title: Separation of Bacteria, Protozoa and Carbon Nanotubes by Density Gradient Centrifugation.
abstract: Sustainable production and use of carbon nanotube (CNT)-enabled materials require efficient assessment of CNT environmental hazards, including the potential for CNT bioaccumulation and biomagnification in environmental receptors. Microbes, as abundant organisms responsible for nutrient cycling in soil and water, are important ecological receptors for studying the effects of CNTs. Quantification of CNT association with microbial cells requires efficient separation of CNT-associated cells from individually dispersed CNTs and CNT agglomerates. Here, we designed, optimized, and demonstrated procedures for separating bacteria (Pseudomonas aeruginosa) from unbound multiwall carbon nanotubes (MWCNTs) and MWCNT agglomerates using sucrose density gradient centrifugation. We demonstrate separation of protozoa (Tetrahymena thermophila) from MWCNTs, bacterial agglomerates, and protozoan fecal pellets by centrifugation in an iodixanol solution. The presence of MWCNTs in the density gradients after centrifugation was determined by quantification of14C-labeled MWCNTs; the recovery of microbes from the density gradient media was confirmed by optical microscopy. Protozoan intracellular contents of MWCNTs and of bacteria were also unaffected by the designed separation process. The optimized methods contribute to improved efficiency and accuracy in quantifying MWCNT association with bacteria and MWCNT accumulation in protozoan cells, thus supporting improved assessment of CNT bioaccumulation.
authors.affiliation: Bren School of Environmental Science and Management, Earth Research Institute and University of California Center for the Environmental Implications of Nanotechnology (UC CEIN), University of California, Santa Barbara, CA 93106, USA; Laboratory of Environmental Toxicology, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 12618, Estonia.
authors.forename: Monika
authors.initials: M
authors.lastname: Mortimer
authors.affiliation: Biosystems and Biomaterials Division, Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
authors.forename: Elijah J
authors.initials: EJ
authors.lastname: Petersen
authors.affiliation: Center for Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory, Livermore, CA 94550, USA.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Buchholz
authors.affiliation: Bren School of Environmental Science and Management, Earth Research Institute and University of California Center for the Environmental Implications of Nanotechnology (UC CEIN), University of California, Santa Barbara, CA 93106, USA.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Holden
keywords: Pseudomonas aeruginosa; Stokes’ law; Tetrahymena thermophila; bioaccumulation; bioconcentration; carbon-14; iodixanol; sucrose