Introduction

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  Biofilm is a multi-cellular bacteria community, which is prevalent in diverse natural and artificial settings. It is best known for its robust adaptability and strong resistance to environmental stress. We intended to utilize and modify this natural multicellular structure to develop a new kind of extracellular scaffold, constructing a stratified protein expression system using the naturally formed graded oxygen concentration within the bacteria biofilm.
  Biofilm formation process can be roughly divided into four stages: bacteria attachment, micro colony formation, biofilm maturation and biofilm dispersion. The best sample for the observation is the matured biofilm, which, according to previous studies, was formed after 48 hours after inoculation with only exception in bubbling method (Bandara HM et al. 2009; Klausen M. et al. 2003.).
  Based on the references (Almeida C. et al. 2011; Rani SA et al. 2007), we've developed three different biofilm formation methods: Bubbling method, Rubber tube method and cell culture plate method. The biofilm formed in the two former ways were observed through cryo-sectioning coupled with normal fluorescence microscopy while confocal laser scanning microscope was employed to obtain the biofilm images of the third method. In addition to the above, we've tested different material's applicability in biofilm formation experiments and introduced autoinducer AI-2 as an accelerator molecule.

How to set up a biofilm reactor in your lab: Shopping list

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Bubbling method:

Large test tubes with plug http://item.taobao.com/item.htm?id=12269690038	$0.43
Rubber tube http://item.taobao.com/item.htm?id=12473511203	$0.32/meter (mailing cost not included) 0.4 meter per usage
Glass slide http://item.taobao.com/item.htm?id=8877150757	$0.94 for 50 slides
Common lab ware: (Test tubes, a shaker, an incubator, pipettes and culturing medium)	We believe you already have them
Total cost	$0.58 per set

Rubber Tube method:

Silicon tube http://item.taobao.com/item.htm?id=10401921234&	$0.32/meter (mailing cost not included) 0.6 meter per usage
Glassware	Need to be made to order, prize may vary
Wriggle pump http://item.taobao.com/item.htm?id=1484983763	$217 (mailing cost not included) can be used on two sets of experiments at the same time
Common lab ware: (Test tubes, a shaker, an incubator, pipettes and culturing medium)	We believe you already have them
Total cost	$218 per 2 sets

For Cell Culture Plate Method

A 24 well, flat bottom Corning? Costar? cell culture plate;Or a 6 well, flat bottom Corning? Costar? cell culture plates	$3
10 cover slips	$0.5
Common lab ware: (Test tubes, a shaker, an incubator, pipettes and culturing medium)	We believe you already have them
Total cost	$3.5 per set!

 

Modeling

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Introduction

Compartment:The biofilm itself is distinguished from the overlying water and the substratum to which it is attached. A mass-transport boundary layer separates the biofilm from the overlying water.

Within each compartment are components: include different types of biomass ,substrates , products. biomass is often divided into active microbial species, inert cells, and extracellular polymeric substances(EPS).

The components can undergo transformation, transport, and transfer processes. For example, substrate is consumed, and this leads to the synthesis of new active biomass.

All process affecting each component in each compartment are mathematically linked together into a mass balance equation that contains rate terms and parameters for each process.

Model Selection:Many kinds of Mathematics models have been founded to describe a system of biofilm. Models of different dimensions (1d, 2d, 3d) focus on different properties of a biofilm. Since we care most about the oxygen concentration gradients perpendicular to the substratum, numerical 1-dimensional dynamic model(N1) would be a proper choice for us.

Compartment

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The biofilm:A biofilm is a gel-like aggregation of microorganisms and other particles embedded in extracellular polymeric substances. A biofilm contains water inside it, but its main physical characteristic is that it is a solid phase. A biofilm normally is anchored to a solid surface called the substratum on one side and in contact with liquid on its other side. Frequently, a mass-transfer boundary layer is included between the bulk liquid and the biofilm itself. Thus, following figure illustrates a biofilm having four compartments: the substratum, the biofilm itself, the boundary layer, and the bulk liquid outside of the biofilm. While it is complex even for a homogeneous biofilm morphology, we assume the biofilm surface is flat and all material below the maximum biofilm thickness as part of the biofilm components, and they have a constant density.

 

the mass-transport boundary layer: Experimental observations clearly indicate strong concentration gradients for solutes just outside the biofilm when these solutes are utilized or produced by the microorganisms in the biofilm. Consequently, the solute concentrations at the biofilm surface and in a completely mixed bulk liquid often are significantly different.So we introduce the mass-transport boundary layer,which is a hypothetical layer of liquid above the biofilm and in which all the resistance to mass transport of dissolved components outside the biofilm occurs.

The bulk liquid: In our experiments, the bulk liquid is large compared to the biofilm. So the simplest way seems to consider it as a boundary condition of the biofilm compartment and specify the concentrations of dissolved. However, dissolved components can exchange between the biofilm and the bulk liquid, and it has a profound impact on the concentrations in the bulk liquid. Thus we include the bulk liquid not only as a boundary condition, but also as a separate, completely mixed compartment, varying according to the inflow, outflow, and the exchanges with the biofilm.

The substratum: In our basic model, the substratum is a separate compartment and impermeable. So it does not have much effect on the biofilm system. However in some bioreactor, the substratum may be permeable, or include organic solids that are biodegraded by attached microorganisms.

Component

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Dissolved components: There are two kind of dissolved components in our model, one is oxygen, the other is substrates where nutrients are inside. They are expressed by inflow concentration of oxygen, inflow concentration of substrates and monod half saturation constant for substrates, monod half saturation constant for oxygen.Diffusion coefficient of oxygen and diffusion coefficient of substrate are also used to characterize the property of these components.

Particulate components: In our model, the particulate components are microbes and EPS. We assume that they are homogeneously mixed in the same proportion in all parts of the biofilm. So can use bacterial density to relate the amount of bacterial and the volume it takes up. And the volume fraction of EPS in Bacterial is specified by a constant.

Process

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Transformation processes usually are biochemical reactions that produce or consume one or more components: e.g., consumption of substrate, production of metabolic end-products, microbial growth and decay, and production of EPS.

The transport processes that regularly are considered in biofilm models are advection, molecular diffusion, and turbulent dispersion. In special cases, transport of charged components by migration in an electric field created is included. The general, 1d expression to model the specific mass flux of a component is calculuted in the direction z

Transfer processes exchange mass of dissolved or particulate components between two compartments. At the interface between the compartments, a continuity condition for the component concentration C and the specific flux j of the exchanged mass must be fulfilled. Continuity means that C and j are the same on both sides of the interface between the compartments. C and j can be calculated at each side of the interface from boundary conditions

 

Results

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Microscopy images

Figure 1 The figure shows the modified DH5alpha's biofilm formed on rubber tube. The pink color was due to the RFP expressed by the bacteria. The picture was obtained with Leica DME microscope, under 40X objective.

Figure 2:
3D CLSM picture of the biofilm formed by E. Coli DH5alpha. The picture was obtained with Zeiss LSM 710NLO confocal laser scanning microscope, using a 20X objective. The colors of the biofilm were not from the fluorescence proteins but artificially added in order to give better resolution.

Figure 3:
CLSM image of the stratified biofilm obtained with the Cell Culture Plate Method. It was formed by our modified E.Coli expressing device BBa_K561003 (you can refer to our Rainbofilm module for more information). The picture was taken by Zeiss LSM510 Meta confocal laser scanning microscope, using a 63? oil-immersion objective.

* Price comparison:
Biofilm reactor from biosurface company CDC Biofilm Reactor: $770 Model DFR 110 Drip Flow Reactor: $989.00 BST Model DK 20-1 Disk Reactor System with stir plate and support stand: $1,040.00

References

1.Bandara HM, Yau JY, Watt RM, Jin LJ, Samaranayake LP. Escherichia coli and its lipopolysaccharide modulate in vitro Candida biofilm formation. J Med Microbiol. 2009 Dec; 58(Pt 12): 1623-31.
2. Klausen M, Heydorn A, Ragas P, Lambertsen L, Aaes-Jorgensen A, Molin S, Tolker-Nielsen T. Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants. Mol Microbiol. 2003 Jun; 48(6): 1511-24.
3.Almeida C, Azevedo NF, Santos S, Keevil CW, Vieira MJ. Discriminating multi-species populations in biofilms with peptide nucleic acid fluorescence in situ hybridization (PNA FISH). PLoS One. 2011 Mar 29; 6(3): e14786.
4.Teal TK, Lies DP, Wold BJ, Newman DK. Spatiometabolic stratification of Shewanella oneidensis biofilms. Appl Environ Microbiol. 2006 Nov; 72(11): 7324-30.
5.Rani SA, Pitts B, Beyenal H, Veluchamy RA, Lewandowski Z, Davison WM, Buckingham-Meyer K, Stewart PS. Spatial patterns of DNA replication, protein synthesis, and oxygen concentration within bacterial biofilms reveal diverse physiological states. J Bacteriol. 2007 Jun; 189(11): 4223-33.
6.Gonzalez Barrios AF, Zuo R, Hashimoto Y, Yang L, Bentley WE, Wood TK. Autoinducer 2 controls biofilm formation in Escherichia coli through a novel motility quorum-sensing regulator (MqsR, B3022). J Bacteriol. 2006 Jan; 188(1): 305-16.
7. Dickschat JS. Quorum sensing and bacterial biofilms. Nat Prod Rep. 2010 Mar; 27(3): 343-69.
8. Lopez D, Vlamakis H, Kolter R. Biofilms. Cold Spring Harb Perspect Biol. 2010 Jul; 2(7): a000398.