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
PCBs: Persistent organic pollutants • 209 congeners • Very Stable • Bioaccumulate • Toxicity concern meta ortho 3 2 2’ 3’ • Sediments/soils = global sinks para 4 4’ 5 6 6’ 5’
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.: >4 Cl subst.: Complete Aerobic PCB Anaerobic PCB mineralization degradation dechlorination Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Oxygen Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl conc. Aerobic bacterium Anaerobic Burkholderia LB400 bacterium DF1 Pore water Biofilm on soil particle
PCBs - Processes in sediment Resuspension Ingestion PCB PCB +O 2 Cl Cl Cl Cl Cl Cl Cl Cl Uptake Fatty acids Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Biological Biological dechlorination degradation > 50 years
Effect of Activated Carbon L. plumulosus N. arenaceodentata Conclusion: • Reduced uptake of PCB in tissue • Reduced bioavailability for tested species Questions: • Reduced bioavailability for bacteria? • Effects on dechlorination rates and products? Millward et al, Environ. Sci. Technol. 2005
PCBs - Processes in sediment Resuspension Ingestion PCB PCB +O 2 ? Cl Cl Cl Cl Cl Cl Cl Cl ? Uptake Fatty acids Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Biological Biological dechlorination degradation
Are PCBs available for bacteria? 7.0 Average chlorine GAC added No GAC 6.5 content Chlorines per biphenyl With GAC 6.0 5.5 5.0 4.5 4.0 0 50 100 150 200 250 300 Time (Days) 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
Are PCBs available for bacteria? 40% ??? Increase Bacteria with no GAC Bacteria 35% Bacteria with GAC Bacteria Alone 30% mono/di- tri/tetra/penta-chlorinated With GAC chlorinated congeners - STALLS 25% congeners GAC without 20% Mol% FULL Bacteria 15% DECHLORINATION 10% 5% 0% C1 C4,10 C7,9 C6 C5,8 C14 C19 C11 C15,17 C16,32 C29,54 C25 C28,31,50 C20,21,33,53 C22,51 C52,73 C43,49,38 C35,104 C44 C41,64,71,72,68 C57,103,40 C67,100 C66,80,93 C90,101 C119,150,112 C136 C151 C124,135,144 106,118,139, 149 C153,132,105,127 C141,179 C176, C130 C138,163,164 C158,186 C187,159,182 C183 C174,181 C177 C156,171,202 C197 C193 C170,190 -5% Decrease -10% Congener Conclusion: •With GAC, full dechlorination possible •Aerobic microbes can now degrade biphenyl rings 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 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 Dual approach : 1. Adsorption of PCBs on activated carbon Conclusion: 2. Biofilm instead of liquid inoculum for bioaugmentation? • Biofilms are present in sediment Objective: • Natural mode of growth Apply biofilm communities to PCB contaminated sediment as a delivery system to enhance dechlorination • Can we utilize this observation?
Technical Approach - Detail Granular activated carbon Anaerobic dechlorinating bacteria PCBs 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 15 Previous Bioremediation Approach Biofilm Bioremediation approach
Technical Approach - Detail Granular activated carbon Anaerobic dechlorinating bacteria PCBs 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 16 Previous Bioremediation Approach Biofilm Bioremediation approach
Direct SYBR Green staining - CLSM Betsey Pitts, MSU/CBE, 2012
Anaerobic Biofilm Formation SYBR Green staining & CLSM Edwards et al, 2016 (In prep)
Effect on dechlorination? Sediment mesocosms from Grasse River, NY • Two types of biofilm inoculum Dechlorination rate * Biofilm enhances the * dechlorination rate * Statistical significance <30% - EPA Standard Edwards et al, 2016 (In prep)
Effect on dechlorination? Mono and di-chlorinated congeners - significantly more in the presence of biofilm compared to GAC and liquid inoculum Increase Dechlorination after 200 days Day 200 * * Day 0 * * * * * * * Decrease * Statistical significance <30% - EPA Standard Edwards et al, 2016 (In prep)
Can the numbers explain? 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? Edwards et al, 2016 (In prep)
Effect of Biofilms? Looking at the microbial populations in the sediment ↓ What is the effect of Biofilm Augmentation? • 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)
Effect of Biofilm Augmentation? Summa mmary: ry: • 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 hat does does thi his mean? ean? Biofilms impact the overall sediment population, NOT only the PCB dechlorinating population. Mec echani hanism? 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)? • Surface area/porosity? • Other? Activated carbon Biochar Fe covered AC Zeolite A. Prieto et al (in prep), 2016
Biofilm based delivery system Coconut biochar Sand Peanut Biochar – B. xenovorans LB400
Summary • 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 → Good solution for anaerobic bioremediation → Complete mineralization?
Fate of PCBs in Mixed Biofilms Aerobic +O 2 CO 2 Burkholderia LB400 Anaerobic Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl DF-1 A. Prieto et al (in prep), 2016
Recontamination of sediment from wastewater? 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 30
Acknowledgements Financial support for Project ER 2135 Kjellerup Lab Students : Freshta Akbari, Sarah Edwards Chiara Draghi (Graduated) Kirstie Coombs, BS (2017) Raymond Jing, Ph.D. (2019) Dr. Ana Prieto, Post Doc. Others involved: Terp Service Day • 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
Thanks for your attention. Contact – Please email: bvk@umd.edu
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