Palladium nanoparticles produced and dispersed by Caldicellulosiruptor saccharolyticus enhance the degradation of contaminants in water

Article abstract of DOI:10.1039/c4ra14991b

This study focused on examining the general applicability of coupling bio-palladium (Pd) nanoparticle generation and bio-H₂ produced by Caldicellulosiruptor saccharolyticus for wastewater treatment under extreme thermophilic conditions. Na₂PdCl₄ was added to cell cultures to achieve a final Pd concentration of 50 mg L^-1. Methyl orange (MO) and diatrizoate were chosen as the contaminants in water. In the cultures with, and without, Pd added, MO (100 mg L^-1) was degraded within 30 min and in over 6 h, respectively. Diatrizoate (20 mg L^-1) was degraded within 10 min in Pd-added cultures. However, no diatrizoate degradation happened without Pd addition. The degradation rates were correlated positively with dissolved hydrogen generated by C. saccharolyticus. Furthermore, the catalytic actions of Pd(0) nanoparticles and cells were distinguished during the degradation process. MO was degraded under the combined action of Pd(0) and hydrogenase. However, Pd(0) was the essential catalyst, and hydrogenase had no effect on the deiodination of diatrizoate within 20 min. Pd(0) particles were dispersed well by the cells of C. saccharolyticus and showed a better catalytic activity than Pd(0) formed without cells. Dissolved hydrogen produced by C. saccharolyticus should be the perfect reduction equivalent for Pd formation and for reducing degradation. Therefore, Pd should be added to C. saccharolyticus cultures to enhance the degradation of contaminants in water. This journal is

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Graphical Abstract

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Palladium nanoparticles produced and dispersed by Caldicellulosiruptor
saccharolyticus enhance the degradation of contaminants in water
Nan Shen1, Xiu-Yang Xia1, Yun Chen1, Hang Zheng2, Yong-Chen Zhong3, Raymond J.
Zeng1,*
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CAS Key Laboratory for Urban Pollutant Conversion, Department of Chemistry,
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University of Science and Technology of China, Hefei, Anhui 230026, People’s
Republic of China
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2 School of Earth Sciences, The University of Queensland, St Lucia 4072, Australia
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Jiansu Zhongheng Architectural D&R Institute Co. Ltd., Yangzhou, Jiangsu,
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People’s Republic of China
* Corresponding author. Tel.: +86 551 63600203, fax: +86 551 63600203, E-mail
address:rzeng@ustc.edu.cn
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Abstract
This study focused on examining the general applicability of coupling bio-palladium
(Pd) nanoparticle generation and bio-H2 produced by Caldicellulosiruptor
saccharolyticus for wastewater treatment under extreme thermophilic conditions.
Na2PdCl4 was added to cell cultures to achieve a final Pd concentration of 50 mg/L.
Methyl orange (MO) and diatrizoate were chosen as the contaminants in water. In the
cultures with, and without, Pd added, MO (100 mg/L) was degraded within 30 min
and in over 6 h, respectively. Diatrizoate (20 mg/L) was degraded within 10 min in
Pd-added cultures. However, no diatrizoate degradation happened without Pd addition.
The degradation rates were correlated positively with dissolved hydrogen generated
by C. saccharolyticus. Furthermore, the catalytic actions of Pd(0) nanoparticles and
cells were distinguished during the degradation process. MO was degraded under the
combined action of Pd(0) and hydrogenase. However, Pd(0) was the essential catalyst,
and hydrogenase had no effect on the deiodination of diatrizoate within 20 min. Pd(0)
particles were dispersed well by the cells of C. saccharolyticus and showed a better
catalytic activity than Pd(0) formed without cells. Dissolved hydrogen produced by C.
saccharolyticus should be the perfect reduction equivalent for Pd formation and for
reducing degradation. Therefore, Pd should be added to C. saccharolyticus cultures to
enhance the degradation of contaminants in water.
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1. Introduction
Nanoparticles (NPs) of Pd(0) have a high catalytic activity in a standard
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hydrogenation reaction . Conventional production methods of these NPs require the
use of a series of toxic and expensive chemical agents, such as NaBH4 and H2. Both
stabilizers and carrier materials are needed to prevent the particles from aggregating in
a solvent. These materials would be released and pollute the environment, resulting in
an overall increase in cost. However, without these materials, Pd(0) could be formed in
bulk instead of the nanoscale. It is known that bulk Pd(0) has a lower catalytic activity
compared to NPs 2. Nanopalladium catalysts can also be synthesized by the
precipitation of Pd on the surface of bacteria, leading to the production of biogenic Pd
nanoparticles 3. This synthesis process is considered a more ‘green’ or
environment-friendly, low-cost technique. Hence, there is growing interest in
synthesizing metal NPs by biological methods.
Bio-Pd(0) formation and hydrogenation reactions need the addition of an
external electron donor, such as hydrogen, in the biosystem. Hydrogen is consistently
identified as one of the most reactive electron donors. To avoid the safety concerns
with the use of hydrogen and to reduce the cost of synthesis, many investigators
prefer to use hydrogen produced by bacteria in situ. Thus, several strains of
hydrogen-producing bacteria were investigated in the reduction of Pd(II) or other
metals 4-6. Hennebel et al. studied how bacteria could be used to produce Pd(0) under
fermentative conditions. In situ hydrogen produced by bacteria was coupled to the
formation of Pd(0). Then Pd(0) was tested for the ability to dehalogenate the
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recalcitrant aqueous pollutants, diatrizoate and trichloroethylene 6. Those studies were
all under mesophilic conditions. Whether extreme thermophilic bacteria have the
same or better ability to reduce metals using in situ hydrogen is not known. How Pd(0)
particles are formed and act under the extreme thermophilic conditions has also not
been reported. Other studies have reported that the biochemical reaction rate of
anaerobic digestion is higher at high temperatures 7, 8. Thus, in this study, the
degradation rate of contaminants and the catalytic activities of Pd(0) particles under
extreme thermophilic conditions and mesophilic conditions were compared.
Caldicellulosiruptor saccharolyticus, isolated from thermal springs, is an
extreme thermophilic fermentation bacterium with an optimum growth temperature of
70 °C. C. saccharolyticus can use saccharides or polysaccharides for growth, and has
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high hydrogen yields (up to 3.5 mol H2/mol glucose) . More importantly, it contains
Ni-Fe hydrogenase bound to the cytoplasmic membrane 10. Ni-Fe hydrogenase is
involved in both the uptake and release of H2 11. When dissolved hydrogen is reduced
by Ni-Fe hydrogenase, the generated electrons could be transferred to other electron
acceptors, such as heavy metals. For example, the reduction of Pd by Escherichia coli
is catalyzed by three hydrogenases 12. Others have reported the extracellular iron
reduction of neutral red by E. coli, mediated by hydrogen and with the aid of a
hydrogenase 13. With these properties (hydrogen production and hydrogenase), C.
saccharolyticus is expected to produce Pd(0) from Pd(II).
In this study, the in situ application of hydrogen produced by C. saccharolyticus
was investigated under extreme thermophilic conditions. Methyl orange (MO) and
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diatrizoate were chosen as the contaminants in water. This study aimed at getting Pd(0)
out of Pd(II), and studying the catalysis in decolorization and deiodination. First, it
was verified that Pd(0) was formed under the action of in situ hydrogen produced by
C. saccharolyticus. Second, the effects of Pd(0) on enhancing decolorization and
deiodination were studied. Third, the catalysis of Pd(0) and hydrogenase in the
processes of decolorization and deiodination was distinguished. Furthermore, the role
of cells in dispersion was confirmed.
2. Materials and methods
2.1. Microorganism and growth medium
Pure culture of C. saccharolyticus (DSM 8903) was purchased from Deutsche
Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany) and
was cultivated in the DSM640-medium as described previously without supply of
cysteine, trypticase and FeCl3 14. Glucose was supplied as the carbon source.
2.2. Experimental setup
2.2.1. Batch experiment setup: with glucose
The experiments were conducted in 165-mL serum bottles with 60 mL DSM640
medium. After being purged with N2 gas to ensure anaerobic conditions, the serum
bottles were sealed with butyl rubber stoppers and aluminum caps, sterilized by
autoclaving under 105 °C for 20 min and then incubated at 70 °C. After reaching
70 °C, each bottle was inoculated with the microbial culture, which was in its
exponential growth phase, to a final optical density (OD620) of 0.03. Two groups of
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experiments, one with Pd(II) added and the other without Pd(II), were conducted at
the same time. The experiments were carried out in triplicate. Normally, a Na2PdCl4
stock solution (500 mg Pd/L) was used to achieve a final Pd(II) concentration of 50
mg/L. Then 100 mg/L of MO or 20 mg/L of diatrizoate were added into the two
groups, respectively. When Pd(II) was reduced to Pd(0) through the action of
hydrogen produced by C. saccharolyticus, the effect of Pd(0) was investigated.
2.2.2. Batch experiment setup: without glucose
To confirm the reducing action of hydrogen produced by C. saccharolyticus, and
distinguish the catalytic action of Pd(0) and cells, glucose was removed from the
medium. Hydrogen or nitrogen was bubbled into the DSM640 medium without
glucose. Thus, four groups of experiments were conducted at the same time: (1) 60%
H2 and Pd(II); (2) 60% H2, cells and Pd(II); (3) N2 and Pd(II); (4) N2, cells and Pd(II).
After autoclaving, 50 mg/L of Pd(II) was added into the serum bottles. Thirty minutes
later, cells in the logarithmic growth phase were centrifuged (6000×g for 8 min) and
collected, and then re-suspended into the medium.
The difference in the catalytic activity between Pd(0) particles produced with and
without cells was investigated in three groups: (1) H2 and Pd(0); (2) H2 and
inactivated cells; (3) H2, activated cells and Pd(0). The headspace contained 60% H2
and 40% N2. Harvested cells were washed with medium and added into the anaerobic
serum bottles until the OD620 value was 1.0.
2.3. Pd reduction and nanopalladium observation
The concentration of Pd(II) was determined by atomic absorption spectrometry
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(AAS, Shimadzu AA-360, Kyoto, Japan). Palladium particles produced by hydrogen
without cells were observed directly by scanning electron microscopy (SEM,
JSM-6700F, JEOL Co., Japan). The palladium particles produced in the presence of
cells were fixed in 5% glutaraldehyde for 12 h at 4 °C, and then dehydrated in
increasing concentrations of ethanol (30%, 50%, 70%, 80%, 95% and 100%) for 15
min in each, and freeze-dried. After the samples were prepared, they were observed
by SEM. Transmission electron microscopy combined with energy dispersive
spectrometry (TEM-EDS) analysis was also performed to observe the morphology of
the cells and particles, and their distribution.
2.4. Chemical analysis
Concentrations of residual MO and the intermediates of MO decolorization were
determined using a HPLC system (LC-1100, Agilent Inc., USA) equipped with a UV
detector and a Hypersil ODS C18 column. Methanol (solution A) and water with 0.1%
(v/v) acetic acid and 0.1% (w/v) ammonium acetate (solution B) were used as the
isocratic mobile phase. The gradient elution program was as follows: 5% (v/v)
solution A and 95% (v/v) solution B at 0 min; 20% solution A and 80% solution B at
15 min; 100% solution A at 30 min; 5% solution A and 95% solution B at 38 min. The
flow rate was 0.8 mL/min, the wavelength of the UV detector was set at 450 nm and
254 nm, and 20 µL of sample were injected for analysis.
The concentration of diatrizoate was monitored by HPLC (LC-1100, Agilent Inc.,
USA) equipped with a UV detector and a Hypersil ODS C18 column. Elution was
performed isocratically at 25 °C and at a flow rate of 1 mL/min with 5% solvent A
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(100% acetonitrile) and 95% solvent B (0.1% formic acid). The injection volume was
50 µL, and the elution program ran within 15 min.
Hydrogen was sampled by a gas-tight syringe (SGE Analytical Science, Australia)
and the hydrogen concentration was determined by a GC (Lunan model SP7890, CN)
equipped with a thermal conductivity detector and a 1.5-m stainless steel column
packed with a 5-Å molecular sieve. The temperatures of the injector, detector, and
column were kept at 80, 100, and 50 °C, respectively. N2 was used as the carrier gas.
3. Results and discussion
3.1. Pd nanoparticle formation
The reduction of Pd(II) and Pd(0) nanoparticle formation could be confirmed by
the results of AAS, TEM images and EDS (Fig. 1). The results of AAS showed that
Pd(II) disappeared completely from the solution (data not shown). The color change
of the medium, from pale yellow to black, indicated that Pd(0) nanoparticles formed
in the system. EDS analysis of the point marked in the TEM image indicated that Pd(0)
was indeed formed in this system. This result also illustrated that hydrogen, as a
reducing power produced by C. saccharolyticus, could reduce Pd(II) to Pd(0) via the
action of hydrogenase, as reported by others 6, 12, 15. Furthermore, the TEM image
illustrated that Pd(0) particles were formed and that the diameter of the Pd(0) particles
was around 10–20 nm. Most of the particles were distributed around the cells.
Initially, the Pd(II) ions spread over the cell culture including the extracellular
polymeric substances (EPS) of C. saccharolyticus. EPS are high-molecular-weight
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polymers that are secreted by microorganisms into the surrounding environment
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Many particles could hide in the EPS. Hydrogen produced by C. saccharolyticus
escaped from the cytomembrane, and then reached the EPS and the bulk solution.
Then Pd(0) formed with the aid of hydrogen. It is possible that the EPS facilitated the
dispersion of the Pd(0) particles. Thus the Pd(0) particles could disperse well without
the addition of a chemical dispersant. The EPS prevented the aggregation of the
Pd(0) particles. Conventional production methods of Pd(0) particles required the use
of a series of toxic or expensive chemical agents, such as stabilizers and reductant 6, 17
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Thus, our study has shown that EPS can act as an environmental bio-dispersant, and
that hydrogen is produced in situ by C. saccharolyticus.
3.2. Enhanced decolorization and dehalogenation
The in situ application of the Pd(0) particles and hydrogen produced by C.
saccharolyticus was investigated in cases of decolorization and dehalogenation. MO
(100 mg/L) was added into all the batches with and without Pd(0) particles. The rates
of decolorization with Pd(0) particles were higher than that without Pd(0) particles
(Fig. 2). MO was completely removed within 30 min in the group with Pd(0) particles.
However, more than 6 h were required to completely remove 100 mg/L of MO in the
group without Pd(0) particles. To eliminate the possible effect of adsorption by Pd(0)
particles, the intermediates of MO decolorization were determined.
4-aminobenzenesulfonic acid (4-ABA) and N’,N-dimethyl-p-phenylenediamine (DPD)
were produced as the intermediates during the decolorization of MO. The
concentrations of both 4-ABA and DPD increased with the decrease of MO (Fig. 2a,
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b). As shown in Fig. 2b, 100 mg/L of MO (0.30 mM) disappeared completely within
30 min, which corresponded to the stoichiometric appearance of 50 mg/L 4-ABA
(0.29 mM) and 40 mg/L DPD (0.29 mM). This result demonstrated that MO was
reduced rather than adsorbed by cells and Pd(0) particles.
The complete decolorization in the group without Pd(0) particles was realized
with the combined action of hydrogen and hydrogenase. This phenomenon was
confirmed in our previous study 18. In the group with Pd(0) particles, the complete
decolorization was the combined effects of hydrogen, hydrogenase and Pd(0) particles.
The nanopalladium enhanced the decolorization of MO. MO decolorization driven by
cells alone needed longer time as other researchers reported. For example, MO (100
mg/L) was completely removed within 7 h by S. oneidensis MR-1 19. A 95%
decolorization by P. luteola was observed at 6 h and 9 h for 100 and 350 mg/L MO,
respectively 20.
Pd(0) particles have been reported frequently as the catalyst of the reduction of
halides, such as trichloroethylene (TCE), chlorophenols and diatrizoate 21-24. This
study chose diatrizoate as the halide to test the catalytic activity of Pd(0) particles in
extreme thermophilic condition. As shown in Fig. 3, 20 mg/L of diatrizoate
disappeared within 10 min in the two groups with Pd(0) particles during different time
slot. However, in the group without Pd(0) particles diatrizoate was not reduced within
20 min. This result indicates that the catalytic activity of Pd(0) particles was essential
in the deiodination, whereas cells had no catalytic effect. Furthermore, the rate of
deiodination was slightly higher when the reaction of deiodination started at 20 h
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compared to 10 h. The time span, i.e. 10 and 20 h, was calculated from the inoculation
time. The hydrogen content in the headspace was 16 mL at 20 h and 10 mL at 10 h.
From the Michaelis-Menten model, it can be seen that the enzymatic catalytic reaction
rate is positively related to the concentrations of catalyst and substrate. In this study,
the catalyst was Pd(0) particles and the substrate was hydrogen. Thus, higher
concentrations of hydrogen accelerated the rate of deiodination.
The rate of deiodination observed in this present study was higher than that
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reported in a study of Citrobacter braakii , which was also able to reduce Pd(II) to
Pd(0) through hydrogen produced in situ under mesophilic conditions. The same
amount of Pd(II) (50 mg/L) was added into both systems. More hydrogen (1.31 mmol
vs. 0.6 mmol in our study) was produced under mesophilic conditions when the
reaction of deiodination started. However, it took over 30 min to remove 20 mg/L of
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diatrizoate in the study of C. braakii , whereas in the present study it took only 10
min. It is likely that temperature played an important role in enhancing the
deiodination process in our experiments. Thus, extreme thermophilic conditions
should be beneficial to deiodination. It is also possible that the extreme thermophilic
fermentation bacterium C. saccharolyticus performed better in coupling bio-Pd
nanoparticle generation and bio-H2 for the deiodination process than many mesophilic
bacteria reported previously.
3.3. Catalysis of hydrogenase and/or Pd(0)
In our previous study, we found that hydrogen could decolorize MO with the
action of hydrogenase 18. Pd(0) enhanced the degradation of MO and also had the
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catalytic activity of hydrogenation, as in the deiodination of diatrizoate. What role did
hydrogenase and Pd(0) play in, respectively, decolorization and deiodination? The
role of hydrogenase and Pd(0) in decolorization and deiodination was determined in
this study. Glucose was removed from the medium, and four groups of experiments
were conducted at the same time. As shown in Fig. 4a, MO (50 mg/L ) was removed
completely within 30 min in the group with H2, cells and Pd(0), as the concentration
of MO was measured only at 30 min. The disappeared MO (50 mg/L) corresponded to
the appearance of 29 mg/L 4-ABA and 23 mg/L DPD, indicating the completed
degradation of MO, rather than adsorption by Pd(0). However, only 25 mg/L of MO
was removed in the group with H2 and cells within 30 min. In the group with N2, cells
and Pd(II), only 10 mg/L of MO was removed, which was a result of adsorption. In
the group with N2 and Pd(II), there was almost no reduction of MO. Pd(II) could not
be reduced to Pd(0) in all the batches without hydrogen in our test. Thus, there was no
catalytic activity of Pd(0) in both groups without hydrogen. It was speculated that
about 20% MO was adsorbed by cells and 30% was catalyzed by hydrogenase, which
was comparable to the catalysis of Pd(0) and hydrogenase within 30 min. Thus, Pd(0)
greatly enhanced the decolorization of MO, and the catalytic activity of hydrogenase
and the adsorption of cells could be instead by the result of the catalytic effect of Pd(0)
particles, because the catalytic activity of Pd(0) particles was sufficiently powerful to
completely remove 50 mg/L of MO within 30 min.
Fig. 4b shows that 20 mg/L of diatrizoate was removed within 5 min in the group
with H2, cells and Pd(0). Diatrizoate was not degraded in the groups without Pd(0)
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formation. About 3 mg/L of diatrizoate was removed in the group with N2, cells and
Pd(II), resulted from adsorption to cells within 20 min. This phenomenon indicated
that Pd(0) was the essential catalyst, and that hydrogenase had no effect in the
deiodination process within 20 min. In other studies, diatrizoate could be effectively
degraded solely by Pd(0). For example, the removal of 20 mg/L diatrizoate by a 10
mg/L Pd suspension and 100% hydrogen was completed after 4 h 21. However, in our
study, 50 mg/L Pd and 60% hydrogen were added. The degradation rate was different,
as the degradation rate of diatrizoate was related positively to the dosage of Pd(0) and
hydrogen. Furthermore, the temperature were different in two studies. Compared with
the catalytic effect in the batches with glucose in the previous section (Fig. 3), it can
be seen that the catalytic effect was better in the batches without glucose in this
section, because the content of hydrogen in the headspace in the batches with glucose
was between 0.45 and 0.71 mM, and was around 2.68 mM in the batches without
glucose. Hydrogen was filled artificially in this section, whereas hydrogen was
produced by cells in the batches with glucose in the previous section. This could also
be explained by the Michaelis-Menten model 18.
3.4 Dispersive action of cells on Pd(0) particles
The difference in size and catalytic activity between Pd(0) particles produced
with and without cells was also investigated in this study. Cells were sterilized after
Pd reduction to eliminate the effect of hydrogenase. This design aimed to confirm the
dispersive action of cells. The Pd(0) particles aggregated together and formed a bulk
in the system without dispersant (Fig. 5a, b). The diameter of the Pd(0) particles
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formed under this condition (chemical-Pd(0)) was between 1 and 2 µm. By contrast,
in the system with the cells as a ‘green’ dispersant, the Pd(0) particles could be finely
dispersed (Fig. 5c, d). The diameter of most Pd(0) particles formed in the presence of
cells was under 100 nm. These Pd(0) particles were polyporous and homogeneous.
Previous research on EPS offered a sufficient theoretical foundation for this study 25-28
that is, the EPS of C. saccharolyticus could adsorb the Pd(0) particles and prevent
them from aggregating together. The Pd(0) particles in the system with cells had a
smaller size and a larger specific surface area compared with the Pd(0) particles in the
system without cells. Thus the Pd(0) particles in the system with cells should
theoretically have a higher catalytic activity. The following experiments with these
systems, i.e. with and without cells, provided an answer.
;
MO was chosen as the contaminant to verify the catalytic activity of the Pd(0)
particles in three groups. As shown in Fig. 6a, only 25 mg/L of MO was removed
within 15 min in the group with H2 and Pd(0) (Fig. 6a), and the tendency of the
intermediates is shown clearly in Fig. 6b, indicating the degradation of MO. Fig. 6c
shows that there were no intermediates detected in the group with H2 and cells. This
result indicates that reduced MO was not degraded but adsorbed by the cells. Not
more than 20 mg/L of MO was adsorbed by cells within 30 min, which indicates that
the adsorption by the cells played only a small role. However, 100 mg/L of MO was
removed completely within 15 min in the group with H2, cells and Pd(0), and the MO
was degraded to 4-ABA and DPD (Fig. 6d). These results confirmed that Pd(0)
particles formed in the group with cells had a higher catalytic activity in the
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decoloration of MO. The cells of C. saccharolyticus were ‘green’ dispersants in this
system.
4. Conclusions
Pd(0) particles were produced from Pd(II) with in situ hydrogen generated by C.
saccharolyticus during glucose fermentation. The degradation of MO and diatrizoate
were both enhanced by Pd addition. The removal of MO was the result of the
combined action of hydrogen, hydrogenase and Pd(0) particles. However, Pd(0)
particles played an essential role in the removal of diatrizoate. Furthermore, the Pd(0)
particles were well dispersed by cells of C. saccharolyticus and showed a better
catalytic activity than chemical Pd(0) without dispersant. Generally speaking, the
addition of Pd would enhance the degradation of contaminants in water.
Acknowledgements
This work was supported by the Natural Science Foundation of China (50978244,
51478447), the Hundred-Talent Program of Chinese Academy of Sciences, the
National Hi-Technology Development 863 Program of China (2011AA060901), the
Program for Changjiang Scholars and Innovative Research Team in University, the
Fundamental Research Funds for the Central Universities (wk2060190040)，and the
Collaborative Innovation Center of Suzhou Nano Science and Technology
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A list of figures
Fig. 1 a, b: TEM image of the Pd(0) particles and cells in C. saccharolyticus with
glucose supplied; c: EDS analysis of the point marked in the TEM image.
Fig. 2 Decolorization profiles of MO and metabolite formation, a: without Pd added;
b: with Pd added in cultures of C. saccharolyticus with glucose supplied.
Fig. 3 Removal of diatrizoate under different growth phase conditions: with and
without Pd added in cultures of C. saccharolyticus with glucose supplied.
Fig. 4 a: Decolorization profiles of MO; b: removal of diatrizoate under different
conditions in cultures of C. saccharolyticus without glucose supplied.
Fig. 5 SEM images of Pd nanoparticales formed under hydrogen supplied. a, b:
without the participation of cells, c, d: with the participation of cells
Fig. 6 a: Decolorization profiles of MO under different conditions, b, c, d: metabolite
formation under different conditions. b: H2+Pd (Pd particles formed with hydrogen
supplied only); c: H2+cell (hydrogen and inactivity cells); d: H2+Pd+cell (Pd particles
formed with cells and hydrogen supplied).
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Fig. 1 a, b: TEM image of the Pd(0) particles and cells in C. saccharolyticus with
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glucose supplied; c: EDS analysis of the point marked in the TEM image.
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Fig. 2 Decolorization profiles of MO and metabolite formation, a: without Pd added;
402
b: with Pd added in cultures of C. saccharolyticus with glucose supplied.
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Fig. 3 Removal of diatrizoate under different growth phase conditions: with and
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without Pd added in cultures of C. saccharolyticus with glucose supplied.
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Fig. 4 a: Decolorization profiles of MO; b: removal of diatrizoate under different
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conditions in cultures of C. saccharolyticus without glucose supplied.
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Fig. 5 SEM images of Pd nanoparticales formed under hydrogen supplied. a, b:
without the participation of cells, c, d: with the participation of cells
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c
b
d
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Fig. 6 a: Decolorization profiles of MO under different conditions, b, c, d: metabolite
formation under different conditions. b: H2+Pd (Pd particles formed with hydrogen
supplied only); c: H2+cell (hydrogen and inactivity cells); d: H2+Pd+cell (Pd particles
formed with cells and hydrogen supplied).
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