A Novel Approach for Characterizing the Ultra-Micro Size-Fraction Community

A Novel Approach for Characterizing the Ultra-Micro Size-Fraction Community

Students: Abdullah Salim, Priscilla Pineda, Isabella Alamilla

Mentors/Supervisors: Andrew Putt, Terry C. Hazen

ABSTRACT

The ultra-micro size-fraction (UMSF) are bacteria that can pass through the 0.2 µm pore membrane filters employed in environmental surveys. Despite being ubiquitous and having high metabolic activity, UMSF remain elusive and largely uncultured. Investigations of UMSF are skewed by difficulties in culturing and a lack of techniques for measuring UMSF biogeochemical signatures. This study measures surface stream UMSF community diversity, and community response to the addition of the synthetic pharmaceutical and cosmetic carbon product cyclodextrin which was injected into half of the thirty-six 100 mL anaerobic microcosms. Microcosms were filled from anaerobically collected rural stream water processed using two methods defined as, single-filtered (1F) and double-filtered (2F). The 1F inoculation involved only filtration with a 0.2 µm pore diameter polyethersulfone RapidFlow^TM filter, and the 2F inoculation involves re-filtering the water through a 0.22 µm Nylon Syringe filter during serum bottle inoculation. Various filtration and DNA extraction techniques were developed and tested for microcosm biofilms and filters while carbon source phenotypic microarrays were performed to determine UMSF community metabolic response. On days 10 and 24, replicate microcosms were destructively sampled to measure pH, acridine orange direct cell counts, and ions with DNA extracted from planktonic cells collected on 0.1 and 0.2 µm filters and bottle biofilms. Isolates were successfully cultured from day 24, microcosm water that was used to inoculate enriched R2A media plates and anaerobic culture tubes. The concentration of 0.1 µm DNA steadily increased over the course of the study with the 1F cyclodextrin samples containing the highest concentration of 0.1 µm DNA. The 1F and 2F cyclodextrin bottle biofilms and 0.2 µm biomass demonstrated consistently higher average DNA concentrations than the control. The optical density growth curves of the day 24 anaerobic tubes showed log phase growth occurred primarily in the control samples from hour 18 to hour 50 with lower average densities among cyclodextrin injected samples. Work is continuing on isolate characterization and 16S rRNA amplicon sequencing using an Illumina® MiSeq while the lab techniques employed here will guide future research into the UMSF and understanding their relationship with man-made carbon sources.