This project examines the role of microbial activities in controlling phosphorus mobility in landscapes prone to saturation-drying cycles, from continuously saturated streams to variably saturated soils. Laboratory and field experiments were conducted to better understand how hydrology and microbes interact to affect phosphorus retention and release. Results showed that stream biofilms accumulated phosphorus, mostly as polyphosphate, in response to external phosphorus loading levels independently of biofilm biomass. Diurnal changes in oxygen levels within biofilms, mimicking natural conditions, promoted phosphorus release under anaerobic conditions and removal under aerobic conditions, likely due to the activity of polyphosphate-accumulating organisms (PAOs). The identification of putative PAO populations in stream biofilms provided insights

1. Hydrological-Microbial Interactions Controlling Landscape Phosphorus Mobility
John 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 of
the landscape prone to different patterns of saturating-drying cycles, from continuously saturated (e.g., streams) to
variably saturated (e.g., soils). The immediate goal of this project is to improve the scientific understanding of how
the interactions between hydrology and microbial processes affect P mobility and retention in the landscape. The
long-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 activities
are focused on polyphosphate (polyP) retention and release by polyP-accumulating organisms (PAOs) in stream
biofilms, 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 a
strong 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; 3
Dept. of Biology & Institute for Great Lakes Research, Central Michigan University; 4 USDA-ARS Pasture Systems and Watershed
Management Research Unit, PA
Acknowledgement
This project is supported by USDA National Institute of Food and Agriculture award no. 2014-67019-21636.
Laboratory Experiments on Stream Biofilms
Bench-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 biological
phosphorus 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 flume
locations, 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 strong
correlation between polyP and TP in these two-week biofilms (right).