**7. Acknowledgement**

270 Biomedical Science, Engineering and Technology

In 2004, Sculptra™ [poly(*L*-lactic acid)] was approved by the FDA as the first injectable facial "volumizer" in the treatment of lipoatrophy due to its significant therapeutic effectiveness (Burgess & Quiroga, 2005). The lipodystrophy syndrome is associated with the usage of highly active antiretroviral therapy (HAART) containing protease inhibitors or nucleoside reverse transcriptase inhibitors for HIV patients. The action mechanism of Sculptra™ is via stimulation of the fibroblastic activity with generation of collagen and other connective tissue fibers. In addition, it acts as dermal matrix adding support by thickening the dermis (Vleggaar & Bauer, 2004). Moreover, PLA can help improve the appearance of scars due to acne, surgery, trauma, or suture (Lowe & Beer, 2005, as cited in Beer & Rendon,

Due to the multiple desirable characteristics of PLA including renewability, biocompatibility, transparency, and thermoplasticity, it is being used or is a potential candidate for many consumer and biomedical applications (Jamshidian et al., 2010). Ever increasing environmental concerns associated with conventional polymers derived from petrochemicals lead to constantly expanding applications for PLA since its discovery in 1932

In previous years, the most negative point of PLA was its higher price as compared with petrochemical-based polymers. Today, by optimizing the LA and PLA production processes, and with increasing PLA demand, a reduction in its price can be achieved. The price of PLA is currently much lower than in previous years. Meanwhile, PLA is mainly synthesized in the industry by ROP employing tin(II) bis(2-ethylhexanoate) (SnOct2) as a catalyst, which has been approved as a food additive by the FDA, but the potential toxicity associated with most tin compounds cannot be ignored for biomedical applications. Scientists all over the world are now exploring novel, well-defined catalysts with good biocompatibility, high catalytic activity, low toxicity, and excellent stereoselectivity. This should remain an everlasting interest area. Finally, the possibility of obtaining 100% biosourced opens the way for PLA to become more independent from petrochemical-based

However, the major disadvantages of PLA such as its poor ductility, slow degradation rate, and poor hydrophilicity somewhat limit its applications. The modification of PLA bulk and surface properties has thus become crucial to increase its applicability. Many of the bulk and surface modification strategies discussed above have been designed to tune the PLA surface properties according to the demands of biomedical applications. Unfortunately, all these established methods for surface modification are somewhat deficient and while they provide control over the wettability, degradation rate, and functionality, it is still compulsory to minimize their negative impact on PLA bulk properties. Thus a combined modification strategy (e.g. irradiation followed by grafting) or a better balance of PLA surface and bulk properties should be sought. Ideally, with respect to a better balance of properties and shorter modification times, one-step approaches need to be developed because it is time-consuming to carry out surface and bulk modifications separately, and the solvents and reagents involved in multiple modification steps tend to affect PLA properties

All these modification strategies aim at tailoring the properties of PLA-based materials for certain applications. Fortunately, more and more encouraging results have been reported,

2006).

**6. Conclusions**

by Carothers at DuPont.

significantly.

polymers, free of environmental and health concerns.

It was supported by the Natural Science of Hubei Province for Distinguished Young Scholars (No. 2008CDB279) and the Key Technologies R & D Program of Hubei Province (20091933), the Fundamental Research Funds for the Central Universities, Huazhong University of Science and Technology (2010JC016) and the National High Technology Research and Development Program ("863" Program) of China (No. 2008AA10Z339). The authors are also grateful to Dr. Wen-bin Zhang from the University of Akron for his valuable suggestions during the preparation of this manuscript.

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*Review* **Kramers' Theory and the Dependence of Enzyme Dynamics on Trehalose-Mediated Viscosity**

#### **José G. Sampedro 1,\*, Miguel A. Rivera-Moran 1 and Salvador Uribe-Carvajal 2**

Received: 29 April 2020; Accepted: 29 May 2020; Published: 11 June 2020

**Abstract:** The disaccharide trehalose is accumulated in the cytoplasm of some organisms in response to harsh environmental conditions. Trehalose biosynthesis and accumulation are important for the survival of such organisms by protecting the structure and function of proteins and membranes. Trehalose affects the dynamics of proteins and water molecules in the bulk and the protein hydration shell. Enzyme catalysis and other processes dependent on protein dynamics are affected by the viscosity generated by trehalose, as described by the Kramers' theory of rate reactions. Enzyme/protein stabilization by trehalose against thermal inactivation/unfolding is also explained by the viscosity mediated hindering of the thermally generated structural dynamics, as described by Kramers' theory. The analysis of the relationship of viscosity–protein dynamics, and its effects on enzyme/protein function and other processes (thermal inactivation and unfolding/folding), is the focus of the present work regarding the disaccharide trehalose as the viscosity generating solute. Finally, trehalose is widely used (alone or in combination with other compounds) in the stabilization of enzymes in the laboratory and in biotechnological applications; hence, considering the effect of viscosity on catalysis and stability of enzymes may help to improve the results of trehalose in its diverse uses/applications.

**Keywords:** trehalose; viscosity; enzymes; protein dynamics; Kramers' theory; protein stabilization; enzyme inhibition

#### **1. Trehalose in Biology**

The disaccharide trehalose has been used as food or therapeutic agent by humans since ancient times [1]. Trehalose is naturally found in the sweet cocoon (the capsule named trehala-manna) that protects the larvae of weevils, insects in the genus *Larinus* [1–4]. Pure trehalose crystals were isolated first from the ergot of rye by Wiggers (1832), then the carbohydrate was identified as the main component of trehala-manna (from which it takes the name trehalose) by Berthelot (1858) and identified as a non-reducing disaccharide formed by two glucose units [5].

Trehalose (α-D-glucopyranosyl-1, 1-α-D-glucopyranoside) was found at high concentrations in diverse organisms [6] and under stress conditions [4], including yeast and fungi [7,8], plants [9], and others [6]. Trehalose, in addition to forming part of cellular structures [6], has been claimed to behave as a reserve carbohydrate as it is usually found in spores of yeast, fungi, and bacteria [4,6–8,10], and also as a biostructure stabilizer, namely of proteins and membranes during harsh environmental conditions [4], and even in the pathogenesis of virulent bacteria (like actinomycetes and mycobacteria) and fungi [11,12]. Currently, the metabolic pathways for trehalose biosynthesis and hydrolysis, and their regulation are well known [8–10,13,14], and in some organisms, trehalose metabolic pathways seem to have a major role in the regulation of other metabolic processes [7,8].

Among the diverse stress conditions where trehalose is synthesized and accumulated, dehydration (or the phenomenon of anhydrobiosis) has been that which has attracted the most interest (because of its potential industrial applications) [5,15–17], namely in food and pharmaceutics. Van Leeuwenhoek (1702) first described the phenomenon in bdelloid rotifers [18,19]. Since then, other organisms have been recognized to endure the absence of water, such as nematodes, brine shrimp cysts, yeast, and some plants [4,6,18].

Experiments have demonstrated the effectiveness of trehalose to preserve the structure of proteins and membranes in the dry state [16,20,21]. Nonetheless, the role of intrinsically disordered proteins (stress proteins) protecting organisms that synthesize little to no trehalose seems to be important [22,23]. In nature, the dehydration process in anhydrobiotic organisms seems to be slow, i.e., water is removed in minute amounts leading to cell desiccation [16]. At the same time, trehalose begins to be synthesized, reaching high concentrations in the cytoplasm, while a small amount is exported (trehalose is required at both surfaces of the plasma membrane) [24]. Although cell survival does correlate with the amount of trehalose present in the cell [16,19], other biochemical responses contributing to anhydrobiosis (or acting synergically with trehalose) seem to take place simultaneously to trehalose accumulation [16], e.g., the synthesis of heat shock proteins [25].

Physically, under optimum growth conditions (nutrients, temperature, etc.) the cell cytoplasm is a crowded space containing a great diversity of molecules, hence inherently highly viscous [26–29]. Then, during dehydration, the accumulation of trehalose increases viscosity even further as water-loss proceeds [30–34]. Hence, viscosity increases up to finally reach the glassy state [34,35]. Here, although the interaction of trehalose with proteins has been subjected to some controversy [36], the effect of trehalose on the maintenance of the integrity of membranes (via the interaction with phospholipid headgroups) is relatively well understood [16,37]. Nonetheless, what is certain is that viscosity (the glassy state) generated by trehalose is high. Here proteins and other bio-structures are "arrested", which in turn preserves both the structure and function of proteins [34,38–40].

In microorganisms, trehalose is also synthesized in the stationary phase of growth. In yeast cultures when nutrients are exhausted, cells enter a quiescent state known as the stationary phase [41], where the metabolic rate is slowed and cell division stops (arrested cell cycle) [42,43]. Notably, during entry to the stationary phase, trehalose is accumulated at high concentrations (up to 20% in dry weight), as well as glycogen (up to 8% in dry weight) [42]. The cells at the stationary phase thus become denser, heavier, and cytoplasm viscosity increases [31,32,42]. In this regard, trehalose seems to have a role in the exit of yeast from the stationary phase besides [42]; however, no specific role of trehalose in cell metabolism under the quiescent state has been described. Instead, cells in the stationary phase display high thermotolerance, possibly due to trehalose accumulation [4,21].

#### **2. Trehalose, Water, and Proteins**

Trehalose/water interaction is the basis for protein stabilization by trehalose [36,44–46]. Water is the medium where most biochemical processes take place and the hydration water has a fundamental role in protein structure and function [47–49]. In this regard, water molecules seem to be more uniformly distributed on the surface of globular proteins when compared to those on intrinsically disordered proteins (IDPs) [48,50]; despite that, the arrangement of tetrahedral water molecules is more disordered on the surface of globular proteins than on IDPs [50]. The above is probably due to the highly irregular surface of globular proteins. Interestingly, the dynamics of hydration water on proteins are different from bulk water molecules [47,48,51–53]. In solution, trehalose molecules display a relatively rigid conformation and slow translational motion (self-diffusion) [46,54]. Besides, trehalose molecules interact through trehalose–trehalose hydrogen bonds [44,55,56]; while trehalose–water hydrogen bonds are weaker [56]. This seems to facilitate vitrification [46], i.e., by allowing water molecules to escape from the system. Additionally, trehalose appears to be physically (preferentially) excluded from protein surfaces [57,58], thus promoting preferential hydration [45,59–61]. This is while maintaining the disordered arrangement of hydration water molecules [50]. Nonetheless, the dynamics

of hydration water appears to be slowed down by trehalose and consequently, dynamics of the amino acid residues in proteins decrease [45,48,61–67], e.g., in C-phycocyanin (CP), trehalose slows down the dynamics of the hydration water by an order of magnitude when compared to the dynamics of bulk water [68]. As a result, protein processes where structural dynamics are a major component become affected by the presence of trehalose in the medium, namely folding, unfolding, thermal and cold inactivation, oligomerization, aggregation, catalysis, and even the equilibrium between catalytic states (Figure 1). The thermodynamic effect of trehalose would be to increase the energetic barrier between different structural states (Figure 1).

**Figure 1.** Energy landscape of diffusion-dependent protein processes. High medium viscosity (η) increases the free energy height (red line) of the interconversion between conformational states in proteins (blue line). (**a**) Structural conformational states in enzyme catalysis, (**b**) conformational equilibrium between the *T* and *R* states in a cooperative enzyme regarding the model of Monod, Wyman, and Changeux [69,70], (**c**) three-state protein unfolding (N ↔ I ↔ U); formation of the intermediary (I) may involve the dissociation of monomers from the oligomer, (**d**) two-state protein unfolding (N ↔ U).

#### **3. Protein Dynamics and Catalysis**

Proteins, like most polymers, display inherent movements inside their structure [71,72]. Some movements participate in the function, e.g., catalysis [71,73], while others are part of the protein vibrational state specific to the medium temperature [74–76]. Indeed, the function of proteins is defined by the dynamics of the three-dimensional structure [77–81]. The inter-conversion between different structural states determines the function of the protein [82]. For example, the motions of the enzyme structure are a major component in catalysis [73,78,83–86]. There exists ample evidence on the role of enzyme structural dynamics in catalysis [73,87–89]; currently, experimental and computational techniques have demonstrated enzymes are highly dynamic entities [90]. Importantly, the rate of the structural dynamics in enzymes whether internal, specific (like loops and domain), as a structural network, or global (collective) has been determined as the limit for the turnover rate (*k*cat) [71,91]. In this regard, protein crystallography has provided the structural details of enzymes at the atomic level [74,92,93]. Unfortunately, such a structure is just a snapshot of the protein structure that yields limited information about function [77,94]. Nonetheless, efforts have resulted in the successful crystallization of enzymes in complex with ligands which demonstrates the existence of structural dynamics related to catalysis [74,95]. In some fortunate cases, the crystallographic structures of enzymes obtained under different conditions allow the depiction of the catalytic cycle [71,73,96–98]; however, it is worth noting that these states usually are a (small) fraction of the infinite conformations the protein structure may sample [75,82]. Experimental methods to study protein dynamics include NMR relaxation, hydrogen-deuterium exchange, fluorescence, UV-Vis spectroscopy, Raman spectroscopy, infrared spectroscopy, and molecular dynamics simulations (MDS) [73,77,99]; nonetheless, the timescale of measurement usually is variable, e.g., MDS ranges in the ns scale and less, while hydrogen-deuterium exchange ranges between ms and s scale [77]. Importantly, most biological processes (such as enzyme catalysis) lay on the μs to ms time scale [71,73,77]. Here protein dynamics have been evaluated by different methods, e.g., in rotary proteins like FoF1-ATPase [100,101]. MDS has emerged as a very helpful technique to study protein dynamics and function [72,88,102]. The position of any given atom in the polypeptide chain may be known with more precision at any given moment [72], although, a high-resolution three-dimensional (3D) structure is a requisite [103]. Still, when the 3D-structure is not available, modeling is an option [104]. However, the in vitro experimental validation of the in silico results is desirable [49,80,105].

In enzymes, it is known that under isobaric conditions the structural dynamics are modulated by variations in temperature (thermal energy) and solvent composition (cosolvents, ions, polymers, etc.) [38,49,76,106–108], i.e., in Michaelian enzymes, some conformational states may be favored as the kinetics of state interconversion are affected [77,109,110]. The diverse conformational states in a "resting" protein/enzyme (in the absence of ligands) in solution result from side-chain fluctuations, movements of loops and secondary structures, structural domains, and collective global arrangements [82]; thus, before catalysis, enzymes probe multiple conformations [77,109,111]. In this sense, even the whole catalytic cycle may occur in some enzymes in the absence of ligands [73,112]. Notably, in the kinetics of cooperative enzymes, the Monod, Wyman, and Changeux model is used on the assumption that enzymes fluctuate between two states with high ("relaxed", *R*) and low substrate affinity ("tight", *T*) [69,113], while binding of ligands, allosteric effectors, and even phosphorylation of some amino acid residues, changes the equilibrium towards the high- or low-affinity state, accordingly [114].

#### **4. Dependence of Enzyme Catalysis on Medium Viscosity (Kramers' Theory)**

The viscosity of the crowded cell cytoplasm is higher than pure water [26,27,115–117]. Viscosity varies by cell type, e.g., in *Saccharomyces cerevisiae* cytoplasm it has been calculated to be ~10 cP [115]. Certainly, the viscosity of the cytoplasm is considerably higher in organisms at the quiescent state and during dehydration, osmotic stress, and at heat-shock as they accumulate viscosity generating solutes such as trehalose, glycerol, and others [115]. In the physics of fluids, viscosity is defined as the friction between molecules or the resistance to flow (molecular diffusion) in a liquid (or gaseous) system [115]. In this environment, the three-dimensional (3D) structure of enzymes fluctuates between different conformations [71,118,119]. Protein structural fluctuations are coupled to the movement of water molecules at the hydration shell, and these in turn, to the dynamics of the molecules present in the bulk solution [49,120]. Recently, studies on the translational diffusion of hydration waters concluded that protein function may be modulated by sugar molecules [48,64]. Hence, the inhibition or hindering of protein motions, whether by a decrease in temperature or by changing the composition of the suspending medium, leads to decreased catalysis [85,121–124].

Specifically, trehalose promotes the hydration of the protein surface (preferential hydration) [60,61,125], but it also diminishes the dynamics of hydration water [61,64]. As a result, the motion of amino acid residues is slowed down, and the rate constant of enzyme structural fluctuations becomes dependent on the viscosity of the medium [47]. Certainly, the effect of medium viscosity on enzyme catalysis has already been considered [118], when the dependence of catalysis on viscosity was rationalized to the inhibition of enzyme fluctuations according to Kramers' theory [106,118,120,123,126–128], as described by Equation (1):

$$k = \frac{\mathbf{A}}{\eta} e^{-\Delta/k\_B T} \tag{1}$$

where Δ is the height of the potential barrier, *k* is the rate constant of catalysis (*k*cat), *A* is a function of structural parameters characterizing the potential energy profile, *k*B is the Boltzmann constant, and η is the viscosity of the medium. Equation (2) is an actualized version of Equation (1) when using the enzyme kinetic parameter *V*max (*V*max = *k*cat·[*E*t]).

$$V\_{\text{max}} = \frac{\mathbf{A}}{\eta} e^{-\Lambda Ul/RT} \tag{2}$$

where −Δ*U* is the height of the potential barrier, and *R* is the gas constant. Notably, Equation (2) has been useful to analyze the inhibition of enzyme catalysis by the presence of trehalose [31,32,129,130]; thus indicating that trehalose does inhibit the rate of catalysis by hindering the structural fluctuations of enzymes during the catalytic cycle [31,32,108,130]. *V*max becomes inversely proportional to the viscosity (*V*max α 1/η) of the medium, and a straight line is usually observed when plotting *V*max versus η−1 [31,32,108,130]; i.e., as viscosity increases, enzyme activity decreases [32]. Importantly, the viscosity generated by trehalose in the bulk solution would affect mainly the largest structural motions involved in catalysis (Figure 1) [31,129,130]. For example, in the plasma membrane H+-ATPase from yeast, the motion of the N-domain (a globular structure of ~16.5 kDa) toward the P-domain is required to transfer the γ-phosphate of ATP to the Asp residue [131]. Notably, the activity of the H+-ATPase decreases linearly as the viscosity generated by trehalose increases (Figure 2a) [31]. This behavior is in agreement with Equation (2), as described by Kramers' theory [31]. Similarly, the H+-ATPase from plants is inhibited linearly by the viscosity generated by the presence of sucrose (the natural disaccharide in plants) [132]. Additionally, the kinetics of pyruvate reduction by lactate dehydrogenase (LDH) from rabbit muscle is dependent on medium viscosity, as LDH catalysis depends on the movement of a loop on the active site [130,133]. The LDH *V*max is dependent on medium viscosity as expected from Kramers' theory (Figure 2b) [130]. Notably, the viscosity generated by trehalose increases the activation energy (*E*a) in both enzymes, H+-ATPase, and LDH [31,130] (Figure 3), as suggested in Figure 1a.

**Figure 2.** Dependence of enzyme catalysis (30 ◦C) on medium viscosity as described by Kramers' theory. (**a**) Viscosity dependence of *V*max of plasma membrane H+-ATPase from *Kluyveromyces lactis*. Adapted with permission from Sampedro et al. [31]. Copyright © 2020, American Society for Microbiology; (**b**) viscosity dependence of *V*max of lactate dehydrogenase (LDH) from rabbit muscle. Adapted with permission from Hernández-Meza and Sampedro [130]. Copyright © 2020, American Chemical Society.

**Figure 3.** Plot of the activation energy (*E*a) of enzyme catalysis versus trehalose concentration. (**a**) Plasma membrane H+-ATPase [31]. Reprinted with permission from Sampedro et al. [31]. Copyright © 2020, American Society for Microbiology; (**b**) LDH [130]. Reprinted with permission from Hernández-Meza and Sampedro [130]. Copyright © 2020, American Chemical Society. The activation energies were calculated as described by the Arrhenius equation (*kcat* = A·*e*−*Ea*/*R*·*T*) using the enzymes *V*max values (*V*max = *k*cat·[*E*t]).

Enzymes do display catalysis dependence on viscosity as described by Kramers' theory even when using viscogens others than trehalose; e.g., a decrease in the rate constants (*k*cat) is observed in the CO2 hydration and HCO3− dehydration by carbonic anhydrase (CA) [134], the ATPase hydrolysis in chloroplast coupling factor (CF1), myosin and meromyosin [135,136], the deacylation step of subtilisin BPN catalyzed ester hydrolysis [137], the thioesterase activity of fatty acid synthetase (FAS) [138], the carboxypeptidase-A (CPA)-catalyzed benzoylglycyl-L-phenyl lactate hydrolysis [139], the cysteine protease activity of human ribosomal protein S4 (RPS4) [140], the DNA polymerizing activity of polymerase β (POLB) [141], the sugar cleaving enzyme oligo-1,6-glucosidase 1 (MalL) [142], the indole-3-glycerol phosphate synthase (IGPS) from *Sulfolobus solfataricus* [105], and in the electron transfer reactions of sulfite oxidase (SO), respiratory and photosynthetic complexes [143–145]. In extreme situations of viscosity, i.e., enzymes embedded in a glass-state environment where viscosity is infinitely high, the absence of protein motions leads halts catalysis [120]. The modulation

of protein/enzyme dynamics by viscosity has been also recently demonstrated by molecular dynamics simulation (MDS) [146] using the small protein factor Xa [147,148], and ssIGPS [105].

Viscosity generating solutes also affects the enzyme Michaelis-Menten constant (*K*m = *k*−1+*k*2 *k*1 ) or the binding (*k*1) and dissociation (*k*−1) of the substrate from the binding site, when in the enzyme a structural diffusion is involved in either of these processes, e.g., glycerol increases the *K*m in phosphorylase B [149], sucrose increases the *K*m of polymerizing myosin [135], and trehalose decreases the *K*m of glucose oxidase (GOx) and glucoamylase from *Aspergillus niger* [150,151]. Therefore, the catalytic efficiency (*k*cat/*K*m) of enzymes varies as substrate affinity is decreased/increased by viscogens [149–151] by hampering structural motions in either ligand binding or dissociation. Interestingly, enzyme activation by trehalose has been reported in thermophilic enzymes like neutral glucoamylase from the mold *Thermomucor indicae-seudaticae* [152]. Similarly, in pyruvate kinase (PK) from *Geobacillus stearothermophilus* (a cooperative enzyme), trehalose increases the velocity of the enzyme reaction (Figure 4a) by increasing substrate affinity (*K*R and *K*T) in both "tight" and "relaxed" (T and R) states, and hindering the population of catalytically unproductive conformational states; nonetheless, trehalose does affect the equilibrium between the R and T states as described in Figure 1b, without changing the enzyme cooperativity (Hill number, *n*) (Figure 4b). This phenomena (enzyme activation) may be explained either by the effect of viscosity on the enzyme structural fluctuations (i.e., in the transition between R and T states) [70], by favoring the population of a given oligomeric state [153], by decreasing the protein volume [154] thus leading to the formation of the binding site [155], by preventing the sample of catalytically unproductive enzyme conformations [85], or by a combination of all of them. In this regard, the compressing effect of protein structure by viscosity generating solutes has already been observed with glycerol [155,156], sucrose [155,157], and trehalose [67,155], e.g., the volume of lysozyme decreases ~2% in the presence of trehalose [67].

Kinetics of protein processes others than catalysis are also affected by viscosity [158] as described by Kramers' theory, some of these are the escape of O2 from respiratory proteins [159] and hemerythrin [160], the heme pocked relaxation in hemoglobin after CO photolysis [161], the dissociation of CO from horse ferrocytochrome *c* (FCC) [162], and the CO binding to horse myoglobin [163].

**Figure 4.** Activation of pyruvate kinase (PK) from *G. stearothermophilus* by trehalose [164]. (**a**) Cooperative kinetics of PK in the absence (-) and presence (•) of trehalose. Considering the model of Monod, Wyman, and Changeux [69] in enzyme kinetics analysis: *V*max = 112 and 118 μmoles PEP·(min·mg protein)−1 in the absence and presence of trehalose, respectively. Affinity constants: *K*T = 328 and 11, and *K*R = 40 × 10−4 and 5 × 10−4 mM phosphoenolpyruvate (PEP) in the absence and presence of trehalose, respectively. The Hill number (*n*) was ≈1.5, both in the absence and presence of trehalose. The ratio [*T*0]/[*R*0] (*L*) increases ~13 times in the presence of trehalose. (**b**) Hill plot of PK cooperative kinetics of data in a (symbols as in a). The slope (*n*) value in both straight lines is ≈1.5. Adapted with permission from Rivera-Moran [164]. Copyright © 2020, M.A. Rivera Moran.

#### **5. The Application of Kramers' Theory on Heat-Mediated Enzyme Inactivation, Protein Folding, and Unfolding**

Heat-shock is another stress condition where trehalose may accumulate in the cytoplasm of some organisms [4,6,8,14,24,129,165], e.g., in yeast, trehalose accumulation (~0.5 M) has been related to the ability of the cells to survive at relatively high temperatures [21,165]. The number of conformational states a protein may sample is related to the surrounding temperature [75]. When raising the temperature, the structural fluctuations increase critically up to unfold/denature the three-dimensional (3D) structure of the protein as described in Figure 1c,d; in the denatured state, the number of possible conformations becomes immense [23,166,167]. The first event to occur in enzymes when undergoing unfolding is the loss of catalytic activity [168–171]. It has been proposed that the high viscosity generated by trehalose stabilizes proteins by inducing preferential hydration of the protein surface [60,61], preserving the hydration shell and decreasing its dynamics [172], increasing surface tension on the trehalose-water phase [59], and hampering protein motions that lead to unfolding [61,129,173]. Hence, thermal stabilization mediated by trehalose has been observed in a diversity of enzymes, such as bovine intestine alkaline phosphatase (BIALP) [174], human brain-type creatine kinase (hBBCK) [175], ornithine carbamoyltransferase (OCTase) [176], polyphenol oxidase (PPO) from yacon roots (*Smallanthus sonchifolius*) [177], rabbit muscle phosphofructokinase (PFK) [153], citrate synthase (CS) [25], cutinase from *Fusarium solani pisi* [178], larval *Manduca sexta* fat body glycogen phosphorylase B (GPb) [179], *Renilla* luciferase (Rluc) from *Renilla reniformis* [180], yeast cytosolic pyrophosphatase (PPi) [181], and others. In this regard, the MDS of thermal inactivation and unfolding of Rluc in the presence of trehalose showed that Rluc is stabilized through the inhibition of the structural fluctuations generated by high temperature [180], while in thermal inactivation of lysozyme and glucoamylase (GA) from *A. niger* and α-amylase (AA) from *Bacillus sp*, it was found that trehalose prevents the loss of protein hydrophobic interactions and helical structure, thus avoiding protein aggregation [150,182,183].

The kinetics of enzyme thermal inactivation in the presence of trehalose has been determined experimentally for some enzymes where usually first-order kinetics is observed [178,184,185]. Kinetic analysis showed that trehalose decreases the inactivation rate constant (*k*i) of xanthine oxidase (XO) from *Arthrobacter* M3 [184], PK from rabbit muscle [186], GOx from *A. niger* [151], and mushroom tyrosinase (MT) from *Agaricus bisporus* [185]. Therefore, trehalose increases the thermal stability of the enzymes by decreasing the inactivation rate constant (*k*i) [129]. Interestingly, thermal inactivation of the plasma membrane H+-ATPase from the yeast *Kluyveromyces lactis* is irreversible, showing biphasic kinetics with the presence of an active intermediary [129,173], i.e., a three-state mechanism (N → I → U) (Figure 1c) with respective inactivation rate constants for each phase, *k*i1, and *k*i2. Notably, physiological concentrations of trehalose stabilize the H+-ATPase by decreasing *k*i1 and *k*i2 [173,187]. The high viscosity generated by trehalose has been proposed as the mechanism of thermal stabilization of H+-ATPase [129,173]. Detailed analysis of the H+-ATPase thermal inactivation kinetics shows that the inactivation rate constants (*k*i1 and *k*i2) are inversely proportional to medium viscosity (*k*i α 1/η) as described by Kramers' theory and Equation (3) [129]:

$$k\_i = \frac{\mathbf{A}}{\eta} e^{-\Delta l I/RT} \tag{3}$$

where *k*i is the first-order inactivation rate constant, η the viscosity of the medium, *A* has the same meaning as in Equation (1) but regarding the enzyme inactivation process, Δ*U* is the potential energy barrier for inactivation, *R*is the gas constant, and *T* is the absolute temperature [129]. The plot of *k*i versus η−1 usually shows a straight line when a diffusive component on the thermal inactivation mechanism of the enzyme exists [129,173,188], e.g., the dissociation of monomers from a large oligomer [188]. Analysis of the decrease of the inactivation rate constant by trehalose shows that trehalose increases the activation energy (*E*a) for thermal inactivation (Figure 1) of AA [183], H+-ATPase [173], and GOx [151].

Particularly, in GOx where catalysis is dependent on the presence of the coenzyme FAD in the active site [151], the thermal inactivation occurs first through the formation of a molten globule and after that FAD is released (Figure 5a) [189], resulting in the irreversible loss of GOx activity [151,189]. Interestingly, trehalose decreases the rate constant of the thermally generated large structural changes hence preventing the formation of the molten globule [151]. Due to the hampering of the FAD release, the inactivation rate constant (*k*i) of GOx decreases [151] (Figure 5b), the *T*1/2 for FAD release is increased to higher temperatures (Figure 5b). In this unfolding mechanism, trehalose seems to act by compacting the protein structure of GOx, thus preventing the release of FAD, and as a consequence, GOx retains the catalytic activity [151]. A similar stabilizing pattern by trehalose was observed in OCTase against thermal inactivation [176].

The dependence of protein unfolding on the viscosity (Figure 1d) generated by trehalose presence has been observed in cytochrome *c* (Cytc) [140], phosphoglycerate kinase (PGK), β-lactoglobulin (βLG) [190], cutinase, and the small protein barstar (Bs) [169]. Like in thermal inactivation, trehalose decreases the rate constant of thermal unfolding (*k*U) by promoting the compactness of the unfolded state [191] and inhibiting large structural fluctuations [180]. Regarding protein folding, the high viscosity generated by trehalose affects the folding rate by inhibiting the structural dynamics as described by Kramers' theory [192–197].

**Figure 5.** Trehalose prevents the release of FAD during thermal inactivation/unfolding of glucose oxidase (GOx) from *Aspergillus niger* [198]. (**a**) Thermal inactivation mechanism of GOx [151,189]; (**b**) Effect of trehalose on the release of FAD from GOx during thermal inactivation; Trehalose (-) 0.0, and (•) 0.6 M. The data were fitted to the Pace equation [199] by non-linear regression; *T*1/2 = 61.3 and 64.3 ◦C in the absence and presence of 0.6 M trehalose, respectively. Adapted with permission from Paz-Alfaro [198]. Copyright © 2020, K.J. Paz-Alfaro.

Finally, the effect of the high viscosity generated by trehalose on macromolecular folding is being currently extended to other important biopolymers such as DNA [200,201] and RNA, e.g., in human telomere sequence [202] and RNA tertiary motif of the GAAA tetraloop receptor (TLR) [203], human telomerase hairpin (hTR HP) and H-type pseudoknot from the beet western yellow virus (BWYV) [204].

In cells, trehalose synthesis and accumulation helps to cope with harsh stress conditions. Trehalose affects enzyme activity (catalysis) and stability by hindering protein mobility through the high viscosity generated. Viscosity-mediated effects of trehalose (and other solutes) on enzymes are explained by Kramers' theory, i.e., the rate constant (*k*) of a given diffusive process in the enzyme structure is inversely proportional to the media viscosity. Currently, trehalose is widely used in enzyme applications (in the laboratory and biotechnology), whether alone or in combination with other compounds; therefore, it would be safe to consider the effect of viscosity on enzyme structural dynamics to improve the results of the use of trehalose.

**Author Contributions:** Conceptualization, J.G.S.; Methodology, J.G.S.; Validation, J.G.S. and M.A.R.-M.; Formal Analysis, J.G.S., M.A.R.-M., and S.U.-C.; Investigation, J.G.S. and M.A.R.-M.; Resources, J.G.S. and S.U.-C.; Data Curation, J.G.S.; Writing—Original Draft Preparation, J.G.S.; Writing—Review & Editing, J.G.S., M.A.R.-M., and S.U.-C.; Visualization, J.G.S.; Supervision, J.G.S.; Project administration, J.G.S.; Funding Acquisition, J.G.S. and S.U.-C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by FAI-UASLP México grant number C19-FAI-05-89.89 to J.G.S. and by a grant to S.U.-C.: DGAPA/PAPIIT Project IN203018.

**Acknowledgments:** M.A.R.-M. is a recipient of a Ph.D. fellowship of CONACyT México.

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

#### **References**

© 2020 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 (http://creativecommons.org/licenses/by/4.0/).


**4. Case studies from Asia and Africa**

#### **4.1. Applications in rural road improvement projects with community initiatives in Myanmar**

#### *4.1.1. Application at flooding area*

#### *<IP_ADDRESS>. Site selection*

In Kayin State of Myanmar, following the recommendations of the Department of Rural Development, village Y was selected for a rural road improvement project. Through meetings with the leader and members of the community and a field survey, a 120‐m length of road prone to flooding, in which the maximum water level was more than 1 m above ground level, was identified for improvement. When flooded, the community must use small boats to gain access to schools, hospitals, and markets. Accidents have occurred, and two school‐age children were killed in past floods.

#### *<IP_ADDRESS>. Design*

To raise the road surface above the maximum water level, an embankment with a Do‐nou retaining wall was designed, as shown in **Figure 11**. The existing ground surface was pro‐ tected with single‐column Do‐nou layers filled with soil mixed with cement. The embank‐ ment was supported with double‐column Do‐nou layers, the inner filled with *in‐situ* soil and the outer with soil mixed cement. The *in‐situ* soil was judged appropriate for mixing with cement for use as filling material into Do‐nou bags and use as the embankment mate‐ rial. The column of Do‐nou layers was <1.5 m in height, and the slope ratio was rise of 1 over run of 0.5. Trials of the mixing ratio of cement and *in‐situ* soil identified an appropri‐ ate ratio of 1:10 by volume.

#### *<IP_ADDRESS>. Construction*

Construction was performed during the dry season, from March to April in 2014, with 49 villagers working 5–6 h/day. With these resources, the length of 120 m was completed, with an embankment and Do‐nou retaining wall (**Figure 12**).

Spot Improvement of Rural Roads Using a Local Resource‐Based Approach: Case Studies from Asia and Africa http://dx.doi.org/10.5772/66109 103

**Figure 11.** Design of road embankment with Do‐nou retaining wall.

**•** Improving the most problematic road sections is only a partial solution;

organized to work together productively.

**4. Case studies from Asia and Africa**

*4.1.1. Application at flooding area*

children were killed in past floods.

ate ratio of 1:10 by volume.

embankment and Do‐nou retaining wall (**Figure 12**).

*<IP_ADDRESS>. Construction*

*<IP_ADDRESS>. Site selection*

*<IP_ADDRESS>. Design*

**Myanmar**

102 International Development

**•** The community must be motivated to improve the roads by themselves and must be

**4.1. Applications in rural road improvement projects with community initiatives in**

In Kayin State of Myanmar, following the recommendations of the Department of Rural Development, village Y was selected for a rural road improvement project. Through meetings with the leader and members of the community and a field survey, a 120‐m length of road prone to flooding, in which the maximum water level was more than 1 m above ground level, was identified for improvement. When flooded, the community must use small boats to gain access to schools, hospitals, and markets. Accidents have occurred, and two school‐age

To raise the road surface above the maximum water level, an embankment with a Do‐nou retaining wall was designed, as shown in **Figure 11**. The existing ground surface was pro‐ tected with single‐column Do‐nou layers filled with soil mixed with cement. The embank‐ ment was supported with double‐column Do‐nou layers, the inner filled with *in‐situ* soil and the outer with soil mixed cement. The *in‐situ* soil was judged appropriate for mixing with cement for use as filling material into Do‐nou bags and use as the embankment mate‐ rial. The column of Do‐nou layers was <1.5 m in height, and the slope ratio was rise of 1 over run of 0.5. Trials of the mixing ratio of cement and *in‐situ* soil identified an appropri‐

Construction was performed during the dry season, from March to April in 2014, with 49 villagers working 5–6 h/day. With these resources, the length of 120 m was completed, with an

**Figure 12.** Construction of road embankment with Do‐nou retaining wall: (a) Before construction; (b) construction of the embankment with Do‐nou retaining wall; (c) after construction.

The cost of construction is summarized in **Table 2**. The largest element was the cost of cement, at 36.6% of the total, followed by the cost of Do‐nou bags, and the transportation of tools and materials. The labour fee was not concern of the community members, and thus, this sum was paid into the Community Fund after construction was completed.

**Table 2.** Cost of construction.

#### *<IP_ADDRESS>. Impact*

One year later, an assessment was made of the construction site during the rainy season. This assessment confirmed that conditions remained good, as shown in **Figure 13**. The inhabitants of village Y subsequently applied the Do‐nou retaining wall construction method to other sections of road, after the project ended. The work was initiated by the community leader, and the material and transportation costs were covered by the Community Fund.

Interviews with villagers revealed the following positive impacts of the project:

**Figure 13.** Road condition 1 year after the completion, during rainy season.

#### *4.1.2. Application at delta*

#### *<IP_ADDRESS>. Site selection*

*<IP_ADDRESS>. Impact*

104 International Development

from class is decreased;

conducted;

project.

**•** Crops can be transported before spoiling;

One year later, an assessment was made of the construction site during the rainy season. This assessment confirmed that conditions remained good, as shown in **Figure 13**. The inhabitants of village Y subsequently applied the Do‐nou retaining wall construction method to other sections of road, after the project ended. The work was initiated by the community leader, and

**•** Students can attend schools from their home even during the rainy season, thus the absence

**•** The community knowledge of Do‐nou technology enables continuous maintenance to be

**•** The community has become better organized through the experience of working on the

the material and transportation costs were covered by the Community Fund.

**•** Patient can be transported to hospital more safely and quickly;

**Figure 13.** Road condition 1 year after the completion, during rainy season.

Interviews with villagers revealed the following positive impacts of the project:

**•** During the rainy season, boats are no longer needed to move around the village;

Ayeyarwady region of Myanmar is located at delta of the river. The sedimentary clay is prevailing. Following recommendations of a local NGO, village K in Ayeyarwady region was selected for a rural road improvement project. The people were used to construct their access roads by spreading the *in‐situ* clay dug out from the adjacent rice paddy. During rainy seasons, the surface becomes muddy so that even bikes and bicycles are not passable, while during the dry season, the road surface becomes firm but rough so the traffic has to pass slowly. Due to the inaccessibility of roads, during rainy seasons, children have to be absent from their classes. In order to enable the access to the neighbouring village all the year, a 1200‐m length of the existing road was targeted for improvement.

#### *<IP_ADDRESS>. Design*

Considering the current major traffic means in village K, that are bikes and bicycles, concrete pavement with 10 cm in thickness was constructed on the existing road surface as shown in **Figure 14**. In order to enable those traffic loads to go by each other, the two lanes with 60 cm in width were built. To protect the embankment from erosion caused by going up and down of the water level surrounding, Do‐nou filled with *in‐situ* clay were laid along the slope. Here, to address the vulnerability to ultraviolet light of Do‐nou bags, Do‐nou laid at 45° gradient was covered with the cohesive *in‐situ* clay soil. The vegetation occurs from the attached clay and the root spreads, which stabilize the slope without confinement effect generated from the bags.

**Figure 14.** Design of concrete pavement and Do‐nou retaining wall.

#### *<IP_ADDRESS>. Construction*

Construction was performed during the dry season, from January to May in 2014, with 20–50 villagers working 6 h/day. During the hottest season from March to May, the community managed to work at night, from 6 o'clock to midnight to avoid the heat (**Figure 15**). With these resources, the length of 1200 m was completed, with the concrete pavement and Do‐nou retaining wall.

The cost of construction is summarized in **Table 3**. The unit cost per m with the cross section as shown in **Figure 14** is USD 12.32. The largest element was the cost of gravel, at 27.7% of the total, followed by the cost of cement, and tools. The labour fee was paid for the skilful workers who assisted concrete pavement work. The community members participated in the road works have no concerns on their labour fee, thus this sum was paid into the Community Fund after construction was completed.

**Figure 15.** Construction at village K: (a) Covering Do‐nou with clay; (b) working at night; (c) bucket brigade for casting concrete.

**Table 3.** Cost of construction.

#### *<IP_ADDRESS>. Impact*

*<IP_ADDRESS>. Construction*

106 International Development

retaining wall.

concrete.

**Table 3.** Cost of construction.

after construction was completed.

Construction was performed during the dry season, from January to May in 2014, with 20–50 villagers working 6 h/day. During the hottest season from March to May, the community managed to work at night, from 6 o'clock to midnight to avoid the heat (**Figure 15**). With these resources, the length of 1200 m was completed, with the concrete pavement and Do‐nou

The cost of construction is summarized in **Table 3**. The unit cost per m with the cross section as shown in **Figure 14** is USD 12.32. The largest element was the cost of gravel, at 27.7% of the total, followed by the cost of cement, and tools. The labour fee was paid for the skilful workers who assisted concrete pavement work. The community members participated in the road works have no concerns on their labour fee, thus this sum was paid into the Community Fund

**Figure 15.** Construction at village K: (a) Covering Do‐nou with clay; (b) working at night; (c) bucket brigade for casting

Labour fee for skilful worker 60 Person day 217.79 1.5 Supervisor: 4.10 USD/day Assistant: 3.28

Labour fee for participants 354.24 2.4 Sum amount agreed with the community,

USD/day

paid to the Community Funds

**Item Quantity Unit Cost (USD) % Note** Gravel 212.2 m3 4088.60 27.7 Sand 85.0 m3 401.80 2.7 Cement (50 kg) 845 Bag 3440.31 23.3 Do‐nou bags 18,000 Bag 1697.40 11.5 Tools – – 3309.18 22.4 Transportation – – 1031.64 7.0

Others – – 242.26 1.7 Total 14,783.22 100.0 From the assessment one year after the completion, it was confirmed that conditions remained good, as shown in **Figure 16**. The interviews with villagers revealed the following positive impacts of the project:

**Figure 16.** Road condition: (a) Before construction; (b) condition 1 year after the completion.

On the other hand, as negative impact, the concerns on the increase in traffic accidents were raised. The training on traffic safety and installing the safety facilities, such as sign boards and bump, should be included in the project.

#### **4.2. Construction of a vented ford in collaboration with the provincial/municipal government, community and NGOs**

#### *4.2.1. Vented ford construction site*

In Nueva Vizcaya Province of the Philippines, the people of village C had suffered from flooding at the intersection of the river and the access road into the town during rainy seasons. Some sections of the road from the town to the village had been gradually paved with concrete by the provincial government, but no intervention had been made at the intersection with the river.

A Japanese NGO had trained the people of village C on the cultivation and marketing of organic agricultural products for income generation. The project had been implemented in close collaboration with local NGOs and provincial/municipal governments. However, the access problem at the intersection had constrained the project, and improvement was beyond the resources of the local governments. Surveying and construction started after the NGO consulted with the authors to seek solutions to this problem.

#### *4.2.2. Vented ford design*

Following a field survey with the municipal engineers, a vented ford with concrete slabs 4.0  m wide and 30.0 m in length was proposed, as shown in **Figure 17**.

The structure followed the basic pattern in the LBT manual [5], with Do‐nou filled with soil and cement substituted for masonry. The objectives were to promote participation by all of the community members and to shorten the construction time. The ratio of cement to gravel was 20% by volume. Layers of Do‐nou were combined vertically with penetrating reinforcement bars. To bear the water pressure at the upstream side, the concrete was cast to form a 20‐cm‐ wide wall between Do‐nou columns serving form.

The reinforced concrete pipes were backfilled with river gravel and with Do‐nou filled with gravel. Concrete pavement was constructed in accordance with the specifications for provincial roads in the Philippines.

Due to the land use near the intersection, the axis of the vented ford could not be made vertical to the river flow, but had to be slanted as shown in **Figure 17a**.

**Figure 17.** Plain and cross‐sectional view of the vented ford: (a) Plain view; (b) longitudinal section view; (c) vertical cross‐sectional view.

#### *4.2.3. Collaboration with provincial/municipal government, community and NGOs*

Three‐way discussions were held between the local government, village C, and the authors' NGO to discuss the design and clarify the role of each through the facilitations of the local and the Japanese NGO. The roles were allocated as shown in **Table 4**, and **Figures 18** and **19**.

**Table 4.** Role and contribution of each party.

the resources of the local governments. Surveying and construction started after the NGO

Following a field survey with the municipal engineers, a vented ford with concrete slabs 4.0

The structure followed the basic pattern in the LBT manual [5], with Do‐nou filled with soil and cement substituted for masonry. The objectives were to promote participation by all of the community members and to shorten the construction time. The ratio of cement to gravel was 20% by volume. Layers of Do‐nou were combined vertically with penetrating reinforcement bars. To bear the water pressure at the upstream side, the concrete was cast to form a 20‐cm‐

The reinforced concrete pipes were backfilled with river gravel and with Do‐nou filled with gravel. Concrete pavement was constructed in accordance with the specifications for provincial

Due to the land use near the intersection, the axis of the vented ford could not be made vertical

**Figure 17.** Plain and cross‐sectional view of the vented ford: (a) Plain view; (b) longitudinal section view; (c) vertical

consulted with the authors to seek solutions to this problem.

wide wall between Do‐nou columns serving form.

to the river flow, but had to be slanted as shown in **Figure 17a**.

m wide and 30.0 m in length was proposed, as shown in **Figure 17**.

*4.2.2. Vented ford design*

108 International Development

roads in the Philippines.

cross‐sectional view.

**Figure 18.** Work items managed using equipment provided by local government: (a) Excavation of the cut‐off‐wall and foundation; (b) transit mixers and water pump during casting concrete; (c) compaction of base course with roller.

**Figure 19.** Work items managed by the community: (a) Leveling the concrete surface; (b) filling the gaps of RC pipes; (c) laying Do‐nou.

The cost details prepared by the authors' NGO are shown in **Table 5**. For 48 days between March and April 2015, 20–30 people worked for 8 h/day on the project.

**Table 5.** Cost of construction.

#### *4.2.4. Maintenance by local governments and communities*

In October of 2015, the area was hit by the strongest typhoon in 15 years. The vented ford overflowed, and sediment was deposited at the upstream side. After the typhoon passed, it was observed that, although the ford was partially damaged, its stability was not affected. To restore the ford's functionality, the provincial government sent an excavator for emergency removal of the sedimentation (**Figure 20**). The neighbouring community voluntarily assisted with the emergency work.

The structure was confirmed to be stable even after the flooding, and the local government and communities took ownership of the ford.

**Figure 20.** Vented ford affected by a typhoon and emergency work done by the provincial government: (a) Overflow‐ ing; (b) deposited sedimentation; (c) removing the sedimentation by the excavator of the provincial government for emergency.

#### *4.2.5. Impact*

**Item Quantity Unit Cost (USD) % Note**

Cement (40 kg) 1620 Bag 8527.68 30.0 Do‐nou bags 9300 Bag 3124.80 11.0

Tools – – 839.55 3.0

Total 28,428.24 100.0

*4.2.4. Maintenance by local governments and communities*

**Table 5.** Cost of construction.

110 International Development

with the emergency work.

emergency.

communities took ownership of the ford.

RC pipe 70 pcs 3998.40 14.1 Inner diameter 0.9 m, thickness 0.1 m

RC bars 85 pcs 462.69 1.6 D10, D12, D16, 1 pcs = 6 m

Fuel 1354 Litre 947.12 3.3 For equipment and truck

Allowances 1380 Person day 7728.00 27.2 Minimum wage per day: USD 5.6 Lunch 1380 Person day 1545.60 5.4 USD 1.12 per person day

Gravel 217.00 m3 – – Provided by municipal government Stones 82.00 m3 – – For foundation, collected on site Transportation – – – – Two trucks with drivers, provided by

Equipment – – – – Excavator, loader, transit mixer, roller with

In October of 2015, the area was hit by the strongest typhoon in 15 years. The vented ford overflowed, and sediment was deposited at the upstream side. After the typhoon passed, it was observed that, although the ford was partially damaged, its stability was not affected. To restore the ford's functionality, the provincial government sent an excavator for emergency removal of the sedimentation (**Figure 20**). The neighbouring community voluntarily assisted

The structure was confirmed to be stable even after the flooding, and the local government and

**Figure 20.** Vented ford affected by a typhoon and emergency work done by the provincial government: (a) Overflow‐ ing; (b) deposited sedimentation; (c) removing the sedimentation by the excavator of the provincial government for

municipal government

operator, provided by municipal government

Gabion net 28 pcs 1254.40 4.4 1 m × 1 m × 2 m

The access to the local town from village C has been improved drastically (**Figure 21**). Recog‐ nizing the effectiveness of vented ford, the provincial government adopted the structure and applied the same construction method at a nearby intersection of the same river and a provincial road.

**Figure 21.** Conditions at the intersection with the river before and after construction: (a) Before the construction; (b) after the construction; (c) the conditions one year after the construction.

#### **4.3. Application to rural road improvement through capacity building of farmer/youth groups in Kenya**

#### *4.3.1. Rural road improvement and impact through capacity building of farmer and youth groups using Do‐nou technology*

Since 2008, Do‐nou technology has been used in Kenya for spot improvement using the local resource base. The approach has been transferred to 125 farmer and youth groups (**Figure 22**). Training and demonstration groups have been held, generally involving 25 members over 10– 15 days. These were organized at road sections identified as problematic by the group. Do‐nou technology has been used to build base courses, retaining walls at both the inlet and the outlet of culverts, and abutments of wooden bridges, as shown in **Figure 23**. In most cases, the drainage system was also improved.

**Figure 22.** Training/demonstration of spot improvement using Do‐nou technology: (a) Training at road side; (b) dem‐ onstration on Do‐nou technology.

**Figure 23.** Applications of Do‐nou technology during training/demonstrations with farmer groups: (a) Before construc‐ tion; (b) after the construction; (c) retaining wall at inlet of culvert; (d) Do‐nou abutment of wooden bridge.

The training/demonstration sessions helped the trainees and neighbouring communities appreciate the benefits gained from the road works, such as better access to schools and hospitals throughout the year, reduction in public transportation fares and reduced loss of agricultural product.

However, after the training was completed, the groups were unable to continue the road projects by themselves, due to difficulties in collecting the necessary materials, in particular, granular soil for use in the bags and wearing course.

Cooperation with and supports from other stakeholders was therefore necessary to enable the community initiatives to continue. Do‐nou technology and the concept of spot improvement using local resource‐based approach must be understood by stakeholders such as government institutes and private road construction clients.

#### *4.3.2. Dissemination of Do‐nou technology in Kenya*

**Figure 24** shows the progress of the dissemination of Do‐nou technology in Kenya.

Initially, to demonstrate improved access to markets of farmers, training and demonstrations were conducted with farmer groups on spot improvement using Do‐nou technology. These training sessions, and the outcome of the technology, were reported to local authorities and to the Ministry of Agriculture, which was keen to promote market‐oriented agriculture. How‐ ever, little support or cooperation was gained to enable the road work to be continued.

Spot Improvement of Rural Roads Using a Local Resource‐Based Approach: Case Studies from Asia and Africa http://dx.doi.org/10.5772/66109 113

**Figure 24.** Progress of the dissemination of Do‐nou technology.

**Figure 23.** Applications of Do‐nou technology during training/demonstrations with farmer groups: (a) Before construc‐

The training/demonstration sessions helped the trainees and neighbouring communities appreciate the benefits gained from the road works, such as better access to schools and hospitals throughout the year, reduction in public transportation fares and reduced loss of

However, after the training was completed, the groups were unable to continue the road projects by themselves, due to difficulties in collecting the necessary materials, in particular,

Cooperation with and supports from other stakeholders was therefore necessary to enable the community initiatives to continue. Do‐nou technology and the concept of spot improvement using local resource‐based approach must be understood by stakeholders such as government

Initially, to demonstrate improved access to markets of farmers, training and demonstrations were conducted with farmer groups on spot improvement using Do‐nou technology. These training sessions, and the outcome of the technology, were reported to local authorities and to the Ministry of Agriculture, which was keen to promote market‐oriented agriculture. How‐ ever, little support or cooperation was gained to enable the road work to be continued.

**Figure 24** shows the progress of the dissemination of Do‐nou technology in Kenya.

tion; (b) after the construction; (c) retaining wall at inlet of culvert; (d) Do‐nou abutment of wooden bridge.

agricultural product.

112 International Development

granular soil for use in the bags and wearing course.

institutes and private road construction clients.

*4.3.2. Dissemination of Do‐nou technology in Kenya*

Some of the farmer groups then established an association that undertook missions to improve rural access roads using their acquired knowledge of Do‐nou technology. It was considered advantageous for the association to negotiate with stakeholders to gain their support and cooperation. However, opportunities for collaboration were limited and ad hoc, because the association was not a qualified contractor.

However, Do‐nou technology was recognized by the Kenya Rural Road Authority following stakeholder meetings and field visits. The International Labour Organization funded a youth employment promotion project, in which youth groups were trained in Do‐nou technology for spot improvement of their residential area. Their representatives participated in the training at the Kisii Training Center (KTC), which is the public training institute for LBT, for a six‐week course on routine maintenance and business management. Ten of the 20 youth groups then established their own company, called Micro and Small Enterprises (MSEs), and began work. With the certification provided by the training course at KTC, the three companies registered as qualified contractors, allowing them to bid for government projects and thus expanding their business opportunities. Contracts for access road construction and other building works were also secured from other sectors apart from road administrators, such as health centers, schools, and churches.

In order to address the issue of youth unemployment meaningfully, the Kenyan government launched the policy, "Access to Government Procurement Opportunities", in which 30% of all government contracts were to be earmarked for youth, women, and persons with disability, without requiring competitive bidding against established firms [10]. The policy helped the youth groups that had learned Do‐nou technology and joined the training course at KTC to further expand their businesses.

Do‐nou technology was also recognized in the Roads 2000 Strategic Plan 2013–2017 [11], and a training course in routine maintenance and Do‐nou technology was established in the KTC program.

In 2015, the Ministry of Transportation and Infrastructure (MOTI) allocated funds amounting to about USD 229,300 to train 120 youth members in the KTC course on routine maintenance and Do‐nou technology. By May of 2016, about 30% of the graduates from the course had either established their own companies or registered as qualified contractors. MOTI is now planning to adopt a specification for Do‐nou technology.

#### *4.3.3. Involving communities in road projects*

Based on the experience of capacity building in Kenya, four patterns for involving the local communities in road projects have been identified. These are summarized in **Figure 25**. Pattern A is the case of a conventional road project using LBT. Patterns B and C reflect the original

purpose of developing and transferring spot improvement using a local resource‐based approach, which was to provide access to communities excluded from conventional road projects executed by government institutes. Both patterns present challenges in the sustaina‐ bility of road projects undertaken by communities themselves. The cohesiveness and self‐ reliance of the groups is key in these patterns. Support and cooperation in the provision of materials help to make the community road projects sustainable and to ensure the continuing accessibility of the rural roads.

In Pattern D, a combination of government policies on youth employment and Do‐nou technology training forms the basis for helping youth launch businesses as contractors. Subsidization of the training fee (about USD 1500 per person) for a comprehensive training course on routine maintenance and business management is necessary. The commitment of those graduating from the course supports the establishment of MSEs and the registration of participants as contractor.


*Edited by Serafeim Polyzos*

Cities are growing as never before and nowadays, it is estimated that at least 50% of the world's population lives in urban areas. This trend is expected to continue and simultaneously the problems in urban areas are anticipated to have an increase. Urbanization constitutes a complex process involving problems with social, economic, environmental and spatial dimensions that need appropriate solutions. This book highlights some of these problems and discusses possible solutions in terms of organisation, planning and management. The purpose of the book is to present selected chapters, of great importance for understanding the urban development issues, written by renowned authors in this scientific field. All the chapters have been thoroughly reviewed and they cover some basic aspects concerning urban sustainability, urban sprawl, urban planning, urban environment, housing and land uses. The editor gratefully acknowledges the assistance of Dr Marius Minea in reviewing two chapters.

ISBN 978-953-51-0442-1

Urban Development

Photo by NosUA / iStock


**Author details**

Vanisree C.R.1 , Mahipal Singh Sankhla2 \*, Prashant Singh3 , Ekta B. Jadhav4 , Rohit Kumar Verma5 , Kumud Kant Awasthi6 , Garima Awasthi6 and Varad Nagar2

1 Department of Forensic Science, Sage University, Indore, Madhya Pradesh, India

2 Department of Forensic Science, Vivekananda Global University, Jaipur, India

3 School of Forensic Science, National Forensic Science University, Gujarat, India

4 Department of Forensic Chemistry and Toxicology, Government Institute of Forensic Science Aurangabad, Maharashtra, India

5 Dr. APJ Abdul Kalam Institute of Forensic Science and Criminology, Bundelkhand University, Jhansi, India

6 Department of Life Sciences, Vivekananda Global University, Jaipur, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.