Grantee Research Project Results
Final Report: Repercussion of Carbon Based Manufactured Nanoparticles on Microbial Processes in Environmental Systems
EPA Grant Number: R831720Title: Repercussion of Carbon Based Manufactured Nanoparticles on Microbial Processes in Environmental Systems
Investigators: Turco, Ronald F. , Filley, Timothy , Applegate, Bruce M.
Institution: Purdue University
EPA Project Officer: Aja, Hayley
Project Period: July 1, 2004 through June 30, 2007
Project Amount: $335,000
RFA: Exploratory Research to Anticipate Future Environmental Issues: Impacts of Manufactured Nanomaterials on Human Health and the Environment (2003) RFA Text | Recipients Lists
Research Category: Nanotechnology , Safer Chemicals , Human Health
Objective:
Objective Area 1: We propose there will be a shift in the structure of soil microbial populations in systems exposed to Carbon Based Manufactured Nanoparticles (CMNP) as the nanomaterial will exert pressure on the microbial population.
Explanation and General Approach: Simply stated, we will determine the size of the starting population and the diversity within the population and monitor the effects of added CMNP on these parameters. Our efforts will be geared to: (1) understand how the introduction of CMNP affects the resident populations; (2) describe how the populations adapt and alters themselves in response to the introduction of CMNP. We propose to measure if this shift takes place and how stable the altered population becomes. The intrinsic features describing activity will be estimated in four ways: 1) we will draw information from the ratio of key fatty acids taken from the phospholipid fatty acids fraction (PLFA) and relate it to a background status of the soil microbial populations; 2) Use genetic approaches (DGGE) with both bacterial and fungal primers; 3) use enzyme assays for dehydrogenase, urease, cellubiase; 4) use respiration and trapping of CO2 to estimate aerobic activity, respectively, in the presence of the CMNP.
Objective Area 2: The long‐term fate of CMNP in the environment, and their entrance into soil and aquatic biogeochemical cycles, will mostly be a function of the activity of the specific oxygenase, ligninase, laccase, and fenton systems resident in microbial populations.
Explanation and General Approach: Enhanced functionalization of CMNP, e.g., hydroxylation, ring cleavage and oxidation, will be largely governed by extracellular enzymatic systems active in the soil; therefore under carbon‐rich residue (plant or sludge) soil conditions (condition of high enzyme activity) we anticipate the greatest chemical modification to the CMNP. Under these conditions the CMNP will enter fully into the soil and sediment biogeochemical carbon cycles through coupling reactions to insoluble macromolecules and humic acids as well as the aquatic biogeochemical cycles through enhanced solubility of the added functionalities or binding to fulvic acids. Using 13C‐fullerenes and degradation products we tracked CMNP carbon to determine how the soil microbial biomass responds to CMNP. We will also assess the degree to which CMNP carbon is assimilated into microbial biomass, or is converted to a form bound with soil carbon. Additionally, we will inoculate various litter forms (wood and leaves) spiked with 13C‐labeled fullerene and fullernol with aggressive decay fungi where our goal is to assess the degree to which CMNP carbon is assimilated into fungal biomass or converted to functionalized forms (free and bound).
Objective Area 3: Water‐borne CMNP represent an, as yet, unassessed toxicological risk to aquatic organisms because of their capacity to physically interact with cell membranes possibly causing harm to the cells.
Explanation and General Approach: We will develop baseline information on the effects of CMNP on the stability, death and re‐growth of bacteria in water using a unique analytical approach to assay the bacterial dynamics. The lux‐gfp based assay allows us to estimate the impact of the CMNP on the processes of respiration and growth and allow us to arrive at the first CMNP structure‐to‐microbial function model. This objective will involve monitoring bacterial bioluminescence to evaluate the impact of CMNP (amount or structure) on bacterial response in aqueous systems.
Summary/Accomplishments (Outputs/Outcomes):
This project has provided fundamental information about the fate and impact of C60 and single walled nanotubes (SWNT) on microorganism in the soil environment. In particular, we have shown: 1) The introduction of low or high levels of C60 (e.g., 1 μg C60 g‐1 soil in aqueous suspension (nC60) or 1000 μg C60 g‐1 soil in granular form) did not negatively affect the functioning of the soil or the makeup of the soil population. This is in contrast to our original hypothesis that, based on information from pure culture studies, carbon nanomaterials would have a negative impact on soil microbial functions and population structure. 2) We have shown the application of the aqueous form of C60 (nC60) to soil across a range of particle sizes (average diameter 51, 78, 108 and 250 nm at concentration of 1 μg g‐1 soil) had little impact on either the biological functioning of the soil or the makeup of the soil population. DGGE banding patterns showed some minor enhancement and inhibition in a number of bands, but the overall structures for both bacteria and eukaryote patterns was not changed by C60. Again, this is in contrast to our original hypothesis that, based on information from pure culture studies, smaller sized carbon nanomaterials would have a negative impact on soil microbial functions and population structure as compared to larger particle sizes. 3) We have shown that solvents used in the manufacture or delivery of fullerenes could have the potential for more harm than the actual nanomaterial if they find their way into soil. High concentration of solvents showed negative effects on the microbial activity and did alter bacterial community composition. We saw suppression of respiration and changes in the population structure related to the solvents. This was somewhat unexpected. We also found that metal contaminants that occur in the production of single wall carbon nanotubes, could like the solvents, suppress microbial functions. 4) In terms of microbial degradation we have found C60 to be relatively stable even when aggressively attacked with a variety of fungi including three brown rot basidiomycetes (Gloeophyllum trabeum, Fomitopsis pinicola, and Postia placenta), two white rot basidiomycetes (Trametes versicolor and Phlebia tremellosa) and one soft rot ascomycete (Cadophora malorum). Detail analysis showed only minor incorporation of the 13C carbon into the biomass. This was in direct contradiction to our hypothesis that because these fungi have been shown to use a variety of extracellular, non‐specific, oxidative degradation mechanisms to degrade natural substances such as wood lignin, wood cellulose, and black carbons, and also anthropogenic substances such as polycyclic aromatic hydrocarbons they would be able to attack C60. 5) In terms of microbial degradation we have found C60‐OH (C60 fullerol) to be less stable when attacked with a variety of fungi. This study, using bulk and compound specific 13C stable isotope ratio mass spectrometry techniques and absorbance analysis, showed two white rot basidiomycete fungi (Phlebia tremellosa and Trametes versicolor) to metabolize and degrade 13C‐labeled C60 fullerol. Both fungal species were able to bleach and oxidize fullerol to CO2 in the presence of wood tissue and without. The fungi were also capable under the conditions tested to incorporate fullerol carbon into biomass, but only in trace amounts. Phlebia tremellosa proved to be, in general, more aggressive towards fullerol degradation than Trametes versicolor without the presence of wood tissue. Detail analysis showed only minor incorporation of the 13C carbon into the biomass. These results are important in that they represent the first report of the fungal degradation, and resulting environmental fates, of these important nanomaterials and suggest that the environmental fate of these nanomaterials will be a complex set of interactions. For example, we know that C60 fullerol can form in light and it now appears that once formed, it can be attacked by fungi. 6) For the biomarker work we concentrated on the effects of SWNTs on the acute toxicity of SWNTs and viability of bioluminescent E. coli O157:H7. Raw SWNTs showed toxic effect on both bioluminescence and viability of the cells. Functionalized SWNTs inhibited the number of viable cells but did not show acute toxicity. This work demonstrated the importance of considering contaminants left over from the manufacturing of the nano materials as we saw a strong correlation between the amount of left‐over metal and negative response.
Details: Carbon‐based nanomaterials are finding their way into many product applications and the release of nanomaterials into the environment is inevitable. An early assessment of their potential environmental impacts, prior to environmental release, is warranted. This work focuses on an assessment of the impacts of fullerene (C60), single‐wall carbon nanotubes (SWNTs) and the degradation product of fullerene, fullerol on soil microbial processes, considered to be a sensitive indicator of the potential for environmental stress. Under objective 2, a major emphasis was placed on the fate of fullerols as early work showed fullerene to be stable to microbial attack. However, work outside of this project demonstrated the importance of sunlight as potential oxidizing agent that will create the derivate materials. For objective 1, a series of the most currently encountered nanomaterials including fullerene C60 or its aqueous suspension (nC60), single‐wall carbon nanotubes (SWNTs), and the C60 oxidation dation products in their raw manufactured form or with unctionalization were introduced to two soils with high or low organic matter contents. Impacts of the applications were evaluated by measuring soil basal respiration, glucose‐induced respiration, total microbial biomass (indicated by phospholipid derived phosphate), or soil enzymatic activities including β‐glucosidase, acid‐phosphatase, dehydrogenase and urease. Community structure was evaluated using fatty acid profiles or denaturing gradient gel electrophoresis (DGGE) of 16S and 18S rDNA fragments amplified by PCR from soil DNA extracts. Our observations show that C60, introduced in dry form or as aqueous suspension, have limited impact on the structure and activity of the soil microbial community. SWNTs have some effects on microbial community structures but they did not exert a major effect on the activities. The response of soil microorganisms was not affect by C60 introduced in organic solvent (toluene or tetrahydrofuran). However the high concentration of solvent showed negative effects on the microbial activity and changed the bacterial community composition. Although some researchers have shown aqueous suspension of C60 as being toxic to bacteria our results did not show any toxic effects from either granular C60 or nC60 in aerobic soil microcosms. We suggest our findings differ from others in terms of toxicity in that we are releasing the C60 into soil that contains both organic matter and salts which control the availability of C60 in soil which will ultimately control the exposure level and toxicity of fullerenes. Work in our laboratory (data not shown) as well as others has shown C60 to have a limited toxicity towards Gram‐negative bacteria when they are grown in cultures with concentrations of salts typical of culture media. We would also expect ionic strength to affect the physical behavior of C60 in aqueous systems. Combined these data suggest that bioavailability of chemicals is important in controlling its environmental impacts. On the other hand, these data do suggest that at 180‐day, the C60 is having at least a minor effect on the fatty acids profile of the resident microorganisms. When exposed to soil, it is suggested that C60 partitions into soil organic matter and this will decrease the solution‐level bioavailability. We suggest, however, that partitioning into soil organic matter is most likely the major factor controlling availability as recent work has shown log Koc values to approach 7.73. This level of sorption indicates the soil will retain most of the applied material, especially at the lower application levels, thus ameliorating any possible toxic effects of the C60 on the biota. Previous studies with experimental designs that resulted in facilitated routes of exposure of C60 to bacteria have indicated some level of toxic response. We hypothesized that an introduction of 1000 μg g‐1 granular C60 would overcome both sorption and salt effects and generate a negative effect if one actually exists. Even at these high application concentrations data suggest that the overall effects of the nanomaterials are limited. However, when PLFA was differentiated between Gram‐positive and Gram‐negative bacteria, the proportion of Gramnegative bacteria in all treated soils were slightly (~5%) higher. For objective 2, this portion of the study examined the degradation potential of C60 fullerenes and fullerols by a range of wood‐rotting fungal species, including three brown rot basidiomycetes (Gloeophyllum trabeum, Fomitopsis pinicola, and Postia placenta), two white rot basidiomycetes (Trametes versicolor and Phlebia tremellosa) and one soft rot ascomycete (Cadophora malorum). These fungi have been shown to use a variety of extracellular, nonspecific, oxidative degradation mechanisms to degrade natural substances such as wood lignin, wood cellulose, and black carbons, and also anthropogenic substances such as polycyclic aromatic hydrocarbons. Bulk and compound specific 13C stable isotope techniques as well as absorbance analysis of fullerol bleaching are used to determine the extent of fullerene and fullerol degradation. Experiments were performed both in the presence of a wood wafer and without, to determine if the presence of wood tissue influences the fungal degradation of these ompounds. In general, the white rot fungi were more aggressive towards degradation of fullerene (measured by 13C content in fungal lipids) and fullerols (measured by production of 13C‐labeled CO2, 13C content in fungal lipids, and production of low molecular weight metabolites). These results are important in that they represent the first report of the fungal degradation, and resulting environmental fates, of these important nanomaterials. White rot and brown rot basidiomycete fungi have very different mechanisms of degradation, and in fact naturally degrade different parts of wood tissue. This study has shown that they also have differing abilities to degrade a very important carbon based manufactured nanomaterial: C60 fullerol. While both types of fungi showed the ability to produce CO2 and build mycelial biomass from fullerol carbon, white rot fungi had the ability to do so to a far greater degree than brown rot fungi. These findings suggest that white rot fungi also had the ability to produce low molecular weight metabolites from fullerol, as evidenced by bleaching of the fullerol in the growth media by the fungi. Brown rot fungi evidently do not have this ability, or if they do, it is to a far lesser degree than that of white rot fungi. The presence of wood tissue also appeared to make a difference in the degradation of fullerol for both types of basidiomycete fungi. Wood tissue enabled the white rot fungus T. versicolor to bleach the fullerol more consistently, however wood tissue did not affect the degradation of fullerol by P. tremellosa. Overall, less fullerol carbon was oxidized to CO2 or incorporated into biomass when wood tissue was present in the system, but this was likely due to the fact that more carbon overall was available in the system when wood wafers were present. The increased bleaching indicates that at least for white rot fungi, the presence of wood tissue activates enzyme systems and enables an increased degradation of the fullerol. Degradation by white rot basidiomycete fungi especially, and to a lesser extent brown rot fungi, is likely to be an important degradation mechanism for fullerols either released into the environment or produced in the environment through oxidation of fullerene. This study indicates that these fungi have the ability, both in the presence of wood tissue and without, to utilize fullerol as both an energy source (mineralization to CO2) and a carbon source (uptake into mycelial biomass). For objective 3, we concentrated on the effects of nanocarbon additions on the viability of bioluminescent E. coli O157:H7. In this study, Single Walled Carbon NanoTubes (SWNTs) were examined. The acute toxicity of raw SWNTs and functionalized SWNT was evaluated using luminescence‐based whole‐cell bioassay, which is widely used as an indicator for bacterial response to toxic compounds. When E. coli‐lux cells were treated with SWNTs for 10 or 30 min, declines in luminescence were observed compared to the control. As‐produced or raw SWNTs showed toxic effect on both bioluminescence and viability of the cells. For all three types of SWNTs examined, the light intensity decreased as the concentration of nanoparticles increased. For the lux system light emission is a measure of the metabolic activity of the cells. The bioluminescence response is directly dependent on an energy input. Therefore, adding glucose to the system will stimulate light production in healthy cells. E. coli‐lux will respond differently to added glucose if the metabolic activity of cells is impaired by a potential toxin. Increase in luminescence was detected for all samples but at different levels. Relative light production compared to the control was calculated and plotted against the concentration for all measurements. After adding glucose, the relative light production dramatically decreased at all concentrations of AP‐SWNT and this implies that the metabolic activity of cells was altered by the tubes or a material with the tube. For both fSWNTs, the relative light production did not change significantly at any concentration regardless the presence of glucose or the incubation time, indicating these nanoparticles do not adversely affect the metabolic activity of cells. Again, the effect of catalyst metals carried in SWNTs should be considered as the metal concentration can reach as high as 15 μg mL‐1. Functionalized SWNTs inhibited the number of viable cells but did not show acute toxicity.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 2 publications | 2 publications in selected types | All 2 journal articles |
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Kim S, Schuler B, Terekhov A, Auer J, Mauer L, Perry L, Applegate B. A bioluminescence-based assay for enumeration of lytic bacteriophage. JOURNAL OF MICROBIOLOGICAL METHODS 2009;79(1):18-22. |
R831720 (Final) |
Exit Exit |
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Schreiner K, Filley T, Blanchette R, Bowen B, Bolskar R, Hockaday W, Masiello C, Raebiger J. White-Rot Basidiomycete-Mediated Decomposition of C-60 Fullerol. ENVIRONMENTAL SCIENCE & TECHNOLOGY 2009;43(9):3162-3168. |
R831720 (Final) R831710 (Final) |
Exit Exit |
Supplemental Keywords:
Microbial Response to Environmental Toxicants, RFA, Scientific Discipline, ENVIRONMENTAL MANAGEMENT, ECOSYSTEMS, Ecosystem Protection/Environmental Exposure & Risk, Sustainable Industry/Business, Sustainable Environment, Ecosystem/Assessment/Indicators, Ecosystem Protection, Technology for Sustainable Environment, Ecological Effects - Environmental Exposure & Risk, Environmental Microbiology, Ecological Monitoring, Ecological Risk Assessment, Risk Assessment, microbial removal and monitoring, aquatic ecosystem, bioassessment, ecosystem assessment, nanotechnology, microbial communities, carbon based manufactured nanoparticles, nanomaterials, ecological assessment, ecological impacts, toxicity, nanoparticles, environmental fateProgress 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.