Grantee Research Project Results
2011 Progress Report: Photochemical and Fungal Transformations of Carbon Nanotubes in the Environment
EPA Grant Number: R834858Title: Photochemical and Fungal Transformations of Carbon Nanotubes in the Environment
Investigators: Jafvert, Chad T. , Fairbrother, D. Howard , Filley, Timothy
Institution: Purdue University , The Johns Hopkins University
EPA Project Officer: Hahn, Intaek
Project Period: August 15, 2010 through August 14, 2013
Project Period Covered by this Report: May 1, 2011 through May 1,2012
Project Amount: $600,000
RFA: Increasing Scientific Data on the Fate, Transport and Behavior of Engineered Nanomaterials in Selected Environmental and Biological Matrices (2010) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Objective:
We have proposed that due to their size, any transformation of carbon nanotubes (CNTs) in the environment is likely dominated by abiotic oxidative and extracellular microbial processes. Consequently, in this study we are investigating the photochemical and fungal mediated transformations that occur to colloidal and solid phase CNTs. Our goal is to identify transformation products, reaction kinetics, and reaction mechanisms, including the effects of coupled photochemical-fungal exposures.
Our objectives are based on two overarching hypotheses: (i) Photochemical and fungal transformations of CNTs will occur and proceed via oxidative processes with important consequences for their overall persistence in the environment, and (ii) that the rate of these reactions will depend on CNT physicochemical properties (e.g., surface properties), environmental conditions (e.g., pH, fungi type), and CNT form (e.g., colloids or immobilized in polymers).
Progress Summary:
Initiated under a previous EPA STAR grant and completed under this grant, we initially examined the production of reactive oxygen species (ROS) as a function of CNT functionalization. Commercially available carboxylated and PEG-functionalized single-walled CNTs were examined and compared to pristine (i.e., unfunctionalized) CNTs. Aqueous colloidal dispersions of both types of functionalized CNTs generated ROS, including singlet oxygen (1O2), superoxide anion (O2.-), and hydroxyl radicals (∙OH) in light within the solar spectrum (λ = 300-410 nm). With surfactant added as a dispersing agent, unfunctionalized SWCNTs produced no measurable ROS within 60 hrs of irradiation, whereas functionalized SWCNTs produced measurable ROS in less than 5 hrs, as detected using the same ROS scavengers.
This initial study indicated the need to further examine the possible role of carbon and metal impurities (added as catalysts for their preparation) in facilitating ROS production, as commercial production of truly pure CNTs (> 99.9%) appears both technically and economically infeasible at the current time.
In ongoing experiments, the types of CNTs investigated include both pristine SWCNT from Carbon Solutions (AP and P2) and from SouthWest Nano Technologies (SG-65); and carboxylated (P3-COOH) and PEG-functionalized SWNTs from Carbon Solutions (P3 and P7, respectively). Also, SG-65 nanotubes that have been functionalized by acid treatment and further purified to remove metal catalysts (used in their preparation) are being examined.
Upon irradiating dispersions of these CNTs with eight black-light phosphor lamps (RPR-3500 (λ = 300-410 nm), the production and concentrations of 1O2, O2-∙, and ∙OH have been quantified using FFA (furfuryl alcohol), XTT (3'-{1-phen ylamino-carbonyl-3,4-tetrazolium}bis(4-methoxy-6-nitro)-benzenesulfonic acid hydrate), and p-chlorobenzoic acid (pCBA), respectively, as ROS scavengers. While FFA decay (associated with 1O2 production) occurred in dispersions of P3-COOH tubes, insignificant decay was observed by the oxidized SG-65 tubes. Employing XTT as a superoxide anion (O2-∙) scavenger indicates that this ROS is produced by both types of CNTs in sunlight. Employing pCBA as a hydroxyl radical (OH) scavenger indicates greater production of OH by P3-COOH than oxidized SG-65.
Some studies were performed with oxidized multiwalled CNTs (MWCNTs) in UVC light at λ = 254 nm. These CNTs - prepared in aqueous colloidal form - aggregate and settle upon UVC irradiation under anoxic conditions, presumably due to decarboxylation, as more flocculation occurs at lower pH values. The time to the onset of settling is linear with light intensity, indicating a one photon process.
Fungal studies during the first year were directed toward establishing toxicity limits of CNT exposure to specific fungi and bacteria, assessing mechanisms to effectively plate CNT/fungi for incubations that allow gas and isotope analysis, and time series of extracellular enzyme activity that may indicate fungal-CNT interaction. Exposing unpurified Carbon Solutions AP-CNT to fungi, resulted in failure of most inocula to grow, including Trametes versicolor. A further complication is that inhibition of growth appears to be also a function of the media type and nutrient status. Fungi growing on malt extract media, for example, resulted in minor inhibition of growth by AP-CNT. Exposure to AP-CNT also has an effect on the morphology of fungi depending on the media on which it is grown.
Reactions in Solar Spectrum Light. To examine the effect of metal impurities on the photoreactivity of SWCNTs, we conducted ROS measurements on two types of single-walled carbon nanotubes: (1) "P3-COOH" are commercially available carboxylated single-walled carbon nanotubes from Carbon Solutions with almost 6% metallic impurities (e.g., mostly nickel); and (2) oxidized SG-65 CNTs. SG-65 tubes are purified unfunctionalized SWCNTs from Southwest Nanotechnologies and contain a high fraction of semi-conducting 6,5 (roll-up angle) small diameter SWCNTs. Fairbrother's group has partially oxidized a batch of these SWCNTs through acid treatment (e.g., HNO3), forming "oxidized SG-65." SEM analysis on oxidized SG-65 confirmed that through oxidation and further purification processes, the metal content has decreased from ~2% to ~1% (e.g., mostly molybdenum). As shown in Figure 1, XPS analysis by co-PI Fairbrother shows an increase in oxygen content from 5% to almost 12% while the carbon content decreased from 95% to 88%.
Figure 1. XPS spectra of carbon 1s and oxygen 1s regions before and after oxidation with
70% nitric acid on SG-65 carbon nanotubes
Upon irradiating dispersions of these CNTs with eight black-light phosphor lamps (RPR-3500 (λ = 300-410 nm), co-PI Jafvert's group quantified the production and concentrations of 1O2, O2-∙, and ∙OH using FFA (furfuryl alcohol), XTT (3'-{1-phen ylamino-carbonyl-3,4-tetrazolium}bis(4- methoxy-6-nitro)-benzenesulfonic acid hydrate), and p-chlorobenzoic acid (pCBA), respectively, as ROS scavengers.
Whereas FFA decay (associated with 1O2 production) occurred in dispersions of P3-COOH tubes, insignificant decay was observed by the oxidized SG-65 tubes (Figure 2).
Figure 2. FFA loss indicating 1O2 production at pH7
in lamp light (λ = 350 ± 50 nm) by aqueous dispersions
of P3-COOH (■), and oxidized SG-6 5 (▲), and the
corresponding dark control samples of P3-COOH □),
and oxidized SG-65 (△).
In a similar experiment, XTT was employed as a superoxide anion scavenger to quantify the production of this ROS. Reaction of XTT with O2.- produces a product that strongly absorbs light at 470 nm, allowing confirmation of superoxide anion production by UV-Vis spectroscopic analysis. Figure 3 shows that aqueous dispersions of P3-COOH and oxidized SG-65 containing XTT generate the reaction product more rapidly than XTT or nanotube control sample, indicating superoxide anion is produced by both types of oxidized tubes. Employing pCBA as a hydroxyl radial (∙OH) scavenger indicates greater production of ∙OH by P3-COOH than oxidized SG-65, but greater production by both types of CNTs compared to all control samples. These data (Figures 2 and 3 and pCBA experiments) are strong preliminary evidence that metal impurity concentrations or species type (i.e., nickel vs. molybdenum), the extent of surface oxidation, or even the tube diameter or roll up angle, control the extent and type of ROS produced under sunlight by carbon nanotubes.
Figure 3. Evidence of O2 production by XTT (0.1 nM)
products formation under lamp light (λ = 350 ± 50 nm)
in aqueous suspenstions of : P3-COOH with XTT (▲),
oxidized SG-65 with XTT (■), and XTT alone (x) and the
corresponding dark controls.
Photochemical Reactions of Oxidized Multiwalled CNTs under UV Irradiation (254 nm). In this portion of the study, multiwalled CNTs (MWCNTs) purchased from NanoLabs, Inc., have been oxidized using a 3:1 ratio of sulfuric acid:nitric acid to covalently modify the surface resulting in 9% oxygen (measured by XPS). These CNTs were used in colloidal form to examine the effect of pH, light intensity, and irradiation time on these dispersions. UV-Vis absorbance and particle size measurements were used to determine the effects of 254 nm UV light. Experiments were accomplished using a Rayonet RPR-100 with 16 lamps (unless specified) to irradiate two 150 mL quartz flasks that were purged with nitrogen to produce anoxic conditions. A rotating carousel was employed to ensure uniform exposure to both flasks. The appropriate phosphate buffer (3 mM) was used to set the pH at 4, 7, and 10.
Figure 4. A typical example of MWCNTs
irridated by UV light, the control (left) which
was wrapped in aluminum foiil and the sample
exposed to light (right) where the CNTs have
aggregated and precipitated to the bottom of
the flask.
A common phenomenon that was observed for all of the oxidized MWCNTs was UV-induced aggregation, as shown in Figure 4. Over the course of irradiation, the particles begin to aggregate until the flocculates reach a size large enough to become visible and settle to the bottom of the flask. This process of decreasing particle stability with irradiation by UV light could be followed as a function of time by monitoring changes in the concentration of CNT particles remaining stable in suspension using UV-Vis absorbance, and examining their average particle size over the course of the experiment. An example is shown in Figure 5, which illustrates a corresponding change in absorbance (open triangles) and change in particle size (filled circles), and both are plotted as a function of irradiation time. The particle size increases over the entire period of irradiation (see right side axis). When the particles reach a certain size limit, approximately 300-400 nm, they begin to settle and the concentration of oxidized MWCNTs remaining in suspension decreases dramatically. The region that is shaded by the diagonal stripes indicates the time period where visual aggregation and sedimentation of MWCNTs occurs. An important factor to note is that even during particle growth, there is minimal change in the absorbance value of the system until the shaded area is reached, corresponding to the peak size limit for aggregation.
Figure 5. Relation of absorbance measurements
to particle size in determining aggregation period
of MWCNTs and pH7 and 3mM phosphate buffer
under UV irridation with 8 bulbs. Absorbance data
is plotted as open triangles and particle size measurements
are plotted as filled circles.
The stability of the oxidized MWCNTs towards aggregation also is affected by solution conditions (e.g., pH) and light intensity (e.g., number of bulbs). MWCNT stability in suspension while under irradiation increased systematically as the pH increased, as shown in Figure 6, corresponding to increased surface charge that aids in electrostatic repulsion. To test the effect of light intensity, experiments were performed at pH 7 under anoxic conditions. The intensity of light was adjusted by changing the number of bulbs used for exposure in the chamber. The carousel was kept constantly rotating to ensure uniform exposure. Figure 7 illustrates the experiments from 16, 8, and 4 bulbs and the results from the absorbance data. This plot showed that decreasing the light intensity in half approximately doubled the length of time needed to reach total CNT aggregation. The results from this particular study indicate that the irradiation and subsequent aggregation of CNTs follows a one-photon process.
 Figure 6. Effect of pH on collodial stability of MWCNTs while under irridation with UV light at 254 nm: pH4 (pink), pH7 (cyan), and pH10 (yellow) |  Figure 7. Effect of bulb intensity on the collidal stability of MWCNTs while under irridation with UV light at 254nm: 16 bulbs (red), 8 bulbs (blue), and 4 bulbs (green) |
Based on these preliminary studies our current efforts are focused on understanding the reasons for the UV-induced aggregation, with the hypothesis that it is mediated by photo-decarboxylation. To test this hypothesis, we are conducting studies with larger reactors where we can irradiate sufficiently large quantities of O-MWCNTs to facilitate analysis of the post-irradiated samples with X-ray photoelectron spectroscopy (XPS), carried out in conjunction with chemical derivatization to assay the distribution of oxygen-containing functional groups.1-4 Similar XPS analysis will be conducted on CNTs irradiated with sunlight.
Initial Studies with Fungi and Carbon Nanotubes. In this first year, we have been working to establish toxicity limits of CNT exposure to specific fungi and bacteria, assessing mechanisms to effectively plate CNT/fungi for incubations that allow gas and isotope analysis, and time series of extracellular enzyme activity that may indicate fungal-CNT interaction. As a cost saving measure we have been experimenting initially with unpurified Carbon Solutions AP-CNT, which has resulted in failure of most inocula to grow. We quickly assessed that this material exerted a strong inhibition to growth. For example, inhibition of fungal growth by CNT is apparent in the series of photographs shown in Figure 8 of Trametes versicolor after a week of growth. In this experiment, plugs of malt extract agar were inoculated with fungi and placed on plates containing thin layers of minimal media. This minimal media was supplemented by the addition of 0.1%, 1%, and 10% unpurified CNT by weight carbon.
Figure 8. T. versicolor growing on media containing 0.1 (upper left), 1.0 (upper right)
, and 10% (lower left) unprufied CNT by mass. CNTs used on these plates are Carbon
Solutions, Inc. AP-CNT. Note confinement of growth to the disk of non-CNT's starter
media at the highest CNT concentrations.
There is a complication associated with this plate toxicity approach in that the inhibition of growth also is a function of the media type and nutrient status. This effect is illustrated in the photoset in Figure 9, which follows the growth of T. versicolor over the period of 5 to 7 days after plating on media supplemented with 5% AP-CNT by carbon mass in either malt agar media (top row) or minimal medium with veratryl alcohol (bottom). In the rightmost column of each row is a control for each media type on day 7 of growth. Fungi growing on malt extract media shows minor inhibition of growth (growth rings are measured with time and biomass is collected at the end) by AP-CNT when compared to control fungi. On minimal media in the presence of AP-CNT, however, they exhibit significant inhibition compared to the control. Results from assays on ß-glucosidase and peroxidases are pending and may shed light on this.
Figure 9. Phoset depicting possible mitigation of AP-CNT toxicity based on media additives. Top row:
malt extract agar +5% ATP-CNT after 5, 6, and 7 days growth, and the control plate of the same media
without ATP-CNT after 7 days. Bottom row: minimal media with veratyl alcohol +5% ATP-CNT after 5,
6, and 7 days, and the corresponding control plate after 7 days growth.
In addition to a decrease in growth rate, AP-CNT exposure also has an effect on the morphology of fungi based upon the media on which it is grown. The photographs in Figure 10 depict T. versicolor after 3 weeks of growth on media containing 5% AP-CNT and nitrogen limited minimal media (top left) or malt extract (upper right); a control without AP-CNT supplementation is show below each plate. The light-confluent growth seen in control nitrogen limited plates (bottom left) is not present in media containing AP-CNT; instead, filamentous branching rhizomorphs are formed. Presence of AP-CNT in malt media appears to induce denser confluent growth when compared to control.
Figure 10. Photo inset exhibiting differences in fungal colony morphology based on media
and AT-CNT supplementation. Nitrogen limited media supports confluent, but very light
growtn (bottom left) malt extract media (bottom right) supports confluent growth of moderate
density. Addition of AP-CNT causes more selective growth with tendril-like rhizomorphs
on nirtigen limited media (top left) while fungus on AP-CNT malt agar chows very dense
confluent growth. (Major graduation = 1cm)
CO2 Measurements. In addition to the initial fungal growth studies, we have been conducting performance tests on gas phase CO2 measurements to develop LOQ (Limit of Quantification) above background, to assess whether it will be necessary to sparge background CO2 from samples that will undergo exposure to light and/or fungi (see future activities section).
One paper has been published in Carbon in 2011, and two additional papers are planned for submission in the next year. One of these will be on photochemical transformation of CNTs under UVC light, and one on changes in functionalization and ROS production by functionalized (i.e., partially oxidized) CNTs. These efforts support EPA's mission by elucidating the environmental fate of carbon-based nanomaterials. A strong international collaboration has been initiated with Professor Kaili Jiang at Tsinghua University and Professor Xinghui Xia at Beijing Normal University in China. In this partnership, professors Xia and Jiang are synthesizing 13C-labeled SWNT. They have provided the first batch (100 atom % 13C) and we hope to receive the remaining materials within the next 4 months. Prof Xia plans to send a student to Purdue to assist with studying the interactions of specific microbes targeted in her lab with 13C-CNTs. In addition, Lee Ferguson at Duke University is collaborating with us to examine reaction products by near infrared fluorescence (NIRF) spectroscopy. A Think-Swiss proposal was written for graduate student, Somi BeigzadehMilani, to work for 1 month in the Laboratory of Prof. Kristophere McNeill at ETH Zurich (one of our international collaborators), however, the proposal to the Office of Science, Technology and Higher Education of Switzerland was not funded.
Future Activities:
Later in the project (years 2-3), we will study transformation of CNTs in polymer composites, and determine if and under what conditions CNTs are released from composites as a result of exposure to light and/or fungi.
Experiments that examine reactivity of CNTs upon long-term solar irradiation will be conducted to examine changes in surface functionalization and elemental composition. Indirect phototransformation of non-functionalized CNTs will be examined. Long-term incubations of fungi with 13C-labeled SWNTs will be performed to assess the ability of the organisms to oxidize the nanomaterials. Both photochemical and fungal experiments will use trace-gas analysis coupled with isotope-ratio-mass-spectrometry to determine CO2 efflux, and X-ray photoelectron spectroscopy (XPS) to determine changes in surface functionalization.
We have experiments ongoing involving: (1) long-term irradiation of both pristine (unfunctionalized) and functionalized SWCNTs under sunlight to examine changes in surface functionalization and elemental composition (i.e., C/O ratios); (2) studies under sunlight where we are adding hydrogen peroxide to study indirect photo-oxidation of CNTs by producing ∙OH photochemically from H2O2; and (3) studies to determine the extent to which (if any) CNTs undergo mineralization when exposed to sunlight or UV-irradiation. In the next year, we will initiate experiments on effects of photolysis on CNT-containing polymer composites in air and in aqueous environments.
New collaborations established with Dr. Eoin Brodie at Lawrence Berkeley National Laboratory have allowed us to expand these studies to include a number of strain of actinomyces that were originally found to degrade black carbon; we believe that the ability of these bacteria to utilize condensed carbon from biochar suggests that they possess enzymes that might be able to transform carbon nanomaterials as well. These experiments are scheduled to run concurrently with those above with a projected completion in Autumn 2012.
We also will begin long-term incubations of fungi with 13C-labeled SWNTs to assess the ability of the organisms to oxidize the nanomaterials. These experiments will use trace-gas analysis coupled with isotope-ratio-mass-spectrometry to determine what amount of CO2 efflux is derived from CNT in the mesocosms. This experiment will be coupled with analysis of phospholipid fatty acids in samples taken from mesocosms to determine the quantity of 13C from the CNTs that is incorporated into fungal biomass. In parallel experiments we will include both the dissolved and precipitate fractions of nanomaterials produced in the previously mentioned photo-irradiation experiments in long-term soil mesocosms. The use of 13C enriched nanomaterials will allow for an examination of the ultimate fate of these nanomaterial fractions in soil environments by analyzing the isotopic composition of phospholipid fatty acids of bacteria and fungi in the soils used.
References:
Langley, L. A.; Fairbrother, D. H., Effect of wet chemical treatments on the distribution of surface oxides on carbonaceous materials. Carbon 2007, 45(1), 47-54.
Smith, B.; Wepasnick, K.; Schrote, K. E.; Cho, H.-H.; Ball, W. P.; Fairbrother, D. H., Influence of Surface Oxides on the Colloidal Stability of Multi-walled Carbon Nanotubes: A Structure-Property Relationship Langmuir 2009, 25(17), 9767-9776.
Wepasnick, K. A.; Smith, B. A.; Bitter, J. L.; Fairbrother, D. H., Chemical and structural characterization of carbon nanotube surfaces. Anal. Bioanal. Chem. 2010, 396(3), 1003-1014.
Wepasnick, K. A.; Smith, B. A.; Schrote, K. E.; Wilson, H. K.; Diegelmann, S. R.; Fairbrother, D. H., Surface and structural characterization of multi-walled carbon nanotubes following different oxidative treatments. Carbon 2011, 49(1), 24-36.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
| Other project views: | All 31 publications | 11 publications in selected types | All 11 journal articles |
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Chen C-Y, Jafvert CT. The role of surface functionalization in the solar light-induced production of reactive oxygen species by single-walled carbon nanotubes in water. Carbon 2011;49(15):5099-5106. |
R834858 (2011) R834858 (Final) |
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Supplemental Keywords:
Carbon nanotubes, nanotechnology, environmental photochemistry, fungi, trace gas analysis, transformation, waterRelevant Websites:
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.