Transcript Balzano et al. 2014, Desal. Water Treat.

THE BIOFOULING ROLE OF MICROBES IN THE
DESALINATION SYSTEM
A/Prof. S.C. Leterme
Current work
Microbiological approach for water treatment
systems:
• pre-treatment efficacy
• biofouling issues
Microbial Loop
Prochlorococcus
Diatoms,
cyanobacteria
Ciliates, protists
Bacteria
EPS – Extracellular polysaccharide
• Produced by all microbial loop
• Especially by bacteria, cyanobacteria,
phytoplankton
• Acts as a biological glue that alters the
surface characteristics and the sticking
properties of organisms/particles
• EPS may serve as a precursor for the
production of transparent exopolymer
particles (TEP), with a size of a few µm.
• Random collision of TEPs forms marine
snow.
Bhaskar et al. 2005. FEMS Microbiol. Ecol. 53: 255
Passow et al. 2012. Environ. Res. Lett. 7 035301
Recent work
Environmental monitoring
(Balzano et al. 2015)
Assessment of pre-treatment efficacy (Balzano et al. 2014)
Focus on microbial communities and TEP production:
• Cross-flow experiments (Le Lan et al. 2015; Jamieson et al 2016; Leterme et al.
2016)
• Isolation and sequencing of bacteria (Jamieson et al 2016)
Identification of the biofouling propensity of seawater through 16s and
18s sequencing (Jamieson et al in prep)
CASE STUDY: Desalination plant of Penneshaw, Kangaroo Island,
South Australia
Penneshaw desalination plant
• No local natural fresh water
sources.
• Conventional reverse osmosis
(RO) technology.
• Minimise the impact of the brine
on the marine environment.
• Output of about 300 KL of fresh
water every day.
Balzano et al. 2014, Desal. Water Treat.
State of the environment at Penneshaw
Investigated seasonal changes
Period of study (July 2012—July 2013) had oligotrophic conditions
The phytoplankton community was dominated by species:
• adapted to grow under low nutrients
• showing mixotrophic behaviour allowing them to complement
photosynthesis with predation
Balzano et al. 2015, Oceanologia
Desalination system and 5 sampling points
Balzano et al. 2014, Desal. Water Treat.
Percentage variation for each pre-treatment step
MP-UV
MMF
15 µm CF
5 µm CF
ANOVA (p< 0.01)
TEP
20% ± 75%
-82% ± 20%
-10% ± 31%
-11% ± 33%
MMF ≠ 15umCF
Phytoplankton
-9% ± 53%
-96% ± 6%
-14% ± 49%
-20% ± 68%
MMF ≠ 15umCF
Bacteria
2% ± 26%
-56% ± 53%
-4% ± 24%
-9% ± 21%
MMF ≠ 15umCF
Viruses
9% ± 40%
-37% ± 53%
-10% ± 23%
-4% ± 33%
Not significant
Within the seawater pre-treatment, the most efficient step was MMF, whereas both MP-UV
irradiation and CF revealed poor efficiency
Balzano et al. 2014, Desal. Water Treat.
Efficacy of the desalination system
All assessed parameters underwent significant variability due to the
seasonal variability of the feedwater.
Lower removal efficiencies were found for viruses and some
phytoplankton species like diatoms
This kind of pre-treatment system is thought to be inefficient during
phytoplankton blooms - no loss in performance
Our results questioned the usefulness of the two sets of
cartridge filters located downstream to MMF in conventional
seawater pre-treatment
Balzano et al. 2014, Desal. Water Treat.
Biofouling propensity of seawater
Current seawater pre-treatment/biofouling treatments do not fully
prevent RO membrane biofouling
Spatial location and seasonal variation lead to variations in biofouling
components and precursors
Biofouling - complex mechanism involving the interaction of
microbial cells with organic molecules (TEP, proteins, lipids)
Better understanding of biofouling components and its precursors in each RO
desalination plant will lead to developing more efficient and targeted
treatments for different water environments
Cross-flow (CF) experiments
6 Sterlitech CF042
membrane cross-flow
cells
DOW Filmtech
©SWR30HR
Flow 1.5 L/min
Pressure 500 psi
7 days
Experiment 1: Raw seawater vs Pre-treated seawater
Experiment 2: Bacteria isolated from RO feed tank
Experiment 3: Diatoms isolated from RO feed tank
Le Lan et al. (2015) Desalination
Difference in seawater and pre-treated seawater
Different communities at T0 and T7d - variations in nutrient
composition, no changes in TEP (except on the membranes)
Large phytoplankton species (>300µm in length) with conic and/or
elongated shape can pass through all pre-treatment steps
Size of microbes vary depending on:
- growth stage
- cell cycle
What role do viruses have in regulating the
composition/abundance of other microbes in the system?
Le Lan et al. (2015) Desalination
Biofilm analysis
• Regulation of the expression of TEP precursors depends on:
- nutrient stress
- microbial community composition
- Temperature
- Pre-treatments
How do those factors impact on:
1. The microbial production of TEP
2. the efficiency of bacteria in decomposing organic matter
such as TEP ?
Le Lan et al. (2015) Desalination
Isolation and sequencing of microbes
• Pre-treated water from RO feed tank for static experiments of
biofilm formation
• Isolated biofouling microbial communities using different Agar
media and extracted genomic DNA for 16S sequencing
39% α-Proteobacteria
38% γ-Proteobacteria
22% Actinobacteria
1% belonged to Flavovacteria or Bacilli lineages
α-Proteobacteria Pseudomonas sp. used for static and CF
experiments
• TEP production reflected the growth stages of the biofilm
• Higher TEP production in CF than in Static conditions
How does shear impact on TEP production?
Jamieson et al. (2016) Env. Sci.: Water Res. Technol.
Biofouling Propensity of seawater
Sequencing of the V4 region of the 16S rRNA gene
and of the V1-V2 region of the 18S rRNA:
• seawater (seasonally)
• fouled membranes (2yr – 4yr old)
No similarity in microbial communities for 16S i.e. prokaryotes
Similarity observed between seawater and fouling communities
for 18S i.e. eukaryotes
1. What causes the difference in communities?
2. What is the role of pre-treatment?
Balzano et al. (2015) AME, Leterme et al. (2016) Desalination
Future work
What role do viruses have in regulating the
composition/abundance of other microbes in the system?
How do pre-teatment impact on:
1. The microbial production of TEP
2. the efficiency of bacteria in decomposing organic matter
such as TEP ?
How does shear impact on TEP production?
Why do we observe similarities only for Eukaryotes? What is the
role of pre-treatment?
Thanks to
• Prof. A. Ellis and Prof. M. Brown
• Dr S. Balzano and Dr C. Le Lan
• Students: Tamar Jamieson (PhD), Ashley D. Hemraj (PhD),
Marie Chabert (HSc), Hugo Compas (MSc), Amelie Prevost
(MSc) and Camille Moreau (MSc)
• at SA Water: Tim Kildea, Mary Drikas, Gavin Ralston, Nick
Nedelkov and Troy Kirby
The sand filter has six layers of media
The layers consists of
• 6 - 12 mm gravel underbed around and nominally 50 mm over top
of underdrains
• 3 - 6 mm gravel layer 100 mm thick
• 1.5 - 3 mm gravel layer 100 mm thick
• 0.3 mm ES Garnet, UC < 1.5, 200 mm thick layer
• 0.45 - 0.55 mm Filter Sand UC < 1.5, 500 mm thick layer
• 0.9 - 1.1 mm Filter Coal, UC <1.5, 300 mm thick layer
The Microbial Loop
• transfer of nutrient between trophic levels
• also takes into account the contributions of
different types of microbes in recycling
nutrients:
• autotrophic microbes are primary producers
• heterotrophic microbes consume dissolved organic
matter (DOM)
AQUATIC MICROBES
• PHOTOSYNTHETIC BACTERIA
• Ex. Cyanobacteria (also called blue-green algae)
• PROTISTS
• 6 groups that are abundant in aquatic environments
Role of viruses in the microbial loop
Viral lysis of microbes accelerates the
rate at which these microbes are
converted to particulate and dissolved
organic matter (i.e. POM and DOM)
This decreases the efficiency of
nutrient transfer to higher trophic
levels
Viruses also lyses Heterotrophic
bacteria to generate even more POM
and DOM
tailed bacteriophage
(Newton et al. In preparation)
Cyanobacteria
• Synechococcus, Prochlorococcus
• two marine genera
• account for ~30% of CO2 fixation, ~50% of O2 production
• less than 1% of ocean mass
• Prochlorococcus
• Most abundant photosynthesiser in the ocean
• 1988!
Prochlorococcus
Scanlan et al. Microbiol. Mol. Biol. Rev. 2009;73:249-299
Flow cytometric signature of microbial populations of (A, B)
Cyanobacteria and algae and (C) Viruses and bacteria
(A) Discrimination according to red
and orange fluorescence
(B) Discrimination according to red
fluorescence and side-scatter
(indicative of cell size)
(C) Discrimination according to
SYBR Green fluorescence
(indicative of DNA content) and
side-scatter
Martínez-Martínez J et al. J. Plankton Res. 2006;28:783-791
Protists - Diatoms
• frustule – two-piece cell wall of silica
• asexual division
• hypotheca template forming epitheca
Odontella spp.
Navicula salinarum
(Vrieling et al. 2009)
Diatoms
• frustule – two-piece cell wall of silica that form a
‘glass box’ containing the cell
• Each valve fits over the other one
• The valves are decorated
with a unique pattern of
nano-size features (i.e.,
pores, channels, ridges,
spikes, spines). Specie
specific.
Odontella spp.
Navicula salinarum
(Vrieling et al. 2009)
Diatom species under light microscopy
Diatoms
• Diatoms are single-celled,
eukaryotic algae which occur in
most marine, estuarine and
freshwater habitats on earth
• They account for 40% of the
total primary production in the
ocean and are the primary
cyclers of silica in the ocean
• The size of the nanopores present at the surface of diatoms varies
with salinity changes
Dinoflagellates
• large group found in marine plankton
• cause phosphorescence and toxic blooms seen in
seawater
• nutritionally complex: autotrophy, heterotrophy,
mixotrophy
• symbiotic forms (zooxanthellae)
• live in association with reef building
corals
Recent work
Standard water quality methods combined with flow cytometry
and molecular methods (16S rRNA sequencing and
fingerprinting) to assess in parallel:
• the physicochemical properties,
• the microbial abundance,
• the active microbial community composition
of the intake waters and along the desalination system
Sampling fortnightly from July 2012- August 2013
Desalination plant of Penneshaw, Kangaroo Island, South
Australia
Balzano et al. 2014, Desal. Water Treat.
Insert Figures from Jamieson et al 2016
Jamieson et al. (2016) Desalination
Water sources in Australia
Major sources of water for urban water utilities in
Australia are dams, rivers, streams, groundwater
bores, desalination plants and recycled water plants.
Many Australians also have their own on-site water
sources, such as rainwater tanks and greywater
systems that supplement mains supply.
Desalination in Australia
125ML/D
143ML/D
150ML/D
270ML/D
250ML/D
450ML/D
Marine Snow = shower of microbe detritus
Made up of organic matter, including dead or dying bacteria, protists, fecal matter, sand
and other inorganic dust
Most trapped particles are more vulnerable to grazers than they would be as free
floating individuals and the falling snow is followed by organisms that feeds on it
The particles are held together
by a sugary mucus transparent
extracellular polysaccharides
(TEPs)
TEP = natural polymers exuded
as waste products mostly by
phytoplankton and bacteria
TEP – Transparent exopolymeric particles
• TEPs are planktonic, organic gel-like
particles that are abundant in aqueous
environments
• These gels allow for the formation of
aggregates made of sinking and
colliding organisms/particles
• Increasing evidence indicates that
TEPs play an active role in the process
of aquatic biofilm formation
Passow et al. 2012. Environ. Res. Lett. 7 035301
Biofilm formation
Immediately upon exposure to
seawater:
(A) organic polymers and
colloids
(B) microgels such as
uncolonized TEPs
(C) protobiofilm
begin to attach to pristine
surfaces.
Single cells of planktonic
bacteria also attach
(D) reversibly
(E) or irreversibly
to conditioned surfaces.
With time (minutes to hours), a contiguous coverage of mature biofilm (F) develops
Bar Zev et al. 2012 PNAS
viruses · mL-1
2.0E+07
Spring
Summer
Autumn
28/01/13
28/04/13
Winter
1.5E+07
1.0E+07
5.0E+06
1.0E+03
1/08/12
30/10/12
27/07/13
Sampling date
Post MP-UV
Post MMF
Spring
Summer
Autumn
Winter
Post 15 µm CF
Post 5 µm CF
1.5E+06
1.0E+06
1.0E+06
1.0E+05
cells · L-1
Bacteria cells · mL-1
2.0E+06
Raw seawater
5.0E+05
0.0E+00
1/08/12
30/10/12
28/01/13
Sampling date
28/04/13
1.0E+04
1.0E+03
1.0E+02
Sampling date
4…
1…
2…
7…
1…
1…
2…
8…
1…
3…
27/07/13
Balzano et al. 2014, Desal. Water Treat.
Spring
Summer
Autumn
Winter
1.0E+05
1.0E+04
Raw seawater
1.0E+03
Post MP-UV
Post MMF
12/09/12
21/11/12
13/02/13
24/04/13
3/07/13
Sampling date
600
Summer
Post 15 µm CF
Post 5 µm CF
Autumn
Winter
500
400
1.0E+06
300
1.0E+05
cells · L-1
1.0E+02
4/07/12
Concentration (µg Xeq · L-1)
Phytoplanktopn cells · L-1
1.0E+06
200
100
0
21/11/12 10/01/13
1/03/13
20/04/13
Sampling date
1.0E+04
1.0E+03
1.0E+02
Sampling date
9/06/13
4…
1…
2…
7…
1…
1…
2…
8…
1…
3…
29/07/13
Balzano et al. 2014, Desal. Water Treat.
Balzano et al. 2014, Oceanologia
Further work currently being done
Seasonal metagenomics analysis of feed water
(16s, 18s) being compared to autopsy of RO
membranes
Investigation of the viscoelasticity of biofilms in
collaboration with Food industry and Medical experts
Phytoplankton abundance in seawater
Pre-treatment
efficacy
p<0.05
Le Lan et al. (2015) Desalination
Biofilm analysis
SEM images of DOW Filmtec(R) SW30HR RO membranes
RO membrane
unused
Fouled RO membrane
Raw seawater
Fouled RO membrane
Pre-treated seawater
After 7 days, the density of biofilm looks similar with bacteria and diatoms present in
both biofilm aggregates of microbes and other particles, salt crystals and a biofilm
formed for both experiments
Microbes aggregate and communicate through Quorum sensing
which would enhance even more biofilm formation
Leterme et al. (2016) Desalination