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Removal of mercury(II), lead(II) and cadmium(II) from aqueous solutions using Rhodobacter sphaeroides SC01
Journal Pre-proof Removal of mercury(II), lead(II) and cadmium(II) from aqueous solutions using Rhodobacter sphaeroides SC01 Yan-Qiu Su, Yang-Juan Zhao, Wei-Jia Zhang, Guo-Cheng Chen, Han Qin, Dai-Rong Qiao, Yang-Er Chen, Yi Cao PII:
S0045-6535(19)32405-1
DOI:
https://doi.org/10.1016/j.chemosphere.2019.125166
Reference:
CHEM 125166
To appear in:
ECSN
Received Date: 8 January 2019 Revised Date:
9 October 2019
Accepted Date: 20 October 2019
Please cite this article as: Su, Y.-Q., Zhao, Y.-J., Zhang, W.-J., Chen, G.-C., Qin, H., Qiao, D.-R., Chen, Y.-E., Cao, Y., Removal of mercury(II), lead(II) and cadmium(II) from aqueous solutions using Rhodobacter sphaeroides SC01, Chemosphere (2019), doi: https://doi.org/10.1016/ j.chemosphere.2019.125166. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
Removal of mercury(II), lead(II) and cadmium(II) from
2
aqueous solutions using Rhodobacter sphaeroides SC01
3 4
Yan-Qiu Sua, Yang-Juan Zhaob, Wei-Jia Zhanga, Guo-Cheng Chena, Han Qina, Dai-Rong
5
Qiaoa, Yang-Er Chenb,*, Yi Caoa,*
6 7
a
8
Life Sciences, Sichuan University, Chengdu, 610064, China
9
b
College of Life Sciences, Sichuan Agricultural University, Ya’an, 625014, China
11
*
Corresponding author.
12
E-mail address: [email protected] (Y.-E. Chen), [email protected] (Y. Cao)
Microbiology and Metabolic Engineering of Key Laboratory of Sichuan Province, College of
10
13 14
ABSTRACT
15
Microorganisms and microbial products can be highly efficient in uptaking soluble and
16
particulate forms of heavy metals, particularly from solutions. In this study, the removal
17
efficiency, oxidative damage, antioxidant system, and the possible removal mechanisms were
18
investigated in Rhodobacter (R.) sphaeroides SC01 under mercury (Hg), lead (Pb) and
19
cadmium (Cd) stress. The results showed that SC01 had the highest removal rates (98%) of
20
Pb among three heavy metals. Compared with Hg and Cd stress, Pb stress resulted in a lower
21
levels of reactive oxygen species (ROS) and cell death. In contrast, the activities of four
22
antioxidant enzymes in SC01 under Pb stress was higher than that of Hg and Cd stress. 1
23
Furthermore, the analysis from fourier transform infrared spectroscopy indicated that
24
complexation of Pb with hydroxyl, amid and phosphate groups was found in SC01 under Pb
25
stress. In addition, X-ray diffraction analysis showed that precipitate of lead phosphate
26
hydroxide was produced on the cell surface in SC01 exposed to Pb stress. Therefore, these
27
results suggested that SC01 had good Pb removal ability by biosorption and precipitation and
28
will be potentially useful for removal of Pb in industrial effluents.
29 30
Keywords:
31
Heavy metal; Rhodobacter sphaeroides; Antioxidant system; Reactive oxygen species;
32
Biosorption
33 34 35
1. Introduction It has been known that heavy metal pollution is a major environment problem in aquatic
36
eco-system
restoration
due
to
its
non-biodegradability,
bioaccumulation,
and
37
biomagnifications in the world (Calvano et al., 2014; Aryal et al., 2015). Many heavy metals
38
are toxic to organisms including humans and other higher animal even at low concentrations.
39
Hg is regarded as one of the most toxic heavy metals found in the environmental
40
conditions including lithosphere, hydrosphere, atmosphere, and biosphere. The main
41
anthropogenic sources of Hg contamination are urban discharge, agricultural materials,
42
mining, combustion and industrial discharges in the environment (Deng et al., 2001, 2011).
43
Pb is also an important environmental contamination because of anthropogenic sources and
44
natural geochemical processes (Dursun et al., 2003). It is widely applied in many important 2
45
industrial applications. Cd is one of heavy metals found in industrial effluents like metal
46
plating, mining, metallurgical alloying, ceramics and so on (Hutton et al., 1987; Nriagu and
47
Pacyna, 1988). Therefore, it has been a potential challenge for how to remove these heavy
48
metal contaminants from the environment, especially for aquatic eco-system.
49
Many technologies have been used to reduce or eliminate heavy metals from waste waters.
50
Among these methods, bioremediation has gained the great attention because it is a more
51
cost-effective and environment-friendly method with less toxic sludge compared with
52
conventional methods (Aryal et al., 2015). Photosynthetic bacteria (PSB) has been thought to
53
be an ideal candidate for bioremediation and has better environmental adaptability than other
54
microorganisms because it can change the metabolic type flexibly with environment change,
55
and use solar radiation as their unique energy source (Giotta et al., 2006; Gadd, 2009; De
56
Philippis et al., 2011). Rhodobacter (R.) sphaeroides, as purple non-sulphur bacterium, has
57
been shown to possess a remarkable ability in growing under aerobic or anaerobic respiration,
58
anoxygenic photosynthesis, and fermentation (Schultz and Weaver, 1982; Kiley and Kaplan,
59
1988; Calvano et al., 2014). Many previous studies have indicated that R. sphaeroides has
60
high tolerance to various heavy metal pollution (Giotta et al., 2006; Panwichian et al., 2011)
61
and thus has been widely used for wastewater treatment to remove heavy metals. Studies
62
about heavy metal removal of R. sphaeroides have been investigated in wastewater and soil
63
polluted by heavy metals including chromate reduction (Nepple et al., 2000), Cd removal
64
(Watanabe et al., 2003; Bai et al., 2008), removal of Cd and zinc (Li et al., 2016a; Peng et al.,
65
2018), and bioremediation of Pb (Li et al., 2016b).
66
It has been known that PSB in the photosynthetic systems and structure is different with 3
67
green plants. Plants usually response to different heavy metal stresses by membrane system
68
(Kreslavski et al., 2017), osmotic regulation substances, and antioxidant defense system
69
(Chen et al., 2015). However, the detailed mechanism is still unclear in ROS accumulation
70
and antioxidant system in PSB under heavy metal stress. Although R. sphaeroides has been
71
used for investigating its removal ability to heavy metals, the study about the removal
72
efficiency and oxidative damage by comparing ROS accumulation and antioxidant defense
73
systems has rarely been reported in PSB under heavy metal stress.
74
Our recent study indicated that R. sphaeroides SC01 showed high Cr resistance and could
75
probably be applied in Cr removal in industrial effluents (Su et al., 2017). To explore the
76
biosprption capacity and mechanism for heavy metals in R. sphaeroides SC01, we compared
77
the growth and structures of cells, the oxidative damage, antioxidant defense system and
78
removal efficiency under three heavy metals stresses. In addition, we further investigated the
79
tolerance to Pb, the changes in functional groups in details and the composition of
80
bioremediation products under Pb stress in SC01. Our results will provide the important
81
theoretical and practical value for the application of SC01 in heavy metal removal in the
82
environment.
83 84
2. Materials and methods
85
2.1. Strain, cultivation and culture conditions
86
The experiments were performed with SC01, which was isolated from the water collected
87
from saline paddy fields in Sichuan province, China. According to our previous study (Su et
88
al., 2017), SC01 was identified as SC01 is deposited at the GDMCC Culture Collection, and 4
89
its collection number is GDMCC1.1264. SC01 was cultured with purple non-sulfur bacteria
90
enriched medium (NH4Cl 1.0 g, NaHCO3 1.0 g, yeast extract 2.0 g CH3COONa 3.0 g,
91
K2HPO4 0.5 g, MgCl2·6H2O 0.2 g, NaCl 5.0 g, CaCl2·2H2O 0.075 g, ferric citrate 0.01 g) in
92
our laboratory. The pH of medium was kept at 7.0 before autoclaving (Su et al., 2017). The
93
strain was grown anaerobically at a light intensity of about 4000 lux given with incandescent
94
lamp at 35 °C. Before heavy metal stress, the same cell density at 680 nm (OD680) was
95
measured by spectrophotometer (Hitachi-U2000, Hitachi, Ltd., Tokyo, Japan). Then, cells of
96
stationary phases were used in different experiments. All other conditions were the same in
97
the illuminating incubator.
98 99
2.2. Heavy metal treatments
100
Different concentrations of Hg2+ (0, 0.2, and 1 mg L-1), Pb2+ (0, 30, and 160 mg L-1) and
101
Cd2+(0, 0.8, and 6 mg L-1) were prepared by diluted different amounts of HgCl2, Pb(NO3)2,
102
and CdCl2 solutions with deionized water. Then, SC01 with the same concentrations at
103
stationary phase was added to the aqueous solutions for different heavy metal stresses. The
104
stress experiments were performed at the same growth condition for 7 d. Cell concentration
105
was recorded daily for the growth curve of SC01.
106 107
2.3. Removal rate of Hg, Pb, and Cd
108
Experiments were performed with 250 mL of reaction mixtures containing different
109
concentrations of Hg2+, Pb2+ and Cd2+ for removal rate analysis in 250 mL flasks. Then, the
110
strain at stationary phase was exposed to the polluted aqueous solution for 7 d in the 5
111
controlled conditions. After 7 d, 50 mL of SC01 solutions was sampled from each flask and
112
then centrifugated at 5,000 rpm for 15 min. After centrifugation, heavy metal concentrations
113
in the supernatant were determined by an inductively coupled plasma mass spectrometry
114
(ICP-MS) (Optimal 2100DV, Perkin Elmer Instruments, Waltham, MA, USA). Three standard
115
curves (Fig. S1) were obtained using 100 PPM, 1 PPM, and 1 PPM standard solution of Hg,
116
Pb, and Cd in 1% HNO3 (Sigma, Co. Ltd. USA), respectively. The quality control of the
117
results was performed using a parallel analysis of Hg, Pb, and Cd national reference materials
118
for water testing (GSB 07-3173-2014, GSB 07-1183-2000, and GSB 07-1185-2000,
119
respectively). The percent of heavy metal removal was calculated using the equations:
120
removal rate = [(C0 - C) / C0] × 100, where C0 is the initial heavy metal concentration (mg L-1)
121
and C is the residual heavy metal concentration (mg L-1). The batch experiments were
122
conducted for triplicate.
123 124
2.4. Oxidative stress measurements
125
H2O2 accumulation in the strain SC01 under heavy metal stress (1 mg L-1 Hg, 160 mg L-1
126
Pb, and 6 mg L-1 Cd) was observed with the fluorescent probe 2, 7-dichlorofluorescin
127
diacetate (DCFH-DA) as described by Su et al. (2017). After heavy metal stress, cells were
128
infiltrated with PBS buffer containing 20 µM DCFH-DA (Sigma) for 40 min in water bath
129
with 37 °C in the dark under vacuum and rinsed at least three times with PBS buffer. Then,
130
the H2O2 imaging of the cells in vivo was visualized with a confocal laser scanning
131
microscopy (emission, 530 nm; excitation, 488 nm).
132
6
133
2.5. Propidium iodide staining
134
The cells were stained with propidium iodide (PI) stain according to the method of Su et
135
al. (2017). The collected cells by centrifugation were washed three times and resuspended
136
with 2 mL of 0.1 M PBS (pH 7.6). Then, SC01 cell suspension (1 mL) was mixed with 0.5
137
µg/mL PI solution (Sigma) gently. The mixture was placed in the dark for 10 min at room
138
temperature and then were rinsed with PBS buffer for three times. Finally, the stained SC01
139
cells were resuspended with PBS buffer (0.1 mL) and subsequently observed immediately
140
with a fluorescence microscope (BX-53 System, Olympus Corporation, Tokyo, Japan) with an
141
excitation wavelength of 546 nm.
142 143
2.6. Cell characterization analysis under Pb stress
144
To describe the characteristics of the bioremediation products, the cells of SC01 were
145
collected before and after 160 mg L-1 Pb stress by centrifugation (8,000 rpm for 10 min).
146
Then, the precipitates were washed two times with ultrapure water and were lyophilized in a
147
vacuum freeze dryer (Scientz-10N, Scientz Biotechnology, Co. Ltd., Ningbo, China). The
148
cellular characteristics of the precipitates were analyzed by Fourier transform infrared FTIR
149
spectroscopy (Nicolet 6700, USA) and X-ray diffraction (XRD, AXIS Ultra DLD, Kratos Ltd.,
150
British) according to the previous method (Li et al., 2016b).
151
The collected precipitates were washed three times with 0.1 M PBS buffer (pH 7.0) and
152
then mixed well with 2.5% (w/v) glutaraldehyde. The mixture was fixed in the refrigerator at
153
4 °C overnight. The fixed cells were dehydrated with different concentration of ethanol (60,
154
70, 80, 90, and 100%) for 15 min. The precipitates were freeze-dried in a vacuum freeze dryer. 7
155
The cells were analyzed by scanning electron microscope (SEM) (JSM-7500F, Electron
156
Optics Laboratory Co. Ltd. Japan) and energy dispersive spectroscopy (SEM-EDS)
157
(JSM-7500F, Electron Optics Laboratory Co. Ltd. Japan) as described by Wang et al. (2001).
158 159
2.7. Statistical analysis
160
All experiments were performed for at least triplicates, and the given results represent as
161
the mean of three values ± standard deviations (SD). SPSS Statistics 19.0 software (IBM,
162
Chicago, IL, USA) and the Duncan’s multiple range test were used for the statistical analyses
163
of data. In the graphic representation, a different letter above the error bar was thought to be
164
significant when P < 0.05.
165 166
3. Results
167
3.1. Growth of R. sphaeroides SC01
168
The growth curve of SC01 was obtained according to biomass production under stressful
169
and non-stressful conditions daily (Fig. S2). No significant differences in biomass production
170
could be detected in the absence of Hg, Pb, and Cd. We found that the value of OD reached
171
the maximal value after 6 d in the control conditions. After 7 d, the value of OD declined
172
significantly compared with the control when three heavy metals were added into culture
173
medium. It is noteworthy that cell concentrations were significantly reduced by about 50%
174
under 1.0 mg L-1 Hg and 6.0 mg L-1 Cd stress, indicating that SC01 suffered significant
175
damage from Hg and Cd stress. In addition, we found that SC01 under Pb stress presented a
176
lower decrease in biomass than that under Hg and Cd stress, suggesting that SC01 suffered 8
177
less damage from Pb stress relative to Hg and Cd stress.
178
To further investigate the degree of heavy metal damage to SC01, the contents of
179
carotenoid and bacteriochlorophyll a were measured under heavy metal stress. The content of
180
carotenoid significantly decreased under Hg, Pb, and Cd stress (Fig. S3A-C). 1.0 mg L-1 Hg
181
treatment resulted in the largest decline in the production of carotenoid compared with Pb
182
(160 mg L-1) and Cd (6 mg L-1). Bacteriochlorophyll a content of SC01 under three heavy
183
metals stresses presented the same results with the growth (Fig. S3D). Compared with the
184
control, heavy metal treatments resulted in the significant decline in bacteriochlorophyll a
185
content. However, bacteriochlorophyll a show a lower decrease under Pb stress compared
186
with Hg and Cd stress.
187 188
3.2. Removal rate of Hg, Pb, and Cd
189
The concentrations of Hg, Pb, and Cd in SC01 were determined in the stationary phase
190
under three heavy metals stresses and shown within the linear range. The specific uptake of
191
three heavy metals presented significant differences in SC01 (Fig. 1). Compared with Hg and
192
Cd stress, Pb treatments with low and high concentrations resulted in the highest percent Pb
193
removal, reaching to 98%. However, the strain exhibited a significant difference in the
194
percent Hg and Pb removal under Hg and Cd stress. These results suggested that SC01 has the
195
best removal rate of Pb relative to Hg and Cd.
196 197 198
3.3. ROS accumulation of SC01 under heavy metal stress To investigate whether SC01 suffered more damage from Hg and Cd stress than Pb stress, 9
199
we measured the contents of the two major ROS species, H2O2 and O2˙¯, produced under three
200
heavy metals stresses (Fig. S4). Compared with the control, three heavy metals stresses
201
resulted in the significant increase in the levels of O2˙¯and H2O2, especially for higher
202
concentrations of heavy metal. In addition, compared with Pb stress, Hg and Cd stress led to
203
the higher accumulation of O2˙¯ and H2O2, especially Hg stress. Furthermore, the level of
204
H2O2 in SC01 was measured in situ by a H2O2-sensitive fluorescent probe (DCFH-DA),
205
which has been successfully used for H2O2 detection in our present study (Su et al., 2017) and
206
plants (Xu et al., 2012; Rico et al., 2013). As expect, the results of fluorescent staining in the
207
non-stressed and stressed strains were similar with the quantitative data of H2O2 (Fig. 2).
208
Compared with the control and the lower concentrations of heavy metal, SC01 accumulated
209
more H2O2 under higher concentrations of heavy metal. Relative to 1.0 mg L-1 Hg and 6.0 mg
210
L-1, 160 mg L-1 Pb resulted in a low accumulation of H2O2. These results suggested that SC01
211
suffered a lower oxidative damage from Pb stress relative to Hg and Cd stress.
212 213
3.4. Cell death under Hg, Pb, and Cd stress
214
To investigate the integrity of cell membrane under three heavy metals stresses, the degree
215
of cell death was measured by PI stains, which is a membrane-impermeable dye and can bind
216
to DNA through entering cells with damaged cell membranes (Williams et al., 1998). We
217
found that the results of PI staining were consistent with the quantitative results of O2˙¯ and
218
H2O2 in SC01 exposed to heavy metal stress (Fig. 3). Compared with the control, the cells of
219
SC01 under three heavy metals stresses presented a high intense red fluorescence, especially
220
at the higher concentration of Hg, Pb, and Cd. Similarly, Pb stress showed the weaker red 10
221
fluorescence compared with Hg and Cd stress. These results indicated that SC01 under Pb
222
stress received less cell death than Hg and Cd stress.
223 224
3.5. Enzymatic and non-enzymatic antioxidant activities under heavy metal stress
225
The activities of four important antioxidant enzymes in SC01 under three heavy metals
226
stresses showed in Fig. S5. Four enzymatic activities presented significant differences among
227
Hg, Pb, and Cd stresses. Compared with the control, three heavy metals stresses resulted in
228
the significant increase in the activities of POD, SOD, CAT, and APX in SC01. In addition,
229
the activities of the antioxidant enzymes (POD, SOD, CAT, and APX) under Pb stress were
230
significantly higher than that of Hg and Cd stress (Fig. S5). These results suggested that SC01
231
could maintain the relative high activities of antioxidant enzymes under Pb stress.
232
The activities of non-enzymatic antioxidants were further investigated in SC01 under
233
three heavy metals stresses (Fig. S6). The AsA and GSH contents significantly decreased,
234
whereas the concentrations of DHA and GSSG showed an obvious increase under three heavy
235
metals stresses compared with the control. The most significant decrease in the contents of
236
ASA and GSH was observed under Hg stress. In contrast, Pb stress led to a lower increase in
237
the contents of DHA and GSSG relative to Hg and Cd stress (Fig. S6 B and D).
238 239
3.6. Cell characterization under Pb stress
240
Based on the above-mentioned results, we proposed that SC01 probably has high
241
resistance to Pb among three heavy metals. In order to find out the resistant mechanisms, the
242
cell characterization was further investigated in SC01 under Pb stress. 11
243
To examine the morphological changes in the cells, SEM analysis was used in SC01
244
exposed to Pb. As shown in Fig. 4A, a slight shrinkage was observed on the cell surface under
245
the normal condition. However, SC01 cells showed a more obvious shrinkage and even a lot
246
of cell debris was observed under 160 mg L-1 Pb for 7 d, compared with the control (Fig. 4B).
247
Furthermore, SEM-EDS analyses indicated that the absorption peak of Pb was clearly
248
observed in Pb-treated SC01 compared with the control (Fig. 4C-D), suggesting that a large
249
amount of Pb was absorbed into the cells by the strain.
250
To further explore the possible interactions between Pb and surface functional groups,
251
FT-IR spectra of SC01 was recorded in the presence and absence of 160 mg L-1 Pb (Fig. 5).
252
Many functional groups on the cell surface were revealed by the pre-adsorption FT-IR spectra.
253
These bands or groups were determined according to the data of previous literatures (Table
254
S1). Although the nearly similar variation tendencies of the spectral curves were observed
255
between the control and Pb-stressed strain, there were some differences in the crests before
256
and after Pb treatment. For example, the band at 3300 cm-1 replaced the band at 3420 cm-1
257
after Pb treatment, which was attributed to the –OH and –NH groups..This difference of 120
258
cm-1 peak (∆) indicated the involvement of –OH and –NH groups in the biosorption process
259
in SC01 (Akar et al., 2005). The similar band shifted from 1080 to 1052 cm-1 indicated
260
involvement of the C–O stretching of carboxyl groups and the bending vibration band of the
261
hydroxyl groups (Tunali et al., 2006), or phosphate groups (symmetric stretching of > PO2 )
262
(Lodeiro et al., 2006; Gabr et al., 2008). Two observed increase in the peaks at 980 cm-1 and
263
535 cm-1 in Pb-treated SC01 could be attributed to an intensity between Pb ions and
264
N-containing bioligands (Kamnev et al., 1997; Akar et al., 2005). These results indicated –OH, 12
265
–NH and phosphoric groups were involved in binding with Pb ions.
266
To further identify Pb(II) compounds formed during the biosorption process, Pb-treated
267
and the control SC01 were scanned by XRD (Fig. 6). Compared with the control, the d-values
268
of the major lines were observed at 2.969 (2θ, 30.0922), which closely correspond to (100.00)
269
planes of Pb Phosphate Hydroxide (Pb10(PO4)6(OH)2, JCPDS card No.051-1648). The result
270
indicated that Pb Phosphate Hydroxide was formed during Pb biosorption by SC01.
271 272
4. Discussion
273
It is well known that heavy metal pollution is becoming one of the most severe
274
environmental and human health hazards. Among these heavy metals, Hg, Pb and Cd were
275
widely applied in many industries and also thought to be the important source of industrial
276
pollution (Orr et al., 2018). Biosorption of heavy metal has been one of the most promising
277
technologies. In recent years, it obtains a great deal of attention due to its potential application
278
in industry in the world (Li et al., 2016b). To explore the application of SC01 in biosorption
279
of heavy metals, we investigated the growth of SC01, the oxidative damage, and the removal
280
efficiency of Hg, Pb and Cd under these heavy metals stresses in the present experiment.
281
Furthermore, the mechanism of biosorption was also studied in SC01 under Pb stress.
282
The growth of PSB has been commonly used as a effective marker under heavy metal
283
stress. The results showed that biomass production of SC01 reached rapidly the maximal
284
values on 6th d under the control conditions, suggested that SC01 has an excellent growth in
285
the cultured medium. Many studies indicated that toxic ions or salt stress conditions can
286
decrease bacterial metabolism and lead to inhibition of growth (Sheng et al., 2005; Upadhyay 13
287
et al., 2011). In consistent with the finding, we found that the growth of SC01 showed a
288
obvious decline under heavy metal stress. In addition, the rapid decline in biomass under high
289
concentration of Hg was probably because this strain is more sensitive to Hg and Cd than Pb.
290
Many studies indicated that PSB usually have different removal rate to different heavy
291
metals under stressful conditions (Giotta et al., 2006; Aryal et al., 2015; Peng et al., 2018).
292
Our recent study showed that SC01 had a high removal efficiency of Cr under high
293
concentration of Cr (Su et al., 2017). In the present experiment, we found that SC01 also
294
exhibited a very high removal rate of Pb under Pb stress. The high removal efficiency of Pb in
295
SC01 was probably due to the high resistance to Pb. Therefore, the good growth and the high
296
removal efficiency of Pb under Pb stress suggested that SC01 has high Pb resistance and will
297
be suitable for the removal of Pb from industrial effluent.
298
Some studies reported that the decline of bacteriochlorophyll a and carotenoid content
299
was usually observed in algae or bacteria response to heavy metal stress (Feng et al., 2007;
300
Hou et al., 2016; Kalaji et al., 2016; Batool et al., 2017; Su et al., 2017). Consistent with these
301
studies, our results showed that the contents of carotenoid and bacteriochlorophyll a gradually
302
decreased with the increase in concentrations of three heavy metals, indicating that three
303
heavy metals could lead to severe damage to the photosystem of SC01. However, we found
304
that the content of bacteriochlorophyll a in SC01 under Pb stress showed a slow decline
305
compared with Hg and Cd stress, suggested that this strain had a high resistance to Pb.
306
It is well known that heavy metal stress can induce ROS generation, and subsequently
307
result in oxidative damage to microorganism (Mathew et al., 2011; Su et al., 2017) or plants
308
(Dixit et al., 2002; Panda and Choudhury, 2005). In consistent with these findings, our study 14
309
found that three heavy metals induced the excessive accumulation of ROS in this strain,
310
especially at high concentrations of Hg and Cd, implying that SC01 suffered severe oxidative
311
damage from high concentrations of ROS under Hg and Cd stress. Compared with Hg and Cd
312
stress, Pb stress resulted in the lower accumulation of ROS in SC01. The reason could be
313
because SC01 had a high resistance to Pb, which was in accordance with the finding obtained
314
from Cr stress (Su et al., 2017). In addition, the severe oxidative damage usually lead to the
315
obvious cell death in plants and PSB under environmental stresses (Su et al., 2017; Chen et al.,
316
2018). In the present study, we found that a large number of cell death was observed in SC01
317
exposed to heavy metal stress, especially at high concentrations of Hg. The results were in
318
agreement with the observed levels of ROS accumulation in the strain under three heavy
319
metals stresses. Therefore, these findings indicated that SC01 suffered less oxidative damage
320
by lowing the levels of ROS under Pb stress relative to Hg and Cd stress.
321
It has been known that many organisms including PSB are able to reduce or eliminate
322
effectively oxidative damage induced by ROS through a complex antioxidant defense system
323
under environmental stresses (Foyer and Shigeoka, 2011; Su et al., 2017). Some studies
324
indicated that activities of antioxidant enzymes enhanced in some plants under low or
325
moderate concentrations of heavy metal stress (Chen et al., 2015, 2018). Consistent with these
326
findings, our results showed that the activities of four antioxidant enzymes significantly
327
increased under three heavy metals stresses, suggesting that SC01 is able to alleviate the
328
oxidative damage through improvement of the activities of the antioxidant enzymatic system
329
under heavy metal stress. However, the activities of four antioxidant enzymes were markedly
330
higher under Pb stress than that of Hg and Cd stress in SC01. The reason may be because 15
331
SC01 has a high resistance to Pb and an excellent removal system.
332
In addition, the AsA-GSH cycle is also a key process in regulating the damage of ROS in
333
different organisms under environmental conditions. AsA and GSH were regarded as the most
334
abundant water soluble antioxidants in plants (Foyer and Shigeoka, 2011). Some studies
335
indicated that the contents of AsA and GSH decreased in plants and PSB under environmental
336
stresses (Su et al., 2017; Chen et al., 2018). In accordance with these reports, our results
337
showed that the levels of AsA and GSH were markedly inhibited in SC01 under three heavy
338
metals stresses. These findings suggested that heavy metal application inhibited the activities
339
of non-enzymatic antioxidants in SC01. In addition, we found that Pb stress resulted in higher
340
contents of AsA and GSH and lower concentrations of DHA and GSSH relative to Hg and Cd
341
stress, indicating that the ROS detoxification system is more effective in SC01 under Pb
342
stress.
343
The changes in cell morphology were usually occurred in microorganisms (fungi, algae,
344
bacteria, etc.) under heavy metal stress. In our study, the obvious membrane indentation or
345
shrinkage of cells were observed under Pb stress. The decline in the surface and volume
346
fraction of cells suggested that SC01 could prevent the harmful effects of Pb by trimming the
347
attachable cell surface in contrast to total cell plane (Neumann et al., 2005). In addition, the
348
dumbbell shaped cells may be a strategy for accumulating more Pb in SC01 under Pb stress
349
(Mohite et al., 2018). The results from EDS spectrum further confirmed the presence of Pb on
350
the surface of cells in SC01.
351
The main bioremediation mechanisms of heavy metal removal using microorganism
352
mainly contain two steps, which are defined as the biosorption and bioaccumulation processes 16
353
(Das et al., 2008; Panwichian et al., 2010; Aryal et al., 2015), respectively. It is the first step
354
that metal ions are absorbed to surface of cells by interactions between heavy metals and
355
functional groups (Li et al., 2016b). This process is a dynamic balance between adsorption
356
and desorption because heavy metal ions bound on the cell surface may be eluted by acids,
357
other ions or chelating agents (Das et al., 2008). A previous study indicated that carboxyl,
358
hydroxyl, carbonyl, amido, and phosphate groups were bound with Pb on the bacterial surface
359
under Pb stress (Bai et al., 2014). Consistently, our results demonstrated that some functional
360
groups including hydroxyl, amido and phosphate groups existed on the surface of cells and
361
were mainly responsible for Pb biosorption in SC01. Therefore, Pb ion could penetrate the
362
cell membrane and subsequently enter into the cell due to active biosorption in the second
363
step (Das et al., 2008; Italiano et al., 2009). After heavy metals entering the cell in the second
364
step, microorganisms would accumulate heavy metals in the cell by enrichment, and remove
365
heavy metals by bioprecipitation as different compounds. Some previous studies have
366
demonstrated
367
Pb10(PO4)6(OH)2, Pb5(PO4)3Cl, and Pb9(PO4)6 were formed during the bioaccumulation
368
process of Pb ion in microorganisms (Levinson and Mahler, 1998; Mire et al., 2004; Bai et al.,
369
2014). In the present experiment, our results showed that only Pb10(PO4)6(OH)2 was formed
370
during the bioaccumulation of Pb ion, suggesting that the strain SC01 could accumulate Pb
371
effectively through the formation of Pb10(PO4)6(OH)2 in the cells. A previous study from Pb
372
contaminated soil reported that the main mechanism for Pb removal is the precipitation
373
formation of inert compounds including lead sulfate and lead sulfide in R. sphaeroides (Li et
374
al., 2016b). However, our results were different from the previous findings from heavy metal
that
Pb
phosphate
compounds
17
including
PbHPO4,
Pb5(PO4)3OH,
375
removal using R. sphaeroides. The main reason may be related to different strains and the
376
concentrations of Pb.
377 378
5. Conclusions
379
In this study, we combined the oxidative damage and antioxidant defense system to
380
estimate the removal ability of SC01 to Hg, Pb and Cd under heavy metal stress. Based on
381
high removal efficiency of Pb, we further investigated the bioremediation mechanisms of Pb
382
in SC01 under Pb stress. Our results showed that Hg and Pb stress led to the highest and the
383
lowest oxidative stress in SC01 according to cell death and ROS accumulation, respectively.
384
In addition, we found that SC01 may be more effective in preventing the oxidative damage
385
caused by excessive ROS through activating the antioxidant defense system under Pb stress.
386
Furthermore, the removal rate of Pb in SC01 reached up to 98% under high concentrations of
387
Pb, which was higher than those of previous studies. Moreover, the results obtained from the
388
analyses of bioremediation mechanisms of Pb revealed that Pb ion could bind with hydroxyl,
389
amido and phosphate groups on the cell surface of SC01, and subsequently formed precipitate
390
of Pb Phosphate Hydroxide during the biosorption process. Based on these results, we
391
propose that SC01 probably has high resistance to Pb and high removal efficiency of Pb
392
through regulating the antioxidant system and the formation of precipitates of Pb Phosphate
393
Hydroxide under stressful conditions, respectively. However, further work is needed to
394
address the detailed bioremediation mechanisms of Pb in vivo and the removal efficiency of
395
Pb contained in industrial wastewaters in SC01.
396 18
397
Acknowledgements
398
This work was supported by the Sichuan Science and Technology Bureau (2018GZ0375;
399
2018TJPT0004), Chengdu Science and Technology Bureau Project (2017-GH02-00071-HZ,
400
2018-YF05-00738-SN), National Infrastructure of Natural Resources for Science and
401
Technology Program of China (NIMR-2018-8-1). We thank Ji-qiu Wen and Guang-zhong Liu
402
(Analytical & Testing Center, Sichuan University) for assistance with XRD analysis. We also
403
thank Dong Wang and Yi He (Analytical & Testing Center, Sichuan University) for SEM
404
image.
405 406
References
407
Akar, T., Tunali, S., Kiran, I., 2005. Botrytis cinerea as a new fungal biosorbent for removal
408 409 410
of Pb(II) from aqueous solutions. Biochem. Eng. J. 25, 227–235. Aryal, M., Liakopoulou-Kyriakides, M., 2015. Bioremoval of heavy metals by bacterial biomass. Environ. Monit. Assess. 187, 4173.
411
Bai, H.J., Zhang, Z.M., Yang, G.E., Li, B.Z., 2008. Bioremediation of cadmium by growing
412
Rhodobacter sphaeroides: kinetic characteristic and mechanism studies. Bioresour.
413
Technol. 99, 7716–7722.
414
Bai, J., Yang, X.H., Du, R.Y., Chen, Y.M., Wang, S.Z., Qiu, R.L., 2014. Biosorption
415
mechanisms involved in immobilization of soil Pb by Bacillus subtilis DBM in a
416
multi-metal-contaminated soil. J. Environ. Sci. 26, 2056–2064.
417
Batool, R., Tabassum, T., Ali, M., 2017. Evaluation of Cr (VI) remediation potential of
418
Eichornia sp in conjunction with chromium-resistant bacterial strains. Trop. J. Pharm. 19
419
Res. 16, 1005–1011.
420
Calvano, C.D., Italiano, F., Catucci, L., Agostiano, A., Cataldi, T.R.I., Palmisano, F., Trotta,
421
M., 2014. Erratum to: The lipidome of the photosynthetic bacterium Rhodobacter
422
sphaeroides R26 is affected by cobalt and chromate ions stress. Biometals. 28, 229.
423
Chen, Y.E., Cui, J.M., Yang, J.C., Zhang, Z.W., Yuan, M., Song, C., Yang, H., Liu, H.M.,
424
Wang, C.Q., Zhang, H.Y., Zeng, X.Y., Yuan, S., 2015. Biomonitoring heavy metal
425
contaminations by moss visible parameters. J. Hazard. Mater. 296, 201–209.
426
Chen, Y.E., Mao, H.T., Ma, J., Wu, N., Zhang, C.M., Su, Y.Q., Zhang, Z.W., Yuan, M., Zhang,
427
H.Y., Zeng, X.Y., Yuan, S., 2018. Biomonitoring chromium III or VI soluble pollution by
428
moss chlorophyll fluorescence. Chemosphere. 194, 220–228.
429 430
Das, N., Vimala, R., Karthika, P., 2008. Biosorption of heavy metals- an overview. Indian J of Biotechnol. 7, 159–169.
431
De Philippis, R., Colica, G., Micheletti, E., 2011. Exopolysaccharide-producing cyanobacteria
432
in heavy metal removal from water: molecular basis and practical applicability of the
433
biosorption process. Appl. Microbiol. Biotechnol. 92, 697–708.
434 435 436 437
Deng, X., Jia, P., 2011. Construction and characterization of a photosynthetic bacterium genetically engineered for Hg2+ uptake. Bioresour. Technol. 102, 3083–3088. Deng, X., Wilson, D.B., 2001. Bioaccumulation of mercury from wastewater by genetically engineered Escherichia coli. Appl. Microbiol. Biotechnol. 56, 276–279.
438
Dixit, V., Pandey, V., Shyam, R., 2002. Chromium ions inactivate electron transport and
439
enhance superoxide generation in vivo in pea (Pisum sativum L. cv. Azad) root
440
mitochondria. Plant Cell Environ. 25, 687–693. 20
441 442
Dursun, A.Y., Uslu, G., Cuci, Y., Aksu, Z., 2003. Bioaccumulation of copper(II), lead(II) and chromium (VI) by growing Aspergillus niger. Process Biochem. 38, 1647–1651.
443
Feng, Y.Z., Yu, Y.C., Wang, Y.M., Lin, X.G., 2007. Biosorption and bioreduction of trivalent
444
aurum by photosynthetic bacteria Rhodobacter capsulatus. Curr. Microbiol. 55,
445
402–408.
446 447
Foyer, C.H., Shigeoka, S., 2011. Understanding oxidative stress and antioxidant functions to enhance photosynthesis. Plant Physiol. 155, 93–100.
448
Gabr, R.M., Hassan, S.H.A., Shoreit, A.A.M., 2008. Biosorption of lead and nickel by living
449
and non-living cells of Pseudomonas aeruginosa ASU 6a. Int. Biodeterior. Biodegrad. 62,
450
195–203.
451
Gadd, G.M., 2009. Biosorption: critical review of scientific rationale, environmental
452
importance and significance for pollution treatment. J. Chem. Technol. Biotechnol. 84,
453
13–28.
454
Giotta, L., Agostiano, A., Italiano, F., Milano, F., Trotta, M., 2006. Heavy metal ion influence
455
on the photosynthetic growth of Rhodobacter sphaeroides. Chemosphere. 62,
456
1490–1499.
457
Hou, S.L., Shu, W.J., Tan, S., Zhao, L., Yin, P.H., 2016. Exploration of the antioxidant system
458
and photosynthetic system of a marine algicidal Bacillus and its effect on four harmful
459
algal bloom species. Can. J. Microbiol. 62, 49–59.
460
Hutton, M., Chaney, R.L., Krishna Murti, C.R., Olade, M.A., Page, A.L., 1987. Group report:
461
cadmium. In: Hutchinson, T.C., Meema, K.M. (Eds.), Lead, Mercury, Cadmium and
462
Arsenic in the Environment. Wiley, New York. 35–41. 21
463
Italiano, F., Buccolieri, A., Giotta, L., Agostiano, A., Valli, L., Milano, F., Trotta, M., 2009.
464
Response of the carotenoidless mutant Rhodobacter sphaeroides growing cells to cobalt
465
andnickel exposure. Int Biodeter Biodegr. 63 (7), 948–957.
466
Kalaji, H.M., Sytar, O., Brestic, M., Samborska, I.A., Cetner, M.D., Carpentier, C., 2016. Risk
467
assessment of urban lake water quality based on in-situ cyanobacterial and total
468
chlorophyll-a monitoring. Pol. J. Environ. Stud. 25, 655–661.
469
Kamnev, A.A., Ristić, M., Antonyuk, L.P., Chernyshev, A.V., Ignatov, V.V., 1997. Fourier
470
transform infrared spectroscopic study of intact cells of the nitrogen-fixing bacterium
471
Azospirillum brasilense. J. Mol. Struct. 408/409, 201–205.
472 473
Kiley, P.J., Kaplan, S., 1988. Molecular genetics of photosynthetic membrane biosynthesis in Rhodobacter sphaeroides. Microbiol. Rev. 52, 50–69.
474
Kreslavski, V.D., Brestic, M., Zharmukhamedov, S.K., Luybimov, V.Y., Lankin, A.V., Jajoo,
475
A., Allakhverdiev, S.I., 2017. Mechanisms of inhibitory effects of polycyclic aromatic
476
hydrocarbons in the photosynthetic primary processes in pea leaves and thylakoid
477
preparations. Plant Biol. 19, 683–688.
478 479
Levinson, H.S., Mahler, I., 1998. Phosphatase activity and lead resistance in Citrobacter freundii and Staphylococcus aureus. FEMS Microbiol. Lett. 161, 135–138.
480
Li, X.M., Peng, W.H., Jia, Y.Y., Lu, L., Fan, W.H., 2017. Removal of cadmium and zinc from
481
contaminated wastewater using Rhodobacter sphaeroides. Water Sci. Technol. 75,
482
2489–2498.
483 484
Li, X.M., Peng, W.H., Jia, Y.Y., Lu, L., Fan, W.H., 2016. Bioremediation of lead contaminated soil with Rhodobacter sphaeroides. Chemosphere. 156, 228–235. 22
485
Lodeiro, P., Barriada, J.L., Herrero, R., Sastre de Vicente, M.E., 2006. The marine macroalga
486
Cystoseira baccata as biosorbent for cadmium(II) and lead(II) removal: Kinetic and
487
equilibrium studies. Environ. Pollut. 142, 264–273.
488 489
Mathew, B.B., Tiwari, A., Jatawa, S.K., 2011. Free radicals and antioxidants: a review. J. Pharm. Pharmacol. 4, 4340–4343.
490
Mire, C.E., Tourjee, J.A., O’Brien, W.F., Ramanujachary, K.V., Hecht, G.B., 2004. Lead
491
precipitation by Vibrio harveyi: evidence for novel quorum-sensing interactions. Appl.
492
Environ. Microbiol. 70, 855–864.
493
Mohite, B.V., Koli, S.H., Patil, S.V., 2018. Heavy metal stress and its consequences on
494
exopolysaccharide (EPS)-producing Pantoea agglomerans. Appl. Biochem. Biotechnol.
495
186, 199–216.
496 497
Nepple, B.B., Kessi, J., Bachofen, R., 2000. Chromate reduction by Rhodobacter sphaeroides. J. Ind. Microbiol. Biotechnol. 25, 198–203.
498
Neumann, G., Veeranagouda, Y., Karegoudar, T.B., Sahin, Ö., Mäusezahl, I., Kabelitz, N.,
499
Kappelmeyer, U., Heipieper, H.J., 2005. Cells of Pseudomonas putida and Enterobacter
500
sp. adapt to toxic organic compounds by increasing their size. Extremophiles. 9,
501
163–168.
502 503
Nriagu, J.O., Pacyna, J.M., 1988. Quantitative assessment of worldwide contamination of air, water and soils by trace metals. Nature. 333, 134–139.
504
Orr, S.E., Barnes, M.C., George, H.S., Joshee, L., Jeon, B., Scircle, A., Black, O., Cizdziel,
505
J.V., Smith, B.E., Bridges, C.C., 2018. Exposure to mixtures of mercury, cadmium, lead,
506
and arsenic alters the disposition of single metals in tissues of Wistar rats. J. Toxicol. 23
507 508 509
Environ. Health Part A. 81, 1–11. Panda, S.K., Choudhury, S., 2005. Chromium stress in plants. Braz. J. Plant Physiol. 17, 95–102.
510
Panwichian, S., Kantachote, D., Wittayaweerasak, B., Mallavarapu, M., 2010. Factors
511
affecting immobilization of heavy metals by purple nonsulfur bacteria isolated from
512
contaminated shrimp ponds. World J. Microbiol. Biotechnol. 26, 2199–2210
513
Peng, W.H., Li, X.M., Song, J.X., Jiang, W., Liu, Y.Y., Fan, W.H., 2018. Bioremediation of
514
cadmium- and zinc-contaminated soil using Rhodobacter sphaeroides. Chemosphere.
515
197, 33–41.
516
Rico, C, Hong, J., Morales, M.I., Zhao, L.J., Barrios, A.C., Zhang, J.Y., Peralta-Videa, J.R.,
517
Gardea-Torresdey, J.L., 2013. Effect of cerium oxide nanoparticles on rice: a study
518
involving the antioxidant defense system and in vivo fluorescence imaging. Environ. Sci.
519
Technol. 47, 5635–5642.
520 521
Schultz, J.E., Weaver, P.F., 1982. Fermentation and anaerobic respiration by Rhodospirillum rubrum and Rhodopseudomonas capsulata. J. Bacteriol. 149, 181–190.
522
Sheng, G.P., Yu, H.Q., Yue, Z.B., 2005. Production of extracellular polymeric substances from
523
Rhodopseudomonas acidophila in the presence of toxic substances. Appl. Microbiol.
524
Biotechnol. 69, 216–222.
525
Su, Y.Q., Zhao, Y.J., Wu, N., Chen, Y.E., Zhang, W.J., Qiao, D.R., Cao, Y., 2017. Chromium
526
removal from solution by five photosynthetic bacteria isolates. Appl. Microbiol.
527
Biotechnol. 102, 1983–1995.
528
Tunali, S., Akar, T., Özcan, A.S., Kiran, I., Özcan, A., 2006. Equilibrium and kinetics of 24
529
biosorption of lead(II) from aqueous solutions by Cephalosporium aphidicola. Sep. Purif.
530
Technol. 47, 105–112.
531
Upadhyay, S.K., Singh, J.S., Singh, D.P., 2011. Exopolysaccharide-producing plant
532
growth-promoting rhizobacteria under salinity condition. Pedosphere. 21, 214–222.
533
Wang, C.L., Lum, A.M., Ozuna, S.C., Clark, D.S., Keasling, J.D., 2001. Aerobic sulfide
534
production and cadmium precipitation by Escherichia coli expressing the Treponema
535
denticola cysteine desulfhydrase gene. Appl. Microbiol. Biotechnol. 56, 425–430.
536
Watanabe, M., Kawahara, K., Sasaki, K., Noparatnaraporn, N., 2003. Biosorption of cadmium
537
ions using a photosynthetic bacterium, Rhodobacter sphaeroides S and a marine
538
photosynthetic bacterium, Rhodovulum sp. and their biosorption kinetics. J. Biosci.
539
Bioeng. 95, 374–378.
540
Williams, S.C., Hong, Y., Danavall, D.C.A., Howard-Jones, M.H., Gibson, D., Frischer, M.E.,
541
Verity, P.G., 1998. Distinguishing between living and nonliving bacteria: evaluation of
542
the vital stain propidium iodide and its combined use with molecular probes in aquatic
543
samples. J. Microbiol. Methods. 32, 225–236.
544
Xu, J., Zhu, Y.Y., Ge, Q., Li, Y.L., Sun, J.H., Zhang, Y., Liu, X.J., 2012. Comparative
545
physiological responses of Solanum nigrum and Solanum torvum to cadmium stress.
546
New Phytol. 196, 125–138.
547
25
1
Figure legends
2
Fig. 1. The percent removal of Hg, Pb, and Cd in R. sphaeroides SC01 under heavy metal stress. The
3
data are showed as the mean ± SD of at least three independent measurements. Different letters above
4
bars mean significant difference (P < 0.05) among different treatments according to Duncan’s multiple
5
range test.
6 7
Fig. 2. In vivo imaging of H2O2 in R. sphaeroides SC01 under Hg, Pb, and Cd stress. The strain SC01
8
was stained with 20 µM of the fluorescent probe 2,7-dichlorofluorescin diacetate (DCFH-DA) as
9
described in the “Materials and methods” section. Bars = 400 µm.
10 11
Fig. 3. The integrity of plasma membrane in R. sphaeroides SC01 under Hg, Pb and Cd stress. The
12
strain was stained with 0.5 µg/mL of propidium iodide (PI) as described in the “Materials and
13
methods” section. Bars = 100 µm.
14 15
Fig. 4. Scanning electron micrograph (A and B) and energy dispersive spectroscopy (C and D) of R.
16
sphaeroides SC01 in the control and 160 mg/L Pb treatment. A and C represented the control. B and D
17
represented 160 mg/L Pb treatment.
18 19
Fig. 5. FTIR spectra of R. sphaeroides SC01 biomass in the control (black line) and 160 mg/L Pb
20
treatment (red line). Wavenumbers from 4000 to 400 cm-1 were showed.
21 22
Fig. 6. X-ray diffraction patterns of R. sphaeroides SC01 in the control (gray line) and 160 mg/L Pb
1
23
treatment (red line).
24
2
Highlights
Among three heavy metals, mercury (Hg) stress resulted in the severe oxidative damage in R. sphaeroides SC01.
The strain SC01 showed high removal efficiency of lead (Pb).
Under Pb stress, the formation of precipitates of Pb phosphate hydroxide helped to improve the resistance to Pb and the removal rates of Pb in R. sphaeroides SC01.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0045-6535(19)32405-1
DOI:
https://doi.org/10.1016/j.chemosphere.2019.125166
Reference:
CHEM 125166
To appear in:
ECSN
Received Date: 8 January 2019 Revised Date:
9 October 2019
Accepted Date: 20 October 2019
Please cite this article as: Su, Y.-Q., Zhao, Y.-J., Zhang, W.-J., Chen, G.-C., Qin, H., Qiao, D.-R., Chen, Y.-E., Cao, Y., Removal of mercury(II), lead(II) and cadmium(II) from aqueous solutions using Rhodobacter sphaeroides SC01, Chemosphere (2019), doi: https://doi.org/10.1016/ j.chemosphere.2019.125166. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
Removal of mercury(II), lead(II) and cadmium(II) from
2
aqueous solutions using Rhodobacter sphaeroides SC01
3 4
Yan-Qiu Sua, Yang-Juan Zhaob, Wei-Jia Zhanga, Guo-Cheng Chena, Han Qina, Dai-Rong
5
Qiaoa, Yang-Er Chenb,*, Yi Caoa,*
6 7
a
8
Life Sciences, Sichuan University, Chengdu, 610064, China
9
b
College of Life Sciences, Sichuan Agricultural University, Ya’an, 625014, China
11
*
Corresponding author.
12
E-mail address: [email protected] (Y.-E. Chen), [email protected] (Y. Cao)
Microbiology and Metabolic Engineering of Key Laboratory of Sichuan Province, College of
10
13 14
ABSTRACT
15
Microorganisms and microbial products can be highly efficient in uptaking soluble and
16
particulate forms of heavy metals, particularly from solutions. In this study, the removal
17
efficiency, oxidative damage, antioxidant system, and the possible removal mechanisms were
18
investigated in Rhodobacter (R.) sphaeroides SC01 under mercury (Hg), lead (Pb) and
19
cadmium (Cd) stress. The results showed that SC01 had the highest removal rates (98%) of
20
Pb among three heavy metals. Compared with Hg and Cd stress, Pb stress resulted in a lower
21
levels of reactive oxygen species (ROS) and cell death. In contrast, the activities of four
22
antioxidant enzymes in SC01 under Pb stress was higher than that of Hg and Cd stress. 1
23
Furthermore, the analysis from fourier transform infrared spectroscopy indicated that
24
complexation of Pb with hydroxyl, amid and phosphate groups was found in SC01 under Pb
25
stress. In addition, X-ray diffraction analysis showed that precipitate of lead phosphate
26
hydroxide was produced on the cell surface in SC01 exposed to Pb stress. Therefore, these
27
results suggested that SC01 had good Pb removal ability by biosorption and precipitation and
28
will be potentially useful for removal of Pb in industrial effluents.
29 30
Keywords:
31
Heavy metal; Rhodobacter sphaeroides; Antioxidant system; Reactive oxygen species;
32
Biosorption
33 34 35
1. Introduction It has been known that heavy metal pollution is a major environment problem in aquatic
36
eco-system
restoration
due
to
its
non-biodegradability,
bioaccumulation,
and
37
biomagnifications in the world (Calvano et al., 2014; Aryal et al., 2015). Many heavy metals
38
are toxic to organisms including humans and other higher animal even at low concentrations.
39
Hg is regarded as one of the most toxic heavy metals found in the environmental
40
conditions including lithosphere, hydrosphere, atmosphere, and biosphere. The main
41
anthropogenic sources of Hg contamination are urban discharge, agricultural materials,
42
mining, combustion and industrial discharges in the environment (Deng et al., 2001, 2011).
43
Pb is also an important environmental contamination because of anthropogenic sources and
44
natural geochemical processes (Dursun et al., 2003). It is widely applied in many important 2
45
industrial applications. Cd is one of heavy metals found in industrial effluents like metal
46
plating, mining, metallurgical alloying, ceramics and so on (Hutton et al., 1987; Nriagu and
47
Pacyna, 1988). Therefore, it has been a potential challenge for how to remove these heavy
48
metal contaminants from the environment, especially for aquatic eco-system.
49
Many technologies have been used to reduce or eliminate heavy metals from waste waters.
50
Among these methods, bioremediation has gained the great attention because it is a more
51
cost-effective and environment-friendly method with less toxic sludge compared with
52
conventional methods (Aryal et al., 2015). Photosynthetic bacteria (PSB) has been thought to
53
be an ideal candidate for bioremediation and has better environmental adaptability than other
54
microorganisms because it can change the metabolic type flexibly with environment change,
55
and use solar radiation as their unique energy source (Giotta et al., 2006; Gadd, 2009; De
56
Philippis et al., 2011). Rhodobacter (R.) sphaeroides, as purple non-sulphur bacterium, has
57
been shown to possess a remarkable ability in growing under aerobic or anaerobic respiration,
58
anoxygenic photosynthesis, and fermentation (Schultz and Weaver, 1982; Kiley and Kaplan,
59
1988; Calvano et al., 2014). Many previous studies have indicated that R. sphaeroides has
60
high tolerance to various heavy metal pollution (Giotta et al., 2006; Panwichian et al., 2011)
61
and thus has been widely used for wastewater treatment to remove heavy metals. Studies
62
about heavy metal removal of R. sphaeroides have been investigated in wastewater and soil
63
polluted by heavy metals including chromate reduction (Nepple et al., 2000), Cd removal
64
(Watanabe et al., 2003; Bai et al., 2008), removal of Cd and zinc (Li et al., 2016a; Peng et al.,
65
2018), and bioremediation of Pb (Li et al., 2016b).
66
It has been known that PSB in the photosynthetic systems and structure is different with 3
67
green plants. Plants usually response to different heavy metal stresses by membrane system
68
(Kreslavski et al., 2017), osmotic regulation substances, and antioxidant defense system
69
(Chen et al., 2015). However, the detailed mechanism is still unclear in ROS accumulation
70
and antioxidant system in PSB under heavy metal stress. Although R. sphaeroides has been
71
used for investigating its removal ability to heavy metals, the study about the removal
72
efficiency and oxidative damage by comparing ROS accumulation and antioxidant defense
73
systems has rarely been reported in PSB under heavy metal stress.
74
Our recent study indicated that R. sphaeroides SC01 showed high Cr resistance and could
75
probably be applied in Cr removal in industrial effluents (Su et al., 2017). To explore the
76
biosprption capacity and mechanism for heavy metals in R. sphaeroides SC01, we compared
77
the growth and structures of cells, the oxidative damage, antioxidant defense system and
78
removal efficiency under three heavy metals stresses. In addition, we further investigated the
79
tolerance to Pb, the changes in functional groups in details and the composition of
80
bioremediation products under Pb stress in SC01. Our results will provide the important
81
theoretical and practical value for the application of SC01 in heavy metal removal in the
82
environment.
83 84
2. Materials and methods
85
2.1. Strain, cultivation and culture conditions
86
The experiments were performed with SC01, which was isolated from the water collected
87
from saline paddy fields in Sichuan province, China. According to our previous study (Su et
88
al., 2017), SC01 was identified as SC01 is deposited at the GDMCC Culture Collection, and 4
89
its collection number is GDMCC1.1264. SC01 was cultured with purple non-sulfur bacteria
90
enriched medium (NH4Cl 1.0 g, NaHCO3 1.0 g, yeast extract 2.0 g CH3COONa 3.0 g,
91
K2HPO4 0.5 g, MgCl2·6H2O 0.2 g, NaCl 5.0 g, CaCl2·2H2O 0.075 g, ferric citrate 0.01 g) in
92
our laboratory. The pH of medium was kept at 7.0 before autoclaving (Su et al., 2017). The
93
strain was grown anaerobically at a light intensity of about 4000 lux given with incandescent
94
lamp at 35 °C. Before heavy metal stress, the same cell density at 680 nm (OD680) was
95
measured by spectrophotometer (Hitachi-U2000, Hitachi, Ltd., Tokyo, Japan). Then, cells of
96
stationary phases were used in different experiments. All other conditions were the same in
97
the illuminating incubator.
98 99
2.2. Heavy metal treatments
100
Different concentrations of Hg2+ (0, 0.2, and 1 mg L-1), Pb2+ (0, 30, and 160 mg L-1) and
101
Cd2+(0, 0.8, and 6 mg L-1) were prepared by diluted different amounts of HgCl2, Pb(NO3)2,
102
and CdCl2 solutions with deionized water. Then, SC01 with the same concentrations at
103
stationary phase was added to the aqueous solutions for different heavy metal stresses. The
104
stress experiments were performed at the same growth condition for 7 d. Cell concentration
105
was recorded daily for the growth curve of SC01.
106 107
2.3. Removal rate of Hg, Pb, and Cd
108
Experiments were performed with 250 mL of reaction mixtures containing different
109
concentrations of Hg2+, Pb2+ and Cd2+ for removal rate analysis in 250 mL flasks. Then, the
110
strain at stationary phase was exposed to the polluted aqueous solution for 7 d in the 5
111
controlled conditions. After 7 d, 50 mL of SC01 solutions was sampled from each flask and
112
then centrifugated at 5,000 rpm for 15 min. After centrifugation, heavy metal concentrations
113
in the supernatant were determined by an inductively coupled plasma mass spectrometry
114
(ICP-MS) (Optimal 2100DV, Perkin Elmer Instruments, Waltham, MA, USA). Three standard
115
curves (Fig. S1) were obtained using 100 PPM, 1 PPM, and 1 PPM standard solution of Hg,
116
Pb, and Cd in 1% HNO3 (Sigma, Co. Ltd. USA), respectively. The quality control of the
117
results was performed using a parallel analysis of Hg, Pb, and Cd national reference materials
118
for water testing (GSB 07-3173-2014, GSB 07-1183-2000, and GSB 07-1185-2000,
119
respectively). The percent of heavy metal removal was calculated using the equations:
120
removal rate = [(C0 - C) / C0] × 100, where C0 is the initial heavy metal concentration (mg L-1)
121
and C is the residual heavy metal concentration (mg L-1). The batch experiments were
122
conducted for triplicate.
123 124
2.4. Oxidative stress measurements
125
H2O2 accumulation in the strain SC01 under heavy metal stress (1 mg L-1 Hg, 160 mg L-1
126
Pb, and 6 mg L-1 Cd) was observed with the fluorescent probe 2, 7-dichlorofluorescin
127
diacetate (DCFH-DA) as described by Su et al. (2017). After heavy metal stress, cells were
128
infiltrated with PBS buffer containing 20 µM DCFH-DA (Sigma) for 40 min in water bath
129
with 37 °C in the dark under vacuum and rinsed at least three times with PBS buffer. Then,
130
the H2O2 imaging of the cells in vivo was visualized with a confocal laser scanning
131
microscopy (emission, 530 nm; excitation, 488 nm).
132
6
133
2.5. Propidium iodide staining
134
The cells were stained with propidium iodide (PI) stain according to the method of Su et
135
al. (2017). The collected cells by centrifugation were washed three times and resuspended
136
with 2 mL of 0.1 M PBS (pH 7.6). Then, SC01 cell suspension (1 mL) was mixed with 0.5
137
µg/mL PI solution (Sigma) gently. The mixture was placed in the dark for 10 min at room
138
temperature and then were rinsed with PBS buffer for three times. Finally, the stained SC01
139
cells were resuspended with PBS buffer (0.1 mL) and subsequently observed immediately
140
with a fluorescence microscope (BX-53 System, Olympus Corporation, Tokyo, Japan) with an
141
excitation wavelength of 546 nm.
142 143
2.6. Cell characterization analysis under Pb stress
144
To describe the characteristics of the bioremediation products, the cells of SC01 were
145
collected before and after 160 mg L-1 Pb stress by centrifugation (8,000 rpm for 10 min).
146
Then, the precipitates were washed two times with ultrapure water and were lyophilized in a
147
vacuum freeze dryer (Scientz-10N, Scientz Biotechnology, Co. Ltd., Ningbo, China). The
148
cellular characteristics of the precipitates were analyzed by Fourier transform infrared FTIR
149
spectroscopy (Nicolet 6700, USA) and X-ray diffraction (XRD, AXIS Ultra DLD, Kratos Ltd.,
150
British) according to the previous method (Li et al., 2016b).
151
The collected precipitates were washed three times with 0.1 M PBS buffer (pH 7.0) and
152
then mixed well with 2.5% (w/v) glutaraldehyde. The mixture was fixed in the refrigerator at
153
4 °C overnight. The fixed cells were dehydrated with different concentration of ethanol (60,
154
70, 80, 90, and 100%) for 15 min. The precipitates were freeze-dried in a vacuum freeze dryer. 7
155
The cells were analyzed by scanning electron microscope (SEM) (JSM-7500F, Electron
156
Optics Laboratory Co. Ltd. Japan) and energy dispersive spectroscopy (SEM-EDS)
157
(JSM-7500F, Electron Optics Laboratory Co. Ltd. Japan) as described by Wang et al. (2001).
158 159
2.7. Statistical analysis
160
All experiments were performed for at least triplicates, and the given results represent as
161
the mean of three values ± standard deviations (SD). SPSS Statistics 19.0 software (IBM,
162
Chicago, IL, USA) and the Duncan’s multiple range test were used for the statistical analyses
163
of data. In the graphic representation, a different letter above the error bar was thought to be
164
significant when P < 0.05.
165 166
3. Results
167
3.1. Growth of R. sphaeroides SC01
168
The growth curve of SC01 was obtained according to biomass production under stressful
169
and non-stressful conditions daily (Fig. S2). No significant differences in biomass production
170
could be detected in the absence of Hg, Pb, and Cd. We found that the value of OD reached
171
the maximal value after 6 d in the control conditions. After 7 d, the value of OD declined
172
significantly compared with the control when three heavy metals were added into culture
173
medium. It is noteworthy that cell concentrations were significantly reduced by about 50%
174
under 1.0 mg L-1 Hg and 6.0 mg L-1 Cd stress, indicating that SC01 suffered significant
175
damage from Hg and Cd stress. In addition, we found that SC01 under Pb stress presented a
176
lower decrease in biomass than that under Hg and Cd stress, suggesting that SC01 suffered 8
177
less damage from Pb stress relative to Hg and Cd stress.
178
To further investigate the degree of heavy metal damage to SC01, the contents of
179
carotenoid and bacteriochlorophyll a were measured under heavy metal stress. The content of
180
carotenoid significantly decreased under Hg, Pb, and Cd stress (Fig. S3A-C). 1.0 mg L-1 Hg
181
treatment resulted in the largest decline in the production of carotenoid compared with Pb
182
(160 mg L-1) and Cd (6 mg L-1). Bacteriochlorophyll a content of SC01 under three heavy
183
metals stresses presented the same results with the growth (Fig. S3D). Compared with the
184
control, heavy metal treatments resulted in the significant decline in bacteriochlorophyll a
185
content. However, bacteriochlorophyll a show a lower decrease under Pb stress compared
186
with Hg and Cd stress.
187 188
3.2. Removal rate of Hg, Pb, and Cd
189
The concentrations of Hg, Pb, and Cd in SC01 were determined in the stationary phase
190
under three heavy metals stresses and shown within the linear range. The specific uptake of
191
three heavy metals presented significant differences in SC01 (Fig. 1). Compared with Hg and
192
Cd stress, Pb treatments with low and high concentrations resulted in the highest percent Pb
193
removal, reaching to 98%. However, the strain exhibited a significant difference in the
194
percent Hg and Pb removal under Hg and Cd stress. These results suggested that SC01 has the
195
best removal rate of Pb relative to Hg and Cd.
196 197 198
3.3. ROS accumulation of SC01 under heavy metal stress To investigate whether SC01 suffered more damage from Hg and Cd stress than Pb stress, 9
199
we measured the contents of the two major ROS species, H2O2 and O2˙¯, produced under three
200
heavy metals stresses (Fig. S4). Compared with the control, three heavy metals stresses
201
resulted in the significant increase in the levels of O2˙¯and H2O2, especially for higher
202
concentrations of heavy metal. In addition, compared with Pb stress, Hg and Cd stress led to
203
the higher accumulation of O2˙¯ and H2O2, especially Hg stress. Furthermore, the level of
204
H2O2 in SC01 was measured in situ by a H2O2-sensitive fluorescent probe (DCFH-DA),
205
which has been successfully used for H2O2 detection in our present study (Su et al., 2017) and
206
plants (Xu et al., 2012; Rico et al., 2013). As expect, the results of fluorescent staining in the
207
non-stressed and stressed strains were similar with the quantitative data of H2O2 (Fig. 2).
208
Compared with the control and the lower concentrations of heavy metal, SC01 accumulated
209
more H2O2 under higher concentrations of heavy metal. Relative to 1.0 mg L-1 Hg and 6.0 mg
210
L-1, 160 mg L-1 Pb resulted in a low accumulation of H2O2. These results suggested that SC01
211
suffered a lower oxidative damage from Pb stress relative to Hg and Cd stress.
212 213
3.4. Cell death under Hg, Pb, and Cd stress
214
To investigate the integrity of cell membrane under three heavy metals stresses, the degree
215
of cell death was measured by PI stains, which is a membrane-impermeable dye and can bind
216
to DNA through entering cells with damaged cell membranes (Williams et al., 1998). We
217
found that the results of PI staining were consistent with the quantitative results of O2˙¯ and
218
H2O2 in SC01 exposed to heavy metal stress (Fig. 3). Compared with the control, the cells of
219
SC01 under three heavy metals stresses presented a high intense red fluorescence, especially
220
at the higher concentration of Hg, Pb, and Cd. Similarly, Pb stress showed the weaker red 10
221
fluorescence compared with Hg and Cd stress. These results indicated that SC01 under Pb
222
stress received less cell death than Hg and Cd stress.
223 224
3.5. Enzymatic and non-enzymatic antioxidant activities under heavy metal stress
225
The activities of four important antioxidant enzymes in SC01 under three heavy metals
226
stresses showed in Fig. S5. Four enzymatic activities presented significant differences among
227
Hg, Pb, and Cd stresses. Compared with the control, three heavy metals stresses resulted in
228
the significant increase in the activities of POD, SOD, CAT, and APX in SC01. In addition,
229
the activities of the antioxidant enzymes (POD, SOD, CAT, and APX) under Pb stress were
230
significantly higher than that of Hg and Cd stress (Fig. S5). These results suggested that SC01
231
could maintain the relative high activities of antioxidant enzymes under Pb stress.
232
The activities of non-enzymatic antioxidants were further investigated in SC01 under
233
three heavy metals stresses (Fig. S6). The AsA and GSH contents significantly decreased,
234
whereas the concentrations of DHA and GSSG showed an obvious increase under three heavy
235
metals stresses compared with the control. The most significant decrease in the contents of
236
ASA and GSH was observed under Hg stress. In contrast, Pb stress led to a lower increase in
237
the contents of DHA and GSSG relative to Hg and Cd stress (Fig. S6 B and D).
238 239
3.6. Cell characterization under Pb stress
240
Based on the above-mentioned results, we proposed that SC01 probably has high
241
resistance to Pb among three heavy metals. In order to find out the resistant mechanisms, the
242
cell characterization was further investigated in SC01 under Pb stress. 11
243
To examine the morphological changes in the cells, SEM analysis was used in SC01
244
exposed to Pb. As shown in Fig. 4A, a slight shrinkage was observed on the cell surface under
245
the normal condition. However, SC01 cells showed a more obvious shrinkage and even a lot
246
of cell debris was observed under 160 mg L-1 Pb for 7 d, compared with the control (Fig. 4B).
247
Furthermore, SEM-EDS analyses indicated that the absorption peak of Pb was clearly
248
observed in Pb-treated SC01 compared with the control (Fig. 4C-D), suggesting that a large
249
amount of Pb was absorbed into the cells by the strain.
250
To further explore the possible interactions between Pb and surface functional groups,
251
FT-IR spectra of SC01 was recorded in the presence and absence of 160 mg L-1 Pb (Fig. 5).
252
Many functional groups on the cell surface were revealed by the pre-adsorption FT-IR spectra.
253
These bands or groups were determined according to the data of previous literatures (Table
254
S1). Although the nearly similar variation tendencies of the spectral curves were observed
255
between the control and Pb-stressed strain, there were some differences in the crests before
256
and after Pb treatment. For example, the band at 3300 cm-1 replaced the band at 3420 cm-1
257
after Pb treatment, which was attributed to the –OH and –NH groups..This difference of 120
258
cm-1 peak (∆) indicated the involvement of –OH and –NH groups in the biosorption process
259
in SC01 (Akar et al., 2005). The similar band shifted from 1080 to 1052 cm-1 indicated
260
involvement of the C–O stretching of carboxyl groups and the bending vibration band of the
261
hydroxyl groups (Tunali et al., 2006), or phosphate groups (symmetric stretching of > PO2 )
262
(Lodeiro et al., 2006; Gabr et al., 2008). Two observed increase in the peaks at 980 cm-1 and
263
535 cm-1 in Pb-treated SC01 could be attributed to an intensity between Pb ions and
264
N-containing bioligands (Kamnev et al., 1997; Akar et al., 2005). These results indicated –OH, 12
265
–NH and phosphoric groups were involved in binding with Pb ions.
266
To further identify Pb(II) compounds formed during the biosorption process, Pb-treated
267
and the control SC01 were scanned by XRD (Fig. 6). Compared with the control, the d-values
268
of the major lines were observed at 2.969 (2θ, 30.0922), which closely correspond to (100.00)
269
planes of Pb Phosphate Hydroxide (Pb10(PO4)6(OH)2, JCPDS card No.051-1648). The result
270
indicated that Pb Phosphate Hydroxide was formed during Pb biosorption by SC01.
271 272
4. Discussion
273
It is well known that heavy metal pollution is becoming one of the most severe
274
environmental and human health hazards. Among these heavy metals, Hg, Pb and Cd were
275
widely applied in many industries and also thought to be the important source of industrial
276
pollution (Orr et al., 2018). Biosorption of heavy metal has been one of the most promising
277
technologies. In recent years, it obtains a great deal of attention due to its potential application
278
in industry in the world (Li et al., 2016b). To explore the application of SC01 in biosorption
279
of heavy metals, we investigated the growth of SC01, the oxidative damage, and the removal
280
efficiency of Hg, Pb and Cd under these heavy metals stresses in the present experiment.
281
Furthermore, the mechanism of biosorption was also studied in SC01 under Pb stress.
282
The growth of PSB has been commonly used as a effective marker under heavy metal
283
stress. The results showed that biomass production of SC01 reached rapidly the maximal
284
values on 6th d under the control conditions, suggested that SC01 has an excellent growth in
285
the cultured medium. Many studies indicated that toxic ions or salt stress conditions can
286
decrease bacterial metabolism and lead to inhibition of growth (Sheng et al., 2005; Upadhyay 13
287
et al., 2011). In consistent with the finding, we found that the growth of SC01 showed a
288
obvious decline under heavy metal stress. In addition, the rapid decline in biomass under high
289
concentration of Hg was probably because this strain is more sensitive to Hg and Cd than Pb.
290
Many studies indicated that PSB usually have different removal rate to different heavy
291
metals under stressful conditions (Giotta et al., 2006; Aryal et al., 2015; Peng et al., 2018).
292
Our recent study showed that SC01 had a high removal efficiency of Cr under high
293
concentration of Cr (Su et al., 2017). In the present experiment, we found that SC01 also
294
exhibited a very high removal rate of Pb under Pb stress. The high removal efficiency of Pb in
295
SC01 was probably due to the high resistance to Pb. Therefore, the good growth and the high
296
removal efficiency of Pb under Pb stress suggested that SC01 has high Pb resistance and will
297
be suitable for the removal of Pb from industrial effluent.
298
Some studies reported that the decline of bacteriochlorophyll a and carotenoid content
299
was usually observed in algae or bacteria response to heavy metal stress (Feng et al., 2007;
300
Hou et al., 2016; Kalaji et al., 2016; Batool et al., 2017; Su et al., 2017). Consistent with these
301
studies, our results showed that the contents of carotenoid and bacteriochlorophyll a gradually
302
decreased with the increase in concentrations of three heavy metals, indicating that three
303
heavy metals could lead to severe damage to the photosystem of SC01. However, we found
304
that the content of bacteriochlorophyll a in SC01 under Pb stress showed a slow decline
305
compared with Hg and Cd stress, suggested that this strain had a high resistance to Pb.
306
It is well known that heavy metal stress can induce ROS generation, and subsequently
307
result in oxidative damage to microorganism (Mathew et al., 2011; Su et al., 2017) or plants
308
(Dixit et al., 2002; Panda and Choudhury, 2005). In consistent with these findings, our study 14
309
found that three heavy metals induced the excessive accumulation of ROS in this strain,
310
especially at high concentrations of Hg and Cd, implying that SC01 suffered severe oxidative
311
damage from high concentrations of ROS under Hg and Cd stress. Compared with Hg and Cd
312
stress, Pb stress resulted in the lower accumulation of ROS in SC01. The reason could be
313
because SC01 had a high resistance to Pb, which was in accordance with the finding obtained
314
from Cr stress (Su et al., 2017). In addition, the severe oxidative damage usually lead to the
315
obvious cell death in plants and PSB under environmental stresses (Su et al., 2017; Chen et al.,
316
2018). In the present study, we found that a large number of cell death was observed in SC01
317
exposed to heavy metal stress, especially at high concentrations of Hg. The results were in
318
agreement with the observed levels of ROS accumulation in the strain under three heavy
319
metals stresses. Therefore, these findings indicated that SC01 suffered less oxidative damage
320
by lowing the levels of ROS under Pb stress relative to Hg and Cd stress.
321
It has been known that many organisms including PSB are able to reduce or eliminate
322
effectively oxidative damage induced by ROS through a complex antioxidant defense system
323
under environmental stresses (Foyer and Shigeoka, 2011; Su et al., 2017). Some studies
324
indicated that activities of antioxidant enzymes enhanced in some plants under low or
325
moderate concentrations of heavy metal stress (Chen et al., 2015, 2018). Consistent with these
326
findings, our results showed that the activities of four antioxidant enzymes significantly
327
increased under three heavy metals stresses, suggesting that SC01 is able to alleviate the
328
oxidative damage through improvement of the activities of the antioxidant enzymatic system
329
under heavy metal stress. However, the activities of four antioxidant enzymes were markedly
330
higher under Pb stress than that of Hg and Cd stress in SC01. The reason may be because 15
331
SC01 has a high resistance to Pb and an excellent removal system.
332
In addition, the AsA-GSH cycle is also a key process in regulating the damage of ROS in
333
different organisms under environmental conditions. AsA and GSH were regarded as the most
334
abundant water soluble antioxidants in plants (Foyer and Shigeoka, 2011). Some studies
335
indicated that the contents of AsA and GSH decreased in plants and PSB under environmental
336
stresses (Su et al., 2017; Chen et al., 2018). In accordance with these reports, our results
337
showed that the levels of AsA and GSH were markedly inhibited in SC01 under three heavy
338
metals stresses. These findings suggested that heavy metal application inhibited the activities
339
of non-enzymatic antioxidants in SC01. In addition, we found that Pb stress resulted in higher
340
contents of AsA and GSH and lower concentrations of DHA and GSSH relative to Hg and Cd
341
stress, indicating that the ROS detoxification system is more effective in SC01 under Pb
342
stress.
343
The changes in cell morphology were usually occurred in microorganisms (fungi, algae,
344
bacteria, etc.) under heavy metal stress. In our study, the obvious membrane indentation or
345
shrinkage of cells were observed under Pb stress. The decline in the surface and volume
346
fraction of cells suggested that SC01 could prevent the harmful effects of Pb by trimming the
347
attachable cell surface in contrast to total cell plane (Neumann et al., 2005). In addition, the
348
dumbbell shaped cells may be a strategy for accumulating more Pb in SC01 under Pb stress
349
(Mohite et al., 2018). The results from EDS spectrum further confirmed the presence of Pb on
350
the surface of cells in SC01.
351
The main bioremediation mechanisms of heavy metal removal using microorganism
352
mainly contain two steps, which are defined as the biosorption and bioaccumulation processes 16
353
(Das et al., 2008; Panwichian et al., 2010; Aryal et al., 2015), respectively. It is the first step
354
that metal ions are absorbed to surface of cells by interactions between heavy metals and
355
functional groups (Li et al., 2016b). This process is a dynamic balance between adsorption
356
and desorption because heavy metal ions bound on the cell surface may be eluted by acids,
357
other ions or chelating agents (Das et al., 2008). A previous study indicated that carboxyl,
358
hydroxyl, carbonyl, amido, and phosphate groups were bound with Pb on the bacterial surface
359
under Pb stress (Bai et al., 2014). Consistently, our results demonstrated that some functional
360
groups including hydroxyl, amido and phosphate groups existed on the surface of cells and
361
were mainly responsible for Pb biosorption in SC01. Therefore, Pb ion could penetrate the
362
cell membrane and subsequently enter into the cell due to active biosorption in the second
363
step (Das et al., 2008; Italiano et al., 2009). After heavy metals entering the cell in the second
364
step, microorganisms would accumulate heavy metals in the cell by enrichment, and remove
365
heavy metals by bioprecipitation as different compounds. Some previous studies have
366
demonstrated
367
Pb10(PO4)6(OH)2, Pb5(PO4)3Cl, and Pb9(PO4)6 were formed during the bioaccumulation
368
process of Pb ion in microorganisms (Levinson and Mahler, 1998; Mire et al., 2004; Bai et al.,
369
2014). In the present experiment, our results showed that only Pb10(PO4)6(OH)2 was formed
370
during the bioaccumulation of Pb ion, suggesting that the strain SC01 could accumulate Pb
371
effectively through the formation of Pb10(PO4)6(OH)2 in the cells. A previous study from Pb
372
contaminated soil reported that the main mechanism for Pb removal is the precipitation
373
formation of inert compounds including lead sulfate and lead sulfide in R. sphaeroides (Li et
374
al., 2016b). However, our results were different from the previous findings from heavy metal
that
Pb
phosphate
compounds
17
including
PbHPO4,
Pb5(PO4)3OH,
375
removal using R. sphaeroides. The main reason may be related to different strains and the
376
concentrations of Pb.
377 378
5. Conclusions
379
In this study, we combined the oxidative damage and antioxidant defense system to
380
estimate the removal ability of SC01 to Hg, Pb and Cd under heavy metal stress. Based on
381
high removal efficiency of Pb, we further investigated the bioremediation mechanisms of Pb
382
in SC01 under Pb stress. Our results showed that Hg and Pb stress led to the highest and the
383
lowest oxidative stress in SC01 according to cell death and ROS accumulation, respectively.
384
In addition, we found that SC01 may be more effective in preventing the oxidative damage
385
caused by excessive ROS through activating the antioxidant defense system under Pb stress.
386
Furthermore, the removal rate of Pb in SC01 reached up to 98% under high concentrations of
387
Pb, which was higher than those of previous studies. Moreover, the results obtained from the
388
analyses of bioremediation mechanisms of Pb revealed that Pb ion could bind with hydroxyl,
389
amido and phosphate groups on the cell surface of SC01, and subsequently formed precipitate
390
of Pb Phosphate Hydroxide during the biosorption process. Based on these results, we
391
propose that SC01 probably has high resistance to Pb and high removal efficiency of Pb
392
through regulating the antioxidant system and the formation of precipitates of Pb Phosphate
393
Hydroxide under stressful conditions, respectively. However, further work is needed to
394
address the detailed bioremediation mechanisms of Pb in vivo and the removal efficiency of
395
Pb contained in industrial wastewaters in SC01.
396 18
397
Acknowledgements
398
This work was supported by the Sichuan Science and Technology Bureau (2018GZ0375;
399
2018TJPT0004), Chengdu Science and Technology Bureau Project (2017-GH02-00071-HZ,
400
2018-YF05-00738-SN), National Infrastructure of Natural Resources for Science and
401
Technology Program of China (NIMR-2018-8-1). We thank Ji-qiu Wen and Guang-zhong Liu
402
(Analytical & Testing Center, Sichuan University) for assistance with XRD analysis. We also
403
thank Dong Wang and Yi He (Analytical & Testing Center, Sichuan University) for SEM
404
image.
405 406
References
407
Akar, T., Tunali, S., Kiran, I., 2005. Botrytis cinerea as a new fungal biosorbent for removal
408 409 410
of Pb(II) from aqueous solutions. Biochem. Eng. J. 25, 227–235. Aryal, M., Liakopoulou-Kyriakides, M., 2015. Bioremoval of heavy metals by bacterial biomass. Environ. Monit. Assess. 187, 4173.
411
Bai, H.J., Zhang, Z.M., Yang, G.E., Li, B.Z., 2008. Bioremediation of cadmium by growing
412
Rhodobacter sphaeroides: kinetic characteristic and mechanism studies. Bioresour.
413
Technol. 99, 7716–7722.
414
Bai, J., Yang, X.H., Du, R.Y., Chen, Y.M., Wang, S.Z., Qiu, R.L., 2014. Biosorption
415
mechanisms involved in immobilization of soil Pb by Bacillus subtilis DBM in a
416
multi-metal-contaminated soil. J. Environ. Sci. 26, 2056–2064.
417
Batool, R., Tabassum, T., Ali, M., 2017. Evaluation of Cr (VI) remediation potential of
418
Eichornia sp in conjunction with chromium-resistant bacterial strains. Trop. J. Pharm. 19
419
Res. 16, 1005–1011.
420
Calvano, C.D., Italiano, F., Catucci, L., Agostiano, A., Cataldi, T.R.I., Palmisano, F., Trotta,
421
M., 2014. Erratum to: The lipidome of the photosynthetic bacterium Rhodobacter
422
sphaeroides R26 is affected by cobalt and chromate ions stress. Biometals. 28, 229.
423
Chen, Y.E., Cui, J.M., Yang, J.C., Zhang, Z.W., Yuan, M., Song, C., Yang, H., Liu, H.M.,
424
Wang, C.Q., Zhang, H.Y., Zeng, X.Y., Yuan, S., 2015. Biomonitoring heavy metal
425
contaminations by moss visible parameters. J. Hazard. Mater. 296, 201–209.
426
Chen, Y.E., Mao, H.T., Ma, J., Wu, N., Zhang, C.M., Su, Y.Q., Zhang, Z.W., Yuan, M., Zhang,
427
H.Y., Zeng, X.Y., Yuan, S., 2018. Biomonitoring chromium III or VI soluble pollution by
428
moss chlorophyll fluorescence. Chemosphere. 194, 220–228.
429 430
Das, N., Vimala, R., Karthika, P., 2008. Biosorption of heavy metals- an overview. Indian J of Biotechnol. 7, 159–169.
431
De Philippis, R., Colica, G., Micheletti, E., 2011. Exopolysaccharide-producing cyanobacteria
432
in heavy metal removal from water: molecular basis and practical applicability of the
433
biosorption process. Appl. Microbiol. Biotechnol. 92, 697–708.
434 435 436 437
Deng, X., Jia, P., 2011. Construction and characterization of a photosynthetic bacterium genetically engineered for Hg2+ uptake. Bioresour. Technol. 102, 3083–3088. Deng, X., Wilson, D.B., 2001. Bioaccumulation of mercury from wastewater by genetically engineered Escherichia coli. Appl. Microbiol. Biotechnol. 56, 276–279.
438
Dixit, V., Pandey, V., Shyam, R., 2002. Chromium ions inactivate electron transport and
439
enhance superoxide generation in vivo in pea (Pisum sativum L. cv. Azad) root
440
mitochondria. Plant Cell Environ. 25, 687–693. 20
441 442
Dursun, A.Y., Uslu, G., Cuci, Y., Aksu, Z., 2003. Bioaccumulation of copper(II), lead(II) and chromium (VI) by growing Aspergillus niger. Process Biochem. 38, 1647–1651.
443
Feng, Y.Z., Yu, Y.C., Wang, Y.M., Lin, X.G., 2007. Biosorption and bioreduction of trivalent
444
aurum by photosynthetic bacteria Rhodobacter capsulatus. Curr. Microbiol. 55,
445
402–408.
446 447
Foyer, C.H., Shigeoka, S., 2011. Understanding oxidative stress and antioxidant functions to enhance photosynthesis. Plant Physiol. 155, 93–100.
448
Gabr, R.M., Hassan, S.H.A., Shoreit, A.A.M., 2008. Biosorption of lead and nickel by living
449
and non-living cells of Pseudomonas aeruginosa ASU 6a. Int. Biodeterior. Biodegrad. 62,
450
195–203.
451
Gadd, G.M., 2009. Biosorption: critical review of scientific rationale, environmental
452
importance and significance for pollution treatment. J. Chem. Technol. Biotechnol. 84,
453
13–28.
454
Giotta, L., Agostiano, A., Italiano, F., Milano, F., Trotta, M., 2006. Heavy metal ion influence
455
on the photosynthetic growth of Rhodobacter sphaeroides. Chemosphere. 62,
456
1490–1499.
457
Hou, S.L., Shu, W.J., Tan, S., Zhao, L., Yin, P.H., 2016. Exploration of the antioxidant system
458
and photosynthetic system of a marine algicidal Bacillus and its effect on four harmful
459
algal bloom species. Can. J. Microbiol. 62, 49–59.
460
Hutton, M., Chaney, R.L., Krishna Murti, C.R., Olade, M.A., Page, A.L., 1987. Group report:
461
cadmium. In: Hutchinson, T.C., Meema, K.M. (Eds.), Lead, Mercury, Cadmium and
462
Arsenic in the Environment. Wiley, New York. 35–41. 21
463
Italiano, F., Buccolieri, A., Giotta, L., Agostiano, A., Valli, L., Milano, F., Trotta, M., 2009.
464
Response of the carotenoidless mutant Rhodobacter sphaeroides growing cells to cobalt
465
andnickel exposure. Int Biodeter Biodegr. 63 (7), 948–957.
466
Kalaji, H.M., Sytar, O., Brestic, M., Samborska, I.A., Cetner, M.D., Carpentier, C., 2016. Risk
467
assessment of urban lake water quality based on in-situ cyanobacterial and total
468
chlorophyll-a monitoring. Pol. J. Environ. Stud. 25, 655–661.
469
Kamnev, A.A., Ristić, M., Antonyuk, L.P., Chernyshev, A.V., Ignatov, V.V., 1997. Fourier
470
transform infrared spectroscopic study of intact cells of the nitrogen-fixing bacterium
471
Azospirillum brasilense. J. Mol. Struct. 408/409, 201–205.
472 473
Kiley, P.J., Kaplan, S., 1988. Molecular genetics of photosynthetic membrane biosynthesis in Rhodobacter sphaeroides. Microbiol. Rev. 52, 50–69.
474
Kreslavski, V.D., Brestic, M., Zharmukhamedov, S.K., Luybimov, V.Y., Lankin, A.V., Jajoo,
475
A., Allakhverdiev, S.I., 2017. Mechanisms of inhibitory effects of polycyclic aromatic
476
hydrocarbons in the photosynthetic primary processes in pea leaves and thylakoid
477
preparations. Plant Biol. 19, 683–688.
478 479
Levinson, H.S., Mahler, I., 1998. Phosphatase activity and lead resistance in Citrobacter freundii and Staphylococcus aureus. FEMS Microbiol. Lett. 161, 135–138.
480
Li, X.M., Peng, W.H., Jia, Y.Y., Lu, L., Fan, W.H., 2017. Removal of cadmium and zinc from
481
contaminated wastewater using Rhodobacter sphaeroides. Water Sci. Technol. 75,
482
2489–2498.
483 484
Li, X.M., Peng, W.H., Jia, Y.Y., Lu, L., Fan, W.H., 2016. Bioremediation of lead contaminated soil with Rhodobacter sphaeroides. Chemosphere. 156, 228–235. 22
485
Lodeiro, P., Barriada, J.L., Herrero, R., Sastre de Vicente, M.E., 2006. The marine macroalga
486
Cystoseira baccata as biosorbent for cadmium(II) and lead(II) removal: Kinetic and
487
equilibrium studies. Environ. Pollut. 142, 264–273.
488 489
Mathew, B.B., Tiwari, A., Jatawa, S.K., 2011. Free radicals and antioxidants: a review. J. Pharm. Pharmacol. 4, 4340–4343.
490
Mire, C.E., Tourjee, J.A., O’Brien, W.F., Ramanujachary, K.V., Hecht, G.B., 2004. Lead
491
precipitation by Vibrio harveyi: evidence for novel quorum-sensing interactions. Appl.
492
Environ. Microbiol. 70, 855–864.
493
Mohite, B.V., Koli, S.H., Patil, S.V., 2018. Heavy metal stress and its consequences on
494
exopolysaccharide (EPS)-producing Pantoea agglomerans. Appl. Biochem. Biotechnol.
495
186, 199–216.
496 497
Nepple, B.B., Kessi, J., Bachofen, R., 2000. Chromate reduction by Rhodobacter sphaeroides. J. Ind. Microbiol. Biotechnol. 25, 198–203.
498
Neumann, G., Veeranagouda, Y., Karegoudar, T.B., Sahin, Ö., Mäusezahl, I., Kabelitz, N.,
499
Kappelmeyer, U., Heipieper, H.J., 2005. Cells of Pseudomonas putida and Enterobacter
500
sp. adapt to toxic organic compounds by increasing their size. Extremophiles. 9,
501
163–168.
502 503
Nriagu, J.O., Pacyna, J.M., 1988. Quantitative assessment of worldwide contamination of air, water and soils by trace metals. Nature. 333, 134–139.
504
Orr, S.E., Barnes, M.C., George, H.S., Joshee, L., Jeon, B., Scircle, A., Black, O., Cizdziel,
505
J.V., Smith, B.E., Bridges, C.C., 2018. Exposure to mixtures of mercury, cadmium, lead,
506
and arsenic alters the disposition of single metals in tissues of Wistar rats. J. Toxicol. 23
507 508 509
Environ. Health Part A. 81, 1–11. Panda, S.K., Choudhury, S., 2005. Chromium stress in plants. Braz. J. Plant Physiol. 17, 95–102.
510
Panwichian, S., Kantachote, D., Wittayaweerasak, B., Mallavarapu, M., 2010. Factors
511
affecting immobilization of heavy metals by purple nonsulfur bacteria isolated from
512
contaminated shrimp ponds. World J. Microbiol. Biotechnol. 26, 2199–2210
513
Peng, W.H., Li, X.M., Song, J.X., Jiang, W., Liu, Y.Y., Fan, W.H., 2018. Bioremediation of
514
cadmium- and zinc-contaminated soil using Rhodobacter sphaeroides. Chemosphere.
515
197, 33–41.
516
Rico, C, Hong, J., Morales, M.I., Zhao, L.J., Barrios, A.C., Zhang, J.Y., Peralta-Videa, J.R.,
517
Gardea-Torresdey, J.L., 2013. Effect of cerium oxide nanoparticles on rice: a study
518
involving the antioxidant defense system and in vivo fluorescence imaging. Environ. Sci.
519
Technol. 47, 5635–5642.
520 521
Schultz, J.E., Weaver, P.F., 1982. Fermentation and anaerobic respiration by Rhodospirillum rubrum and Rhodopseudomonas capsulata. J. Bacteriol. 149, 181–190.
522
Sheng, G.P., Yu, H.Q., Yue, Z.B., 2005. Production of extracellular polymeric substances from
523
Rhodopseudomonas acidophila in the presence of toxic substances. Appl. Microbiol.
524
Biotechnol. 69, 216–222.
525
Su, Y.Q., Zhao, Y.J., Wu, N., Chen, Y.E., Zhang, W.J., Qiao, D.R., Cao, Y., 2017. Chromium
526
removal from solution by five photosynthetic bacteria isolates. Appl. Microbiol.
527
Biotechnol. 102, 1983–1995.
528
Tunali, S., Akar, T., Özcan, A.S., Kiran, I., Özcan, A., 2006. Equilibrium and kinetics of 24
529
biosorption of lead(II) from aqueous solutions by Cephalosporium aphidicola. Sep. Purif.
530
Technol. 47, 105–112.
531
Upadhyay, S.K., Singh, J.S., Singh, D.P., 2011. Exopolysaccharide-producing plant
532
growth-promoting rhizobacteria under salinity condition. Pedosphere. 21, 214–222.
533
Wang, C.L., Lum, A.M., Ozuna, S.C., Clark, D.S., Keasling, J.D., 2001. Aerobic sulfide
534
production and cadmium precipitation by Escherichia coli expressing the Treponema
535
denticola cysteine desulfhydrase gene. Appl. Microbiol. Biotechnol. 56, 425–430.
536
Watanabe, M., Kawahara, K., Sasaki, K., Noparatnaraporn, N., 2003. Biosorption of cadmium
537
ions using a photosynthetic bacterium, Rhodobacter sphaeroides S and a marine
538
photosynthetic bacterium, Rhodovulum sp. and their biosorption kinetics. J. Biosci.
539
Bioeng. 95, 374–378.
540
Williams, S.C., Hong, Y., Danavall, D.C.A., Howard-Jones, M.H., Gibson, D., Frischer, M.E.,
541
Verity, P.G., 1998. Distinguishing between living and nonliving bacteria: evaluation of
542
the vital stain propidium iodide and its combined use with molecular probes in aquatic
543
samples. J. Microbiol. Methods. 32, 225–236.
544
Xu, J., Zhu, Y.Y., Ge, Q., Li, Y.L., Sun, J.H., Zhang, Y., Liu, X.J., 2012. Comparative
545
physiological responses of Solanum nigrum and Solanum torvum to cadmium stress.
546
New Phytol. 196, 125–138.
547
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1
Figure legends
2
Fig. 1. The percent removal of Hg, Pb, and Cd in R. sphaeroides SC01 under heavy metal stress. The
3
data are showed as the mean ± SD of at least three independent measurements. Different letters above
4
bars mean significant difference (P < 0.05) among different treatments according to Duncan’s multiple
5
range test.
6 7
Fig. 2. In vivo imaging of H2O2 in R. sphaeroides SC01 under Hg, Pb, and Cd stress. The strain SC01
8
was stained with 20 µM of the fluorescent probe 2,7-dichlorofluorescin diacetate (DCFH-DA) as
9
described in the “Materials and methods” section. Bars = 400 µm.
10 11
Fig. 3. The integrity of plasma membrane in R. sphaeroides SC01 under Hg, Pb and Cd stress. The
12
strain was stained with 0.5 µg/mL of propidium iodide (PI) as described in the “Materials and
13
methods” section. Bars = 100 µm.
14 15
Fig. 4. Scanning electron micrograph (A and B) and energy dispersive spectroscopy (C and D) of R.
16
sphaeroides SC01 in the control and 160 mg/L Pb treatment. A and C represented the control. B and D
17
represented 160 mg/L Pb treatment.
18 19
Fig. 5. FTIR spectra of R. sphaeroides SC01 biomass in the control (black line) and 160 mg/L Pb
20
treatment (red line). Wavenumbers from 4000 to 400 cm-1 were showed.
21 22
Fig. 6. X-ray diffraction patterns of R. sphaeroides SC01 in the control (gray line) and 160 mg/L Pb
1
23
treatment (red line).
24
2
Highlights
Among three heavy metals, mercury (Hg) stress resulted in the severe oxidative damage in R. sphaeroides SC01.
The strain SC01 showed high removal efficiency of lead (Pb).
Under Pb stress, the formation of precipitates of Pb phosphate hydroxide helped to improve the resistance to Pb and the removal rates of Pb in R. sphaeroides SC01.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: