Is a Mineral Surface Critical to Rapid and Successful Anaerobic Benzene Biodegradation? Kathlyne Hyde (PhD Candidate) , Derek Peak, Kris Bradshaw, and Steven D. Siciliano Presenting Members SEIMA SustainTech March 22 2018 Integrity • Excellence • Responsibility
What we know about our PHC contaminated sites... • Soils in which we have learned primarily have adsorbed P. When P conc is increased, precipitation of phosphorus minerals is highly favorable. (Siciliano et al. 2016) 2- , Fe 3+ , Citrate, and PO 4 - , SO 4 3- . NO 3 Image courtesy of L. Moelhman
Linking soil mineralogy and microbiology What happens when we inject these solutions? How can we understand our sites better to cater the amendment for the best possible outcome?
Mineralogical Effects Mineralogy controls reactive ions in soil solution and groundwater. Has a direct effect on key nutrient availability, particularly orthophosphate equilibrium for precipitation and dissolution reactions. Orthophosphate availability will directly control microbial function and biomass, thereby affecting the degradative community.
Orthophosphate • Limiting nutrient for microbial growth • Highly reactive in soils Adsorption complexes 2- and H 2 PO 4 • - Dominate forms are HPO 4 • Outer-sphere • Inner-sphere Bidentate mononuclear and monodentate mononuclear bonding of orthophosphate to hematite surface groups (Hanrahan et al., 2015)
Representative Minerals • Reactive Surface: Hematite • Iron oxide α -Fe 2 O 3 Mostly singly coordinated oxygen in the hexagonal orienta tion • • PZC ~ 8.2 = positively charge surface at neutral pH • Unique ability as a reactive surface with electron transfer • Using hematite for experiments opens up the possibility for understanding and using other adjuncts for in-situ remediation. (Sparks, 2013; Trainor et al., 2004)
Representative Minerals • Semi-Reactive Surface: Corundum • Aluminum oxide α -Al 2 O 3 • Doubly coordinated oxygen in the hexagonal orientation • PZC 4-6 or 8-10 (Trainor et al., 2004)
Enrichment Culture: The Ulrich Culture • Oil sands process affected water • Mixed culture • Nitrate – reducing benzene – degraders • Common Genera: Azoarcuz & Thauera Possible benzene degradation pathways 1) Methylation 2) Hydroxylation 3) Carboxylation Many metabolic processes Benzene DL-Benzylsuccinic acid Cultures courtesy of Dr. Ania Ulrich, University of Alberta
Hypotheses 1) Hematite (reactive surface – inner-sphere) and corundum (partially reactive surface – outer-sphere) will have similar capacities for adsorbing orthophosphate. 1) Benzene degradation rates will increase in the presence of hematite due to unique community biofilm formation. 2) Orthophosphate adsorbed to mineral surfaces via inner- sphere and outer-sphere complexes is accessible to bacteria – specifically hydrocarbon degraders. 3) The orientation of a benzene molecule differs between aqueous and adsorbed phases, thus possibly making it more accessible for bacteria to use as a carbon source.
Laboratory Experiments 1) Adsorption isotherms to determine surface coverage of orthophosphate. 2) Incubate benzene degrading cultures under nitrate reducing conditions (two exp). 3) ATR-FTIR spectroscopy to investigate benzene adsorption on hematite.
Adsorption isotherms (22 ° C) g L -1 mineral • 1 • Increasing the orthophosphate concentration and subsampling • Constant pH • Measure solution P via colorimetric techniques
Adsorption Isotherms (22 ͦ C) 1600 Corundum pH 6.2 Corundum pH 7.2 1400 Hematite pH 6.2 Hematite pH 7.2 Adsorbed PO 4 -P (mg kg -1 ) 1200 1000 800 600 400 ~80-90% monolayer surface coverage 200 2- pH 6.2 HPO 4 2- & H 2 PO 4 - pH 7.2 HPO 4 0 0.0 0.5 1.0 1.5 2.0 2.5 Solution PO 4 -P (mg L -1 )
Experiment 1: Microbial Kinetics • Original media (high P) • Media only sterile controls and inoculated • 4 mM P • Hematite sterile controls and inoculated • FeS • With high P and low P • Low P media • 400 μM (~80% monolayer • 3% v/v inoculant coverage) • 10 g L -1 hematite
- and NO 2 - Concentrations – Evidence of Active Denitrifiers NO 3 14 80 Hematite No Hematite 12 60 10 Nitrate Conc (mg L -1 ) Nitrite Conc (mg L -1 ) 8 40 6 20 4 2 0 0 Hematite No Hematite 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Day Day Increasing nitrite and depletion in Decreasing nitrate in hematite cultures hematite cultures
Faster benzene degradation in hematite cultures
Dialysis Tubing Incubation Experiment • Were the increased degradation rates due to the mineral surface or the hematite changing the solution chemistry? • Use of dialysis tubing to separate the microbes from the mineral allowing for 3- , to pass nutrients, such as PO 4 freely.
Dialysis Tubing Experimental Design Treatments and sterile controls: • Media only • Dialysis tubing • Hematite • Corundum • Microbes inside or outside of tubing
Microbes Microbes Microbes with separated Microbes with separated from Dialysis tubing Media Only Corundum from Hematite Hematite Corundum Microbes out Microbes in Microbes out Microbes in Microbes in or out
Samples are rotating at 14 rpm
The first inoculation had no degradation across treatments over 160 days. https://www.cartoonstock.com/directory/f/failure.asp
We replenished and re-inoculated the cultures H 2 S production = sulfate reducers Normal pale pink for nitrate reducers
Energetically, nitrate is significantly more favourable than sulfate. Figure modified from A. Ulrich and E. Edwards, (2003). Physiological and molecular characterization of anaerobic benzene-degrading mixed cultures. Environmental Microbiology. 5: 92 – 102.
Dialysis tubing likely stalled degradation 50 Sterile Control Sterile Control Media only Media only Dialysis (microbes out) Dialysis (microbes out) Hematite (microbes in) Hematite (microbes in) 40 Benzene (mg/L) Sterile Cont Media only Dialysis (mi 30 Media on 20 Dialysis ( Hematite 0 10 20 30 40 50 Days Dialysis (m Hematite (
ATR-FTIR Theory • Natural vibrations of molecules • Stretching, bending, twisting, etc. • The vibrations have an electrical field, when the infrared radiation electrical field matches that of the molecule, it increases the amplitude of the vibration. • IR active vibrations (peaks in a Figure 1-2. Molecular vibrations of carbon dioxide. spectrum) indicates there is a change in the dipole moment (unequally shared electrons) (Phillips, 2015)
Predicting relaxed orientations of benzene on hematite (Dzade, Roldan, and Leeuw, 2014) Parallel π bonding Slant π π bonding Vertical weak hydrogen H-bond bonding
ATR-FTIR Spectra: Benzene H O H Benzene images from: https://www.masterorganicchemistry.com/2017/02/23/rules-for-aromaticity/
Conclusions • The orthophosphate adsorption capacities of hematite and corundum are not drastically different, despite having different types of complexation. • The presence of hematite enhanced microbial benzene degradation (likely by denitrifying bacteria) when compared to media controls with no hematite in the 1 L cultures. • Dialysis tubing may be stalling and changing the active consortia. • Benzene’s dipole moments differ between pure state (l), in water ( aq), and when adsorbed to hematite. The differences in benzene’s molecular state may influence bioavailability to hydrocarbon degrading consortia.
What does this research mean for industry remediation efforts? Aim to demystify the microbial-mineral interactions when amendment solutions are used. Contribute to finding out why some sites remediation efforts work better than others by linking microbiology and mineralogical effects.
Acknowledgments Supervisors Dr. Steven Siciliano Dr. Derek Peak Lab Technician Alix Schebel Undergraduate Summer Student Samantha Chomyshen Environmental Toxicology Lab Group Environmental Chemistry Lab Group Integrity • Excellence • Responsibility
Questions? Integrity • Excellence • Responsibility
ATR-FTIR set up Hematite deposit on diamond crystal with aqueous benzene solution.
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