Susan Springthorpe Richard Kibbee Turki Abujamel Alain Stintzi

Susan Springthorpe
Richard Kibbee
Turki Abujamel
Alain Stintzi
Andy Campbell
Erin Gorman
Ian Douglas
Project precursor
 E. coli lost cultivability in treated water equally before and 




after disinfectant addition
Something in the settled water affecting growth
Assumed it might be the coagulant residual (alum) since always present in slight excess
Controlled experiments showed the bacteria accumulated Al and growth shut down to give VBNC cells
If E. coli affected, why not many other bacteria?
Could there be an effect on biological filtration?
Water Science & Technology: Water Supply—WSTWS Vol 10 No 3 pp 269–
276 © IWA Publishing 2010 doi:10.2166/ws.2010.902
Introduction & background
 Settled water inevitably contains low level of residual 




coagulant
Most coagulants used are Fe‐ or Al‐based
Fe is essential element for bacterial metabolism
Al has no recognized biological function and is a universal toxin albeit at differing levels
What, if any, are the negative effects of Al or Fe on microbial populations on biological filters?
Free Al can be accumulated by less tolerant bacteria and lead to a shut‐down in some cellular functions: possibly ultimately to bacterial death
Hypotheses
 That use of a Fe‐based coagulant would lead to more genetic and functional diversity of active microorganisms in biological filters than for an Al‐based coagulant
 That as a result of more diversity and potential for biological activity, there might be lower levels of disinfectant by‐products formed
Objectives
 To determine the structure of microbial populations on biological filters operating in parallel when water is treated upstream by either Fe‐based or Al‐based coagulants.
 To examine the relative microbial diversity of these populations
 To evaluate the DBPFP (disinfectant by‐product formation potential) for the samples to determine if lower levels of DBP are formed following use of Fe‐based compared with Al‐based coagulants.
Location & set‐up
 City of Ottawa, Ottawa, Canada
 Passive biofiltration of settled water on main filter beds
 Side‐by‐side pilot plant allowing direct comparison of alternative treatments on single input source
 4 new columns; dual media, sampling ports in each
 Primary comparison intended was on alum vs ferric based coagulation
 Secondary comparisons on different media and on brand new media and old media harvested from full scale plants
4 columns; Diameter 6”
• Filter 1 (Harvested Sand & Anthracite from full scale plant –
Alum as coagulant)
• Filter 2 (Harvested Sand& Anthracite from full scale plant –
Ferric as coagulant)
• Filter 3 (New sand and GAC – Alum as coagulant)
• Filter 4 (New sand and GAC – Ferric as coagulant)
Operation and Matrix
 Approximately a one‐year period
 Pilot plant operated continuously with regular 




backwashing on a cycle similar to the full scale plant
Samples were collected approximately monthly; extracted immediately or frozen at ‐80°C
2 Coagulants: ferric or alum
2 media ages: new or harvested from full scale filters (old)
2media types: sand or anthracite (old); sand or activated carbon (new)
Thus 8 bins of data
Outcomes evaluated
 Types of microorganisms present based on 16S rDNA
(metagenomic sequence data from barcoded samples)
 Most numerous types of microbes present – shown through different taxa summaries
 Diversity of microbial populations  Disinfectant by‐product formation potential (DBPFP) [not measured at all sampling periods]
 Total HAA
 Total THM
Limitations
 Only small amounts of media can be used for DNA extraction; possibility for heterogeneity; should be minimized because regular backwashing
 16S rDNA only gives types present, does not show their relative activities (transcriptomes needed)
 Background levels of bacterial DNA on starting materials significant; even for brand new media
 Inactive types might or not disappear so current data could be somewhat confounded
Methods
 Sampling both media types for each column
 DNA extraction of biofilm microbial communities from media (Fast‐DNA Spin Kit (MP Biomedicals) using the FastPrep bead beater
 Sequencing barcoded samples in the Illumina HiSeq 2500 at Next Generation Sequencing Facility; Centre for Applied Genomics (Hospital for Sick Children, Toronto, ON)
 For more detail on the metagenomic analyses, see
 Abujamel T, Cadnum JL, Jury LA, Sunkesula VCK, Kundrapu S, et al. (2013). PLoS ONE 8(10): e76269. doi:10.1371/journal.pone. 0076269
 DBPFP analysis using standard methods at City of Ottawa
Taxa Summaries
 Data far too diverse to show as is
 Most prominent categories depicted for each level
 Phylum [47]
 Class [131]
 Order [275]
 Family [486]
 Genus [956]
Phylum
Relative Abundance
1.0
0.8
0.6
0.4
0.2
A
lu
m
C
A arb
lu
m New
C
A
a
lu
m rbO
S
ld
A and
lu
m Ne
Sa w
Iro nd
n C Ol
ar d
Iro bN
n C ew
Iro arb
nS
O
an ld
Iro dN
nS ew
an
dO
ld
0.0
Others
Elusimicrobia
Firmicutes
Verrucomicrobia
Armatimonadetes
OD1
Cyanobacteria
Actinobacteria
Nitrospirae
Planctomycetes
Chloroflexi
Bacteroidetes
Acidobacteria
Proteobacteria
Class
Relative Abundance
1.0
0.8
0.6
0.4
0.2
Iro
ld
dO
an
nS
dN
ew
ld
an
bO
nS
ar
Ir o
I ro
nC
bN
ew
ld
ar
nC
Iro
Sa
nd
O
ew
N
A
lu
m
Sa
m
lu
A
nd
bO
ar
C
m
lu
A
A
lu
m
C
ar
bN
ew
ld
0.0
Others
Sphingobacteriia
DA052
SL56
Deltaproteobacteria
TK17
Acidobacteriia
Chloracidobacteria
Acidimicrobiia
Acidobacteria-6
S085
Actinobacteria
Nitrospira
Saprospirae
Anaerolineae
Planctomycetia
Cytophagia
Solibacteres
Alphaproteobacteria
Gammaproteobacteria
Betaproteobacteria
Order
Relative Abundance
1.0
0.8
0.6
0.4
0.2
ld
dO
ew
an
dN
nS
an
I ro
Iro
nS
ar
bO
ld
ew
nC
Ir o
ar
bN
O
nd
nC
I ro
Sa
m
lu
A
ld
ew
N
ld
Sa
m
lu
A
nd
bO
ar
C
m
lu
A
A
lu
m
C
ar
bN
ew
0.0
Others
Acidimicrobiales
iii1-15
envOPS12
Chromatiales
Sphingomonadales
WCHB1-50
SBR1031
Actinomycetales
Gemmatales
Planctomycetales
Nitrospirales
Saprospirales
Rhodospirillales
Gallionellales
Rhizobiales
Rhodocyclales
Cytophagales
Xanthomonadales
Solibacterales
Burkholderiales
Family
Relative Abundance
1.0
0.8
0.6
0.4
0.2
lu
A
A
lu
m
C
ar
b
m N ew
C
A
ar
lu
bO
m
Sa
ld
nd
A
lu
N
m
S a ew
Ir o n d
nC Ol
ar d
I ro b N
nC ew
Ir o a r
nS bO
a n ld
dN
Ir o
ew
nS
an
dO
ld
0.0
Others
Caulobacteraceae
Ellin6075
Sphingomonadaceae
Bradyrhizobiaceae
ACK-M1
oc28
Hyphomicrobiaceae
Isosphaeraceae
Planctomycetaceae
Nitrospiraceae
Rhizobiaceae
Chitinophagaceae
Acetobacteraceae
Gallionellaceae
Solibacteraceae
Rhodocyclaceae
Comamonadaceae
Cytophagaceae
Sinobacteraceae
Oxalobacteraceae
Genus
Relative Abundance
1.0
0.8
0.6
0.4
0.2
A
A
lu
m
C
ar
b
lu
m New
C
A
ar
lu
b
m
S a O ld
nd
A
lu
m Ne
w
Sa
I ro n d
nC O l
ar d
Ir o b N
n C ew
Iro ar
nS b O
an l d
dN
Iro
nS
ew
an
dO
ld
0.0
Other
Rhodoplanes
Methylibium
Synechococcus
Aquicella
Ramlibacter
Polynucleobacter
Variovorax
Gemmata
Legionella
Hyphomicrobium
Novosphingobium
Bradyrhizobium
Phenylobacterium
Neoasaia
Steroidobacter
Planctomyces
Nitrospira
Sulfuritalea
Gallionella
Candidatus Solibacter
Diversity of Microbial Populations
 α population diversity/richness calculated by two measures ‐ Chao1 and Shannon
 Shows greater diversity for Fe vs Al
 β diversity – within group diversity
 Shows no significant differences within groups
Diversity/Richness of Microbial Populations
Linear Discriminant Analysis of Assigned Bacteria Taxa
α diversity
Chao1
NS
800
600
400
200
n
Iro
Shannon
A
lu
m
in
um
0
P = 0.003
7
6
5
lu
m
Iro
n
in
um
4
A
Shannon Index
Chao1 Estimated OTUs
per 3,890 Reads
1000
Alum vs Ferric DBPFP Trial ‐ October 1‐4, 2012 Total THM and Total HAA Concentrations from Pilot Filters #1 and #2 Dosed at 1.60mg/L T= 0, 24, 48 and 72hrs
30
25
0 24 48 72
20
ug/
15
0 24 48 72
0 24 48 72
10
5
0 24 48 72
0
Alum THM ug/L
Ferric THM ug/L
*Both filter columns anthracite and sand
Alum HAA ug/L
Ferric HAA ug/L
Total HAA Concentrations from Pilot Plant Filter Effluent #1, #3 and #4 Chlorine Dosage: 1.70mg/L Coagulant Dosage: Alum 34.50mg/L Ferric 50.31mg/L 30.00
25.00
20.00
15.00
10.00
5.00
0.00
Filter #1 Alum
Anthracite
Filter #1 Alum
Anthracite Dup
Filter #3 Alum GAC
Filter #3 Alum GAC
Dup
Filter #4 Ferric GAC
Filter #4 Ferric GAC
Dup
Blank
Total THM Concentrations from Pilot Plant Filter Effluent #1, #3 and #4 Chlorine Dosage: 1.70mg/L Coagulant Dosage: Alum 34.50mg/L Ferric 50.31mg/L 30
25
µg/L
20
15
10
5
0
Summary and Discussion
 Demonstrable differences between alum and ferric coagulants for 



microbial populations
Greater diversity for ferric coagulation treatment
Lower HAA and THM for ferric treatment
Does the ferric coagulant :
 Improve coagulation?
 Promote beneficial biological activity?
 Both – perhaps by reducing microbial death of the active biodegraders?
 Not restricted to filtration – also can have activity during settling
Bacteria and extracellular bacterial products known to contribute to DBP formation
 Water Research 2013, 47, (8), 2701‐2709.
 Environmental Science & Technology 2012, 46, (20), 11361‐11369.
 Our studies submitted for publication implicating dead cells
Gaps/Further studies needed
 The role of whole bacterial cells – both live and dead versus 



extracellular products such as biofilm matrix and egested endotoxins/cell wall components in DBP formation and degradation
More understanding of the types of microorganisms that are active degraders of TTHM and HAA
DBP formation and degradation in distributed water post disinfection
Studies on other types of biofiltration
The value of sand vs anthracite microbial populations
 Seguin et al. (2014) Disinfection byproduct formation during filter cycle: implications for water treatment. Submitted