Microbial Community Sequencing as an Approach to Addressing Bioremediation

Challenges in the Canadian Arctic

Charles W. Greer, Etienne Yergeau, David Juck, Terry Bell, Don Kovanen, Andrew Tam and Drew Craig

CFS-Alert

•  Year round operation

•  800 km from North Pole

•  Arctic desert -170 mm/yr

CFS-Alert, Nunavut

Bioremediation Challenges in the Arctic

•  Extreme environment (polar desert) - temperature - water activity - nutrient status

• Operational window - approximately 2 months/year

•  Logistics - remoteness - simple, practical approaches required

Objectives

• evaluate the potential for on site bioremediation of petroleum hydrocarbon contaminated soil. • examine parameters to enhance on-site biotreatment:

• different fertilizer types • different fertilizer concentrations

• monitor on-site and in situ biotreatment performance: (metagenomic and stable isotope probing (SIP) analyses)

• microbial community composition • microbial degradation activity • changes in microbial population composition and

functional (degradation) gene content

Deciphering the

Microbial “Black Box”

The microbial community is responsible for contaminant degradation and genomics tools can help decipher the who, what, where and how

• knowing who’s there provides important information on potential capabilities

• knowing what key genes are present identifies degradation potential

• showing which genes are functional indicates who is actively degrading

Initial Laboratory Evaluation

• different fertilizers had a significant effect • mineralization activity with nutrients good at 4°C • virtually no activity in unfertilized soil

Mineralization Determines the potential of the indigenous microbial community to degrade a representative hydrocarbon substrate

Diesel Degradation in Laboratory Mesocosms • extensive degradation with fertilizer addition • temperature effect with degradation greater at 22°C than at 4°C

Hydrocarbon degradation and

mineralization

Microbial Community Structure

• decrease in diversity upon contamination

• high initial numbers of Gammaproteobacteria, decreased over time

• increase over time in Actinobacteria

• main Gammaproteobacteria are Pseudomonas species • low numbers of Pseudomonas in pristine soil • increased dramatically following contamination

Quantitative PCR

•  alkB (Ps)-soil hydrocarbon: r = 0.738, P=0.00945 •  alkB1 (Rh)-soil hydrocarbon: r = -0.703, P=0.0234

Quantitative RT-PCR (who is actively degrading) • Pseudomonas alkane and aromatic degradation genes Most dominant in t=0 and 1 mo soils, decreasing thereafter • Rhodococcus alkane degradation genes dominate from 1 mo to 1 year after

Metagenomic Analysis of hydrocarbon degradation genes

NT- ctrl in back VT - bioventing stacks

TN - turning of soil

OR- application of ORC

Examining the Effects of Fertilization on

In Situ Bioremediation

Effect of MAP Amendment

•  Slight decrease in biomass in pristine environment •  Significant increase in biomass in contaminated

environment –  Providing missing nutrients permitted significant growth

0

1

2

3

4

5

6

7

8

9

10

Pristine Contaminated

Ex

tra

cted

DN

A (

µg

/g s

oil

)

No MAP

14N MAP

15N MAP

alkB Copies

•  alkB gene important in biodegradation of alkanes •  Increased number of copies

–  contaminated vs pristine sites –  amended vs unamended control after 2 months

0

3000

6000

9000

12000

15000

Pristine Contaminated

Co

pie

s a

lkB

/ n

g D

NA

No MAP

14N MAP

15N MAP

16S rRNA gene Taxonomic Distribution

alkB gene Taxonomic Distribution

Contaminated Uncontaminated

Contaminated Uncontaminated

Actinobacteria

Alphaproteobacteria

Betaproteobacteria

Gammaproteobacteria

Deltaproteobacteria

Nitrospira

Gemmatimonadetes

Acidobacteria

Verrucomicrobia

Planctomycetes

Bacteroidetes

Others (Firmicutes, TM7,

WS3, Chloroflexi, OD1,

OP10, Cyanobacteria)

Overall Diversity in MAP-Treated Soils

Conclusions (what metagenomic sequencing has taught us)

•  The microbial community structure changed dramatically in response to contamination and during remediation, serving as an indicator of bioremediation performance

•  Fertilizer addition had a positive impact on hydrocarbon biodegradation (on site and in situ)

•  Key bacteria (eg. Pseudomonas), known to be hydrocarbon degraders,were initial responders after fertilizer addition

•  Known hydrocarbon degradation genes (i.e. alkB, ndoB) showed increased numbers, which corresponded to increased degradation activity

•  Predominant hydrocarbon degraders were Pseudomonas spp. while nutrients available: other bacteria, such as Rhodococcus spp. became dominant when degradation activity and nutrient concentrations decreased.

Acknowledgements

•  8 Wing Trenton Environment Office and CFS-Alert staff

•  Federal Contaminated Sites Action Plan (FCSAP)

•  NRCan Program for Energy Research and

Development (PERD)