**Lipases Immobilization for Effective Synthesis of Biodiesel Starting from Coffee Waste Oils**

## **Valerio Ferrario, Harumi Veny, Elisabetta De Angelis, Luciano Navarini, Cynthia Ebert and Lucia Gardossi**

**Abstract:** Immobilized lipases were applied to the enzymatic conversion of oils from spent coffee ground into biodiesel. Two lipases were selected for the study because of their conformational behavior analysed by Molecular Dynamics (MD) simulations taking into account that immobilization conditions affect conformational behavior of the lipases and ultimately, their efficiency upon immobilization. The enzymatic synthesis of biodiesel was initially carried out on a model substrate (triolein) in order to select the most promising immobilized biocatalysts. The results indicate that oils can be converted quantitatively within hours. The role of the nature of the immobilization support emerged as a key factor affecting reaction rate, most probably because of partition and mass transfer barriers occurring with hydrophilic solid supports. Finally, oil from spent coffee ground was transformed into biodiesel with yields ranging from 55% to 72%. The synthesis is of particular interest in the perspective of developing sustainable processes for the production of bio-fuels from food wastes and renewable materials. The enzymatic synthesis of biodiesel is carried out under mild conditions, with stoichiometric amounts of substrates (oil and methanol) and the removal of free fatty acids is not required.

Reprinted from *Biomolecules*. Cite as: Ferrario, V.; Veny, H.; De Angelis, E.; Navarini, L.; Ebert, C.; Gardossi, L. Lipases Immobilization for Effective Synthesis of Biodiesel Starting from Coffee Waste Oils. *Biomolecules* **2013**, *3*, 514-534.

### **1. Introduction**

Next generation biofuels is about utilization of non-food based feedstock and more sustainable process technology. The biodiesel industrial production worldwide is predominantly by chemical transesterification from food based feedstock. This is because with chemical transesterification the highest yield can be achieved in a relatively short reaction time. However, the current major cost of biodiesel production is from the feedstock [1]. Therefore, various non food feedstocks, such as waste oil and non edible oil, have been considered and studied in view of their potential use as raw materials [2–6].

In most cases the chemical process is performed by an alkaline catalyst, so that acid impurities and especially free fatty acids (FFA) contained in feedstock must be removed before transesterification to avoid the formation of saponification products. For instance, a basic solution is mixed with the extracted oil so that additional pre-treatments steps are introduced in the productive cycle [7]. In this regard, lipase-catalyzed transesterification of non food feedstock is becoming more attractive for the biodiesel industry, not only because it is sustainable and environmental friendly, but also because the free fatty acids can be esterified by lipases. Recently, attention has been given to oil extracted from coffee waste and examples of chemical conversion of oil from spent coffee ground into biodiesel have been reported [8,9]. The coffee grounds are mainly composed of proteins, carbohydrates, and lipids. It must be underlined that coffee is one of the largest agricultural products and, according to the U.S. Department of Agriculture, the world's coffee production is 16.34 billion pounds per year [8].

The spent coffee grounds contain, on an average, 15% (w/w) oil, which can be extracted by solvents such as trichloroethylene [10], *n*-hexane, ether and dichloromethane [10,11] with yields ranging from 6%–28% (w/w) [9,10]. This is quite significant as compared to other major biodiesel feedstock such as rapeseed oil (37%–50%), palm oil (20%), and soybean oil (20%) [12]. Furthermore, the biodiesel from coffee possesses better stability than biodiesel from other sources due to its high antioxidant content, which hinders the rancimat process [13,14].

The work of Kondamundi reports the extraction of 15% (w/w) of oil from spent ground, which was dried prior to extraction in order to reduce the moisture content. A small amount of FFA, monoglycerides (MG), and diglycerides (DG) was also observed in the oil but a conversion in biodiesel of 100% was obtained by chemical transesterification. The oil and biodiesel formed in that process were found to be stable over one month without any observable physical changes and analysis demonstrated that biodiesel obtained from spent coffee grounds is a strong candidate as an alternative to diesel [8].

One additional issue in the development of an economically sustainable production of biodiesel by enzymatic transesterification is represented by the cost of the biocatalyst. The economic impact of biocatalysts can be reduced by immobilizing the lipases on solid supports and then recycling them. Therefore, it is important to select immobilized lipases that not only express high activity but also allow for repeated use thanks to improved stability.

Lipases are one of the classes of enzymes most largely employed in industry also because of their potential to work in non-aqueous environments [15] and they are applied at industrial scale for the transesterification of fats and oils in the food sector [16]. However, to the best of our knowledge, there is still a lack of immobilized lipases commercially available and suitable for application in biodiesel synthesis [17].

Indeed, in most cases, the lipase catalyzed synthesis of biodiesel has been studied by employing biocatalysts originally developed for interesterification of food oils [7]. However, the latter process implies the application of lipases in highly hydrophobic environments whereas biodiesel synthesis involves the use of relatively high percentages of hydrophilic short chain alcohols that have inactivating effects on enzymes [18,19]. Moreover, as the reaction proceeds, glycerol is produced. Consequently, the biocatalyst will behave quite differently in the two processes. Phenomena, such as partition of hydrophilic components, will deserve explicit attention and investigation when planning a methanolysis process.

By analyzing the immobilized lipases available on the market, Lipozyme TL IM (lipase from *Thermomyces lanuginosa*) results probably the most widely applied at industrial scale in triglyceride transformations in the food sector. It is immobilized by spraying the liquid lipase concentrate onto silica particles together with food grade granulation additives. After subsequent drying in fluid beds, the granules are ready for use in interesterification of triglycerides. However, Lipozyme TL IM will disintegrate when dispersed into water or hydrophilic media [20].

In the present work, the enzymatic synthesis of biodiesel was approached by studying immobilized biocatalysts specifically developed for this application. A particular attention was devoted to the nature of the immobilization carrier and the prevention of aggregation and methanol aspecific adsorption, phenomena that might cause enzyme inactivation. Finally, the selected biocatalysts were applied in the transesterification of oil extracted from spent coffee ground. Factors affecting the efficiency of lipase immobilization were also analyzed.

#### **2. Results and Discussion**

#### *2.1. Selection of Lipases and Immobilization Supports*

The present work follows our previous detailed investigation of structural and conformational properties of a series of lipases [21]. Lipases have been evolved for transforming insoluble hydrophobic substrates so that their surface present unusual features that make these proteins adapt for approaching lipophilic surfaces. Accommodation and transformation of bulky triglycerides is strictly related to the accessibility of the active site. The latter, as also the surrounding superficial area, is hydrophobic. A flexible protein domain, called lid, shields the opening of the active site when the protein is exposed to a hydrophilic environment, thus occluding the substrate access. Therefore, in principle, the immobilized lipase should maintain an open active conformation throughout the alcoholysis process despite the presence of polar hydrophilic components, such as the alcohol and the glycerol, in the reaction mixture.

The present study focuses attention on two lipases that demonstrated different conformational behavior when exposed to a hydrophilic environment, namely lipase B from *Candida antarctica*  (CaLB) and *Burkholderia* (*Pseudomonas*) *cepacia* (PcL) (Figure 1). P revious MD simulations indicated that, upon exposure to water, the accessibility of the active site of these lipases is affected only at a minor extent when exposed to polar (e.g., aqueous) media [21].

CaLB is characterized by a small lid [22] and we have previously demonstrated that after 10 ns of MD simulations in explicit water the small lid domain undergoes only some modest conformational changes. Moreover, because of the small size of the lid, there is no closing of the active site. Indeed, the final conformation presents no significant difference in the hydrophobic surface exposed to the bulky aqueous medium [21].

Regarding PcL, MD simulations demonstrated that a ȕ-hairpin domain contributes to the stabilization of a "putative" second lid of PcL in its open conformation by forming two hydrogen bonds between Asn257-Thr224 and between Gln262-Gln215. Only the first one is lost during 20 ns MD in water, so that the upper part of the hairpin moves away from the Į-helix but this second putative lid does not change its position considerably and does not occlude the active site [21].

Here, we show (Figure 2) the starting open conformation and the result after 20 ns MD simulations. No significant variation of the superficial domains occurs when the protein is embedded in water and, most importantly, the conformation achieved leads to a partial coverage of the active site. This can be deduced from the mapping of the hydrophobic/hydrophilic areas performed by GRID analysis [23], which reveals how a considerable part of the hydrophobic active site remains exposed to the solvent (Figure 2b).

**Figure 1.** Tridimensional models of CaLB (**a**) (PDB code 1TCA) and PcL (**b**) (PDB code 1YS1). The structures are colored according to their secondary structures; lids are highlighted in red. PcL (**b**) has the second "putative" lid highlighted in green and the Ca2+ ion represented as orange sphere.

**Figure 2.** GRID analysis of hydrophobic (yellow) and hydrophilic (blue) surface of PcL. On the left: open conformation corresponding to the crystal structure obtained in the presence of an inhibitor (PDB code 1YS1). On the right: partially closed conformation computed after 20 ns MD simulation in explicit water.

From the point of view of substrate specificity, lipase B from *Candida antarctica* (CaLB) shows a preference towards short and medium size fatty acids, although it has been investigated extensively also in biodiesel applications [24,25]. It is recognized that the active site of CaLB is situated in the

core of the protein, the binding site has a funnelǦlike shape and highly hydrophobic amino acid residues envelop the cavity inner walls. Consequently, CaLB has, compared to other lipases, a very limited available space in the active site pocket that explains its substrate specificity [26].

Lipase from *Burkholderia cepacia* (PcL), instead, displays a marked specificity for long chain fatty acids and therefore, it appears to be a promising lipase for applications in biodiesel synthesis, as also reported in the literature [27].

In the present investigation, the selection and study of immobilized lipases suitable for biodiesel synthesis was guided by several considerations. Firstly, it must be taken into account that the enzymatic synthesis of biodiesel, unlike oil interesterification, involves the use of relatively high percentages of hydrophilic short chain alcohols that have inactivating effects on enzymes [19]. Moreover, as the reaction proceeds, glycerol is produced. Therefore, the immobilization of lipases for biodiesel synthesis should aim at preventing any detrimental interaction between the biocatalysts and these hydrophilic components, such as aspecific adsorption that might accentuate enzyme inactivation. Moreover, hydrophilic particles generally aggregate in hydrophobic media. On that basis, hydrophobic supports should be preferred.

However, it must be underlined that lipases recognize and accept very hydrophobic substrates that interact with the lipophilic area corresponding to the opening of the active site. As a continuation of our previous work, we have simulated the behavior of PcL at a water-octane interface (Figure 3) by carrying out a molecular dynamic (MD) simulation using a *coarse grained force field* (MARTINI) [28]. At the beginning of the MD simulation the opening of the active site (yellow dots) is oriented towards the aqueous phase; at the end, the lipase orients the opening of the active site towards the hydrophobic phase. This simulation suggests that the hydrophobic region of the enzyme will be also responsible for the establishment of hydrophobic interactions with hydrophobic supports. Indeed, this feature enables the exploitation of polymeric resins for achieving lipase purification and immobilization in one single step starting from crude enzymatic solutions, where lipases represent the only hydrophobic component [29]. Therefore, such interactions would promote an unfavorable orientation of the enzyme and scarce substrate accessibility.

On that basis, it seems difficult to define an ideal support for lipase immobilization *a priori*.

In order to elucidate the effect of the hydrophilic/hydrophobic nature of the carriers, the present study evaluated three different biocatalysts immobilized on highly and medium hydrophobic organic resins specifically developed by Sprin S.p.A. (Trieste, Italy) for biodiesel synthesis (Table 1). Then, the biocatalysts were compared with two preparations immobilized on hydrophilic siliceous carriers, namely diatomaceous earth (Celite). Table 1 reports the characterization of all the biocatalysts using standard assays.

A commercial formulation of PcL, Lipase PS-IM, was initially considered. It is commercialized by Amano and it consists in lipase PcL adsorbed on diatomaceous earth powder. However, despite the high hydrolytic activity expressed by the enzyme, the preparation was unsuitable for the methanolysis because the hydrophilic powder formed aggregates upon addition of the methanol to the oil.

**Figure 3.** Molecular dynamic (MD) simulation of PcL embedded in an explicit bi-phasic system octane-water. The hydrophobic side of the enzyme, corresponding to the opening of the active site, is highlighted in yellow beads. The octane phase is in grey points in the figure. At the starting point of the simulation (**a**) the water-octane interface is not completely defined; during the simulation the interface was formed (**b**) and the enzyme is oriented with its active site towards the octane part.

**Table 1.** Activity of immobilized lipases tested in the study. CaL = lipase B from *Candida antarctica*. PcL = lipase from *Pseudomonas cepacia.*

a The residual water content in the final immobilized preparation was determined on aluminum plates. A known amount of biocatalyst is dried at 110 °C for 6 h. Solvent content is defined as the % of weight loss after drying. b Synthetic activity: transesterification of vinyl acetate with 1-phenyl-1-ethanol. c Synthetic activity: esterification of lauric acid with 1-propanol. d Hydrolysis of tributyrin.

In light of the aggregation observed using the fine Celite particles, the immobilization of CaLB on hydrophilic carrier was carried out by selecting a commercial porous product, Celite® R-640 (Fluka), which consists in calcined Celite extruded in the form of big porous rods (5 mm high with a diameter of 3 mm). Porosity confers to the immobilization matrix a remarkable ability of adsorbing more than 100% of water (w/w) as well as a cumulative pore volume of 0.8 cm3 /g [30]. Immobilization was carried out by following a protocol previously developed in our lab for application of biocatalysts in low water media [31].

CaL-S and PcL-S are lipases adsorbed on highly hydrophobic styrenic resin whereas CaL-M corresponds to CaLB covalently immobilized on a methacrylic polymer [32]. It appears clear that there is no linear correlation between hydrolytic and synthetic activity. It must be also underlined that no direct comparison is possible between the hydrolytic activity of CaL-S and CaL-M since all enzymes immobilized through physical adsorption undergo partial leaching when suspended in aqueous media and this interfere with kinetic evaluation.

Moreover, the different substrate specificity of CaLB and PcL makes impracticable any direct comparison between the two enzymes.

#### *2.2. Methanolysis of Triolein and Effect of Methanol Concentration*

Methanolysis was conducted at 30 °C by using PcL-S (10% w/w of triolein) and by adding three equivalents of methanol obtaining 13% of conversion after 6 h and only 35% after 26 h. The data confirm the severe inactivating effect of methanol. Therefore, in the following experiments, multistep additions of methanol were used, by adding one equivalent molar of methanol at 0, 100 and 200 min. Proton NMR of the crude mixture (see Electronic Supplementary Information, Figure S1) showed the disappearance of the triglyceride peak already after 6 h of reaction. However, upon recycling, the biocatalyst displayed less than 40% of the initial activity. The recycles were carried out by introducing fresh triolein in each cycle and by determining the residual methyl oleate content prior to addition of methanol. Therefore, the PcL-S preparation, although very active, appeared unsuitable for multiple applications in biodiesel synthesis.

This unexpected behavior might be ascribable to different factors deserving specific investigations, which, however, are out of the scope of the present work. Since PcL displayed such poor efficiency when immobilised on the same support used for CaLB, the cause might reside either in the inherent properties of the protein or in the composition of the crude enzymatic formulation used for the immobilization. PcL is commercialized as a powder containing roughly 1% of protein diluted with cyclodextrins. The effect of this large amount of additives on the final immobilized formulation deserves further attention, although, it must be underlined that purification steps would cause an unacceptable increase of the cost of the biocatalyst.

One further point of attention is represented by the presence of a calcium ion in the structure of PcL (Figure 1b), which strongly affects the protein stability and this might be lost upon recycling [33].

Finally, a specific effect of some micro-components of triglyceride mixture on PcL stability cannot be excluded. Actually, several compounds generated by atmospheric oxidation of triglycerides, such as hydroperoxides and secondary products, may cause significant lipase inactivation in low water medium. Activity decay is usually time-dependent and shows a non-linear dependence on inactivating compound concentration [34].

When methanolysis was catalyzed by CaLB immobilized on Celite® R-640 (10% w/w of triolein) conversions lower than 5% were observed after 48 h. By adding 2.5% w/w of enzyme at the end of the reaction, there was no improvement in the conversion. It must be underlined that the biocatalyst was able to catalyze tributirin hydrolysis and propil laurate synthesis as shown in Table 1. The data indicate that the stop of reaction is not due to inactivation of enzyme but rather to some other factor connected to the reaction system. Most probably, the hydrophilic and hydrated pores (15% water by weight) of Celite® R-640 do not favor the partition of the oil towards the lipase.

Finally, the two preparations CaL-S and CaL-M immobilized on polymeric organic resins were monitored at different methanol to triolein molar ratios. Figure 4 reports the percentage of activity of the two CaLB formulations, defined as percentage of methyl ester yield evaluated after 25 h of reaction (theoretical maximum conversion in methyl ester is 33%, 66% and 100% for methanol to triolein molar ratio of 1:1, 2:1 and 3:1, respectively). Results indicate that the lipase activity of both CaL-S and CaL-M decreased with the increase of methanol concentration. By using a molar ratio of 3:1 the enzyme inactivation is so fast and severe that the reaction stops after 29% and 5% of conversion, respectively. CaL-S appears more resistant to the inactivating effect of methanol, since a conversion of 54% is achieved at a molar ratio of 2:1.

**Figure 4.** Effect of methanol concentration on CaL-S (star) and CaL-M (square) expressed as the conversion in methyl ester achieved after 25 h. The transesterification of triolein was carried out at 30 °C with molar ratios of 1:1, 2:1 and 3:1. Therefore, theoretical maximum conversions achievable were 33%, 66% and 100%, respectively.

The different behavior of the two preparations is visible also from the kinetic profile of the reactions carried out at 1:1 molar ratio of triolein and methanol and 10% of biocatalyst (w/w % of triolein) (Figure 5). Apparently, the CaL-M preparation is less efficient even at such low concentration of methanol. In fact, as expected, initial rates are similar since comparable enzymatic units were employed. However the reaction profile of CaL-M indicates that the reaction slows down and, surprisingly, a lower final conversion is achieved, although the thermodynamic equilibrium of the reaction does not depend on the biocatalyst by definition. No further increase of the yield could be achieved upon addition of fresh enzyme and this would suggest that the poorer performance of CaL-M is not due to enzyme inactivation.

**Figure 5.** Reaction profiles (24 h) of transesterification of triolein using a 1:1 molar ratio of oil and methanol (30 °C). CaL-S (star) and CaL-M (square). The maximum theoretical yield is 33%.

CaL-S and CaL-M show a good dispersion of the polymeric supports throughout the reaction with no formation of aggregates. Figure 6 illustrates the appearance of the reaction system at the end of transesterification with stepwise additions of methanol (molar ratio 1:1).

**Figure 6.** The appearance of the reaction system at the end of the methanolysis using the three preparations of CaLB considered in the study.

Methanolysis reaction (molar ratio 1:1) using CaL-S gives 24% of conversion after 6 h. It must be underlined that we also carried out methanolysis under the same conditions by using *Novozyme 435*  obtaining about 10% of formation of methyl oleate.

As reported before, the transesterification catalyzed by CaL-S and CaL-M did not achieve a quantitative conversion of the triglyceride. In order to verify whether some methanol was lost during the reaction and the sampling procedure, the transesterification was conducted for 48 h under similar operating conditions but without sampling. The final yield of methyl oleate was 29% for CaL-S but the NMR analysis proved no presence of residual methanol in the reaction medium (see Electronic Supplementary Information, Figure S2). The data suggest that during the transesterification process, some methanol is adsorbed on the immobilization support and this phenomenon is more pronounced in the case of CaL-M, as indicated by the lower conversion achieved. Therefore, the nature of the support appears to play a major role, since an increased local concentration of methanol is expected to be detrimental for the stability of the lipase. This is also in accordance with the lower efficiency of CaL-M observed at higher methanol concentration, as illustrated in Figure 5 above.

The effect of the nature of the immobilization carrier on the partition of hydrophilic components was studied by measuring the water activity of the multiphase reaction systems. Water activity was measured before and after the addition of the immobilized enzymes by means of a hygrometer and after 24 h equilibration as described previously [30,35]. The initial reaction system was a mixture of triolein and methanol with molar ratio 1:1. The values of water activity of the different reaction systems are shown in Table 2.

**Table 2.** Water activity of reaction systems in the presence of different preparations of immobilized CaLB.

All the supports here considered are porous and have the ability to bind a significant amount of water (from 50%–100% of their weight) although the residual water (evaluated by weight variation upon drying) resulted to be <5% (w/w) in CaL-S and CaL-M (Table 1). Results show that no immobilized enzyme brings free water to the reaction system that could cause competing hydrolytic reactions [36].

On the contrary, the hydrophilic carriers adsorb some water from the reactants, as indicated by the decrease of aw values, even in the case of Celite R-640, which contains a residual 15% of water in its pores. Significantly, CaL-S, which is the most hydrophobic support, can bind a lower amount of water and, most probably, less methanol as well.

In order to investigate in more detail the effect of methanol on CaL-S, the kinetic profile of the methanolysis at different concentrations of methanol was studied. Figure 7 shows how, by working with three equivalents of methanol, the inactivating effect is visible already after the first hour of reaction. The residual activity of CaL-S after 1 h of reaction at a methanol/triglyceride molar ratio of 3:1 was assayed also by using the method of propil laurate synthesis (see materials and methods). Data indicates that the activity of CaL-S decreases from the initial 3450–1600 (U/g dry), thus confirming the partial inactivation of the enzyme. In the other two cases, the reaction proceeded until the equilibrium was reached and no appreciable variation of conversion was observed by adding fresh enzyme at the end of the reaction (2.5% w/w). This also confirms that the stopping of the reaction was not due to lipase inactivation.

**Figure 7.** Effect of methanol concentration on CaL-S evaluated at 30 °C by monitoring the transesterification of triolein at different methanol/oil molar ratio.

#### *2.3. Recyclability of Cal-S*

The stability study was performed using a methanol to oil molar ratio of 1:1 and by stopping the reaction after 4 h in each cycle and measuring the conversion in terms of formation of methyl oleate. Then, the biocatalyst was recovered and reused without further treatment/rinsing by simply introducing fresh triolein and methanol. It must be noted that for each cycle, after introducing fresh triolein, the residual methyl oleate content deriving from the previous cycle was determined prior to the addition of methanol and the starting of the methanolysis. The results indicate that CaLB on styrene is stable after 10 cycles, without any rinsing treatment (Figure 8). Moreover, the biocatalyst has shown its stability even after 22 days of exposure to the reaction conditions. Therefore, no appreciable inhibition due to oil, glycerol or product was detected. Importantly, no aggregation of the particle was observed. The behavior of the preparation indicates that the styrenic resin prevents the undesirable hydrophilic interactions (glycerol and methanol adsorption, protein aggregation, particle aggregation) which are likely to occur in the hydrophobic oils.

Notably, Liù and co-workers reported how CaLB Novozymes 435 undergoes a severe inactivation under comparable conditions, with a loss of roughly 10% of activity after each synthetic cycle [24].

#### *2.4. Enzymatic Methanolysis of Oil Extracted from Espresso Spent Coffee Ground*

As a final step in the investigation of lipase catalyzed methanolysis, the immobilized CaLB was applied to the transformation of a non-edible oil from food waste. The oil was extracted from espresso spent coffee ground properly dried [8] according to the procedure already reported [37]. The oil from spent coffee ground appears as a transparent homogeneous amber colored liquid (see Electronic Supplementary Information, Figure S3). Table 3 illustrates the composition in terms of fatty acids determined by GC-MS and compared to the oil used in the work of Kondamundi. Of course, fatty acids can vary not only as a function of the type of coffee used but also as consequence of the method used for coffee brewing.

**Figure 8.** Recyclability of CalB immobilized on styrenic support expressed as the percentage of methyl oleate formed after 4 h at 30 °C in the transesterification of triolein with methanol (1:1 molar ratio).

**Table 3.** Composition of the oil from espresso spent coffee ground in terms of fatty acids and compared to the oil used in the work of Kondamundi *et al*. [8].

The transesterification was carried out using CaL-S on oil from espresso spent coffee ground without pretreatment of free fatty acid and at 30 °C.

The reaction was monitored via HPLC by evaluating the formation of the methyl esters of the main fatty acids (palmitic, oleic, linoleic, linolenic, stearic, arachidonic). Because of the complexity of the reaction mixture, a semi-quantitative evaluation of the conversion was obtained by summing the areas of these products of the conversion. About 55% of biodiesel yield was achieved by performing one single addition of three equivalents of methanol at the beginning of the reaction whereas multistep additions of one equivalent of methanol (time = 0, 100 min, 200 min) led to 72% conversion after 30 h of reaction. No optimization of the process (e.g., range of time for addition of methanol) was carried out.

The decrement of the H1 -NMR signals relative to the triglycerides was used as a further indicator of the proceeding of the reaction. The area of the protons at 2.5 ppm (signal related to two protons in Į-position to carbonyl group of fatty acid) and the area of the protons at 3.6 ppm (signal related to the three protons of methyl esters) indicate that the conversion is nearly quantitative within 24 h (Figure 9).

**Figure 9.** H1 -NMR spectra of the oil from espresso spent coffee ground (**a**) and of the reaction mixture after 5 h (**b**) and 24 h (**c**).

The GC-MS analysis of the products obtained from the methanolysis (see Electronic Supplementary Information, Figure S4) confirms the formation of the methyl esters of all the fatty acids present in the oil from spent coffee ground, as reported in Table 3. Therefore, CaLB demonstrates to be a versatile biocatalyst, accepting all the different fatty acids present in the oil from spent coffee ground.

It must be underlined that the chemical (KOH) methanolysis of oil from spent coffee ground reported by Kondamudi required the use of an excess of 40 volumes of methanol and was carried out under reflux at 70 °C. The process described in the present manuscript is carried out at 30 °C and requires no excess of methanol, so that the only co-product of the reaction is the glycerol. Moreover, at the end of the reaction the biocatalyst can be simply filtrated and no acid must be used to neutralize the reaction mixture.

#### **3. Experimental Sections**

#### *3.1. Enzymes*

Native CaLB employed during this study was Lipozyme CalB L from Novozyme (DK). The commercial preparation is constituted by CaLB (11 mg/mL) diluted in a solution of glycerol, sorbitol and salts to stabilize the protein. The pH value of the enzymatic solution is about 4.2.

Native PcL was Lipase PS "AMANO" SD from AMANO (Japan). The commercial preparation is a yellowish lyophilized powder constituted by PcL (about 1%) diluted with dextrins.

Immobilized CaL-M (methacrylic) [32] and CaL-S (styrenic) were kindly donated by Sprin S.p.A. (Trieste, Italy). Organic polymeric resins are in the form of beads with particle size of 300– 500 ȝm. Characterization is reported in Table 1.

The residual water content in the final immobilized preparation was determined on aluminum plates. A known amount of biocatalyst was dried at 110 °C for 6 h. Water content is defined as the % of weight loss after drying.

### *3.2. Immobilization of CalB on Celite®R-640*

The immobilization was done according to the procedure already reported [31]. The Lipozyme solution was adjusted to pH 8 prior to immobilization. 2.5 mL of enzymatic solution, 1 g of dry Celite® R640 and 12 mL of dry toluene were mixed and kept at 25 °C for one day. The organic solvent was removed and the enzymatic preparation was washed with acetone on a büchner filter under reduced pressure for three times (support/solvent ratio 1/2 w/v). The residual water content was then evaluated as reported above.

#### *3.3. Chemicals*

Triolein (99%), methyl esters standards and all other chemical reagents are from Sigma Aldrich.

#### *3.4. Monitoring the Formation of Fatty acid Methyl Ester (Biodiesel)*

Methyl oleate formed from transesterification of triolein was measured by Gilson HPLC equipped with an auto injector. The column was ODS Hypersil SUPERCHROM set at isothermal oven temperature of 54 °C. The detector was UV/VIS-155 with dual wavelength of 200 nm and 210 nm. The mobile phase was 100% Acetonitrile and 0.05% of TFA with flow of 1 mL/min. Biodiesel (methyl esters of fatty acids-FAME) was analyzed by following methods from ref. [10]. Biodiesel yield was calculated by summing the areas of methyl esters present in the reaction mixture and on the basis of calibration curves built up with standards FAME.

#### *3.5. Synthetic Activity of PcL*

Activity was determined by transesterification reaction of vinyl acetate with 1-phenyl-1-ethanol, which is a standard synthetic method reported by AMANO Enzymes company. Transesterification reaction was carried out at 25 °C and mixing at 200 rpm. 1-phenylethyl acetate formed during the reaction was measured by Gilson HPLC equipped with an auto injector. The column was ODS Hypersil SUPERCHROM set at isothermal oven temperature of 30 °C. The detector was a Gilson UV/VIS-155 with dual wavelength of 210 nm and 260 nm. The mobile phase was 40% acetonitrile and 0.05% of TFA and 60% H2O with a flow of 1 mL/min.

#### *3.6. Synthetic Activity of CaLB*

The assay allows the determination of enzymatic activity through the synthesis of propyl laurate formed by enzymatic esterification of lauric acid and 1-propanol. One unit corresponds to the amount of enzyme that produces 1 mol of propyl laurate in one minute at 55 °C without solvent. In a vial of 20 mL, 1.2 g of lauric acid and 0.36 g of 1-propanol were added. The reaction mixture was thermostated at 55 °C and mixed at 250 rpm in order to allow the melting of lauric acid. A withdrawal of 0.1 mL was diluted in 1.3 mL of hexane. 0.2 mL of this solution was added to 1.6 mL of hexane and analyzed by HPLC (*t* = 0). The reaction was started by adding 40–50 mg of biocatalyst to the main solution and maintained under continuous stirring at 250 rpm. Withdrawals were performed at different times and analysed by HPLC.

#### *3.7. Hydrolytics Activity of Lipases*

The activity of enzymatic preparations was assayed by following the tributyrin hydrolysis and by titrating, with 0.1 M sodium hydroxide, the butyric acid that is released during the hydrolysis. An emulsion composed by 1.5 mL tributyrin, 5.1 mL gum arabic emulsifier (0.6% w/v) and 23.4 mL water was prepared in order to obtain a final molarity of tributyrin of 0.17 M. Successively, 2 mL of K-phosphate buffer (0.1 M, pH 7.0) were added to 30 mL of tributyrin emulsion and the mixture was incubated in a thermostated vessel at 30 °C, equipped with a mechanical stirrer. After pH stabilization, about 50 mg of immobilized protein were added. The consumption of 0.1 M sodium hydroxide was monitored for 15–20 min. One unit of activity was defined as the amount of immobilized enzyme required to produce 1 ȝmol of butyric acid per min at 30 °C.

#### *3.8. Water Activity*

Water activity of the reaction system was determined at 30 °C by using a hygrometer (DARAI-Trieste, Italy) as previously described [30]. Measurements were carried out by sealing the sensor into the open end of 5 mL glasses vials, thermostated at 30 °C, until constant reading. All samples were previously equilibrated for 24 h. The hygrometer was firstly calibrated for 24 h with saturated salt solution of MgCl2 (aw = 0.3273), NaCl (aw = 0.7532) and NH4NH2 (aw = 0.927) using ultrapure water [30,35]. The values of standard salt solution from calibrated hygrometer were 0.39, 0.74 and 0.87.

#### *3.9. Monitoring the Methanolysis by HPLC*

Methyl esters formed from the transesterification were evaluated by means of a Gilson HPLC equipped with auto injector. The column was ODS Hypersil SUPERCHROM set at isothermal oven temperature of 54 °C. The detector was UV/VIS-155 with dual wavelength of 200 nm and 210 nm. The mobile phase was 100% acetonitrile and 0.05% of TFA with flow of 1 mL/min. Biodiesel (FAME) content and its standards were analyzed by following methods from [38]. Biodiesel yield was calculated based on total area of the methyl ester mixture in the final product.

#### *3.10. Lipase Catalyzed Methanolysis*

Transesterification was carried out in 10 mL screw capped vials in orbital shaker with temperature control. The temperature was set at 30 °C with shaking rate of 250 rpm. Prior to addition of methanol, the immobilized lipase (10% w/w of oil) was mixed with the feedstock (2 g) for a few minutes. 10 L samples were withdrawn and analyzed by HPLC.

#### *3.11. Recycling of CaL-S*

The stability study was performed using a methanol to oil molar ratio of 1:1, stopping the reaction after 4 hours in each cycle and by measuring the conversion in terms of formation of methyl oleate. Then, the biocatalyst was recovered and reused without further treatment/rinsing by simply introducing fresh triolein and methanol. For each cycle, in order to avoid overestimation of the product concentration, after introducing fresh triolein, the residual methyl oleate content deriving from the previous cycle was determined prior to the addition of methanol and the starting of the new methanolysis cycle. The reactions were performed at 30 °C.

#### *3.12.* 1 H-NMR

Spectra were recorded on a NMR Varian 200 Gemini, operating at 200 MHz. Samples were prepared by diluting 10 L of the reaction mixture in 700 L of CDCl3.

#### *3.13. Extraction of Oil and GC-MS Analysis of Fatty Acids*

The spent coffee ground was dried [8] and extracted according to the procedure already reported [37], obtaining yields around 20% (w/w). Characterization was performed according to a method previously described [39] by mean of GC MS Agilent technologies 6890 N and Agilent 5973 MSD as detector. The column was a Zebron ZB-FFAP Capillary Column 60 m × 0.25 mm × 0.25 ȝm. For the analysis, the temperature raised from 170–235 °C by increasing 2 °C/min. Gas phase was helium at constant flow of 1.3 mL/min.

#### *3.14. Computational Study: Molecular Dynamic Simulations*

The molecular dynamic (MD) simulations to analyse the behaviour of lipases upon water exposure were performed using the software GROMACS 4 with the GROMOS-96 53a6 force field. The crystal structure of *Pseudomonas cepacia* lipase (PDB 1YS1) and the crystal structure of *Candida antarctica* lipase B (PDB 1TCA) were implemented in the force field in *gro* file format by using the tool pdb2gmx of the GROMACS software package, which also adds the necessary hydrogen atoms. The proteins were solvated separately with explicit SPC water in a virtual box of 343 nm3 each. The molecular dynamic simulations were performed in an NPT environment simulating the temperature of 300 K and keeping the pressure constant (Berendsen thermostat and pressure); the cut-off for electrostatic interactions was set to 1.4 nm and the limit for the Van der Waals interactions set to 1.4 nm. For the minimization procedures, the PME (Particle Mesh Ewald) algorithm was used for the calculation of the electrostatic interactions, setting the limit at 1.0 nm. Before the MD simulation, the systems were minimized at least for 10,000 steps.

The MD simulation of *Pseudomonas cepacia* lipase at water-octane interface was performed using the software GROMACS 4 with the MARTINI force field. Crystal structure of *Pseudomonas cepacia* lipase (PDB 1YS1) was implemented in the MARTINI force field in *gro* file format by using the necessary scripts available on the MARTINI web site [40] and the DSSP program [41] for the necessary secondary structure definition. The protein was solvated with explicit water-octane 1:1 mixture in virtual cubic boxes of 512 nm3 . The MD simulation was performed in a NPT environment and in conditions specified above. The time step for integration was set to 4 fs (usually this value is set to 2 fs). Minimization procedures were performed before the MD simulation using a cut-off for electrostatic interactions and by setting the threshold at 1.4 nm. The steepest descendent algorithm was used. Minimization was performed for at least 10,000 steps.

#### *3.15. Computational Study: GRID Mapping of Surface of Proteins*

The GRID analysis [32] was performed on the output of the dinamised crystal structure of *Pseudomonas cepacia* lipase (PDB code 1YS1) by choosing a cage big enough to include the whole protein. The grid nodes were set every 0.5 Å. The probes used for the calculation of the molecular interaction fields were DRY (hydrophobic probe) and WATER (H2O probe). Isopotential surfaces were visualized by setting energy values of í0.3 kcalmolí1 for the DRY probe and í2.5 kcalmolí1 for the WATER probe.

#### **4. Conclusions**

The present study shows that immobilization plays a major role in determining the efficiency of lipases in the methanolysis of oils. Information on the structure and conformation of lipases provides a rational basis for the selection of biocatalysts and their immobilization. *Ad hoc* immobilization methodologies are necessary for fully exploiting the catalytic potential of lipases while favoring effective mass transfer and partition of substrates/products.

Lipase B from *Candida antartica* (CaLB) displayed the highest efficiency when immobilized on the most hydrophobic supports (CaL-S). The biocatalyst showed good stability after 10 cycles of reaction in presence of equimolar concentration of methanol. Hydrophilic immobilization supports demonstrated to be less suitable for reactions involving the bulky and hydrophobic triglycerides, most probably because of unfavorable partition phenomena. Moreover, data suggest that these porous carriers are more prone to absorb water or other hydrophilic components such as methanol, which affect negatively the stability of lipases.

The study of methanolysis of oil from spent coffee ground indicates that new types of non-edible oils can be considered for developing competitive and sustainable enzymatic biodiesel synthesis. Immobilized CaLB was able to catalyze the conversion of all different fatty acids present in the raw material. Optimization studies will be required for implementing appropriate feed-batch processes to decrease the reaction time and for the evaluation of various types of oils from different coffee wastes.

#### **Acknowledgments**

Harumy Veny is grateful to ICS-UNIDO (Trieste, Italy) for a fellowship. Thanks are due to Sara Cantone and Diana Fattor (SPRIN S.p.A., Technology for Sustainable Chemistry, Trieste, Italy) for enzyme samples and useful discussions.

#### **References**


**5. Medical test: Cardiac fitness index based on treadmill HR variation**

In this procedure, the cardiac fitness model consists of a first-order differential-equation system model, describing the heart rate (HR) response (y) to exertion (exercise, jogging etc) monitored in terms of the work-load, where y is defined as follows:

$$y = \frac{HR(t) - HR(rest)}{HR(rest)}\tag{1}$$

The subject is exercised on the treadmill for a period of time *te* (minutes) at a constant work load (W), while the HR(t) (and hence y) is monitored, as displayed in fig 15. Now we develop a model to simulate (i) the y(t) response during exercise, i.e. during *t ≤ te*, and (ii) thereafter for y(t) decay, after the termination of exercise. In a way, *te* represents the exercise endurance of the subject [6].

For a person, the model equation for HR response is represented by:

$$\frac{dy}{dt} + k\_1 y = \mathbb{C}\_0 \mathcal{W} \tag{2}$$

For *t ≤ te*, the solution is given by:

$$y = \frac{y\_e(1 - e^{-k\_1 t})}{(1 - e^{-k\_1 t\_s})} \tag{3}$$

For the recovery period *(t ≥ te )*, the solution of eqn. (2) is :

$$y = y\_{\epsilon}e^{-k\_2(t-t\_{\epsilon})} \tag{4}$$

where *k1* and *k2*, are the model parameters, which can serve as cardiac-fitness parameters (in min-1).

### **Non-dimensional Cardiac Fitness Index:**

A typical y(t) response is illustrated in Fig 15.

Fig. 15. Graph of y (the computed HR response) vs. t during treadmill exercise *t=te* and during the recover of period *t=te.*

The parameters *k1* and *k2* can be continued into a single nondimensional cardiac-fitness index (CFI):

$$\text{CFI} = k\_1 k\_2 t\_e \,\text{ ${}^2$ }\tag{5}$$

According to this formulation of CFI, a healthier subject has (i) greater *k1* (i.e., slower rate-ofincrease of HR during exercise (ii) greater *k2* (i.e., faster rate-of-decrease of HR after exercise) (iii) greater *te* (i.e., exercise endurance), and hence (iv) higher value of CFI.

Table 1. CFI values for athletic, fit and unfit subjects.

where *k1* and *k2*, are the model parameters, which can serve as cardiac-fitness parameters

Fig. 15. Graph of y (the computed HR response) vs. t during treadmill exercise *t=te* and

The parameters *k1* and *k2* can be continued into a single nondimensional cardiac-fitness

According to this formulation of CFI, a healthier subject has (i) greater *k1* (i.e., slower rate-ofincrease of HR during exercise (ii) greater *k2* (i.e., faster rate-of-decrease of HR after exercise)

(iii) greater *te* (i.e., exercise endurance), and hence (iv) higher value of CFI.

Table 1. CFI values for athletic, fit and unfit subjects.

*y e y e*

For the recovery period *(t ≥ te )*, the solution of eqn. (2) is :

**Non-dimensional Cardiac Fitness Index:**  A typical y(t) response is illustrated in Fig 15.

during the recover of period *t=te.*

index (CFI):

(in min-1).

1 1 (1 ) (1 )*e k t e k t*

− = − (3)

2 ( )*e k tt e y ye*− − = (4)

2 *CFI k k t* = 1 2 *e* (5)

− −

Now, we need to evaluate CFI for a big spectrum of patients, and then compute its distribution curve, to determine the efficiency of this index, in order to yield distinct separation of CFI ranges for healthy subjects and unfit patients. This CFI can also be employed to assess improvement in cardiac fitness following cardiac rehabilitation regime. This CFI is non-dimensional, and it can be useful to clinicians as they are able to predict the heart condition or fitness performance of a person by referring to the value of a single index value.


*3.3. Water-Land-Food-Carbon" Coupling Coordination Analysis*

From Figures 9 and 10, the coupling coordination degree of "water-land-food-carbon" of all provinces, municipalities and autonomous regions in China from 2000 to 2020 showed a trend of decreasing before increasing, and the lag of "water-land-food-carbon" system was improved. The coupling coordination in Northeast China first decreased and then increased, among which, the coupling coordination in Heilongjiang was higher than the other two provinces and changed from moderate coordination (0.55) to good coordination (0.74) with a rate of 35.15%; Jilin changed from low coordination (0.50) to good coordination (0.62); Liaoning changed between low coordination and reluctant coordination (0.44→0.38→0.46→0.38→0.49). In North China, Beijing's coupling coordination changed more steadily, all in a low coordination state; Inner Mongolia was between low and reluctant coordination (0.44→0.38→0.46→0.38→0.49) during 2000–2015, and the coupling coordination was higher than the other four provinces and municipalities in 2020, reaching a good coordination state (0.62). Anhui in East China changed from low coordination (0.50) to good coordination (0.57). Except for Anhui, the areas of Jiangsu, Zhejiang, Jiangxi and Shandong increased on the whole and were basically between low coordination and reluctant coordination, while Shanghai and Fujian decreased on the whole and the coupling coordination changed from low coordination (0.41, 0.44) to reluctant coordination (0.36, 0.39). Henan, Hubei and Hunan in Central China had less changes and were basically in the state of low coordination (0.48, 0.48, 0.49). Guangdong, Guangxi and Hainan in South China developed from low coordination (0.42, 0.45, 0.43) to reluctant coordination (0.36, 0.36, 0.31) from 2000 to 2005; from 2005 to 2020, Guangdong and Guangxi developed into low coordination (0.41, 0.43), and Hainan was still in reluctant coordination, but with a relatively large increase of 11.42%. Shaanxi, Gansu, Ningxia and Xinjiang in Northwest China all changed from low coordination (0.45, 0.43, 0.48, 0.45) to reluctant coordination, and then to low coordination before increasing to low coordination (0.45, 0.47, 0.48, 0.49); Qinghai changes were more volatile, developing from reluctant coordination (0.39) to verge of disorder (0.28, 0.30) before rising to low coordination (0.45). The coupling coordination of Tibet in Southwest China was relatively low, developing from reluctant coordination (0.39) to verge of disorder (0.28) from 2000 to 2005, changing to reluctant coordination (0.36) from 2005 to 2020, and showing an overall decreasing trend (0.39→0.36); Ningxia and Shaanxi showed a decreasing trend but were basically in a low coordination state; Gansu, Qinghai and Yunnan were in low coordination. The "water-land-food-carbon" system showed lagging development.

**Figure 9.** "Water–Land–Food–Carbon" coupling coordination degree. Note: BJ, TJ, HB, SX, IM, LN, JL, HLJ, SH, JS, ZJ, AH, FJ, JX, SD, HEN, HUB, HUN, GD, GX, HAN, CQ, SC, GZ, YN, ZX, SAX, GS, QH, NX, XJ is the abbreviation of Beijing, Tianjin, Hebei, Shanxi, Inner Mongolia, Liaoning, Jilin, Heilongjiang, Shanghai, Jiangsu, Zhejiang, Anhui, Fujian, Jiangxi, Shandong, Henan, Hubei, Hunan, Guangdong, Guangxi, Hainan, Chongqing, Sichuan, Guizhou, Yunnan, Tibet, Shannxi, Gansu, Qinghai, Ningxia, Xinjiang.

**Figure 10.** Development trend of coupling degree and coupling coordination degree of "Water–Land– Food–Carbon".

#### **4. Discussion**

#### *4.1. Theoretical Implications of GTCL*

At present, the Chinese government aims to promote green development and the overall green transition of economic and social development. The purpose of this paper is to realize a green, low-carbon, efficient and intensive spatial pattern of sustainable development of cultivated land-use based on the coupling coordination effect of "waterland-food-carbon" (Figure 2). From the perspective of a geographical pattern, the cultivated land-use system is not only a basic material space for economic and social development, but also an important part of rural habitat. In the process of promoting GTCL, priority shall be given to complex adaptive relationships between the whole and the local (element sharing and system coupling), conservation and development (sustainable use and food security), and equity and well-being (intergenerational balance and ecological welfare). For this reason, there is an urgent need to conduct theoretical and practical research on GTCL from the perspective of resource-factor linkage. GTCL is an inherent requirement for driving regional high-quality development, which refers to the sustainable use of cultivated land gradually shifting away from the dependence on high factor consumption and agroecological damage to a green development approach in which economic growth and resource conservation, carbon emission reduction and farmland ecosystem improvement are reinforcing each other. GTCL is not only the essential requirement of the natural properties of land, but also the inevitable requirement of sustainable utilization of resources and ecological civilization construction, as well as the value requirement of realizing the green transition of economic and social development in an all-round way. To promote GTCL, attention must be paid to the conceptual innovation from "independent" means of production to "systematic" resource community, as well as the transition from extensive use and excessive mode to a large-scale, intensive and sustainable way, thus moving towards the unification of value goals from farmers' personal interests to social public interests. Thus, there is a recognition that exploring the WLFC nexus under this framework (Figure 2) is a crucial first step toward effectively implementing its emerging policies for GDCL. For example, policymakers can better formulate legally binding development and protection objectives by analyzing the interaction between WLFC subsystems, and promote the combination of relevant policies and broader socio-economic activities in a timely manner, which will be beneficial to the common realization of the objectives of sustainable use of cultivated land, increase of food production, economic transformation, optimization of resources and environment, and mitigation of climate change.

#### *4.2. Impact of GTCL on the WLFC Nexus*

A compelling reason for analyzing GTCL from the "water-land-food-carbon" perspective is that the degree of water-soil matching, cultivated land reuse, grain yield, cultivated land-use and carbon emissions play a crucial role in achieving sustainable cultivated land-use. "Waterland-food-carbon" is the external condition that leads to changes in GTCL; GTCL is the internal basis that influences the synergy of "WLFC", and the external cause works through the internal cause. According to the analysis above, the development strategies in different periods have different emphasis on "WLFC". From 2000 to 2020, industrial pollution caused by the rapidly developing of industrialization had a negative impact on the resources and environment, the modern agricultural construction was started after the abolition of agricultural tax, and urban sprawl in the period of rapid urbanization led to the massive loss of cultivated land. After that, cultivated land protection policies were integrated into a larger natural resources management framework at national level, which improved the coupling coordination degree of WLFC. Furthermore, the in-depth adjustment of the global industrial chain, supply chain and value chain represented by grain trade has produced a transmission effect on many uncertain risks. However, the policy framework of cultivated land protection has been constantly improved, including regulations, policies, subsidies, standards, technical measures, etc. [49]. Overall, the "W"-shaped fluctuating uptrend of GTCL is observed. That is naturally why the average values of the comprehensive transition index exhibit decrease first and then increase with a

repeatedly periodic dynamic process. Other compelling reasons include the positive effects of the green transition in soil conservation, soil carbon sequestration, soil organic matter levels, water and nutrient retention, and biodiversity support, especially for farmland and pastures with low productivity or environmental sensitivity. In general, when the degree of soil-water matching is close to the critical range, green use plays a dominant role in controlling grain yield and carbon emissions, e.g., the Xinjiang Uygur Autonomous Region and the Inner Mongolia Autonomous Region in Northwest China (Figures 3e and 6); if GTCL is not properly regulated, then no matter how high the degree of land-water matching is, it cannot further increase grain yield or reduce carbon emissions, e.g., Zhejiang Province and Fujian Province in East China (Figure 8a,b and Figure 9). Since the coupling of "water-land-food-carbon" is complex and involves the overlapping combination of virtual resource flows and remote utilization patterns in the context of cross-food trade, the examination of the coupling coordination between the WLFC subsystems will help to deeply analyze the external effects of GTCL, mainly from two perspectives. First, the cultivated land-use system is a complex giant system with coupling interactions of single or multiple subsystems in the process of green transition, and its transition goal, transition direction, transition path and its internal mechanism are mainly governed by water, land, food and carbon. Second, whether it is the degree of water-land matching, cultivated land reuse and grain yield or carbon emission, they are all influencing factors in the process of GTCL. Based on the spatial and temporal patterns of GTCL and the results of "water-land-food-carbon" coupling coordination analysis, the influence of external factors can be fully reflected by monitoring data. Relying on the macroscopic analysis model established on the basis of the transition mechanism, the "details" of the process that are difficult to quantify can be greatly reduced, thus ensuring the scientific and reasonable research results.


**4. Applications**

Due to its high chemical reactivity and rapid oxidation, porous silicon was being utilized for device isolation. By the end of the 1980s, porous silicon had also been used for other purposes like the realization of SOI substrates and the formation of a buffer layer in epitaxial growth of compound semiconductors on silicon substrates. However, it was only after the discovery of strong photoluminescence in porous silicon that the material attracted broad attention. Since then porous silicon has been used for the fabrication of gas sensors, humidity sensors, biosensors, light emitting structures, optical waveguides, distributed Bragg reflectors, Fabry-Pérot resonators, photonic crystals, flat panel displays, optical and acoustic filters, ultrasound generators, and many other devices. Even though optoelectronics has remained the main research area of porous silicon, recently, the material has found application in other areas like medicine, diagnostics, cosmetics, consumer care, and nutrition. In contrast to the conventional chip-based applications, these new areas rely on porous silicon powders and independent structures. In this section, we briefly discuss the application domains of porous silicon and the reader is encouraged to refer to the literature dedicated to porous silicon applications (for example, see [78]).


*4.3. Theoretical Approaches Explaining the Impact of Green Marketing on Customer Behavior*

This section summarizes different theoretical approaches toward desirable customer behavior with green marketing. We investigate theoretical reasoning in customer behavior theories concerning approaches to green marketing, enabling us to determine the biases and possible development directions of green marketing research.

Of the 166 documents, 66 (39.76%) provided a theory or model explaining customer behavior and green marketing as its antecedent. The remainder of the articles discussed green marketing's impact on customer behavior without explicitly choosing the theory or model (Table 3).

The theory of planned behavior is the most famous theoretical perspective on customer behavior in green marketing. This perspective considers attitudes, subjective norms, and perceived behavioral control as predictors of customer behavior. Although the recent marketing literature [69,70] criticizes the theory of planned behavior, this framework has profoundly influenced the evolution of customer behavior literature. Green marketing tools through the lens of the theory of planned behavior were introduced in the study by Testa et al. [45]. The authors focused on the effectiveness of one particular green marketing tool, eco-labeling. Since then, the popularity of the theory of planned behavior has been gradually increasing. For example, in a recent research, Ch et al. [8] emphasized that the theory of planned behavior strongly supports green purchasing behavior.

The other theoretical perspective, the theory of reasoned action, was also widely employed for research into adjusting customer behavior through the tools of green marketing. We have accumulated evidence of the use of this theoretical foundation in the studies of the impact of eco-labeling [45], green appeal [47], perception of green products [49], etc. The

repeated applications of the theory of reasoned action are related to the aforementioned theory of planned behavior, as the theory of reasoned action originated as an improved version of the theory of planned behavior.

The other scholars have chosen stimulus-organism-response, cognition-affect-behavior, environmentally conscious customer behavior, four forces models, attitude-behaviorcontext, attribution, regulatory focus, or value-belief-norm theories as conceptual frameworks. Most research was based on one theory, but some scholars combined two or more theoretical perspectives. For example, Liao [56] confirmed that the combination of signaling and attitude-behavior-context theories could explain the moderation of green marketing on customers' purchase intention. However, some authors [2,68,69] did not exploit any theories.


*Edited by Chatchawal Wongchoosuk*

Intelligent system is an advanced machine that can perceive, learn, and solve the problems with a great accuracy. Technologies with intelligent system are currently available in the market and used in real-world applications, i.e., self-driving cars, Siri, Alexa, Facebook, and so on. To exceed human cognitive capabilities, the important keys rely on the development of sensors and algorithms. Therefore, the insight into artificial intelligence (AI) methods becomes a fundamental building block for design and construction of intelligent system with particular applications. This book aims to describe the AI systems ranging from the basic knowledge, i.e., algorithm and mathematical models of AI techniques, fundamentals of machine learning, genetic algorithm, and fuzzy logic, to the current state-of-the-art applications, such as smart road and biomedical applications.

ISBN 978-1-78923-606-4

Intelligent System

Published in London, UK © 2018 IntechOpen © sakkmesterke / iStock


*1.6. Stem Cell Targeted Therapy*

Stem cell therapy has recently gained further attention and momentum as a promising approach to curing T1D through transplantation of (differentiated) stem cell-derived beta cells that are capable of producing insulin in vivo [145]. This form of regenerative medicine influenced therapy relies on the transplantation of autologous stem cell grafts which can act in immunomodulation or assist in insulin production [146]. The aim of this form of therapy is to provide longitudinal resolve to patients suffering from T1D. This also contributes to the mission of precision medicine by providing a long-term solution to patient complications and removing the reliance on expensive, short term therapies such as insulin injections, which can be difficult to obtain and manage across the stratified sociocultural spectrum in the United States and globally [147]. The value added by clinical translation of stem cell therapy for T1D supersedes previous treatments in the temporal dimension due to its ability to provide functioning beta cells to the patient longitudinally, thus providing for a prolonged period of insulin production during which the patient may not need to rely on other drugs or therapies. Very quickly stem cell therapy can then alter the social landscape of treatment for T1D and similar autoimmune diseases through multiple dimensions by, providing relief to the patient both physiologically and financially. Due to the complexity of autoimmune diseases and their turbulent nature, it is the hope of many scientists that stem cell therapy can provide a long-term resolution to such diseases. It is evident that stem cell therapy has the potential to introduce a great deal of paradigm shifts in the current approach to treatment of autoimmune disease such as T1D.

Currently, there are various approaches to stem cell-based transplantation and therapy for T1D that are being explored in the clinic [148–150]. These approaches often involve immunosuppression of the patient to tolerable levels and subsequent transplantation of the autologous stem cells in the patient to avoid immune rejection and further patient autoreactivity [151]. One such study observed the role of autologous nonmyeloablative hematopoietic stem cell transplantation (AHST). Following this treatment, all but 1 of the 15 patients of various gender and age 14 to 31 were able to remain insulin independent for at least 6 months. The study also showed increased C-peptide levels and decreased anti-GAD antibodies, which is a clinically used biomarker for the diagnosis of T1D [148]. This clinical study is evidence of the longitudinal improvement in patient symptoms and physiological complications that result from T1D. Furthermore, it highlights the impact of a combined approach in which immunosuppression followed by AHST is considered the standard. Another study performing autologous stem cell transplantation for the treatment of T1D used mesenchymal stromal stem cells derived from patient umbilical cord, which were transplanted for treatment of T1D [150]. In this study, improvement in C-peptide levels and reduction in insulin dependence was observed for all patients who underwent treatment. This study highlights another type of stem cell, in this case mesenchymal stromal cells, that can be clinically used in transplantation models for diabetic patients. However, it is evident that these models may require imaging tools and modalities that

can allow for the visualization of such parameters as transplant density, biodistribution, viability, and immunogenicity [152–154]. This can permit both short term and longitudinal monitoring of the transplant in the patient and allow for timely intervention, as is the case in many post-transplant graft loss incidents [155]. It can also allow for the visualization of certain molecular (bio)markers that can indicate the existence of specific cellular states or functions [156–158].

Presently, the main modalities used for molecular imaging of stem cells and stem cell transplants are magnetic resonance imaging (MRI) or magnetic particle imaging (MPI) [159,160]. Each of these modalities rely on the utilization of superparamagnetic iron oxide nanoparticles (SPIONs) for targeted molecular imaging of extracellular and intracellular markers. These SPIONs have previously been altered for targeting of immune cells, islet cells, and stem cells amongst many other cell targets [161–163]. They have also been explored for monitoring cell transplants longitudinally [164]. This is particularly useful in the context of providing tools for monitoring of stem cell transplants for the treatment of T1D because of the possibility for post-transplant rejection, due to host immune rejection, issues with cell transplant procedure, or cell viability post-transplant in the patient. The use of nanoparticles to monitoring stem cell transplants during treatment of autoimmune diseases such as T1D opens new doors to paradigms of theranostics and precision medicine, as autologous stem cells are typically used for transplantation and therapeutic purposes. The dynamic array of moieties in the domain of radionuclides, small nucleic acids, and antibodies that can conjugate to nanoparticles provide a platform for targeting specific cells from a heterogenous distribution and performing various forms of combined therapy and imaging (theranostics) [165]. In the context of guiding and improving therapeutic outcomes, nanoparticle-based tracking of stem cells has provided a far more effective and reliable method in vivo when compared to conventional methods such as labeling cells with organic dye or directly labeling them with fluorescent probes, because of their optimal magnetic and optical properties. Although there are a wide variety of structural platforms and nanomaterials with which to engineer theranostic nanoparticles such as silicon and quantum dots, the most biocompatible and clinically used tool for contrast enhancement and targeted therapy are SPIONs. Several studies have highlighted the ability of SPIONs to image and track stem cells post-transplantation in both mice and humans. One such study labeled MSCs with SPIONs and encapsulated these in collagen-based microcapsules for monitoring of the cells post-transplantation [166]. Although this study focused on use of SPIONs for monitoring MSC transplantation for the treatment of myocardial infarctions, SPIONs can also be used for the labeling of differentiated beta cells derived from iPSCs for monitoring of transplant thereof in vivo [167]. Wang et al. have shown imaging of endogenous beta-cell mass through targeting of the glucagon like peptide 1 receptor (GLP-1R) [168]. This was done through conjugation of the exendin 4 to magnetic nanoparticles and subsequent injection of this probe in mice. The group was able to show specific accumulation of the probe in GLP-1R expressing endogenous beta cells and indicated the correlation between reduced signal interference with decreasing beta cell mass over time. This approach can be extrapolated to instances of imaging GLP-1R-expressing, stem cell-derived beta cell transplantations. This also provides a mechanism for the direct targeting of endogenous and transplanted beta cells, regardless of origin, to deliver interventional nanodrugs and therapeutic molecules in vivo. Additionally, these SPIONs have enabled the use of an emerging imaging modality of MPI. Prior studies have performed MPI of human islet cells labeled with dextran-coated SPIONs and transplanted under the left kidney capsule of mice [164]. However, the limitations of MPI result mainly from its inability to decipher viable vs. non-viable cell transplants, especially after a brief period of time where dead cells and their nanoparticles can undergo degradation and generate false positive signals that do not originate from live cells [169]. Despite these limitations, nanoparticles are gaining popularity in their use for monitoring such cell transplants and are continuously being explored as a platform.


**2.** *In vitro* **models**

#### **2.1 Proliferation assays**

Proliferation assays are assays used to check for cellular metabolism in active metabolic cells. They help to ascertain the viability of cells when treated with NPs to check for the toxic effects of the particles on the cells. One of the commonly used assays is the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. It is a colorimetric assay where 3-[4,5-dimethylthiazole-2-yl]-2,5 diphenyltetrazolium bromide (MTT) is enzymatically reduced by mitochondrial succinate dehydrogenase to formazan crystals, an insoluble product, in the mitochondria of live cells. The breakdown is spectrophotometrically measured to estimate cell viability. In this assay, there is a linear relationship between the color formed when the breakdown to formazan occurs or the absorbance when measured and the viability of the cells. It is a very sensitive and quantitative assay [13, 14]. The MTT assay proves to be advantageous because it produces results quickly under a maximum of 3 to 4 hours. It is also reproducible and does not require manipulation of the target cell [15].

#### In Vitro*,* In Vivo *and* Ex Vivo *Models for Toxicity Evaluation of Nanoparticles: Advantages… DOI: http://dx.doi.org/10.5772/intechopen.111806*

The Alamar Blue Assay is also a colorimetric indicator assay characterized by the reduction of a blue, non-fluorescent dye to a pink-colored substance known as Resazurin [16]. It is both a qualitative and quantitative assay where colorimetric measurement can be taken or physical observation of color change in assessing cell viability or treatment toxicity. It is taken at a wavelength of 570 and 600 nm or 540 and 630 nm using the spectrophotometer. It is excited and emitted at wavelengths of 530–560 and 590 nm respectively [17]. Alamar Blue assay is highly sensitive, requires low cost, it's easy and safe to use, non-radioactive, and can be used for a large number of sample. It can also be used for both quantitative and qualitative analysis. Another method used to assess proliferation is the incorporation of [3 H] thymidine into the DNA of proliferating cells during the S phase of the cell cycle, with the use of autoradiography [18]. As the cells proliferate, new DNA strands are formed. The tritiated thymidine, a radioactive nucleoside, then enters into the new chromosomal strands as the cells divide [19]. [3 H] thymidine incorporation method is also widely used in immunological studies because of its high throughput and direct measurement of cell proliferation. It is also sensitive [19]. Another commonly used assay, clonogenic assay is used to assess cell survival and reproductive ability after treatment. The ability of a single cell to reproduce a colony is checked [20].

As shown in **Figure 2**, MTT assay has its limitations that affect the interpretation of data. Due to the formation of formazan, an insoluble dye, in MTT assay, other assays like XTT (2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) are used. With XTT, in the presence of mitochondrial dehydrogenase enzymes, a soluble water product is produced. However, both assays also faced the limitation of reaction under other conditions like acidic media, additives found in culture media, polyphenols, the presence of NPs and O− 2. These gives a false positive result of cell viability [22–24]. In a study carried out on Chinese hamster ovary cells (CHO-K1) after exposure to nano-TiO2, which increases the formation of O− 2, MTT and XTT assay were found to produce an inaccurate analysis of cell viability, because of the ability of the superoxide produced by the NPs to reduce the two salts [25]. Other factors such as cell number, the concentration of the MTT reagent, the treatment applied to the cells, and the toxic effect of the MTT itself has to be critically considered when analyzing the results from the assay. When prostate cancer cell lines (PC-3) were treated with polyethylene glycol-coated gold NPs (Au-PEG-NPs) at a concentration of 5 nm, there was an observed significant optical interference with the MTT assay. However, this effect could be minimized by washing the cells and removing the supernatant before MTT incubation and using appropriate blanks [21].

A study on the *in vitro* assessment of the toxicity of carbon based NPs HiPco single-walled carbon nanotubes (SWCNT), arc discharge SWCNT and Printex 90 carbon black NPs was done to initiate a more reliable method with the use of clonogenic assay [26]. This study was performed to avoid the interaction of carbon based NPs with the indicator dyes used in colorimetric assays, leading to inaccurate interpretations on toxicity studies [27, 28]. Graphene oxide and TiO2 NPs have also been observed to interact with the dyes [29, 30]. This assay rules out the possibility of the nanoparticle reacting with the assay itself. It is recommended as a very useful tool for testing cytotoxicity since colony number and size are taken into account. This makes it an effective differentiating tool between cell viability and cell proliferation [26]. It is also a sensitive method since colony size depicts the division rate and proliferation after the cells are treated [31].

#### **Figure 2.**

*Factors affecting the final optical density (OD) measurements in the MTT assay. These include the concentration of MTT reagent and the proportion that actually enters the cell, cellular metabolic activity (which is highly dependent on a multitude of variables including treatments to the cells, biological effect of culture media, cell density, and impedance of cell metabolism due to toxic effects of MTT), cell number, timing of formazan crystals extrusion (which could impede further MTT uptake), chemical interference such as abiotic reduction of MTT by culture media, the tested treatment, or released cellular content, optical interference by all the background components, time of incubating cells with MTT reagent and/or tested treatment, and ultimately the optical measurement. Chemical structure of MTT and formazan are illustrated inside the cell: MTT consists of a tetrazole ring core containing four nitrogen atoms (1) surrounded by three aromatic rings including two phenyl moieties (2) and one thiazolyl ring (3). Reduction of MTT results in disruption of the core tetrazole ring and the formation of formazan. Red arrows and the "-" sign indicate disruption of MTT reduction on the normal metabolic activity of the cells and the impeding effect of the formazan crystals (when presenting on the cell surface) on further uptake of MTT reagent by cells [21].*

For Alamar Blue assay, because of its photosensitivity, it is affected by light exposure and has to be done in the dark. Cell density is also a factor that can affect the assay reading, hence cells must be cultured in high population to prevent slow growth and allow adequate dye reduction. The assay is also limited to a pH range of 7.0 and 7.4, and optimum temperature of 37°C. Longer incubation times could also lead to the reduced dye being bleached to a pink color. This quenches the fluorescence and could cause misinterpretation of the fluorescent signals. There is also the need for a positive and negative control to rule out non-specific interaction with the chemistry [17]. Specifically, with the use of nanomaterials, it is necessary to check toxicity using a combination of other assays because of the diversity and physiochemical properties of the nanomaterials, which could affect the cell and also interfere with the chemistry of the assay [32, 33].

The [3 H] thymidine proliferation method is toxic and also requires radioactive facilities, which can be very expensive. Also, there is the need for proper waste management of the radioactive materials used with this method [34, 35]. It has also been found to interfere with the target cells by inducing cell cycle arrest, apoptosis and fragmenting the DNA [34, 36, 37].

One of the major cons of Clonogenic assay is that it is time-consuming as it takes about 10–14 days to perform. This is quite long compared to the other assays described above which take only a few hours to a day [38].

In Vitro*,* In Vivo *and* Ex Vivo *Models for Toxicity Evaluation of Nanoparticles: Advantages… DOI: http://dx.doi.org/10.5772/intechopen.111806*

#### **2.2 Apoptosis assays**

Apoptosis Assay is another assay carried out to evaluate the cytotoxicity of nanomaterials. It measures the extent of DNA damage and cell death when cells are treated. The release of free radicals due to oxidative stress is a pointer to DNA damage and nanomaterials have been found to elicit such reactions [39, 40]. One of the methods used to study apoptosis is the Annexin-V assay. This assay works on the principle of annexin-V binding to phosphatidylserine (PS) which is externalized on the plasma membrane due to activation of caspase-dependent pathway. In combination with Propidium Iodide (PI) which stains the nucleus indicating the last stage of cell death, both dead and apoptotic cells can be identified [41, 42]. Annexin-V and PI assays have been used to detect apoptosis in HeLa and human HepG2 hepatoma cells treated with gold and silica NPs respectively [43, 44]. Annexin-V does not penetrate the cell but only binds to phosphatidylserine on the extracellular membrane of the cell; hence it does not cause cell damage or affect the intracellular components of the cell. It is highly sensitive as it binds to only PS amidst other molecules on the cell surface. It produces bright fluorescent signals and it's easy to perform [41]. There are different options available for its labeling and also it can be performed both *in vitro* and *in vivo* [45].

Comet assay is used to determine DNA damage, single- and double-stranded DNA breaks and the mutagenicity of treatment both *in vivo* and *in vitro* [46–50]. It is one of the commonly used assay to assess the genotoxic effects of engineered nanomaterials like TiO2, SiO2, Zinc oxide NPs [51–55]. Damaged bases are detected when nucleoids are incubated with endonuclease III and formamidopyrimidine DNA glycosylase (FPG), specific to oxidized pyrimidine and purines, respectively [56]. Comet assay is a sensitive technique that is quick, cheap and easy to do [57]. It has been used both *in vitro* and *in vivo* and hence can be used to check the toxic effects of treatment in a particular organ or tissue and on any animal model [49, 58]. It can also be done on the first or specific site of contact.

TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) assay is a commonly, wisely used tool for the detection of DNA damage caused by both apoptosis, DNA fragmentation and necrosis, caused by exposure to toxic materials [59–61]. When DNA fragmentation occurs, the 3′ hydroxyl termina becomes free. Then the enzyme terminal deoxynucleotidyl transferase incorporates labeled dUTP into the free end, which causes the staining observed in the assay [62]. It can be detected by light microscopy, fluorescence microscopy or flow cytometry [63]. TUNEL assay is fast as it can be completed within 3 hours. It has high sensitivity and versatile with the use of various techniques, as single cells can be detected using fluorescence microscopy or few cells using flow cytometry [63, 64].

Flow Cytometry is a very common and versatile technique used in the toxicity analysis of nanomaterials when applied to cells. It works on the principle of detecting fluorescently labeled cells when light beams are passed through the cell suspension. The technique analyses both the size and granularity of the cell populations, which gives a characteristic of the type of cells being studied [65]. With the use of flow cytometry, large populations can be analyzed with valid statistical results and this is done within a short period of time. This method can be used to evaluate the uptake of NPs by the cells, cell death, and expression of certain proteins [66].

However, these assays have their own set of limitations. For example, Annexin-V assay cannot be effectively used to obtain high throughput due to strong background signals when there are unbound labeled annexin-V. It can be difficult to optically

image tissues when stained with annexin-v because of its short lifetime and slow diffusion in the body [67]. It can produce false positive results when it binds to negatively charged aldehyde adducts [68]. Also, Engineered NPs have been found to interfere and hinder the detection of DNA damaged from oxidation with the comet assay when ions are released when the particles are dissolved [69]. Cell toxicity, even in the absence of DNA damage, can lead to false-positive results in Comet Assay. There are also variability of protocols from the assay which can make it difficult to compare results with other laboratories, as there is no standardization [57]. For TUNEL assay, there is the limitation of non-specific staining because it labels all the free 3′ hydroxyl termini end irrespective of the cause of it. Hence, it can label nonapoptotic cells, which gives a false positive result. This means it can stain cells undergoing DNA repair, cells secretes factors causing proliferation in neighboring cells, or cells damaged through other means [70]. It is also costly. For flow cytometry, freshly prepared samples are required. If samples are kept for a long period of time before analysis, it could affect the cell properties and the analysis. Also, the use of the flow machine requires competent and skillful hands to accurately analyze and interpret the data [71]. NPs smaller than 100 nm experience low light scattering which could reduce the sensitivity of the detection [72].