Impact of Pristine Graphene on Intestinal Microbiota Assessed Using a Bioreactor-Rotary Cell Culture System.

The increased use of graphene in consumer products such as food contact materials requires a thorough understanding of its effects on the gastrointestinal commensal bacterial population. During the first phase of study, three representative commensal bacterial species (, , and ) were exposed to different concentrations (1, 10, and 100 μg/mL) of pristine graphene for 3, 6, and 24 h in the Bioreactor Rotary Cell Culture System (BRCCS) which allowed a continuous interaction of intestinal microbiota with the pristine graphene without precipitation of test material. The results showed that pristine graphene had dose-dependent effects on the growth of selective bacteria. To study the interaction of graphene with more diverse consortia of intestinal microbiota, fresh fecal samples from laboratory rats were used. Rat fecal slurry (3%) was maintained in an anaerobic environment and treated with different concentrations (1, 10, and 100 μg/mL) of pristine graphene for 3, 6, and 24 h. Counts of viable aerobic and anaerobic bacteria were assessed and fecal slurries were also collected for microbial population shift analysis using quantitative real-time PCR, as well as 16s rRNA sequencing. The results showed a significant two-fold increase in both aerobic and anaerobic bacterial counts (expressed as colony forming unit; CFU) during the first 3 h of exposure to all pristine graphene concentrations. However, 24 h of continuous exposure resulted in a 120% decrease in the CFU of aerobic bacteria at the highest concentration and the anaerobic bacteria CFU remained unchanged. Multivariate analysis of the q-PCR data showed that the exposure time, as well as the graphene concentrations, impacted the bacterial population abundance. Community analysis of graphene-treated fecal samples by 16S sequencing revealed significant alteration of 15 taxonomic groups, including 9 species. The increased abundance of butyrate-producing bacteria (, , and ) was correlated with an increase of the short-chain fatty acid, butyric acid after exposure to graphene. These results clearly indicate that graphene may cause adverse effects on the intestinal microbiome at the doses equal to 100 μg/mL. Further experiments using intestinal explants (nonanimal model) could reveal the mechanisms by which graphene could perturb the microbe-host intestinal mucosa homeostasis.