Received 10 December 2015 Accepted 29 February 2016Accepted manuscript posted online 4 March 2016

Presented By

Swathy J.R06.08.2016

Vyūha - 'to arrange troops in a battle array'

• a multi-tier defensive formation• counter-measure targeted specifically against each Vyūha

Biofilms of today’s paper…Biofilms of today’s paper…

Crystalline or lithifying biofilms - Biofilms with mineral

deposits

Problems caused,

•Reduce heat exchange efficiency in cooling towers•Water flow in pipes•Membrane clogs•Pathogens

General views of microbialites from the Alchichica Lake. (a) Overview of the lake surrounded by white emerged subfossil microbialites mainly composed of hydrated Mg carbonates, namely hydromagnesite. (b and c) Closer views of white subfossil microbialites from the lake bank and dark microbialites covered by biofilms below the lake surface. (d) Green microbialite fragment sampled between 6 and 8 m water depth.

Maximum intensity projection of stacked CLSM images obtained on RNAlater-fixed sample (a and c) or fresh sample (b and d) of microbialites from 14 m depth stained with calcein (not embedded). Images were obtained with a sequential excitation at 405, 488 and 543 nm, and fluorescence emission collected in the ranges 425–475, 500–530 and 560–660 nm. The white arrow indicates a cell strongly labeled by calcein.

Emmanuelle Gérard et. al., Specific carbonate–microbe interactions in the modern microbialites of Lake Alchichica (Mexico), The ISME Journal (2013) 7, 1997–2009; doi:10.1038/ismej.2013.81; published

online 27 June 2013

Top view of a colony of an undomesticated strain of wild-type B. subtilis (NCIB 3610). The colonies were grown on solid biomineralization-promoting medium without or with a calcium source at 30 °C, in a CO2-enriched environment.

(a) Images representing the thickness of the calcium carbonate buildup underneath the wrinkles of wild-type and lcfA mutant. The images were obtained from the microCT by 2D slice cutting through the biofilm. (b) The surface morphology of a calcite mineral. An environmental scanning electron micrograph (ESEM) image of the calcite mineral extracted from the wild-type strain.

Yaara Oppenheimer-Shaanan et. al., Spatio-temporal assembly of functional mineral scaffolds within microbial biofilms, npj Biofilms and Microbiomes (2016) 2, 15031;

doi:10.1038/npjbiofilms.2015.31; published online 2 March 2016

In this paper

• The paper describes a strategy to use the biofilm’s own source to disrupt it.

• The carbonate scaffold formed inside the biofilm enhances the chlorine penetration.

FIG 1 PAO1 biofilm morphology after no treatment (a), after in situ calcite biomineralization (b), and after calcite particle deposition (c). Images are overlays of calcite deposits (blue) and biofilm biomass (green) (b and c). (Left) Three-dimensional opacity views; (right) orthogonal sections.

P. aeruginosa PAO1-gfp3 days old biofilm

CaCO3 medium (15mM CaCl2 and

NaHCO3)10 ml/h, 12 h, pH

7.6

CaCO3 medium (0.5M CaCO3 and

NaHCO3)1 ml

Confocal microscopy 488 nm

argon laser/63X

Imaging Calcite biomineralization and particle deposition in biofilmsImaging Calcite biomineralization and particle deposition in biofilmsResultsResults

Images were obtained after 190 s of Cy5 transport, when the concentration profiles reached steady state. D is the Cy5 effective diffusion coefficient calculated from the concentration-time distribution. The effective diffusion coefficient is not available (N.A.) for the particle deposition case (c) because the one-dimensional diffusion model cannot uniquely distinguish Cy5 propagation through the particle layer and the underlying biofilm on the basis of the resolution of the available data.

Image: wikipedia

Biofilm permeability assay - Biofilm permeability assay - Cy5 transport in PAO1 biofilm coloniesCy5 transport in PAO1 biofilm colonies

(Top) Planar heat maps of Cy5 intensity in and around a biofilm colony. Black lines, the edges of the biofilm colonies. Bars 10 m. (Bottom) Curves for Cy5 penetration into an untreated biofilm

One dimensional diffusion model

De= Diffusion coefficientC = Radially averaged Cy5 conc. from fluorescence intensityr = distance from biofilm surfaceαC= First order removal of Cy5T = time

30.2% increase in diffusion coefficientR

adia

lly a

vera

ged c o

nce

ntra

t ion

(Top) Planar confocal images with live/dead staining. Dead cells (stained by PI) appear in red, and live cells appear in green. (Bottom) Planar killing heat maps. Chlorine killing is limited to the biofilm surface in untreated biofilms (a), occurs much more deeply in biofilms after biomineralization (b), and is significantly hindered in biofilms after particle deposition (c). (d) Killing efficacy for each treatment. Error bars are standard deviations from triplicate experiments.

Chlorine killing patterns in biofilms after biomineralization and Chlorine killing patterns in biofilms after biomineralization and particle deposition particle deposition

100 ppm NaClO / 30 min

Rinse

Stain

Image

Top view confocal micrographs of chlorine killing patterns in non-treated biofilms (a), biofilms after biomineralization (b), and biofilms after particle deposition (c). Live cells appear in green and dead cells appear in red. Chlorine killing reaches deeper in biofilms after biomineralization (b) and was limited at biofilm surface after particle deposition (c). Scale grid = 23 µm.

Enhanced solute transport induced by biomineralization increases biofilm

killing by chlorine

3D opacity views of Chlorine killing 3D opacity views of Chlorine killing patterns in biofilmspatterns in biofilms

DiscussionDiscussion

• Insitu biomineralisation : Enhanced penetration• Particle deposition : Hinders solute transport

1.Biomineralisation gradually perforate the biofilm architechture

2.Access throughout the biofilm is obtained by the mineralization

3.Minerals introduce channels/pores inside and reshape the internal structure.

4.Decreases local cell density.

The combined effect of insitu biomineralisation and particle deposition has not

been discussed in this work.

Effect of other scale forming minerals like phosphates and sulfates are not

tested

Future directionFuture direction

• Effect of Ag+ penetration into biofilms grown in natural waters

supplemented with CO32- can be studied.

• A time lapse confocal/raman imaging facility available by Dr.

Kamalesh can be a tool to visualize this ?

Thank youThank you