Alginate Encapsulated Nanoparticle-Microorganism System for

Trichloroethylene Remediation

Outline

Nanoparticles NZVI and TCE Need Statement, Objectives, and Hypotheses Prior Work Research Phase I : TCE Degradation with NZVI Research Phase II : TCE degradation with Pseudomonas and

NZVI Research Phase III : TCE degradation with Dehalococcoides

and NZVI Conclusions and Future Work Acknowledgements

2

3

Introduction

Global Nano Market

US$ in Billion (2015)Source: NSF

4

Nanoparticles (NPs)5

(NPs) < 100 nm

NPs : Colloidal particles, Reactive species

NPs Classification

Metallic (gold, iron, nickel, cobalt)

Semiconductor (quantum dots)

Polymeric (poly (L-lactic acid or PLA))

Others (Clay NPs)

ASTM International, 2006; Liu et al., 2006

Applications of Nanoparticles

Natural processes: Ubiquitous

Cosmetics: TiO2, ZnO

Hand Sanitizers: Ag NPs

Lipsticks as pigments: Iron oxide NPs

Dental fillers, prosthetic implants, drug carriers: SiO2 NPs

Environmental applications

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NPs for Environmental Remediation

Water quality improvements

Treatment - contaminated soils, sediments and solid wastes

Halogenated/ Polychlorinated hydrocarbons, certain pesticides

(DDT, lindane)

Hydrocarbons, dyes, and other inorganic anions such as nitrate,

perchlorate, dichromate, and arsenate

US EPA, 2007; Thompson et al., 2010; Zhang, 2003

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Nanoparticles Contaminant ReferenceNano Zeolites Toluene, nitrogen dioxide Song et al. (2004)

Carbon nanotubes (CNTs) p-nitrophenol, benzene, toluene, heavy metal ions,dimethylbenzene

Jin et al. (2007)

Bimetallic nanoparticles (Pd/Fe)

PCBs, chlorinated methane, ethene, Trichloroethylene (TCE)

Xu and Bhattacharyya (2005)Xu and Zhao (2007)Wang and Zhang (1997)Kim et al. (2010)

Ni/Fe and Pd/Au nanoparticles

TCE and PCBs He and Zhao (2005)

TiO2 photocatalyst Heavy metal ionsPentachlorophenol (PCP)

Pena et al. (2005)Quan et al. (2005)

NZVI Herbicides (Alachlor) Thompson et al. (2010)

NZVI Nitrate Bezbaruah et al. (2009)

NZVI TCE Bezbaruah et al. (2011)

NPs for Water Remediation8

Why NZVI?

Non-toxic and inexpensive

Feº is a potential reducing agent

Reactive species

High surface area

Can reductively degrade contaminants

Image Credit: Matheson, and Tratnyek, 1994

Fe0 Fe 2+ + 2e-

R-X + 2e- + H+ R-H + X-

Bezbaruah et al. 2009; Thompson 2010; Zhang 2003

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Contaminant

Trichloroethylene (TCE)

Solvent / Degreasing agent

WHY TCE?

Ranked as one of the most hazardous compounds (worst 10%)

Most commonly detected volatile organic in groundwater

Suspect carcinogen /Endocrine toxicant/Kidney toxicant

EPA permissible levels: 5 ppb

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U.S. EPA, 2011

TCE Contaminated Sites

61% NPL sites contaminated with TCE

Valley City, ND

Baytown township groundwater contamination site (Minnesota) – monitored by MPCA

Hibbing, Minnesota

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USEPA 2007

In-situ Injection

Injection Well

Groundwater Flow

TCE Contaminated plume

NZVI

Aquifer

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Present Study

Challenges

High mobility causes NZVI to sediment out in the aquifer pores and become unavailable for contaminant remediation

Existing technology for TCE removal is not efficient enough

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More on TCE Remediation Methods15

Physical Methods: Expensive/Maintenance

Chemical Methods: By-product generation, short life span

Bioremediation: Slow/ Time consuming

There is a need to develop a more efficient method

Objectives

To engineer a metal-microorganism system for TCE remediation

Specific Objectives

Encapsulation of NZVI (in Ca-alginate capsules)

TCE remediation with encapsulated NZVI

Encapsulation of TCE degrading bacteria

TCE remediation with encapsulated bacteria

TCE remediation with NZVI-microorganism system

Quantify the reaction kinetics of TCE degradation

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Hypotheses17

NZVI can reduce TCE as the first step in the degradation process and then the microorganisms can preferentially take over the process

NZVI-microorganism system will effectively dechlorinate TCE to benign end products

TCE Degradation Mechanism - NZVI

C=C Cl

Cl H

Cl

Fe0

Cl-

e-

C=C H

Cl H

ClFe0

Cl-

e-

C=C H

Cl H

H

Fe0

Cl-

e-

C=C H

H H

H

TCE DCE

VC

Ethene

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Model Microorganisms

Pseudomonas putida F1 strain

Capable of oxidizing organic contaminants

Dehalococcoides sp.

Reductive dechlorination

Converts TCE benign products (ethene)

Some strains convert TCE VC

D. BAV1 : TCE Ethene

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www.miller-mccune.com

http://gtresearchnews.gatech.edu/

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Prior Work

P. putida F1 Interactions with NZVI

Shabnam, 2011

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E. coli 8739 interactions with NZVI 22

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Experimental Design

Experimental Design24

Microorganisms

Pp F1D.

BAV1

Encapsulated Microorganisms

Combined NZVI-Microorganism System

TCE Degradation

NZVI

Bare Encapsulated

Encapsulated NZVI

Research Phase I

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Research Phase I

Encapsulated NZVI System

Research Phase I

Synthesis of NZVI

Preparation of Ca-alginate Capsules (Reactors)

Encapsulation of NZVI

Batch Experiments:

Diffusion Studies

TCE Degradation Studies

Summary

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Synthesis of NZVI

NZVI-synthesized by borohydroxide reduction method 2Fe2+ + BH4

- + 3H2O 2Fe0↓ + H2BO3- + 4H+ + 2H2

TEM image of clustered nanoparticles

Krajangpan et al. 2009; Bezbaruah et al. 2009; Bezbaruah et al. 2011

a

b

a and b: Dried NZVI

XRD spectrum of NZVI

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Encapsulation

“Encapsulation” confining compounds within a matrix or

membrane in particulate form

Encapsulated particles/compounds are free to move

“Entrapment” embedding of compounds within a matrix

Entrapped particles/compounds are not free to move

Mobility and settlement problems can be overcome using

NZVI

(Bezbaruah et al., 2009; Bezbaruah et al., 2011)

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Why alginate?

Bio-degradable

Non-toxic

Porous

Inexpensive

Used in bacterial cell immobilization

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Hill and Khan, 2008

Preparation of Capsules

4 g Maltodextrin6 mL DI water

0.25 g CaCl2

50 mL alginate

Peristaltic pump

Stirrer

6 cm b

a: Capsules

30 mg NZVI

1.33% Iron loss

a

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Bezbaruah et al., 2011

b

b: Encapsulated NZVI

Batch Experiments

DIFFUSION STUDIES

Empty alginate capsules used

45 mL reactors containing 30 and 40 mg TCE /L

Shaken in a rotary shaker

Samples (40 µL) collected over specific time intervals

Experiments done in triplicates

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Capsules

TCE Solution

40 mL Vial

Diffusion Study Results

Bulk TCE concentration decreased and then began to level off ~45 min

Establishes that: NO major mass transfer barrier for contaminant diffusion through Ca-alginate capsules

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TCE Degradation Batch Studies33

Encapsulated NZVI

Samples collected over time

2 h (Rotated)Blank Control Analysis

Capsules

TCE Solution

TCE Solution

TCE Solution+ Bare NZVI

TCE Degradation Batch Study

TCE removal efficiency of 88-90% using bare NZVI

TCE removal efficiency of 89-91% using encapsulated NZVI

Bare and encapsulated systems performed almost the same

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TCE Degradation Kinetics

TCE degradation followed First Order Kinetics for both bare and encapsulated NZVI

No significant difference Bare and Encapsulated NZVI (95% CI, Two Way ANOVA )

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Batch Initial TCE concentration

mg L-1

Reaction rate constant R2

k obs

10-2 min-1

ksa

10-3 L m-2 min-1

Bare NZVI 1 2.92 1.6 0.968910 2.35 1.3 0.980130 1.53 0.8 0.989740 2.24 1.2 0.9868

Encapsulated NZVI

1 3.23 1.7 0.983210 2.45 1.3 0.949130 1.92 1.0 0.992140 2.21 1.2 0.9425

Bezbaruah et al., 2011

Encapsulated NZVI: pH Observations

pH changed from 6 to 9 as the reaction proceeded

Similar results irrespective of concentration

No NZVI – No pH change

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Shelf-life Study

Conducted over 6 months Shelf-life 4 months

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Research Phase I-Summary

Encapsulated NZVI degraded 89-91% of TCE

Shelf-life of 4 months

TCE removal with NZVI followed First Order Kinetics

Potential scope for groundwater applications

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Work Remaining39

Microorganisms

PpF1D.

BAV1

Encapsulated Microorganisms

Combined NZVI-Microorganism system

TCE Degradation

NZVI

Bare Encapsulated

Encapsulated NZVI

Experimental Design40

Microorganisms

PpF1D.

BAV1

Encapsulated Microorganisms

Combined NZVI-Microorganism system

TCE Degradation

NZVI

Bare Encapsulated

Encapsulated NZVI

Research Phase II

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Combined NZVI-Microorganism System

TCE Degrading Bacteria42

Microorganism Method of Removal End Product Reference

Pseudomonas putida F1 Direct OxidationCo-metabolism

ETH (Sun and Wood 1996); (Kim et al. 2010b); (Radway et al. 1998)

Clostridium bifermentans DPH-120 Co-metabolism c-DCE (Chang et al. 2000)

Dehalospirillum multivorans Direct Oxidation c-DCE (Neuman et al.1994)

Dehalococcoides ethenogenes 195 Reductive dechlorination

ETH (Maymo-Gatell et al. 1999), (Fennell et al. 2001)

Dehalococcoides BAV1 Reductive dechlorination ETH (Krajmalnik-Brown et al. 2004; Maymo-Gatell et al. 1999),(He et al. 2003)

Dehalococcoides VS Reductive dechlorination ETH (Cupples et al. 2003)

Dehalococcoides FL2 Reductive dechlorination ETH (El Fantroussi et al. 1998)

Model Microorganisms

Pseudomonas putida F1 strain

Capable of oxidizing organic contaminants

Dehalococcoides sp.

Reductive dechlorination

Converts TCE benign products (ethene)

Some strains convert TCE VC

D. BAV1 : TCE Ethene

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www.miller-mccune.com

http://gtresearchnews.gatech.edu/

44

Research Phase II

Combined NZVI-Pp F1 System

Research Phase II

Growth Study

Encapsulated Pp F1 for TCE degradation

Encapsulated NZVI for TCE degradation in media

Combined NZVI- Pp F1 system

TCE re-dosing Experiment

Summary

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Pseudomonas putida F1 strain

Culture: obtained from ATCC

Optimal growth temperature: 300C

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www.miller-mccune.com

Growth Curve- Pseudomonas putida F1

P. putida incubated at 30oC in TSB media and shaken for 24 h

1 mL aliquot (0-24 h )

Serial dilution and nutrient agar plating

Incubated for 24 h at 30oC

Plate count and plotting of Viable Cells/mL vs Time

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Growth Curve

Bacteria harvested at 12 h for TCE degradation study

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Viable cell count = Number of colonies * (1/Dilution) * (1/Volume)

Growth Curve – With NZVI

Initial 3-h Lag Phase

5-25 h Stationary/Growth

26-35 h Death Phase

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Pseudomonas putida F1 for TCE Degradation

1mL of overnight culture (12 h) encapsulated

Encapsulated Pp F1added to TCE in TSB media (10 mg/L)

Reactors shaken end-over end

Aliquots withdrawn at regular intervals

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Blank P. Pp F1+TCE

Pp F1 for TCE Degradation

Encapsulated P. pF1: TCE removal efficiency of 70%

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NZVI in Growth Media

Purpose: To check whether NZVI behaves differently in media

Protocol:

0.75g/L NZVI +25 mL TCE solution amber vial (10mg/L TCE)

Rotated end-over-end at 28 rpm

Samples collected over time

Result: NZVI degrades TCE in media

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Blank NZVI + TCE (in media)

Batch Experiments53

Purpose: Metal-microorganism system for TCE removal

Batch Study Protocol:

Encapsulated NZVI -P. putida F1+TCE (in media)

Rotated – 3h

TCE analysis

Encapsulated NZVI

Encapsulated P.putida F1

TCE Solution

40 mL Vial

Re-dosing Experiments

Purpose: To understand the individual roles of NZVI and Pp F1

Rotated End-over-end for 3h

Remove TCE solution

Add new TCE solution

Rotated End-over-end 36 h

Aliquots

Analysis

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Results

TCE degradation 0-3h (NZVI dominated)

TCE degradation 3-36h (Bacteria dominated)

Add new TCE Solution

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NZVI Re-dosing

No TCE removal after 3 h

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Kinetic Studies57

E-NZVI: Encapsulated NZVI, E-B: Encapsulated Bacteria, C-NZVI: Combined System (NZVI dominated), C-B: Combined system (bacteria dominated)

Reaction order E-NZVI E-B C-NZVI C-B

Zero

Kobs

mg·L-1·min-1 5.92 2.0 6.05 3.5

R2 0.8764 0.57 0.90 0.84

1st

Kobs

min-1 2.33 4.0 2.47 5.0

R2 0.980 0.66 0.96 0.90

2nd Kobs

L·mg-1·min-1 2.31 8.0 2.80 9.0

R2 0.778 0.74 0.78 0.93

Research Phase II- Summary

Metal-Microorganism system was successfully designed

Pp F1 and NZVI were encapsulated separately

TCE was completely removed after 3 h (~ 100%) (NZVI dominated) and ~70% (bacteria dominated) in 36 h after re-dosing

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Work Remaining59

TCE Degradation

NZVI

Bare Encapsulated

Encapsulated NZVI

Microorganisms

PpF1D.

BAV1

Encapsulated Microorganisms

Combined NZVI-microorganism system

Research Phase III

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Research Phase III

Combined NZVI-D BAV1 System

Research Phase III

Growth Study

Encapsulated DBAV1for TCE degradation

Encapsulated NZVI for TCE degradation in media

Combined NZVI- DBAV1system

TCE re-dosing Experiment

Summary

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Dehalococcoides BAV162

Anaerobic bacteria

Method: Reductive dechlorination

Capable of reducing TCE ethane

Culture obtained from ATCC

D.BAV1 growth Studies63

D.BAV1 incubated at 22±2 o C in (MSM) media and shaken

1 mL aliquot at regular interval (0-36 h )

Serial dilution and nutrient agar plating

Incubated for 24 h at 22±2 oC

Plate count and plotting of Viable Cells/mL vs Time

D. BAV1 Encapsulation64

TCE Degradation Batch Studies65

Combined metal-microorganismEncapsulated D.BAV1

Encapsulated NZVI (re-dosed)Encapsulated NZVI

pH-ORP Data Acquisition System66

pH-ORP Change

ORP- Reducing conditions

Increase at 3 h – exposure to air

pH- initial increase (reaction with NZVI)

Constant throughout the reaction

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Kinetic Study68

Reaction order E-NZVI E-B C-NZVI C-B

Zero Kobs

(mg·L-1·min-1)

5.83 6.6 6.01 5.00

R2 0.863 0.33 0.89 0.82

1st

Kobs

(min-1)

2.76 4.90 2.93 3.80

R2 0.912 0.92 0.93 0.86

2nd

Kobs

(L·mg-1·min-1)

8.35 2.85 1.08 6.05

R2 0.636 0.63 0.64 0.41

Research Phase III -Summary

D BAV1and NZVI were co-encapsulated

TCE was completely removed after 3 h (~ 100%)

(NZVI dominated)

After re-dosing TCE removal ~ 100%

(Bacteria dominated)

First order kinetics

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Conclusions

Encapsulation of NZVI in alginate polymer : viable for TCE

dechlorination

Encapsulation of microorganisms in alginate polymer :

efficient technique for TCE removal

DCE and VC: not detected

Combined metal-microorganism was successful with the

advantages of both NZVI and microorganisms

Potential for in-situ remediation applications (e.g., PRBs)

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Future Directions

Interferences of other groundwater contaminants (electrons acceptors like nitrate and dissolved oxygen)

Shelf-life of the combined NZVI-microorganism system

The roles of NZVI and bacteria in a combined system

Make it versatile (Other Contaminants)

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Acknowledgements

ND Water Resource Research Institute

Department of Civil Engineering

Dr. Achintya Bezbaruah

Dr. Eakalak Khan

Dr. Senay Simsek

Dr. G. Padmanabhan

NRG Group

Members of the Environmental Engineering Laboratory

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Thank you!

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