Conductive magnetite nanoparticles accelerate the microbial reductive dechlorination of trichloroethene by promoting interspecies electron transfer processes

Article abstract of DOI:10.1002/cssc.201200748

Playing your part: Conductive magnetite nanoparticles accelerate the microbial reductive dechlorination of trichloroethene (TCE), an ubiquitous and toxic subsurface contaminant. The stimulatory effect most likely results from the nanoparticles promoting t

Full text of DOI:10.1002/cssc.201200748

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DOI: 10.1002/cssc.201200748
Conductive Magnetite Nanoparticles Accelerate the
Microbial Reductive Dechlorination of Trichloroethene by
Promoting Interspecies Electron Transfer Processes
[a]
[a]
[a]
[b]
Federico Aulenta,* Simona Rossetti, Stefano Amalfitano, Mauro Majone, and
Valter Tandoi
[a]
Microbes thrive on the energy they gain and conserve by
moving” electrons from low-potential electron donors to
dizing acetate using a polarized graphite electrode (anode) as
[6]
“
direct extracellular electron acceptor, and an anaerobic mixed
culture (hereafter named “RD-culture”) previously shown to be
capable of dechlorinating TCE [to cis-dichloroethene (cis-DCE),
vinyl chloride (VC), and ethene] with a polarized graphite elec-
trode (cathode) serving as direct extracellular electron
higher-potential electron acceptors occurring in natural envi-
ronments. Many types of natural donors and acceptors are
freely diffusible gases or soluble species that are easily trans-
ported and metabolized within living cells. On the other hand,
certain microorganisms (often referred to as “electroactive” or
[7,8]
donor.
“
electrochemically-active”) are capable of respiring insoluble
To this aim, an initial set of batch experiments was conduct-
ed in which small samples of the two cultures (5 mL of RD-cul-
ture and 1 mL of MES-culture) were transferred into 120 mL
anaerobic serum bottles containing anaerobic mineral
electron donors or acceptors, including solid-state electrodes
[
1–3]
in microbial electrochemical systems (MESs).
To do this,
these electroactive bacteria employ various strategies, ranging
from the use of electron-transfer proteins located on the outer
membrane (e.g., cytochromes), conductive microbial appen-
dages (e.g., nanowires), to soluble redox shuttles (e.g., pyocya-
[9]
medium,
amended with acetate (1.5 mmol) and TCE
(0.03 mmol), and supplemented with either 15 mL of a filtered
(0.2 mm) suspension of magnetite nanoparticles or 15 mL of fil-
tered (0.2 mm) deionized water. The final liquid volume in the
bottles was 75 mL. Magnetite nanoparticles were prepared as
[
4]
nin).
Although electroactive microorganisms have found applica-
tions as electrocatalysts in a number of different bioelectro-
chemical processes (e.g., from bio-energy generation to
groundwater bioremediation), the ecological and evolutionary
bases of extracellular electron transfer (EET) remain poorly elu-
cidated, and practical strategies for boosting EET only margin-
ally explored. Recent studies have suggested that, in sedimen-
tary environments, microorganisms with EET capabilities may
take advantage of the electric currents running through con-
ductive minerals, which connect spatially segregated bio-geo-
[10]
described by Kang and colleagues. The filtered suspension
used in the batch experiments contained approximately 1.5ꢀ
7
10 particles per mL (Supporting Information, Figure S1), with
À1
a total Fe concentration of 0.16 mmolL (as determined by
ICP–MS). Flow cytometry revealed the presence of two domi-
nant morphotypes of individual particles in the filtered suspen-
sion, with average diameters of 95Æ7 nm and 119Æ8 nm.
Scanning electron microscopy (SEM; Figure S2) confirmed that
the diameter of particles was in the range 80–150 nm. Finally,
energy dispersive X-ray (EDX) spectra confirmed that the nano-
particles were abundant in iron and oxygen (Figure S2). Upon
setup, all of the serum bottles were incubated statically, in the
dark, at room temperature (21–258C).
[
5]
chemical redox processes.
Here, we evaluate the possibility that electrically conductive
magnetite (Fe O ) nanoparticles can enhance the reductive
3
4
dechlorination (RD) of trichloroethene (TCE), an ubiquitous
groundwater pollutant, by allowing electrons to be trans-
ferred—extracellularly—from acetate-oxidizing microorganisms
to TCE-dechlorinating microorganisms. More specifically, we ex-
plore if such an interspecies electron transfer (IET) can occur
between an anaerobic mixed culture (hereafter named “MES-
culture”) previously shown to be capable of anaerobically oxi-
As shown in Figure 1, regardless of the presence of magnet-
ite nanoparticles, TCE was dechlorinated to cis-DCE and,
though to a lower extent, VC. This dechlorination pathway is
consistent with Desulfitobacterium spp. (a bacterial group capa-
[11]
ble of dechlorinating TCE to cis-DCE ) and Dehalococcoides
spp. (a bacterial group capable of dechlorinating TCE to VC
[12]
and ethene ) being the dominating members of the RD-cul-
ture. Even though at the end of incubation (day 38) the extent
of dechlorination was nearly the same (Figure 2a), bottles con-
taining magnetite nanoparticles exhibited a 2.3-fold higher
rate of TCE dechlorination (Figure 2b). Although a greater
stimulatory effect was observed on cis-DCE formation, VC for-
mation was positively influenced by the presence of conduc-
tive nanoparticles (Figure 2b, insert), also, clearly indicating the
occurrence of specific interaction(s) between Dehalococcoides
spp. and magnetite. Importantly, abiotic control experiments
carried out in the presence of magnetite nanoparticles allowed
[
a] Dr. F. Aulenta, Dr. S. Rossetti, Dr. S. Amalfitano, Dr. V. Tandoi
Water Research Institute
National Research Council (IRSA-CNR)
via Salaria km 29.300, 00015 Monterotondo (RM) (Italy)
Fax: (+39)0690672787
E-mail: aulenta@irsa.cnr.it
[b] Prof. M. Majone
Department of Chemistry
Sapienza University of Rome
Piazzale Aldo Moro 5, 00185 Rome (Italy)
Supporting Information for this article is available on the WWW under
http://dx.doi.org/10.1002/cssc.201200748.
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À1
Figure 1. Time course of TCE dechlorination with acetate (20 mmolL ) as
electron donor in anaerobic serum bottles a) supplemented, or b) not sup-
plemented with conductive magnetite nanoparticles. Error bars (when larger
than symbols) represent Æ1 standard deviation of replicated experiments.
^
: TCE,
&
: cis-DCE, ~: VC.
Figure 2. a) Cumulative respiratory electrons channeled to TCE dechlorina-
tion, and b) maximum rates of TCE dechlorination for the batch experiments
depicted in Figure 1. The effect of magnetite nanoparticles on the maximum
rate of VC formation is highlighted as an insert in (b). Error bars (when
larger than symbols) represent Æ1 standard deviation of replicated experi-
to rule out that the stimulatory effect was due to the particles
directly catalyzing the reductive dechlorination of TCE (Fig-
ure 2a and b).
ments.
&
:+magnetite, ^: Àmagnetite, ~:+magnetite (abiotic).
Even though the magnetite nanoparticles were added to
À1
the bottles in relatively small amounts (<10 mgL as total
II
III
iron), the redox cycling of soluble iron species [Fe /Fe ] possi-
bly produced from their dissolution could have contributed to
IET between the MES-culture and the RD-culture. To verify this
possibility a batch experiment was conducted in which an in-
oculum consisting of the MES-culture and the RD-culture was
supplemented with either a filtered suspension of magnetite
periments was conducted. These new experiments included
four different treatments (A–D), as depicted in Figure 3. When
anaerobic serum bottles (total volume 75 mL) were incubated
with the RD-culture (2 mL to a final liquid volume of 37 mL)
and magnetite nanoparticles (2 mL), but in the absence of the
MES-culture (treatment B), the rate of TCE dechlorination (in
À1
III
À1
nanoparticles (0.16 mmolL
as total iron), soluble Fe
the presence of 20 mmolL of acetate) was nearly indistin-
À1
(
(
(
0.16 mmolL ferric chloride), or no iron-based amendments
guishable from that observed in bottles containing only the
RD-culture (treatment A; Figure 3). This trend did not change
after the bottles were subjected to a second feeding cycle (i.e.,
a new spike of TCE after the first one had been completely
consumed; Figure 3). More specifically, even though the rates
of TCE dechlorination during the second feeding cycle were all
substantially higher than those observed during the first feed-
ing cycle, likely due to the growth of dechlorinating (and non-
dechlorinating) microorganisms, the increase was nearly identi-
cal for treatments A and B. This result confirmed that the sus-
pension of nanoparticles was not capable to directly stimulate
the metabolism of dechlorinating microorganisms, for example
by providing some key (trace) elements.
control test) in the presence of TCE (0.03 mmol) and acetate
1.5 mmol) (Figure S3). The rate of TCE dechlorination in the
III
presence of soluble Fe was very similar to that observed in
the control tests, and substantially lower (by a factor of ca. 2)
than that observed in the presence of magnetite nanoparticles.
These results are a clear indication that soluble iron species
did not serve as electron shuttles in IET between members of
the two cultures.
Alternatively, to verify the hypothesis that magnetite nano-
particles served primarily as “conduits” of electrons between
dechlorinating (i.e., the RD-culture) and non-dechlorinating
(
i.e., the MES-culture) microorganisms, a new set of batch ex-
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As shown in Figure 3, the most striking increase in the rate
of TCE dechlorination (a factor of 1.4 in the first feeding cycle
and 1.6 in the second feeding cycle) was noticed when the
RD-culture, the MES-culture, and the magnetite nanoparticles
were simultaneously present in the serum bottle. This finding
is consistent with the hypothesis that conductive nanoparticles
wire up acetate-oxidizing bacteria to TCE-dechlorinating bacte-
ria, resulting in more efficient IET and, in turn, cooperative me-
tabolism (Figure 4). TCE dechlorination was negligible in con-
trol experiments in the presence of the RD-culture, the MES-in-
oculum, and the magnetite nanoparticles, but in the absence
of acetate (data not shown), demonstrating that IET was ulti-
mately driven by catabolic acetate utilization. Similar results
were recently reported by Kato and colleagues, who observed
that supplementation of soil microbes with conductive nano-
particles (in the presence of an excess of carbon source) result-
ed in the acceleration of methanogenesis in terms of lag time
and production rate, while supplementation with an insulative
Figure 3. Effect of magnetite nanoparticles and the MES-culture on the max-
imum rate of TCE dechlorination by the RD-culture, in the presence of ace-
À1
tate (20 mmolL ). Error bars (when larger than symbols) represent Æ1 stan-
dard deviation of replicated experiments. Treatment A: RD-culture, treat-
ment B: RD-culture + magnetite, treatment C: RD-culture + MES-culture,
treatment D: RD-culture + MES-culture + magnetite.
nd feeding cycle.
&
: 1st feeding cycle, :
&
[20]
2
iron oxide did not. Since the presence of conductive nano-
particles stimulated the growth of Geobacter species, the au-
thors suggested that this microorganism grew under syntro-
phic association with methanogens, and IET could occur via
electric currents flowing through the conductive minerals. In
a very elegant co-culture investigation, the same research
group reported that magnetite nanoparticles facilitated IET
from Geobacter sulfurreducens to Thiobacillus denitrificans, ac-
Bottles which contained both the RD-culture (2 mL) and the
MES-culture (2 mL) but not magnetite (treatment C), displayed
slightly higher, though not statistically different, dechlorination
rates (by a factor of 1.1 to 1.2) compared to bottles containing
the RD-culture only (Figure 3). In principle, this finding could
indicate the existence of a cooperative metabolism between
microorganisms in the RD-culture and those in the MES-cul-
ture, resulting in a (although limited) stimulatory effect on the
reductive dechlorination of TCE. Considering that the MES-cul-
ture had not been previously exposed to TCE and did not ex-
hibit any dechlorinating activity (data not shown), it is possible
that the stimulatory effect resulted from a direct electron ex-
change between microorganisms in the MES-culture and the
RD-culture, taking place via conductive biological appendages
[21]
complishing acetate oxidation coupled to nitrate reduction.
[
13,14]
(e.g., extracellular cytochromes and nanowires).
Such a direct (i.e., microbe-to-microbe) IET mechanism has
been recently documented in co-cultures of Geobacter species
with other “electroactive” microorganisms. As an example, eth-
anol oxidation coupled to fumarate reduction, by an adaptively
evolved co-culture of G. metallireducens and G. sulfurreducens
was found to proceed via direct IET, rather than via diffusion of
[
15]
reduced carriers such as H or formate. Interestingly, the co-
2
culture formed microbial aggregates exhibiting a remarkable
electrical conductivity, further supporting the occurrence of
direct IET. Direct IET also appeared as an important process in
multi-species aggregates from a methanogenic digester in
Figure 4. Conceptual illustration of the proposed interspecies electron trans-
fer between dechlorinating and non-dechlorinating microorganisms,
through conductive magnetite nanoparticles.
[16]
which Geobacter and Methanosaeta species predominated.
The occurrence of Geobacter spp. in the MES-culture used in
this study was probable. Indeed, the predominance of Geo-
bacter spp. in acetate-fed bioelectrochemical systems has been
documented in a large and ever-increasing number of publica-
In conclusion, this study confirms the hypothesis that mag-
netite nanoparticles accelerate the reductive transformation of
TCE by promoting cooperative metabolisms among dechlori-
nating and non-dechlorinating microorganisms, based on IET
processes. Considering that conductive minerals are ubiquitous
in subsurface environments, these processes are likely to occur
naturally at many contaminated sites and are expected to con-
tribute greatly to a wide number of geochemical cycles, also
involving the degradation of contaminants. Moreover, this
[
17–19]
tions.
Furthermore, a molecular characterization of the
MES-inoculum by fluorescent in situ hybridization (FISH) con-
firmed that d-proteobacteria (the phylum of Geobacter) were
the dominating members of the culture, accounting for about
50% of total bacteria.
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study raises the intriguing possibility that conductive magnet-
ite nanoparticles can be employed in engineered groundwater
2008 Program. We thank Antonella Di Battista and Katherine An-
tonia Kozar Barrios for their skilful assistance with the batch
experiments.
[
22]
remediation systems such as chemical or bio-electrochemi-
[
23]
cal permeable reactive barriers to improve the kinetics of
contaminants transformation. Finally, the application of mag-
netite nanoparticles is envisaged as a promising strategy to
boost microbe–electrode interactions and extracellular electron
transfer processes in the emerging bioelectrochemical appli-
cations.
Keywords: bioremediation
electron transfer · nanoparticles
·
bacteria
·
dechlorination
·
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1
Analytical methods: Volatile components (TCE, cis-DCE, VC,
ethene, methane) were quantified by injecting 100 mL of serum
bottle headspace (taken with a gas-tight syringe) into a Perkin–
Elmer GC 8500 gas chromatograph (2 mꢀ2 mm glass column
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2
À1
tector temperature 2608C).
Received: October 8, 2012
Revised: November 21, 2012
Published online on February 10, 2013
Acknowledgements
We gratefully acknowledge funding from the Italian Ministry of
Education, University and Research (MIUR) through the PRIN
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