Ecology of activated sludge

1
Ecology of activated sludge
Victor Kunin
2
Phil Hugen-holtz
Falk Warnecke
Hector Garcia Martin
Suzan Yilmaz
Victor Kunin
Natalia Ivanova
Trina McMahon (UW) Linda Blackall (UQ) Project
powered by DOE
3
Metagenomes in pipeline

• Wastewater sludge (EBPR)
• Termite gut
• Guerrero Negro Hypersaline mat
• Antarctic subglacial lake Vostok

4
Ecology?

• population structure
• predator-pray interactions
• biogeography

5
Microbial ecology

• Local population structure
• Global metapopulation structure
• Biogeography
• Dispersal mechanisms
• Survival strategies of the species
• Phage-host interactions
• Can we link an unculturable bacterium to its
  phage by (meta)genomic sequence?
• Ecosystem resilience

6
Enhanced Biological Phosphorus Removal (EBPR)

• Excessive P affects water quality and ecosystem
  balance through eutrophication.

7
Enhanced Biological Phosphorus Removal (EBPR)

• P is the key pollutant.
• P limitations in released water very effective

N,C
N,C,P
8
US and Australian (OZ) samples

Madison
Brisbane

9
Community composition
What organisms live there?
10
Bacterial biogeography

• Everything is everywhere and the environment
  selects
• Microbial species are ubiquitous
• Beijerinck, 1913
• Microbial populations have astronomical sizes
• Microbes travel well
• 1018 microbes are estimated to cross continents
  per year by airlift
• Identical 16S rRNA molecules are found as far
  apart as polar oceans

• Undetected biogeography -poor methods
• Travel takes time, methods are insufficient to
  detect divergence
• Geographical isolation exists in hot spring
  bacteria
• Whitaker et al, 2003, Papke et al, 2003

What is the biogeography of Accumulibacter?
11
Any geographic isolation of strains?
Geographic isolation
12
How do they move?

• CAP is globally dispersed
• Never observed outside of sludge
• How do they move?

13
Metabolic reconstruction of CAP
-gtLooks like it can grow in C,N,P-limited
habitats -gtoligotrophic ancestry or current
lifestyle?
14
Looking for environmental reservoirs


PCR detection using 16S rRNA gene Polyphosphokina
se gene




Contra Costa wastewater treatment plant
15
CAP environmental reservoirs

Aquatic samples mostly yes Terrestrial samples
mostly no

Metapopulation
Previous map
16
In-strain variation
Dominant strain is near clonal
17
Monoculture

• Population structure
• One clonal strain dominates
• No homologous recombination
• The bulk of cells in the system are identical
• Vulnerable ecosystem!
• Explains the crashes of the system
• Waste water engineers bad influent
• No bad influent in bioreactors, still crashes

Kill the winner
18
Genomic imprints of virus-host interactions
gt95 nt identity across most of the genome

• Major differences include
• EPS gene cassettes
• defence against predation
• important for settling in EBPR
• CRISPR elements

19
CRISPR elements
1 GTTTGCCGCCGTGATGGCGGCTTAGAAATCGATGATCATCGACAGG
AGCAGATCGCCCAC 61 GTTTGCCGCCGTGATGGCGGCTTAGAAACAG
TGTCGCTCAGTCCGCCGACCAGATTCTTC 121
GTTTGCCGCCGTGATGGCGGCTTAGAAATCAACCGGAATCGCGTCTGCTT
GTTCGAGGTC 181 GTTTGCCGCCGTGATGGCGGCTTAGAAATCGACGT
GCCGAGCGACGACAGTTGCGATGCG 241 GTTTGCCGCCGTGATGGCGG
CTTAGAAAACATCGTGGCGCGCCTTGATGAGCGCCTGCTC 301
GTTTGCCGCCGTGATGGCGGCTTAGAAATGCGCAGGCACCGCAGCGCCCA
GGCCACCGAC 361 GTTTGCCGCCGTGATGGCGGCTTAGAAAGTGCAGG
GCGAGGCGGCACGTGAATATCCCGA 421 GTTTGCCGCCGTGATGGCGG
CTTAGAAAACGAATCTGGTCTGGCCCAGGCTGCAAGTCCT 481
GTTTGCCGCCGTGATGGCGGCTTAGAAATATCATGACCACCAATCGGTAT
ACATGATCCT
CRISPR - a mechanism to keep the record of and
destroy invasive elements EPS - mechanical
defence against direct contact with phages Viral
influence Genomic - CRISPR/EPS Population
structure - non-recombining, clonal dominant
strains
20
Phage metagenome

• Virion phages metagenome was sequenced
• Loads of phages
• Some phages have deep coverage - high abundance
• CRISPR spacers hit phages
• 8 in US sludge
• 2 in OZ sludge
• Some phage contigs are hit by multiple bacterial
  CRISPR spacers
• Linking unculturable host and its phage

While dispersal of the host is global, adaptation
to phages is local
21
Expression studies

• An array with viral and microbial genes was
  prepared
• The expression was monitored for 3 months
• Any active viruses?

Phages apply a constant pressure on the bacterial
community
22
Low complexity engineered systems

• Most cells in ecosystem are virtually clonal,
  non-recombining cells
• Yogurt production
• EPS cassettes CRISPR are exchanged in
  yogurt-producing strains of Streptococcus
  thermophilus
• Internal instability due to phages - common to
  all low-complexity engineered systems?

23
ConclusionsEcogenomics

• Community population structure
• Domination of a clonal asexual strain
• Global population structure
• Global dispersal
• Dispersal strategies lifestyle
• Phage pressure
• Linking a unculturable host to its phage
• Constant phage expression
• Ecosystem vulnerability
• Skewed strain abundance
• Potential stabilization / diagnostics / cure of
  failing plants
• May be common to many low-complexity engineered
  systems

24
Acknowledgments
Shaomei He S. Brook Peterson Katherine D. McMahon

• Falk Warnecke
• Hector Garcia Martin
• Philip Hugenholtz

Matthew Haynes Forest Rohwer
Linda Blackall