 1,5 DPC method. The AAS was
calibrated prior to total chromium analysis using 1–5 mg/L Cr(VI)
concentration prepared from Cr(VI) stock.
2.5.3. Characterization of metals in the slag dumps
The composition of metallic species and chromium levels in slag,
dust and leachate sample was measured to evaluate the need for de-
velopment of remediation steps to prevent mobilization of Cr(VI) in
waste rock dumps. Metallic species in leachate were measured using the
Inductively Coupled Plasma–Atomic Emission Spectroscopy (ICP–AES)
(Perkin–Elmer, Massachusetts, USA). The solid samples were digested
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Ecotoxicology and Environmental Safety 172 (2019) 281–289
by fusion with lithium metaborate. 0.3 g of sample was carefully mixed
with 4 g lithium metaborate in platinum crucible. The crucible con-
taining samples was heated to 1000 °C for 4 h. After cooling the sample,
fused mass was heated with C HCl at boiling point until dissolved
(Burman et al., 1978; FDA USGS, 2004). The solution was filtered
through Whatman paper No 42 having diameter 2.5 cm. The final vo-
lume of the solution was made up to 100 mL by adding distilled water
for analysis by ICP–AES.
2.6. Biomass analysis
Quantitative analysis of biomass was conducted gravimetrically.
Biomass samples were collected by withdrawing 5 mL from the ex-
perimental batches at regular time intervals (0, 6, 14, 24 h). The sample
was centrifuged at a centrifugal force of 3220×g for 10 min. The re-
maining pellet was then re-suspended in 1 mL distilled water. The re-
suspended solution was then filtered through a pre-weighed Whatman
filter paper (No. 1). The filter paper was then dried at 75–80 °C and
cooled in desiccator and weighed. The samples were dried until a
constant weight was obtained. Total suspended cell weight was calcu-
lated from the difference in weight of the filter paper before and after
filtration (Molokwane, 2010).
3. Reduction kinetics and model development
The reaction rate used in this study was developed earlier based on
the assumption that each Cr(VI) reducing living cell in the reactor
produces a certain amount of enzyme (E′) and that deactivating the cell
deprives the system of that particular amount of enzyme (Shen and
Wang, 1994). Assuming that the remaining active enzyme Ea is pro-
portional to the amount of live cells (X) in the system (Ea∝X), then the
reaction rate catalysed by the enzymes could be considered as being
directly proportional to the reaction rate catalysed by cells. Based on
these assumptions, Shen and Wang (1994) derived the Cr(VI) reduction
rate law of a closed batch system, represented by the specific Cr(VI)
reducing activity of living cells in the system:
dC k C Co C
= mc Xo
dt Kc + C Rc (1)
−3
where C = Cr(VI) concentration (ML ) at any time t (T), kmc =
maximum specific Cr(VI) reduction rate coefficient (T−1), Xo = initial
viable cell concentration (ML−3), Kc = half velocity constant (ML−3),
and the term (Co-C)/Rc represents cells killed due to exposure to Cr(VI)
such that the term brackets is the concentration of active cells available
at time t:
X = Xo
Co C
Rc (2)
where X = viable cell concentration (ML−3) at any time t, Co = initial
Cr(VI) concentration (ML−3), and Rc = finite Cr(VI) reduction capacity
(grams Cr(VI) reduced/gram of cells deactivated) (MM−1). However, it
was observed that the Cr(VI) reduction rate at different initial Cr(VI)
concentrations (Co) produced different values of the maximum specific
Cr(VI) reduction rate coefficient (kmc).
One possible explanation for this phenomenon is that, due to the
fast cell growth rate under aerobic conditions, electrons are released in
excess such that Cr(VI) reduction in such a system is non–competitive.
A non–competitive Cr(VI) reduction expression was therefore proposed
in which increased Co values decreased the impact of the kmc re-
presented by Eq. (3) (Molokwane, 2010):
dC kmc C Co C
= Xo
dt C
1 + Ko Kc + C Rc
(3)
I
−3
where KI = coefficient of inhibition (ML ). When cells grown without
oxygen, the cell growth rate was much slower. Eq. (3) applied under
N. Bansal et al. Ecotoxicology and Environmental Safety 172 (2019) 281–289

non–inhibitory conditions, but after a certain value Cr was exceeded, Table 1

the reactor was affected by the exponential function of inhibition Ferrochrome slag analysis results by ICP-AES.
coefficient K as shown in the modified Eq. (3) (Eq. (4)): Element Slag Dust Leachate
(mg/g) (mg/g) (mg/g)
dC kmc C Co C
= (1 Xo
dt K Cr / Co) (K
c C) Rc (4) Ti 2.9 2.7 n/a
Al 44.1 48.4 n/a
where, K = competitive inhibition factor (dimensionless), Cr = the Cr Fe 75.9 79.9 n/a
(VI) inhibition threshold concentration (ML−3). In this case, the in- Mn 2.9 3.8 0.3
Mg 74.9 122 97
hibition kinetic is still non-competitive until the concentration exceeds
Ca 20 19.5 162
a certain threshold concentration (Cr). Above the threshold concentra- Cr 97.2 93.4 n/a
tion Cr, the reaction rate responded to the initial Cr(VI) concentration Ni 2.6 1.3 n/a
(Co) as shown in Eq. (4). Zr 18.9 0.9 n/a
Ba 16.0 29.3 n/a
Cu 0.2 0.4 n/a
4. Results and discussion Zn 9.9 22.9 0.2
Pb 45.9 48.3 n/a
4.1. Bacterial culture identification
*n/a: below detection limit.
Culture purification and species identification was conducted using
the 16S rRNA genotype fingerprinting at independent labs in Pretoria
researchers also reported Pseudomonas aeruginosa is a promising chro-
(South Africa). The sequenced genomic DNA was compared to se-
mium reducing bacteria at high chromium concentration (Ozturk et al.,
quences in the NCBI (National Centre for Biotechnology Information)
2012; Xu et al., 2009). Klebsiella oxytoca also showed the high tolerance
gene bank using the BLAST search. The results from the genotype
and reduction, potential to reduce Cr(VI) under aerobic (Garavaglia
characterization of natural consortium formed under aerobic and
et al., 2010) as well as anaerobic conditions (Baldi et al., 2001). Alca-
anaerobic conditions showed the predominance of three aerobic
ligenes faecalis isolated from chromium containing waste showed the Cr
Serratia marescens (X1,X2), Pseudomonas aeruginosa (X3), Alcaligenes
(VI) reduction capability under wide range of pH and temperature
faecalis (X5) and one anaerobic Klebsiella oxytoca (X4), gram-negative
(Biswas et al., 2015; Shakoori et al., 2010). Serratia marescens has also
bacteria (Fig. 1).
been reported to reduce Cr(VI) anaerobic conditions (De Bruijn and
It was assumed here that the gram-negative, bacteria played a cri-
Mondaca, 2000).
tical role in the Cr(VI) reducing activity of the sludge culture. Earlier

Fig. 1. Phylogenetic tree of bacteria identified in the BLAST search using the 16S rRNA sequences.

284
N. Bansal et al.
Fig. 2. Evaluation of aerobic Cr(VI) reduction at initial Cr(VI) ranging between
20 and 400 mg/L in the absence of Fe species (a); Aerobic reduction of 50 mg/L
Cr(VI) reduction in the presence of Fe(II) ranging between 5 and 100 mg/L (b);
Aerobic reduction of 50 mg/L Cr(VI) in the presence of Fe(III) ranging between
5 and 100 mg/L (c).
4.2. Mass balances
Total chromium was measured in sacrificial runs of 48 h to check
the mass balance between added Cr(VI) and total chromium after 48 h.
The results showed total recovery of all chromium species as total
chromium after 48 h with an error range of ± 5%. For cultures where
Cr(VI) was completely removed but almost 100% total chromium was
indicated, this confirmed that chromium was still present in the system
as Cr(III). Losses within the first hour of incubation were constant re-
gardless of the initial concentration used and were attributed to ad-
sorption to cell surfaces.
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Ecotoxicology and Environmental Safety 172 (2019) 281–289
Fig. 3. Anaerobic reduction of Cr(VI) in initial concentrations of Cr(VI) ranging
between 20 and 100 mg/L in the absence of Fe species (a); Anaerobic reduction
of 50 mg/L Cr(VI) in the presence of Fe(II) ranging from 5 to 100 mg/L (b);
Anaerobic reduction of 50 mg/L Cr(VI) in the presence of Fe(III) ranging from 5
to 100 mg/L (c).
4.3. Characterization of metals in tailing dump slag, dust, leachate
The elemental analysis of metal in slag, dust and lechate samples
was conducted to determine the typical range of chromium and iron
concentration in an actual slag dump to serve as guide for concentra-
tions to be used in the experiments. The values determined from an
actual slag dump samples for chromium and iron are shown in Table 1.
The metals in Table 1 were detected as oxides of the parent metals.
N. Bansal et al. Ecotoxicology and Environmental Safety 172 (2019) 281–289

Table 2
Optimum kinetic parameters using the non-competitive inhibition model (Eq. (3)) for aerobic conditions and using the competitive inhibition model (Eq. (4)) under
anaerobic conditions.
Conditions Concentrations (mg/L) Aerobic Anaerobic

kmc 1/h Kc mg/L Rc mg/mg Ki mg/L Xo mg/L kmc 1/h Kc mg/L Rc mg/mg K Cr mg/L Xo mg/L

a 20 0.8 700 0.022 7.5 5780 0.12 180 0.01 10 2.5 2240
50 0.8 700 0.022 7.5 5940 0.12 180 0.01 10 2.5 2240
100 0.8 700 0.022 7.5 6220 0.12 180 0.01 10 2.5 2160
200 0.8 700 0.022 7.5 5720 0.12 180 0.01 10 2.5 2195
b 5 0.85 1200 0.02 6.8 5000 0.12 180 0.01 10 2.5 2240
20 0.85 1200 0.02 6.8 5000 0.3 280 0.025 13 20 2360
50 0.85 1200 0.02 6.8 6000 0.1 280 0.028 13 20 2310
75 0.85 1200 0.02 6.8 6000 0.1 280 0.015 13 20 2340
100 0.85 1200 0.02 6.8 6500 0.1 280 0.012 13 20 2280
c 5 0.26 1280 0.008 9.5 4000 0.1 280 0.012 13 20 2260
20 0.26 1280 0.008 9.5 3800 0 400 0 13 20 2360
50 0.26 1280 0.008 9.5 4000 0 400 0 13 20 2360
75 0.26 1280 0.008 9.5 4000 0 400 0 13 20 2360
100 0.26 1280 0.008 9.5 3900 0 400 0 13 20 2360

a: Cr (VI) concentrations, b: Fe(II) concentrations with 50 mg/L chromium, Fe(III) concentrations with 50 mg/L chromium.

Looking at iron and chromium in the Table, it is shown that the prac-
tical experimental range should span the 75 mg/L Fe and 100 mg/L Cr.
4.4. Mixed culture batch performance
4.4.1. Aerobic reduction experiments
Chromium reduction experiments without the presence of iron
showed a 100% reduction of up to 100 mg/L initial concentration of Cr
(VI) within a period of 24 h. Higher concentrations of Cr(VI) were re-
duced for a period up to 48 h before reduction slowed down sig-
nificantly (Fig. 2a). This can be attributed to the toxicity level of the
chromium bacteria being reached. The relatively low concentration of
carbon source used can also indicate that the bacterial cells were de-
pleted of an energy source after about 24 h.
For aerobic reduction experiments in the presence of Fe(II) a large
initial decline of Cr(VI) was seen after the first couple of seconds of the
experiment after Fe(II) was added. This can be attributed to the oxi-
dation-reduction that occurs between Cr(VI) and Fe(II) as both ions are
highly reactive, where Fe(II) act as electron donor. Higher initial Fe(II)
concentrations proved to have a greater effect on the total reduction.
An initial Cr(VI) concentration of 50 mg/L was fully reduced within six
hours in 50–100 mg/L Fe(II) and within 24 h in lower concentrations
(Fig. 2b). Aerobic reduction of Cr(VI) in the presence Fe(III) indicated
that different concentrations of ferric ion did not influence overall Cr
(VI) reduction. It is, however observed that the overall reduction was
much slower, which can indicate that the presence of Fe(III) in the
system has an inhibiting effect on Cr(VI) reduction (Fig. 2c).
4.4.2. Anaerobic reduction experiments
It was observed during the anaerobic reduction that 100% of Cr(VI)
at an initial concentration of 20 mg/L was achieved within 48 h. Higher
initial concentrations showed a fast initial drop in Cr(VI) reduction,
before the reduction slowed dramatically (Fig. 3a). This can be attrib-
uted to the toxicity levels of the Cr(VI) reducing bacteria being reached
(Chirwa and Wang, 2000; Kourtev et al., 2006) or depletion of carbon
source.
As with the anaerobic reduction of Cr(VI) a fast initial drop in Cr(VI)
was observed in the presence of Fe(II). It was observed that higher
initial concentrations of Fe(II) allowed faster overall reduction of Cr(VI)
to take place with full reduction of 50 mg/L Cr(VI) being achieved in
24 h in the presence of 100 mg/L Fe(II) (Fig. 3b). Under anaerobic
conditions, Cr(VI) reduction rate increased from the lowest Fe(III) ex-
posure of 10 mg Fe(III)/L up to the value of 75 mg Fe(III)/L (Fig. 3c).
After 75 mg/L Fe(III), Cr(VI) reduction rate stabilised. Earlier studies
indicated a decrease in Cr(VI) reduction rate after 100 mg/L Fe(III)
showing an inhibitory effect or competition for electrons and Fe(III)
thermodynamically requires electrons to convert to Fe(II) under redu-
cing conditions (Coetzee et al., 2018). The Cr(VI) reduction capacity
beyond the 100 mg Cr(VI)/L threshold was attributed to an inhibiting
effect of Fe(III) in the system or the depletion of carbon source.
4.5. Abiotic reduction
Abiotic reduction of Cr(VI) with heat-killed cultures showed that
there was no abiotic reduction of Cr(VI) taking place aerobically or
anaerobically. The results were confirmed using azide killed and cell
free controls which also showed negative results.
4.6. Batch pH measurements
The pH of the resultant mixture was measured over a period of 24 h.
It was found that in the reduction experiment with and without iron the
pH between the different batches were similar and remained constant
(6–7) over the course of the experiment. Earlier, researchers showed, Cr
(OH)3 is the predominant species in the pH range 5.5–10.5, this cor-
relates with the area where the majority of the biological reaction occur
(Ball and Nordstrom, 1998; Richard and Bourg, 1991).
4.7. Kinetic results
4.7.1. Parameter estimation
Parameter estimation on the kinetic equations was conducted by
using the Computer Program for the Identification and Simulation of
Aquatic Systems AQUASIM 2.01 (Reichert, 1998). The model compar-
ison with experimental data was conducted by minimizing the sum of
squares error between the measured data points and the calculated data
points using the Secant function in AQUASIM.
4.7.2. Aerobic batch kinetics
After fitting the model to the anaerobic and aerobic batch data,
optimum kinetic parameters were obtained for different Fe(II) and Fe
(III) concentrations as shown in Table 2. The results indicate that the
maximum specific reaction rate coefficient was consistent over the
range of Cr(VI) concentrations. Parameter estimation for aerobic study
described the suggested model fits the data for the current consortium.
This model can then be used to predict behaviour and reduction at
other concentrations also. Fig. 4(a,b,c) show modelled plot fits well
with the experimental value for the aerobic batch experiments. The
results showed that all constants are constant for various concentration
of Cr(VI), Fe(II) and Fe(III) for respective aerobic study (Table 2).

286
N. Bansal et al.
Fig. 4. Aerobic Cr(VI) reduction at different concentrations in the absence of Fe
species (line plots indicate model simulations) (a); Aerobic Cr(VI) reduction at a
fixed Cr(VI) concentration (50 mg/L) and varying Fe(II) concentrations (mod-
elled plots) (b); Aerobic Cr(VI) reduction at a fixed Cr(VI) concentration
(50 mg/L) and varying Fe(III) (modelled plots) (c).
Schlautman and Han (2001) showed that the oxidation–reduction
reaction between Cr(VI) and Fe(II) can be a fast reaction occurring
within a matter of seconds. A large initial drop in Cr(VI) was observed
after Fe(II) was added to the system. Analytical methods to determine
Fe(II) concentration showed that there was no Fe(II) available in the
system as fast chemical reaction between Cr(VI) and Fe(II) oxidises
approximately all Fe(II) into Fe(III). This is expected as the Cr(VI) is in
excess and the Fe(II), the limiting reagent in the chemical reduction. It
was thus predicted that the initial drop in Cr(VI) was due to the che-
mical reaction and the remaining Cr(VI) was reduced biologically.
The kinetic results of Fe(II) batches indicated that the maximum
specific rate coefficient, kmc was similar to the pure batch. The
287
Ecotoxicology and Environmental Safety 172 (2019) 281–289
Fig. 5. Anaerobic reduction at different concentrations in the absence of Fe
species (line plots indicate model simulations) (a); Anaerobic reduction at fixed
Cr(VI) concentration (50 mg/L) and varying Fe(II) concentration (modelled
plots) (b); Anaerobic reduction of Cr(VI) at different concentrations of Fe(III)
(modelled plots) (c).
parameters between the different Fe(II) concentrations were similar,
with a good fit of the experimental data. This indicates that the re-
duction rate is governed by biological reaction only, and that the che-
mical effects are negligible. The modelled maximum specific rate
coefficient, kmc, was significantly lower for the aerobic reduction of Cr
(VI) in the presence of Fe(III). This may indicate that the presence of Fe
(III) in the system has an inhibition effect on the reduction of Cr(VI).
This lower rate of reduction can also be attributed to a much lower
initial biomass concentration found in the system. The difference in
initial Cr(VI) concentrations are due to the rapid reaction with Fe(II)
present in the system.
4.7.3. Anaerobic batch kinetics
The cell inactivation model showed that the pseudo mixed-order
non-competitive reaction kinetic model fitted the experimental anae-
robic data well. Like aerobic parameter estimation, anaerobic kinetic
results also showed the well fitted graphs with the suggest model
N. Bansal et al.
Fig. 5(a,b,c). The modelled maximum specific rate coefficient, kmc, for
the anaerobic reduction experiment was much lower than the aerobic
reaction (Table 2). This is expected due to the lower initial biomass
concentration that was achieved after growth.
Similar reaction rate parameter data was observed with anaerobic
Cr(VI) reduction in the presence of Fe(II) which indicates that the
chemical effects of Fe(II) only plays a role in the initial reduction of Cr
(VI). Anaerobic reduction in the presence of Fe(III) show a decrease in
the maximum specific rate coefficient, kmc. This indicates that the
presence of Fe(III) in the system may have an inhibition effect on the
reduction performance of the anaerobic Cr(VI) reducing bacteria. The
modelled data between the different Fe(III) concentrations are similar
and fit the experimental data well, which indicates that the reaction is
governed by bacterial activity only.
5. Conclusion
In this study, aerobic and anaerobic microbial cultures dominated
by Pseudomonas aeruginosa, Serratia marescens, Alcaligenes faecal,
Klebsiella oxytoca were found to withstand high level of Fe(II) and Fe
(III), and were capable of reducing Cr(VI) at concentrations, up to
50 mg/L Cr(VI) in 6 h for aerobic and 24 h in anaerobic conditions.
50 mg/L Cr(VI) was fully reduced in the presence of 50–100 mg/L Fe(II)
for aerobic condition and in presence of 100 mg/L Fe(II) for anaerobic
condition. Presence of Fe(III) did not influence the reduction of Cr(VI)
in both aerobic and anaerobic conditions. The results demonstrate that
introducing such cultures in a Cr(VI) contaminated environment such
as the ferrochrome mining tailing dams could help stabilize the chro-
mium species in the tailing dam. This could be achieved by the re-
ductive potential of ferrous ion which serves as the electron donor in
the Fe/Cr redox couple. In South Africa, total chromium in the range of
18–1104 mg/L has been observed in mining dump site (Olobatoke and
Mathuthu, 2016) of which 30–100 mg/L could be Cr(VI). In the aerobic
system 100% immobilization of Cr(VI) was observed within 24 h of
100 mg/L Cr(VI), while complete reduction of 20 mg/L Cr(VI) was
achieved within 48 h in the anaerobic system. In the current study, a
minimum carbon source concentration of 1 g/L was used. In real site
applications, only a minimum of carbon sources would be needed to
avoid further polluting the environment with the carbon sources which
could create a high oxygen demand in receiving water bodies.
The study conducted under anaerobic conditions is most relevant to
the target application in mine tailing dumps site as the environment
would be mostly cut off from contact with atmospheric water, even
though operation under anaerobic conditions showed a lower Cr(VI)
reduction rate coefficient and a higher susceptibility to Cr(VI) toxicity.
In spite of the slower removal rate under anaerobic conditions, the
principle removal mechanisms appear to be the same as in the aerobic
systems. The trends of removal and the limited removal capacity as Cr
(VI) reduction is evident in both systems.
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