Classification of polyphosphate-accumulating bacteria in benthic biofilms

Open Access
Locke, Nicholas Albert
Graduate Program:
Environmental Engineering
Master of Science
Document Type:
Master Thesis
Date of Defense:
July 08, 2015
Committee Members:
  • John Michael Regan, Thesis Advisor
  • Anthony Buda, Thesis Advisor
  • William D Burgos, Thesis Advisor
  • polyphosphate accumulating organisms
  • phosphorus
  • eutrophication
  • nonpoint source pollution
Polyphosphate accumulating organisms (PAOs) are microorganisms known to store excess phosphorus (P) as polyphosphate (poly-P) in environments subject to alternating aerobic and anaerobic conditions. There has been considerable research on PAOs in biological wastewater treatment systems, but very little investigation of these microbes in freshwater systems. We hypothesize that putative PAOs in benthic biofilms of shallow streams where daily light cycles induce alternating aerobic and anaerobic conditions are similar to those found in EBPR. To test this hypothesis, cells with poly-P inclusions were isolated, classified, and described. Eight benthic biofilms taken from a first-order stream in Mahantango Creek Watershed (Klingerstown, PA) represented high and low P loadings from a series of four flumes and were found to contain 0.39 - 6.19% PAOs. A second set of eight benthic biofilms from locations selected by Carrick and Price (2011) were from third-order streams in Pennsylvania and contained 11-48% putative PAOs based on flow cytometry particle counts. Putative PAOs of the second sample set represented 61 unique operational taxonomic units with taxonomic classification including members of Actinomycetales, Bacillales, Ignavibacteriales, Sphingomonadales, Burkholderiales, and Rhodocyclales. Streams with similar putative PAO species richness were found to have similar trophic states. Canonical correlation analysis showed that some PAO groups were directly related to pH, temperature, conductivity, or stream cover. Four probes targeting the predominant PAO groups of Ignavibacterium, Thauera, Comamonadaceae, and Pseudomonas allowed the colocalization of poly-P and group identity. Members of Thauera and Comamonadaceae were confirmed as PAOs. These results greatly expand our understanding of in-stream PAO-facilitated P cycling and will inform P fate and transport models to better simulate in-stream P cycling in lotic systems.