Bioremediation of polychlorinated biphenyls (PCBs) using biofilms

Kjellerup--1

Bioremediation of polychlorinated biphenyls (PCBs) using biofilms

Birthe Veno Kjellerup, Ph.D.University of Maryland at College ParkDepartment 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 ResearchFuture Research • Questions

PCB contaminated soil

PCBs: Persistent organic pollutants

• 209 congeners

• Very Stable

• Bioaccumulate

orthoorthometameta

parapara2233

44

55 66

2’2’ 3’3’

4’4’

5’5’6’6’

• Bioaccumulate

• Toxicity concern

• Sediments/soils = global sinks

Microbial transformation of PCBs

An environmental legacy of PCBs

Estimated 0.6-1.2 billion kg worldwide

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

<4 Cl subst.: Aerobic PCB degradation

Cl Cl Cl Cl

Cl Cl

Cl Cl Cl Cl

Cl Cl

Cl ClCl Cl

>4 Cl subst.: Anaerobic PCB dechlorination

Complete mineralization

Aerobic bacterium Burkholderia LB400

Oxygen conc.

Pore water Biofilm on soil particle

Cl Cl Cl ClCl Cl Cl ClCl ClCl Cl

Anaerobic bacterium DF1

Bioremediation of polychlorinated biphenyls (PCBs) using biofilms

Kjellerup--2

PCBs - Processes in sediment

Resuspension

Biological dechlorination

Ingestion

Biological degradation

+O2

ClCl

ClCl

ClCl

Fatty acids

PCB PCB

ClCl

ClCl

ClCl

ClCl

ClCl

ClCl

ClCl

ClCl

ClCl

Uptake

> 50 years

Effect of Activated Carbon

L. plumulosus

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?

N. arenaceodentata

PCBs - Processes in sediment

Resuspension

Biological dechlorination

Ingestion

Biological degradation

+O2

ClCl

ClCl

ClCl

Fatty acids

PCB PCB

ClCl

ClCl

ClCl

ClCl

ClCl

ClCl

ClCl

ClCl

ClCl

Uptake ? ?

Are PCBs available for bacteria?

5.0

5.5

6.0

6.5

7.0

lori

nes

pe

r b

iph

enyl No GAC

With GAC

Average chlorine content

GAC added

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

0 50 100 150 200 250 300

Time (Days)

Ch

l

15%

20%

25%

30%

35%

40%

Mo

l%

Increase

Bacteria With GAC

Bacteria Alone

mono/di-chlorinated congeners

tri/tetra/penta-chlorinated congeners - STALLS

FULL

???

GAC without Bacteria

Bacteria with no GACBacteria with GAC

Are PCBs available for bacteria?

-10%

-5%

0%

5%

10%

C1

C4,1

0

C7,

9

C6

C5,

8

C14

C19

C11

C15,1

7

C16,3

2

C29,5

4

C25

C28

,31,5

0

C20,2

1,33,5

3

C22,5

1

C52,7

3

C43

,49,3

8

C35,1

04

C44

C41,6

4,7

1,72,6

8

C57,1

03,4

0

C67,1

00

C66

,80,9

3

C90,1

01

C119,1

50,1

12

C13

6

C15

1

C124,1

35,1

44

106,1

18,1

39, 1

49

C153,1

32,1

05,1

27

C141,1

79

C17

6, C

130

C138,1

63,1

64

C158,1

86

C187,1

59,1

82

C18

3

C174,1

81

C17

7

C156,1

71,2

02

C19

7

C19

3

C170,1

90

Congener

M

Decrease

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

DECHLORINATION

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

Bioremediation of polychlorinated biphenyls (PCBs) using biofilms

Kjellerup--3

Biofilm on GAC in sediment

Dechlorinating bacteria

5 µm

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

• Can we utilize this observation?

Biofilm on GAC in sediment

Dechlorinating bacteria

5 µm

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

• Can we utilize this observation?

Dual approach: 1. Adsorption of PCBs on activated carbon2. Biofilm instead of liquid inoculum for bioaugmentation?

Objective:Apply biofilm communities to PCB contaminated sediment as a delivery system to enhance dechlorination

ClCl

ClClCl Cl

ClClCl ClClCl

ClClCl Cl

ClClCl ClClCl

ClClCl Cl

ClClCl Cl

ClCl

ClClCl Cl

ClClCl Cl

ClCl

ClClCl Cl

ClClCl ClClCl

ClClCl Cl

ClClCl ClClCl

ClClCl Cl

ClClCl Cl

ClCl

ClClCl Cl

ClClCl Cl

Granular activated carbon

Anaerobic dechlorinating bacteria

PCBs

Technical Approach - Detail

15

ClClCl

ClCl

ClClCl Cl

ClClCl Cl

ClCl

ClClCl Cl

ClClCl Cl

ClCl

ClClCl Cl

ClClCl Cl

ClCl

ClClCl Cl

ClClCl Cl

ClCl

ClClCl Cl

ClClCl Cl

ClCl

ClClCl Cl

ClClCl Cl

ClCl

ClClCl Cl

ClClCl Cl

ClCl

ClClCl Cl

ClClCl Cl

Previous Bioremediation Approach Biofilm Bioremediation approach

ClCl

ClClCl Cl

ClClCl ClClCl

ClClCl Cl

ClClCl ClClCl

ClClCl Cl

ClClCl Cl

ClCl

ClClCl Cl

ClClCl Cl

Technical Approach - Detail

Granular activated carbon

Anaerobic dechlorinating bacteria

PCBs

16

ClCl

ClClCl

ClClCl

ClClCl

Cl

ClClCl

ClClCl

ClClCl

ClClCl

ClCl

ClClCl Cl

ClClCl Cl

ClCl

ClClCl Cl

ClClCl Cl

ClCl

ClClCl Cl

ClClCl Cl

ClCl

ClClCl Cl

ClClCl Cl

Previous Bioremediation Approach Biofilm Bioremediation approach

Direct SYBR Green staining - CLSM

Betsey Pitts, MSU/CBE, 2012Betsey Pitts, MSU/CBE, 2012

Anaerobic Biofilm Formation

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

Bioremediation of polychlorinated biphenyls (PCBs) using biofilms

Kjellerup--4

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 daysIncrease

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

Day 200

Effect on dechlorination?

* * *** **

Decrease

Day 0

* 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

Can the numbers explain?

gbacteria are similar over time Not responsible for the

difference in activity

Reason: Diversity? Mode of growth?

Edwards et al, 2016 (In prep)

Looking at the microbial populations in the sediment↓What is the effect of Biofilm Augmentation?

Effect of Biofilms?

• Approach: Multiplex 16S rRNA gene seq. - Illumina MiSeq

Edwards et al, 2016 (In prep)

Influence of Biofilms?

Edwards et al, 2016 (In prep)

Influence of Biofilms?

Edwards et al, 2016 (In prep)

Bioremediation of polychlorinated biphenyls (PCBs) using biofilms

Kjellerup--5

Effect of Biofilm Augmentation?

Summary:• Enhanced PCB dechlorination

• Sediment Population analysis:- Other Chloroflexi than “usual suspects” are involved

(Dehalococcoides and DF1)(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?

Edwards et al, 2016 (In prep)

Biofilm based delivery system

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

• Electrical conductivity? • Sorption (kinetics)?

Activated carbon Biochar Fe covered AC Zeolite

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

A. Prieto et al (in prep), 2016

Biofilm based delivery systemSandCoconut biochar

Peanut Biochar – B. xenovorans LB400

• Dechlorinating biofilms can effectively be cultured under anaerobic conditions

• Application of anaerobic biofilms as a delivery vehicle

Summary

enhances dechlorination of PCBs in sediment

• Biofilms are robust and can be maintained in sediment

→ Good solution for anaerobic bioremediation→ Complete mineralization?

Aerobic

BurkholderiaLB400

Anaerobic

+O2CO2

Fate of PCBs in Mixed Biofilms

ClCl

ClClCl Cl

ClClCl Cl DF-1

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

Bioremediation of polychlorinated biphenyls (PCBs) using biofilms

Kjellerup--6

Financial support for Project ER 2135

Students:Freshta Akbari, Sarah EdwardsChiara Draghi (Graduated)Kirstie Coombs, BS (2017)Raymond Jing, Ph.D. (2019)

Kjellerup Lab

Acknowledgements

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

Raymond Jing, Ph.D. (2019)Dr. Ana Prieto, Post Doc.

Terp Service Day

Thanks for your attention.

Contact – Please email: bvk@umd.edu