hydrological-microbial interactions controlling landscape phosphorus mobility

Hydrological-Microbial Interactions Controlling

Landscape Phosphorus Mobility

Jay ReganEnvironmental Engineering

USDA AFRI and NIWQP PD Meeting, October 12, 2016

M. Todd WalterBiological and Environmental Engr.

Hunter CarrickBiology

Anthony BudaUSDA-ARS Pasture Systems & Watershed Mgmt. Research Unit

How do microbial activities control P mobility in agroecosystems under fluctuating redox conditions?

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Field sampling:- Stream biofilms (diel

redox fluctuations)

- Soils across soil topographic index gradient (i.e., variable saturation frequencies)

Soil column sampling (controlled conditions)

Primarily focused on:

• Dissimilatory iron-reducing bacteria (DIRB)

• Polyphosphate-accumulating organisms (PAOs)

Dissimilatory iron-reducing bacteria use iron oxides under anaerobic conditions, which liberates P

Hypothesis – DIRB contribute to increased dissolved P under saturated (i.e., anaerobic) conditions

Modified from Weber et al (2006) Nature Reviews Microbiol.

Polyphosphate-accumulating organisms alternate between P release (anaerobic) and P uptake (aerobic)

Anaerobic

Glycogen

ATPPHA

Poly-P

PO43-

Acetic AcidCell Maint.

Aerobic

PO43-

Poly-P

Glycogen

ATPPHA

Cell Growth

H20½O2

PHA - Polyhydroxyalkanoate

Hypothesis – PAOs contribute to increased dissolved P under saturated (i.e., anaerobic) conditions and decreased dissolved P under unsaturated (i.e., aerobic) conditions

Molecular characterization of PAO populations in PA stream biofilms

Field tests on stream biofilm development with variable P loads

Bench tests on stream biofilms under diel redox cycles

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Emphases of this project report

Bench tests on stream biofilms under diel redox cycles

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• Stream biofilms collected• Cyclic aerobic and anaerobic conditions

imposed to mimic diel conditions in phototroph-dominated biofilms*

• Monitored soluble P, Fe, S, K, Mg, Mn• Measured PAO (DAPI staining and

fluorescence microscopy) in biofilms

*Diel oxygen trends in shallow lake sediments. (Carlton and Wetzel (1988) Limnology and Oceanography)

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Aerobic and anaerobic cycles induced P release during anaerobic periods (not observed in aerobic controls)

Saia et al. (In Review)

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Ca and K (counter ions used in polyphosphate granules) fluctuated with P, but Fe2+ and S (not shown) were stable


P trend not due to iron-reducing bacteria

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At the end of the experiment, more polyP-containing cells were detected in the aerobic biofilm


P trend may be due to PAO… P release from biofilm to water during anaerobic (i.e., night) conditions, uptake in biofilm during aerobic (i.e., light) conditions

Stream biofilms developed with variable P loads

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• Stream biofilms established using in situ enrichment system (vial with P-loaded agar and porous porcelain cap)

• Six different P loadings (incl. ambient)• Collected and analyzed for biomass and various

forms of P

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Increased P loadings did not affect biofilm biomass (Chl-a proxy), but did increase P storage as polyP

Taylor et al. (In Preparation)

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In biofilm P extracts, the polyP fraction increased from 12% at ambient P loading to 45% at max. P loading

Taylor et al. (In Preparation)

PIP – particulate inorganic PPOP – particulate organic P

Stream biofilms accumulate excess P largely as polyP

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Developing a method with lead nitrate staining and NanoSIMS quantification of polyP granules in diatoms

Molecular characterization of PAO in PA stream biofilms

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• Stream biofilms collected from six streams representing a range of geochemical conditions

Molecular characterization of PAO in PA stream biofilms

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• Intracellular polyP stained with DAPI• Biofilms dispersed• Putative PAOs separated with flow

cytometry and cell sorting• 16S rRNA genes of sorted cell fractions

sequenced with MiSeq

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Common Groups:• Ignavibacterium

album• Comamonadaceae• Thauera sp.• Pseudomonas

Sequencing of sorted (yellow) cells showed putative PAO populations

Locke et al. (In Preparation)

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0 100 200 300 400 500 600 700

Chlorophyll-a Measurement (mg/m2)

OligotrophicMesotrophic

Eutrophic

PAO community structures were similar among biofilms from oligotrophic streams (Burkholderiales dominated)


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0 100 200 300 400 500 600 700



Eutrophic


Similarly, PAO community structures were similar between biofilms from eutrophic streams

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0 100 200 300 400 500 600 700



Eutrophic


Interestingly, PAO similar to those that predominate many wastewater treatment systems was rare in all biofilm samples

Summary of stream biofilm studies

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• Polyphosphate-accumulating organisms are present in stream biofilm communities

• These PAO contribute to P storage within the biofilm and mobility within the biofilm and overlying waters

• PAO populations in stream biofilms are quite dissimilar from those enriched in enhanced biological phosphorus removal systems… physiology largely uncharacterized

Soil column studies to uncouple DIRB and PAO contributions to P uptake/release under variable saturation conditions

Analysis of P trends and metagenomic analysis of microbial communities

Soil sampling across a range of soil topographic index conditions

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Ongoing research on soil microbial processes

Acknowledgements

Nicholas Locke, Claudia Rojas, Miranda Stockton

USDA AFRI Grant #2014-67019-21636

Sheila Saia

Shayna TaylorKyle Elkin

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Based on flow cytometry data, putative PAOs comprised 13-38% of biofilm-derived particles (cells)
