Bioremediation of Polychlorinated Biphenyls (PCBs) …...Why are PCBs of concern? • Bioaccumulates and biomagnifies in the food chain • Present in lipophilic tissue, blood and

Bioremediation of polychlorinated biphenyls (PCBs) using biofilms

Birthe Veno Kjellerup, Ph.D.

University of Maryland at College Park Department of Civil & Environmental Engineering

AGENDA

• Presence and concerns of PCBs? • Biological fate of PCBs? • Bioremediation using activated carbon • Biofilms in bioremediation • Aerobic-anaerobic biofilms in soil • Future Research • Questions

PCB contaminated soil

ortho meta

para 2 3

4

5 6

2’ 3’

4’

5’ 6’

PCBs: Persistent organic pollutants

• 209 congeners

• Very Stable

• Bioaccumulate

• Toxicity concern

• Sediments/soils = global sinks

Presenter

Presentation Notes

Research focuses on transformation of PCBs in marine and estuary sediments structurally PCBs consist of biphenyl w/chlorine substitutions - 209 permutations/120 manufactured will be refering to o,m,p positions throughout talk in US comercially produced/released as Arochlors since 20s/450 million kilograms or one billion pounds have been released very stable - microbial anaerobic dechlorination only described in past decade - most of those studies concentrated on the freshwater environment and because they are hydrophobic - sorb to soil/sediments affinity towards fat tissue - issue for ecology and hyman health 1st indicator of health problem - late 60s - dermal toxicity in Yusho, Japan and Yaching, Taiwan - chloroacne as a result of eating oil contaminated w/PCBs (probably due to traces of polychlorodibenzofurans) 2nd indicator of health problem - 70s - carcinogenesis generally attributed to coplanar dioxin-like congeners 3rd indicator of health - 80s - teratogenicity development resulting in low I.Q. neuruotoxicity has been observed in rats including ortho-substituted most recently: estrogenic effect: estrogen analogs cause affect regulation of estrogen-associated responses synergistic interactions of chemical mixtures enhances estrogenic effect,in some cases over a thousand-fold effect

Microbial transformation of PCBs

Estimated 0.6-1.2 billion kg worldwide

An environmental legacy of PCBs

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Why are PCBs of concern?

• Bioaccumulates and biomagnifies in the food chain

• Present in lipophilic tissue, blood and breast milk

• Toxicological effects: Cancer, problems with endocrine and reproductive organs as well as immunological issues

• Humans: Source - ingestion (sea food, meat, poultry etc.)

The Microbial Fate of PCBs

Aerobic bacterium Burkholderia LB400

Oxygen conc.

Pore water Biofilm on soil particle

<4 Cl subst.: Aerobic PCB degradation

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl

Cl Cl

Cl Cl

Cl Cl

Anaerobic bacterium DF1

>4 Cl subst.: Anaerobic PCB dechlorination

Complete

mineralization

Presenter

Presentation Notes

Lesser chlorinated PCBs – oxidative ring cleavage via hydroxylation and subsequent oxidation of one of the rings via a dihydroxybiphenyl dioxygenase yielding chlorobenzoate. More chlorianted PCB – sequenctial reductive dechloration of chlorine groups, likely used as terminal electron acceptors for oxidation of a reduced carbon compound such as acetate or formate. Complementary processes in the environment

PCBs - Processes in sediment

Resuspension

Biological dechlorination

Ingestion

Biological degradation

+O2

Cl Cl

Cl Cl

Cl Cl

Fatty acids

PCB PCB

Cl Cl

Cl Cl

Cl Cl

Cl Cl

Cl Cl

Cl Cl

Cl Cl

Cl Cl

Cl Cl

Uptake

> 50 years

Effect of Activated Carbon

Millward et al, Environ. Sci. Technol. 2005

Conclusion: • Reduced uptake of PCB in tissue • Reduced bioavailability for tested species

Questions: • Reduced bioavailability for bacteria? • Effects on dechlorination rates and products?

L. plumulosus

N. arenaceodentata

PCBs - Processes in sediment

Resuspension

Biological dechlorination

Ingestion

Biological degradation

+O2

Cl Cl

Cl Cl

Cl Cl

Fatty acids

PCB PCB

Cl Cl

Cl Cl

Cl Cl

Cl Cl

Cl Cl

Cl Cl

Cl Cl

Cl Cl

Cl Cl

Uptake ? ?

Are PCBs available for bacteria?

Conclusion: • Dechlorination of Aroclor 1260 in sediment with GAC ⇒ No effect of GAC based on average chlorine content

Kjellerup et al, Water Res (2014), Apr 1;52C:1-10

4.0

4.5

5.0

5.5

6.0

6.5

7.0

0 50 100 150 200 250 300

Time (Days)

Ch

lori

nes

per

bip

hen

yl

No GACWith GAC

Average chlorine content

GAC added

-10%

-5%

0%

5%

10%

15%

20%

25%

30%

35%

40%

C1

C4,

10

C7,

9

C6

C5,

8

C14

C19

C11

C15

,17

C16

,32

C29

,54

C25

C28

,31,

50

C20

,21,

33,5

3

C22

,51

C52

,73

C43

,49,

38

C35

,104

C44

C41

,64,

71,7

2,68

C57

,103

,40

C67

,100

C66

,80,

93

C90

,101

C11

9,15

0,11

2

C13

6

C15

1

C12

4,13

5,14

4

106,

118,

139,

149

C15

3,13

2,10

5,12

7

C14

1,17

9

C17

6, C

130

C13

8,16

3,16

4

C15

8,18

6

C18

7,15

9,18

2

C18

3

C17

4,18

1

C17

7

C15

6,17

1,20

2

C19

7

C19

3

C17

0,19

0

Congener

Mo

l%

Increase

Decrease

Conclusion: •With GAC, full dechlorination possible •Aerobic microbes can now degrade biphenyl rings

Bacteria With GAC

Bacteria Alone

mono/di-chlorinated congeners

tri/tetra/penta-chlorinated congeners - STALLS

FULL DECHLORINATION

???

GAC without Bacteria

Bacteria with no GAC Bacteria with GAC

Are PCBs available for bacteria?

Kjellerup et al, Water Res. 2014

Different bacterial populations? Screening of bacterial diversity using DHPLC and primers targeting dechlorinating bacteria (16S rRNA)

Conclusion: • Dominant dechlorinating phylo-types are the same → Not

responsible for difference in dechlorination Kjellerup et al, Water Res (2014), Apr 1;52C:1-10

Biofilm on GAC in sediment

5 µm

Dechlorinating bacteria

Conclusion: • Biofilms are present in sediment • Natural mode of growth

• Can we utilize this observation?

Biofilm on GAC in sediment

5 µm

Dechlorinating bacteria

Conclusion: • Biofilms are present in sediment • Natural mode of growth

• Can we utilize this observation?

Dual approach: 1. Adsorption of PCBs on activated carbon 2. Biofilm instead of liquid inoculum for bioaugmentation? Objective: Apply biofilm communities to PCB contaminated sediment as a delivery system to enhance dechlorination

15

Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Granular activated carbon

Anaerobic dechlorinating bacteria

PCBs

Previous Bioremediation Approach

Technical Approach - Detail

Biofilm Bioremediation approach

16

Cl Cl

Cl Cl Cl

Cl Cl Cl

Cl Cl Cl

Cl

Cl Cl Cl

Cl Cl Cl

Cl Cl Cl

Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Technical Approach - Detail

Previous Bioremediation Approach Biofilm Bioremediation approach

Granular activated carbon

Anaerobic dechlorinating bacteria

PCBs

Direct SYBR Green staining - CLSM

Betsey Pitts, MSU/CBE, 2012

Anaerobic Biofilm Formation

SYBR Green staining & CLSM Edwards et al, 2016 (In prep)

Sediment mesocosms from Grasse River, NY • Two types of biofilm inoculum

Dechlorination rate

Biofilm enhances the dechlorination rate

Effect on dechlorination?

* *

* Statistical significance <30% - EPA Standard Edwards et al, 2016 (In prep)

*

*

*

* * * * * *

Dechlorination after 200 days Increase

Decrease

Mono and di-chlorinated congeners - significantly more in the presence of biofilm compared to GAC and liquid inoculum

Day 0

Day 200

Effect on dechlorination?

* Statistical significance <30% - EPA Standard Edwards et al, 2016 (In prep)

Can the numbers of bacteria explain the difference in activity? • Anaerobic DF1 biofilm

The numbers of dechlorinating bacteria are similar over time ⇒ Not responsible for the

difference in activity Reason: ⇒ Diversity? ⇒ Mode of growth?

Can the numbers explain?

Edwards et al, 2016 (In prep)

Looking at the microbial populations in the sediment ↓ What is the effect of Biofilm Augmentation? • Approach: Multiplex 16S rRNA gene seq. - Illumina MiSeq

Effect of Biofilms?


Influence of Biofilms?


Influence of Biofilms?


Effect of Biofilm Augmentation?

Summary: • Enhanced PCB dechlorination • Sediment Population analysis: - Other Chloroflexi than “usual suspects” are involved

(Dehalococcoides and DF1) - 18 groups of bacteria show 2+ fold upregulation → Related to contaminated sediment/soil (anaerobic) What does this mean? Biofilms impact the overall sediment population, NOT only the PCB dechlorinating population. Mechanism?


Biofilm based delivery system

Activated carbon Biochar Fe covered AC Zeolite

Ongoing research activities: Identification of the mechanism responsible for increased activity of GAC-Biofilm based bioaugmentation

• Electrical conductivity? • Sorption (kinetics)? • Surface area/porosity? • Other?

A. Prieto et al (in prep), 2016

Biofilm based delivery system Sand Coconut biochar

Peanut Biochar – B. xenovorans LB400

• Dechlorinating biofilms can effectively be cultured under anaerobic conditions

• Application of anaerobic biofilms as a delivery vehicle enhances dechlorination of PCBs in sediment

• Biofilms are robust and can be maintained in sediment

Summary

→ Good solution for anaerobic bioremediation → Complete mineralization?

Aerobic

Cl Cl

Cl Cl Cl Cl

Cl Cl Cl Cl

Burkholderia LB400

DF-1

Anaerobic

+O2 CO2

Fate of PCBs in Mixed Biofilms

A. Prieto et al (in prep), 2016

Recontamination of sediment from wastewater?

30

Outfall 002 Outfall 001

Average 4.25 5.09

STDEV 0.51 0.39

Outfall 002 Outfall 001

Average 4.25 5.09

STDEV 0.51 0.39

Others involved: • Kevin R. Sowers, Ph.D. IMET, UMBC • Ms. Betsy Pitts, M.Sc., Center for Biofilm Engineering,

Montana State University • Dr. Recep Avci, Ph.D., Image and Chemical Analysis

Laboratory, Montana State University

• Natasha Andrada & Upal Ghosh, Ph.D., UMD/UMBC

Financial support for Project ER 2135

Students: Freshta Akbari, Sarah Edwards Chiara Draghi (Graduated) Kirstie Coombs, BS (2017) Raymond Jing, Ph.D. (2019) Dr. Ana Prieto, Post Doc.

Kjellerup Lab

Acknowledgements

Terp Service Day

Thanks for your attention.

Contact – Please email: [email protected]

Documents

Bioremediation of Polychlorinated Biphenyls (PCBs) …...Why are PCBs of concern? • Bioaccumulates and biomagnifies in the food chain • Present in lipophilic tissue, blood and