BIOREMEDIATION, MICROBIAL ECOLOGY AND FUNCTIONAL

METAGENOMICS

Don Cowan

Centre for Microbial Ecology and Genomics

University of Pretoria

don.cowan@up.ac.za

The University of Pretoria campus

Centre for Microbial Ecology and Genomics

The CMEG team (and friends)14 postdocs, 9 postdocs, 6 MScs

The Centre for Microbial Ecology and Genomics

CMEG core technologies

Microbial phylogenetics:

[DGGE, TRFLP, 454]

Genome sequencing and analysis

Quantitative microbial ecology

Protein crystallography

Protein chemistry and enzymology

Gene cloning and expression

Functional metagenomics

Gene discovery

Applied enzymology

Stress response proteins

New CAZymebiocatalysts

Desert soilmetagenome

analyses

Psychrophilegenomes

Hot and cold desert soil

ecosystems

Extreme environment research

Antarctic cold desert soils Ethiopian haloalkaline lake waters Terrestrial thermal sites

Namibian hot desert soils Sub-Antarctic peat bogs Kenyan alkalophilic lakes

The application principles of bioremediation

1. Isolate organism by enrichment culture, amplify and applyProof of functional degradation capacityCapacity to use ex situ fermentation to generate high

biomass Simple application for local surface remediationWell suited for niche applications (ex situ liquid stream

processing)

• BUT, subject to serious limitations Restricted to culturable species (0.1-10%) Isolated organisms often do not compete in situ

The application principles of bioremediation

2. Stimulating endogenous capacity

Low cost supplementation options

Not dependent on single organism (can take advantage of complex consortia, inter-dependent and synergistic interactions)

But, must understand in situ limiting factors

Requires the use of metagenomic methods

What is metagenomics?

“The concept of the metagenome: the composite of all organisms, their genetic elements and their functional capacities, in a sample, a site or an environment”

A typical soil metagenome....

100 – 10,000 bacterial species (3-10Mbp genomes)

100 – 1,000 fungal species (0.1 – 5 Gbp genomes)

10 – 100 invertebrate species (0.1 – 5Gbp genomes)

1000 – 1,000,000 phage /virus genomes (40 – 200Kbp)

Accessing the metagenome

Cell recovery, lysis and DNA purification

Separation of cells from substrate/matrix prior to lysis

Reduced background contamination (humic acids)

Allows cell sorting (size fractionation)

Low cell recovery efficiency with adsorbent matrices (clays)

In situ DNA extraction

Good representivity of species diversity

Co-extraction of contaminants and inhibitors

Losses of DNA by adsorption to cationic minerals

Estimating microbial diversity

‘Universal’ phylogenetic markers 16S rRNA gene (bacteria and archaea)

18S rRNA gene, ITS sequence (lower eukaryotes)

Phylogenetic marker gene amplicon analysis Clone library synthesis, clone

selection, ARDRA de-replication, insert sequencing)

Amplicon sequencing (MiSeq or 454)

Phylogenetic tree construction

(Semi)-quantitative metagenomics

Methods for quantitative and comparative phylogenetics

DGGE (Denaturing Gradient Gel Electrophoresis)

ARISA (Automated Ribosomal Intergenic Spacer Analysis)

T-RFLP (Terminal Restriction Fragment Length Polymorphism)

DGGE patterns of multiple samples

T-RF patterns from successive year sampling

Using DGGE and T-RFLP analysis to follow microbial community changes

Soil bacterial community structure patterns through an annual cycle

2D MDS plot of community changesSemi-quantitative

analysis of changes in individual phylotypes

through an annual cycle

Using 16S amplicon sequencing to monitor community changes

3-year soil bacterial community profile changes after nutrient amendment

Techniques of functional metagenomics1. Capturing novel genes

Metagenomic Expression Libraries Capturing functional genes

Access to uncultured majority

Multiple screening technologies

A powerful tool for synthetic genomics

Cowan, DA, Stafford, W. (2007) Metagenomic methods for determining active microorganisms and genes in bioremediation and biotransformation processes. Ch. 58 in ASM Manual of Environmental Microbiology, 3rd Edition. Editors: CJ Hurst, RL. Crawford, JL. Garland, DA Lipson, AL Mills, LD Stetzenbach., ASM Press, New York. 1310 pp. ISBN: 978-1-55581-379-6

Techniques of functional metagenomics 2. Linking phylotype to function

SIP (Stable isotope probing) In situ identification of specific functional species

Enrich with enriched substrate (e.g., 13C hydrocarbon)

Extract metagenome

Separate heavy (labelled) DNA fraction using density gradient centrifugation

Phylogenetic analysis of labelled DNA yields metabolically active phylotypes

Techniques of functional metagenomics 3. Assessing degradative potential

Gene-specific libraries Metagenomic DNA extraction

PCR amplification with gene-specific primer sets

Amplicon sequence analysis

Analysis of the distribution of key functional genes (e.g., assessing degradative capacity of a community)

Techniques of functional metagenomics 4. Phylogeny and genomics capacity

Metagenome sequencing Metagenomic DNA extraction

Complete NG sequencing (e.g., 20-30Gbp from HiSeq)

Contig assembly

Dominant genomes (with phylogenetic affiliations)

Gene and pathway diversity (biodegradative capacity)

What can metagenomics contribute to bioremediation?

Pre-assessment of biodegradative capacity of an environment

Assessing in situ microbial diversity (baseline study)

Estimating functional capacity

What can metagenomics contribute to bioremediation?

Monitoring in situ biodegradation performance

Monitoring supplementation processes

Monitoring community (or target species) stability

What can metagenomics contribute to bioremediation?

Assisted selection of functional strains/communities

Phylogenetic analysis in enrichment cultures

Rapid assessment of Functional capacity (SIP analysis)

What can metagenomics contribute to bioremediation?

Assisted selection of functional strains/communities

Phylogenetic analysis in enrichment cultures

Functional capacity (SIP analysis)

What can metagenomics contribute to bioremediation?

Driving synthetic biology

Synthesis of new polyfunctional biodegradative strains

Identification of new biodegradative pathways

Impediments to use

Ignorance – awareness of molecular ecological tools may not exist in some industries

Perceptions – seen as a research activity rather than a set of diagnostic tools

Costs – perceptions of high operating costs against a background of low investment

Timescales – methods not real-time

Thank you

Acknowledging the support and assistance of:

The University of Pretoria, SAThe University of the Western Cape, SAThe National Research Foundation of South Africa

THANK YOU