The invention relates to a method for preparation of a Lactobacillus rhamnosus GG probiotic bacterial biofilm on a surface of gold and bioelectrode obtained by this method.

Microbial bioelectrochemical systems (BESs) are an intensively developing field on the boundary of biology and electrochemistry. They utilise the ability of microorganisms inhabiting anode or cathode to transform chemical energy into electrical energy [1 -3]. Such a process is possible due to a transfer of electrons originating from the metabolic pathway of these organisms, which are called electrochemically active microorganisms [1]. So far, the BESs have found application in many fields, particularly in biosensor construction. One example is the use of bacteria of the Acetobacter family for construction of a bioelectrode being a part of a hydrogen peroxide-sensitive sensor [4]. Another example consists in monitoring of glucose level using sensors with immobilised Escherichia coli bacteria [5], which have found application in electricity production and sewage treatment too [6]. Also, other bacterial species, such as Shewanella, Streptococcus, Pseudomonas, have found application in construction of electrodes used in hydrogen production, water desalination, or in chemical synthesis [7- 11].

Microbial fuel cells (MFCs) are known type of BESs. Due to the increasing need of energy production from renewable sources, they enj oy a still increasing interest [12-13]. MFC systems are based on a biofilm formed on the anode, i.e. a layer of bacteria, which inhabit the surface of the electrode and are covered with an extracellular matrix composed of organic and inorganic substances, and the whole exhibits an electrochemical activity. Moreover, occurrence of plankton organisms in the electrolyte is possible too, which have also an electrochemical activity and may find application in construction of cells. The operational principle of a microbe-based cell consists in the fact that the electrons released as a result of oxidation of organic compounds by bacteria are transferred to the anode, and then to the cathode, causing a current flow in the system [14]. The electron transfer may occur in several ways. At present, four basic mechanisms of the electron transfer from the biofilm to the anode surface are being distinguished in the literature [13,15]: a) transfer via artificial mediators (mainly synthetic dyes); b) abiotic oxidation (on the surface of the electrode) of reduced organic compounds formed as a result of bacterial fermentation; c) transfer through natural mediators, produced by the bacteria; d) direct electron transfer to the anode.

The actual mechanism depends on many factors, such as the electrode type, the bacterial strain, as well as the type of electrolyte used.

In known examples of bioelectrochemical systems, pathogenic or potentially pathogenic microorganisms are typically used. One of such strains is Escherichia coli, which, despite it occurs in human bacterial flora, is known of its ability to cause diseases of, among others, alimentary system.

[16]. Other group of bacteria used in BES is constituted by those obtained from sewage, river deposits or soils [7,10]. Thus, application of these strains poses a risk of numerous hazards, therefore, provision of solutions based on the use of microorganisms friendly both for humans and the environment, is desirable.

One of such solution may be the use of a Lactobacillus rhamnosus strain. It belongs to the gram- positive bacteria, occurring in the form of bacilli, and having a characteristic feature of forming chains. Some strains, e.g. Lactobacillus rhamnosus GG, are used for production of probiotics and yoghurts [17]. However, despite their advantageous properties, they have not found application in bioelectrochemistry hitherto, as they do not form biofilms on metal surfaces. A particular issue which requires solving consists in proposing modification of the surface of metallic electrodes so as to enable formation of a bacterial film by this strain.

An important aspect of construction of components of a biological system for electrochemical applications consists in the type and structure of the electrode. Both the material of the electrode and the electrode's morphology may have a significant impact on colonisation of the surface by microorganisms. At present, carbon materials (graphite [1, 18], carbon fiber, carbon paper, carbon brushes [1]), which may be subjected to numerous modifications, are most commonly used for the anode construction. They have the advantage of a low cost, however, adhesion of bacteria to their surfaces may be impeded because of a too low roughness of the electrode. In consequence, performance of such a fuel cell or sensor will be insufficient. That is why new materials are still being sought for, which will allow for increasing the yield of the generated electric current, and simultaneously, will not be toxic for the bacteria inhabiting them. Such metals as copper, silver or gold could be a promising alternative for carbonic materials, however, their bacteriostatic or even bactericidal properties are commonly known [19-21]. To enable adhesion and multiplication of micro-organisms on the surface of such metals, its modification is necessary.

The main goal of the present invention is to provide a method for production of a Lactobacillus rhamnosus biofilm on a surface of gold.

Surprisingly, the problems defined above have been solved in the present invention. The invention relates to a method for preparation of a bacterial biofilm on a metal surface, characterised in that:

a) the surface made of gold is covered with a polymer layer having a permanent positive charge (polycation)

b) the surface of gold modified with the polycation, obtained in step a), is colonised by bacteria of the Lactobacillus rhamnosus GG strain by submerging it in a suspension of these bacteria.

Preferably, the polycation used in the method according to the invention is a polymer selected from the group including a polymer obtained by polymerisation of 3- methacryloylaminopropyltrimethylammonium chloride or a polysaccharide modified with glycidyltrimethylammonium chloride, preferably dextran, pullulan or inulin.

Preferably, in step a) of the method according to the invention, a polymer layer is applied onto a gold surface by submerging it in a solution containing positively charged polymer molecules ("layer by layer" technique).

Preferably, in step b) of the method according to the invention, the colonisation by Lactobacillus rhamnosus GG bacteria is carried out by submerging the surface in a suspension of these bacteria in a culture medium, particularly MRS, preferably for approx. 2 h.

Preferably, in step b) of the method according to the invention, the colonised surface is left additionally in the culture medium for from 24 to 72 hours.

Another subject of the invention is a bioelectrode, characterised in that it has a surface made of gold and covered with a layer of a polymer having a permanent positive charge (polycation) colonised by bacteria of the Lactobacillus rhamnosus GG strain.

Preferably, the polycation is a polymer selected from the group including a polymer obtained by polymerisation of 3-methacryloylaminopropyltrimethylammonium chloride or a polysaccharide modified with glycidyltrimethylammonium chloride, preferably dextran, pullulan or inulin.

Preferably, the bioelectrode according to the invention was obtained by the aforementioned method.

Bioelectrodes according to the invention may find application in production of fuel cells or biosensors.

In the method according to the invention, it is possible to obtain a Lactobacillus rhamnosus biofilm, particularly that of the Lactobacillus rhamnosus GG strain, on a surface of gold, which has been pre-modified by covering it with a layer of a polycation, preferably obtained by the LbL method. Surprisingly, the formation of a polycationic layer on a surface of gold allowed for adhesion of Lactobacillus rhamnosus GG bacteria on the so-modified metal surface. Due to this fact, the negative impact of a noble metal on the survivability of the microorganisms was not observed. Surprisingly, the bacteria were still proliferating and forming a bacterial biofilm on the metal surface covered with the polycation. Also, it allowed for maintaining the electrochemical activity by the Lactobacillus rhamnosus GG strain, enabling to obtain a bioelectrode according to the invention.

According to the invention, by "biofilm", a colony of alive bacteria is understood, maintaining an electrochemical activity manifesting itself in a possibility to affect the potential of the so-obtained metal electrode, preferably gold electrode.

According to the invention, the metal surface, particularly gold, is being covered with a layer of polycations. In context of the invention, "polycations" are all known polymers having a permanent positive charge, resulting from the presence of positively charged groups in their structure, for instance, quaternary amines, for example trimethylammonium amines. Polymers suitable for use according to the invention may be of natural origin, such as cationic polysaccharides, or of synthetic origin. Examples of cationic polysaccharides are dextran, pullulan, inulin, or other polysaccharide of natural origin, transformed as a result of an attachment of a proper amine. On the other hand, examples of synthetic polycations suitable for use according to the invention are constituted by products of polymerisation of proper amino derivatives of methacrylic or acrylic acid. Usually, cationic polymers exhibit a high toxicity in solutions (analogically as cationic surfactants), however, there are some exceptions in the group of natural polycations. These compounds are often used for changing the properties of surfaces characterised by a positive charge.

The method suitable for use according to the invention, which allows the polycation layer to be applied to the metal surface, is the "layer by layer" technique. In this technique, a material with a given surface potential charge is submerged in a solution containing macromolecules having the opposite charge. As a result of interactions {La., electrostatic) between the surface and the adhering molecules, spontaneous formation of a layer of these molecules occurs on the surface. Also, formation of multilayers is possible, by submerging the material alternately in solutions containing polymers with positive and negative surface potentials, with intermediary stages of washing the surface with water. Modification of the surface of the material using layer by layer method preferably may change its properties (e.g. a hydrophobic surface may become hydrophilic).

For a better understanding of the essence of the invention, its description is supplemented with the following embodiments and appended Figures.

Figure 1 shows a schematic diagram of the bioelectrode according to the invention described in Example 5 with a bacterial biofilm of L. rhamnosus, obtained on the basis of a copper strip covered with a thin layer of gold and a polycation applied by the LbL method.

Figure 2 shows SEM microphotographs of bacterial films obtained on various substrates: Cu - copper plate; Cu/DEX-G - copper strip with an LbL layer of a cationic derivative of dextran applied; Cu/Au - copper strip covered with a layer of gold; Cu/Au/DEX-G - copper strip covered with a layer of gold and an LbL layer of a cationic derivative of dextran; Cu/Au/P APTAC - copper strip covered with a layer of gold and an LbL layer of a cationic derivative MAPTAC; Cu/Au/INU-G - copper strip covered with a layer of gold and an LbL layer of a cationic derivative of inulin; Cu/Au/PUL-G - copper strip covered with a layer of gold and an LbL layer of a cationic derivative of pullulan.

Figure 3 shows exemplary voltammetric curves for the studied electrodes, (a) comparison of CV curves for electrodes without a bacterial film (Cu/Au and Cu/Au/dex) and with a biofilm of L. rhamnosus (alive) recorded in a medium; (b) CV curves recorded for the electrode with a biofilm in a mixed electrolyte (medium: KNO3) for various scanning rates.

Example 1. Synthesis of dextran modified with glycidyltrimethylammonium chloride (GTMAC)

[22]

2 g of dextran 40 kDa was dissolved in 100 ml of distilled water, then 0.4 g of NaOH and 12 ml of GTMAC (90% aqueous solution) were added. The so-obtained solution was mixed at a temperature of 60°C for 4 hours. Then, the polymer solution was purified (dialysed) for 5 days, using a cellulosic tube permeable for particles smaller than 12 kDa. After that time, the polymer was isolated using lyophilisation. The degree of modification amounted to approx. 0.5 GTMAC molecules per each glucose unit.

Example 2. Synthesis of pullulan modified with GTMAC [22]

2 g of pullulan 200 kDa was dissolved in 100 ml of distilled water, then 0.4 g of NaOH and 24 ml of GTMAC (90% aqueous solution) were added. The so-obtained solution was mixed at a temperature of 60°C for 4 hours. Then, the polymer solution was dialysed for 5 days, using a cellulosic tube permeable for particles smaller than 12 kDa. After that time, the polymer was isolated using lyophilisation. The degree of modification amounted to approx. 0.5 GTMAC molecules per each glucose unit.

Example 3. Synthesis of inulin modified with GTMAC [22]

2 g of inulin 5 kDa was dissolved in 100 ml of distilled water, then 0.4 g of NaOH and 24 ml of GTMAC (90% aqueous solution) were added. The so-obtained solution was mixed at a temperature of 60°C for 4 hours. Then, the polymer solution was purified for 5 days, using a cellulosic tube permeable for particles smaller than 3 kDa. After that time, the polymer was isolated using lyophilisation. The degree of modification amounted to approx. 0.5 GTMAC molecules per each glucose unit.

Example 4. Synthesis of a polymer formed as a result of polymerisation of 3- methacryloylaminopropyltrimethylammonium chloride

Argon was bubbled through 6 ml of distilled water for 0.5 h to remove oxygen. Then, 6 g of 50% solution of MAPTAC monomer (methacrylamidopropyltrimethyloammonium chloride) and 15 mg of the initiator 4,4'-azobis(4-cyanovaleric acid) were added to this volume. The whole solution was mixed at a temperature of 70°C for 4 h. The obtained mixture was dialysed to distilled water for 5 days. The polymer was isolated using a lyophilisator.

Example 5. Construction and forming of the electrode

a) Preparation of the surface of the electrode covered with gold

Properly prepared copper foil was used as a starting material. Degreased samples were subjected to polishing (electrochemical and chemical), and then, they were washed with distilled water and ethanol and dried in air. The so-prepared samples were sputtered with a thin layer of gold (approx. 20 nm), and then thickened chronopotentiometrically. The process was carried out in a conventional three-electrode system, where the sputtered sample was the working electrode, and platinum grids constituted the counter electrode and the comparative electrode. Commercially available gold solution (Auruna®5000), containing 7 g/dm ³ gold was used as the electrolyte. The process was carried out with a given current value (1.5 niA/cm ² ) for 300 s. Then, the sample was washed with distilled water and ethanol, and dried in air.

b) Modification of the electrode surface with polycations - layer by layer method

Solutions of polycations obtained in Examples 1 -4 were prepared, using a phosphate buffer saline (PBS) with a pH = 7.4 and a concentration of 0.1 g/dm ³ as the solvent. The pre-prepared metallic substrates were submersed in corresponding polycation solutions for 15 min, then washed with distilled water and placed in a PBS solution.

c) Formation of Lactobacillus rhamnosus biofilm on metal electrodes

First of all, microbiological medium containing 20 g/dm ³ of glucose was prepared, by dissolving 12.75 g of MRS Broth in 250 ml of distilled water and adding 250 μΐ of Tween ^® 80. Then, the solution was autoclaved at a temperature of 121 °C for 15 min. Content of a probiotic capsule (Dicoflor®60, Bayer) was introduced into the so-prepared medium and shaken at a temperature of 37°C for 24 h. Unmodified and modified (using the layer by layer method) substrates were washed with the medium solution and placed in sterile Petri dishes. Then, a medium solution containing bacteria was being applied on them and left for approx. 2 h to inoculate the bacteria on the surface of electrodes. After this time, the excess of microorganisms was washed away with the PBS and medium solution, and the substrates were placed in tubes with the medium. The so-prepared samples were left for 24 h or 72 h, to facilitate the formation of the bacterial biofilm. A schematic diagram of the electrode formed is shown in Fig. 1.

Example 6. Confirmation of colonisation of the electrode by electron microscopy To confirm the colonisation of the electrodes by microorganisms, the samples were fixed according to the procedure described below. The electrodes after the culturing of bacteria were washed with the PBS solution, than place in 3% solution of glutaraldehyde in a phosphate buffer for approx. 30 min. Then, they were washed with the PBS solution and water. The bacteria were dehydrated in an alcohol series with the following increasing concentrations: 60% < 70% < 80% <90% <100%. Each step lasted for 5 min. The final dehydration and drying was carried out using hexamethyldisilazane. The samples were submersed in this solution for approx. 1 min, then dried in air. The so-prepared electrodes were sputtered with gold and their surfaces were observed by using a scanning electron microscope (SEM). Fig. 2 shows microphotographs of the obtained samples.

The presented microphotographs confirm that L. rhamnosus bacteria adhere, divide and form a bacterial film only on copper electrodes covered with a layer of gold and modified with cationic derivatives of polymers. Copper electrodes both pure, and covered only with polymer or only with gold do not affect the growth of bacteria advantageously. In SEM photographs, a smaller amount of adhered bacteria is clearly visible. Moreover, the bacterial cells are damaged and they do not form chains characteristic for this species of bacteria. It confirms ultimately that the layered structure of an electrode (metal-gold- cationic polymer derivative) is most favourable for the biofilm formation of this bacterial strain.

Example 7. Confirmation of the electrochemical activity of the obtained electrode

The prepared gold electrodes covered with a layer of polycation and colonised by the bacterial biofilm were subjected to initial electrochemical tests. Cyclic voltammetry (CV) measurements were carried out to check whether the tested electrodes give a current response. Two electrolytes were used for the tests: (i) the culture medium and (ii) a mixture of the culture medium and 0.1 M KNO3 solution (1 :1 v/v). Voltammograms were recorded for the range of potentials of -0.6-0.5 V against the saturated calomel electrode (SCE). The curves were recorded for a gold electrode not covered with the polymer, an electrode with an LbL dextran layer, and an electrode with a bacterial biofilm. The results presented in Fig. 3 prove that - as opposed to uncolonised electrodes - the L. rhamnosus bacteria forming a biofilm on the gold electrode covered with a polymer layer give a current response. Two peaks (for oxidation and reduction) are evident, corresponding to electrode reactions occurring in the system.

The voltammograms shown in Fig. 3 confirm that the biofilm formed on the described electrodes exhibits an electrochemical activity. It forms a good ground for use of such a system, e.g. In construction of electrodes for fuel cells or biosensors. References

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