Electrochemical behaviour and surface chemical composition of 70Cu-30Ni alloy after short-term immersion in artificial seawater and filtered natural seawater

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Exopolymers are primarily composed of high molecular weight polysaccharides (10 to 30 kDa) and generally have heteropolymeric composition, that consist of a mixture of neutral and charged sugar residues with organic or inorganic substituent that greatly affect their physical and biological properties. Microbial exopolysaccharides are considered to play an important role in biofilm formation, pathogen persistence, and have several applications in the food and medical industries [45], [55], [61], [63]. The most well-known exopolysaccharides present inside biofilms are presented in Table I.4 [63].

Extracellular proteins

The biofilm matrix can contain considerable amount of proteins that, in some cases, may overtake the polysaccharide content on a mass basis. The proteins in the matrix, such as the cell surface-associated and extracellular carbohydrate-binding proteins (lectins), are involved in the formation and stabilization of the polysaccharide matrix, and constitute a link between the bacterial surface and EPS. Proteins rich in acidic amino acids, including aspartic and glutamic acid, contain carboxylic groups that contribute to the anionic properties of EPS [64].
Bacteria produce a large variety of enzymes, which can be categorized as ectoenzymes (associated with the cell but expressed outside the cytoplasmic membrane) and extracellular enzymes (present in the biofilm matrix). Some extracellular enzymes have been detected in biofilms, many of which include polysaccharidases, proteases, lipases, esterases, peptidases, glycosidases, phosphatases, and oxidoreductases [64]. Extracellular enzymes can be retained in the biofilm matrix by their interactions with exopolysaccharides, enhancing the thermostability of the enzymes and their resistance to proteolysis. Furthermore, extracellular redox enzymes play a role in MIC, while other extracellular enzymes are of commercial interest and are produced on a large scale industrially. They can be, additionally, involved in the degradation of structural EPS to promote the detachment of bacteria from biofilms. The presence of such degrading enzymes makes the matrix an external digestive system that can potentially degrade EPS components in case of nutrients starvation; the resulting products can then be taken up and used as carbon and energy sources. Exopolysaccharides are degraded mainly by hydrolases and lyases, but degradation is generally slow. The chemical properties of the biofilm matrix, such as the presence of different types of binding sites within macromolecules forming this matrix, enhance close association between EPS enzymes and exogenous substrates, thus enabling enzymatic reactions [55], [64].

Extracellular nucleic acids

Although deoxyribonucleic acid (DNA) was initially considered as residual material from lysed cells, nowadays it is considered to play an important role in the formation of the biofilm structure (nucleases can be regulators of biofilm formation), and to be integral part of the matrix and of the biofilm mode of life. Lysed cells are not the only source of extracellular DNA; active excretion of DNA cannot be excluded. The localization of extracellular DNA can vary widely between biofilms; in some biofilms, it forms a grid-like structure whereas in others, it forms a filamentous network [55], [63]. Nucleic acids are polyanionic due to the phosphate residues in the nucleotide moieties [64].

Surfactants and lipids

Lipids are also found in the matrix. Furthermore, some bacteria produce extracellular lipids with surface-active properties, referred to the ability of a molecule to alter the interface between two different phases. Substances with surface-active properties (surfactants) are amphiphilic molecules with both hydrophilic and hydrophobic or lipophilic (generally hydrocarbon) moieties able to display a variety of surface activities that allow solubilisation of hydrophobic substrates [55], [65]. Biosurfactant is a substance that is synthesized by microorganisms (mostly bacteria and yeasts) and that is surface active. It may be either extracellularly released into the environment or localized on surfaces, being associated with the cell membrane. Microorganisms produce them to increase the hydrophobicity of the cell and help them to survive in hydrophobic environment. Additionally, they can mediate the solubilisation of hydrophobic compounds in their environment, these compounds being then used as substrates. Biosurfactants can have antibacterial and antifungal properties, and are important for bacterial attachment and detachment from oil droplets. Biosurfactants generated by microorganisms at the air water interface reduce surface tension, influencing the gas exchange between oceans and the atmosphere. In addition, they stabilize emulsions, promote foam and are generally non-toxic and biodegradable [55], [65].

Interaction of EPS with metals

In order to understand the interactions between bacteria and a solid surface, all the factors that may be involved on these interactions must be considered [34]. Firstly, it has been demonstrated that the chemical composition of the substratum to which microorganisms attach is an important factor during the early stages of biofilm accumulation and may influence the biofilm formation rate and the cell distribution during the first hours of exposure [9].
Another important factor to be considered is the EPS because the EPS molecules provide the forces responsible for cohesion of the biofilm and its adhesion to the substratum [34]. Biofilm cohesion and adhesion are provided by weak interactions such as van der Waals forces, electrostatic interactions, and hydrogen bonds [57]. The cell wall is involved in the adhesion process only in the afterwards stages. Microbial adhesion can be considered mainly as an abiotic process, which can also occur when the cells are dead. This means that the cells do not need to be viable for adhesion [34].
Exopolysaccharides and other biopolymers bind a wide variety of metals such as Pb, Sr, Zn, Cd, Co, Cu, Mn, Mg, Fe, Ag, and Ni [45], [62], with various degrees of specificity and affinity [66]. The binding affinity in the complex depends principally on the ion size/charge ratio, and other factors such as EPS composition, physical gel state, pH, ionic salinity, and carbohydrate/protein ratio [62], [66].
The functional groups of microbial EPS may be either negatively or positively charged at near neutral pH values [64]. Two types of mechanisms may be involved in the EPS metal-binding capacity: a) ion exchange due to the high amount of negatively charged functional groups in EPS; and b) complexation with negatively charged functional groups, such as hydroxyl groups, amino groups, carboxylic groups, phosphoryl and sulphate groups [59], [62], [66], [67]. It has been reported that exopolymers of aquatic microorganisms act as polyanions under natural conditions by forming salt bridges with carboxylic groups of acidic polymers such as uronic acids, or by forming electrostatic bonds with hydroxyl groups on polymers containing neutral carbohydrates [66]. . Therefore, the adsorptive affinity of certain metals has been often correlated with the content of uronic acids in the exopolymers [45]. .
The influence of pH on the metal binding to exopolymer emphasizes the involvement of H+ ions. At low pH the availability of negatively charged sites such as carboxylic groups is strongly decreased, and consequently a small number of metallic cations can be bound. Whereas at higher pH, metal binding is enhanced by a proportional increase in the number of ionized acidic groups that are free to bind ions and therefore tend to favour their chelation. The maximum binding affinities occur near the pH of seawater, generally between 8.0 and 8.2. This may vary considerably within the localized microenvironments in the biofilm due to the photosynthetic and respiratory activities of microorganisms [45], [62]. Another potential factor that may influence metal chelation is the modification of EPS by UV irradiation. Laboratory studies have shown that there is an enhancement of available carboxylic groups after exposure to UV-irradiation, which may increase their potential for binding metallic ions in EPS. [62].

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Composition and physico-chemical properties

Serum albumin is probably one of the most studied globular proteins. It is commonly used as a model protein in several research areas such as molecular biology, food industry, medicine and environment, among others; it corresponds to the most abundant protein in blood plasma. Serum albumin function is associated with the binding and transport of several small molecules such as fatty acids, dyes, metals, and amino acids [73], [74].
Bovine serum albumin (BSA) has a molecular weight of 66.43 kDa (66430 g.mol-1), and a density in aqueous solution of 1.3 g.cm-3, slightly varying with the pH. The isoelectric point of this protein is reported to be 4.5 and 5.6; thus in neutral solution, BSA is negatively charged as a whole molecule. The amino acid composition of BSA has been published for the first time in 1975 by J. R. Brown [75] and later by Hirayama et al. [76] in 1990. The amino acid composition of BSA proposed by these authors is presented in Table I.5.

Bacterial culture (Pseudomonas NCIMB 2021)

Pseudomonas is considered the most prevalent species in industrial water and seawater, they have been found to be involved in the corrosion process of several metallic materials. Aerobic Pseudomonas strains are recognized to be the pioneer colonizer in the process of biofilm formation, and their primary role appears to create an oxygen-free environment to harbor the SRB. Furthermore, it is subsequently found that these strains are aerobic slime-formers and often grow in a patchy distribution over the metal surface and exclude oxygen via respiration; thereby creating oxygen concentration cells or ion concentration cells [68].
Pseudomonas NCIMB 2021 is a marine bacterium that has been intensively studied with the aim of solving problems related to biofouling. This strain was first isolated by Fletcher and Floodgate from the Menai Straits, Anglesey in Wale [78], [79].
The extracellular polymeric substances (EPS) of Pseudomonas NCIMB 2021 were isolated by Fletcher in 1980, using ethanol precipitation. It was reported that the EPS consisted of 50-80% protein and a carbohydrate fraction containing mannose, glucose, glucosamine, rhamnose, galactose and ribose, but no uronic acids. However, the presence of acidic groups in the carbohydrate fraction of this marine pseudomonas was confirmed by Christensen, who also showed that this organism produces one type of polysaccharides in the exponential growth phase and a different type in the stationary phase [79].
Fletcher and Loeb compared the adhesion of the marine pseudomonas to hydrophobic and hydrophilic surfaces. Bacteria were most abundant on hydrophobic surfaces, and the number of attached cell decreased with the wettability of those surfaces. Fletcher concluded that bacteria have both active and passive attachment mechanisms [80].

Electrochemical measurements

When a metallic material takes part as electronic conductor in the presence of an aqueous media, the corrosion phenomenon occurs and is usually of electrochemical nature, and therefore the use of electrochemical techniques is essential for the study of this process.
The use of electrochemical techniques for the study of corrosion has the advantage of providing results in a relatively short period of time, in comparison with the traditional weight loss measurements, and allows elucidating mechanistic information about the processes being studied [26]. The basis of electrochemical techniques consists in maintaining constant all the parameters that determine the state of the metal/solution interface (e.g. temperature, pressure, area), and i) applying an input potential (DC or AC), and measuring the output current (or vice versa); or ii) measuring the spontaneous potential or current fluctuations of the system without applying any perturbation [26], [95].

Table of contents :

I. Background
I.1. Cooling water systems
I.1.1. Importance of cooling water systems
I.1.2. Types of cooling water systems and heat exchangers in power supply facilities
I.1.3. Materials
I.1.4. Operating problems in cooling water systems
I.1.4.1. Scaling
I.1.4.2. Fouling
I.1.4.3. Biofouling and Microbially Influenced Corrosion (MIC)
I.2. Biofilms
I.2.1. Introduction
I.2.2. Conditioning films
I.2.3. EPS and biofilm architecture
I.2.3.1. Exopolysaccharides
I.2.3.2. Extracellular proteins
I.2.3.3. Extracellular nucleic acids
I.2.3.4. Surfactants and lipids
I.2.3.5. Interaction of EPS with metals
I.3. Bovine Serum Albumin (BSA)
I.3.1. Composition and physico-chemical properties
I.3.2. Structure/conformation
I.4. Bacterial culture (Pseudomonas NCIMB 2021)
I.5. Conclusions
II. Experimental
II.1. Materials, electrolytes, biomolecules, and bacteria
II.1.1. Metallic materials
II.1.2. Electrolytes
II.1.3. Biomolecules
II.1.3.1. BSA
II.1.3.2. EPS extracted from Pseudomonas NCIMB 2021
II.2. Experimental techniques
II.2.1. Electrochemical measurements
II.2.1.1. Electrochemical cell
II.2.1.2. Instrumentation
II.2.1.3. Ecorr vs time
II.2.1.4. Polarisation curves
II.2.1.5. Electrochemical Impedance Spectroscopy (EIS)
II. Principle
II. Impedance data analysis
II.2.2. Surface analysis
II.2.2.1. X-ray Photoelectron Spectroscopy (XPS)
II. Principle
II. Instrumentation
II. Data processing
II.2.2.2. Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS)
II. Principle
II. Instrumentation
III. Electrochemical behaviour and surface chemical composition of 70Cu-30Ni alloy after short-term immersion in artificial seawater and filtered natural seawater 
III.1. Results
III.1.1. Static conditions
III.1.1.1. Electrochemical measurements
III.1.1.2. Surface analysis
III.1.2. Effect of hydrodynamics
III.1.2.1. Comparison static conditions/under flow and stirring
III. Electrochemical measurements
III. Surface analysis
III.1.2.2. Electrochemical measurements using a RRE
III. Theory for a Rotating Ring Electrode
III. Experimental results obtained with a RRE
III.2. Discussion
III.2.1. Impedance model for 70Cu-30Ni
III.2.2. Surface layers models (combined XPS and ToF-SIMS)
III.2.3. Conclusions
IV. Influence of biomolecules adsorption on the electrochemical behaviour and the surface chemical composition of 70Cu-30Ni alloy in seawater
IV.1. Results
IV.1.1. Bovine Serum Albumin (BSA)
IV.1.1.1. Static conditions
IV. Electrochemical measurements
IV. Surface analysis
IV.1.1.2. Effect of hydrodynamics
IV. Effect of hydrodynamics on the surface chemical composition (comparison static conditions/under flow and stirring)
IV. Electrochemical measurements using a RRE
IV.1.2. EPS from Pseudomonas NCIMB 2021
IV.1.2.1. Electrochemical measurements
IV.1.2.2. Surface analysis
IV.2. Discussion
IV.2.1. Analysis of impedance data
IV.2.2. Surface layer models (combined XPS and ToF-SIMS)
IV.2.3. Organic layers (XPS)
IV.3. Conclusions
V. Influence of biomolecules adsorption on the electrochemical behaviour and the surface chemical composition of passive materials (304L stainless steel, Ti) 
V.1. Electrochemical measurements
V.1.1. Titanium
V.1.2. 304L stainless steel
V.2. Surface analysis
V.2.1. Titanium
V.2.2. 304L stainless steel
V.3. Discussion
V.3.1. Surface layers models (combined XPS and ToF-SIMS)
V.3.2. Analysis of impedance data
V.3.3. Organic layer
V.4. Conclusions
VI. General conclusions and future work


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