Title slideProfessor, Microbial Interactions Laboratory, Department
of Environmental Health Sciences,
Arnold School of Public Health, Univ. South Carolina Columbia, SC
29208;
[email protected]
The Biofilm: Efficient, Adaptable, & Structured
Alan W. Decho
• What is a biofilm?
• Key processes within biofilm EPS
• Influences of biofilms on the biofouling process?
• Insights for biofilm-control/ manipulation
WHY BACTERIA FORM BIOFILMS
Bacteria- a lonely life?
3. BIND, LOCALIZE & CONCENTRATE:
Diversity of Natural Biofilms: Why is a biofilm so resilient?
• thousands of OTU’s (species, strains?)
• high Metagenomic diversity (often undescribed genes)
• abundant/rare Species: Ecological “Insurance”
BIOFILMS IN MARINE SYSTEMS
• Open-water Aggregates (marine snow), colloids, marine snow oil, C
flux to ocean bottom
• Biomineralization (CaCO3)
• Sediments – sand grains mats; (sediment stability, alter optical
properties)
• Biofouling of Surfaces: intertidal, subtidal
Are biofilms friendly places to live?
• Abundant agonistic/antagonistic interactions;
1mm layer
-Release planktonic cells
Functional groups on EPS (FT-IR)
Wavenumbers (cm-1)
Ab so
rb an
EPS Sorptive bind organics, metals; - assume many shapes,
configurations - Physical form of matrix is adaptable
1. EPS Matrix loaded with potentially-sorptive Functional
Groups
Composition: polysaccharides, proteins, DNA, lipids
2. EPS Ultrastructure
EPS allows bacteria to: ‘localize, protect, and coordinate
processes efficiently;
AFM of EPS Gel in SW (30 ppt)
Decho
Signal molecule
(i.e. low density)
Signal conc prop to cell #s;
Diffusion signal reaches ‘threshold concn
Coordinate gene express & physiol activ.
“Common to Many Bacteria
QS A change in gene expression
Natural Bacteria (in biofilms) live in ‘clusters or groups’ This
facilitates QS
Bacterial cells
Biofilm formation;
Light production;
Antibiotic prod.
How does QS work? acyl-homoserine lactones (AHLs)
O
O
Substitute at C3 position
6 5 4 3 2
At ‘threshold concentration’ (10-100 nM) signal binds to receptor
protein (luxR)
Signal / luxR complex activates (gene) operon
Allows bacteria to coordinate activities more powerful
effects!
Many different AHLs: acyl chain-length specificity of signal
C4
C10
C8
C6
3-oxo-C4
3-oxo-C10
3-oxo-C8
3-oxo-C6
3-oxo-C12
Organic extraction
2. EPS Localizes Signaling within Biofilms Can Cell-Clusters
communicate with other Clusters
What distances can cells ‘talk’ to each other?
O
O
Diel Cycle: pH 6.5 (NIGHT) – 9.6 (DAY)
C6-AHL
AHLs are pH susceptible: acidic (day) and alkaline (night)
conditions
T(1/2) of AHL vs pH
0
10
20
30
40
50
AHL
AHL
Chart1
33.5
2.4
34.2
2.3
28.3
3.1
23.4
4.6
26.6
10.9
22.5
36.9
AHL
Sheet1
pH
C6
C7
C8
C10
C12
C14
c6
c7
c8
c10
c12
c14
6.2
79.8
150.3
89.6
53.1
63.9
75.3
0.01
0.01
0.01
0.01
0.01
0.001
7.2
33.5
34.2
28.3
23.4
26.6
22.5
0.01
0.01
0.01
0.01
0.02
0.01
8.2
4.2
4.2
5.7
8.7
22.4
35.5
0.02
0.02
0.03
0.02
0.01
0.01
8.7
2.4
2.3
3.1
4.6
10.9
36.9
0.02
0.02
0.02
0.01
0.01
0.02
9.55
0.52
0.5
0.73
1.15
1.1
11.4
0.09
0.06
0.18
0.04
0.11
0.09
pH
C6
C7
C8
C10
C12
C14
c6
c7
c8
c10
c12
c14
7.2
33.5
34.2
28.3
23.4
26.6
22.5
0.01
0.01
0.01
0.01
0.02
0.01
8.7
2.4
2.3
3.1
4.6
10.9
36.9
0.02
0.02
0.02
0.01
0.01
0.02
Sheet1
AHL
Sheet2
Sheet3
• 3D - EPS polymers translucent; Good penetration of photons; •
High-O2 , oxidants, (during daylight; 300x overlying water!)
EPS
Modified AHL
5. Photo-induced Oxidant Effects on C6-AHL
CH3 NH
66 7
40 60 80 100 120 140 160 180 200 220 240 m/z0
100
168.1156.1 213.8186.2
MS of Compound 4: Note m/z = 113.0 fragment ion represents
oxo-substituted side chain. Formula C10H15NO4 further confirmed by
accurate mass measurement using Q-TOF. Confirmed by comparison of
retention time with 3-oxo-C6-AHL std.
3-oxo-C6-AHL
C6-HSL pH 5 photolysis for 24 hr
3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00
15.00 16.00 17.00 18.00 19.00 20.00 Time0
100
Four major ‘-oxo-’ products
C6-AHL was changed into an oxo-C6 different AHL via environ
conditions: = Environ SENSOR?
5. EPS: localize ‘Vesicle Minefields’ in biofilms
Contain: • Signals: protect signals outside of cells?
• e-enzymes, • antibiotics; • large plasmids;
When desiccated biofilms are rehydrated, bacterial
cells/extracellular QS quickly ( 1 hr) regain activity – common
process!
‘Hydrated’ Wet Mat ‘Desiccated’ Dry Mat
AHLs remain intact. ‘EPS GLASS’ formation preserves ‘hydrated’
steric conformation of cell membranes, enzymes and signals
(AHLs)
Hydrophobic Shield by amphiphilic EPS
Hydrophilic biofilm- allows exchange
Biofilm fouling of Sediments & Sensor Devices-Shallow
Oceans
EPS Changes ‘Optical Properties’:
-Enhanced ‘forward scattering of photons (light);
Biofilms cause detectable changes in photon absorption &
scattering
(Office Naval Res., Environ. Optics Program [Decho et al. 2003.
Limnol Oceangr.]
Biofilms & Concentration of Larval Settlement cues by EPS
Image adapted from: Aldred et al. 2018. Proceeds Royal Soc B
Surface
EPS Cues and other natural products are localized/ concentrated by
diffusion- slowing properties of EPS. Process is likely variable
over small spatial scales.
Lower concentrations of cues in water directly over biofilm
Overlying Water
Nanotechnology:
Biofilm 61% +2.6
Biofilms are ‘Environmental Sponges’ for Nanoparticles?
In estuarine mesocosm, 60% of added gold NPs were sequestered in
biofilms
• Can Au-NPs can be added to biofilm, accumulate, then ‘heated’
using infared-irradiation kill cells, ‘burn EPS’
-surfaces can be easily functionalized; -IR penetrates seawater
well, non-damaging; - non-toxic; -approx. 500 atoms per NP;
A. Can Au-NPs be used to alter biofilms?
EPS is a ‘barrier’ and/or ‘sponge’ for NPs
Experimentally compare EPS density: ‘tight-gel’ vs ‘loose-slime’
measure diffusion & penetration of NPs in EPS matrix
Larger- nanoparticles
Au NP w/ coating
Ongoing Work: NP Penetration into EPS
HYPOTHESIS: EPS ‘Density’ & ‘pore-space size’
Water Pore-space
Use Super-Resolution Microscopy- diffusion rates of fluor NP
through EPS
C. Create Self-sustaining Inhibitory Biofilms - inhibitory to
larval settlement: ‘PROTECTIVE BIOFILMS’
• Macrofouling of surfaces is a large driver in compromising ocean
vessels and ocean-located devices.
• Can surfaces be designed to select for biofilms, which inhibit
larval settlement?
• Only ‘minimal biofilm fouling’ is present! -no
macrofouling.
• e.g. Fish skin, macro-algae leaves
Grand Challenge: What happens at the small scale counts! Biofilm
Adaptability!
Future biofilm analyses:
-EPS Chem Comp changes (Raman confocal)
-Small-Scale Heterog. of EPS (new in situ image appr.)
-QS Inhibition- biofilm ctrl Biofilm
Clean surface Macrofouled surface
Alan W. Decho Microbial Interactions Laboratory,
Dept. Environ. Health Sciences, Arnold School of Public Health,
University of South Carolina
NSF –BME Prog. ONR – CoBOP Prog. (past) NIH – NIAID Prog. NASA –
Exobiology Prog.
ICMCF Conf. 2018, Melbourne, FL.
Slide Number 1
Slide Number 2
Diversity of Natural Biofilms: Why is a biofilm so resilient?
Biofilms in marine systems
Slide Number 7
Slide Number 9
Slide Number 11
Many different AHLs: acyl chain-length specificity of signal
Slide Number 14
Slide Number 15
Slide Number 16
AHLs are pH susceptible: acidic (day) and alkaline (night)
conditions
4. Effects of ‘Photo-Oxidants’ on biofilm AHLs
5. Photo-induced Oxidant Effects on C6-AHL
5. EPS: localize ‘Vesicle Minefields’ in biofilms
6. EPS Protection against ‘Desiccation’
Slide Number 22
Biofilms & Concentration of Larval Settlement cues by EPS
Insights for ‘Control’ of biofilms & biofouling?Nanotechnology:
Can we target Bacterial Communication Processes and EPS
matrix!
Slide Number 26
Slide Number 27
Slide Number 29
C. Create Self-sustaining Inhibitory Biofilms - inhibitory to
larval settlement: ‘Protective Biofilms’
Grand Challenge: What happens at the small scale counts! Biofilm
Adaptability!
Thanks…..to Sergey and ICMCF!
