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
Saia et al. (In Review)
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
Saia et al. (In Review)
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)
Locke et al. (In Preparation)
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0 100 200 300 400 500 600 700
Chlorophyll-a Measurement (mg/m2)
OligotrophicMesotrophic
Eutrophic
Locke et al. (In Preparation)
Similarly, PAO community structures were similar between biofilms from eutrophic streams
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0 100 200 300 400 500 600 700
Chlorophyll-a Measurement (mg/m2)
OligotrophicMesotrophic
Eutrophic
Locke et al. (In Preparation)
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)
Locke et al. (In Preparation)
