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Microbial Transformation of Multi-walled Carbon Nanotubes by Mycobacterium vanbaalenii PYR-1 Yaqi You, Kamol K. Das, Huiyuan Guo, Che-Wei Chang, Maria Navas-Moreno, James W. Chan, Paul Verburg, Simon R. Poulson, Xilong Wang, Baoshan Xing, and Yu Yang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04523 • Publication Date (Web): 12 Jan 2017 Downloaded from http://pubs.acs.org on January 12, 2017
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Environmental Science & Technology
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Microbial Transformation of Multi-walled Carbon Nanotubes by Mycobacterium
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vanbaalenii PYR-1
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Yaqi You1*, Kamol Das1, Huiyuan Guo2, Che-Wei Chang3, Maria Navas-Moreno3, James W.
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Chan3, Paul Verburg4, Simon R. Poulson5, Xilong Wang6, Baoshan Xing2, Yu Yang1*
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Department of Civil and Environmental Engineering, University of Nevada Reno, MS258, 1664
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N. Virginia Street, Reno, Nevada 89557, USA; 2
Stockbridge School of Agriculture, University of Massachusetts Amherst, 410 Paige Laboratory,
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Amherst, MA 01003, USA; 3
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Center for Biophotonics, University of California Davis Medical Center, 2700 Stockton Blvd
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Suite 1400, Sacramento, CA 95817, USA; 4
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Department of Natural Resources and Environmental Science, University of Nevada Reno, MS186, 1664 N. Virginia Street, Reno, Nevada 89557, USA;
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Department of Geological Sciences & Engineering, University of Nevada Reno, MS172, 1664 N. Virginia Street, Reno, Nevada 89557, USA;
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Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences, Peking University, Beijing 100871, China.
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*To whom correspondence should be addressed:
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[email protected] or
[email protected]; phone: 775-682-6609.
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ABSTRACT
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Carbonaceous nanomaterials are widely used in industry and consumer products, but
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concerns have been raised regarding their release into the environment and subsequent impacts
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on ecosystems and human health. Although many efforts have been devoted to understanding the
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environmental fate of carbonaceous nanomaterials, information about their microbial
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transformation is still rare. In this study, we found that within 1 month a polycyclic aromatic
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hydrocarbon-degrading bacterium, Mycobacterium vanbaalenii PYR-1, was able to degrade both
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pristine and carboxyl-functionalized multi-walled carbon nanotubes (p-MWCNT and c-
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MWCNT), as demonstrated by consistent results from high resolution transmission electron
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microscopy, Raman spectroscopy, and confocal Raman microspectroscopy. Statistical analysis of
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Raman spectra identified significant increase in the density of disordered or amorphous carbon in
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p-MWCNT and c-MWCNT after biodegradation. Microbial respiration further suggested
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potential mineralization of MWCNTs within about 1 month. All of our analyses consistently
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showed higher degradation or mineralization of c-MWCNT compared to p-MWCNT. These
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results highlight the potential of using bacteria in engineered systems to remove residual
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carbonaceous nanomaterials and reduce risk of human exposure and environmental impact.
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Meanwhile, our finding suggests possible transformation of carbonaceous nanomaterials by
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polycyclic aromatic hydrocarbon-degrading bacteria in the natural environment, which should be
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accounted for in predicting environmental fate of these emerging contaminants and in
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nanotechnology risk regulation.
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INTRODUCTION
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Due to their unique physicochemical properties, carbon nanotubes (CNTs) have attracted
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worldwide commercial interests in diverse applications for both industry and consumer
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products.1-2 Current global production capacity of CNTs has surpassed 5,000,000 kg per year,
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and is increasing by about 500,000 kg annually.2 Such rapid growth in production and
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widespread use of CNTs has raised concerns about their impact on human and environmental
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health following their release into the environment.3-6
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Although many efforts have been made to understand their environmental fate,
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information about biotransformation of CNTs in the environment is still scarce.7 Several studies
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have reported in vitro transformation of single-walled carbon nanotubes (SWCNTs) and multi-
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walled carbon nanotubes (MWCNTs), catalyzed by plant-derived horseradish peroxidase,8-10
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manganese peroxidase from white-rot fungus,11 and human myeloperoxidase and eosinophil
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peroxidase.12,13 Given the extremely high abundance and diversity of microbes in the
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environment and their versatile metabolic potentials, microbial degradation of CNTs is very
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likely the major biotransformation process in the environment, and deserves more
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investigation.14 Recently, Zhang et al.15 reported mineralization of as much as 6.8% of
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MWCNTs within 7 days by a bacterial community through co-metabolism with extra carbon
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sources, and identified Burkholderia kururiensis, Delftia acidovorans and Stenotrophomonas
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maltophilia as three potential key players. Parks et al.16 reported that after 6 months, less than
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0.1% of SWCNTs were mineralized by the white-rot fungus Trametes versicolor or by
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microbiota from polychlorinated biphenyl-contaminated sediments or aerated wastewater
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treatment plant sludge. These dissimilar degradation rates warrant more efforts toward a better
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understanding of microbial transformation of CNTs.
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A promising group of candidate microbes for CNT biodegradation are bacteria capable of
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degrading polycyclic aromatic hydrocarbons (PAHs), which share similar structural skeletons
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with CNTs. Previous studies have unambiguously demonstrated that species of Mycobacterium
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can utilize PAHs as the sole source of carbon and energy.14,17 It is possible that those bacteria can
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also transform CNTs, and even utilize CNTs as their carbon and energy source. To address this
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question, we utilized a model PAH-degrading bacterium, Mycobacterium vanbaalenii PYR-1,
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and studied its interaction with pristine (p-MWCNT) or carboxyl-functionalized MWCNT (c-
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MWCNT) under various incubation conditions. Multiple analytical methods, including
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transmission electron microscopy (TEM), Raman spectroscopy and confocal Raman
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microspectroscopy, were applied to characterize physicochemical changes in MWCNTs after
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reaction with this bacterium. Potential mineralization of MWCNTs was investigated using a
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variety of conditions. By comparing results from p-MWCNT and c-MWCNT, we explored the
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role of surface functionalization in biodegradation of MWCNTs by PYR-1.
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MATERRIALS AND METHODS
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MWCNTs preparation
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Research-grade MWCNTs were used in this study: 1) pristine MWCNT (p-MWCNT)
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(Nanocyl NC3150); 2) carboxyl-functionalized MWCNT (c-MWCNT) (Nanocyl NC3151).
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According to the manufacturer (http://www.nanocyl.com/product/), both MWCNTs have an
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average diameter of 9.5 nm and an average length of 95.0%
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carbon and 80.0% carbon, 30 spectra.
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A point-scan confocal Raman system25,26 was utilized to construct Raman maps of D- and
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G-bands, from which the distribution of ID/IG ratios were calculated. Samples were prepared as
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aforementioned, and 20 µL aliquots were drop-casted on 70% ethanol-cleaned quartz cover slips
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(0.2 mm thick, Technical Glass Products) and air-dried overnight. A 785 nm diode laser beam
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(0.3 mW power) was used for excitation, with an acquisition time of 1 sec per spectrum. Each
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map was generated by scanning a 6 µm × 6 µm region, which resulted in 400 Raman spectra (1
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spectrum per 0.3 µm × 0.3 µm). For each sample, 3-6 regions were examined.
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Measurement of CO2 from different incubations
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We measured CO2 evolved from bacteria incubated with different external carbon
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sources. After M. vanbaalenii PYR-1 cultures (MSM + 1% glucose + 5 mM pyerne) reached a
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density of 109 cell/mL, they were harvested, washed three times with phosphate-buffered saline
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(pH 7.0), and re-suspended in autoclaved MilliQ water for respiration measurement. Respiration
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experiments were set up in duplicate or triplicate in 473 mL polypropylene jars, with each jar
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containing approximately 4×109 bacterial cells and 111.17 mL one of the following media: 1)
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MSM (pH 7.0), 2) MSM + 10 mg p-MWCNT, 3) MSM + 10 mg c-MWCNT, 4) MSM + 1%
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glucose and 5 mM pyrene + 1 mg p-MWCNT, 5) MSM + 1% glucose and 5 mM pyrene + 1 mg
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c-MWCNT. Medium (1) served as a negative control without external carbon source; medium
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(2) or (3) represented a condition with MWCNTs being the only external carbon source; medium
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(4) or (5) represented a positive control containing both energetic carbon sources and low-dose
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MWCNTs. Respiration jars were wrapped with aluminum foil and incubated aerobically at
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20−25°C for about 32 days. The jar lids were then tightened and sealed with parafilm to keep
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gas-tight; headspace CO2 level was continuously monitored for 3 days using a Vaisala GMP343
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probe that was inserted through the lid. CO2-based estimation of mineralization is detailed in the
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SI.
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Data analyses and statistics
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Bacterial growth kinetics was analyzed by fitting experimental data to the modified
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Logistic or Gompertz equations27 using R (3.2.4). Raman spectra were analyzed using R and
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MATLAB (more details in the SI). Statistical analyses were performed with R. Levene’s test was
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conducted to check homogeneity of variance, after which data were compared using ANOVA or
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Welch t-test, as well as Mann-Whitney U test.
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RESULTS AND DISCUSSION
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M. vanbaalenii PYR-1 growth
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For each medium condition and each concentration of both p- and c-MWCNT, growth
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trends from two independent batches were consistent (Figure S3). Contrast to other reports on
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CNT’s concentration-dependent cytotoxicity on microorganisms,22 the presence of either p-
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MWCNT or c-MWCNT stimulated M. vanbaalenii PYR-1 growth, when glucose alone or both
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glucose and pyrene served as the primary carbon source. For instance, after 312-h growth,
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bacterial cultures in the glucose medium containing 25 ppm p-MWCNT or c-MWCNT reached
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an OD600 reading 3.0 or 1.7 times that of cultures grown in the same medium without MWCNTs,
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respectively (Figure S3, A1 and B1). Similarly, after 312-h growth, bacterial cultures in the
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glucose plus pyrene medium containing 5 ppm p-MWCNT or c-MWCNT reached an OD600
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reading 1.4 or 1.6 times that of cultures grown in the same medium without MWCNTs,
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respectively (Figure S3, A1 and B1). The addition of MWCNTs triggered earlier exponential
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growth of bacterial cultures. Growth kinetics was fitted with the Logistic model (eq. 1) or the
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modified Logistic model incorporating population decline (eq. 2) that are commonly used for
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proliferating populations:27
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y=
y=
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A ⎧⎪ ⎡ 4µ ⎤⎫⎪ ⎨1+ exp⎢ m (λ −t)+2⎥⎬ ⎢⎣ A ⎥⎦⎪⎭ ⎪⎩
(eq. 1)
Aexp(−dt) ⎧⎪ ⎡ 4µ ⎤⎫⎪ ⎨1+ exp⎢ m (λ −t)+2⎥⎬ ⎢⎣ A ⎥⎦⎭⎪ ⎩⎪
(eq. 2)
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where A (-) is the maximal population size, µm (h-1) is the maximum specific growth rate, λ (h) is
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the lag time, and d (h-1) is the decline rate (fitted parameters shown in Table S2). Using cultures
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grown in each medium without MWCNTs as reference, we assessed the impact of MWCNTs on
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PYR-1’s lag time. As shown in Figure 1, the addition of 25 ppm p-MWCNT or c-MWCNT to
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the glucose medium resulted in a 46.20% or 39.05% decrease in the lag time, respectively.
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Similar patterns were observed for cultures in the glucose plus pyrene medium and for cultures
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exposed to MWCNTs at other concentrations (Figure 1 and Table S2). When PYR-1 was
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incubated without glucose or pyrene, no significant growth was observed within 2 months, no
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matter whether MWCNTs were present or not (Figure S3). These results indicate that MWCNTs
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could stimulate M. vanbaalenii PYR-1 growth when more labile carbon source(s) was present,
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but MWCNTs alone were insufficient to support population proliferation.
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Several recent studies have shown enhanced cellular growth of Escherichia coli,
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Staphylococcus aureus and Bacillus subtilis by 10 ppm SWCNTs or MWCNTs, and suggested
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that bacterial cells attached on CNT bundles could produce extracellular polymeric substances
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(EPS) and form biofilms mitigating CNT toxicity.28-30 Carbonaceous nanomaterials other than
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CNTs, such as graphene oxide, have also been reported to enhance bacterial growth through
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promoting cell attachment, proliferation and biofilm formation.31 EPS as a protective barrier was
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also reported for Shewanella oneidensis under the stress of metal nanoparticles.23 In this study,
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M. vanbaalenii PYR-1 cells aggregated with MWCNTs (Figure S4 and Figure S5) and formed
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biofilms encapsulating MWCNTs (Figure S6), a phenomenon previously observed for
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Salmonella exposed to SWCNT.22 Mycobacterial strains are known to attach and form biofilms
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on PAH particles with their extremely hydrophobic rigid cell envelope, and can modify their cell
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wall composition in response to carbon sources.32 We thus speculate that MWCNTs may induce
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biofilm formation by PYR-1 (SEM images shown in Figure S6) and consequently shorten the lag
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time of its exponential growth, although more research is needed. The small amount of
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amorphous carbon on the surface of MWCNTs (Table S1), although insufficient to serve as the
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sole carbon/energy source, might also contribute to the observed growth stimulation.
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Alternatively, MWCNTs can potentially up-regulate key genes involved in this bacterium’s
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growth. For example, MWCNTs are known to promote plant growth through up-regulating genes
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for cell division/cell wall formation and water transport.33 Similar mechanisms could have
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enhanced M. vanbaalenii PYR-1 growth (e.g., up-regulation of DNA metabolism shown in
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Figure S7), but to determine the exact mechanism needs further investigation. In any case, a
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majority of PYR-1 cells attached on MWCNTs remained alive with intact cellar integrity (green
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cells in Figure S5) and cell morphology (Figure S6), whereas losing cellular integrity is believed
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to be one major mechanism for CNT’s bacterial cytotoxicity.7,34
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Biotransformation of MWCNTs
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TEM imaging of initial p- and c-MWCNT revealed sharp-edge tubular structures and
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crystal lattices (10 layers on average according to HRTEM; Table S1), without substantial
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amorphous materials on the surface (Figure 2 and Figure S8; Table S1). The same morphology
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was observed for MWCNTs after abiotic incubation (Figure 2 and Figure S8). In contrast, a
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variety of drastic morphological changes were detected in both p-MWCNT and c-MWCNT
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incubated with M. vanbaalenii PYR-1 for 25 days, regardless of the growth medium (containing
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glucose or both glucose and pyrene). We observed shorter and thinner nanotubes and highly
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disordered tubular structure with kinks and bends (Figure S8, A3-D3), as well as nanotubes with
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broken ends (Figure S9). Moreover, we observed exfoliated graphitic flakes with substantial
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amorphous materials on the surface, likely residues from incomplete degradation of graphite
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lattices (Figure 2, A2, A5, B2, B5; Figure S8, A4-D4). These TEM observations indicated
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degradation of MWCNTs by PYR-1, likely through oxidization of crystal lattices and subsequent
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exfoliation of graphitic walls. Similar structural transformations were observed in degradation of
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CNTs by horseradish peroxidase, microglial cells, and a microbial consortium, where CTNs
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were broken down to shorter nanotubes or deformed through a layer-by-layer mode.9,15,35
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As another line of evidence for degradation, Raman spectra were analyzed for MWCNTs
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incubated with M. vanbaalenii PYR-1, and compared to initial and abiotically incubated
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MWCNTs. Incubations using the glucose medium (Figure 3 and Figure 4) or the glucose plus
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pyrene medium (Figure S10 and Figure S11) yielded similar results. As shown in Figure 3,
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Raman spectra were similar for initial p- and c-MWCNT, displaying two fingerprint peaks as the
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tangential-mode G-band (∼1580 cm-1) and the disorder-induced D-band (∼1340 cm-1).36 The
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intensity ratio of D-band to G-band (ID/IG) for p- and c-MWCNT was measured as 1.07±0.05
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(n=67) and 1.16±0.11 (n=48) (Figure 4), consistent with other report on the same MWCNTs.37
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Surface functionalization of p-MWCNT with carboxylic functional groups (Table S1) slightly
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increases the ID/IG value (p
