Hydrological-Microbial Interactions Controlling Landscape Phosphorus MobilityJohn M. Regan1, Nicholas Locke1, M. Todd Walter2, Sheila Saia2, Hunter J. Carrick3, Shayna M. Taylor3, and Anthony R. Buda4

Introduction Field Experiments on Stream Biofilms

Conclusions and Future Work

Identification of Polyphosphate-Accumulating Organisms (PAOs)

This project focuses on phosphorus (P) mobilization-retention roles played by microbial activities unique to parts ofthe landscape prone to different patterns of saturating-drying cycles, from continuously saturated (e.g., streams) tovariably saturated (e.g., soils). The immediate goal of this project is to improve the scientific understanding of howthe interactions between hydrology and microbial processes affect P mobility and retention in the landscape. Thelong-term objective is to develop better land- and water-management strategies that capitalize on improved hydro-microbiological insights for reducing nonpoint source nutrient enrichment of freshwater bodies. Current activitiesare focused on polyphosphate (polyP) retention and release by polyP-accumulating organisms (PAOs) in streambiofilms, in which oxygenic phototrophy and respiration induce diurnal aerobic and anaerobic microenvironments.

We carried out a series of experiments in July-August of 2014 that provided a direct link between watershed P-loading and in-stream storage of total P (TP) and polyP in resident stream biofilms. Vials fitted with porous caps dispensed six levels of P-loading (0-1,000 ug P/L/day) at four sequential flumes situated within the FD-36 experimental watershed (Fig 5). After 14 days of deployment, microbiology techniques were used to measure chlorophyll (biomass proxy) and P storage by pro- and eukaryotic components of the biofilm assemblage. Results showed no difference biomass distribution at each location and P loading, but astrong correlation between P storage and P loading as well as biofilm polyP and TP (Fig 6).

• Bacterial PAO populations from stream biofilms were quite dissimilar from those typically enriched in wastewater treatment plants with enhanced biological phosphorus removal. FISH and polyP co-localization confirmed the identification of bacteria with this phenotype, and the method is being expanded to the analysis of soil communities from the FD-36 watershed.

• Stream biofilms appear to accumulate excess P, predominantly as polyP, as a function of external P-loading and independent of biofilm biomass levels. The role of taxonomic composition on storage is ongoing.

• Diurnal oxic and anoxic conditions, imposed on the bulk water in these experiments but arising naturally in biofilm microenvironments due to oxygenic phototrophy and respiration, promoted P release and removal in stream biofilms, likely due to PAO activity.

1 Dept. of Civil & Environmental Engineering, Penn State University; 2 Dept. of Biological & Environmental Engineering, Cornell University; 3Dept. of Biology & Institute for Great Lakes Research, Central Michigan University; 4 USDA-ARS Pasture Systems and Watershed

Management Research Unit, PA

AcknowledgementThis project is supported by USDA National Institute of Food and Agriculture award no. 2014-67019-21636.

Laboratory Experiments on Stream BiofilmsBench-top experiments were conducted using stream biofilms to determine the conditions in which these microbial assemblages take up and release P. Established biofilms were transferred to the lab (Fig 7) and subjected to various treatments. Systems with diurnal aerobic and anaerobic cycling showed P release under anaerobic conditions and P reductions during aeration, suggesting involvement of PAOs. Cation (i.e., Ca, K, and Mg) uptake and release mimicked phosphate cycling, which could be a result of known cation accumulation by PAOs and/or chemical precipitation of cation-P. Fe and S were not correlated with changes in P or redox conditions.

Fig 2. DAPI stains poly-P yellow, and PAO were separated by fluorescence-activated cell sorting.

Fig 3. Putative PAO communities showed similarities within stream productivity groupings.Benthic biofilm PAO populations were distinct from those found in enhanced biologicalphosphorus removal wastewater treatment systems (denoted with red box).

Fig 1. Biofilms collected from 1 – E. Hickory Cr., 2 – Cowanesque R., 3 – Red Clay Cr., 4 – Cooks Cr., 5 – Penns Cr., 6 – Spring Cr.

Fig 4. PAOs in East Hickory Ck. identified by FISH with probe RHO-PAO. Images show (a) nucleic acids, (b) poly-P granules, (c) RHO-PAO probe, and (d) EUB-338 mix probe

Fig 5. The FD-36 Experimental Watershed image with flumelocations, and deployment of vials with porous caps.

Fig 8. Observed (points) and modeled (lines) concentrations of (A) P, (B) Ca, (C) K, (D) Mg, (E) Fe2+, and (F) total S in the surrounding water as a function of time for treatment 1 (T1; alternating anaerobic/aerobic conditions) and treatment 2 (T2; aerobic conditions only). Black bar indicates periods when T1 was anaerobic, and non-marked periods for T1 were aerobic.

Fig 7. Biofilms from Cascadilla Cr. Watershed, NY were taken to the laboratory for testing.

Surprisingly little is known about PAOs in natural environments. We collected benthic biofilms from six PA streams representing a range of conditions and productivities (Fig 1). DAPI was used to stain intracellular polyP yellow (Fig 2). We then used flow cytometry with cell sorting to separate putative PAOs based on their DAPI-imparted yellow fluorescence (Fig 2). These sorted cells were sequenced using MiSeq 16S rRNA gene sequencing (Fig 3), and sequences of putative PAO populations were used to design oligonucleotide probes for microscopic colocalization of DAPI-stained polyP and fluorescent in situ hybridization (FISH) targeting the putative PAO (Fig 4). Fig 6. While productivity was not affected by P loading (not shown), biofilm

TP increased with increasing P loading (left), and there was a strongcorrelation between polyP and TP in these two-week biofilms (right).