Applied Microbiology and Biotechnology

https://doi.org/10.1007/s00253-017-8690-x

ENVIRONMENTAL BIOTECHNOLOGY

Chromium removal from solution by five photosynthetic

bacteria isolates
Yan-Qiu Su 1,2 & Yang-Juan Zhao 3 & Nan Wu 3 & Yang-Er Chen 3 & Wei-Jia Zhang 1 & Dai-Rong Qiao 1 & Yi Cao 1

Received: 26 September 2017 / Revised: 1 December 2017 / Accepted: 2 December 2017

# Springer-Verlag GmbH Germany, part of Springer Nature 2017

Abstract
Biological method has been recognized as a low-cost and ecofriendly approach for removing heavy metals from aqueous wastes.
In this study, the ability of five photosynthetic bacteria isolates (strains labeled SC01, HN02, SC05, JS01, and YN01) was
examined for their ability to remove Cr from Cr-containing solutions. Furthermore, the possible removal mechanisms were
elucidated by comparing chromium removal rates, antioxidant reaction, and accumulation of reactive oxygen species (ROS).
Among the five bacteria, strains SC01 and SC05 presented the highest removal rates of chromium ions and the activity of
cysteine desulfhydrase under Cr stress. They also showed lower levels of ROS and cell death than the other three bacteria strains
under Cr stress. In addition, total bacteriochlorophyll content and activities of six antioxidant enzymes in SC01 were highest
among these selected strains. On the contrary, strain HN02 presented the lowest level of Cr removal and the lowest activities of
antioxidant enzymes. It also exhibited the highest level of ROS under Cr(VI) stress. Overall, these results show that the strains
SC01 and SC05 have good Cr removal ability and could be used for removal of Cr in industrial effluents.

Keywords Chromium . Photosynthetic bacteria . Removal . Antioxidant enzymes . Reactive oxygen species

Introduction et al. 2011). Therefore, Cr contamination of soil and water has

Chromium (Cr) is one of the most toxic heavy metals on earth
and a widespread environmental contaminant released mainly
as a pollutant in its industrial use (Panda and Choudhury
2005). Heavy metals are well recognized as environmental
pollutions because of their undegradability and persistence
in soil and water (Gupta et al. 2000; Sytar et al. 2016). In
nature, the stable forms of Cr are hexavalent Cr(VI) and triva-
lent Cr(III) species. Unlike Cr(III), Cr(VI) is a non-essential
metal for living organisms. Cr(VI) is 100 times more toxic to
living organisms than the trivalent form (Hayat et al. 2012).
Chromate is freely soluble in water and has been widely re-
ported to be toxic to both prokaryotes and eukaryotes (Parker
* Yi Cao
caoyi_01@163.com
1
Microbiology and Metabolic Engineering of Key Laboratory of
Sichuan Province, College of Life Sciences, Sichuan University,
Chengdu, China
2
Tongwei Group Co. Ltd, Chengdu, Chengdu, China
3
College of Life Sciences, Sichuan Agricultural University,
Ya’an, China
received much attention.
Conventional methods for the removal of heavy metals
from waste waters, such as chemical precipitation, ion ex-
change, and active carbon adsorption, have many significant
disadvantages, which mainly include high costs, incomplete
metal removal, and other waste production (Cho and Kim
2003; Göksungur et al. 2005). Therefore, a cost-effective
and eco-friendly method for heavy metal decontamination of
waters and soil has been long sought. Recently, the removal of
heavy metal cations from wastewaters by living or non-living
biomass has been shown to be effective (Chojnacka 2010).
The major benefits of this method include low operation costs,
disposal of low quantities of sewage sludge after treatment,
simple operation, and high efficiency in the treatment of dilute
effluents (Norton et al. 2004; Orhan et al. 2006).
Photosynthetic bacteria (PSB) found in some illuminated
anaerobic aquatic environments have been exploited for envi-
ronmental protection and bioremediation of some organic-
and heavy metal-polluted environments (Takeno et al. 1999;
Nagadomi et al. 2000). Some studies have showed that the
selectivity in the metal removal of PSB is a major advantage to
their use for removal of metal pollutants from wastewater
effluents (Gadd 2009; De Philippis et al. 2011). Therefore, it

is very important to choose appropriate PSB for selective re-
moval of metals from effluents. Although PSB have been
previously studied for their ability to remove heavy metals
(Feng et al. 2007; Bai et al. 2008), little has been reported
on Cr removal by PSB and antioxidant defense systems in
PSB under Cr stress.
Among the microorganisms used for heavy metal removal,
photosynthetic bacteria have been widely investigated by
some authors (Feng et al. 2007; Bai et al. 2008; Colica et al.
2012). Photosynthetic bacteria are an autotrophic microbe,
which can change the metabolic type flexibly enough along
with environment change, and thus has better environmental
adaptability than other microorganisms (Giotta et al. 2006). It
is mainly used to treat breeding wastewater in aquaculture.
However, the treatment of heavy metal wastewater is less
reported in photosynthetic bacteria, especially the treatment
of chromium ion.
It is well known that the photosynthetic systems of photo-
synthetic bacteria are different with green plants. Under heavy
metal stress, the response of plants usually involves membrane
system (Kreslavski et al. 2017), photosynthetic system (Kato
et al. 2012), antioxidant defense system, and osmotic regula-
tion substances (Chen et al. 2015). How, the detailed response
of PSB is unclear in antioxidant system and photosynthetic
systems under heavy metal stress. In this study, the ability of
five photosynthetic bacteria to remove Cr from solutions con-
taining Cr was evaluated. The possible mechanisms of five
PSB were also studied by comparing their removal rate of
chromium, reactive oxygen species (ROS) accumulation, and
antioxidant system under Cr stress.
Materials and methods
Microorganism and culture conditions
The experiments were carried out with five PSB strains SC01,
HN02, SC05, JS01, and YN01. SC01 was isolated from water
collected from saline paddy fields in Sichuan province, China.
HN02 was isolated from seawater in Hainan province, China.
SC05 was isolated from water collected from Hailuogou
Valley in Sichuan province, China. JS01 was isolated from
water collected from pond in Jiangsu province, China.
YN01 was isolated from water collected from shrimp pond
in Charong province, Vietnam. Five strains were firstly iden-
tified to belong to PSB according to morphological and phys-
iological characteristics. Then, we further indicated that
strains SC01 and SC05 are Rhodobacter spheroids and
Rhodocyclaceae thauera through molecular identification, re-
spectively. SC01 and SC05 strains are deposited at the
GDMCC Culture Collection. Their collection numbers are
GDMCC1.1264 (strain SC01) and GDMCC1.1265 (strain
Appl Microbiol Biotechnol
SC05). Then, all strains were cultured by our laboratory and
were used in subsequent experiments.
The cultures were performed in purple non-sulfur bacteria-
enriched medium (250 mL) containing NH4Cl 1.0 g, K2HPO4
0.5 g, MgCl2·6H2O 0.2 g, NaCl 5.0 g, CaCl2·2H2O 0.075 g,
NaHCO3 1.0 g, CH3COONa 3.0 g, ferric citrate 0.01 g, and
yeast extract 2.0 g. The pH of medium was adjusted to 7.2
with NaOH before autoclaving (Feng et al. 2007). The bacte-
ria were cultured anaerobically at 30–40 °C under continuous
illumination with incandescent lamps at a light intensity of
about 3000 lx. Before the Cr(VI) stress experiment, the cell
concentration was adjusted to the same biomass by measuring
OD value at 680 nm. Then, cells in stationary phases were
collected to compare the Cr(VI) removal capabilities and dif-
ferent parameters.
Measurements of pigments, total protein, proline,
and soluble sugar
One hundred milliliter samples were centrifuged at 4000 rpm
for 10 min at 4 °C. The pellets were resuspended in 80%
acetone, and then sonicated for 30 × 5 s (4-s interval;
200 W) with an ultrasonic cell crusher. After centrifugation
at 10,000 rpm for 10 min at 4 °C to remove the cell debris,
absorbance of the supernatant was read at 645 and 663 nm
using a spectrophotometer (Hitachi-U2000, Hitachi, Ltd.,
Tokyo, Japan). The concentration of chlorophyll (Chl) a was
calculated using the equations: Chl a (mg g−1) = (12.7 ×
OD663 – 2.69 × OD645) × V (80% acetone; mL) / W (wet
weight; g) (Hou et al. 2016). Carotenoid was extracted from
five PSB using methanol/acetone (v/v, 2:3) solvent according
to the previous method (Saejung and Apaiwong 2015). The
absorption spectrum of carotenoid extract was recorded using
an ultraviolet-visible spectrophotometer (A560, Shanghai
AOE Instruments Co., Ltd., China). The total protein content
was assayed by the Lowry method (Lowry et al. 1951) using
bovine serum albumin (BSA) as a standard. Soluble sugar was
extracted in boiling water, and its concentration was deter-
mined by the method of Thomas (1977). Proline extract
(0.1 g) was extracted in 3% sulfosalicylic acid (5 mL), and
then, 2 mL of glacial acetic acid and 2 mL of ninhydrin re-
agent were added. The reaction mixture was boiled for 30 min,
and readings were taken at 520 nm in a UV spectrophotometer
as described previously (Bates et al. 1973).
Measurements of lipid peroxidation and electrolyte
leakage
Lipid peroxidation was estimated by assaying the
malondialdehyde (MDA) content as described previously
(Chen et al. 2015) with some modifications. Fifty milliliter
samples were centrifuged at 4 °C for 10 min at 4000 rpm.
After removing the supernatant, 3 mL of 5% TCA was added
Appl Microbiol Biotechnol
to the sedimented cell and the cells were broken by sonication.
The sonicate was centrifuged at 12,000 rpm centrifugation for
10 min at 4 °C. To the supernatant, 2 mL 0.67% thiobarbituric
acid was added. The mixture was boiled for 30 min, and its
OD values (OD535–OD600) were determined. The extinction
coefficient was 155 mM−1 cm−1. Electrolyte leakage (EL)
values were measured using a conductivity meter (DDSJ-
308A, Shanghai Precision Instruments Co., Ltd., China) as
described by Chen et al. (2015). The relative EL was calcu-
lated as the ratio of the initial conductivity to the absolute
conductivity (boiled at 95 °C for 30 min).
Determination of enzymes and non-enzymatic
antioxidants
For analysis of enzymatic and non-enzymatic antioxidant ac-
tivities, PSB cells were collected by centrifugation at
4000 rpm for 10 min at 4 °C. After PBS (0.1 mol L−1, pH
7.8) was added, cells were sonicated with a sonicator in an ice
bath. Then, the sonicate was centrifuged at 12,000 rpm for
10 min at 4 °C, and the supernatant was stored at − 80 °C until
use. Cysteine desulfhydrase was determined according to the
method of Oguri et al. (2012). The activities of peroxidase
(POD), superoxide dismutase (SOD), catalase (CAT), ascor-
bate peroxidase (APX), glutathione peroxidase (GPX), and
glutathione reductase (GR) were assayed according to the
methods of Egley et al. (1983), Giannopotitis and Ries
(1977), Cakmak and Marschner (1992), Nakano and Asada
(1981), Flohe and Gunzler (1984), and Foyer and Halliwell
(1976), respectively.
Measurements of reduced ascorbic acid/dehydroascorbate
(AsA/DHA) in 5% (w/v) of trichloroacetic acid were per-
formed according to the previous method (Cakmak and
Marschner 1992). The amounts of reduced glutathione
(GSH) and oxidized glutathione (GSSG) were measured using
the GSH and GSSG Assay Kit (S0053; Haimen Beyotime).
Cr(VI) removal experiments
Ten milliliters of each of the five pure PSB cultures in their
logarithmic and stationary growth phases was added to
K2Cr2O7 solution at different initial concentrations and purple
non-sulfur bacteria-enriched medium. Experiments were per-
formed with 250 mL of reaction mixtures containing different
Cr6+ concentrations (0, 25, 50, 100 mg Cr L−1) in 250-mL
flasks. Then, the mixture was cultured anaerobically at 30–
40 °C under continuous illumination about 3000 lx. After
12 days, the biomass was separated from Cr solutions by
centrifugation. Then, the pellet and the supernatant were col-
lected and analyzed. Then, the Cr concentration of the super-
natant was measured by using an inductively coupled plasma
mass spectrometry (ICP-MS) (Optimal 2100DV, PerkinElmer
Instruments, Waltham, MA, USA). For Cr content
determination, three individual samples for each treatment
were measured. The percent Cr removal was calculated using
the equations: percent Cr(VI) removal = [(C0 − C) / C0] × 100,
where C0 is the initial Cr ion concentration (mg L−1) and C is
the residual Cr ion concentration (mg L−1).
Detection of reactive oxygen species
To measure the superoxide (O2˙ˉ) content, the PSB cells (0.3 g
fresh weight) were suspended in 65 mM potassium phosphate
buffer (3 mL; pH 7.8) and sonicated in an ice bath. After cen-
trifugation of the sonicate (12,000 rpm for 10 min), the O2˙ˉ
was determined in the supernatant by the method described by
Elstner and Heupel (1976). To determine the concentration of
H2O2 in the sonicate, 5 mL of 0.1% (m/v) trichloroacetic acid
(TCA) was added to the PSB cells (3 g fresh weight) and son-
icated. The sonicate was centrifuged at 12,000 rpm for 20 min
at 4 °C. After centrifugation, 0.5 mL of the supernatant was
added to 1 mL of 10 mM PBS buffer (pH 7.0) and 1 mL of 1 M
KI. The absorbance of this mixture was then read at 390 nm as
described by Okuda et al. (1991).
The Cr-induced H2O2 accumulated by each of the five
strains was monitored using the probe 2,7-dichlorofluorescin
diacetate (DCFH-DA) according to the methods of Lee et al.
(2011) and Xu et al. (2012) with minor modifications. The
treated strains were incubated and infiltrated in the dark under
vacuum with 20 μM DCFH-DA (Sigma) in PBS buffer for
40 min in water bath with 37 °C and washed three times in
PSB buffer. Then, the in vivo imaging of H2O2 in the cells was
obtained using a confocal laser scanning microscopy (excita-
tion, 488 nm; emission, 530 nm).
Propidium iodide staining
To assess the extent of cell death among five PSB exposed to
the stress of 100 mg Cr per liter, the cells were stained with
propidium iodide (PI) stain, as previously described by
Yamaguchi and Nasu (1997). PSB cells were collected by
centrifugation at 4000 rpm for 10 min at 4 °C. Then, PSB
cells were washed two times and resuspended with 2 mL of
PBS (0.1 M, pH 7.6). Then, PI solution (0.5 μg mL−1, Sigma)
was added into the 1 mL of PSB cell suspension and mixed
gently. After staining for 10 min at room temperature in the
dark, the samples were washed for three times with PBS buff-
er. Then, the stained PSB cells were resuspended with 0.1 mL
PBS and subsequently examined immediately with a fluores-
cence microscope (BX-53 System, Olympus Corporation,
Tokyo, Japan) under an excitation wavelength of 546 nm.
Statistical analysis
All experiments were repeated at least three times. All results
are shown as averages ± standard deviations (SD). The results

were analyzed statistically using SPSS Statistics 19.0 software
(IBM, Chicago, IL, USA), using the Duncan’s multiple range
test. In the graphic representation, differences in the heights of
bars headed by different letters were considered significant at
P < 0.05.
Results
Growth of the five photosynthetic bacteria strains
The growth of each of the five photosynthetic bacterial strains
was determined in terms of color and biomass production
under standard conditions. Each of the five strains was cul-
tured for 1, 3, 5, and 9 days, because no change in color could
be detected after 9 days. The five strains reached the stationary
phase after 10 days. At days 3 and 5, the color of strains SC01,
SC05, and YN01 was deeper than that of strain HN02 and
JS01, especially that of strain HN02 (Fig. 1). Biomass deter-
minations in terms of OD of strains SC01 and SC05 were high
on day 14 (Fig. 2). Strain HN02 attained the lowest values in
the logarithmic and stationary phases, indicating that strains
SC01 and SC05 produced the most biomass and grew best.
Fig. 1 The phenotypes of five
photosynthetic bacterial strains
(SC01, HN02, SC05, JS01, and
YN01) under normal growth
conditions. Only the phenotypes
of five strains cultured for 1, 3, 5,
and 9 days are presented. OD
values are shown below each
image
Appl Microbiol Biotechnol
We determined biomass production of each of the five pho-
tosynthetic bacteria strains in the logarithmic and stationary
phases under Cr stress with results shown in Table 1. In the
presence of 25 mg Cr per liter, slight differences in biomass
production were noted among the five strains. On exposure to
50 and to 100 mg Cr per liter, strain SC01 and strain SC05
reproduced more biomass than the other three strains. It is
noteworthy that in the presence of 100 mg Cr per liter, biomass
production by strain HN02 was significantly reduced, indicat-
ing that this strain suffered significant damage from Cr stress.
Pigment content, total protein content, proline,
and soluble sugar in five strains under Cr stress
Strains SC01 and HN02 under non-stressed conditions showed
the highest and lowest production of carotenoid in the station-
ary phase, respectively (Fig. 3). At 50 and 100 mg Cr per liter,
significantly less carotenoid was found in strain HN02 com-
pared to the other four strains. Similarly, bacteriochlorophyll a
content of the five strains under Cr stress also showed the nearly
identical results with carotenoid production (Fig. 4a). Although
the total protein content showed no significant difference
among the five strains under non-stressed conditions, treatment
Appl Microbiol Biotechnol

that in the respective controls (Fig. 4c, d). Compared to the

other four strains, strain HN02 maintained higher concentra-
tions of proline and soluble sugar under Cr stress, especially
at Cr concentrations of 50 and 100 mg per liter.

ROS accumulation and lipid peroxidation in the five

strains under Cr stress

To investigate whether strains SC01 and SC05 accumulated

Fig. 2 The growth curve of five photosynthetic bacterial strains (SC01,
HN02, SC05, JS01, and YN01) under normal growth conditions. All
error bars correspond to the standard deviation of three independent
biological replicates (n = 3)
with 100 mg Cr per liter resulted in a significant decrease in the
five strains (Fig. 4b). Relative to the other four strains, strain
SC01 retained more total protein at a stress of 100 mg Cr per
liter. Furthermore, the concentration of proline and soluble sug-
ar increased significantly with the Cr treatment compared to
fewer ROS compared to the other three strains, we analyzed
the level of the two major ROS species, O2˙ˉ and H2O2, pro-
duced under Cr stress. There was obvious difference among
the five strains (Fig. 5a, b). The levels of O2˙ˉ and H2O2 were
upregulated significantly in the five strains exposed to Cr
stress compared to the controls, especially at 100 mg Cr per
liter. In a comparison of the five strains, the O2˙ˉ and H2O2
accumulation was more pronounced in strain HN02 than in
strain SC01. The production of H2O2 by the five strains was
also studied in situ by using stains that were sensitive to H2O2.
The DCFH-DA fluorescent probe has been successfully used
for H2O2 detection in plants (Xu et al. 2012; Rico et al. 2013).
In the present study, the responses of the non-stressed and
stressed strains to H2O2 production were as expected. Strain
HN02 accumulated more H2O2 than the other four strains at a

Table 1 Biomass of photosynthetic bacterial strains (SC01, HN02, SC05, JS01, and YN01) in both the logarithmic and stationary phases under normal
growth conditions and Cr stress

Strains Concentration Days

(mg L−1)
1 2 3 4 5 6 7 8 9 10 11 12

SC01 0 0.80 0.94 1.01 1.13 1.17 1.31 1.32 1.39 1.48 1.50 1.52 1.52
25 0.80 0.8 0.97 1.04 1.11 1.19 1.23 1.27 1.27 1.32 1.39 1.39
50 0.80 0.84 0.89 0.89 0.94 0.99 1.01 1.03 1.04 1.08 1.18 1.18
100 0.80 0.85 0.88 0.85 0.86 0.88 0.92 0.97 1.01 1.03 1.05 1.05
HN02 0 0.79 0.91 1.07 1.13 1.21 1.28 1.38 1.30 1.30 1.35 1.32 1.32
25 0.79 0.85 0.88 1.00 1.17 1.22 1.23 1.29 1.30 1.31 1.32 1.32
50 0.79 0.79 0.86 0.8 0.90 0.94 0.97 1.01 1.03 1.02 1.04 1.04
100 0.79 0.79 0.80 0.80 0.80 0.85 0.86 0.91 0.92 0.93 0.93 0.93
SC05 0 0.80 0.95 1.21 1.23 1.25 1.27 1.29 1.30 1.33 1.38 1.39 1.49
25 0.80 0.98 1.1 1.17 1.23 1.24 1.34 1.35 1.38 1.40 1.41 1.41
50 0.80 0.81 0.8 0.83 0.89 0.96 0.96 0.97 1.03 1.05 1.11 1.11
100 0.80 0.81 0.8 0.82 0.86 0.86 0.90 0.95 1.00 1.01 1.04 1.04
JS01 0 0.80 0.99 1.11 1.21 1.23 1.26 1.40 1.38 1.40 1.41 1.46 1.46
25 0.80 0.91 1.09 1.11 1.19 1.25 1.28 1.30 1.34 1.36 1.36 1.37
50 0.80 0.89 0.92 0.96 0.98 1.03 1.05 1.07 1.08 1.09 1.09 1.09
100 0.80 0.86 0.87 0.85 0.84 0.84 0.88 0.93 0.94 0.96 0.99 0.99
YN01 0 0.80 0.91 1.02 1.11 1.18 1.36 1.37 1.41 1.42 1.41 1.42 1.42
25 0.80 0.8 0.99 1.10 1.14 1.19 1.29 1.31 1.33 1.35 1.35 1.35
50 0.80 0.85 0.85 0.90 0.93 0.97 1.05 1.05 1.06 1.07 1.07 1.07
100 0.80 0.84 0.83 0.81 0.82 0.82 0.86 0.86 0.89 0.94 0.99 0.99

The OD values are means ± SD from three independent biological replicates

 Appl Microbiol Biotechnol

Fig. 3 Carotenoid production of

five photosynthetic bacterial
strains (SC01, HN02, SC05,
JS01, and YN01) under Cr stress.
The data are presented as the
mean of at least three independent
experiments. a Control. b
25 mg L−1 Cr stress. c 50 mg L−1
Cr stress. d 100 mg L−1 Cr stress

stress of 100 mg Cr per liter (Fig. 6). Similar trends in H2O2
production were shown by the other strains by treatment with
100 mg Cr per liter. These results suggest that Cr stress result-
ed in greater and more rapid ROS accumulation by strain
HN02.
The degree of cell damage from oxidative stress by Cr in
the five strains was determined by measuring the amount of
malondialdehyde (MDA) formed and the electrolyte leakage
(EL). Although MDA production and EL were not significant-
ly different in the five strains in the absence of Cr stress, these
two parameters increased significantly in the presence of
100 mg Cr per liter (Fig. 5c, d). Compared to strain HN02,
strains SC01 and SC05 produced less MDA and EL under Cr
stress.

Fig. 4 Chlorophyll a (a), total

protein (b), proline (c), and
soluble sugar (d) of five
photosynthetic bacterial strains
(SC01, HN02, SC05, JS01, and
YN01) under Cr stress. Bars
represent standard deviations
(SD) of three independent bio-
logical replicates. Values follow-
ed by different letters are signifi-
cantly different when P < 0.05
according to Duncan’s multiple
range test
Appl Microbiol Biotechnol

Fig. 5 Superoxide anion radical

(O2˙ˉ) production rate (a),
hydrogen peroxide (H2O2)
content (b), the malondialdehyde
(MDA) content (c), and electro-
lyte leakage (d) of five photosyn-
thetic bacterial strains (SC01,
HN02, SC05, JS01, and YN01)
under Cr stress. The data are pre-
sented as the mean ± SD of at
least three independent experi-
ments. Values followed by differ-
ent letters are significantly differ-
ent at P < 0.05 according to
Duncan’s multiple range test

The integrity of the plasma membrane was tested using PI
staining. PI is a membrane-impermeable dye and can only
enter cells with damaged cell membranes (i.e., dead cells),
where it binds to DNA (Williams et al. 1998). Consistent with
the quantitative effect of ROS accumulation by the five strains
in response to 100 mg Cr per liter, PI staining in terms of a red
fluorescence was more intense with the cells of strain HN02
than with the other four strains (Fig. 7). Correspondingly,
strains SC01 and SC05 showed the weakest fluorescence
among the five strains upon staining with PI. These results
indicate that strains SC01 and SC05 received less oxidative
damage than the other three strains.
Parameters of oxidative stress of the five strains
The differences observed among the five strains regarding the
Cr-induced ROS and cell death led us to investigate the activ-
ities of antioxidant enzymes and non-enzymatic antioxidants.
The activities of several important antioxidant enzymes
among five strains showed no significant difference when
the five strains were cultured in fresh medium without Cr for
12 days (Fig. 8). When Cr stress was imposed, the activities of
six enzymes showed significant change among the five
strains. The analysis of enzymatic activities in the five strains
showed a significant increase in POD, SOD, APX, GPX, and
GR at a Cr stress of 25 mg L−1. The most significant increase
in activity of these five enzymes was noted in strain SC01.
However, Cr stress at 100 mg Cr L−1 decreased the activities
of the POD, SOD, APX, CAT, and GR enzymes. Compared to
the control, strains SC01 and HN02 showed a weak and strong
reduction of the six antioxidant enzyme activities at a Cr stress
of 100 mg L−1, respectively. It is noteworthy that of all the
enzyme activities studied, that of CAT declined significantly
to lower levels under Cr stress. The most significant decrease
in CAT activity was noted in strain HN02 exposed to 100 mg
Cr per liter.

Fig. 6 In vivo imaging of H2O2 in

five photosynthetic bacterial
strains (SC01, HN02, SC05,
JS01, and YN01) at a Cr(VI)
stress of 0 and 100 mg L−1. The
five strains were stained with
20 μM of the fluorescent probe
2,7-dichlorofluorescin diacetate
(DCFH-DA) as described in the
BMaterials and methods^ section.
Bars = 400 μm
 Appl Microbiol Biotechnol

Fig. 7 The plasma membrane integrity of five photosynthetic bacterial strains (SC01, HN02, SC05, JS01, and YN01) at a Cr(VI) stress of 0 and
100 mg L−1. The five strains were stained with 0.5 μg mL−1 of propidium iodide (PI) as described in the BMaterials and methods^ section. Bars = 100 μm

The determination of the non-enzymatic antioxidants
(ASA/DHA and GSH/GSSG) in the strains showed a reduc-
tion in ASA and GSH under Cr stress, whereas the determi-
nation of DHA and GSSG showed a significant increase
(Fig. 9). The most significant decrease in ASA and GSH con-
tent took place in strain HN02 (almost a 50% reduction). The
ASA and GSH content in strains SC01 and SC05 was higher
than that in the other three strains under Cr stress, especially at
100 mg per liter.
Cr accumulation and its effects on biomass in five PSB
The Cr(VI) content of the five photosynthetic bacteria in-
creased at higher concentrations of K2Cr2O7 in the logarithmic

Fig. 8 Activities of antioxidant

enzymes from five photosynthetic
bacterial strains (SC01, HN02,
SC05, JS01, and YN01) exposed
to Cr stress. Peroxidase (POD, a);
superoxide dismutase (SOD, b);
catalase (CAT, c); ascorbate
peroxidase (APX, d); guaiacol
peroxidase (GPX, e); glutathione
reductase (GR, f). The data are
presented as the mean ± SD of
three replicates. Values followed
by different letters are
significantly different at P < 0.05
according to Duncan’s multiple
range test
Appl Microbiol Biotechnol
Fig. 9 Activities of non-
enzymatic antioxidants from five
photosynthetic bacterial strains
(SC01, HN02, SC05, JS01, and
YN01) exposed to Cr stress. AsA
(a) and DHA (b) reduced ascorbic
acid and dehydroascorbate, re-
spectively; GSH (c) and GSSG
(d) reduced and oxidized gluta-
thione, respectively. The data are
presented as the mean ± SD of
three replicates. Values with a
common letter are statistically
different at P < 0.05 according to
Duncan’s multiple range test
and stationary phases (data not shown). The specific Cr(VI)
uptake by the five strains also showed clear differences
(Fig. 10a). At a stress of 25 mg Cr per liter, the percent
Cr(VI) removal in both the logarithmic and stationary phases
was nearly the same in the five strains. However, at a stress of
50 and 100 mg Cr per liter, strains SC01 and SC05 exhibited a
notable increase in the percent Cr removal, whereas the other
three strains, especially strain HN02, exhibited a slight de-
crease in the percent Cr removal. These results indicate that
strain SC01 exhibited the best metal removal percentage.
To study the basis for the high Cr(VI) removal percentage
in strains SC01 and SC05 further, we compared the cysteine
desulfhydrase (CDS) activity in the five photobacterial strains.
The CDS activity in the five strains was not significantly dif-
ferent in the absence of Cr(VI) stress (Fig. 10b). However, at a
Cr(VI) stress of 25 and of 50 mg L−1, the CDS activity in
SC01, SC05, JS01, and YN01 increased significantly com-
pared to the corresponding controls. The CDS activity in
strain HN02 decreased significantly under Cr(VI) stress, es-
pecially at 100 mg Cr(VI) per liter, compared to the corre-
sponding control. Thus, compared to the other four strains,
the low activity of CDS under Cr(VI) stress of strain HN02
suggests that it had weak Cr(VI) removal efficiency.
Discussion
It is well known that high concentrations of heavy metals
present in aquatic systems pose serious problems to aquatic
life. Among these heavy metals, Cr is widely employed by
many industries and may be an important source of industrial
pollution, making its removal from industrial effluent very
important. Biological removal of heavy metals from solution
by bacteria, algae, yeasts, and fungi is an economic and
ecofriendly method. Thus, metal-resistant biosorbents (e.g.,
bacteria) are considered very important agents in the removal
of heavy metal from wastewater. In the present study, we
compared the removal efficiency of five selected photosyn-
thetic bacterial strains (SC01, HN02, SC05, JS01, and YN01)
by investigating their growth, their antioxidant defense, and
the accumulation of ROS by them. Our aim was to find a
photosynthetic bacterial strain with high chromium resistance,
which can be employed in removal of Cr from industrial
effluents.
In this study, we firstly compared the growth of the five
strains in the absence of Cr stress. The results showed that
strains SC01 and SC05 grew best compared to other test
strains under the same conditions. Differences in growth
among our five test strains may be the result of differences
in their growth environment. However, we found that our five
strains reached stationary phase at nearly the same time.
Therefore, the growth of these strains needs to be further in-
vestigated. Some studies indicated that toxic ions or salt stress
conditions can decrease bacterial metabolism and lead to in-
hibition of growth (Sheng et al. 2005; Upadhyay et al. 2011).
In the present study, the growth under chromium stress of
strains SC01 and SC05 was better than that of the other
strains, suggesting that these two strains were relatively resis-
tant to chromium. Although in some studies in the literature
the removal of metals was expressed in terms of a q value

Fig. 10 The precent removal of Cr (a) and activity of cysteine
desulfhydrase of photosynthetic bacterial strains SC01, HN02, SC05,
JS01, and YN01 (b) under Cr stress. The data are presented as the
mean ± SD of at least three independent determinations. Values
followed by different letters are significantly different at P < 0.05
according to Duncan’s multiple range test
(Feng et al. 2007; Colica et al. 2012), percent removal is also
usually applied to evaluate heavy metal removal performance
(Nunkaew et al. 2015). Our results showed that strain SC01
exhibited a high percent Cr removal, even at a high concen-
tration of Cr. The high removal efficiency of Cr may contrib-
ute to the high resistance to Cr in strain SC01. The high value
in percent removal is consistent with the CDS activity, which
increased significantly in the Cr-exposed strains SC01 and
SC05. Previous studies have indicated that CDS plays an im-
portant role in the removal of heavy metals by information of
precipitation (Fukamachi et al. 2002; Auger et al. 2005).
Therefore, the high activity of CDS may reflect the high re-
moval of Cr in strains SC01 and SC05.
Reduction in pigment content is a commonly observed
phenomenon in plants exposed to different stresses including
heavy metal stress (Allakhverdiev et al. 2008; Din et al., 2011;
Brestic et al. 2016). Some studies also indicate that chloro-
phyll a and cartenoid content could decrease in algae or bac-
teria in the removal of heavy metals (Feng et al. 2007; Hou
et al. 2016; Kalaji et al. 2016; Batool et al. 2017). Consistent
Appl Microbiol Biotechnol
with these reports, our results indicated that high concentra-
tion of Cr markedly decreases the bacteriochlorophyll a con-
tent, suggesting that Cr stress could result in severe damage to
the photosystem of PSB. However, we found that bacterio-
chlorophyll a content of strains SC01 and SC05 decreased
slowly compared to the other three strains under Cr stress,
implying that strains SC01 and SC05 have high resistance to
Cr. Carotenoid could protect the plasma membrane of PSB by
eliminating the oxygen-free radicals (Feng et al. 2007). Thus,
the high content of carotenoid in strain SC01 indicated that
SC01 strain had an effective protection mechanism against
oxidative damage and high metal tolerance.
Proline and soluble sugar, important osmotic regulators, usu-
ally accumulate under strong stress in plants (Xu and Huang,
2010). We found that strains SC01 and SC05 accumulated rel-
atively less soluble sugar and proline, indicating that strains
SC01 and SC05 could improve their Cr resistance by changing
probably the levels of organic solutes as well as plants exposed
to environmental stresses (Misra and Gupta 2005).
Many studies have demonstrated that Cr can induce the
production of ROS, which usually cause oxidative damage
to plants (Dixit et al. 2002; Panda and Choudhury 2005).
The toxicity of Cr(VI) is attributed to its partial reduction to
the highly unstable Cr(VI) radical inside the cells, which leads
to the formation of ROS (Mala et al. 2015). This study showed
that, as expected, Cr stress markedly induced ROS accumula-
tion in five PSB, especially at high Cr concentration, suggest-
ing that PSB could produce high concentrations of ROS and
suffer severe oxidative damage under Cr stress. Furthermore,
some previous studies also indicated that resistant plants usu-
ally accumulate lower levels of ROS than susceptible plants
under environmental stresses (Asthir et al. 2010; Chen et al.
2015). In accordance with these reports in plants, our results
showed that in our strains SC01 and SC05, the accumulation
of ROS remained low under Cr stress. Under stress, ROS are
reportedly involved in lipid peroxidation, which in turn results
in cell membrane injury (Smirnoff 1993). In the present study,
we observed that levels of lipid peroxidation and the electro-
lyte leakage were lower in strain SC01 than the other strains.
Low MDA content means high antioxidative ability, reflecting
low oxidative damage to the cells and high resistance to dam-
age by Cr (Shao et al. 2005). This finding was consistent with
the observed levels of ROS accumulation in the five strains.
These results suggested that strains SC01 and SC05 among
five PSB had high Cr resistance and are suitable for the re-
moval of Cr from industrial effluent.
To protect themselves against oxidative damage caused by
oxygen-free radicals and harmful substances, different organ-
isms have developed a highly complex antioxidant defense
system, which includes antioxidant enzymes such as SOD,
POD, CAT, APX, GPX, and GR and non-enzymatic antioxi-
dants such as ascorbate (AsA) and reduced glutathione (GSH)
(Foyer and Shigeoka 2011; Luo et al. 2013). Some previous
Appl Microbiol Biotechnol
studies indicated that activities of SOD, CAT, GPX, and GR
markedly increase in resistant plants (Asthir et al. 2010;
Hameed et al. 2012; Chen et al. 2015). In the present study,
pronounced differences in six antioxidant enzyme activities
were found in five PSB. After Cr treatment, the activities of
POD, APX, GPX, and GR markedly increased in strains SC01
and SC05 compared to the other strains, suggesting that these
two strains have a more effective antioxidant defense system in
regulating the levels of ROS. An analogous result has been
reported in algal cells, in which an antioxidant activity system
aids in the disposal of external stresses (Hou et al. 2016). CAT,
one of the key enzymes of the defense system, can transform
hydrogen peroxide into oxygen and water, thereby preventing
cell damage by hydrogen peroxide. In this study, the large de-
crease in CAT activity in strain HN02 suggests that this strain
suffers severe oxidative damage and is weakly Cr resistance.
The results from the study of the antioxidant enzyme activities
suggest that the antioxidants were directly involved in resisting
Cr stress in the PSB cells. This conclusion is in agreement with
previous studies (Zhang et al. 2014; Hou et al. 2016).
The AsA-GSH cycle is important in the protection against
the effect of ROS in different cell compartments. AsA and
GSH are the most abundant and best characterized water-
soluble antioxidants in plants (Foyer and Shigeoka 2011).
Our results showed that the cells of strains SC01 and SC01
contained higher levels of AsA and GSH than the other
strains, suggesting that in these two strains, the ROS detoxi-
fication system is effective under Cr stress. These results were
consistent with the catalytic activities of APX and GR, which
are major enzymes in the Halliwell-Asada pathway (AsA-
GSH cycle) (Lin et al. 2009). The increased GR activity was
consistent with high ascorbate production, leading to high
APX activity and then, ultimately, to low oxidative stress in
SC01 and SC05. We thus concluded that strains SC01 and
SC05 could lower the oxidative damage caused by ROS
through efficient ROS-scavenging systems and subsequently
enhance the resistance to Cr stress, finally improving the re-
moval efficiency of Cr.
In summary, this study examined, for the first time, the Cr
removal ability of five selected photosynthetic bacteria in
terms of the percent Cr removal, the antioxidant enzyme sys-
tem, and ROS accumulation under Cr stress. Our results indi-
cated that strains SC01 and SC05 may be more effective in
preventing the oxidative damage caused by excessive ROS
through the activity of antioxidant enzymes and non-
enzymatic antioxidants. Extensive growth and a high percent
of Cr removal were observed by strains SC01 and SC05 when
exposed to Cr stress. The maximum percent removal of Cr
arrived to 85 and 81% in strains SC01 and SC05 exposed to
100 mg Cr per liter, respectively. Based on these results, we
propose that SC01 and SC05 may have high Cr removal effi-
ciency and may be applicable in the removal of Cr from aque-
ous solutions. The use of strains SC01 and SC05 for removal
of many heavy metals like Cr could contribute to reduce the
environmental impact of the mining activities and improve its
production costs. However, further research is needed in order
to elucidate the possibility to remove the Cr contained in in-
dustrial wastewaters and the detailed mechanisms of Cr re-
moval in these two strains.
Acknowledgements This work was supported by the Sichuan Science
and Technology Bureau (2014GXZ0005, 2017JCPT0001) and National
Infrastructure of Natural Resources for Science and Technology Program
of China (NIMR-2016-8).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
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