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 of 14C-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.

Increasing evidence indicates low bioaccumulation of carbon nanotubes

“…In one study on the trophic transfer of MWCNTs from bacteria to protozoa, the BMF was below 1 (ranging from 0.01 to 0.04) for all conditions tested 89, 90 . There are also some studies which have demonstrated the capacity for metal-based ENMs such as gold (Au), cerium dioxide, lanthanum oxide and titanium oxide nanoparticles to be transferred along a food chain 91–97 .…”

A density gradient centrifugation method for rapid separation of nanoTiO2 and TiO2 aggregates from microalgal cells in complex mixtures with mercury

“…Here we present a novel method for separation of the nanoTiO 2 and their aggregates from green microalga Chlamydomonas reinhardtii and subsequent determination of the bioaccumulated Hg during exposure to mixtures of Hg and nanoTiO 2 . This method is adaptation of a previous methodology successfully used to separate bacterium Pseudomonas aeruginosa from unbound multiwall carbon nanotubes (MWCNTs) and MWCNT aggregates [9] .…”

Challenges in characterizing the environmental fate and effects of carbon nanotubes and inorganic nanomaterials in aquatic systems

“…116 In the case of bacteria and protozoa, an approach employing separation of 14 C-labeled functionalized MWCNTs by density gradient centrifugation was described. 117 The bacterium Pseudomonas aeruginosa was found to adsorb the functionalized MWCNTs at concentrations of 0.18 ± 0.04 and 21.9 ± 4.2 μg per mg dry mass at respective nominal concentrations of 0.01 and 1 mg L −1 . In the same study, an accumulation of up to 0.9 ± 0.3 μg of functionalized MWCNT per mg dry mass was recorded in the protozoan Tetrahymena thermophila following trophic transfer via MWCNT-encrusted Pseudomonas aeruginosa cells, while up to 3 ± 1 μg of functionalized MWCNT per mg dry mass was detected following direct uptake.…”

“…123 Current methodologies for isolating CNTs and their transformed forms from complex matrices are still limited, whilst techniques for accurately characterizing and quantifying CNTs also remain underdeveloped. 23,98,117,[124][125][126] Furthermore, the presence of low CNT concentrations in environmental matrices and the difficulty in distinguishing natural sources of carbon from those corresponding to CNTs compound the challenges of separation and analysis. The challenge of determining CNT uptake and accumulation in organisms and environmental samples highlights the necessity of using radiolabeling in laboratory studies.…”