**6. Glass greenhouses**

Glasshouses are the best protected indoor area for growing vegetables in regions where climatic conditions are unfavourable for their growth and development in either open field or plastic-glazed greenhouses. They are the best environment for controlling the climatic conditions and the mineral feed of the crops during their vegetative period. At the same time, also, the warm season crop vegetative period could be prolonged when compared with growing such crops in open field. Furthermore, the modern glass greenhouses are perfect with regard to applying modern technologies and scientific developments in growing crops. Of course, the economic factor is of utmost importance for commercial crop production. So, both short and extensive financial plans have to be made. Glass greenhouses are built mostly in cold climates, but as they provide the environment where the climatic conditions can be completely controlled, they are also a good choice for growing vegetables even in warm climate regions. Modern vegetable crops growing in glass greenhouses are very intensive from the perspective of capital investments, application of the latest production technologies, know-how, labor, energy, substrate, hydroponics. The greatest issue in modern greenhouses is the high cost of energy. There have been, however, some significant shifts toward reducing the cost of heating. The Venlo greenhouse type [8] is probably the most-built type worldwide and the most economical one among the professional vegetable crop growers. The design of the Venlo greenhouse is suitable for all sorts of crops and under various climatic conditions. This type of greenhouses was developed in the Netherlands and was named after the region and the place of Venlo where it was first made (southeast the Netherlands, toward the German border). The whole region has one of the largest concentrations of greenhouse horticulture.

The Venlo greenhouse is the galvanized steel frame structure, as the best and most durable material that can deal with the weight of the entire greenhouse and the roof frame. The roof frame can be made of aluminium or galvanized steel of various profiles. The roof frame also carries the windows/openings that in the Venlo glasshouse can be opened from both sides and make 20–30% of the total glass

### *Advantages of Growing Vegetable Crops in Modern Greenhouses DOI: http://dx.doi.org/10.5772/intechopen.101469*

surface area. As the frame structure consists of a great number of columns of various sizes and dimensions, there is actually much shade inside the greenhouse that affects the crops. For that reason, it is advisable for the entire frame structure to be perforated and painted in white so as to increase the indoor lighting. The crops that are mostly grown in greenhouses are as follows: tomatoes, peppers, eggplants, cucumber, melons and small watermelons (up 1 kg), lettuce, spinach, grapes, raspberries, blackberries, strawberries, blueberries, and, of course, seedlings of vegetables and flowers and various edible berry fruit. Often grown in greenhouses are also broccoli, cauliflower, leek, bananas, ginger. Actually, all horticultural varieties can be grown in greenhouses, but it would be best to check first how cost-effective it could be, that is, to do a feasibility study. If a feasibility study has to be done as a guaranty for the bank loan or a subsidy, then such a study should be detailed and done by a recognized firm or a consultant. Since greenhouses provide the environment for almost complete control of the selected crop production, the grown crops are safe for human consumption. That means they meet the best standards with regard to both safety and quality. Furthermore, there is a minimal environmental pollution. Other benefits are that the early crops come to market sooner and also, high yields are achieved. For instance, in hydroponic tomato production (on the rockwool growing substrate), the yield can be even up to 100 kg/m2 , or 1000 t/ha per annum. The fact is that the yield (or the production rate) in the modern glass greenhouse can be 10 times as much as in the open field. High yields are achieved through intensive growing methods and by prolonging the production season. Besides the vegetable quality and the environment pollution reduction, the yield should be high also because of a return of the investment and payments of additional costs. The majority of the glass greenhouses manufacturers put on offer glasshouses and the horticultural production technology, which may provide optimal production under the given market conditions, so that the additional costs would pay out through the earnings from the additional production. Consumers in most of the developed countries expect to find good-quality vegetable produce grown in glass greenhouses on their respective markets. In most cases, it is the improved quality of the vegetable produce that gives a motive for starting the glasshouse vegetable production. For different types of produce and different markets, there are also different parameters for food quality. The quality parameters for the fruit and vegetables required on the market may include the following: taste, flavor, aroma, size, color, stage of ripeness, firmness or softness—texture, shelf life, the absence of any spots or defects, minimum or no waste, no signs of pests or disease, with or without leaves, and many more. One of the most important standards in the primary agricultural production, which is also a quality guarantee of both the primary agricultural production and the produce itself, is the GLOBALG.A. P. standard/certificate. According to the construction and engineering characteristics, appearance and purpose, there are several types of glass greenhouses that could be single-span, multi-span, lean-to, gabled.


**2. Experimental**

#### **2.1. Analytical equipments**

**NMR measurements:** The NMR spectra were recorded on a Bruker 800 MHz NMR spectrometer. The HTPB samples (10 % (w/v) for the 1H NMR and 30 % (w/v) for the 13C{1H} NMR analysis) were recorded in CDCl3 at room temperature. The 1H NMR acquisition parameters were: spectral width = 16 ppm, acquisition time =2 s, relaxation delay = 1 s, pulse width = 90 °, and number of scans =1000. Similarly, 13C{1H} NMR spectra were recorded using spectral width = 220 ppm, acquisition time = 2 s, relaxation delay = 10 s, pulse width = 90 °, and number of scans = 300. HMQC spectra of HTPB samples (30 % (w/v) in CDCl3 at room temperature) were recorded on a Bruker 500 MHz with a 5 mm inverse Z-gradient probe. Spectral widths: F2 (1H)=8000 Hz, F1(13C)=27500 Hz. Time domains : (1H)=1024 and (13C) =515, acquisition time (1H) =0.23s, delay (1H) =2s. In processing, the FID was zero-filled to 32 K data points and the resulting 32 K time domain was Fourier transformed. Additionally, Gaussian apodization was also applied in both 1H and 13C domains. **Viscosity measurements:** A Brookfield HADV-II+ programmable rotational type viscometer equipped with a motorized stand (helipath stand) was used to perform isothermal viscosity measurement at different temperatures. The temperature was controlled by a thermostatic temperature control bath (Brookfield). The temperature control accuracy was ± 0.5 °C. Polymer samples were sheared at different shear rate (rpm). The spindle used for binder slurry was AB-4, whereas for propellant slurry, T-E was used. For each experiment, data was collected after one complete revolution. For each successive revolution, total 10 readings, each at an interval of one second were recorded at the set rotational speed by using Wingather Software. The average viscosity value was calculated and used for data analysis and modelling. **DSC experiments**: Mettler FP-900 thermal analysis system equipped with FP-85 standard cell and FP90 central processor was used for DSC measurement. The heat flow and temperature calibration of DSC were carried out using pure indium metal as per the procedure recommended by the manufacturer (ΔH = 26.7 J/g, MP = 158.9 °C). All experiments were carried out in an air atmosphere at different heating rates, ranging from 2-15 °C/min. Aluminum sample pans (40 μL) were used for the DSC experiments. Almost constant sample mass of 5 ± 1 mg was used. **Tensile properties**: The tensile stress-strain measurements were performed at room temperature, using samples previously kept at 23±2 °C and relative humidity of 50± 5% for 48 hrs, according to ASTM D 618. Elastomeric test specimens were punched from the cured slab using a die prepared in accordance with ASTM D 412-68. Tensile testing was performed in an Instron Universal Testing Machine (UTM) using dumb-bell shaped specimens of cured PUs as well as propellants. A 100 kg load was applied at a crosshead speed of 50 mm/min. Hardness was measured by a Shore A Durometer as per the standard procedure.

#### **2.2. Synthesis of PUs**

230 Polyurethane

gas producing fuel. It is physically and chemically compatible with the conventional oxidizers and other ingredients at normal storage conditions. As it contains mostly carbon and hydrogen, during combustion, it is decomposed to give large volume of stable molecules like carbon monoxide, carbon dioxide, and water vapours increasing the specific impulse of the rocket motor. Additionally, PU obtained from HTPB offers many advantages over conventional polyether and polyester based urethane systems. Properties exhibited by polyurethanes(PUs) prepared from HTPB include (a) excellent hydrolytic stability, (b) low water absorption, (c) excellent low temperature flexibility, (d) high compatibility with fillers and extenders, and (e) formulation flexibility (Sadeghi et al., 2006). Of late, there is a growing demand of segmented HTPB PUs as these PUs have a unique combination of toughness, durability and flexibility, biocompatibility and biostability that makes them suitable materials for use in a diverse range of biomedical applications (Poussard et al., 2004). HTPB based pervaporation membrane technology is the current wave of innovation.

The polymer chemo-rheology and thermo-oxidative degradation are the two relevant key areas of interest, where in-depth knowledge is essential for the effective performance assessment of PU propellants. Chemo-rhelogy is related with the PU processiblity, whereas thermo-oxidation is related to the stability and combustion performance. The information of change on viscosity during the curing process is critical in modelling the PU flow behaviour. Though extensive works have been carried out on this topic in the last decade (Muthiah et al., 1992, Lakshmi & Athithan, 1999, Singh et al., 2002 & Mahanta et al., 2007), it is still a fascinating research area at present. The thermal decomposition of HTPB has been studied exclusively in inert atmosphere (Panicker & Ninan, 1997). However, thermo-oxidative degradation in air, which is the most relevant in view of combustion of the polymer, has not been studied thoroughly. Additionally, the HTPB prepolymer being the decisive component in HTPB PUs, characterization of this polymer (HTPB) at macro as well as micro levels has been of paramount importance in last decade. Two types of HTPB prepolymer are currently in use: i.e., free radical HTPB and anionic HTPB. The free radical grade HTPB is widely used in composite PU propellants because of its low cost and wide availability. The current chapter is focused on prepolymer characterization, rheology, and oxidative degradation of

**NMR measurements:** The NMR spectra were recorded on a Bruker 800 MHz NMR spectrometer. The HTPB samples (10 % (w/v) for the 1H NMR and 30 % (w/v) for the 13C{1H} NMR analysis) were recorded in CDCl3 at room temperature. The 1H NMR acquisition parameters were: spectral width = 16 ppm, acquisition time =2 s, relaxation delay = 1 s, pulse width = 90 °, and number of scans =1000. Similarly, 13C{1H} NMR spectra were recorded using spectral width = 220 ppm, acquisition time = 2 s, relaxation delay = 10 s, pulse width =

It has introduced a new dimension to PU elastomeric technology.

the polymer and the PU systems.

**2.1. Analytical equipments**

**2. Experimental**

#### *2.2.1. Unfilled PUs: PU-I and PU-II*

The basic compositions that were studied in the present work are shown in Table 1. The binder system studied consists of PU formed by reacting mixture of alcohols [(HTPB, OH value = 42 mg KOH/g), Butanediol (BDO, OH value = 1232 mg KOH/g) as chain extender and trimethylol propane (TMP, OH value = 1227 mg KOH/g) as cross linking agent] with toluene diisocyanate (TDI, purity > 99 percent and a mixture of 2, 4 and 2, 6-isomers in 80:20 ratio). The BDO and TMP were mixed in a fixed ratio (2:1) and dried under vacuum to reduce the moisture content (< than 0.25%) of the mixture. The mixture thus obtained had the hydroxyl value of 1242 mg of KOH/g.

HTPB-Polyurethane: A Versatile Fuel Binder for Composite Solid Propellant 233

), and thus, ܯഥ of the

NMR spectra. Although 13C NMR spectroscopy is good in terms of a wider range of chemical shifts and thus offering less possibility of overlapping peaks, problems associated with questionable assignments occasionally arise from steric-sensitive environments in the carbon skeleton. Additionally, the Nuclear Overhauser Enhancement (NOE) of different types of carbon is usually not equal and the wide spin-lattice relaxation time (t1) range makes quantitative measurements of carbon signals difficult. A combination of NMR techniques such as 1H, 13C{1H}, 13C{1H}-DEPT (Distortionless Enhancement By Polarization Transfer) and 1H/13C-HMQC (Hetero-nuclear Multiple Quantum Coherence) permit assignments of all 1H and 13C resonance peaks. To our knowledge, hitherto the actual physical characteristics of the HTPB are not precisely known, particularly its absolute number-average molecular weight (ܯഥ). In the current work, we have examined the 1H and 13C NMR spectra of HTPB in order to precisely

polymer. A typical 13C{1H} NMR spectrum (200MHz, CDCl3) of free radical HTPB prepolymer is shown in Fig.1. For convenience, resonances in the spectrum can be divided in to three distinct regions, *i.e.* (a) an olefinic region: δ 113-144, (b) a carbon bearing hydroxyl end group region: δ 56 – 65, and (c) an aliphatic region: δ 24-44. However, due to complex nature of the prepolymer, a complete assignment of all signals was not possible. The, methine and methylene carbons were distinguished by using the DEPT technique.

The 13C{1H}-DEPT spectrum of the polymer, recorded in CDCl3, is depicted in Fig.2.

**Figure 1.** 13C{1H} NMR (CDCl3, 200 MHz) spectrum of free radical HTPB prepolymer.

determine its number-average degree of polymerization (ܦܲതതതത

\*Hard segment content= ሼሾݓ்ூ ݓைା்ெሿΤ ሽ ݓ௧௧ ൈ ͳͲͲǡ ݓ ൌ ݓ݄݁݅݃ݐǤ

**Table 1.** The basic composition of the one step PUs.

The PUs were prepared in bulk by one step procedure. Mixing was carried out in a pilot mixer with facility for circulation of hot/cold water around the mixer jacket. The HTPB and BDO-TMP mixture were taken in the pilot mixer and stirred for 10 minutes. The calculated amount of TDI was added to the mixer, and the contents were stirred for 20 minutes at 40 ± 1 °C. The binder slurry was cast in to a Teflon coated mould and cured at 60 °C for 3 days.

#### *2.2.2. Filled PUs (propellant): PU-IIp*

The basic propellant composition that uses 68% ammonium perchlorate (AP) and 18% aluminum (Al) powder was taken up for study. AP (with purity > 99%) was used in bimodal distribution (3:1) having average particle size 280 m and 49 m, respectively. Particle size of AP and Al powder (mean diameter = 33.51 μm) were measured by a CILAS Particle Size Analyzer-1180 model. Dioctyl adipate (saponification value = 300 mg KOH/g) was used as a plasticizer. The mixing was carried out in two phases. In the first phase, all the ingredients, except the curing agent, were premixed thoroughly for about 3 h at 38 2 °C. Hot water was circulated through the jacket of the mixer bowl to keep a constant temperature throughout the mixing cycle. A homogeneous test of the slurry was carried out after completion of the premix to confirm the uniform dispersion of AP and Al powder. In the second phase of mixing, a calculated amount of curing agent, *i.e.* toluene diisocyanate (TDI) was added to the premixed slurry, and further mixed for 40 minutes at 40 1 °C. The propellant slurry was cast in to the Teflon coated mould and cured at 60 °C for 5 days.


2.4.1. Water Footprint

The total water footprint *Wf* was calculated as the sum of the water consumption within systems (direct water use, *Wd*) and the water footprint of energy consumed (indirect water use, *Wind*) according to Equation (1):

$$\mathcal{W}\_f = \mathcal{W}\_d + \mathcal{W}\_{ind} = k \cdot \mathcal{W}\_{\text{cv}} + \mathcal{C}\_{\mathcal{W}, \text{cl}} E\_{\text{cl}}.\tag{1}$$

In the present evaluation, we did not account for water pollution impacts (so-called grey water), but only for blue water footprint, which measures the consumptive use of surface and ground water.

Direct water consumption only occurs in CT configurations due to evaporation loss, drift and makeup-water requirements. Evaporated quantities were calculated with TRNSYS [33] using *type51b*. Additional water losses due to bleed o ff and drift were quantified as in [9] using a multiplicative coe fficient *k* on the evaporated water *Wev*, taking *k* = 2 as a reasonable estimate [47]. The footprint calculation approach and the data sources reported in [9] were used to derive the indirect water consumption rate *CW,el* for each reference city based on the national electricity production mix reported in Table 2, elaborated from the WorldBank database [48].

The total electricity demand *Eel* was determined as the sum of the energy required for each component simulated in TRNSYS. The chiller performance was considered in the energy consumption calculation by using the corresponding TRNSYS types. In-built TRNSYS performance data files were used to evaluate the EER and consequently the energy consumption, which is related to the cooling water temperature returning from the heat rejection device (DC or CT) as well as the temperature of chilled outlet water. For the absorption chiller, the inlet hot water temperature was also introduced as parameter to determine the EER. As a result, the yearly average EER values obtained from simulations in the climate regions of concern ranged between 0.52 and 0.55 for absorption cooling systems, and between 5.24 and 9.61 for compression cooling systems.

#### 2.4.2. Carbon Footprint and Primary Energy Demand Calculation

Carbon footprint has been defined as "the quantity of GHGs expressed in terms of CO2 equivalent mass emitted into the atmosphere by an individual, organization, process, product or event from within a specified boundary" [49].

As in the case of water footprint, di fferences in the carbon footprint of the configurations examined are exclusively bound to electricity consumption, since none of the air conditioning alternatives examined implies any direct fuel consumption. Carbon footprint was thus calculated according to Equation (2).

$$\text{CO2}\_f = \text{CO2}\_{\text{ind}} = \text{C}\_{\text{CO2}, \text{el}} \text{E}\_{\text{el}}.\tag{2}$$

On the other hand, based on the data sources used in this study (see [9]), carbon footprint coe fficients for electricity consumption *CCO2,el* were estimated with a life cycle approach (i.e., all *CO2eq* emissions consumption from extraction to plant construction were considered).

In a similar manner to [50], in this study it was assumed that the changes in direct carbon equivalent emissions from refrigerant leaks induced by switching from vapour compression units to absorption cooling systems were negligible compared to the emissions of greenhouse gases embodied in purchased electricity.

The primary energy consumption associated with purchased electricity was calculated according to Equation (3):

$$PED = C\_{PED,el} E\_{el} \,. \tag{3}$$

Site-to-source energy conversion factors *CPED*,*el* reported in Table 2 were obtained with the methodology and data sources discussed in [9,51] based on national energy mix data reported in Table 2.

#### *2.5. Basis for Economic Assessment*

The life cycle cost was used as a basis for economic assessment. According to the scheme proposed in [46], life cycle costs of buildings under an energy efficiency assessment framework may include initial, operation, repair, spare, downtime, loss, maintenance (corrective, preventive and predictive) and disposal costs. Based on available data, only initial (capital) costs of installations and operational costs of electricity and of water were considered.

For each configuration, the life cycle cost (LCC) for an interest rate of 10% and a lifetime of 10 years was calculated using the standard formula:

$$LCC = C\_{op} \left(\frac{q^n - 1}{q^n \cdot i}\right) + C\_{cap} \tag{4}$$

where:

$$- \qquad q = 1 + i.$$

A payback analysis was also later introduced to compare the economic feasibility between ABS and FC configurations. The payback period (PB) was evaluated using the following formula:

$$PB = \frac{\mathbb{C}\_{p,ABS} - \mathbb{C}\_{p,FC}}{\mathbb{C}\_{o,FC} - \mathbb{C}\_{o,ABS}},$$

where:

One should bear in mind that the simple payback time calculated according to Equation (5) allows a direct comparison between ABS and FC alternatives, but does not account for interest rates, which may lead to slightly different results with respect to LCC-based comparisons.

Capital cost estimates for absorption cooling systems, mechanical vapour compression cooling systems, dry coolers and cooling towers were obtained from the cost functions reported in [9], summarized in Table 3.

**Table 3.** Equipment cost functions used in this work.

The operational expenses for different configurations were mainly determined by electricity consumption and—for CT configurations—by water consumption. It was not possible to retrieve electricity and industrial water prices all over the world. To obtain an approximate estimate for reasonable ranges, Western European values were derived from [9] as guide values, and are reported in Table 4.

**Table 4.** Prices of electricity and water used in this work.

## **3. Results and Discussion**

#### *3.1. Average Total Cabin Cooling Load*

Figure 5 shows the total cooling load, averaged over operating hours of one year, for the reference electric cabin in the climate zones of interest. On a yearly basis, heat transfer through the cabin envelope led to lower cooling loads, particularly in colder climates. However, the difference between the average cooling load in climate 7 (cold) and in climate 1A (hot) Was less than 3% of the total cooling load.

**Figure 5.** Simulated average cabin load over 16 climate zones.


**4.3 Tumours with clear cell and vacuolated cell pattern**

The differential diagnosis of tumours of intraoral SGs comprising of clear cells and vacuolated cell pattern include – Epithelial–Myoepithelial carcinoma (EMC), Myoepithelial tumours such as myoepithelioma (ME) and myoepithelial carcinoma (MC), clear cell carcinoma (CCC), mucoepidermoid carcinoma (MEC), acinic cell carcinoma (AciCC), secretory carcinoma (SC).

#### **Figure 9.**

*9a: Showing cells arranged in a predominant papillary architecture with fine fibrovascular core in a case of acinic cell carcinoma. The cells have fine vacuolated cytoplasm (May-Grunwald Giemsa stain x 4), 9b: Corresponding histology showing branching papillae (arrow) with fibrovascular core (Haematoxylin & Eosin x 4).*

*Cytopathology of Intraoral Salivary Gland Tumours and Tumour-Like Lesions… DOI: http://dx.doi.org/10.5772/intechopen.98872*

## *4.3.1 Epithelial–myoepithelial carcinoma (EMC)*

It is a an unusual tumour of major salivary gland predominantly occurring in parotid (60–80%) but can also be seen in minor SGS [20]. Palate is the most common site of occurrence and clinical presentation can be a ulcerative nodular lesion.

**Key cytological features –** The smears are cellular comprising of biphasic population of epithelial and myoepithelial cells. The myoepithelial cells are seen in loosly cohesive sheets, clusters and spheres with fragile, pale to clear delicate glycogen rich cytoplasm that disperses in background resulting in many naked or stripped nuclei. The nucleus of myoepithelial cells is round to oval with open chromatin and small distinct nucleoli (**Figure 10a**). Epithelial cells are seen usually in tight cohesive clusters. Sometimes mild nuclear atypia can be encountered. Hyaline stromal globules and basement membrane-like material can also be seen. The biphasic pattern of EMC can be demonstrated by using IHC on cell blocks with low molecular weight keratin and epithelial membrane antigen (EMA) for highlighting the duct cells and with smooth muscle actin (SMA), calponin, p63 and S-100 for highlighting the myoepithelial cells component [37]. Points of differentiation of EMC with other tumours with clear and vacuolated cells are discussed with each individual tumour**.**

#### *4.3.2 Myoepithelioma (ME)*

**Myoepithelioma (ME)** is a benign tumour that can occur in minor SGs with palate being the most common site. The myoepithelial cells in ME have pale to clear cytoplasm (**Figure 5b**), resembling myoepithelial cells of EMC. However, the myoepithelial cells of EMC are larger than that of ME due to presence of abundant glycogen [21]. Also, ME lacks the biphasic pattern of EMC (**Figure 10a**). ME may undergo transformation into myoepithelial carcinoma (MC). However, atypical cytological features such as nuclear pleomorphism, coarse chromatin with prominent nucleoli with background necrosis and mitotic activity seen in MC can differentiate between the two [21]. Differentiation of ME from cellular PA with predominant myoepithelial component is already discussed at 4.1.1e.

#### *4.3.3 Clear cell carcinoma (CCC)*

**Clear cell carcinoma (CCC)** of the SG is a rare low-grade malignancy that occurs primarily in intraoral minor salivary glands predominantly in the palate [38].

#### **Figure 10.**

*10a: Smear showing admixture of dual population of cells comprising of epithelial and myoepithelial cells in a case of epithelial myoepithelial carcinoma (Haematoxylin & Eosin x 40), 10b:s Smear showing a cluster of cells with multilayering of round to polygonal oncocytic cells (arrow) with round nucleus and abundant dense granular cytoplasm in a case of an oncocytoma (May-Grunwald Giemsa stain x 40)*

Epithelial cells of CCC are seen in clusters and sheets with prominent cell borders, uniform round to ovoid nuclei, granular chromatin and abundant glycogen rich clear cytoplasm**.** The cells of **CCC** may resemble myoepithelial cells of EMC. Unlike EMC, CCC lack evidence of either ductal or myoepithelial differentiation.

Clear cells can be encountered in MEC of minor SGs. However, identification of other accompanying cells (intermediate and epidermoid cells) in MEC gives clues to the diagnosis [34]. Mucin in cells of MEC can also be demonstrated by mucicarmine stain on cell blocks.


*Article* **Exploitation of Enzymes for the Production of Biofuels: Electrochemical Determination of Kinetic Parameters of LPMOs**

**Dimitrios Zouraris 1 , Anthi Karnaouri 2 , Raphaela Xydou 1 , Evangelos Topakas 2 and Antonis Karantonis 1,\***

**Abstract:** Lytic polysaccharide monooxygenases (LPMOs) consist of a class of enzymes that boost the release of oxidised products from plant biomass, in an approach that is more eco-friendly than the traditional ones, employing harsh chemicals. Since LPMOs are redox enzymes, they could possibly be exploited by immobilisation on electrode surfaces. Such an approach requires knowledge of kinetic and thermodynamic information for the interaction of the enzyme with the electrode surface. In this work, a novel methodology is applied for the determination of such parameters for an LPMO from the filamentous fungus *Thermothelomyces thermophila*, *Mt*LPMO9H.

**Keywords:** LPMO; Fourier Transform ac Voltammetry (FTacV); cyclic voltammetry; Direct Electron Transfer (DET)

**Citation:** Zouraris, D.; Karnaouri, A.; Xydou, R.; Topakas, E.; Karantonis, A. Exploitation of Enzymes for the Production of Biofuels: Electrochemical Determination of Kinetic Parameters of LPMOs. *Appl. Sci.* **2021**, *11*, 4715. https://doi.org/10.3390/app11114715

Received: 23 April 2021 Accepted: 18 May 2021 Published: 21 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

The exploitation of lignocellulosic biomass, such as wood and agricultural residues, towards the production of fermentable sugars for the production of second-generation biofuels in an environmentally friendly way, requires of enzymatic degradation. This step has low energy requirements, selectivity, mild processing environment and no need of harsh chemicals, in contrast to classical chemical methods, and it is catalyzed by a combination of hydrolytic and oxidative enzymes [1,2]. Regarding the oxidation reactions, performing these steps in an electrochemical reactor, i.e., electroenzymatic synthesis of sugars, is a promising approach for the production of such chemicals. In principle, when electrolysis is combined with enzyme reactions, a redox enzyme is maintained in its active state through an electrode process. This can be achieved by oxidation of or a reduction in a cofactor or a mediator on the electrode surface, or by direct electron transfer to the enzyme. Therefore, applying enzyme immobilization onto the electrode and combining bioelectrocatalysis with traditional enzymatic hydrolysis offer perspectives for developing novel processes for the production of biofuels and other value-added materials from lignocellulose.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that have the capacity to cleave polysaccharides via an oxidative mechanism [3]. They are classified as Auxiliary Activities (AAs) in the Carbohydrate Active enZyme database (CAZy). More specifically, they are divided into seven families (AA9–11 and AA13–16) based on their sequence similarities, with the majority of those that are studied thoroughly belonging to the AA9 and AA10 families [4]. LPMOs act synergistically with other biomassdegrading enzymes and significantly boost the hydrolytic performance of cellulase and hemicellulase cocktails, primarily by improving the accessibility of the enzymes to the substrate [5,6].

Until recently, the main theory regarding the mechanism of action of LPMOs involved the introduction of a single oxygen atom from molecular oxygen into the substrate, while

taking electrons from an external electron donor for their action [7]. More recently, Bissaro et al., while examining an LPMO belonging to the AA10 family, supported the idea that the chemically relevant co-substrate of LPMO catalyzed oxidation is H2O2 [8]. Regardless of the mechanism of the oxidative cleavage of the polysaccharidic substrate, in both cases, the reaction is initiated when the active center of the enzyme takes an electron from an external electron donor. The nature of the donors used in laboratory experimental setups include ascorbic acid, cysteine and reduced glutathione, but also a wide range of plant-derived phenols [9,10], lignin compounds [11] and photocatalytic and enzymatic systems [12]. The reported mechanisms for the initial reduction in LPMOs do not implicate any chemical steps other than a one-step, one-electron reaction.

One interesting point regarding these enzymes, which could possibly be an advantage for their electrochemical exploitation, is that their copper-containing active center is not "buried" inside their structure but can be found on a solvent-exposed, flat surface [13]. This could facilitate their use as electrode coatings, by directly providing them with electrons from an electrode surface, while immobilized on the electrode surface. Other than their study, this characteristic could lead to the exploitation of the bioelectrocatalytic action of LPMOs towards the production of oxidised products (oligosaccharides and glucans) from biomass, in concert with hydrolytic enzymes, in the absence of any external reducing agent. This means that by providing electrons directly from an electrode surface to an immobilised LPMO, the enzyme could be utilised in bioconversions through bioelectrocatalysis focusing on product life cycles. The developed bioconversions will be carried out in predominantly aqueous media using enzymes, and therefore be characterised by a limited use of toxic reagents or solvents, requiring only an ambient temperature, in the absence of any reducing agent [14].

In order to test if the above task is feasible, a parameter that should be mentioned is the standard electron transfer rate constant (*k* 0 ) between the immobilized electrode and the enzyme. This electron transfer rate should be rather fast, so that the electrocatalytic current measured represents the rate of substrate turnover at the enzyme active site [15]. Among the electroanalytical methods, a technique that is very promising for the study of immobilised LPMOs is that of large amplitude fast Fourier transform alternating current voltammetry (FTacV), with which one can study both free and immobilised species, eliminating capacitance currents, a problem common in immobilised-on-electrode species [16–19]. In the past, this technique has been employed for the determination of the formal potential of immobilised LPMOs [20], as well as for the study of the direct electron transfer of multi-copper laccase like oxidases [6] and the interaction with epinephrine as a substrate [21]. Recently, a methodology for the extraction of the transfer coefficient *a* and the *k* 0 for immobilised species has been proposed, but has yet to be applied experimentally [17]. In this work, this particular methology shall be employed for the extraction of the *k* 0 an LPMO from the filamentous fungus *Thermothelomyces thermophila*, *Mt*LPMO9H, and the calulated value of *k* 0 shall be verified by numerical simulation. To our knowledge, *k* 0 values for the interaction of an immobilized LPMO with an electrode surface is reported for the first time.

#### **2. Materials and Methods**

Voltammetric experiments were performed in a single-compartment three-electrode cell consisting of a working electrode, a Pt wire as a counter electrode, and a Ag|AgCl, KCl sat. reference electrode (+0.197 V vs. NHE). The aqueous solutions of about 5 mL consisted of 0.5 M acetate buffer (pH 6) as a supporting electrolyte.

The working electrode consisted of a cobalt functionalised multi-walled carbon nanotubes modified glassy carbon (GC) electrode, of 3 mm diameter. The immobilisation matrix was prepared as follows: A total of 1 g of commercial functionalised multi-wall carbon nanotubes (MWCNTs) (Hongwu Material, item No: C933-MC, Guangzhou, China) were added in a 150 mL solution with 20 % *v*/*v*. The suspension was stirred for 20 min at 120 ◦C for further functionalization, until the liquid phase was evaporated. The solution

was left to dry overnight at 90 ◦C until dry powder was collected. The powder was washed, then centrifuged 5 times with deionised water until the supernatant had a pH value of '7.0. This step ensured the removal of acetic acid excess. The obtained functionalised commercial MWCNTs (f-MWCNT) were left to dry at 90 ◦C. Subsequently, the f-MWCNTs were mixed with 150 mL deionised water and 0.4227 g of (CH3COO)2Co·4H2O (MERCK) leading to a loading 10 atom% Co on f-MWCNTs, which was then subjected to sonication at a low frequency (20 kHz) using a Hielscher (UIP500hd) sonicator (probe tip diameter of 22 mm) for 1 h. The solution was further left to dry and centrifuged (as described above) for the collection of the final product. The cobalt-functionalised MWCNTs (Co-f-MWCNTs) were stored in a desiccator at room temperature.

For the immobilisation of the enzyme on the electrode, the following procedure was followed: A total of 0.005 g of Co-f-MWCNTs were suspended in 500 µL of NafionTM perfluorinated resin solution (concentration 5 wt% in lower aliphatic alcohols and water, Aldrich). Then, 8 µL of the enzyme were mixed with 2 µL of Co-f-MWCNTs suspension. Finally, 2 µL of the resulting mix were left to dry on the GC electrode surface.

*Mt*LPMO9H was produced and purified to homogeneity as previously described [22]. After the purification the enzyme was divided in aliquots of 500 µL, and stored at −15 ◦C until use. Each aliquot was stored for a maximum time of 2 days at 4 ◦C after its first use. The concentration of the purified enzyme was about 5.5 mg/mL, identified by the method introduced by Lowry et al. [23].

#### **3. Results**

#### *3.1. Cyclic Voltammetry*

As a first step in the examination of the interaction of the immobilised *Mt*LPMO9H with the electrode surface via direct electron transfer, cyclic voltammetry was performed. *Mt*LPMO9H was immobilised on a Co-f-MWCNTs modified GC electrode and the potential was scanned between 0.6 and −0.1 V at a scan rate of 0.1 V s−1 . The voltammogram is presented in Figure 1 (black curve). Two rather faint peaks appear, one during the cathodic scan appearing at 0.252 V and one during the anodic scan at 0.281 V. Capacitance currents are evident in this voltammogram. The peak separation is estimated at 0.029 V, indicating a quasi-reversible reaction. The cyclic voltammetry of the Co-f-MWCNTs modified GC electrode in the absence of the enzyme is also present in Figure 1 (red curve). In this case, no redox peaks are detected, indicating that the redox peaks can indeed be attributed to the redox reaction of the enzyme and not any interference of the matrix.

In order to extract the kinetic constant *k* 0 and the transfer coefficient *a* from a cyclic voltammogram, assuming Butler–Volmer kinetics [24], one would take advantage of the correlation of the anodic and cathodic peak shift by varying the scan rate. However, these equations do not take the capacitance current and how it might affect the dependence of the peak shift into account. Moreover, at the higher scan rates which would be necessary in such a case, the capacitance would be more dominant, thus rendering the analysis even more difficult. One option would be to subtract the capacitance current from the total current in order to have only the Faradaic component and base the analysis on that alone. However, uncertainty rises for the precision of this approach in cases of non-ideal capacitance. The same uncertainty arises in case one would attempt to extract other magnitudes from the cyclic voltammogram, such as the surface coverage of the enzyme immobilised on the electrode surface.

**Figure 1.** Experimental results: Cyclic voltammetry of *Mt*LPMO9H immobilised on a Co-f-MWCNTs modified GC electrode (black) and Co-f-MWCNTs modified GC electrode without enzyme (red) at 25 ◦C. Experimental conditions: acetate buffer 500 mM (pH 6) and *v* = 0.1 V s−1 .

Nonetheless, an interesting first approach to making an estimation of the value of the *k* 0 would be to solve the equations for the immobilised species numerically in the case of cyclic voltammetry for different standard electron transfer kinetic constants. In order to do so, the problem has to be formulated, by considering an electrochemical reaction having formal electrode potential *E* 00 , where both oxidised (EOx) and reduced (ERed) form of the enzyme are immobilised on the electrode surface

$$\mathbf{E\_{Ox}} + \mathbf{e} \xrightleftharpoons[k\_{\rm a}]{k\_{\rm c}} \mathbf{E\_{Red}} \tag{1}$$

where *k*c the cathodic kinetic constant and *k*a the anodic one. The units for both kinetic constants are 1/s. Let us denote as Γ 0 the initial surface concentration of EOx species and assume a Butler–Volmer law for the rate constants

$$k\_{\mathbb{C}} = k^0 e^{-\frac{aF}{RT}(E - E^0)} \tag{2}$$

$$k\_{\mathbf{a}} = k^0 e^{\frac{(1-a)F}{RT}(E - E^{0\prime})} \tag{3}$$

Thus, the current density expression of the Faradaic current during the redox reaction of the immobilised species can be given from Equation (4) as a funtion of the surface coverage of the Ox species *θ* = ΓOx Γ 0 , with ΓOx the surface coverage of the EOx species

$$\dot{\mathbf{r}}\_{\rm F} = \Gamma^{0} F k^{0} [(1 - \theta) e^{\frac{(1 - a)F}{RT}(E - E^{0\prime})} - \theta e^{-\frac{aF}{RT}(E - E^{0\prime})}] \tag{4}$$

where *F* is the Faraday constant and the coverage is found by solving the following differential equation,

$$\frac{d\theta}{dt} = \frac{i\_{\text{F}}}{F} \tag{5}$$

Assuming a cyclic voltammetry experiment where the potential starts from an initial value *E*I and is scanned linearly towards more cathodic potentials against time at a certain scan rate *v* , and the scan is reversed at a time *t*R, the applied potential can be expressed as in Equation (6)

$$E = E\_\mathbf{I} - vt\_\mathbf{R} + v|t - t\_\mathbf{R}|\tag{6}$$

In order to also include capacitance in the problem, we shall assume a contribution to the current, which can be expressed as follows

$$i\_{\mathbb{C}} = \mathbb{C}\_{\text{dl}} \frac{\text{d}E}{\text{d}t} \tag{7}$$

where *C*dl is the specific capacitance of the double layer. The total current density, *i*, shall be expressed as a sum of the Faradaic and the capacitance contributions,

$$i(t) = i\_{\rm F}(t) + \mathcal{C}\_{\rm dl} \left( v \frac{t - t\_{\rm R}}{|t - t\_{\rm R}|} \right) \tag{8}$$

By solving the problem numerically, the cyclic voltammograms are presented in Figure 2, for different *k* 0 values. The other parameteres used for this example are Γ 0 = 5.1 × 10−6 mol m−2 , *C*dl = 1 F m−2 , *E* 00 = 0 V, *v* = 0.1 V s−1 . For the different *k* 0 values, the peak separation values are presented in Table 1. From these values, one would expect that based on the peak separation of the experimental results at 0.1 V s−1 at 0.029 V, a rough estimation of the *k* 0 value should be around 5 s−1 .

**Figure 2.** Computational results: Cyclic voltammetry for different *k* 0 values. Parameter values: Γ 0 = 5.1 × 10−6 mol m−2 , *C*dl = 1 F m−2 , *E* 00 = 0 V, *v* = 0.1 V s−1 and *k* 0 = 100 (black), 20 (red), 10 (blue), 5 (green), 2 (orange) and 1 s−1 (orange).

#### *3.2. FT Alternating Current Voltammetry*

Before attempting to extract the *k* 0 , FTacV was performed on a Co-f-MWCNTs modified GC electrode with and without *Mt*LPMO9H. The cathodic 5th harmonic in acetate buffer 500 mM, pH 6 for *A*0 = 0.2 V and *f* = 5 is presented in Figure 3. In the case of the immobilised enzyme (black curve) a set of intense peaks around the principle peak at 0.25 V. On the other hand, in the absence of the *Mt*LPMO9H, a faint peak of small amplitude appears at 0.2 V (red curve), leading to the conclusion that the high amplitude harmonic is indeed due to the enzyme and not the immobilization matrix.

Then, FTacV was performed so as to apply the methodology introduced [17] for the extraction of the *k* 0 . At this point, the methodology is summarized:

**Figure 3.** Experimental results: 5th harmonics of *Mt*LPMO9Himmobilised on a Co-f-MWCNTs modified GC electrode (black) and Co-f-MWCNTs modified GC electrode without the enzyme (red) at 25 ◦C. Experimental conditions: acetate buffer 500 mM, pH 6, *A*0 = 0.20 V and *f* = 5.

Therefore, as a first step, FTacV was performed for two amplitudes, 0.2 and 0.25 V, at frequencies of 5, 3, 1, 0.5, 0.2 and 0.1 Hz. The scan rate was adjusted at each case accordingly in order to fullfill the slow scan rate approximation and render the experiment independent of this factor [16,19]. The cathodic scan of the 4th and the 5th harmonics are presented in Figures 4 and 5, respectively, for *A*0 = 0.2 and 0.25 V. Looking at both the amplitudes employed, there is no intense deformation in the harmonics that would suggest a transfer coefficient that would deviate beyond the range of 0.4–0.6; thus, regarding the dominant peak [17], no significant errors would be inserted in the preliminary analysis in case an *a* = 0.5 is assumed. Moreover, both 4th and 5th harmonic seem free of capacitance contribution.

As a second step, the trend of the main observable, i.e., the peak height of the principle peaks, is to be observed. In both cases, typical 4th and 5th harmonics for quasireversible reactions of immobilised species are presented for the higher applied frequencies (Figures 4a,c and 5a,c). Apparently, as the frequency is decreased, the amplitude of the harmonics decreases, as predicted theoretically. The trend of the normalized peak height of the principle peaks, *I*p,*h*/ *f* , on frequency is presented in Figures 4b,d and 5b,d. By observing these figures, it is evident that a saturation in the peak height of the dominant peak is observed for a frequency of 0.1 Hz. This indicates that reversibility is reached by that point, and the height of the dominant peak of the reversible harmonic, *I* (*rev*) p,*h* has been determined.

As a next step, a quasi-reversible harmonic is chosen and the ratio *I*p,*h*/*I* (rev) p,*h* is calculated, where *I*p,*h* is the normalised peak current for a quasi-reversible harmonic and *I* (rev) p,*h* the normalised peak current for the reversible harmonic at a certain *A*0 and *a*. For all the combinations of amplitudes and harmonics, the chosen frequency for the quasi-reversible harmonic is that of 5 Hz, while for the peak of the reversible harmonic, it is 0.1 Hz. Let us now use the example of *I*p,4/*I* (rev) p,4 and *I*p,5/*I* (rev) p,5 for 0.25 V. The respective estimated values are 0.57 and 0.6, respectively.

As a final step, these values shall be used for the estimation of the *k* 0 from the nomograms of *I*p,*h*/*I* (rev) p,*h* against the log(*k* 0 *f* −1 ). These nomograms are calculated for *a* = 0.5 (Figure 6a for the 4th harmonic and Figure 6b for the 5th). The same procedure is done for the amplitude of *A*0 = 0.2 V. By taking an average of the extracted values, the *k* 0 is estimated at 4.6 ± 2.6 s−1 .

**Figure 4.** Experimental results: (**a**) 4th harmonics, (**b**) normalised peak height of the 4th harmonic, (**c**) 5th harmonics and (**d**) normalised peak height of the 5th harmonic, of *Mt*LPMO9H immobilised on a Co-f-MWCNTs modified GC electrode at 25 ◦C. Experimental conditions: acetate buffer 500 mM, pH 6, *A*0 = 0.2 V, and *f* = 5 (black), 3 (red), 1 (blue), 0.5 (green), 0.2 (orange) and 0.1 (magenta) Hz.

**Figure 5.** Experimental results: (**a**) 4th harmonics, (**b**) normalised peak height of the 4th harmonic, (**c**) 5th harmonics and (**d**) normalised peak height of the 5th harmonic, of *Mt*LPMO9H immobilised on a Co-f-MWCNTs modified GC electrode at 25 ◦C. Experimental conditions: acetate buffer 500 mM, pH 6, *A*0 = 0.25 V, and *f* = 5 (black), 3 (red), 1 (blue), 0.5 (green), 0.2 (orange) and 0.1 (magenta) Hz.

**Figure 6.** Computational results: Dependence of the normalised principal peak height for the (**a**) 4th and (**b**) 5th harmonic of an immobilised species. Parameters: *a* = 0.5 and *A*0 = 0.2 (black) and 0.25 V (red).

In order to validate the value of *k* 0 , found experimentally, FTacV experiments were simulated numerically for *k* 0 equal to 4.6 s−1 and *a* equal to 0.57. In order to do so, Equation (5) was solved numerically where the potential ramp is perturbed by a sinusoidal of amplitude *A*0 and frequency *f* ,

$$E = E\_\mathrm{I} - vt\_\mathrm{R} + v|t - t\_\mathrm{R}| + A\_0 \sin 2\pi ft \tag{9}$$

and the harmonics where calculated from the current obtained by multiplying the current density, Equation (4) with the electrode surface *S*. Apparently, both the surface area *S* and the surface concentration of the oxidized form of the enzyme Γ 0 are unknown and are expected to vary between different immobilisation experiments. Therefore, the factor Γ 0*S* was chosen by trial-and-error. The simulation results (red curves) together with the corresponding experimental plots (black curves) for *A*0 = 0.2, Γ 0*S* = 3 × 10−13 mol and *A*0 = 0.25 V, Γ 0*S* = 1.2 × 10−12 mol and *f* = 1 Hz, are presented in Figure 7a,b, respectively. A rather good agreement is observed, implying a satisfactory estimation of *k* 0 .

**Figure 7.** Experimental (black) versus computational (red dashed line) of the 5th harmonic of *Mt*LPMO9H immobilised on a Co-f-MWCNTs modified GC electrode at 25 ◦C. Experimental conditions: acetate buffer 500 mM, pH 6, *f* = 1 Hz and (**a**) *A*0 = 0.2 and (b) 0.25 V. Computational parameters: a=0.57, *f* = 1 Hz, (a) *A*0 = 0.2 V, Γ 0*S* = 3 × 10−13 mol and (**b**) *A*0 = 0.25 V, Γ 0*S* = 1.2 × 10−12 mol.

#### **4. Discussion**

*Mt*LPMO9H was immobilised on an electrode surface, ensuring that direct electron transfer is indeed possible for this enzyme with the immobilisation method used with an indication that, for the conventional scan, rated when using cyclic voltammetry, the kinetics were quasi-reversible. The signal was governed by capacitance, a common feature for immobilised enzymes on electrode surfaces. Despite this, still, an initial estimation of the standard electron transfer rate constant was intented through the computational reproduction of cyclic voltammograms. From the peak separation, it was predicted that the *k* 0 would be around 5 s−1 , assuming an *a* equal to 0.5.

Then FTacV was performed for two different amplitudes as various frequencies in order to assure that both quasi-reversibility and reversibility are achieved in order to apply the methodology to extract the *k* 0 of immobilised species using FTacV. The extracted value was at 4.6 ± 2.6 s−1 being in good accordance with what was expected from cyclic voltammetry.

In order to validate the *k* 0 value obtained experimentally, FTacV experiments were simulated numerically and a rather good agreement was found for transfer coefficient *a* equal to 0.57. In order to approximate the peak heights of the voltammograms, different values of Γ 0*S* were used for the two different amplitudes. At this point, the validity of the Γ 0*S* used shall be ascertained. Since the enzyme concentration is 5.5 mg mL−1 and 8 µL were utilised, and they were dilluted in 10 µL to a final concentration of 4.4 mg mL−1 . From this, 2 µL were used, and, taking into consideration that the molecular weight of the enzyme is 65 kDa [22], the maximum moles that can be 1.35 × 10−10. Considering that not all of the enzymes would be bound to the matrix and from what was bound, not all would be oriented in such a way so as to interact with the enzyme, the values used for

the simulation seem to be valid. Nevertheless, it has to be pointed out that the proposed experimental methodology for obtaining kinetic parameters does not require knowledge of the surface concentration of the immobilised species or the the value of the surface area.

In general, it is common practice to compare computational with experimental results in order to ascertain the validity of the parameters used for the computational ones. The computational fits tend to reproduce most of the features of the voltammogram, just as in this case, where the basic features of the harmonics presented are fitted rather well [25,26]. Regarding the precision of the comparison of the computational harmonics with the experimental ones, it should also be taken into account that the above analysis is valid only if the assumptions of the model are closely met, where the immobilised species must behave as a Langmuirian monolayer and a dispersion of the electrochemical transfer rates should be absent. Otherwise, where kinetic or thermodynamic dispersion is present, more elaborate models have to be considered [27–29]. The influence of kinetic and thermodynamic dispersion might affect the waveform of the voltammograms [30], and other theories such as that of Marcus–Hush can applied, instead of the Butler–Volmer law [31]. Yet, in this case, the Butler–Volmer law seems to be rather sufficient for this case.

To conclude, with the *k* 0 value that was estimated, it seems that, under the right conditions, it is feasible to render the system reversible and take advantage of it for bioelectrocatalytic purposes. Thus, it could be proven to be rather resourceful in such applications.

**Author Contributions:** Conceptualization, D.Z. and A.K. (Antonis Karantonis); methodology, D.Z.; software, A.K. (Antonis Karantonis); validation, D.Z. and A.K. (Antonis Karantonis); formal analysis, R.X., D.Z.; investigation, D.Z.; resources, A.K. (Antonis Karantonis), E.T.; data curation, D.Z.; writing original draft preparation, D.Z., A.K. (Antonis Karantonis); writing—review and editing, A.K. (Antonis Karantonis), A.K. (Anthi Karnaouri), E.T.; supervision, A.K. (Antonis Karantonis), E.T.; project administration, A.K. (Antonis Karantonis); funding acquisition, A.K. (Antonis Karantonis). All authors have read and agreed to the published version of the manuscript.

**Funding:** The research work was supported by the Hellenic Foundation for Research and Innovation (H.F.R.I.) under the "First Call for H.F.R.I. Research Projects to support Faculty members and Researchers and the procurement of high-cost research equipment grant" (Project Number: HFRI-FM17-3090).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data sharing not applicable.

**Acknowledgments:** Chr. Argirusis and P. Pandis are aknowledged for providing the Co-f-MWCNTs.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**