Although much progress has been made in the use of renewable energy in recent years, fossil fuels (especially oil and gas) still meet most of the global energy demand, and they will continue to be the dominant source of energy worldwide over the next few decades (1).

Petroleum is a naturally occurring material found in various geological formations (reservoirs) worldwide. Crude oil, the liquid part of petroleum, is primarily composed of hydrocarbons (2). However, it may also include compounds of nitrogen, sulfur, oxygen and metals (3). Because crude oil in reservoirs is found as a mixture containing varying constituents and proportions, each crude oil has its own unique properties. The most important specified properties are density and sulfur content (4). The density of crude oil is reported in terms of American Petroleum Institute (API) gravity (specific gravity). Based on the API gravity, crude oils can be classified into light, medium, heavy and extra heavy oils (3). Depending on the amount of sulfur content (elemental sulfur or sulfur compounds such as hydrogen sulfide), the crude oil is categorised as ‘sweet’ or ‘sour’. In addition to chemical composition and physical properties, crude oil typically is also identified by underground reservoir (4). Reservoir characteristics (depth, temperature, pressure and other factors) vary significantly from one location to another, even in the same geologic formation (5, 6). The fact that microbial community composition and reservoir conditions vary dramatically not only between the different geographical areas, but also among different oil fields in the same region, makes each oil reservoir ecosystem unique.

Despite the extreme environmental conditions in the oil-bearing formations (i.e. anoxic, high temperature, high salinity), many microorganisms are capable of surviving in the oil and water phases of the oil wells (7, 8). Oil fields harbour mainly facultative aerobic and strictly anaerobic microorganisms due to the low redox potential in the reservoirs (8). These ecosystems contain different types of microbial communities (such as mesophiles, thermophiles and halophiles) which adapt to the reservoir conditions (9). Bacterial and archaeal groups identified in oil fields include sulfate-reducing bacteria (10), sulfur-oxidising bacteria (11), methanogens (12), fermentative microorganisms (13), acetogens (14), nitrate reducers (15), manganese and iron reducers (16) and hydrocarbon degraders (17). Among these microbes, sulfate-reducing bacteria have attracted much attention due to their detrimental effects such as reservoir souring and biocorrosion (7). In addition, different members of the oil microbial community are involved in syntrophic interactions. Fermenting bacteria and methanogenic archaea are involved in methanogenic hydrocarbon biodegradation through their close syntrophic associations (18). This microbial process is undesirable in oil reservoirs because it causes a decrease in oil quality and value (19). Syntrophic microorganisms in oil reservoirs also play important roles in the global biogeochemical cycling of sulfur, carbon and nitrogen. For instance, sulfate-reducing bacteria and sulfur-oxidising bacteria, the key drivers in sulfur transformations, are involved in the sulfur cycle (11). Thus, knowledge of the microbial groups and microbial dynamics in oil fields enable us to obtain detailed insights into the microbial ecology of oil associated environments.

Understanding the microbial ecology of oil reservoirs is crucial to the petroleum industry because the success of oilfield operations is strongly influenced by the activity of microorganisms. Oil microbes with different metabolic capabilities have significant negative and positive impacts on the petroleum resources and the extraction processes (7). Microbial activity may lead to severe problems such as reservoir souring and microbial corrosion. Reservoir souring, which is characterised by an increase in production of H₂S in the reservoir fluids, most commonly occurs when sulfidogenic microorganisms reduce sulfate to sulfide, a toxic and corrosive product (20). Undesirable accumulation of sulfide minerals in reservoirs is one of the major challenging problems in oil production because it causes plugging of reservoirs, decreasing the oil quality and value and increasing the refining costs. Moreover, exposure to H₂S can be dangerous in terms of worker health and safety due to its high toxicity. Additionally, the produced H₂S promotes corrosion of the metallic equipment and structures used for oil production and processing (21). Another destructive phenomenon is biocorrosion, which is defined as microbial attack on the surface of the metal infrastructure leading to disruption of the material (22). In addition to sulfate-reducing bacteria, which play a major role in biocorrosion, other corrosive microbes, such as acetogenic bacteria and methanogenic archaea, are also associated with corrosion failures (23). Biocorrosion is a great concern because it leads to loss of material, large economic losses and safety issues in the oil industry (24). In contrast, hydrocarbon-degrading bacteria may be used for environmental clean-up processes (6). Bacterial degradation of hydrocarbons was carried out by both aerobic (for example, Rhodococcus sp., Sphingomonas sp., Pseudomonas putida, Pseudomonas stutzeri, Acinetobacter sp.) and anaerobic bacteria (such as Fe(III)-reducing bacteria, sulfate-reducing bacteria) (6, 17). Furthermore, microbial products such as biopolymers and biosurfactants can be used for facilitating oil movement in a widely used technology, known as microbial enhanced oil recovery (MEOR) (1). Compared with other conventional oil recovery techniques, MEOR has advantages such as low cost, wide application, high efficiency and low environmental pollution (25). Therefore, diversity, metabolic processes and habitat conditions of microbial communities in oil reservoirs should be investigated, so that their negative effects can be decreased and their positive effects can be exploited.

This study aimed to determine the bacterial community composition and to identify the predominant community members in produced water from oil fields located in the Diyarbakır region in Turkey. To this end, we used PCR-DGGE to analyse 20 produced water samples from the Diyarbakır region. There are limited studies on produced water from the Diyarbakır region and this paper represents the only in situ study available. The results of this study provide not only new data about the microbial ecology of the Diyarbakır oil fields, but also information on the bacterial populations which may have potential roles in terms of increasing or decreasing the efficiency of industrial applications.

2.1 Sampling Procedure

2.2 DNA Extraction

2.3 Polymerase Chain Reaction Amplification

2.4 Denaturing Gradient Gel Electrophoresis

2.5 Comparative Sequence Analysis

3.1 Molecular Analysis of Bacterial Communities

In order to increase our knowledge about microbial diversity, culture-dependent and molecular-based approaches are used for describing the diversity of microbes. Molecular-based approaches such as PCR-DGGE methodology, which is a useful tool for monitoring the genetic diversity of complex microbial populations (26), provide valuable information about the microbial community structure and dynamics in nature. For these reasons, PCR-DGGE fingerprinting analysis of environmental samples was used in this study.

The choice of appropriate primers for PCR amplification is a crucial step to accurately characterise the microbial communities. In this study, primer pair (341F-GC/907R), targeting the V3-V5 region of the 16S rRNA gene fragment, was selected due to its suitability for DGGE analysis of bacterial populations in environmental samples (26). This primer pair designed by Muyzer et al. (27, 28) has been used predominantly for microbial community analysis (26).

The DNA yield obtained from produced water samples was very low. It is known that crude oil samples contain low amounts of biomass which makes DNA isolation difficult to achieve (46). In this study, the permit included taking up to 500 ml of oil/water mixture from each sampling point so that only ca. 25 ml of each produced water sample could be obtained. In this scope, the low sample volumes of produced water separated from the oil/water mixture may be a reason for the low amount of DNA. It was reported in other studies that higher sample volumes (100–4000 ml) of produced water were used for DNA isolation (35, 47–50). The low DNA yield affected the efficiency of the PCR technique and for this reason, a two-step PCR was applied to the produced water samples. Thus, a sufficient amount of PCR product for DGGE for the produced water samples could be obtained.

Bacterial communities associated with the produced waters was analysed by the PCR-DGGE approach. Although numerous bands were visible on the DGGE gel, only dominant bands could be excised and sequenced. Most of the sequences retrieved from produced water samples were related to unclassified bacteria. Different studies on oil reservoir microbiota have also shown that oil fields harbour new and still unidentified microbial species. For example, Lenchi et al. described microbial communities in production and injection waters from the Algerian oil fields. In their study, they detected that a large number of unclassified bacterial and archaeal sequences were found in the water samples (51). Furthermore, uncultured bacteria such as uncultured Sphingomonas sp. and uncultured Caenispirillum sp. clone Ppss_Ma27 were detected in our study. This result is consistent with the fact that the vast majority of microorganisms are uncultured and do not grow under laboratory conditions as stated by Lewis et al. (52). In order to isolate more microbes, an appropriate identification laboratory protocol should be followed. At this point, different strategies such as mimicking natural conditions via decreased nutrient, extended incubation times, the modification of isolating media formulations and different incubation parameters (for example, temperature) were suggested for the cultivation of microorganisms (53). For instance, pollutant degrader Sphingomonas, which seemed to be previously uncultured by nutrient-rich methods, could be isolated from crude oil contaminated soil by using an in situ method that mimics the original environment (54). In addition, culture-dependent investigation should also be supported by molecular techniques.

Based on the sequences, organisms related to known mesophilic bacteria were predominant in the produced water samples. In addition, some organisms related to thermophilic bacteria (Aeribacillus pallidus, Marinobacter sp. Trimyema-2, Phenylobacterium lituiforme and Thermithiobacillus sp.) were also identified. Bacteria having different metabolic capabilities (denitrifying, biodegrading and sulfur removing bacteria) were also detected. In addition, bacteria which may cause biocorrosion on steel surfaces were detected.

The dominant bacterial phylum was the Proteobacteria. The members of this phylum were also frequently found in many other studies on microbial diversity of oil field produced waters (55–58). Moreover, it was stated that Proteobacteria are ubiquitous in oil reservoirs over all temperature ranges (59).

In this study, among the detected genera in produced water samples that potentially contain hydrocarbon degrading bacteria were Aeribacillus, Acinetobacter, Sphingomonas, Marinobacter and Phenylobacterium. It has been known for years that the species belonging to these genera are capable of degrading hydrocarbons (6, 17, 60, 61). In addition, G. daeguensis, a hydrocarbonoclastic bacterium, was detected in produced water sample with a 100% sequence similarity. G. daeguensis has also been demonstrated as a potential microbe for bioremediation due to its hydrocarbon degradation ability (44). Further investigations are needed because our current knowledge of the metabolic capability of G. daeguensis is limited. Moreover, sulfur-oxidising Thermithiobacillus sp. was also identified in produced water sample. Sulfur-oxidising bacteria, which oxidise the sulfur compounds produced by the activity of sulfate-reducing bacteria in oil reservoirs, may play a key role in the oil industry because they can be utilised to resolve processing problems such as reservoir souring (11).

Pseudomonas was the dominant genus detected among the produced water samples. Pseudomonas stutzeri was the species identified in five produced water samples. P. stutzeri was previously isolated not only from formation water, produced from the petroleum wells in Adıyaman (62), but also oil-contaminated soils in Batman petroleum refinery, Turkey (63). These two areas are close to the Diyarbakır region from where the samples in this study were collected and these findings show that P. stutzeri is distributed widely in south-eastern Turkey. In other different geographical areas, this species was also isolated from oil-associated environments, such as oil field production water (64), oil sludge (65) and oil contaminated soil (66). However, although P. stutzeri is often isolated from oil reservoirs, the origin of P. stutzeri in oil reservoirs is a debatable issue. Because oil reservoirs have low redox potentials and contain little oxygen, anaerobic microorganisms are considered as truly indigenous to oil reservoirs (67). In this regard, it is believed that P. stutzeri, most of whose strains are aerobes, is an exogenous organism inoculated into oil reservoirs during the oil production processes. Even if strains of P. stutzeri are introduced into oil reservoirs with injected fluids, they should adapt to the physicochemical characteristics of the reservoir to survive. At this point, it has been proposed that extreme reservoir conditions may act as special factors for the evolution of P. stutzeri, thereby forming mutant strains (68). Furthermore, P. stutzeri, being found in a wide variety of habitats, is known for its diverse metabolism. Some strains of P. stutzeri are capable of denitrification, degradation of aromatic compounds and nitrogen fixation (32). These metabolic features make P. stutzeri highly attractive for biotechnological processes, such as reservoir souring control (69), microbial enhanced oil recovery (64) and bioremediation of oil-polluted environments (65).

In undisturbed oil reservoirs, microorganisms are found in different phases such as reservoir fluid containing crude oil and formation water, and rock surfaces. While planktonic microbes thrive in the water phase, sessile microbes may attach to oil or rock surfaces (59). In addition, biofilm may form on the metal surfaces of the pipes in the oil-producing wells (70). Oil microbiome studies focus mainly on the analysis of the water phase due to its easy sampling. However, it should be noted that the water phase itself contains only a minor portion of the microbes found in the oil reservoir (59). On the other hand, the sampling of sessile microbes is likely to be more challenging (70).

This study reported for the first time the bacterial community composition of produced water from Diyarbakır oil reservoirs as obtained by DGGE analysis of PCR-amplified 16S rRNA gene fragments. DGGE analysis of produced water samples demonstrated that the majority of the bacterial sequences belonged to unclassified bacteria, indicating that oil reservoirs harbour still undescribed microbial species. Among the classified bacteria, the members of Proteobacteria were more abundant. Pseudomonas was the dominant genus detected in the produced water. Although the members of Pseudomonas were known as exogenous organisms inoculated into oil reservoirs, Pseudomonas stutzeri was found in five produced water samples. Bacteria having different metabolic capabilities (denitrifying, biodegrading and sulfur removing bacteria) were also detected. It can be stated that the metabolic capacities of these bacteria make them potential candidates for utilising in biodegradation, bioremediation, the improvement of oil quality and oil recovery processes. The knowledge of the bacterial community composition in oil reservoirs of the Diyarbakır region obtained in this study will be of great interest for both scientific research and applications in the oil industry. To build on the data presented in this study, metagenomic analyses should be performed to explore the undescribed microbes.

Footwear is the most commonly worn apparel in daily life, and its design features must prioritise anatomy, comfort and hygiene. For this reason, it is important to develop sustainable improvements to footwear’s functional properties.

Footwear that carries the body’s weight during the day can affect foot health physically, chemically and microbiologically. Continuous contact with the external environment exposes footwear to microorganisms during normal use. All sorts of footwear play a role in the transport, spread and contamination of pathogenic or non-pathogenic microorganisms (1).

There are different microorganisms in every part of the human body. Sweat is regularly secreted from the body under normal conditions. It contains 98% water and urea, uric acid, fatty acid, lactic acid and sulfates (2). The feet have more sweat glands than other parts of the body. Sweat secreted from feet during the usage of footwear is decomposed by means of foot microbiota; as a result, bad odours emerge in footwear. Brevibacterium linens, Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli are some microorganisms that make footwear unhygienic. As a result of the breakdown of amino acids in sweat and skin by these microorganisms, bad odour arises in feet, socks and footwear (3, 4).

Nowadays, in addition to individual foot care and hygiene for odour prevention in shoes, commercial materials with various deodorising and antimicrobial effects are also employed (5). Footwear insock is a thin layer of materials put into the shoe after manufacture to cover the insole. It directly contacts the sole of the wearer’s foot and can provide a more sanitary environment when specially treated for antimicrobial purposes (6).

Spray drying is an advantageous way to encapsulate active substances and essential oils. Spray drying is a common and accepted encapsulation method for industrial applications. With this method, it is possible to mass produce capsules. The distribution of particles is uniform (7–10).

Microencapsulation technology has been used for the application of orange oil to textiles and leathers, being an economically viable, fast and efficient method by combining core and shell materials, desirable perceptual and functional characteristics, and also allowing functional substances to be released in a controlled manner. This technique has also been used to microencapsulate a wide range of active, functional, sensitive or volatile substances (11–14). Tea tree oil containing melamine formaldehyde microcapsules, essential oils (eucalyptus, lavender or oregano), polyurethane dispersions containing photoactive antimicrobial agents, zinc oxide and silver nanoparticles are some substances that protect upper leathers from the harmful propagation of microorganisms (3, 15). In addition, aromas confined to microcapsules are also used to prevent bad odours in footwear (16, 17). Application of antibacterial and aromatic materials onto footwear insocks to control bad odours is good for foot hygiene and desired shoe comfort.

The use of orange oil presents as an ecological alternative to synthetic chemicals, attracting the attention of the scientific community to the development of eco-friendly antimicrobials. In this study, microparticles were produced by a spray drying method after the emulsions with orange oil and chitosan were prepared in different ratios. Microparticles manufactured in this way were then transferred to the surface of the footwear insock leathers using a finishing process. Afterward, some tests and analyses were performed on microparticle coated footwear insock leather samples to evaluate the effectiveness of the microparticles, their presence on the leather surface and their antimicrobial properties.

Pharmaceutical grade cold pressed orange oil was donated from Ephesus, Turkey. Chitosan was purchased from Acros Organics^TM (Belgium). Analytical grade chemicals were used in the analyses. Insock leathers, without dye and ready for experimental application, were donated from Ata Dilek Leather (Izmir, Turkey).

For the microparticle production, chitosan (shell material) was added to 1% w/w aqueous acetic acid for preparing the chitosan solution. This solution was stirred at 45°C by using a magnetic stirrer until wholly dissolved. During the pre-emulsion preparation, orange oil (core material) was gradually mixed into the chitosan solution and stirred for 1 min at 10,000 rpm. The surfactants as compound emulsifiers used for pre-emulsion preparations were Tween 40 with Span 20 at the ratio of 8:2 w/w. Then, the microparticles were prepared by using an SD Basic spray dryer (Labplant, UK) with nozzle diameter of 0.5 mm. The orange oil to chitosan ratios in the four encapsulating compounds came to 1:1, 1:1.33, 1:1.67 and 1:2 w/w. The ingredients of the formulations in the spray-drying process are shown in Table I. Homogeneous emulsions were fed to the spray dryer under the following conditions: pump speed 12 ml min⁻¹, outlet air temperature 114°C and inlet air temperature 175°C.

The microparticles’ morphology was examined by a Quanta 250 FEG scanning electron microscope (FEI, USA) at 2 kV accelerating voltage. Before coating in an argon atmosphere with gold-palladium by a K550X sputter coating machine (Quorum Emitech, UK), the samples were mounted onto an aluminium stub. The grain side of leathers coated with microparticles was examined by a TM1000 tabletop scanning electron microscope (Hitachi, Japan) after coating with gold-palladium.

The FTIR spectra of the spray-dried microparticles and leathers with microparticles were procured by a Spectrum 100 FTIR attenuated total reflectance (ATR) spectrometer (PerkinElmer, USA). The measurements were made using four scans with a resolution of 4 cm⁻¹ between 4000 cm⁻¹ to 650 cm⁻¹ wavenumber ranges at room temperature.

Encapsulation efficiencies of microparticles were calculated as the amount of orange oil (core material) encapsulated in the microparticles. The encapsulation efficiency was calculated using Equation (i) (18).

(i)

A solvent extraction method was used to determine total oil content. A 0.1 g measurement of orange oil loaded microparticles was dissolved in 10 ml of 1% acetic acid solution at room temperature for 45 min. Released orange oil which was obtained from the completely dissolved microparticles was placed in a beaker containing 50 ml n -hexane for extraction 45 min. So as to determine the total amount of orange oil in the microparticles, this extract was filtered through a syringe filter (0.22 μm). Orange oil content in the filtrate was measured using a UV-1800 UV-vis spectrophotometer (Shimadzu, Japan) at 202 nm in triplicate. Surface oil content was also determined by the same solvent extraction method described above, except for a dissolving process in 1% acetic acid solution (11).

In vitro release studies of microparticles and microparticle loaded leathers were carried out at a speed of 100 rpm in phosphate-buffered saline (PBS) and methanol at 37°C. 1 mg orange oil loaded microparticles was suspended in beakers containing 4 ml methanol and 16 ml of PBS. Insock leathers with 2.5 cm² area were placed in beakers containing 16 ml methanol and 16 ml of PBS for in vitro release studies of microparticle loaded leathers. At suitable time intervals, the medium in the beakers was filtered through a 0.22 μm syringe filter. Sink conditions were maintained in the receptor compartment during in vitro release studies. The released amount of orange oil was analysed by UV method, as previously described, for 5 h. Experiments were performed five times.

A spraying pistol with nozzle diameter of 0.5 mm was used to apply microparticles to the insock leathers during the finishing process. Spray-dried microparticles were added to the finishing recipe as 20 g m⁻² (19). The basic finishing recipe for the insock leathers is given in Table II (20).

The efficacy of microparticle coated insock leathers against test microorganisms Staphylococcus aureus ATCC^® 6538^TM, Escherichia coli ATCC^® 25922^TM, Candida albicans ATCC^® 10231^TM, Klebsiella pneumoniae ATCC^® 4352^TM and Bacillus subtilis ATCC^® 6633^TM was examined by agar disc diffusion method (21–24). Test microorganisms were placed into an incubator for incubation at 37°C for 18 h in the Mueller Hinton broth (MHB) medium. Then, microorganisms were inoculated in petri dishes containing 10⁵ colony forming unit (CFU) ml⁻¹ of Mueller Hinton agar (MHA) medium. Next, microparticle coated insock leather samples with 12.7 mm diameter were placed into the petri dishes (20, 25). All petri dishes were placed into an incubator for incubation at 37°C for 24 h, and inhibition zones were measured to determine antibacterial activity.

In this study, orange oil microparticles were successfully prepared by spray drying method. This method is a simple, viable method to obtain microparticles, suitable to prevent active substance biological activity loss, avoiding exposure to elevated heating and to organic solvents.

3.1 Surface Appearance of Microparticles and Microparticle Coated Insock Leathers

3.2 Fourier Transform Infrared Spectroscopy Studies

3.3 Encapsulation Efficiency

3.4 In Vitro Release Studies of Microparticles and Microparticle Coated Insock Leathers

3.5 Microbiologic Studies on Microparticle Coated Insock Leathers

During the usage of footwear, perspiration and bacterial activity negatively impact foot health and generate bad odours from both the feet and footwear. Shoe production using natural and non-toxic materials that prevent or inhibit bad odours and bacterial activity is one solution to this hygienic problem. Likewise, the successful application of microparticles that release for a long time on footwear insock leather is an important alternative to existing toxic products.

Our research found that emulsions with orange oil and chitosan have natural antibacterial activity. These emulsions, when successfully converted into encapsulated powders by a spray drying method, produce a core-shell material. SEM images showed how an effective finishing process was used to apply laboratory produced microparticles to the surface of footwear insock leather. Microbiological tests performed on microparticle coated leathers proved that footwear insock leathers were fortified with antibacterial properties.

These findings demonstrate that application of orange oil-chitosan microparticles onto footwear insock leather surfaces is an alternative natural method to control hygiene and eliminate bad odours. Non-toxic, functional, leather shoes can incorporate such natural materials in their manufacture and maintenance. This production improvement would thus contribute to people’s foot health, hygiene and comfort.

Metalloenzymes have been prominent in the field of enzyme engineering since its emergence some 40 years ago, at the birth of protein and enzyme engineering (1, 2). Metal ions or cofactors in solution have an intrinsic chemistry that can be catalytic and these are accessible to detailed mechanistic study. These properties mean that co-localisation of substrate and metal within a peptidic scaffold can be sufficient in forming an ArM, without further influence from the protein on the catalytic mechanism. With the advent of modern protein engineering and design technologies, ArMs were developed by incorporating metal binding sites in or adjacent to hydrophobic pockets. While the resulting ArMs were active, they often displayed low efficiency and specificity. Therefore, directed evolution (i.e. iterative rounds of mutagenesis and selection for activity, Figure 1) has become a key step in creating enzymes with new and useful properties. The choice of starting point for such a forced evolution campaign, in this case the metal-protein complex formed initially, is of great importance. Since any particular enzyme follows a unique evolutionary trajectory as new mutations move it along the fitness landscape towards (potentially local) maxima, choice of the starting point may directly predetermine the result. By nature of the selection process, it is further possible, that trajectories leading to the global maximum fitness fall beneath the cut-off limit for further evolution, becoming inaccessible. For instance, a mutation introduced in the first round of mutagenesis may lead to a destabilisation of the protein at assay conditions, causing that initial variant to be discarded through selection. However, a compensating mutation to that variant in a subsequent round of mutagenesis could result in an enzyme which is stable, active and closer to a global fitness maximum. Finally, not every method of generating ArMs may be compatible with current methods for directed evolution and therefore limit the extent of evolution that can be achieved.

Fig. 1.

In this perspective, different routes towards ArMs are considered in the context of the starting protein scaffold as well as the type of catalytic centre and reactions involved. Advances in ArMs have recently been reviewed and the reader is referred to these for further details of the strategies used to find new systems (3–5). This article aims to provide an overview of the strengths and weakness of these different approaches and to provide a perspective of some challenges that remain.

One particular area that will greatly impact chemical production on this planet is synthetic biology. Replacing synthetic catalysts, acting on petrochemical feedstocks in non-aqueous solvents, with biocatalytic systems working in water with simple carbon neutral feedstocks (carbon dioxide even?) is clearly highly desirable. But why engineer new enzymes, particularly using expensive and relatively scarce transition metals, when the ability to find new catalysts amongst gene products from all corners of the biological world has developed at staggering pace (6–8)? As a consequence of the latter, any target chemical can conceivably be obtained by recombining pre-existing metabolic pathways (9). What will new and unnatural metalloenzymes provide?

One clear feature is orthogonality: the objective of introducing functionality into a cell that has no counterpart in the natural world could provide chemistry that biology cannot currently catalyse, alkene metathesis for example. As there is a limit to the number of additional transformations a viable cell will perform, these orthogonal reactions may allow access to much shorter, and therefore more efficient, pathways. If not for a synthetic purpose, one could also imagine orthogonal catalytic chemistry providing a diagnostic or reporter output without interference from the host endogenous processes. For it to be truly orthogonal, it is difficult to imagine evolving a new enzyme based around metals already abundant in nature and already used as catalysts in biology. The transition metals used by nature are very carefully controlled by acquisition and regulatory networks that ensure catalytic metal ions are not free to operate outside the endogenous metabolism. Therefore, there is significant advantage in trying to introduce metals that biology currently has no evolved means of metabolising. This work therefore focuses primarily on non-biological transition metal cofactors as a route to introducing novel orthogonal activity into a biologically viable system.

Natural evolution has provided numerous examples of metal ions used by enzymes for a plethora of different catalytic purposes. Rigorous mechanistic and structural biochemistry has advanced understanding of the mechanistic detail of metalloenzyme activity significantly, to the point that a few underpinning principles can be identified, linking protein structure and thermodynamics to catalytic activity of metal centres. Together with the knowledge garnered from extensive research on transition metal catalysts, it is possible to establish key properties desirable for novel ArMs.

3.1 Considerations on Protein-Substrate Interactions

3.2 Considerations on Metal Chemistry in Proteins

3.3 The Optimal Method of ArM Formation

ArMs are generated either from the combination of an unnatural transition metal cofactor being introduced into a protein scaffold or a natural metalloprotein being evolved in a laboratory to enhance or alter its natural catalytic reactivity. A detailed review of the field of the directed evolution of natural metalloproteins is out of the scope of this perspective. However, the engineering and evolutionary approaches developed by Frances Arnold and applied to haem metalloproteins (for example, cytochrome P450) are particularly noteworthy and applicable when evolving unnatural metal-protein hybrid catalysts (27–29). Four successful strategies have been employed to localise an unnatural metal to a well-defined location within a protein matrix.

4.1 Metal Ion Substitution in Natural Enzymes

4.2 Supramolecular, Non-Covalent Binding of Tagged Complexes

4.3 Covalent Anchoring Through Metal Ligands

4.4 Direct Activation by Metal Coordination to Protein Side Chains

Significant advances in the incorporation of organometallic complexes into proteins in order to generate ArMs have been made. The studies highlighted above reliably create hybrid molecules where the stability and turnover number of the metal centre is higher than the comparable small molecule organometallic complex in aqueous solution. Maybe unsurprisingly, the propensity for side reactions and catalyst decomposition is lowered once the complex is in a hydrophobic protein environment, already showcasing the usefulness of these hybrid systems. However, the question remains, as to whether or not these strategies make full use of the protein component. The unique and numerous demands of ArMs call for a highly integrated approach. To date, most of the work described in the literature attempts to exploit the chemistry of metal ions and their complexes in a protein scaffold but with limited influence from the protein on any catalytic activity because metal-protein coordination is largely indirect and so cooperativity is limited.

The potential for synthetic organometallic chemistry to deliver cofactors which utilise ligand chemistry not available to naturally evolved systems can vastly expand the orthogonal catalysis available in synthetic biological applications. Using such molecules to embed novel metal-peptide hybrid complexes in protein scaffolds allows for three-dimensional and electronic control around the metal centre that reduces the need for intricate synthetic catalyst generation. Instead, control of the steric and electronic environment around the metal ion can be delivered via the protein coordination sphere, particularly where a direct coordination bond is used to anchor the metal ion to the protein. When combined with the ability to efficiently evolve enzymes, a sophisticated organometallic precursor complex together with a suitable apoprotein could potentially give rise to a number of diverse reactivities. Therefore, new problems in catalysis could be addressed in a faster and more specific manner than with small molecule catalysts. Together with non-covalent contributions to catalysis and the intermolecular interactions that pre-organise substrates and stabilise transition states, such a system contains many readily evolvable components.

The majority of protein scaffolds selected for ArM construction have been chosen because of their apparent engineerability. However, in most cases the focus seems to lie solely on the peptidic component with little consideration for evolution of the metal complex. Although methods of selection and directed evolution have been applied, these are often operating on already well-defined protein scaffolds that carry an abiotic cofactor but not a direct protein-metal complex, which inevitably limits the scope for evolution. Arguably it is desirable, therefore, to select for a promiscuous and versatile protein starting point which is not constrained by one energy minima but instead can potentially offer numerous distinct metal-binding environments, both in terms of direct coordination and through secondary, intramolecular spheres of influence, ultimately generating differential catalytic ArM activity.

Performing catalysis with exogenous metal complexes within cellular environments has enormous potential applications in medicinal chemistry and synthetic biology. Given the potential difficulties associated with cell-uptake, minimising deactivation, overcoming toxicity of exogenous metal ions and precise localisation of metal cofactors in cells, the idea of using traditionally inert organometallic complexes has obvious advantages in that reactive promiscuity is reduced. As pointed out above, such complexes would be designed to have a latent catalytic activity which emerges once the metal complex is bound to a protein. The design challenges raised by this approach are not just as a result of a need to control the electronic and three-dimensional steric coordination sphere of the metal ion, but also to limit ligand exchange processes, restricting lability of a precursor complex (in the cellular milieu) until it reaches a specific protein target. Since the metal-ligand exchange processes for 4d and 5d metal complexes are typically slow, they are particularly attractive from this point of view but are hard to predict ab initio.

In conclusion, in order to optimise the chemistry and biochemistry of ArMs, directed evolutionary campaigns coupled with high throughput screening methods rather than individually-designed synthetic strategies are much more likely to generate optimised orthogonal catalysts for new and efficient metabolic processes. Direct coordination between metal ions and enzymes is essential in order to deliver truly interdependent systems, ideally where entatic states deliver enhanced reactivity, efficiency and selectivity that cannot easily be replicated in conventional, synthetic metal catalysis. Going forward, methods of generating ArMs should be evaluated and developed for both their ability to be used in directed evolution procedures and the extent to which the protein scaffold participates in the activity of the metal complex.

A cooling tower is a heat dissipation unit which cools bulk water in industrial systems. Cooling towers provide cooling by spraying the heated water coming from the system onto a fill material and rejecting the heat to the open atmosphere (1). The cooled water returns to a basin to recirculate again through the system. Common uses of wet cooling towers include air conditioner systems, manufacturing facilities, telecommunication devices and power plants. Such man-made installations provide an ideal environment for bacterial growth similar to an incubator, supported by water temperatures ranging between 24°C and 38°C (2–4). The heated water comes from the source to the heat exchanger that allows the exchange of heat between two liquids at different temperatures by indirect contact inside water jacketed tubes (5).

Wet cooling towers providing cool water for heating, ventilating and air conditioning (HVAC) systems are known to be subject to contamination. Organic and inorganic substances in bulk water are deposited on the water contact surfaces, reducing the heat transfer significantly and threatening the operating stability of the whole system. Established biofilms offer cleaning challenges because they are resistant to most chemical and physical cleaning protocols and they also reduce the heat transfer efficiency (6, 7). HVAC systems are responsible for about half of the energy consumed in modern buildings and industrial facilities. Therefore, biofouling is always a significant issue for heat exchangers and should be taken into account during heat exchanger design and production. As a solution, altering the surface properties could be an effective approach to reduce biofouling in such hard-to-reach articulated systems (2, 8, 9).

A biofilm layer is a community formed by bacterial cells living in a polymeric matrix that they produce; a functional partnership adhered to a living or inanimate surface organised by microorganisms in a dense exopolymer matrix. The capability of microbes to stick to a substratum and to produce a biofilm layer has great significance in a diversity of cooling towers, where fouling can act as a perpetual source of contamination. Biofilm layer must be kept to a minimum in order to prolong the operating life of man-made water systems and facilitate control of pathogens. Disinfectants may be used for this purpose (4, 9).

Industrial cooling towers can be manufactured from different materials. Generally, towers are made of reinforced concrete or fibreglass, stainless steel, wood or reinforced plastic sheets. The fill material is generally made of plastic sheets (polypropylene, polyethylene or polyvinylchloride) where heat dissipation occurs. For corrosion resistance, towers are specially treated, painted and covered with a protective film layer (7). In the case of corrosive water or atmospheric conditions, the use of plastic towers is recommended. But heat exchanger units are made of stainless steel or copper for better thermal conduction (5). The critical issue that affects cooling is the aggregation of deposits over the heat exchanger surfaces which includes biofouling. Conventional steel heat exchangers may have corrosion or deposits may have formed on the heat exchanger tubes. Both of these factors reduce the heat transfer rate (10). To solve this problem, novel anti-fouling coatings are considered. Nano-silica can be used in the form of liquid composites in many matrices as coating materials. Nano-silica is used in the textile and automotive industries because of its self-cleaning, abrasion resistant, hydrophobic and oleophobic features. It is known that nano-silica is able to create low-cost, hard and tough coatings which are resistant to wear and weathering (11).

Although biofilm formation on plastic fill surfaces in wet cooling towers has been studied widely, no studies were found on biofilm formation on steel heat exchangers in cooling towers. As coating of heat exchangers is not common, the aim of the current work was to limit tenacious biofouling on heat exchangers using a nano-silica coating, which will lead to longer material life, better cooling of water and less clogging in closed-loop systems.

A brand new fabricated closed-loop cooling tower was monitored for six months. A real size, fully working closed-loop cooling tower system was kept in operation by the manufacturer during the experimental period at the factory test laboratory. The system was filled with distributed network water. Regular blowdown was implemented to limit the concentration of dissolved solids. In a circulation rig, hot process water was kept separate from the cooling water in a closed-loop system (Figure 1). For the experiments, a half portion of the stainless steel (316 SS) heat exchanger tubes were coated with nano-silica and the other part was left without coating. Coating was done by coaxial electrospraying before assembly and left to cure in air for 24 hours. Coaxial electrospraying has several implicit advantages such as high encapsulation efficiency and uniform particle distribution. The coating thickness was between 4–6 μm. Before coating, the heat exchanger tubes were sprayed with 96% ethanol to remove any dirt, oil or grime. This application made the bonding of the coating stronger.

Fig. 1.

Silica in powder form is hydrophilic. To produce hydrophobic nano-silica, the silica particles were transformed by fluorination to confer hydrophobicity. The final particle size was about 40 nm. The aqueous form of the nano-silica coating contains ethanol as solvent to keep it in liquid form before use. The final nano-silica product was supplied by a local company. After curing, the coating was solid on the surfaces, and no colour change, shedding or weight loss were observed on any of the coated test surfaces after the experimental period. The stability of the coating was tested in a different study by the present author (9) and the mean overall adhesion capability of the coating was recorded as 1.6 using a pull-off adhesion tester, which matches very well with the general rating of adhesion. Water was circulated over the stainless steel (316 SS) heat exchanger tubes, where natural biofilm formation was allowed to occur. Sampling of the biofilm required dismantling the outer shell of the heat exchanger unit every month. The system temperature water was kept constant at 37°C using an electrical heating unit to eliminate temperature fluctuation which might influence biofilm formation over time.

Pipe segments were cut monthly from the heat exchanger using an angle grinder, kept in a container filled with system water and brought quickly to the laboratory for analysis. LIVE/DEAD^® Bac Light^TM Bacterial Viability Kit (Invitrogen^TM, Thermo Fisher Scientific, USA) dye was added immediately to cover the surfaces completely to stain the actively respiring and dead bacteria. After 15 min, the surfaces were rinsed with sterile bi-distilled water to remove unattached cells, air dried, covered with immersion oil and cover slip, then examined in the dark. This was repeated every month until the study finished at the sixth month. An epifluorescence microscope (Eclipse 80i, Nikon Instruments Inc, Japan) was used to visualise the biofilm cells in situ. The camera enables counting and taking images of bacteria on solid surfaces, with the signals displayed on the computer monitor. Counting and recording were carried out using special software (NIS-Elements, Nikon Instruments Inc, Japan). Signals obtained from 20 randomly selected regions were recorded. Images were saved for later analysis.

The LIVE/DEAD^® kit stains dead cells red and live cells green in colour. The LIVE/DEAD^® test kit contains two DNA-binding dyes, propidium iodide and SYTO^® 9. These dyes differ in their spectral properties and their ability to enter the living bacterial cell. The first dye in the kit is SYTO^® 9, which can pass through the membrane of all bacteria and stain the cells green. Propidium iodide only enters into cells with a damaged cell membrane, allowing them to appear red under fluorescent light. The number of viable and dead bacteria on surfaces can be determined in a single step using a dual emission filter cube (Chroma Technology GmbH, Germany).

For both parameters over the six-month duration of the experiment, the difference between the average bacterial numbers were compared by two-way analysis of variance. A follow-up post-hoc analysis was done in order to determine differences. The difference was considered significant when p < 0.05. SPSS^® Version 18.0 software (IBM Corp, USA) was used for the statistical analyses.

The bacterial numbers from the LIVE/DEAD^® test kit were analysed in situ on the surfaces using the manufacturer’s software during the experimental period for six months. The results are given in Table I. The number of signals per cm² were calculated using the magnification factor. Since the raw data were too scattered, the values are given in the logarithmic (log₁₀) base for better comparison. The logarithmic reduction was clearly significant starting from the first sampling.

The total bacterial numbers on coated tubes were recorded as 49,090 cell cm⁻² after the initial month, and 13,016,957 cell cm⁻² on uncoated surfaces after the first month. The results distinctly showed that this type of coating reduces biofouling formation on heat exchanger surfaces from the start of the experimental set-up. The numbers of surface associated bacteria on uncoated control tubes gradually increased and reached 1.28 × 10¹² cell cm⁻² after the sixth month, at which time the biomass on nano-silica coated tubes was 6.3 × 10⁴ cell cm⁻². No significant rise (p < 0.05) of bacterial numbers on nano-coated heat exchanger tubes was recorded during the six-month period in terms of total biofilm counts. This outcome demonstrates that a nano-silica coating can clearly reduce the bacterial biofilm layers on coated heat exchanger surfaces.

As expected, nano-silica coating slowed down the adhesion and colonisation of bacteria on the substrata thanks to its strong hydrophobic properties. The pH, dissolved oxygen, total dissolved matter and temperature values of the water in the system during the six-month test period were recorded and are given in Table II. The values in Table II were important to monitor circulating water due to the blowdown regime.

It is known that even with conventional cleaning and disinfection regimens, there is a problem fighting against biofilm formation and development of microbial resistance (12). Based on previous studies conducted in this field (13, 14), it is impossible to eliminate the formation of biofilm layers on surfaces, but biofilm formation can be reduced (9, 15, 16). For this purpose, it is possible to modify surfaces with different coatings. The nano-hydrophobic coating changes the surface properties of the material and supports less biofilm formation (16–18). Hydrophobic coatings limit the wettability of the surface, making it difficult for organic and inorganic matter or microorganisms to adhere; and even if they do, they can easily be detached from the surface by physical factors such as laminar or turbulent water shear stress (19).

The issue of antimicrobial coatings has been extensively studied (20–24). The problem with these products is development of bacterial resistance against the agent (11, 25). Even antibiotic-containing coatings have been reported to promote biofilm formation (26). Silver compounds combined with silica, silane and titanium coatings in particular gave antimicrobial properties but the problem of toxicity in medical devices was mentioned (27). In industrial use, the resistance of microorganisms is at the top of the list as a disadvantage (28). In addition, silver compounds in water systems will reach the aquatic environment and appear as a separate environmental problem.

It is also emphasised that anti-biofilm coatings are very important for preventing the formation of a biofilm layer at an early stage (29, 30). However, studies conducted to date are mostly aimed at solving clinical problems and have been done in vitro with pure cultures (15, 17, 18, 31–33). Using monospecies biofilms is a sterile approach and cannot represent mixed cultures in the natural environment and their interaction with each other. Sol-gel products and superhydrophobic coatings which are more strongly water repellent (31, 34) have also been tried. It was observed that the life of these coatings was not as long as hydrophobic coatings. On the other hand, the high cost of superhydrophobic coatings was a drawback. Contrary to hydrophobic coatings, some hydrophilic coatings were also found to be effective against biofouling. Holberg et al. (8) reported that biocide-free silicone coatings showed promising real-life performance on fresh water-cooled heat exchangers and also performed well in laboratory tests.

Ding et al. (35) tested an environmentally friendly antifouling coating product, butenolide, which was designed for controlled release from biodegradable polyurethane. The anti-fouling effect was shown by in situ tests. The main target was marine biofouling, especially larval settlement on surfaces. Since the adhesion of fouling organisms relies on a microbial biofilm layer, inhibition of primer settlement is crucial. Hu et al. (36) sprayed bacterial-anti-adhesive modified polystyrene microspheres to construct bacterially-anti-adhesive surfaces. It can be used on any surface thanks to the lotus effect. It was reported as robust and durable on surfaces. Similar surface engineering strategies focus on altering the physicochemical properties of the material surface. In general, reduced efficacy of regular disinfectants leads to progress in development of antimicrobial surfaces and coatings (37, 38).

This is the first report of a nano-silica coating on a stainless steel cooling tower heat exchanger. The study showed that the nano-silica coating significantly reduced bacterial fouling on surfaces. There are many similar surfaces with biofouling problems which have contact with water and require a solution. Nano-silica has proven to be effective at reducing the formation of biofilms on surfaces and can be applied as a cost-effective, effortless, non-toxic, readily available material. Due to growing restraints on environmental release of biocidal agents and the growing restrictions on the use of disinfectants in man-made water systems, as well as demand to decrease the cost of system maintenance, different ways to limit biofilms in man-made water systems hold much expectation.