Studies over the past ten years have made it apparent that various commercially-relevant nanomaterials have harmful impacts on the environment. Unfortunately, there is a void of knowledge about the environmental implications of cellulose-based nanomaterials (nanocellulose). At the same time, the market for cellulose-based nanomaterials is expected to exceed a billion dollars by 2020. Given the estimated size of this market and the expected chemical and biological stability of nanocellulose there is a need to evaluate the environmental implications of these nanomaterials. Even though cellulose is generally considered to be an environmentally-friendly material given its omnipresence in woods, fibers, and tunicate animals, nanocellulose is both physically and chemically very different. These differences mean that it cannot be assumed that nanocellulose is as biodegradable and environmentally benign as cellulose in its native state. Thus, the proposed research will evaluate biodegradation (i.e., will nanocellulose naturally break down) and toxicity (i.e., is nanocellulose harmful to some organisms) utilizing bacterial communities relevant to wastewater treatment plants (WWTPs) and impacted water environments. WWTP microbes are especially relevant because as production of nanocellulose escalates, concentrations entering WWTPs will correspondingly increase. The study will focus on two hypotheses: Hypothesis One: Bacterial biodegradation of nanocellulose will be influenced by its surface properties (e.g., charge, hydrophobicity, and steric hindrance imparted by surface functional groups). Hypothesis Two: Negatively charged nanocellulose materials are non-toxic, while those possessing positively-charged surface modifications will be more toxic because they adhere to or disrupt cellular membranes (which are negatively charged). To test these hypotheses we have developed a research plan consisting of two integrated research tasks: Task 1. Evaluate the biodegradability of nanocellulose, and Task 2. Evaluate the toxicity and stress responses elicited by nanocellulose. Because very little is known about the environmental implications of nanocellulose production and use, the laboratory research efforts defined by Tasks 1 & 2 will be complemented by the parallel development of a life cycle assessment (LCA) inventory module for an undergraduate LCA course (ENGR 3134) at Virginia Tech. In this effort, undergraduate students taking ENGR 3134 will produce inventories that consider nanocellulose production and use.

INTELLECTUAL MERIT: The proposed one year investigation is high risk/high reward and will establish critical baseline information on nanocellulose biodegradation and toxicity potential. Nanocellulose holds great promise as a potentially "green" nanomaterial. However, it is critical that this assumption be validated, especially as major production facilities are now going online. The physicochemical state (e.g., surface moieties, surface charge, aggregation state) of varying preparations of nanocellulose will be linked both to its biodegradability and microbial toxicity using complex microbial communities (anaerobic digester and wetland sediment) relevant to environments most likely to be impacted by disposal or other release. As the core material of nanocellulose is thought to be "inert", the approach could eventually provide a means to isolate the effects of surface chemistry and the behavior of nanomaterials as a whole in governing their environmental implications. This investigation will also advance the fundamental knowledge base of anaerobic cellulose degradation, a key biogeochemical process important for critical issues such as climate change and the development of alternative biofuels.

BROADER IMPACTS: Two Ph.D. student researchers will be funded by this project and will gain interdisciplinary training across fields of nanotechnology, sustainable biomaterials, environmental microbiology, environmental engineering and application of molecular tools. The project will have institutional impact by catalyzing interdisciplinary collaboration between the VT Institute for Critical Technology and Applied Science (ICTAS) Sustainable Nanotechnology (SuN) and Water Sustainability research thrusts, which will provide complementary graduate student support. Interdisciplinary graduate education will also be enhanced via the companion VT SuN Interdisciplinary Graduate Education Program (IGEP), which will also provide opportunities for student support. The proposed LCA inventory will serve as an integral component of the undergraduate Virginia Tech Green Engineering program, providing a hands-on opportunity for in-class and independent undergraduate researchers, while also establishing a paradigm by which the "green" nature of nanotechnologies can objectively be assessed. Outreach efforts to women and economically underrepresented groups will be made to support community education as well as to aid in recruiting Ph.D. and undergraduate researchers to assist in this project.

Project Report

The nanotechnology industry is booming across the U.S. and globally. As with the dawn of any new industry, there is the promise of numerous benefits to society. At the same time, it is important to learn from history and to be proactive in anticipating unintended negative consequences of nanotechnology. An example is the addition of lead to gasoline, which acted as an excellent anti-knocking agent, but also polluted the environment with a highly toxic metal. In the case of nanotechnology, nanocellulose is touted as "green" because it is sourced from trees and is comprised of the natural organic polymer: cellulose. The overarching goal of this project was to objectively assess the biodegradability and toxicity of nanocellulose, as these are two critical aspects defining truly "green" materials. A range of nanocelluloses were synthesized, each with unique surface properties: TEMPO, HCl hydrolyzed, sulfuric acid hydrolyzed, cationic, and Jeffamine-treated. Microcrystalline cellulose was used as a control that is much larger than the nano-form. Each nanomaterial was then fed to two different cultures of bacteria that were adapted to biodegradation of microcrystalline cellulose as a food source: a wetland-derived culture and an anaerobic digester-derived culture. The biodegradation of the nanocellulose was measured by examining the nanocellulose using RAMAN spectroscopy and by measuring the disappearance of glucose, which is the building block of cellulose. Shifts in the microbial community structure were monitored by using next-generation DNA sequencing to examine all of the bacteria and archaebacteria present and also quantitative polymerase chain reaction to examine how many bacteria were present with time and also the levels of genes associated with cellulose biodegradation (cel48 gene). RNA was also monitored to examine gene expression over short term exposure times to the nanocellulose as an indicator of toxicity. It was found that the nanocelluloses did in fact display different biodegradation patterns than the control microcrystalline cellulose. This was most apparent when comparing the TEMPO nanocellulose, which did not carry any functional groups, with microcrystalline cellulose. The bacterial growth patterns were distinct for these two forms of nanocellulose. The surface functionalization also had an effect. For example, the cationic functionalized and HCl hydrolyzed nanocelluloses resulted in the greatest enrichment of cellulose-degrading bacteria, according to the cel48 to bacterial 16S rRNA gene ratios. Finally, the composition of the microbial culture also had an effect. The biodegradation patterns were different for the wetland-derived and anaerobic digester-derived cultures. RAMAN spectroscopy confirmed that there were different biodegradation patterns for the different functionalized forms, and that different bacterial cultures attacked the nanocelluloses differently. Currently metagenomic data is under examination to determine if different kinds of cellulose enzymes were stimulated by the different kinds of nanocellulose, and if these are distinct relative to those stimulated by natural forms of cellulose. In terms of Intellectual Merit, this research project is the first to our knowledge to directly examine the biodegradability of nanocellulose and the effect of functional groups. It provides an important proof-of-concept, indicating that nanocellulose does biodegrade differently than natural celluloses and that this should be examined more closely to ensure that the materials are fully biodegradable and non-toxic in the environment. Also, the project provides a framework for the concept of guiding green manufacturing, by informing which functional groups are least toxic and likely to influence biodegradation. Various disciplines can draw from and advance this work, including nanosciences, sustainable biomaterials, wood science, green manufacturing, environmental science, environmental engineering, and cellular and molecular biology. In terms of Broader Impacts, this project provided an ideal platform for training of graduate and undergraduate students on an interdisciplinary topic with importance to society. Three graduate students participated and completed the training for the Interdisciplinary Graduate Education Program (IGEP) in Sustainable Nanotechnology (SuN). Four undergraduates also received hands-on training through this project. Modules were also developed for ENGR 3134 Environmental Life Cycle Analysis, which is a core course in the undergraduate minor in Green Engineering at Virginia Tech. Material was also incorporated into GRAD 5134, a core course of the SuN IGEP program. The research was presented at the International Water Association Microbial Ecology in Water Engineering Conference, the Association for Environmental Engineering and Science Professors Conference, and the Sustainable Nanotechnology Organization Conferences. Presentations were also given at local venues to enhance outreach, including the Civil and Environmental Engineering Research Day and the Virginia Science Festival. Three journal publications related to this work were published, and one is in preparation and expected to be submitted in 2015. Blogs about the project were posted to the Virginia Tech Sustainable Nanotechnology website. The Virginia Tech Institute for Critical Technology and Applied Sciences (ICTAS) and the associated ICTAS SuN Center also partnered in this work.

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