Rapid and Successful Anaerobic Benzene Biodegradation? Kathlyne Hyde - - PowerPoint PPT Presentation

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Presenting Members

Is a Mineral Surface Critical to Rapid and Successful Anaerobic Benzene Biodegradation?

Kathlyne Hyde (PhD Candidate), Derek Peak, Kris Bradshaw, and Steven D. Siciliano

SEIMA SustainTech March 22 2018

What we know about our PHC contaminated sites...

NO3

• , SO4

2- , Fe3+, Citrate, and PO4 3-.

Image courtesy of L. Moelhman

• Soils in which we have learned

primarily have adsorbed P. When P conc is increased, precipitation

• f phosphorus minerals is highly
• favorable. (Siciliano et al. 2016)

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?

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.

Mineralogical Effects

Orthophosphate

• Limiting nutrient for microbial growth
• Highly reactive in soils
• Dominate forms are HPO4

2- and H2PO4

• (Hanrahan et al., 2015)

Adsorption complexes

• Outer-sphere
• Inner-sphere

Bidentate mononuclear and monodentate mononuclear bonding of orthophosphate to hematite surface groups

Representative Minerals

(Sparks, 2013; Trainor et al., 2004)

• Reactive Surface: Hematite
• Iron oxide α-Fe2O3
• Mostly singly coordinated oxygen in the hexagonal orientation
• 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.

Representative Minerals

(Trainor et al., 2004)

• Semi-Reactive Surface: Corundum
• Aluminum oxide α-Al2O3
• Doubly coordinated oxygen in the hexagonal orientation
• PZC 4-6 or 8-10

Enrichment Culture: The Ulrich Culture

Many metabolic processes

• Oil sands process affected water
• Mixed culture
• Nitrate – reducing benzene – degraders
• Common Genera: Azoarcuz & Thauera

Cultures courtesy of Dr. Ania Ulrich, University of Alberta

Possible benzene degradation pathways Methylation 1) Hydroxylation 2) Carboxylation 3)

Benzene DL-Benzylsuccinic acid

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

• f 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.

Hypotheses

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)

1

• g L-1 mineral

Increasing the

• rthophosphate

concentration and subsampling Constant pH

• Measure solution P via
• colorimetric techniques

Solution PO4-P (mg L-1)

0.0 0.5 1.0 1.5 2.0 2.5

Adsorbed PO4-P (mg kg-1)

200 400 600 800 1000 1200 1400 1600 Corundum pH 6.2 Corundum pH 7.2 Hematite pH 6.2 Hematite pH 7.2

Adsorption Isotherms (22 ͦ C)

~80-90% monolayer surface coverage

pH 6.2 HPO4

2-

pH 7.2 HPO4

2- & H2PO4

Experiment 1: Microbial Kinetics

• Media only sterile controls and inoculated
• Hematite sterile controls and inoculated
• With high P and low P
• 3% v/v inoculant
• Original media (high P)
• 4 mM P
• FeS
• Low P media
• 400 μM (~80% monolayer

coverage)

• 10 g L-1 hematite

NO3

• and NO2
• Concentrations – Evidence of Active Denitrifiers

Day

5 10 15 20 25 30

Nitrite Conc (mg L-1)

2 4 6 8 10 12 14 Hematite No Hematite

Day

5 10 15 20 25 30

Nitrate Conc (mg L-1)

20 40 60 80 Hematite No Hematite

Decreasing nitrate in hematite cultures Increasing nitrite and depletion in hematite cultures

Faster benzene degradation in hematite cultures

• 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 nutrients, such as PO4

3-, to pass

freely.

Dialysis Tubing Incubation Experiment

Treatments and sterile controls:

• Media only
• Dialysis tubing
• Hematite
• Corundum
• Microbes inside or outside of tubing

Dialysis Tubing Experimental Design

Media Only Dialysis tubing Microbes with Corundum Microbes separated from Corundum Microbes with Hematite Microbes separated from Hematite

Microbes in Microbes in Microbes out Microbes out Microbes in

• r out

Samples are rotating at 14 rpm

https://www.cartoonstock.com/directory/f/failure.asp

The first inoculation had no degradation across treatments over 160 days.

We replenished and re-inoculated the cultures

H2S 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

• f anaerobic benzene-degrading mixed cultures. Environmental Microbiology. 5: 92 – 102.

10 20 30 40 50 20 30 40 50 Days Benzene (mg/L)

Sterile Control Media only Dialysis (microbes out) Hematite (microbes in)

Dialysis tubing likely stalled degradation

Sterile Cont Media only Dialysis (mi Media on Dialysis ( Hematite Dialysis (m Hematite (

Sterile Control Media only Dialysis (microbes out) Hematite (microbes in)

ATR-FTIR Theory

Figure 1-2. Molecular vibrations of carbon dioxide.

(Phillips, 2015)

• 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

spectrum) indicates there is a change in the dipole moment (unequally shared electrons)

Predicting relaxed orientations of benzene on hematite (Dzade, Roldan, and Leeuw, 2014)

Parallel

π bonding

Slant

π bonding

Vertical weak hydrogen bonding π

H-bond

ATR-FTIR Spectra: Benzene

Benzene images from: https://www.masterorganicchemistry.com/2017/02/23/rules-for-aromaticity/

O H H

• 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. Conclusions

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.

What does this research mean for industry remediation efforts?

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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

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Questions?

ATR-FTIR set up

Hematite deposit on diamond crystal with aqueous benzene solution.