**5.1 Materials and methods**

#### **5.1.1 AISI 304 stainless steel**

Tests were performed using AISI 304 Stainless Steel in rectangular samples and pretreated by mechanically polishing with abrasive paper of increasingly finer grit between 800 and 2000 μm and finally chemical cleaning. The samples as working electrode were mounted in a tetrafluoroethylene-perfluoroalkylvinylether copolymer (PFA), sleeve sample holder except for an exposed test area of 12 cm2 (Stoica et al., 2010b).

#### **5.1.2 Biocide solution**

The biocide (*Neoseptal*) is a commercial disinfectant, effective against all types of microorganisms in food industry manufactured by Dr.Weigert (Germany) based on H2O2 (dihydrogen dioxide – 30% wt.). Fresh solutions of *Neoseptal* were prepared by dilution of commercially *Neoseptal* biocide. The concentration of *Neoseptal* was 0.2% performed at 20±2°C and acted for at least 30 min.

#### **5.1.3 Fungi strains**

Three fungal types were tested: a) *Aspergillus niger* ATCC 16404 (provided by the "Ion Cantacuzino" Institute (Bucharest-Romania), with the spores concentration of 1.41x107spores/mL; b) *Candida mycoderma* isolated from spoiled wine, of suspension containing 1.38x107 cells/mL; c) *Saccharomyces cerevisiae* (Pakmaya, Rompak), with a cell number in the suspensions of 1.40x107 cells/mL. The cells concentrations were measured using a Thoma cytometer. An aliquot volume of fungal suspension (5 mL) was used in the electrochemical experiments (50 mL as total volume).

#### **5.1.4 Electrochemical measurements**

104 Food Industrial Processes – Methods and Equipment

European market for biocidal products and their active substances such that product authorisation content in one Member State can be recognized in other Member States; (ii) to provide a high level of protection for people, animals and the environment (from the use of biocidal products) through risk assessment. These objectives requires the submission and evaluation of data relating to substances' chemistry, toxicity to humans, and toxicity and fate in the environment and (iii) to ensure products are sufficiently effective against the target species. The Registration REACH - a new chemical regulation was implemented by the EU on June 1st 2007 and is being implemented in stages to be completed by 1 st June 2018. The main objective of REACH is a high level of protection for human health and the environment, while maintaining the competitiveness and innovation of EU chemicals industry. REACH provides a single regulatory framework for the control of chemicals, replacing the previous patchwork of controls, and ensures that information on the properties of chemicals is transmitted down the supply chain, thus enabling them to be

**5. Study of electrochemical behaviour of AISI 304 stainless steel immersed in** 

In this study the corrosion behaviour of AISI 304 Stainless Steel (*SS*) treated by biocide *Neoseptal* solution (noted as *Neo*) and in mixtures consisting of *Neo* with fungal suspension (*Neo - Aspergillus niger, Neo - Candida mycoderma* and *Neo - Saccharomyces cerevisiae*) was

Tests were performed using AISI 304 Stainless Steel in rectangular samples and pretreated by mechanically polishing with abrasive paper of increasingly finer grit between 800 and 2000 μm and finally chemical cleaning. The samples as working electrode were mounted in a tetrafluoroethylene-perfluoroalkylvinylether copolymer (PFA), sleeve sample holder

The biocide (*Neoseptal*) is a commercial disinfectant, effective against all types of microorganisms in food industry manufactured by Dr.Weigert (Germany) based on H2O2 (dihydrogen dioxide – 30% wt.). Fresh solutions of *Neoseptal* were prepared by dilution of commercially *Neoseptal* biocide. The concentration of *Neoseptal* was 0.2% performed at

Three fungal types were tested: a) *Aspergillus niger* ATCC 16404 (provided by the "Ion Cantacuzino" Institute (Bucharest-Romania), with the spores concentration of 1.41x107spores/mL; b) *Candida mycoderma* isolated from spoiled wine, of suspension containing 1.38x107 cells/mL; c) *Saccharomyces cerevisiae* (Pakmaya, Rompak), with a cell number in the suspensions of 1.40x107 cells/mL. The cells concentrations were measured using a Thoma cytometer. An aliquot volume of fungal suspension (5 mL) was used in the

**mixtures consisting of** *Neoseptal* **and fungal suspensions** 

except for an exposed test area of 12 cm2 (Stoica et al., 2010b).

electrochemical experiments (50 mL as total volume).

safely handled.

98

investigated.

**5.1 Materials and methods 5.1.1 AISI 304 stainless steel** 

**5.1.2 Biocide solution** 

**5.1.3 Fungi strains** 

20±2°C and acted for at least 30 min.

Tests were performed on the corrosion behaviour of samples of *SS* in *Neo* with and without fungal suspensions. All electrochemical measurements were carried out in a glass electrochemical-cell (Metrohm, Switzerland) equipped with three electrodes, at room temperature (22±1ºC).The working electrode was AISI 304 Stainless Steel type, the counter electrode (CE) was a Platinum foil and as the reference electrode (RE) was a saturated calomel electrode (SCE). The entire three-electrode assembly was placed in a Faraday cage to limit the noise disturbance and then connected to potentiostat-galvanostat Bio-Logic SP-150 (France). The investigations are carried out using EC-Lab® Express v 9.46 software. The electrochemical measurements were carried out using the Linear Polarization Technique (LP). The polarization measurements were initiated after 60 seconds immersions to access an equilibrium open potential on the sample surface. In the preliminary study the potential was tested for different ranges, as ±2V; ±1.0 V (SCE) a variation of scan rate between 100÷10 mV/s. There were no large modifications of the results in the polarization curves tested. The following measurements recorded for a potential of ±1.5V (SCE) at the 20 mVs-1 were take into consideration because of larger scan rate appeared in the distorsion of the curves also for higher potential range. At the same time all solutions were examined for conductivity measurements, using the Metrohm 712 Conductometer.

### **5.2 Results and discussions**

#### **5.2.1 Electroactivity of working solutions**

The electrochemical behaviour of *SS* samples in *Neo* with and without fungal suspensions was investigated at room temperature. Figure 3 shows the polarization curves obtained for *SS* immersed in *Neo* and also in different fungal suspensions.

Fig. 3. Polarization curves of SS in *Neo* (1) and different fungal suspensions: *Aspergillus niger*  (2), *Candida mycoderma* (3) and *Saccharomyces cerevisiae* (4).

A larger range passivation of the processes could be observed in both electrochemical reactions (anodic and cathodic). From the polarization curves (Fig. 3, curves 2-4), that the potential *Ecorr* values show a more negative range for all fungal suspensions tested, whereas for *Neo Ecorr* the value is situated at a positive potential range (Fig. 3, curve 1). Electrochemical parameters for *Neo* (Fig. 3, curve 1) are *Ecorr +*390 mV and corrosion current

Electrochemical Behaviour of AISI 304 Stainless Steel

*niger.*

Solution Time

*Neo - Aspergillus niger*

(min.)

that the processes that occur on *SS* surface are not similar.

*Ecorr*  (mV)

*Neo* biocide is a depolarization agent for *SS* surfaces.

*jcorr* (μA/cm2)

Table 2. Tafel parameters of *SS* immersed in *Neo* and *Neo - Aspergillus niger*.

Immersed in Mixtures Consisting by Biocide and Fungal Suspensions 107

corresponding to the *Neo - Aspergillus niger* and curve 1 for the only *Neo* biocide suggests

Table 2 shows the electrochemical parameters of *SS* immersed in *Neo* and in *Neo - Aspergillus* 

*Neo* 3 390 24 182 405 1722 0.0207

As it can be observed from the Table 2 the potential *Ecorr* value of *SS* immersed in *Neo* is +390 mV(SCE). By adding the *Aspergillus niger* suspension in *Neo*, the spores are exposed initially to a chemical attack. The spores wall due to its affinity to the active molecules oxidizing – H2O2, may restrict acces to the plasma cells membrane and to the cytoplasm. The oxidizing molecules have a damaging action to the spores wall, more or less crossed, then act at a cytoplasmic level where it causes the first serious damage. The spores wall penetration is physically a charge transfer between the wall (regarded as electrode) and the spores cytoplasm (regarded as electrolyte) and has as effect the charge modification of the double layer near the spores surface and also shift in potential. Under these circumstances, the wall can be crossed by a net current which raises with increasing the concentration of ionic species participating in the process. The application of electrical potential exposes the *Aspergillus niger* spores (chemically attacked) to the aggression of the electric field. The potential effects manifest through dielectric breakdown and migration of the cytoplasma, followed by electrolytic contamination of the *Neo* biocide. At the same time, the electrical applied potential leads to the occurrence of some oxidations and reductions followed by a potential *Ecorr* shift in the negative range with an amplitude of about 94 mV (*i.e*. from +390 mV (SCE) at +296 mV) (SCE). During the experiment the *Ecorr* values increases slightly, through a shift in positive range with an amplitude of about 8 mV to the end of the exposure. The shift of potential *Ecorr*, suggests that the *Aspergillus niger* suspension added in

The electrochemical system *Neo - Aspergillus niger* indicate a current flow which generating the orderly movement between the surface and biocide solution whithout an interference. Morever ther is a current resulting after oxidation of some biomolecules in solution (not attacked intracytoplasmic) and a current after oxidation of intracytoplasmic electrolysis products, but go into solution because of the dielectric breakdown. This fact shown that in *Neo - Aspergillus niger* the current which flows through working electrode has a higher intensity than the current which flows through working electrode in *Neo* only. The addition of *Aspergillus niger* suspension is characterized by an increase of current density with 46 μA/cm2, *i.e.* from 24 μA/cm2 to 70 μA/cm2. During the experiment, a slight breakthrough

*βc*  (mV/dec.)

3 296 70 203 478 595 0.0608 6 263 80 198 536 580 0.0691 9 257 77 201 497 562 0.0668 12 247 77 198 509 553 0.0666 30 239 76 189 519 573 0.0657

*βa*  (mV/dec.)

*Rp* (Ω·cm2)

*Vcorr* (mm/y)

101

density, *icorr* 24 µA/cm2. The potential *Ecorr* and *icorr* values are -316 mV respectively 30.27 µA/cm2 for *Aspergillus niger* suspension and -338 mV, respectively 37 µA/cm2 for *Candida mycoderma* suspension. In case of *Saccharomyces cerevisiae* suspension a – 511 mV value for *Ecorr* respectively 70.40 µA/cm2 for *icorr* were obtained. These data confirm on the one hand the electroactivity behaviour of the tested biocide solutions and on the other hand a high electrons density, in the electrochemical system which contains only fungal suspensions. Just like electrons in wires, these ions contribute to the transport charge in the electric field and thus to the current flow. In this situation the system responds through a current predominant anodic and it can speed up the metal dissolution. A more current density is produced by *Saccharomyces cerevisiae* suspension (Fig. 3, curve 4). This fact could explain a more intense sensibility of these cells for the applied potential, due to their size. These cells appear bigger in comparison to *Aspergillus niger* spores and *Candida mycoderma* cells (Bui et al., 2008). Some authors consider that the larger cells are more sensitive to lower electric potential field, thus stronger than the smaller cells (Teissie et al., 1999; Kotnik et al., 2001). The applied potential for the acceleration of corrosion tests and biocide can produce a permeabilization of the plasma membrane and appear an electrochemical contamination in mixtures, due to the intracellular ions transit out of cells.

#### **5.2.2 The corrosion effect of mixtures solutions**

The electrochemical behaviour of the *SS* in the *Neo* with fungal suspension (without nitrogen) was evaluated at room temperature at different contact times after adding the fungal suspensions. The polarization curves are presented in Figures 4-6. Using Tafel fit. and *Rp* fit. analysis tool, made it possible to obtain the electrochemical parameters like corrosion potential (*Ecorr*), current density (*jcorr*), polarization resistance (*Rp*), corrosion rate (*Vcorr*) . The results are presented in Tables 2-4. Figure 4 presents the variation of *Ecorr* potential values during polarization curves of *SS* immersed in *Neo* and in *Neo - Aspergillus niger* at different contact time from the immersion of samples.

Fig. 4. Polarization curves of *SS* immersed in *Neo* (1) and *Neo - Aspergillus niger* at 3 min. (2); 6 min.(3); 9 min.(4); 12 min. (5) and 30 min. (6).

The potential *Ecorr* values are in a negative range for the *Neo - Aspergillus niger* (Fig. 4, curves 2-6), whereas *Ecorr* value is situated in the positive range for *Neo* (Fig. 4, curve 1). The absence of the parallelism of cathodic and anodic branches between the set of curves 2-6

density, *icorr* 24 µA/cm2. The potential *Ecorr* and *icorr* values are -316 mV respectively 30.27 µA/cm2 for *Aspergillus niger* suspension and -338 mV, respectively 37 µA/cm2 for *Candida mycoderma* suspension. In case of *Saccharomyces cerevisiae* suspension a – 511 mV value for *Ecorr* respectively 70.40 µA/cm2 for *icorr* were obtained. These data confirm on the one hand the electroactivity behaviour of the tested biocide solutions and on the other hand a high electrons density, in the electrochemical system which contains only fungal suspensions. Just like electrons in wires, these ions contribute to the transport charge in the electric field and thus to the current flow. In this situation the system responds through a current predominant anodic and it can speed up the metal dissolution. A more current density is produced by *Saccharomyces cerevisiae* suspension (Fig. 3, curve 4). This fact could explain a more intense sensibility of these cells for the applied potential, due to their size. These cells appear bigger in comparison to *Aspergillus niger* spores and *Candida mycoderma* cells (Bui et al., 2008). Some authors consider that the larger cells are more sensitive to lower electric potential field, thus stronger than the smaller cells (Teissie et al., 1999; Kotnik et al., 2001). The applied potential for the acceleration of corrosion tests and biocide can produce a permeabilization of the plasma membrane and appear an electrochemical contamination in

The electrochemical behaviour of the *SS* in the *Neo* with fungal suspension (without nitrogen) was evaluated at room temperature at different contact times after adding the fungal suspensions. The polarization curves are presented in Figures 4-6. Using Tafel fit. and *Rp* fit. analysis tool, made it possible to obtain the electrochemical parameters like corrosion potential (*Ecorr*), current density (*jcorr*), polarization resistance (*Rp*), corrosion rate (*Vcorr*) . The results are presented in Tables 2-4. Figure 4 presents the variation of *Ecorr* potential values during polarization curves of *SS* immersed in *Neo* and in *Neo - Aspergillus niger* at different

Fig. 4. Polarization curves of *SS* immersed in *Neo* (1) and *Neo - Aspergillus niger* at 3 min. (2);

The potential *Ecorr* values are in a negative range for the *Neo - Aspergillus niger* (Fig. 4, curves 2-6), whereas *Ecorr* value is situated in the positive range for *Neo* (Fig. 4, curve 1). The absence of the parallelism of cathodic and anodic branches between the set of curves 2-6

mixtures, due to the intracellular ions transit out of cells.

**5.2.2 The corrosion effect of mixtures solutions** 

100

contact time from the immersion of samples.

6 min.(3); 9 min.(4); 12 min. (5) and 30 min. (6).

corresponding to the *Neo - Aspergillus niger* and curve 1 for the only *Neo* biocide suggests that the processes that occur on *SS* surface are not similar.

Table 2 shows the electrochemical parameters of *SS* immersed in *Neo* and in *Neo - Aspergillus niger.*


Table 2. Tafel parameters of *SS* immersed in *Neo* and *Neo - Aspergillus niger*.

As it can be observed from the Table 2 the potential *Ecorr* value of *SS* immersed in *Neo* is +390 mV(SCE). By adding the *Aspergillus niger* suspension in *Neo*, the spores are exposed initially to a chemical attack. The spores wall due to its affinity to the active molecules oxidizing – H2O2, may restrict acces to the plasma cells membrane and to the cytoplasm. The oxidizing molecules have a damaging action to the spores wall, more or less crossed, then act at a cytoplasmic level where it causes the first serious damage. The spores wall penetration is physically a charge transfer between the wall (regarded as electrode) and the spores cytoplasm (regarded as electrolyte) and has as effect the charge modification of the double layer near the spores surface and also shift in potential. Under these circumstances, the wall can be crossed by a net current which raises with increasing the concentration of ionic species participating in the process. The application of electrical potential exposes the *Aspergillus niger* spores (chemically attacked) to the aggression of the electric field. The potential effects manifest through dielectric breakdown and migration of the cytoplasma, followed by electrolytic contamination of the *Neo* biocide. At the same time, the electrical applied potential leads to the occurrence of some oxidations and reductions followed by a potential *Ecorr* shift in the negative range with an amplitude of about 94 mV (*i.e*. from +390 mV (SCE) at +296 mV) (SCE). During the experiment the *Ecorr* values increases slightly, through a shift in positive range with an amplitude of about 8 mV to the end of the exposure. The shift of potential *Ecorr*, suggests that the *Aspergillus niger* suspension added in *Neo* biocide is a depolarization agent for *SS* surfaces.

The electrochemical system *Neo - Aspergillus niger* indicate a current flow which generating the orderly movement between the surface and biocide solution whithout an interference. Morever ther is a current resulting after oxidation of some biomolecules in solution (not attacked intracytoplasmic) and a current after oxidation of intracytoplasmic electrolysis products, but go into solution because of the dielectric breakdown. This fact shown that in *Neo - Aspergillus niger* the current which flows through working electrode has a higher intensity than the current which flows through working electrode in *Neo* only. The addition of *Aspergillus niger* suspension is characterized by an increase of current density with 46 μA/cm2, *i.e.* from 24 μA/cm2 to 70 μA/cm2. During the experiment, a slight breakthrough

Electrochemical Behaviour of AISI 304 Stainless Steel

*Ecorr* (mV)

good agreement to *Neo - Aspergillus niger.*

Time (min.)

*mycoderma.*

Solution

*Neo - Candida mycoderma*

the anodic reactions.

Immersed in Mixtures Consisting by Biocide and Fungal Suspensions 109

*Candida mycoderma* are not similar with the processes that occur on *SS* immersed in *Neo* in

Table 3 presents the electrochemical parameters of *SS* immersed in *Neo* and *Neo - Candida* 

*Neo* 3 390 24 182 405 1722 0.0207

Similar to the electrochemical system (which contains *Neo* - *Aspergillus niger)* the *Neo - Candida mycoderma* system random currents circulate, whose intensity is determined by different source. The applied electric potential exposes the *Candida mycoderma* cells to the aggression of the electric field. The experimental potential effects manifest through dielectric breakdown and migration of the cytoplasmic content, followed by an electrolytic contamination of the *Neo* biocide. At the same time, the potential leads to the occurrence of some oxidations and reductions, followed by a potential *Ecorr* shift in negative range with an amplitude of about 96 mV *i.e*. from +390 mV (SCE) at +294 mV (SCE). During exposure was observed that the corrosion potential fluctuated suggesting alternative passivation and activation of the surface and conducting its the spontaneous reversible oxidations. The shift of *Ecorr* suggests that the *Candida mycoderma* suspension added in *Neo* biocide is a depolarization agent for *SS* surface. The addition of *Candida mycoderma* fungal suspension is characterized by an increase of current density with 34 μA/cm2, *i.e.* from 24 μA/cm2 to 58 μA/cm2. During the experiment, the current density showed a reduction value after at 6 minutes (Table 3). This suggests the initiation of a partial surface passivation process, but which is insignificantly in an amplitude of 47 μA/cm2 at exposure end (Table 3). In electrochemical system of *Neo - Candida mycoderma*, the polarization is confirmed by the shift in the cathodic (βc) and anodic (βa) Tafel slopes (Stoica et al., 2010b). As it can be observed from the Table 3, *Neo - Candida mycoderma* moves the cathodic (βc) slope from 182 mV/dec. to 196 mV/dec. and the anodic (βa) slope from 405 mV/dec. to 488 mV/dec. The shift of the Tafel slopes observed throughout the experiment (Table 3) reveals that the *Candida mycoderma* fungal suspension induces a corrosion mechanism on metallic surface (Stoica et al., 2010b). These results suggest that the *Neo - Candida mycoderma* controlling predominantly

There is a shift of the potential *Ecorr,* the increase of *jcorr* and the decrease of polarization rezistance, during experiment (Figure 5 curves and Table 3). The results indicate a synergic effect between H2O2 *Candida mycoderma* cells and apllied electric potential, which manifests through a distorted current predominantly anodic. The predominant anodic mechanism observed in this case, is in good agreement with experimental results previously reported in

case of H2O2 – *Aspergillus niger* – applied electric potential.

*βc*  (mV/dec.)

3 294 58 196 488 746 0.0503 6 289 50 195 484 827 0.0437 9 296 41 194 566 961 0.0354 12 276 33 161 488 1222 0.0285 30 281 88 185 546 949 0.0334

*βa*  (mV/dec.)

*Rp* (Ω·cm2)

*Vcorr* (mm/y)

103

*jcorr* (μA/cm2)

Table 3. Tafel parameters of *SS* immersed in *Neo* and *Neo - Candida mycoderma.* 

corrosion current density was observed with an amplitude of about 10 μA/cm2 at 6 minutes from exposure and after that it begins to decrease slightly up to the end of the exposure (Table 2). The decrease of current density suggests that the absorption of OH- ions occurs and a possible surface passivation may appear. However, the current density does not return to the initial value and it maintains at levels three times higher than the initial value.

An increasing content of hydrogen and oxygen from oxidation of biomolecules by biocide substance could appear in the electrochemical system which contains *Neo - Aspergillus niger*. A large flow of electrons have as effect the cell current increase an electrode polarization. This polarization is confirmed by the shift in the cathodic (βc) and anodic (βa) Tafel slopes (Stoica et al., 2010b). As it can be observed from the Table 2, the *Neo - Aspergillus niger* move the cathodic (βc) slope from 182 mV/dec. to 203 mV/dec. and the anodic (βa) slope from 405 mV/dec. to 478 mV/dec. The shift of the Tafel slopes observed throughout the experiment (Table 2) reveals that the *Aspergillus niger* fungal suspension induces a corrosion mechanism on metallic surface (Stoica et al., 2010b). These results suggest that the *Neo - Aspergillus niger* controlling predominantly the anodic reactions.

There is a shift of the potential *Ecorr* and the increase of *jcorr* and the decrease of polarization resistance, during experiment from the Figure's 4 curves and from the Table's 2 data. These results indicate a synergic effect between H2O2, *Aspergillus niger* spores and applied electric potential. This fact can substantially accelerate the corrosion process of metallic surfaces immersed in the mixture consisting of biocide and fungal suspension. The predominant anodic parameters could be an answer of the degradation metallic surfaces and this phenomenon is in good agreement with data previously reported in literature (Stoica et al*,* 2010b).

Figure 5 presents polarization curves of *SS* immersed in *Neo* and *Neo - Candida mycoderma* at different contact time.

Fig. 5. Polarization curves of *SS* immersed in *Neo* (1) and *Neo - Candida mycoderma* at 3 min. (2); 6 min.(3); 9 min.(4); 12 min. (5) and 30 min. (6).

In Figure 5, it can be observed that the potential *Ecorr* values are in negative range for the *Neo - Candida mycoderma* (curves 2-6), whereas *Ecorr* value is situated in the positive range for *Neo* (Fig. 5, curve 1). Absence the parallelism of cathodic and anodic branches between the set of curves 2-6 which corresponding the *Neo - Candida mycoderma* and curve 1 which corresponding the *Neo* suggests, that the processes that occur on *SS* immersed in *Neo -* 

corrosion current density was observed with an amplitude of about 10 μA/cm2 at 6 minutes from exposure and after that it begins to decrease slightly up to the end of the exposure (Table 2). The decrease of current density suggests that the absorption of OH- ions occurs and a possible surface passivation may appear. However, the current density does not return to the initial value and it maintains at levels three times higher than the initial value. An increasing content of hydrogen and oxygen from oxidation of biomolecules by biocide substance could appear in the electrochemical system which contains *Neo - Aspergillus niger*. A large flow of electrons have as effect the cell current increase an electrode polarization. This polarization is confirmed by the shift in the cathodic (βc) and anodic (βa) Tafel slopes (Stoica et al., 2010b). As it can be observed from the Table 2, the *Neo - Aspergillus niger* move the cathodic (βc) slope from 182 mV/dec. to 203 mV/dec. and the anodic (βa) slope from 405 mV/dec. to 478 mV/dec. The shift of the Tafel slopes observed throughout the experiment (Table 2) reveals that the *Aspergillus niger* fungal suspension induces a corrosion mechanism on metallic surface (Stoica et al., 2010b). These results suggest that the *Neo - Aspergillus niger*

There is a shift of the potential *Ecorr* and the increase of *jcorr* and the decrease of polarization resistance, during experiment from the Figure's 4 curves and from the Table's 2 data. These results indicate a synergic effect between H2O2, *Aspergillus niger* spores and applied electric potential. This fact can substantially accelerate the corrosion process of metallic surfaces immersed in the mixture consisting of biocide and fungal suspension. The predominant anodic parameters could be an answer of the degradation metallic surfaces and this phenomenon is in good agreement with data previously reported in literature (Stoica et al*,*

Figure 5 presents polarization curves of *SS* immersed in *Neo* and *Neo - Candida mycoderma* at

Fig. 5. Polarization curves of *SS* immersed in *Neo* (1) and *Neo - Candida mycoderma* at 3 min.

In Figure 5, it can be observed that the potential *Ecorr* values are in negative range for the *Neo - Candida mycoderma* (curves 2-6), whereas *Ecorr* value is situated in the positive range for *Neo* (Fig. 5, curve 1). Absence the parallelism of cathodic and anodic branches between the set of curves 2-6 which corresponding the *Neo - Candida mycoderma* and curve 1 which corresponding the *Neo* suggests, that the processes that occur on *SS* immersed in *Neo -* 

controlling predominantly the anodic reactions.

(2); 6 min.(3); 9 min.(4); 12 min. (5) and 30 min. (6).

2010b).

102

different contact time.

*Candida mycoderma* are not similar with the processes that occur on *SS* immersed in *Neo* in good agreement to *Neo - Aspergillus niger.*

Table 3 presents the electrochemical parameters of *SS* immersed in *Neo* and *Neo - Candida mycoderma.*


Table 3. Tafel parameters of *SS* immersed in *Neo* and *Neo - Candida mycoderma.* 

Similar to the electrochemical system (which contains *Neo* - *Aspergillus niger)* the *Neo - Candida mycoderma* system random currents circulate, whose intensity is determined by different source. The applied electric potential exposes the *Candida mycoderma* cells to the aggression of the electric field. The experimental potential effects manifest through dielectric breakdown and migration of the cytoplasmic content, followed by an electrolytic contamination of the *Neo* biocide. At the same time, the potential leads to the occurrence of some oxidations and reductions, followed by a potential *Ecorr* shift in negative range with an amplitude of about 96 mV *i.e*. from +390 mV (SCE) at +294 mV (SCE). During exposure was observed that the corrosion potential fluctuated suggesting alternative passivation and activation of the surface and conducting its the spontaneous reversible oxidations. The shift of *Ecorr* suggests that the *Candida mycoderma* suspension added in *Neo* biocide is a depolarization agent for *SS* surface. The addition of *Candida mycoderma* fungal suspension is characterized by an increase of current density with 34 μA/cm2, *i.e.* from 24 μA/cm2 to 58 μA/cm2. During the experiment, the current density showed a reduction value after at 6 minutes (Table 3). This suggests the initiation of a partial surface passivation process, but which is insignificantly in an amplitude of 47 μA/cm2 at exposure end (Table 3). In electrochemical system of *Neo - Candida mycoderma*, the polarization is confirmed by the shift in the cathodic (βc) and anodic (βa) Tafel slopes (Stoica et al., 2010b). As it can be observed from the Table 3, *Neo - Candida mycoderma* moves the cathodic (βc) slope from 182 mV/dec. to 196 mV/dec. and the anodic (βa) slope from 405 mV/dec. to 488 mV/dec. The shift of the Tafel slopes observed throughout the experiment (Table 3) reveals that the *Candida mycoderma* fungal suspension induces a corrosion mechanism on metallic surface (Stoica et al., 2010b). These results suggest that the *Neo - Candida mycoderma* controlling predominantly the anodic reactions.

There is a shift of the potential *Ecorr,* the increase of *jcorr* and the decrease of polarization rezistance, during experiment (Figure 5 curves and Table 3). The results indicate a synergic effect between H2O2 *Candida mycoderma* cells and apllied electric potential, which manifests through a distorted current predominantly anodic. The predominant anodic mechanism observed in this case, is in good agreement with experimental results previously reported in case of H2O2 – *Aspergillus niger* – applied electric potential.

Electrochemical Behaviour of AISI 304 Stainless Steel

depolarization agent for *SS* surface.

*Candida mycoderma* – applied potential.

**5.2.3 Conductivity variations of solutions** 

Immersed in Mixtures Consisting by Biocide and Fungal Suspensions 111

105

*Ecorr* suggests that the *Saccharomyces cerevisiae* suspension added in *Neo* biocide is a

The addition of *Saccharomyces cerevisiae* fungal suspension is characterized by an increase of current density with 90 μA/cm2, *i.e.* from 24 μA/cm2 to 114 μA/cm2. During the experiment, the current density showed a value reduction after 6 minutes. This suggests of initiation of partial surface passivation process with insignificantly the amplitude of about 1 μA/cm2 at exposure end (Table 4). In electrochemical system *Neo-Saccharomyces cerevisiae*, the polarization is confirmed by the shift in the cathodic (βc) and anodic (βa) Tafel slopes (Stoica et al., 2010b). The cathodic (βc) slope from 182 mV/dec. to 247 mV/dec. and the anodic (βa) slope from 405 mV/dec. to 449 mV/dec., for the first 3 minutes from exposure moves (Table 4). The shift of the Tafel slopes observed throughout the experiment reveals that the *Saccharomyces cerevisiae* fungal suspension induces a corrosion mechanism on metallic surfaces (Stoica et al., 2010b) and suggests that *Neo-Saccharomyces cerevisiae* system

There is a shift of the potential *Ecorr,* the increase of *jcorr* and the decrease of polarization rezistance, during the experiment (Fig. 6 and Table 4). The results indicate a synergistic effect between H2O2 *Saccharomyces cerevisiae* cells and apllied potential. The predominant anodic mechanism observed in this case, is in good agreement with experimental results previously reported for the systems: H2O2 – *Aspergillus niger* – electric potential and H2O2 –

Conductivity is simply the ability of a liquid to conduct electricity, which is related directly to the concentration of ions in the liquid. Figure 7 gives the variation of solutions

Fig. 7. Conductivity variation in *Neo* (series 1) and *Neo* with different fungal suspension: *Aspergillus niger* (series 2), *Candida mycoderma* (series 3) and *Saccharomyces cerevisiae* (series 4). The presence of the fungal suspensions can be easily observed in the *Neo* biocide (Fig. 7, series 2-4) with a strong effect on the conductivity values in comparison with those observed for *Neo* without fungal cells (Fig. 7, series 1). This fact could suggest that in *Neo* with fungal cells an electrolytic contamination is produced, which can be explained through synergistic action between H2O2 and the applied potential inside the fungal cells, although the ions efflux remains difficult to elucidate (Sukhorukov et al., 2007). As a result the mixtures

controls the cathodic and anodic reactions, being predominantly anodic.

conductivity of *Neo* compared with *Neo* with fungal suspensions tested.

Figure 6 presents the polarization curves of *SS* immersed in *Neo* and *Neo - Saccharomyces cerevisiae* at different contact time.

Fig. 6. Polarization curves of *SS* immersed in *Neo* (1) and *Neo - Saccharomyces cerevisiae* at 3 min. (2); 6 min.(3); 9 min.(4); 12 min. (5) and 30 min. (6).

The potential *Ecorr* values are in negative range for the *Neo - Saccharomyces cerevisiae* (Fig. 6, curves 2-6), whereas *Ecorr* value is situated in the positive range for *Neo* (Fig. 5, curve 1). The absence of the parallelism of cathodic and anodic branches between the curves 2-6 corresponding the *Neo - Saccharomyces cerevisiae* and curve 1 corresponding the *Neo* suggests that the processes are not similar with the processes that occur on *SS* immersed in *Neo - Aspergillus niger* and *Neo - Candida mycoderma* system*.*

Table 4 reveals the electrochemical parameters of *SS* immersed in *Neo* and *Neo - Saccharomyces cerevisiae.*


Table 4. Tafel parameters of *SS* immersed in *Neo* and *Neo-Saccharomyces cerevisiae.*

Similar to electrochemical systems (*Neo - Aspergillus niger* and *Neo - Candida mycoderma)*, through electrochemical system *Neo - Saccharomyces cerevisiae*, the random currents circulate, by their source of origin. The applicated potential exposes the *Saccharomyces cerevisiae* cells to the aggression of the electric field. The experimental potential effects manifest through dielectric breakdown and migration of the cytoplasmic content, followed by an electrolytic contamination of the *Neo* biocide. At the same time, the potential lead to the occurrence of some oxidations and reductions, followed by a potential *Ecorr* shiftting in negative range with an amplitude of about 180 mV *i.e*. from +390 mV (SCE) at +213 mV (SCE). During the exposure the corrosion potential fluctuated suggesting alternative passivation and activation of the surface and conducting its spontaneous reversible oxidations. The shift of

Figure 6 presents the polarization curves of *SS* immersed in *Neo* and *Neo - Saccharomyces* 

Fig. 6. Polarization curves of *SS* immersed in *Neo* (1) and *Neo - Saccharomyces cerevisiae* at 3

The potential *Ecorr* values are in negative range for the *Neo - Saccharomyces cerevisiae* (Fig. 6, curves 2-6), whereas *Ecorr* value is situated in the positive range for *Neo* (Fig. 5, curve 1). The absence of the parallelism of cathodic and anodic branches between the curves 2-6 corresponding the *Neo - Saccharomyces cerevisiae* and curve 1 corresponding the *Neo* suggests that the processes are not similar with the processes that occur on *SS* immersed in *Neo -* 

Table 4 reveals the electrochemical parameters of *SS* immersed in *Neo* and *Neo - Saccharomyces* 

*βc*  (mV/dec.)

3 213 114 247 449 418 0.0993 6 216 98 238 492 475 0.0852 9 210 95 236 482 507 0.0824 12 205 93 236 514 507 0.0802 30 198 94 236 495 499 0.0816

*Neo* 3 390 24 182 405 1722 0.0207

Similar to electrochemical systems (*Neo - Aspergillus niger* and *Neo - Candida mycoderma)*, through electrochemical system *Neo - Saccharomyces cerevisiae*, the random currents circulate, by their source of origin. The applicated potential exposes the *Saccharomyces cerevisiae* cells to the aggression of the electric field. The experimental potential effects manifest through dielectric breakdown and migration of the cytoplasmic content, followed by an electrolytic contamination of the *Neo* biocide. At the same time, the potential lead to the occurrence of some oxidations and reductions, followed by a potential *Ecorr* shiftting in negative range with an amplitude of about 180 mV *i.e*. from +390 mV (SCE) at +213 mV (SCE). During the exposure the corrosion potential fluctuated suggesting alternative passivation and activation of the surface and conducting its spontaneous reversible oxidations. The shift of

*βa*  (mV/dec.)

*Rp* (Ω·cm2)

*Vcorr* (mm/y)

*jcorr* (μA/cm2)

Table 4. Tafel parameters of *SS* immersed in *Neo* and *Neo-Saccharomyces cerevisiae.*

min. (2); 6 min.(3); 9 min.(4); 12 min. (5) and 30 min. (6).

*Aspergillus niger* and *Neo - Candida mycoderma* system*.*

*Ecorr* (mV)

*cerevisiae.*

104

*Neo-Saccharomyces cerevisiae*

Solution Time

(min.)

*cerevisiae* at different contact time.

*Ecorr* suggests that the *Saccharomyces cerevisiae* suspension added in *Neo* biocide is a depolarization agent for *SS* surface.

The addition of *Saccharomyces cerevisiae* fungal suspension is characterized by an increase of current density with 90 μA/cm2, *i.e.* from 24 μA/cm2 to 114 μA/cm2. During the experiment, the current density showed a value reduction after 6 minutes. This suggests of initiation of partial surface passivation process with insignificantly the amplitude of about 1 μA/cm2 at exposure end (Table 4). In electrochemical system *Neo-Saccharomyces cerevisiae*, the polarization is confirmed by the shift in the cathodic (βc) and anodic (βa) Tafel slopes (Stoica et al., 2010b). The cathodic (βc) slope from 182 mV/dec. to 247 mV/dec. and the anodic (βa) slope from 405 mV/dec. to 449 mV/dec., for the first 3 minutes from exposure moves (Table 4). The shift of the Tafel slopes observed throughout the experiment reveals that the *Saccharomyces cerevisiae* fungal suspension induces a corrosion mechanism on metallic surfaces (Stoica et al., 2010b) and suggests that *Neo-Saccharomyces cerevisiae* system controls the cathodic and anodic reactions, being predominantly anodic.

There is a shift of the potential *Ecorr,* the increase of *jcorr* and the decrease of polarization rezistance, during the experiment (Fig. 6 and Table 4). The results indicate a synergistic effect between H2O2 *Saccharomyces cerevisiae* cells and apllied potential. The predominant anodic mechanism observed in this case, is in good agreement with experimental results previously reported for the systems: H2O2 – *Aspergillus niger* – electric potential and H2O2 – *Candida mycoderma* – applied potential.

#### **5.2.3 Conductivity variations of solutions**

Conductivity is simply the ability of a liquid to conduct electricity, which is related directly to the concentration of ions in the liquid. Figure 7 gives the variation of solutions conductivity of *Neo* compared with *Neo* with fungal suspensions tested.

Fig. 7. Conductivity variation in *Neo* (series 1) and *Neo* with different fungal suspension: *Aspergillus niger* (series 2), *Candida mycoderma* (series 3) and *Saccharomyces cerevisiae* (series 4).

The presence of the fungal suspensions can be easily observed in the *Neo* biocide (Fig. 7, series 2-4) with a strong effect on the conductivity values in comparison with those observed for *Neo* without fungal cells (Fig. 7, series 1). This fact could suggest that in *Neo* with fungal cells an electrolytic contamination is produced, which can be explained through synergistic action between H2O2 and the applied potential inside the fungal cells, although the ions efflux remains difficult to elucidate (Sukhorukov et al., 2007). As a result the mixtures

Electrochemical Behaviour of AISI 304 Stainless Steel

and *Saccharomyces cerevisiae* (series 4).

and safety food bioprocessing industry.

**5.3 Remarks** 

Immersed in Mixtures Consisting by Biocide and Fungal Suspensions 113

107

containing H2O2 from *Neo* biocide with *Aspergillus niger* respectively *Saccharomyces cerevisiae*. This fact suggests that during food line disinfection the corrosion rate of AISI 304 Stainless Steel surfaces exceeds 0.02 mm/y (Fontana, 1987) and reduces the equipments lifetime. It is possible that the killing action showed in these experiments is due to other chemical reactions occurring while the electrical currents pass through the liquid medium. The results obtained could suggest that an electrochemical disinfection is quite attractive as a promising alternative technology. The conductivity and potential sensors constantly monitor the concentration of the biocide solutions with cells contamination are benefic in cleaning the food processing. The *SS* surface behaviour is complex and requires further investigation for

its understanding under the action of different biocides and fungal suspensions.

Fig. 9. *Vcorr* values of *SS* immersed in *Neo* (1) and trend *Vcorr* values of *SS* immersed in *Neo* with fungal suspensions (10% vol.): *Aspergillus niger* (series 2), *Candida mycoderma* (series 3)

The work deals with the corrosion behaviour of AISI 304 Stainless Steel into biocide solution (*Neoseptal* with hydrogen peroxide as active substance) through artificial contamination with three (10% vol.) fungal suspensions as: *Aspergillus niger*, *Candida mycoderma* and *Saccharomyces cerevisiae*. At the applied electrical potential the biocide can work better within the fungal cells, and thus disturbing the present microorganisms homeostasis in several ways such as increasing the environmental conductivity of solutions and the corrosion rate of metallic support. A synergic effect achieved through the mixture of biocide, fungal suspension and applied electric potential, is more destructive than each parameters by its self. The *Ecorr* values of AISI 304 Stainless Steel in the mixtures decreased during the contact time after artificial contamination. The fungal suspension has a significant influence on the synergic effect of the AISI 304 Stainless Steel corrosion in the following order: *Saccharomyces cerevisiae>Aspergillus niger*>*Candida mycoderma*. A more influence on the synergic effect of the surfaces immersed at the mixture consisting on biocide *Neoseptal* solution with *Saccharomyces cerevisiae* could be explained through the less resistance at the chemical attack from biocide. The synergic effect between the active substance of the disinfectant, fungal suspensions and the applied electric potential should taken into account for the hygienic

consisting of *Neo* biocide with all fungal suspensions are very conductive systems and they can accelerate the corrosion of AISI 304 stainless steel immersed in them.

#### **5.2.4 Synergic of working parameters**

The corrosion behaviour on *SS* was tested in the biocide solution with and without fungal suspensions. The working parameters taken into account in accelerated corrosion tests (LP) were: nature and size of cells, cellular density of suspensions and conductivity of solutions, applied potential and pretreatment of working electrode of *SS*. Eletctrochemical parameters can justify the shift of *Ecorr* potential in the anodic direction and increase of *icorr* for mixtures consisting by *Neo* biocide with fungal suspensions. The corrosion behaviour of *SS* surface is specific to each system and it is made evident through *Rp* (polarization resistance) and *Vcorr* (corrosion rate) variations from Tables 2 - 4.

Figure 8 shows the *Rp* values of *SS* immersed in *Neo* and *Neo* with fungal suspension at different contact time after immersion.

Fig. 8. *Rp* values of *SS* immersed in *Neo* and *Neo* with fungal suspension: *Aspergillus niger*  (series 2), *Candida mycoderma* (series 3) and *Saccharomyces cerevisiae* (series 4).

The *Rp* values are lower in *Neo* with fungal suspension than in *Neo* only. The order of magnitude of *Rp* is 104 Ω.cm2 for *Neo*. In case of *Neo - Aspergillus niger* is observed a greatly decrease of *Rp* with the increase in time. In *Neo* - *Candida mycoderma* system the *Rp* values decrease up to minute 3 and afterwards a significant increase was obtained up to minute 12 followed by a lower decrease at final contact time (Fig. 8, series 3). In *Neo* - *Saccharomyces cerevisiae* a significant decrease in the *Rp* values was observed (Fig. 8, series 4). The order of magnitude of *Rp* is 103 Ω.cm2 in *Neo* with fungal suspensions.Thus, the *SS* surface is more susceptible in electrochemical system containing H2O2 from *Neo* biocide with *Aspergillus niger* suspension, respectively *Saccharomyces cerevisiae* suspension.

Figure 9 presents the *Vcorr* values of *SS* immersed in *Neo* and *Neo* with fungal suspension at different contact time after immersion.

In case of *Neo - Aspergillus niger* the *Vcorr* values increase more in time up to minutes 6 and afterwards a smaller decrease was obtained up to minutes 30 (Fig. 9, series 2). The *Vcorr* values decrease more in *Neo* - *Candida mycoderma* system up to 12 minutes followed by an increase at final contact time (Fig. 9, series 3). The highest increase of *Vcorr* values was obtained when *Saccharomyces cerevisiae* was added in *Neo*, at the beginning of the measurements and then there was a slight decrease up to minute 12 from immersion contact time (Fig. 9, series 4). Thus, the *SS* surface is more corrosive in electrochemical system containing H2O2 from *Neo* biocide with *Aspergillus niger* respectively *Saccharomyces cerevisiae*. This fact suggests that during food line disinfection the corrosion rate of AISI 304 Stainless Steel surfaces exceeds 0.02 mm/y (Fontana, 1987) and reduces the equipments lifetime. It is possible that the killing action showed in these experiments is due to other chemical reactions occurring while the electrical currents pass through the liquid medium. The results obtained could suggest that an electrochemical disinfection is quite attractive as a promising alternative technology. The conductivity and potential sensors constantly monitor the concentration of the biocide solutions with cells contamination are benefic in cleaning the food processing. The *SS* surface behaviour is complex and requires further investigation for its understanding under the action of different biocides and fungal suspensions.

Fig. 9. *Vcorr* values of *SS* immersed in *Neo* (1) and trend *Vcorr* values of *SS* immersed in *Neo* with fungal suspensions (10% vol.): *Aspergillus niger* (series 2), *Candida mycoderma* (series 3) and *Saccharomyces cerevisiae* (series 4).

#### **5.3 Remarks**

112 Food Industrial Processes – Methods and Equipment

consisting of *Neo* biocide with all fungal suspensions are very conductive systems and they

The corrosion behaviour on *SS* was tested in the biocide solution with and without fungal suspensions. The working parameters taken into account in accelerated corrosion tests (LP) were: nature and size of cells, cellular density of suspensions and conductivity of solutions, applied potential and pretreatment of working electrode of *SS*. Eletctrochemical parameters can justify the shift of *Ecorr* potential in the anodic direction and increase of *icorr* for mixtures consisting by *Neo* biocide with fungal suspensions. The corrosion behaviour of *SS* surface is specific to each system and it is made evident through *Rp* (polarization resistance) and *Vcorr*

Figure 8 shows the *Rp* values of *SS* immersed in *Neo* and *Neo* with fungal suspension at

Fig. 8. *Rp* values of *SS* immersed in *Neo* and *Neo* with fungal suspension: *Aspergillus niger* 

The *Rp* values are lower in *Neo* with fungal suspension than in *Neo* only. The order of magnitude of *Rp* is 104 Ω.cm2 for *Neo*. In case of *Neo - Aspergillus niger* is observed a greatly decrease of *Rp* with the increase in time. In *Neo* - *Candida mycoderma* system the *Rp* values decrease up to minute 3 and afterwards a significant increase was obtained up to minute 12 followed by a lower decrease at final contact time (Fig. 8, series 3). In *Neo* - *Saccharomyces cerevisiae* a significant decrease in the *Rp* values was observed (Fig. 8, series 4). The order of magnitude of *Rp* is 103 Ω.cm2 in *Neo* with fungal suspensions.Thus, the *SS* surface is more susceptible in electrochemical system containing H2O2 from *Neo* biocide with *Aspergillus* 

Figure 9 presents the *Vcorr* values of *SS* immersed in *Neo* and *Neo* with fungal suspension at

In case of *Neo - Aspergillus niger* the *Vcorr* values increase more in time up to minutes 6 and afterwards a smaller decrease was obtained up to minutes 30 (Fig. 9, series 2). The *Vcorr* values decrease more in *Neo* - *Candida mycoderma* system up to 12 minutes followed by an increase at final contact time (Fig. 9, series 3). The highest increase of *Vcorr* values was obtained when *Saccharomyces cerevisiae* was added in *Neo*, at the beginning of the measurements and then there was a slight decrease up to minute 12 from immersion contact time (Fig. 9, series 4). Thus, the *SS* surface is more corrosive in electrochemical system

(series 2), *Candida mycoderma* (series 3) and *Saccharomyces cerevisiae* (series 4).

*niger* suspension, respectively *Saccharomyces cerevisiae* suspension.

can accelerate the corrosion of AISI 304 stainless steel immersed in them.

**5.2.4 Synergic of working parameters** 

106

(corrosion rate) variations from Tables 2 - 4.

different contact time after immersion.

different contact time after immersion.

The work deals with the corrosion behaviour of AISI 304 Stainless Steel into biocide solution (*Neoseptal* with hydrogen peroxide as active substance) through artificial contamination with three (10% vol.) fungal suspensions as: *Aspergillus niger*, *Candida mycoderma* and *Saccharomyces cerevisiae*. At the applied electrical potential the biocide can work better within the fungal cells, and thus disturbing the present microorganisms homeostasis in several ways such as increasing the environmental conductivity of solutions and the corrosion rate of metallic support. A synergic effect achieved through the mixture of biocide, fungal suspension and applied electric potential, is more destructive than each parameters by its self. The *Ecorr* values of AISI 304 Stainless Steel in the mixtures decreased during the contact time after artificial contamination. The fungal suspension has a significant influence on the synergic effect of the AISI 304 Stainless Steel corrosion in the following order: *Saccharomyces cerevisiae>Aspergillus niger*>*Candida mycoderma*. A more influence on the synergic effect of the surfaces immersed at the mixture consisting on biocide *Neoseptal* solution with *Saccharomyces cerevisiae* could be explained through the less resistance at the chemical attack from biocide. The synergic effect between the active substance of the disinfectant, fungal suspensions and the applied electric potential should taken into account for the hygienic and safety food bioprocessing industry.

Electrochemical Behaviour of AISI 304 Stainless Steel

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