RESEARCH PAPER

https://doi.org/10.1071/EN21155

Effect of dissimilatory iron reduction and Carex DOM on CrVI

reduction by Enterobacter sp. PY16 isolated from wetland soil
Yao ShuA,B, Xiaofeng GongA,B,* , Yuanhang LiA,B, Yuheng SunA,B, Danni NiuA,B and Hongting YeA,B

Environmental context. Dissimilatory iron reduction and Carex-produced dissolved organic matter (DOM) have an important
influence on Cr(VI) reduction by the Fe(III)-reducing bacterium Enterobacter. The role of Carex DOM and ferrihydrite in the
biotransformation of Cr(VI) by the bacterial isolate was investigated. The findings should help underpin the remediation and
detoxification of chromium in anaerobic environments, and provide promising insights into the quaternary system of bacterium/
Fe(III)/Cr(VI)/DOM.

For full list of author affiliations and

ABSTRACT
declarations see end of paper
Rationale. FeIII oxides and organic matter are important factors influencing CrVI degradation
*Correspondence to:
in wetland soils. However, it remains unclear how they interact in anaerobic systems.
Xiaofeng Gong Methodology. In this study, a strain of iron-reducing bacterium was isolated from Poyang
School of Resources & Environment, Lake Wetland and identified as Enterobacter sp. PY16 (PY16) by PCR-16S-rDNA sequence
Nanchang University, Nanchang 330031,
analysis. Moreover, microbial reduction of FeIII/CrVI by PY16 and their mutual transformation
China
Email: xfgong@ncu.edu.cn in the quaternionic system of PY16/ferrihydrite/CrVI/dissolved organic matter (DOM,
extracted from Carex cinerascens) were investigated. Results. The results showed that
Handling Editor: PY16 could directly participate in the reduction of ferrihydrite and CrVI. The rate of CrVI
Kevin Wilkinson reduction decreased with the increase of initial CrVI concentration, while it was enhanced
by 2.78–42.99% in the presence of ferrihydrite. Moreover, 15 mg L−1 CrVI was almost
eliminated after 72 h and the content of FeII increased by 78.21 mg L−1 in the presence of
DOM. Discussion. The fastest CrVI reduction rate occurred when ferrihydrite and DOM
coexisted in the system, mainly because the promoting effect of DOM on ferrihydrite
synergistically promoted CrVI reduction. DOM and FeII produced during the ferrihydrite
reduction process served as electron shuttles that promoted CrVI reduction by a biochemical
redox pathway. However, the electron transfer and donation capacity of DOMox/DOMred
and FeIII/FeII in the reaction process still need to be further studied. Implications for future
research. The results underscored the importance of FeIII oxides and DOM on microbial
CrVI reduction, thus providing a valuable technique to remove and detoxify chromium in
wetland soils.

Keywords: chemical reduction, CrVI reduction, dissmilatory iron reduction, DOM, electron
transfer, Enterobacter sp., iron reducing bacteria, microbial reduction, PY16.

Received: 30 November 2021

Accepted: 26 March 2022
Published: 1 June 2022
Cite this:
Shu Y et al. (2022)
Environmental Chemistry
19(1), 13–22. doi:10.1071/EN21155
© 2022 The Author(s) (or their
employer(s)). Published by
CSIRO Publishing.
Introduction
Chromium (Cr) is a common and hazardous contaminant that is frequently detected in
sediments and groundwater (Jiang et al. 2019; Rebhi et al. 2019). Cr generally exists in
the form of hexavalent chromium (CrVI) and trivalent chromium (CrIII) in the natural
environment. CrVI is readily water-soluble and more harmful than CrIII owing to its strong
oxidation capacity (Rajyalaxmi et al. 2019). Therefore, the reduction of CrVI to its less
harmful form CrIII can be a practical approach towards remediation (Vaiopoulou and
Gikas 2012). Compared with the chemical method, microbial CrVI reduction is currently
considered as a promising remediation method (Sani et al. 2002) due to the benefits of
low operating costs and hardly any secondary pollution.
Y. Shu et al.
Iron-reducing bacteria (IRB) are microorganisms that
can utilise a carbon source such as organic matter as
an electron donor and FeIII oxide as an electron acceptor
to reduce FeIII to FeII, and obtain energy to support its
own metabolism through dissimilatory iron reduction
(Honetschlagerova et al. 2018). Since Lovley et al. (1987)
first isolated Geobacter metalli-reducens GS-15 from river
sediments, more and more IRB have been discovered
(Cummings et al. 2000; Roh et al. 2002; Liu et al. 2020),
among which the Geobacter and Shewawnella were the most
extensively studied (Hu and Li 2018). However, few studies
have reported the isolation and characterisation of FeIII-
reducing bacteria in Poyang Lake Wetland where the sea­
sonal water levels fluctuate wildly.
In addition to changing the form of iron, IRB can also result
in the degradation of some heavy metals and toxic pollutants,
such as chromium (Dai et al. 2018). There is increasing evi­
dence suggesting that IRB can microbiologically reduce CrVI
through enzymatic reduction reactions (Parmar et al. 2000).
But high levels of CrVI can produce inhibitory effects and cyto­
toxicity on cell growth (Parker et al. 2011). Fortunately, micro­
bial dissimilatory iron reduction has been proved to promote
CrVI reduction by IRB (Wielinga et al. 2001). It was found that
the ferrous iron (FeII), produced through a dissimilatory iron
reduction process, is a more potent reductant enhancing CrVI
reduction (Carvalho et al. 2019). Compared to enzymatic pro­
cesses, the redox cycles of FeII/FeIII can significantly stimulate
the reduction of CrVI to CrIII (Huang et al. 2016).
Dissolved organic matter (DOM) refers to the water-
soluble organic matter that can pass through a 0.45 μm mem­
brane, and is known to serve as an electron shuttle during
redox reaction (Nurmi and Tratnyeka 2002). Moreover,
DOM usually affects the state of FeIII oxide and CrVI oxide
by facilitating electron transfer in reduction reactions (Bolan
et al. 2003). However, the impact of ferrihydrite and Carex
DOM on CrVI biotransformation by Enterobacter sp. is not yet
very clear. The simultaneous transformation of CrVI in a
quaternionic system of Enterobacter sp. PY16 (PY16)/ferri­
hydrite/CrVI/Carex DOM is expected to be further studied.
Therefore, this research aimed to study the capacity of
iron-reducing bacterium Enterobacter sp. PY16 isolated from
Poyang Lake Wetland to remediate CrVI. PY16 was used as
the driving force of electron transfer. Artificial amorphous
ferrihydrite and Cr2O72− were used as the terminal electron
acceptors. In addition, the influence of ferrihydrite and
Carex DOM on CrVI reduction by PY16 concerning its prob­
able mechanism were investigated to provide a theoretical
basis for the purification of the contaminants.
Table 1. Basic properties of the sediments.
Sample Fe (g kg−1)
Amorphous Fe Free Fe
Sediment 1.23 ± 0.23 6.38 ± 0.25
14
Environmental Chemistry
Materials and methods
Sample collection
The soil sample was collected from lakeshore sediments in
Nanjishan Nature Reserve, Poyang Lake. The main charac­
teristics are shown in Table 1.
Chemicals and DOM preparation
The composition of the media (g L−1) used was as follows: LB
media (peptone, 10; beef extract, 5; NaCl, 5); Ferric citrate
medium (ferric citrate, 3.3; NH4Cl, 1; CaCl2·2H2O, 0.07;
MgSO4·7H2O, 0.6; K2HPO4·3H2O, 0.722; KH2PO4, 0.25; glu­
cose, 10); CrVI-reducing medium (glucose, 20; KCl, 0.1; NaCl,
0.1; CaCl2, 0.1; NaH2PO4, 0.8; tryptone, 4; corresponding
K2Cr2O7 solution). The pH of the medium was about 7.0.
The medium was sterilised by autoclave before use (121°C
for 20 min). Artificial ferrihydrite was prepared following
previous studies and stored at 4°C (Wang et al. 2014).
K2Cr2O7 solution was prepared using ultra-pure water and
stored at 4°C. Carex DOM was extracted from Carex, a kind
of plant growing in the wetland of Poyang Lake. The extrac­
tion method used referred to previous research (Shen et al.
2019). The prepared Carex DOM solutions were stored at 4°C.
Isolation and identification of the strain
The wetland soil from Poyang Lake was selected as the
source of FeIII-reducing bacteria. Dried soil (10 g), artificial
ferrihydrite (1 mL, 8.7 g L−1), and some pure water were
added into a 100 mL serum bottle, incubated at 30°C for a
week and the supernatant was obtained as inoculum. The
diluted inoculum was coated onto ferric citrate medium agar
patches and cultivated for 3–4 days. Single colonies were
then harvested and inoculated into a liquid ferric citrate
medium for 24 h. The strains with the strongest FeIII reduc­
tion capacity were then re-streaked and further purified, a
process that was repeated several times. The suspension was
prepared using LB medium and suspended at 0.8–0.9
(OD600) by sterile water. 16S rRNA gene sequence analysis
was used to identify the isolated strain, carried out by
KeChang Biotechnology Company (NanChang, China).
MEGA 7.0 was used to analyse the phylogentic tree.
CrVI reduction experiments
Serum bottles were used as reaction vessels and inoculated
with 2% of bacterial suspension or 1 mL of ferrihydrite or
1 mL of Carex DOM. All the treatments were incubated
pH Organic matter (g kg−1)
Total Fe
12.64 ± 0.09 5.72 ± 0.30 28.24 ± 0.68
www.publish.csiro.au/en Environmental Chemistry

anaerobically in the dark at 35°C for better cell growth,
conducted in triplicate. Batch experiments for exploring
effects of ferrihydrite and Carex DOM on CrVI degradation
were performed as follows: (1) Reduction and tolerance of the
PY16 to different concentrations of CrVI was tested. (2) Effect
of ferrihydrite on CrVI reduction by the PY16 was tested.
(3) Mutual coupling of ferrihydrite/Carex DOM on CrVI reduc­
tion was tested. During the reaction process, OD600, FeII con­
centration and CrVI concentration were measured, respectively.
Analytical method
DOM concentration was expressed by water-soluble organic
carbon (DOC) and measured using a total organic carbon
(TOC) meter (Multi N/C-2100, Jena, Germany). Biomass
accumulation OD600 was measured using a spectrophotometer
(TU-1901, China). FeII concentration was determined by the
o-phenanthroline spectrophotometric method (Li et al. 2017).
CrVI concentration was determined by the diphenyl carbo­
hydrazide spectrophotometric method (Peng et al. 2015).
The morphological changes of the PY16 and the oxida­
tion state of Cr after reaction were detected by scanning
electron microscopy coupled with energy dispersive spec­
trometry (SEM-EDS) and X-ray photoelectron spectroscopy
(XPS) (Li et al. 2019), carried out by SuLin Technology
Company (HuBei, China). Sample pretreatment procedures
followed the previous research of Zhang et al. (2018a).
Briefly, the suspension after the reaction was fixed with
2.5% glutaraldehyde and then continuously dehydrated
with 25, 50, 75, 95 and 100% ethanol. Samples were then
dried and sputter-coated with Pt before being analysed.
Statistical analyses
SPSS 26.0 and Excel 2018 were used for statistical analysis
of experimental data, and Origin 8.5 software was used for
plotting. XPS peak 41 software was used to analysis the
XPS data.
Results and discussion
Isolation and identification of IRB
After several solid–liquid separations, PY16 was selected as
the strain in this experiment due to its strong reducing
ability of ferrihydrite. According to its 16S rRNA sequencing
results, PY16 was similar to the known strain of Enterobacter
asburiae strain JCM6051 (NR_024640.1) with a genetic
homology of 97.47% (Fig. 1). Therefore, PY16 was analo­
gous to Enterobacter species and was tentatively designated
as Enterobacter sp. PY16.
Up to now, previous studies related to understanding
dissimilatory iron reduction mainly focus on Geobacter
and Shewanella and are thus considered to be the model
bacteria. The process of iron reduction mediated by
Enterobacteria has rarely been reported. The result indicated
that Enterobacter sp. had a potential iron-reducing function,
which was similar to previous studies suggesting that pure
strains belonging to Enterobacter sp. can utilise FeIII oxides
(Liu and Wang 2016). PY16 could use glucose as a carbon
source and ferrihydrite as a terminal electron acceptor to
support its own growth and metabolism through dissimila­
tory iron reduction.

NR 117679.1 Enterobacter cloacae strain DSM 30054 16S ribosomal RNA partial sequence
–
66 NR–102794.2 Enterobacter cloacae strain ATCC 13047 16S ribosomal RNA complete sequence
NR 113615.1 Enterobacter cloacae strain NBRC 13535 16S ribosomal RNA partial sequence
78 –
NR 028912.1 Enterobacter cloacae strain 279–56 16S ribosomal RNA partial sequence
98 –
NR 044978.1 Enterobacter cloacae subsp. dissolvens strain LMG 2683 16S ribosomal RNA partial sequence
–
21 NR 118011.1 Enterobacter cloacae subsp. dissolvens strain ATCC 23373 16S ribosomal RNA partial sequence
–
PY16
NR 024640.1 Enterobacter asburiae strain JCM6051 16S ribosomal RNA partial sequence
75 –
21
27 NR 145647.1 Enterobacter asburiae strain JM-458 16S ribosomal RNA partial sequence
–
27 NR 146667.2 Enterobacter tabaci strain YIM Hb-3 16S ribosomal RNA partial sequence
–
NR 044977.1 Enterobacter cancerogenus strain LMG 2693 16S ribosomal RNA partial sequence
–
77 NR 042349.1 Enterobacter ludwigii strain EN-119 16S ribosomal RNA partial sequence
–
35 NR 116756.1 Enterobacter cancerogenus strain LMG 2693 16S ribosomal RNA partial sequence
–
NR 118568.1 Enterobacter cloacae strain ATCC 13047 16S ribosomal RNA partial sequence
–
32 NR 126208.1 Enterobacter hormaechei subsp. xiangfangensis strain 10-17 16S ribosomal RNA partial sequence
–
30 NR 148649.1 Enterobacter bugandensis strain 247BMC 16S ribosomal RNA partial sequence
–
NR 117547.1 Enterobacter soli ATCC BAA-2102 strain LF7 16S ribosomal RNA partial sequence
–
44 NR 028993.1 Enterobacter kobei strain CIP 105566 16S ribosomal RNA partial sequence
–
99 NR 113321.1 Enterobacter kobei strain JCM 8580 16S ribosomal RNA partial sequence
–

0.001

Fig. 1. Phylogenetic tree of PY16.

15
Y. Shu et al. Environmental Chemistry

CrVI tolerance and CrVI reduction of PY16 Fig. 2b depicts the CrVI reduction trend of PY16 exposed
to various initial CrVI concentrations. The majority of CrVI
Direct microbial reduction of CrVI by IRB via an extracellular
(85.66 ± 4.27%) was reduced within 2 days when the CrVI
electron transfer (EET) pathway can be an effective method
concentration was 5 mg L−1. The CrVI reduction rate was
(Cheng et al. 2020). The tolerance of strain PY16 to CrVI is
only 41.76 ± 15.89% in 2 days and 86.53 ± 11.00% in
shown in Fig. 2a. The PY16 proliferated and attained a
7 days when the CrVI concentration was 10 mg L−1, whereas
maximum OD600 of 0.65 ± 0.03 after continuous cultivation
it was less than 40% in 7 days when the CrVI concentration
for 2 days without CrVI. When the CrVI concentration was
exceeded 15 mg L−1. The phenomenon illustrated that PY16
5 mg L−1, the cells entered a logarithmic growth phase at a
had microbial CrVI reduction capacity, mainly through a
similar rate as compared with the control group (0 mg L−1).
chromate reductase enzymatic reaction (Masaki et al.
The maximum OD600 of 0.76 ± 0.01 was observed at the
2015). However, the toxicity of CrVI restrained cell growth
third day of cultivation, which was even higher than that of
and metabolic activity of the PY16, which also reduced the
the control group. The finding indicated that PY16 had the
activity and reduction ability of the chromate reductase,
best growth at 5 mg L−1 of CrVI, with no toxic effect.
ultimately inhibiting CrVI reduction (Rager et al. 2019).
However, the growth of PY16 was inhibited with varying
CrVI contents, ranging from 10 to 30 mg L−1. Meanwhile,
the OD600 was maintained at a low level of about 0.1 within Effect of dissimilatory iron reduction on
7 days. The results illustrated that the tolerance and reduc­ enhancing CrVI reduction by PY16
ing capacity of PY16 decreased when exposed to high levels CrVI reduction with the addition of ferrihydrite was investi­
of CrVI. The toxic effects of CrVI might inhibit cell growth gated as shown in Fig. 3a, the CrVI reduction rates in the
and metabolic activities, thus inhibiting the reduction abil­ systems coexisting with 500 mg L−1 of ferrihydrite and dif­
ity (Gutiérrez et al. 2010). ferent concentrations of CrVI. THe CrVI content was almost

(a) (b)
1.0
0 mg L–1 CrVI 15 mg L–1 CrVI 5 mg L–1 CrVI 20 mg L–1 CrVI
0.9 5 mg L–1 CrVI 20 mg L–1 CrVI 10 mg L–1 CrVI 30 mg L–1 CrVI
10 mg L–1 CrVI 30 mg L–1 CrVI 100 15 mg L–1 CrVI
0.8
CrVI removal rate (%)

0.7
80
0.6
OD600

0.5 60
0.4
40
0.3
0.2
20
0.1
0.0 0
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
Incubation time (day) Incubation time (day)

Fig. 2. Cell growth (a) and CrVI reduction properties (b) of PY16 exposed to different CrVI concentrations.

(a) (b)
5 mg L–1 CrVI
–1
20 mg L Cr
VI
0 mg L–1 CrVI 15 mg L–1 CrVI
120 10 mg L–1 CrVI 30 mg L–1 CrVI 5 mg L–1 CrVI 20 mg L–1 CrVI
80
15 mg L–1 CrVI –1
10 mg L Cr
VI
30 mg L–1 CrVI
100
CrVI removal rate (%)

60
FeII (mg L–1)

80

60 40

40
20
20

0 0
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
Incubation time (day) Incubation time (day)

Fig. 3. CrVI reduction (a) and FeII accumulation (b) byPY16 during CrVI reduction in the presence of ferrihydrite.

16
www.publish.csiro.au/en Environmental Chemistry

eliminated within 1 day when the CrVI concentration was This might be due to the microbial dissimilatory iron reduc­
5 mg L−1. Moreover, CrVI reduction rates were 97.31 ± 1.16% tion induced by the PY16 which could reduce ferrihydrite
and 81.33 ± 12.14% when the CrVI concentrations were 10 into FeII, and the resulting FeII quickly reacted with CrVI
and 15 mg L−1, respectively. However, the CrVI reduction through a chemical reduction pathway. During this redox
rate did not increase apparently when the CrVI concentration cycle, CrVI was reduced into CrIII, whereas the FeII was
exceeded 20 mg L−1. This result indicated that ferrihydrite oxidised back into FeIII (Lan et al. 2005). Therefore, the
could hardly remove the toxic impact of CrVI on microor­ FeII content was very low at the beginning of the cultivation
ganisms in the presence of high levels of CrVI. Fig. 3b shows process. Nevertheless, CrVI was almost eliminated with an
the FeII accumulation in systems with different CrVI concen­ extension of culture time, thus causing the rapid increase of
trations. In the system without CrVI, ferrihydrite could be FeII. In addition, there was hardly any FeII content when the
rapidly converted into FeII by PY16, and accumulated CrVI concentration exceeded 15 mg L−1, and the CrVI was not
78.88 ± 2.96 mg L−1 of FeII after 7 days. When CrVI concen­ completely removed.
trations were 5 and 10 mg L−1, FeII appeared slowly at A comparison of CrVI reduction characteristics with/
the initial incubation stage and began to increase rapidly without ferrihydrite are shown in Fig. 4. As shown in Fig. 4,
after 2 days of incubation, reaching 60.94 ± 7.40 and the growth of the PY16 decreased to varying degrees under
41.28 ± 8.97 mg L−1 after 7 days of incubation, respectively. 10–30 mg L−1 of CrVI, especially when the CrVI concentration

III
Without Fe 100
100 Adding FeIII
1.0
OD600 without FeIII
II
Fe concentration
80
80 0.8
CrVI removal rate (%)

FeII (mg L–1)

60 0.6 60
OD600

40 0.4 40

20 0.2 20

0 0.0 0 Fig. 4. Comparison of CrVI reduction characteris­

0 5 10 15 20 30 tics of PY16 with or without ferrihydrite at different
CrVI concentration (mg L–1) CrVI concentrations after 7 days.

(a) (b)
VI
strain + Cr
VI
strain + Cr + Fe
III
strain + CrVI + FeIII
120 VI VI III VI III
strain + Cr + DOM strain + Cr + Fe + DOM strain + Cr + Fe + DOM
CrVI + DOM 100 strain + FeIII
III
100 strain + Fe + DOM
CrVI removal rate (%)

FeIII + DOM
80
80
FeII (mg L–1)

60
60

40 40

20 20

0 0
0 12 24 36 48 60 72 0 12 24 36 48 60 72
Incubation time (h) Incubation time (h)

Fig. 5. CrVI reduction by PY16/DOM (100 mg L−1)/FeIII (500 mg L−1) (a); FeII accumulation by PY16/DOM
(100 mg L−1)/FeIII (500 mg L−1)/CrVI (15 mg L−1) (b).

17
Y. Shu et al. Environmental Chemistry

exceeded 15 mg L−1, which was consistent with the results
of Fig. 2 mentioned above. Compared with the control group
without ferrihydrite, the rates of CrVI reduction were
enhanced by 2.78–42.99% after 7 days with the addition of
ferrihydrite. This result was similar to a previous study
reported by Liu et al. (2020) that CrVI removal rates by
Shewanella oneidensis MR-1 were increased by 20–30% com­
pared to that of FeIII-free controls. Also, Du et al. (2018)
reported a 12% increase in CrVI removal rates by Shewanella
oneidensis MR-1 in comparison to the FeIII-free controls. In
the reaction system, the PY16 transferred electrons to ferri­
hydrite prior to transferring electrons to CrVI. Therefore, FeII

(a) (b)

10 mm 1 mm

(c) (d )

10 mm 1 mm

(e) (f )
2500 2500

C O

2000 O 2000

1500 1500
cps (eV)

cps (eV)

1000 1000
Fe
C
500 Fe P 500
Na Cl P Fe
N Fe CrNa
Si Cl
K Fe N K Ca Cr Fe
0 0
0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9
keV keV

Fig. 6. SEM-EDS images of PY16 after 7 days of incubation from experiments in Fig. 3. SEM image of the PY16 exposed to
0 mg L−1 CrVI (a, b); 15 mg L−1 CrVI (c, d). EDS image of PY16 exposed to 0 mg L−1 CrVI (e) and 15 mg L−1 CrVI (f).

18
www.publish.csiro.au/en
was hardly detected when CrVI was not completely reduced.
Compared to direct microbial CrVI reduction by PY16, the
redox cycles of FeII/FeIII during dissimilatory iron reduction
had a promoting effect on CrVI reduction (Huang et al.
2016). The presence of ferrihydrite might also prevent direct
contact between PY16 and CrVI, thus alleviating the cyto­
toxicity of CrVI.
Effect of DOM on enhancing CrVI reduction
by PY16
The properties of CrVI reduction with/without DOM by
PY16 is shown in Fig. 5a. Carex DOM had no noticeable
impact on FeIII/CrVI reduction without the involvement of
microorganisms (Fig. 5a, b). Without the presence of Carex
DOM and ferrihydrite, the addition of PY16 improved the
CrVI reduction rate, nearly 40% of CrVI transformed to CrIII
within 72 h. CrVI was almost eliminated within 72 h with
Carex DOM or ferrihydrite added separately. This phenom­
enon is likely due to DOM which can be used as an electron
shuttle to improve microbial CrVI reduction (Wittbrodt and
Palmer 1997). In addition, the CrVI removal rate in the
Carex DOM and ferrihydrite coexisting system was much
faster than that with Carex DOM or ferrihydrite alone,
which showed the great influence of Carex DOM on micro­
bial CrVI reduction (Choudhary et al. 2017).
Fig. 5b shows FeII accumulation during bacterial culture.
There was more FeII content during microbial dissimilatory
iron reduction systems without CrVI. FeII appeared immedi­
ately in the systems with Carex DOM, but decreased with
the presence of CrVI that could consume the FeII by redox
actions. In CrVI reduction and ferrihydrite/CrVI reduction
systems with Carex DOM, the accumulated amount of FeII
was enhanced by 75.64 and 78.21 mg L−1, respectively, indi­
cating that Carex DOM promoted the dissimilatory iron
reduction process. The quinones in DOM could act as electron
shuttles that accept electrons from IRB and transfer them to
terminal electron acceptors such as FeIII oxides and CrVI, thus
promoting dissimilatory iron reduction and microbial CrVI
reduction (Nurmi and Tratnyek 2002). Carex DOM promoted
FeII generation by promoting the dissimilatory iron reduction
process, thus promoting the redox cycles of FeII and CrVI.
SEM-EDS and XPS analysis
SEM was used to study the morphology of Fe/Cr after
reaction. The cells of the PY16 were short rod-shaped,
with a smooth surface and regular edge after 7 days of
incubation without CrVI (Fig. 6a, b). The mineral surface
was porous and adsorbed with multiple bacteria, possibly
due to dissimilatory iron reduction. However, the mineral
after the reaction was agglomerated with quite a few bacteria
on the surface in the system with 15 mg L−1 CrVI (Fig. 6c, d).
The probable reason might be the formation of an amorphous
chromium precipitate on the surface (Zhu et al. 2008). Hence,
Environmental Chemistry
the cells may agglomerate inside the deposit. The results also
indicated that cellular growth of the PY16 was inhibited with
15 mg L−1 CrVI, which is consistent with the above results.
The EDS study revealed that there were Cr and Fe absorption
peaks on the cell surface (Fig. 6e, f), indicating that a series of
microbial FeIII/CrVI reductions had occurred. Compared with
the CrVI-free group, the absorption peak of C decreased
significantly in the system with 15 mg L−1 CrVI, while the
absorption peak of Fe increased. This might be due to the
decrease of biomass adsorbed on the mineral surface in the
presence of CrVI, resulting in the decrease of the C peak,
which is consistent with the results observed by SEM. In
addition, the rate of dissimilatory iron reduction in the
presence of CrVI might be slower than that of the CrVI-free
group, so the absorption peak of Fe was slightly higher.
The reduced state of CrVI in the reaction system was
characterised by XPS (Kumar et al. 2015). The full XPS
spectrum of PY16 after 7 days exposed to 5 or 15 mg L−1
CrVI is depicted in Fig. 7. There were Cr 2p and Fe 2p peaks,
and the peak intensity increased when the CrVI concentra­
tion increased. There were two peaks for Cr 2p (Cr 2p3/2
and Cr 2p1/2) (Fig. 8a, b). The peaks at 578.0 ± 0.1 eV
could correspond to Cr 2p3/2 from CrVI. Meanwhile, the
peaks at 576.6 ± 0.1 eV corresponded to Cr 2p3/2 from
CrIII. These results indicated that there were Cr2O3 and Cr
(OH)3 precipitates in the product after reaction (Ai et al.
2008; Zhang et al. 2018b). As described above (Fig. 2), CrVI
was almost eliminated within 7 days with 5 mg L−1 CrVI.
Therefore, the peak of CrVI (588.1 eV) disappeared at this
point (Fig. 8a), possibly being K2Cr2O7 (588.7eV), which
was consistent with the above results. The peak of CrVI
(588.1 eV) appeared with 15 mg L−1 CrVI when the removal
rate was 81.33 ± 12.14% within 7 days (Fig. 8b), because
there was still unreacted Cr2O72− in the system. Fig. 8c, d
shows XPS spectra of Fe 2p after Cr adsorption. The typical
peaks were assigned to Fe 2p1/2 (725.0 eV) and Fe 2p3/2
(711.5 and 713.2 eV), indicating that reduction of FeIII was
600 000
0 mg L–1 CrVI
500 000
5 mg L–1 CrVI
15 mg L–1 CrVI
O 1s
400 000
Counts (s)
300 000 Fe 2p
C 1s
200 000 Cr 2p
N 1s
100 000
0
0 200 400 600 800 1000 1200
Binding energy (eV)
Fig. 7. XPS spectra of PY16 after 7 days.
19
Y. Shu et al. Environmental Chemistry

involved in the reduction of CrVI by strain PY16. Meanwhile, Since the reduction of FeII/CrVI was instantaneous, there
the spectra of the Fe 2p had two peaks, FeIII and FeII was hardly any FeII (710.2 eV) in the Fe 2p spectra.
(710.2 eV) (Fig. 8c), which is due to the further reduction
of FeIII by strain PY16 after CrVI reduction. The peak of CrVI
Mechanisms in the system of PY16/ferrihydrite/
(588.1 eV) appeared under 15 mg L−1 of CrVI when the
CrVI/Carex DOM
removal rate was 81.33 ± 12.14% within 7 days, and there
was no FeII (710.2 eV), because the CrVI was not completely
reduced. During the process, strain PY16 preferentially The proposed mechanism in a quaternionic system of
reduced FeIII to FeII, and then the FeII reduced CrVI to CrIII. PY16/ferrihydrite/CrVI/Carex DOM is summarised in Fig. 9.

(a) (b)
26 400
25 800 CrVI 578.1
26 200
26 000
25 600 25 800
CrIII 586.9 CrIII 586.9
CrIII 576.8 25 600
25 400 25 400
CrIII 576.8
Counts (s)

Counts (s)
25 200
VI
Cr 578.1 25 000
25 200 CrVI 588.1
24 800
24 600
25 000 24 400
24 200
24 800 24 000
23 800
23 600
24 600
565 570 575 580 585 590 595 565 570 575 580 585 590 595
Binding energy (eV) Binding energy (eV)
(c) (d )
60 000
50 000
55 000
FeIII 711.5 FeII 725 FeII 725
45 000 FeIII 711.5
III
50 000 Fe 713.2
II FeIII 713.2
Fe 710.2
Counts (s)

45 000
Counts (s)

40 000

40 000
35 000
35 000
30 000
30 000
25 000
25 000

20 000 20 000
700 710 720 730 740 700 710 720 730 740
Binding energy (eV) Binding energy (eV)

Fig. 8. High-resolution XPS spectra of Cr 2p of PY16 after 7 days of exposure to 5 mg L−1 CrVI (a); 15 mg L−1 CrVI (b); XPS
spectra of Fe 2p of strain PY16 after 7 days of exposure to 5 mg L−1 CrVI (c); 15 mg L−1 CrVI (d).

(CH2O)n FeIII CrO42–

DOMox
e–
e– (B) e– e
–

e– PY16
e– (A) e– FeII Cr(OH)3/Cr2O3
CrO42– Fig. 9. Proposed mechanism in the quaternionic
– e– system of PY16/ferrihydrite/CrVI/Carex DOM. (a):
CO2 e DOMred the cycles of DOMox/DOMred; (b): the cycles of
Cr(OH)3/Cr2O3
FeII/FeIII.

20
www.publish.csiro.au/en
As described above, the PY16 has direct FeIII/CrVI reduction
capacity mainly through direct enzymatic reactions. But these
enzymatic reactions of PY16 through the EET pathway can be
inhibited by high levels of CrVI. Apart from enzymatic pro­
cesses, Carex DOM and FeII, produced during the dissimila­
tory iron reduction process, act as electron shuttles between
the PY16 and CrVI, thus reducing CrVI to CrIII by a chemical
redox pathway (Cummings et al. 2007). Firstly, Carex DOM
obtains electrons from the respiratory chain of the PY16 and is
reduced to DOMred. The DOMred then transfers electrons to
FeIII/CrVI, and transforms itself to an oxidised state DOMox,
the is coupled with FeIII being reduced to FeII and CrVI being
reduced to CrIII. The resulting FeII can also reduce CrVI by
redox reaction. The reduction efficiency of FeII/FeIII and
DOMox/DOMred combined was stronger than that of a single
cycle due to a higher electron transport capability.
Conclusions
In summary, this work demonstrated the significance of
ferrihydrite and Carex DOM in the electron transfer reaction
of microbial CrVI reduction by Enterobacter sp. PY16 iso­
lated from Poyang Lake Wetland. Results showed that PY16
could microbiologically reduce FeIII oxide ferrihydrite and
CrVI directly. However, high levels of CrVI could cause a
cytotoxic and inhibitory effect, thus decreasing the CrVI
reduction rate. Fortunately, Carex DOM and the FeII pro­
duced during the dissimilatory iron reduction process acted
as electron shuttles that significantly enhanced CrVI reduc­
tion through a chemical redox pathway. Carex DOM and
ferrihydrite had a comparable promoting effect on CrVI
reduction, and when they were added simultaneously, the
reduction rate was the fastest, indicating that their abiotic
coupling action might exhibit a higher redox function, thus
significantly promoting CrVI reduction by the PY16. The
SEM-EDS and XPS results showed that the resulting CrIII
might be precipitated as Cr2O3 or Cr(OH)3. Therefore, it is
feasible that studies on the effects of DOM and dissimilatory
iron reduction on microbial CrVI reduction by Enterobacter
sp. may present a valuable theoretical basis for the effect of
chromium detoxification.
Data Availability
All data are presented within the manuscript. If you have
any requirement, please contact: xfgong@ncu.edu.cn.
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Declaration of funding. The financial support of this study from the National Natural Science Foundation of China (No. 41761095) is gratefully
acknowledged.
Author affiliations
A
School of Resources & Environment, Nanchang University, Nanchang 330031, China.
B
Key Laboratory of Poyang Lake Environment and Resource Utilization, Nanchang University, Ministry of Education, Nanchang 330031, China.
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