Interactions between bacteria and solid surfaces in relation to bacterial transport in porous media

Research output: Thesisinternal PhD, WU

Abstract

Interactions between bacteria and solid surfaces strongly influence the behaviour of bacteria in natural and engineered ecosystems. Many biofilm reactors and terrestrial environments are porous media. The purpose of the research presented in this thesis is to gain a better insight into the basic mechanims of bacterial adhesion and transport in such systems. This knowledge is essential for bacterial adhesion science in general, and important for practical applications such as the bioremediation of contaminated soils, sediments and groundwaters and the treatment of industrial waste streams in fixed bed biofilm reactors.

Model systems
Assessment of cell-substratum interactions in bacterial adhesion require materials with defined properties and an experimental set-up that allows a reproducible quantification of the transport of bacterial cells from bulk liquid to substratum. We performed our studies with model systems and under welldefined conditions in which these prerequisites are met.

Characteristics of solids and bacteria . Negatively charged teflon and glass were used as the model substrata. Eight coryneform bacteria and four pseudomonads were selected. These organisms have different negative cell surface charges at pH 7 (chapter 2). The hydrophobicity of solids and bacteria were determined by measuring the contact angle of drops of water on flat pieces of solid and on dried bacterial lawns. Glass is hydrophilic and teflon extremely hydrophobic. The hydrophobicities of dried bacteria varied between strongly hydrophilic and extremely hydrophobic. However, evidence was found that the drying of the bacteria changes their surface properties, especially in the case of amphiphilic cell-coatings. Therefore, a further characterization of the bacterial cell-surfaces was performed under hydrated conditions by measuring the isoelectric point of the bacterial strains (Chapter 4). An isoelectric point smaller than 2.8 correlated with the presence of anionic polysaccharides on the cell surface. On the basis of the isoelectric point, the water contact angle and adhesion data, three types of macromolecular cell-coatings were indentified: (i) non-polysaccharide macromolecules, like lipids and/or proteins, (ii) amphiphilic macromolecules, i.e., combinations of hydrophilic polysaccharides with either lipids or hydrophobic polysaccharides, and (iii) anionic hydrophilic polysaccharides.

Experimental set-ups. Two types of experimental set-ups were used: (i) static batch systems containing bacterial suspension and flat pieces of surface and (ii) dynamic model porous media that consisted of water-saturated columns packed with spherical substrata to which the bacterial suspensions were applied. Transport of cells from bulk liquid to substratum is much more efficient in dynamic columns than in static systems: transport is controlled by convection and diffusion under dynamic conditions (chapters 2 and 3) but by diffusion only in static systems (chapter 2).

Discrimination between method-de pendent and method-independent effects . A comparison of the static and dynamic deposition results demonstrated the absence of methodical influences on adhesion in 68 % of the cases tested. The deviating deposition behaviour of the other cases could be attributed to methodical effects resulting from specific cell characteristics, i.e., to the presence of capsular polymers, an ability to aggregate, large sizes, or a tendency to desorb after passing through an air-liquid interface. Interestingly, a strong stimulation of adhesion upon passing through an air-liquid interface occured for bacteria coated with amphiphilic compounds. Although important for specific practical applications, all method-dependent cases were excluded from further studies. The cases for which such effects are absent were subjected to further studies in order to reveal generally occurring adhesion mechanisms.

Interactions between bacteria and solid surfaces .
Adhesion is generally irreversible with respect to lowering cell concentration (chapter 3). Hence, analyses of the interactions in adhesion requires a kinetic approach. We quantified the interactions in terms of activation Gibbs energies for adhesion and detachment as determined from experimental adhesion and detachment rates. The adhesion efficiency α, which is the probability of a cell to attach upon reaching a substratum, is related to this activation energy for adhesion: activation energy = -In α.

Interactions at high ionic strength (0.1 M) . The activation energies for adhesion varied between 0 and 5 kT ( k (J K -1) is the Boltzmann constant; 1 kT = 4 x 10 -211 at room temperature) at an ionic strength of 0.1 M. A Gibbs energy barrier, located between cell and substratum and several hundreds of kT high, is created by the DLVO (Derjaguin, Landau, Verwey and Overbeek) interactions (chapters 2 and 3). This barrier cannot be surmounted by whole cells. However, bacterial cell surface macromolecules can penetrate this energy barrier and hence reach the substratum at high ionic strength. Therefore, the interactions between the cell surface macromolecules and the substratum, which are generally called steric interactions, determine adhesion at high ionic strength. The following two types of steric interactions generally occur: (i) bridging, that promotes adhesion and causes a lowering of the activation energy of adhesion, and (ii) steric hindrance that inhibits adhesion and therefore increases the activation energy of adhesion.

Mechanisms of adhesion at high ionic strength (0.1 M). The various mechanisms of adhesion (chapter 3) as deduced for the different combinations of types of cell-surface coatings and solid substrata (chapter 4) are summarized in Fig. 1 (the capitals given below refer to this figure). Long range electrostatic interactions as described by the DLVO theory (A and B) create strong repulsive barriers which cannot be passed by whole cells. In addition, deep secondary minima exist for glass but not for teflon. The secondary minima on glass result from strong Van der Waals attraction and are sufficiently deep for irreversible adhesion. Lipid or protein (non-polysaccharide) cell surface macromolecules cause strong attractive bridging on the hydrophobic surface (teflon) (C). On glass they slightly inhibit adhesion and permit adhesion in the secondary DLVO minimum as was demonstrated to occur for two hydrophobic coryneforms (D). Bacteria with an amphiphilic cell surface may adhere by bridging on a hydrophobic surface (E) whereas strong steric hindrance prevents secondary minimum adhesion on glass (F). The adhesion of bacterial cells coated with anionic polysaccharides is strongly inhibited on both surfaces (G and H). The values of the activation energy for adhesion and the adhesion efficiency for the different adhesion mechanisms are summarized in Table 1.

Activation energy for detachment. This activation energy exceeds 5 kT for irreversible adhering bacteria (chapter 3). The greatest resilience against detachment exists for hydrophobic bacteria on hydrophobic substrata. For hydrophobic/hydrophilic and hydrophilic/hydrophilic bacterium/substratum combinations, binding mechanisms not related to hydrophobicity inhibit the detachment.

Contribution of DLVO and steric interactions at various ionic strengths . The effect of DLVO and steric interactions on adhesion as a function of the ionic strength (chapter 5) is illustrated in Fig. 2. Steric interactions dominate at high ionic strength (0.1 W: adhesion is at maximum and even 100% efficient (a = 1) in the case of bridging. Long range electrostatic repulsion, as described by the DLVO model, starts to exert its influence when the ionic strength is reduced to below a critical value Is (Fig. 2). These interactions dominate the adhesion at an ionic strength of 0.0001 M. The value of 1. is determined by the distance over which the cell-surface macromolecules penetrate the electrostatic barrier. Between the bacterial strains tested, the extension of cell surface macromolecules varies between 5 nm and 80 nm, which correspond to /,-values varying between 0.1 and 0.001 M. A cell-substratum separation of 165 nm can even be bridged by a flagellated Pseudomonas putida strain. The practical consequence of these findings is that studying and controlling bacterial adhesion in groundwater and waste water should include the assessment of both DLVO and steric interactions since the ionic strength of these environments varies around 0.01 M.

Bacterial transport in porous media.
Bacterial deposition on spherical glass and teflon collectors was studied in vertical downflow columns (chapters 6 and 7). Deposition was analyzed in terms of the clean bed collision efficiency a, (the probability of a cell to attach upon reaching a cell-free substratum), and a blocking factor 8 (the ratio of the area blocked by an attached cell to the geometric area of a cell).

Deposition in porous media at high ionic strength (0.1 M). The influence of the bacterial cell coating and substratum type on the initial adhesion efficiency is similar as found for the static batch systems (Fig. Is Table 1). However, the dynamic column system is superior to the static batch system, since it provides a means to also determine the influence of cell-cell interactions on bacterial deposition. Cell-cell repulsion, enhancing blocking, increases with decreasing (more negative) charge of the cell and with increasing hydrophilicity of the cell-surface macromolecules.

Deposition in porous media at varying ionic strengths. The initial efficiency (α o ) decreased with decreasing 1 , for 1 -values smaller than the criticial level Is in a similar way as found for the static batch systems. The level of B in creases about one order of magnitude upon changing 1 from 0.1 M to 0.001 M.

Control of bacterial transport and the occurrence of pore-clogging. The effect of cell- solid interaction (as inferred from the initial adhesion efficiency) on bacterial transport in porous media is illustrated in Figure 3. Depths of penetration into a porous medium may be as high as 50 m at low ionic strength. At high ionic strength, this penetration depth varies between about 0.25 m and 20 m depending on the type of cell-coating.

Maximum control of microbial mobility in porous media can be reached in systems for which B and α o , are high at high ionic strength: the high B -value corresponds to strong cell-cell repulsion which minimizes the occurrence of multilayer adhesion and pore-clogging (Fig. 4). The dependencies of α o and B on the ionic strength allow manipulation of deposition by varying the ionic strength.

An analysis of literature data on dispersal of Bacillus cells in columns packed with coarse sand (chapter 6) demonstrated that the collision-blocking model also applies to natural systems. Hence, deposition of microbes during their transport through engineered or natural porous media is adequately described by the collision-blocking model for strongly blocking cells or weakly blocking cells at low coverage conditions.

Concluding remarks
In this study we have entered the interface between microbiology and physical chemistry. It was demonstrated that the interactions between, at first sight complex biological systems like bacteria, and solid surfaces can be well analyzed and understood in terms of basic physico-chemical principles. A major accomplishment of the research presented here is the quantification of the steric interactions between cell-surface macromolecules and solid surfaces. Combining these with the effects of long range Van der Waals and electrostatic interactions provided a complete classification of the generally occurring mechanisms of bacterial adhesion. A second important achievement is the quantitative formulation of bacterial deposition during transport in porous media. This makes it possible to describe and predict bacterial transport in natural and engineered porous media with basic particle deposition parameters, which in turn, are related to the properties of the bacterial cells.

Several challenges for further research emerged during our research, The analyses of the steric interactions in bacterial adhesion presented in this study are strictly empirical. A more fundamental approach could be the investigation of the adhesion of better defined organisms, like Iipopolysaccharide mutants of Salmonella typhimurium and various Rhizobium species. These mutants are fully characterized and have cell-surface macromolecules varying in chain-length and chemical composition. In some cases, these cell-surface polymers are even commercially available. Combining experimental studies with these defined materials with theoretical investigations by computer-simulation promises to provide further insights into the steric interactions in bacterial adhesion.

Another important topic for further research is the role of cell-cell interactions in the performance of bacterial deposition. This is of great practical relevance for the application of bacteria in porous media since it is a key-factor in the occurrence of pore-clogging. Furthermore, these interactions may also be of relevance for bacterial biofilm formation. Bacterial deposition techniques using isolated cells may serve as tools to characterize these cell-cell interactions which may then be related to the performance of the biofilm.

Revealing the fundamentals of bacterial deposition is a key step towards the application of these organsisms in the bioremediation of contaminated soils and groundwaters. However, bacterial adhesion is not the only factor in a successful bioremediation procedure. The interactions between contaminants and the solid matix are also important. In fact, many pollutants adsorb very strongly to the solid phase and become only slowly available for biodegradation. We also investigated this problem using the porous medium model systems presented in this study. That work is not included in this thesis and will be presented in separate papers to be published in the near future.

This research is a symbiosis between the discplines of physical and colloid chemistry and microbiology that produced knowledge about the interactions between bacteria and solid surfaces which could not have been revealed without this cooperation. Hopefully, the highly interesting and economically important interface between microbiology and physical chemistry will be further explored in future.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • Zehnder, A.J.B., Promotor
  • Lyklema, J., Promotor, External person
  • Norde, W., Promotor, External person
Award date25 May 1994
Place of PublicationWageningen
Publisher
Print ISBNs9789054852520
DOIs
Publication statusPublished - 25 May 1994

Keywords

  • microbiology
  • waste treatment
  • waste water treatment
  • surfaces
  • interface
  • applications
  • electricity
  • magnetism
  • surface phenomena
  • electrostatics
  • electromagnetism

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