Thursday, May 5, 2016

Biofilms and Drinking Water

Clean drinking water is an essential in any modern society. As has been well known, even the well managed drinking water may be contaminated as a result of the presence of biofilms[1]. External contaminants are carefully restricted but the growth of biofilms is almost inevitable. The growth is often fostered by the pipes themselves with iron based pipes providing a fertile ground for the biofilm[2]. Biofilms are the development of an integrated bio mass resulting from the growth of bacteria and other microorganisms. The biofilm aggregates and builds into a large mass which can degrade pipes, inhibit flow, and initiate bio hazards if the fluid such as water is to be used in a potable manner. Biofilms are a significant cost in the operations and maintenance of various water flow mechanisms in residential and commercial facilities. 

We have prepared an analysis of biofilms and their inhibition in remodified drinking water systems.

There are many nano bacteriostatic mechanisms for surface treatment have been demonstrated to inhibit bacteria and the resultant biofilm growth. The application of additive nano-Se appliqués or other extractive nano-surfacing have been shown to inhibit biofilm growth via the bacteriostatic actions. The nano technology can be applied to the repair and maintenance of existing distribution systems.

This analysis focuses specifically on the use of the nano technology to public water supply systems. Specifically, it addresses the issue of system remediation and the need for a low cost and fast method to purge the biofilm contaminants as well as shielding other contaminants such as lead from public water supply systems. The proposal focuses on the use of PVC treated with the nano surfacing technology as well and the development of the insertion and installation methodologies to achieve a very low cost remediation system.
The problem being proposed for study here is the mitigation and inhibition of biofilms in the transport and distribution of water in public water distribution systems. We demonstrate the typical system below.

As the above demonstrates water is generally collected from aquifers, or other storage areas, and at times directly from flowing bodies. It is then treated and purified and perhaps stored in local facilities. Then as demand occurs it is distributed across a local network. It is that local network which is extensive and in many cases aged that biofilms occur. It in this network that seeks to examine the efficacy and cost efficiency of deploying PVC nano-treated insertions. The proposed installation is demonstrated below:
The process is simple: 1. Purge old pipe and clean with a standard "pigging" device and then repurge for all removed biofilm. 2. Insert nano treated PVC sleeve for a new pipe.

Preliminary analysis indicates that this nano treatment will inhibit regrowth of biofilms for extended periods and further the PVC sleeve will inhibit outflow of such elements as lead from the old pipe. The primary purpose of this study therefore is to validate this approach using the existing nano technology based upon data and sample obtained from actual systems in situ.

Biofilms are created by the adhesion of bacterial aggregates on the surfaces of various fluid processing, transport and containment mechanisms. The basic physics surrounding this phenomenon was presented by van Loosdrecht et al and it is based upon the construct of surface energy. The small biological particles can adhere to surfaces and one attaching can create via extracellular membrane extension the foundation for a developing biofilm. The problems with this biofilm are significant in a variety of areas such as oil pipelines as discussed by AlAbbas et al and in desalination as discussed by Elimechem et al in desalination plants. Srey et al provide an excellent survey of the impact of biofilms in the food industry. A typical biofilm encrustation is shown below:

Note the significant growth of biofilm. In the reference by Pervical et al they have extensive discussions regarding the development process in potable water. The effect of chlorine in the water does diminish the growth slightly but it is a common factor not only in loss of flow but in contamination. Fundamentally the process is some three steps as shown below:
The details on the above are also in Kanematsu and Barry. The first step is the development of a protein film on the surface. Then the bacteria reversibly attach and finally the bacteria irreversibly attach and additional biofilm mass grows to cover the bacteria. Another more detailed view will incorporate a combine reversible and then irreversible process initiated by the coating of the surface by a protein like substance shown below.

Note in the above we must have the layer irreversibly adsorbed to allow an initial reversible bacterium to attach. We will explain this later when examining the issues regarding forces. From Fleming and Ridgeway, we have:

The term “biofouling” is referred to as the undesired development of microbial layers on surfaces. This operationally defined term has been adapted from heat exchanger technology where “fouling” is defined generally as the undesired deposition of material on surfaces, including:

 – Scaling, mineral fouling: deposition of inorganic material precipitating on a surface
– Organic fouling: deposition of organic substances (e.g. oil, proteins, humic substances)
– Particle fouling: deposition of, e.g., silica, clay, humic substances and other particles
– Biofouling: adhesion of microorganisms to surfaces and biofilm development

The conditioning film shown above is essential. It is a laying down of proteins and water which adhere to the surface via an adsorption process. We generally suspect that such an adsorption is due to the van der Walls forces from the surface to the structure of the specific proteins. We will expand this discussion latter. Upon completion of the surfacing then the proteins extending from the bacillus manage to penetrate this barrier and also become attached via van der Waals forces. Darouiche discusses the impact of biofilms on medical implants as well. He notes:

The essential factor in the evolution and persistence of infection is the formation of biofilm around implanted devices. Soon after insertion, a conditioning layer composed of host-derived adhesins (including fibrinogen, fibronectin, and collagen) forms on the surface of the implant and invites the adherence of free-floating (planktonic) organisms. Bacterial cell division, recruitment of additional planktonic organisms, and secretion of bacterial products (such as the glycocalyx) follow.

As Batte et al note:

Most of the pipes used in drinking water distribution systems are made of plastic (PVC, PE, etc.) or metal (copper, cast iron) which can become highly corroded (Figure 11). A recent survey of public distribution system pipes in France showed that a large proportion of them are PVC (40%), while the rest are grey iron (22%) or ductile iron (20%) (Cador 2002).

They continue:

The effects of the organic nutrients released by plastic pipes on bacterial growth in drinking water have long been questioned. Organic additives which leach out of plastic have a measurable impact on biofilm accumulation, and are known to promote the multiplication of opportunistic, pathogenic bacteria in laboratory tests. However, no field studies have looked at these events…The lack of information on biofilm dynamics is a limiting factor in managing the quality of water in distribution system and conducting drinking water surveys. In spite of the difficulty of gaining access to the inner surfaces of distribution pipes, biofilm measurement on pipe walls is indispensable if more information on the water contamination risks is to be obtained. New methods need to be developed, adapted, evaluated and optimized. Such methods will create important advantages: continuous, non-destructive, simple, in situ, online information on biofilm location and development.

From Preedy et al we have:

Biofilms are defined as a layer or layers of cells adhered to a substratum which are generally embedded in an organic biological matrix, i.e., extracellular polymeric substances (EPS). It is due to biofilm formation that many bacteria survive in highly diverse and adverse environments as a result of the polymicrobial ecosystem....Not surprisingly, biofilms have formed on a variety of surfaces and are not only restricted to attachment at a solid—liquid interface but have been observed at solid—air and liquid—liquid interfaces, with some having beneficial results as well as detrimental; for example, in industry biofilms are used successfully to separate coal particles from mineral matter. On the other hand, biofilms have been known to cause biofouling reducing mass and heat transfer and effectively increasing corrosion; also from a medical point of view, biofilm colonized implanted medical devices often lead to implant failure.

Current technological areas focus on several areas. The areas are:

Nano Surface Enhancements: These are nanotechnology enhanced titanium surfaces which demonstrate reduction in bacterial infection potential and also demonstrate enhanced tissue and bone growth ensuring improved human acceptance.

Surface Bactericidals for Intracorporeal Applications: These are nanotechnologies for surface coatings of various catheters and the like that result in dramatically reduced risks of infection by inhibiting bacterial growth.   

Selenium Enhanced Bactericidals: This is a selenium based product which enables the control of bacterial growth. It appears to function as a bacteriostatic agent. Combined with a bactericidal agent the combination may affect dramatic control for long periods of bacteria on surfaces. This area of product development appears to have several areas of application: (1) Those applications which can be seen to be applied directly to the skin (cosmetics, wound dressing, etc.), and (2) Those applications which can be used in clinical and consumer applications to treat surfaces for anti-bacterial purposes, (3) The control of growth on various surfaces of harmful flora or fauna.

Treatments have developed an approach to mitigate the growth of biofilm. This is via the treatment of the surface by nano processing. The treatments may be either by addition of materials such as nano Se or by the selective deletion of surfaces to create a similar nano surfacing effect by the use of lipase and other similar surface treatments. For example, nano Se has been demonstrated to slow down S. aureus proliferation at a dose-dependent rate. Increased lag time (in 40 and 20 μg/mL doses) would allow for the body’s immune system to attack bacteria before exponential growth. We demonstrate some of these results below:

In the above we note that the regrowth of bacteria is dramatically reduced by the application of a Se surface at concentrations of 20-40 micrograms/milliliter. It has also been observed that surface coatings of a density of 100 ng/sq cm of 20-70 nm diameter nano Se on the surface are also adequate. The question we pose herein is; what is the physical process that causes this to occur? Bacterial efficacy has been demonstrated as shown below:

·       Gram positive Staphylococcus epidermidis was decreased by several logs on SeNP-coated paper towels

·       SeNP coatings have also reduced gram negative Pseudomonas aeruginosa, E. coli, MRSA, and ampicillin resistant E. coli

We now examine the physical processes which may account for this twofold process. Namely:

1. Nano Se coatings and lipase nano processing of surfaces tend to create a bacteriostatic environment.

2. Nano Se coatings and nano surface processing tend to create an environment that enhances tissue adhesion.

These appear to be contradictory results. It would appear that both processes are controlled by the same physical mechanism. Yet the outcomes are dramatically different. We attempt to explain some of these effects. However, it should be noted that in our analysis the explanation is yet far from clear.

The following is a brief discussion of some of the basic principles and specific technologies. Details are contained in the papers by Webster and his team at Northeastern and Brown University. We have also examined the literature in general and provides a summary regarding that as well.

The principal basis for the technology is understanding surface energy as relates to bacterial adhesion and subsequent biofilm growth. We demonstrate the basic principle below for a eukaryotic cell using the approach by Webster. On a smooth surface we have with cells a fibronectin, a glycoprotein, which binds integrins. This allows the pathogen cell to attach to the surface and commence biofilm growth.

The issue then is to create a surface which is not conducive to the binding. This can be accomplished by manipulating the surface energy by mechanical means. We can show that the protein absorption is proportional to the surface energy. We can briefly examine van der Waals forces as discussed by Butt and Kappl. Let us first consider simple Coulomb forces. We can consider three types of surface to external adhesion for vdW. They are shown below:

 Later we shall see that many argue for the simple connection of inverse square where one surface is positive and the other is negative. This is a simple vdW approach. However, the other two can be equally valid depending on the nature of the molecules connecting. Namely in the case of proteins the protein structure can be quite complex depending on the specific amino acid construction. Note that for proteins the bonds generally are inverse fourth power strength due to the dipole-monopole configuration. There may even be cases in certain protein structures where the bonds are inverse sixth power[3].

The adhesion of bacteria to surfaces is a complicated and yet to be satisfactorily answered phenomenon. There are several theories and we will examine one herein. We use the DLVO approach which is a force or energy approach. Alternative approaches using thermodynamically defined terms and Gibbs Free Energy, G, have also been proposed but they do not seem to provide adequate answers. Let us first review some general principles.

The DLVO (Derjaguin, Landau, Vervey, Overbeek) approach uses the two forces; van der Waals and Ionic. The paper by Trefaly and Borkovec is an excellent summary of this and we shall follow its approach.

Now the surface may be seen as below with these two forces:

Note that in close we have an attraction due to van der Waals and then at a distance we have the double layer effect. The scales are not precise but just descriptive. The vdW force is much stronger but there is a positive "barrier" between it and the outer layer. Brownian motion can get a bacterium close to the surface and catch it reversibly in the ionic or DL area. However, to have an irreversible bond something must get to the vdW section, a much stronger section.

Now the bacterium sends out a filament to try to bond to the surface via the vdW forces. It must penetrate the barrier and then bond. In the Boland et al paper we have an example of such bonding showing the extending filaments:
 They discuss what they term cellulose binding domains, CBD, areas of the protein which do the binding in this case to cellulose. They state:

These CBDs have been classified into 10 families (I-X) on the basis of amino acid sequence homology. The amino acid sequences of CBDs in C. cellulovorans and C. josui show high homology with those from other cellulolytic genera such as Bacillus. CBDs in this family contain several highly conserved amino acid sequences:

1. Tryptophane-asparate-phenylalanine-asparagine-asparate-glycine-threonine
2. Isoleucine-alanine-alanine-isoleucine-proline-glutamine
3. Isoleucine-leucine-phenylalanine-valine-glycine

We can then ask; what if we roughen the surface, what will that do? The specific answer is not known and even less understood conceptually. A logical conclusion is that by roughing the surface we increase the positive side by moving the inner vdW in and out and thus make it more difficult to adhere. The Thermodynamic argument is a hand waving discussion of surface energy. But we tried that argument above without success on adhesion of human tissue cells.

As Bok noted in his Thesis:

The forces that govern microbial deposition, adhesion and detachment are still not fully understood, and difficult to relate with each other. In a previous study we successfully investigated the characteristic shear force to prevent adhesion of microbial strains. In the current research we used a more systematic approach by including not only the shear forces to prevent adhesion, but also those that stimulate detachment of adhering bacteria, as well as theoretical adhesion forces calculated using the extended DLVO theory. …

1) A strong hydrodynamic shear force to prevent adhesion relates to a strong hydrodynamic shear force to detach an adhering organism.

2) A weak hydrodynamic shear force to detach adhering bacteria implies that more bacteria will be stimulated to detach by a passing air-liquid interface through the flow chamber….

3) DLVO interactions determine the characteristic hydrodynamic shear forces to prevent adhesion and to detach adhering micro-organisms as well as the detachment induced by a passing air-liquid interface.

Thus from the above experimental analyses the DLVO has some merit but it clearly does not describe the entire process. There are significant issues still outstanding to be explained theoretically. Bacterial adhesion and the formation of biofilms is still in the process of being fully understood. Kanematsu and Barry provide an exceptionally strong discussion here but we must resort to experimental data for phenomenological insight. Boland et al also provide a substantial discussion on this but fail to provide a strong analytical basis. Their analysis is useful to better understand some of the phenomenology.

The thermodynamic paradigm is based upon certain principles that aggregate large collections of common particles like gas, steam, or a fluid. Thermodynamic principles work in the large like those used in reactors or distillation columns or heat exchangers. We shall review some of these principles and then demonstrate their lack of efficacy in this model.

For example, when considering the process of wetting, one can generally use thermodynamic and surface tension methods. There is a homogeneity on the surface and on the wetting materials. Tran and Webster (2013) have provided an interesting analysis for nano scale wetting. They explain it via the Wenzel and also the Cassie-Baxter models. They all involve surface tension as is done in the core Young's analysis[4].

van Loosdrecht et al were one of the first to explain the adhesion via thermodynamic principles. Then they state:

The Concept of Short-Range Interactions

If adhesion is performed at constant pressure and temperature, and if the molecular composition of the surface does not change, all G's can be replaced by the corresponding interfacial tensions. This concept is restricted to those cases where bacteria and the solid surface are in direct contact and the original phase boundaries are replaced by a new one, namely, the bacterium solid interface. When this new interface is formed, interfacial tensions may be used for a direct estimation of the adhesion Gibbs energy. …

The Concept of Long-Range Interactions

The DLVO theory for colloidal stability can be used to calculate the interaction Gibbs energy between a particle and a surface as a function of the separation distance (H). The balance of interracial Gibbs energies … is the basic premise of this theory. The net interaction Gibbs energy is interpreted in terms of Van der Waals interactions (which are usually attractive) and an electric interaction due to the overlap of the electrical double layers at the charged surfaces. The most important parameters determining the van der Waals interaction are the Hamaker constant, which is a material property, the distance (H) between bacterium and substratum, and the geometry of the system. …

alAbas et al demonstrate oil pipeline biofilm as below:

Now in contrast alAbas et al note:

Thermodynamic approach

The thermodynamic approach assumes the system is in equilibrium and the bacterial attachment is a reversible process. The interfacial free energies between the interacting surfaces are compared and calculated, …. This comparison is expressed in the so-called free energy of adhesion. …The microbial adhesion will be favorable when the change in G, is negative (< 0) and will not be energetically favorable if … positive.

They then continue:

DLVO Approach: The drawback of the thermodynamics approach is that it ignores the electrical double-layer interaction with the bacteria… This assumption is invalid as the bacterial cells have a surface-negative or-positive charge. In contrast, the DLVO approach displays a balance between attractive Lifshitz- van der Waals… and repulsive or attractive electrostatic forces … These two forces are function of the distance (d) between the bacteria and surface. In order to calculate the adhesion free energy … the electrostatic interactions between surfaces should be included. The inclusion of electrostatic interactions requires that the zeta potentials of the interacting surfaces be measured, in addition to measuring contact angles...Extended DLVO approach: The extended DLVO theory relates the origin of hydrophobic interactions in microbial adhesion and includes four fundamental interaction energies: Lifshitz-van der Waals, electrostatic, Lewis acid-base, and Brownian motion forces …

 The above approach makes semi-macro thermodynamic assumptions. Specifically, a large mass of surface, liquid and biofilm concentrate. In fact, the dynamics of the process are totally overlooked. This is the general failing of thermodynamic approach; they assume some form of steady state along with homogeneity. In reality we have a dynamic process in a highly heterogeneous environment. We briefly discuss the technology to be employed. The details are contained in the references by Webster discussed herein.

Nano surface treatments can be accomplished by treating the surface itself or adding nano materials to the surface. The result is a stable nano surface that inhibits bacterial growth and ensuing biofilm development. The Gecko has nano fibrils on its feet that allow it to climb any surface by means of van der Waals attraction as we see in nano material surfaces. The production of nano Se is performed via a proprietary process but fundamentally is the following:

Glutathione +NaOH + Se --à Nano Se

As Mendonca et al note, using reference to Webster's work, the details of surface energy effects and adhesion or lack thereof:

The changes in initial protein–surface interaction are believed to control osteoblast adhesion. This is a critical aspect of the osseointegration process. When implants come into contact with a biological environment, protein adsorption (e.g. plasma fibronectin) that occurs immediately will mediate subsequent cell attachment and proliferation. Cell binding to protein domains of adhesive extracellular matrix proteins involves receptors termed integrin receptors that transmit signals through a collection of proteins on the cytoplasmic face of the contact, termed focal contacts. … Webster and colleagues observed an increased vitronectin adsorption on nanostructured surfaces when compared to conventional surfaces. They also found an increased osteoblast adhesion when compared to other cell types, such as fibroblasts, on the nanosurfaces. … Surface roughness at the nanoscale is an important determinant of protein interactions that ultimately direct cell activity in control of tissue formation at implant surfaces.

To obtain a proper nano surface there are two methods. The additive method uses nano Se which can be made at specific nano size and in a very well controlled and defined distribution so as to assure the proper surface energy. The second approach is the deletive approach whereby a nano surfacing has a process that removes materials in such a controlled manner so as to achieve the same desired surface energy.

Nano Selenium has been demonstrated as highly effective. In addition, as we demonstrate below it is also safe and sustains the effect on the surface for an extended period of time.

Why Se? The reasons are as follows:
  •  Essential micronutrient metalloid, and component of several key antioxidants, detoxifying and metabolic enzymes, in form of selenocysteine, selenomethionine
  • Two allotropes: red (bioactive) and grey (crystalline) and  Strong associations with reduction of Reactive Oxygen Species1,2,3 (ROS) as well as  Cofactor of glutathione peroxidase
  • Antibacterial activity to a broad range of pathogenic strains
  •   Nano Se can be produced at specific nano diameters with minimal dispersion,  Spherical in shape
  • Monodisperse—size distribution fits within one bell curve and negatively charged (uncoated)

The deletive approach used extraction mechanisms which produce similar effects to the additive mechanism of nano Se. The advantage of such an approach is that it does not add anything to the material. The disadvantage in certain active biological surfaces such as human skin is that it causes immunological effects. However, its used in stable media such as PEEK, Titanium, steel, and other materials used for water flow and containment is that it can be readily effected and at low cost. There are numerous processes to implement nanoscale surface features on metallic or polymeric surfaces. We then utilize one of our processes to create such nanoscale features: Anodization or Chemical etching. The deletive approach provides comparable results to that for Se coatings.

There have been a variety of biofilm inhibition methods. As Garrett et al note:

Bacterial adhesion has become a significant problem in industry and in the domicile, and much research has been done for deeper understanding of the processes involved. A generic biological model of bacterial adhesion and population growth called the bacterial biofilm growth cycle, has been described and modified many times.

The biofilm growth cycle encompasses bacterial adhesion at all levels, starting with the initial physical attraction of bacteria to a substrate, and ending with the eventual liberation of cell clusters from the biofilm matrix. When describing bacterial adhesion one is simply describing one or more stages of biofilm development, neglecting the fact that the population may not reach maturity. This article provides an overview of bacterial adhesion, cites examples of how bacterial adhesion affects industry and summarizes methods and instrumentation used to improve our understanding of the adhesive properties of bacteria.

The NRC report states[5]:

The pipe surface itself can influence the composition and activity of biofilm populations. Studies have shown that biofilms developed more quickly on iron pipe surfaces than on plastic PVC pipes, despite the fact that adequate corrosion control was applied, the water was biologically treated to reduce AOC levels, and chlorine residuals were consistently maintained…In addition to influencing the development of biofilms, the pipe surface has also been shown to affect the
composition of the microbial communities presents in the biofilm. Iron pipes supported a more diverse microbial population than did PVC pipes. The purpose of these studies is not to indicate that certain pipe materials are preferred over another but to demonstrate the importance of considering the type of materials that come into contact with potable water. 

We examined several issues. Specifically:

1. What is a Biofilm? This we have answered by reference to various studies.

2. How do biofilms form? The answer to this may often depend but it is clearly a dynamic process.

3. What is the physical phenomenon that allows biofilms to adhere and have strong adsorption? This is a work in progress. We believe the thermodynamic approach is problematic at best. It is necessary to consider more detailed dynamic physical phenomenon. We make some suggestions here.

4. What is the effect of nano-surfacing on biofilms? This appears to be uncertain at best. There are contrasting phenomenological results.

5. Why does nano-surfacing enhance adsorption of certain eukaryotic cells such as bone and ligaments while inhibiting the adsorption of prokaryotic cells such as bacteria? This appears not to have been examined.

6. How can nano-surfacing be optimized to minimize biofilms? Argument from surface energy have been proposed but are problematic.

These questions can and have been answered in part but there remains a set of uncertainties that challenge the effective utilization of nano technologies.

We can possibly argue the following explanation from what we have developed herein.

1. The first coating of a surface is by the protein layer. Generally, this is done by some local van der Waals forces since the proteins are close to the surface and are well known to exhibit such forces. Also the protein layer seems to be a prerequisite for adhesion. However, the type of protein layer may very well depend on the surface structure. They structure of proteins vary widely and perhaps if we adjust the nanostructure we selectively change the type of protein adhering to the surface.

2. We know that bacteria seem phenomenologically to require proteins to adhere for them in turn to reversibly adhere to the proteins. This the proteins must be electrostatically and vdW wise strongly attracted to the surface and the cell.

3. After a reversible adhesion then we seem to have the appearance of protein filaments extruding from the bacteria and down through the protein layer, most likely using the protein to overcome the barrier wall normally between van der Waals and electronic forces. Once the filament hits the surface then it adheres irreversibly and the biofilm commences growth.

4. The supposition is that by changing the roughness of the surface we change the types of proteins or the nature of their adhesions on the surface. There does not appear to be any research determining this one way or the other at this time.

The challenge of this analysis is shown below. On the one hand certain biologicals adhere on roughness and others are repelled. One of the continuing questions is why, and what is the fundamental physical reason.

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[1] See EPA Report 1992.
 [2] See NRC 1998 report in detail.
[3] See Petsko and Ringe, Protein Structure and Function, Sinauer (Sunderland, MA) 2004. Pp 8-11.
 [4] See Butt et al, pp161-180.
[5] See p 230-234.