**1. Introduction**

Cellulose, a linear polysaccharide of glucose linked by β-1,4-glycosidic linkages, is the most abundant biopolymer on Earth and is found in the cell walls of plants. Cellulose consists of long chains of glucose tightly packed together due to H-bonds and constitutes the chief load-bearing polysaccharide. It is embedded in a matrix of pectins and hemicelluloses, and is additionally impregnated by lignin in some instances [1]. Cellulases are grouped into endoglucanases (EC: <IP_ADDRESS>), that randomly hydrolyse internal β-1,4-glycosidic bonds and exoglucanases (cellobiohydrolase, EC: <IP_ADDRESS>) that processively release mainly cellobiose from the reducing or non-reducing chain

extremity [2]. Processive endoglucanases that possess the properties of both endo- and exocellulases have also been described [3,4].

Based on amino acid sequence similarity, cellulases are classified into different glycosyl hydrolase (GH) families [5,6]. For example, endocellulases span the GH-families, 5–10, 12, 26, 44, 45, 48, 51, 61, 74, and 124, whereas exocellulase members are found in the GH families, 5, 6, and 9 (CAZy database, available online: http://www.cazy.org/Glycoside-Hydrolases.html). Most cellulases involved in the degradation of cellulose deriving from plant lignocellulosic biomass are produced by bacteria, archaea, fungi, and protozoa [7]. Some bacteria, oomycetes, protozoa, sea squirts, the fungus *Microdochium nivale* [8] and especially plants synthesize cellulose for growth and development, and, hence, require cellulases to degrade, modify, and remodel cellulose [9]. Some microorganisms (bacteria, fungi, protozoa) that live in a symbiotic relationship within the guts of phytophagous organisms also produce cellulases [10]. Later it was discovered that, apart from cellulolytic enzymes from symbionts, invertebrates also possess endogenous cellulases secreted by salivary glands and the gut [11]. Until recently, it was considered that cellulose catabolism was limited to heterotrophic organisms and higher plants (for remodeling cellulose). However, in 2012, it was experimentally established that the photosynthetic microalga *Chlamydomonas reinhardtii* (Cr) can utilize cellulose for growth in the absence/limitation of other C-sources by secreting endocellulases [12]. The alga combines features of both plants and animals (it is considered a "planimal" [12]), and has a genome characterized by an expansion of transporter gene families, indicative of an adaptation to life in soil environments [13].

In view of the biotechnological applications of novel cellulases in the degradation of lignocellulosic biomass to produce biofuel, here, we bioinformatically analyze, for the first time, cellulases from three microalgal species whose complete genomes have been published and compared [14,15]. We choose cellulase homologs from microalgae with increasing multicellularity (unicellular alga *C. reinhardtii*; colonial algae *Gonium pectorale*, Gp, with 16 and *Volvox carteri*, Vc, with 2000–6000 cells) and compare their sequences with different cellulases from diverse taxonomic groups. We model all the microalgal cellulase homologs and analyze in detail conserved motifs and their phylogenetic relationship, arrangement of different domains, and active-site architecture in addition to examining carbohydrate-binding modules (CBMs) and linker regions. We conclude this study by determining the expression levels of three cellulases in Gp in a control condition and after the addition of crystalline cellulose substrate (filter paper) to the growth medium.

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

The present work is based on the discovery that the photosynthetic microalga, *C. reinhardtii*, can secrete cellulases into the medium under CO2-limiting conditions, although cellulase secretion was not detected in the closely related Chlorella kessleri [12]. Interestingly, Chlorella has cellulose, whereas Cr, Gp, and Vc do not have cellulose in their cell walls [16]. In the present paper, we discuss the sequence and structural analysis of cellulases from three members of Chlorophyceae (Cr, Gp, and Vc) with increasing cellular complexity (from single cells to colonies).

#### *2.1. Algal Cellulases Belong to Glycosyl Hydrolase Family 9*

The structurally and functionally important conserved residues show that all algal sequences of catalytic domains (CDs) belong to the inverting GH9 family of CAZymes (Carbohydrate-Active Enzymes) with (α/α)6-barrel topology. Glucanases, belonging to the GH family 9, are considered the most conserved cellulases and are widely distributed among bacteria, fungi, amoebozoa, invertebrate metazoans, mosses, ferns, gymnosperms, and angiosperms [17]. Three conserved regions are identified in the CDs of algal cellulases (Figure 1 lower panels, Supplementary Figure S1a), consistent with the motifs/patterns of GH9 cellulases reported from across diverse taxonomic groups [17]. The variation of amino acids at each position within each region is compared between microalgal (Figure 1, lower panels) and all other GH9 cellulases described (Figure 1, upper panels) [17]. Region I of microalgal cellulases contains the characteristic DAGD motif where, in addition to

H-bonding of acidic residues with water (Figure 1, lower panel and asterisks), the C-terminal D acts as the catalytic base that extracts a proton from the nucleophilic water and the N-terminal D acts as an essential supporting residue [3,18–20]. The pattern corresponding to Region I, ([LVS]-x-[GK]-G-[WFYLM]-[YHF]-D-[ACGS]-G-[DSN]-X(2)-[KMR]-[FAILY]-X-[FWYLQTV]-[APTN S]-[MLGAQS]) has now been included in the PROSITE database (PS60032) [21]. Interestingly, in Region I of GH9 from all other organisms, D (catalytic base) is replaced by an N in the Angiosperm *Medicago* and G in few sea-squirt isoenzymes [17]; however, the activities of these enzymes have not been determined. In Region I of GH9 from all other organisms, two G and a K residues are also conserved, however, their role in catalysis has not yet been elucidated (Figure 1, upper panel).

The comparison of Region II (PROSITE pattern, PS00592) reveals that, although H and R residues (Figure 1, upper panels) are involved in substrate-binding via H-bonding [18,19], both residues are not conserved. The H is replaced by V in Vc and by S in *Panesthia cribrata* (Metazoa), whereas R is replaced by K and S in microalgae and by A or G in many GH9 cellulases [17]. An interesting finding about Region II of algal cellulases is the presence of an extra four residue sequence (PT[PTA][YSG]) (Figure 1, lower middle panel), which is missing in non-algal GH9 enzymes, with the exception being two cellulase homologs (CrCel9D and Gp KXZ44756) (Supplementary Figure S1a). The PROSITE pattern, PS00592, has now been revised to [HLY]-[AILMV]-[FIL]-G-x-[NSTW]-x(2,4)-[SCTV]-[FY]-[LIVMFY]-[SITV]-G-x(1,5)-[GSY]-x(2)-[AFPSTY]- [FLPSV]-x(2)-[AILPQVM]-[HV]-[DHLS]-[KRS] [21].

Residues in Region III are involved in substrate-binding and catalysis (Figure 1, asterisks), with fully conserved E acting as an acid that protonates the leaving group [19,22] and stabilizes the positively-charged oxocarbonium transition-state [18,23]. The fully conserved nucleophilic D forms H-bonds with the residues of the active-site loop, comprising of regions I and II, to bring it in proper alignment [18].

**Figure 1.** Variation in amino acids at each position within three conserved motifs of microalgal cellulases compared to other consensus sequences of GH9 cellulases from across the taxonomic groups. The sequences from different taxonomic groups were chosen, as mentioned in Figure 2. Upper panels, sequences from [17]; lower panels, microalgal sequences (this study). The gaps are denoted by dashes. \*, catalytic, and binding residues. Blue residues, variations in algal sequences. The extra four residues in Region II is found in all algal cellulases, except CrCel9D and Gp KXZ44756. "X" refers to extra residues in Region II not shown by [17]. The pattern corresponding to Region I updates the PROSITE Database.

#### *2.2. Algal Cellulases Are Closest to Invertebrate Metazoan GH9 Enzymes*

The percentage identity matrix (Supplementary Figure S1b) and the phylogenetic analysis (Figure 2) of CD regions (such as blue highlighted regions, Supplementary Figure S1a) from GH9 cellulases reveal that algal cellulases are closer to invertebrate metazoan enzymes than to plants. The identity matrix of GH9 family cellulases from selected groups show that algal enzymes have the highest (33–42%) and lowest (17–36%) sequence identity, with proteins from invertebrates and bacteria, respectively, whereas the identity with cellulases from plants is 27–33% (Supplementary Figure S1b).

Full length sequences of GH9 cellulases have other domains, such as CBM, immunoglobulin (Ig-like), fibronectin type III (Fn3-like), and dockerin, which can produce a bias during phylogenetic analysis [17]. To accurately determine the similarity, all cellulases were truncated to comprise only CDs (Supplementary Figure S2) and the phylogenetic tree was then constructed. The phylogenetic analysis revealed that truncated GH9 cellulases cluster together within a taxonomic group and that algal cellulases are closer to invertebrate metazoan enzymes (Figure 2), as suggested by the identity matrix (Supplementary Figure S1b). Notably, the GH9 belonging to the representatives of a specific (Sub)Kingdom (notably, Eubacteria, Fungi, Metazoa, Plantae) cluster together, except for *Ciona savignyi*, which does not form a cluster with the other representatives of Chordata *Branchiostoma floridae* and *Ciona intestinalis*, but clusters, instead, with arthropods. *C. intestinalis* forms a separate branch where the two cellulases analyzed are found together. The only representative of the Kingdom Protista (Phylum Amoebozoa), *Dictyostelium discoideum*, clusters in a sister clade to the one formed by bacteria. Interestingly, the cellulases from the green microalgae, *G. pectorale*, *C. reinhardtii*, *V. carteri*, and *Chlorella zofingiensis*, do not cluster with GH9 from Plantae, but instead form separate groups. This is consistent with the hypothesis that GH9 genes are related by vertical descent and not by horizontal gene transfer [17]. Particularly, the *G. pectoral*, GH9 KXZ44756, is sister to *C. reinhardtii* CrCel9D, which is the gene described as being strictly induced by crystalline cellulose [12].

**Figure 2.** Maximum likelihood phylogenetic tree of GH9 cellulases (built using catalytic domains, CD in the protein sequences; see Supplementary Figure S2) from different species (100 bootstraps). The circles refer to the bootstraps (range 0.6–1; the size of the circles is proportional to the bootstrap values). The name of the species analyzed and their accession numbers are indicated in the tree. The *V. carteri* cellulases are indicated VC2958622 and VC2952174. The different colors represent the different taxonomic groups, i.e., either (Sub) Kingdoms, Phyla, Divisions, or Orders.


**Platelet-rich Plasma (PRP) in Orthopedics and Traumatology — Review**

Pencho Kossev and Tsvetan Sokolov

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61326

#### **Abstract**

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[148] Walter C, Al-Nawas B, du Bois A, et al: Incidence of bisphosphonate-associated os‐

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172 Immunopathology and Immunomodulation

In the last few years various methods are being applied in the use of platelet-rich plasma (PRP) during treatment in different orthopedic disease and sports trauma. They allow im‐ provement of local biological condition and regeneration of different types of tissues. PRP is a modern treatment strategy with worldwide recognition. There is a high concen‐ tration of platelet growth factors in small amounts of plasma. PRP and its various forms have become one of the best methods to support the healing process of various tissues. PRP is used in regenerative medicine, because it provides two of three components (growth factors and scaffolds) necessary for complete tissue regeneration. The particular reason for the appearance of lesions is important in order to select an appropriate treat‐ ment method and technical application. Main indications are acute and chronic wounds, pseudarthrosis, ligament and muscle injuries, some tendinopathies, osteoarthritis, chon‐ dral injuries.

**Keywords:** Platelet rich plasma, platelets, platelet growth factors, chronic skin wounds

#### **1. Introduction**

Platelet-rich plasma (PRP) is a modern treatment strategy with worldwide recognition. PRP was introduced in the 1950s and is currently used in multiple fields of medicine. There is high concentration of platelet growth factors in small amounts of plasma. This works for offering an ''ideal environment'' for tissue regeneration and is part of the so-called biological therapy. The PRP implementation is an autologous therapy, which eliminates the risk of blood-borne infections. PRP and its various forms has become one of the best methods to support the healing process of various tissues. The term "regenerative medicine" was introduced in the 1990s. Many studies related to stem cells, growth factors, and extracellular matrix support the development of this new treatment philosophy. It differs from classic tissue engineering,

© 2015 The Author(s). Licensee InTech. 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.

where tissue regenerates ex vivo. Here, the major idea is regeneration of fully functional tissue on the spot, while damaged tissue is provided for by cellular elements and the process is managed by local factors. PRP appears to be the main source of autologous products in regenerative medicine and a true precursor and foundation of the healing process along with scaffold and stem cells [1]. PRP is used in regenerative medicine, because it provides two of the three components (growth factors and scaffolds), necessary for complete tissue regenera‐ tion. This review discusses the state-of the-art-studies on PRP action mechanisms in surgical and non-surgical implementation (treatment of tendon injuries, cartilage damage, muscle trauma, cartilage and bone pathologies, and wound healing).


**Author details**

Rafael Vasconcelos Oliveira and Valfredo Azevedo Lemos *Universidade Estadual do Sudoeste da Bahia, Laboratório de Química Analítica (LQA), Campus de Jequié, Jequié, Bahia, Brazil*

#### **5. References**

276 Polyurethane

Cd

Pb

\* µg g-1.

**4. Conclusion**

laboratory.

**Author details**

Rafael Vasconcelos Oliveira and Valfredo Azevedo Lemos

*Laboratório de Química Analítica (LQA), Campus de Jequié, Jequié, Bahia, Brazil*

*Universidade Estadual do Sudoeste da Bahia,*

**Element Process of extraction Enrichment**

Solid phase extraction

Ionic liquid dispersive

liquid–liquid microextraction

Cd Dispersive liquid-liquid

**factor**

Pb 35 1.0 14.0

**Limit of detection (�g L-1)**

30 0.8 9.0

Pb Solid phase extraction 2.1 ----- Yalcinkaya et al, 2011)

microextraction 55 0.4 ----- (Rojas et al., 2011)

Cd Solid phase extraction 19 0.77 ----- (Tang & Hu, 2011)

Cd Ion-flotation Separation 45 1.2 ----- (Tavallali et al., 2011)

Cd Cloud point extraction 29 0.1\* ----- (Moghimi & Tajodini,

Pb Cloud point extraction 56 and 42 1.14 ----- (Shah *et al.*, 2011)

Pb Solid phase extraction 50 0.65 ----- (Melek et al., 2006)

**Table 4.** Analytical characteristics of various procedures for the determination of Cd and Pb by FAAS,

The PUF grafted with Me-BTAP was successfully applied to the preconcentration of cadmium and lead. The sorbent showed characteristics that are desirable for materials used in solid phase extraction systems, such as resistance to swelling and changes in pH, low resistance to flow passage and simplicity in preparation. Moreover, when applied to the preconcentration system, the solid phase provided a simple and sensitive method for the determination of cadmium and lead by FAAS. The synthesized material is a good alternative for the determination of these elements. The extraction of other elements will be tested using this sorbent, and further work in this area is currently being conducted in our

**Sample volume (mL)**

40 1.5 20.0 (Soylak & Yilmaz,

2011)

2010)

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**Chapter 13**

© 2012 El-Shahawi et al., licensee InTech. This is an open access chapter 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.

© 2012 El-Shahawi et al., licensee InTech. This is a paper 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.

**Fast, Selective Removal and Determination of**

**Total Bismuth (III) and (V) in Water by Procaine**

**Hydrochloride Immobilized Polyurethane Foam**

Bismuth is found in nature in trivalent state as bismuthinite, Bi2S3, bismite, Bi2O3 and bismuth sulfide- telluric, Bi2Te2S. It is also found as a secondary component in some lead, copper and tin minerals [1]. Bismuth (V) compounds do not exist in solution and are important in the view of pharmaceutical analytical chemistry [1]. In the Earth's crust, bismuth presents at trace concentration (8 μg Kg-1) while, bismuth minerals rarely occur alone and are almost associated with other ores [2]. Bismuth appears to be environmentally significant because its physical and chemical properties have led it to be used in different areas of life. Pamphlett et al, 2000 [3] have reported that, bismuth compounds after oral intake enter the nervous system of mice, in particular, in motor neurons [3]. Hence, bismuth

The development of selective, separation, pre-concentration and determination method for bismuth at sub-micro levels is a challenging problem because of its extremely low concentrations in natural samples and of its strong interference from the sample matrices. Several methods e.g. hydride generation atomic absorption spectrometry [4], electro thermal atomic absorption spectrometry [5], atomic fluorescence spectrometry [6], hydride generation atomic absorption spectrometry [7], and cathotic and anodic adsorptive stripping voltammetry [8 - 10] have been reported for bismuth determination. Most of these methods require preconcentration of bismuth for precise determination because most analytical

**Packed Column Prior to Inductively Coupled**

**Plasma – Optical Emission Spectrometry**

M.S. El-Shahawi, A.A. Al-Sibaai, H.M. Al-Saidi and E.A. Assirey

Additional information is available at the end of the chapter

species are included in the list of potential toxins [3].

techniques do not possess adequate sensitivity for direct determination.

http://dx.doi.org/10.5772/47962

**1. Introduction**

**Fast, Selective Removal and Determination of Total Bismuth (III) and (V) in Water by Procaine Hydrochloride Immobilized Polyurethane Foam Packed Column Prior to Inductively Coupled Plasma – Optical Emission Spectrometry**

M.S. El-Shahawi, A.A. Al-Sibaai, H.M. Al-Saidi and E.A. Assirey

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/47962

**1. Introduction**

280 Polyurethane

No.4, p.p.459-463.

p.p.159-165.

Teixeira, L. S. G., Bezerra, M. D., Lemos, V. A., Dos Santos, H. C., De Jesus, D. S. & Costa, A. C. S. (2005). Determination of copper, iron, nickel, and zinc in ethanol fuel by flame atomic absorption spectrometry using on-line preconcentration system. *Separation*

Tuzen, M., Citak, D. & Soylak, M. (2008). 5-Chloro-2-hydroxyaniline-copper(II) coprecipitation system for preconcentration and separation of lead(II) and chromium(III) at trace levels. *Journal of Hazardous Materials*, Vol.158, No.1, p.p.137-141. Tzvetkova, P., Vassileva, P. & Nickolov, R. (2010). Modified silica gel with 5-amino-1,3,4 thiadiazole-2-thiol for heavy metal ions removal. *Journal of Porous Materials*, Vol.17,

Yalcinkaya, O., Kalfa, O. M. & Turker, A. R. (2011). Preconcentration of Trace Copper, Cobalt and Lead from Various Samples by Hybrid Nano Sorbent and Determination by

Zambrzycka, E., Roszko, D., Lesniewska, B., Wilczewska, A. Z. & Godlewska-Zylkiewicz, B. (2011). Studies of ion-imprinted polymers for solid-phase extraction of ruthenium from environmental samples before its determination by electrothermal atomic absorption spectrometry. *Spectrochimica Acta Part B-Atomic Spectroscopy*, Vol.66, No.7, p.p.508-516. Zeeb, M. & Sadeghi, M. (2011). Modified ionic liquid cold-induced aggregation dispersive liquid-liquid microextraction followed by atomic absorption spectrometry for trace determination of zinc in water and food samples. *Microchimica Acta*, Vol.175, No.1-2,

Zhang, L., Li, Z. H., Du, X. H. & Chang, X. J. (2011). Activated carbon functionalized with 1 amino-2-naphthol-4-sulfonate as a selective solid-phase sorbent for the extraction of

*Science and Technology*, Vol.40, No.12, p.p.2555-2565.

FAAS. *Current Analytical Chemistry*, Vol.7, No.3, p.p.225-234.

gold(III). *Microchimica Acta*, Vol.174, No.3-4, p.p.391-398.

Bismuth is found in nature in trivalent state as bismuthinite, Bi2S3, bismite, Bi2O3 and bismuth sulfide- telluric, Bi2Te2S. It is also found as a secondary component in some lead, copper and tin minerals [1]. Bismuth (V) compounds do not exist in solution and are important in the view of pharmaceutical analytical chemistry [1]. In the Earth's crust, bismuth presents at trace concentration (8 μg Kg-1) while, bismuth minerals rarely occur alone and are almost associated with other ores [2]. Bismuth appears to be environmentally significant because its physical and chemical properties have led it to be used in different areas of life. Pamphlett et al, 2000 [3] have reported that, bismuth compounds after oral intake enter the nervous system of mice, in particular, in motor neurons [3]. Hence, bismuth species are included in the list of potential toxins [3].

The development of selective, separation, pre-concentration and determination method for bismuth at sub-micro levels is a challenging problem because of its extremely low concentrations in natural samples and of its strong interference from the sample matrices. Several methods e.g. hydride generation atomic absorption spectrometry [4], electro thermal atomic absorption spectrometry [5], atomic fluorescence spectrometry [6], hydride generation atomic absorption spectrometry [7], and cathotic and anodic adsorptive stripping voltammetry [8 - 10] have been reported for bismuth determination. Most of these methods require preconcentration of bismuth for precise determination because most analytical techniques do not possess adequate sensitivity for direct determination.

© 2012 El-Shahawi et al., licensee InTech. This is an open access chapter 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. © 2012 El-Shahawi et al., licensee InTech. This is a paper 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.

Solvent extraction in the presence of co-extractant ligands e.g. bis (2, 4, 4-trimethyl pentyl) monothiophosphinic acid [11], pyrolidine dithocarbamate [12] etc has received considerable attention. However, these methods are too expensive, suffer from the use of large volumes of toxic organic solvents, and time-consuming. Thus, recent years have seen considerable attention on preconcentration and/ or monitoring of trace and ultra trace concentrations of bismuth by low cost procedures in a variety of samples e.g. fresh, marine and industrial wastewater [13]. Solid phase extraction (SPE) techniques have provided excellent alternative approach to liquid – liquid extraction for bismuth preconcentration prior to analyte determination step [14 -18].

Polyurethane foams (PUFs) sorbent represent an excellent solid sorbent material due to their high available surface area, cellular and membrane structure and extremely low cost [19]. Thus, several liquid solid separation involving PUFs methods have been employed successfully for separation and sensitive determination of trace and ultra trace levels of metal ions including bismuth (III) [19-29]. The membrane like structure and the available surface area of the PUFs make it a suitable stationary phase and a column filling material [25, 27]. Thus, the main objectives of the present chapter are focused on: i. developing of a low cost method for the removal of bismuth(III) and (V) species after reduction of the latter to tri valence state employing PUFs impregnated PQ+.Cl- ; ii. Studying the kinetics, and thermodynamic characteristics of bismuth (ІІІ) sorption by trioctylamine plasticized PQ+.Cltreated PUFs and finally iii. Application of the developed method in packed column for complete removal and / or determination of bismuth (III &V) species in wastewater by PQ+.Cl treated PUFs sorbent.


*9.1.3 Minimization of CO2 footprint*

Circular bioeconomy could decarbonize energy systems and reduce CO2 footprint. Substituting Haber-Bosch fertilizer with digestate for example avoids the 7 tons of CO2 emitted for each ton of fertilizer produced. Studies replaced fossil fuel with biogas in the transport sector. Biogas caused only 21–36% of the GHG emissions of fossilbased fuels [162], and biomethane reduced CO2 footprint by 49–84% [163]. Emissions linked to AD production of renewable energy and biofertilizer were lower than those for production of fossil energy and mineral fertilizer [164]. A study of combined heat and power (CHP) production found that pressurized AD systems achieved a direct CO2 footprint of about 13 kgCO2/MW hf. This footprint is quite low when compared to the 700 kgCO2/MW hel direct CO2 footprint of conventional CHP systems [165]. In the analysis of sorghum cultivation as an energy crop, inorganic fertilizer accounted for 51% of GHG emissions; digestate decreased carbon footprint by 11%. Digestate was then recommended for sustainable energy crop production and GHG mitigation [166]. Furthermore, a study from Switzerland quantified their national biomass availability and confirmed its potential for biofertilizer provision and climate change mitigation. The authors concluded that by 2050, circular bioeconomy application of AD would save significant amounts of mineral fertilizer (over 10 kt/a), and GHG emissions (38 kt/a of CO2eq) [167].


**2.3 Stanford Sleepiness Scale (SSS)**

122 Sleep Disorders

It is used especially in the assessment of type and of frequency about the tendency to fall asleep, micro-sleep episodes and monotony intolerance at work (especially in monitoring activities) and to capture the possibility of active participation in road traffic and other social

The *Epworth Sleepiness Scale* (ESS), the *Stanford Sleepiness Scale* (SSS), the *Multiple Sleep Latency Test* (MSLT) and the *Maintenance Wakefulness Test* (MWT) are among the methods that are most widely used for the investigation of daytime sleepiness in sleep disorders. The ESS reflects the global and subjective severity of daytime sleepiness in eight different situations and activities of daily living. The SSS is, however, to capture subjective circadian fluctuations of daytime sleepiness. To objective capture electrophysiological and standardized tests are often, such as the MSLT and the MWT used to determine the degree

If, on the basis of questionnaire data and medical history of sleeping on the basis of suspicion that a pathological daytime sleepiness (Table 1) exists, then objective analysis

The Epworth Sleepiness Scale (ESS) of Johns (1991) is very often used as a screening method for detecting the global daytime sleepiness and fall asleep in sleep disorders, especially used in hypersomnias. It is asked retrospectively, how high is the probability to fall asleep in eight everyday situations. The scale has a 4-step response format, in which values between 0 and 3 (0 = never to 3 = strongly agree) must be marked and results are added up a total

Following Johns (1991, 1992, Johns & Hocking 1997) a cut-off value ≥ 11 indicates a pathological daytime sleepiness. Standardization studies for the German-speaking countries were presented by Büttner et al. (2004c) and Sauter and colleagues (2007). The study found that 85% of healthy persons achieved a total value < 10, which corresponds to the calculated cut-off values in other studies (Johns 1991, Johns & Hocking 1997). The test-retest reliability of the ESS was calculated by Johns (1994) and based on a survey after five months in 87 healthy medical students. It was rtt = .82 (p <.001), even the quality of internal consistency

The ESS has in spite of it being subjective and a global assessment of daytime sleepiness (Johns 2000) has a very good validity. At a cut-off value > 10 it shows a high sensitivity of 93.5% and - high specificity 98.4%. The ESS is thus a highly reliable and valid procedure. The short implementation time and simple evaluation makes it very economical and cost effective. In addition, it can also be used for measuring the effectiveness of nCPAP therapy. Nevertheless the ESS does not lend itself to capture gradually different levels of sleepiness (Sangal et al. 1997b) and that four of the eight items have very low selectivity (Rühle et al.

Central nervous system activation

situations (Walsleben 1992, Weeß 2011).

of alertness on the basis of tonic activation.

Table 1. Sleepiness functions

maximum value of 24.

2005).

**2.2 Epworth Sleepiness Scale (ESS)**

was confirmed (Cronbach's alpha = .88 (p <.001).

methods can be used to measure sleepiness-related functions.

Vigilance

Selected Attention Divided Attention The Stanford Sleepiness Scale (SSS) of Hoddes et al. (1973) is a scale on which momentary alertness can be assessed on a grading of 1 to 7 and thus serves to assess the circadian variations in daytime sleepiness. The scale describes gradual gradations of awareness; it varies between very alert and drowsy conditions. The alertness descriptions are also described, each with typical sensations (e.g. *some slack*, *slows*, *woozy*) characterized. Studies on the sensitivity of the scale showed that ratings in 15-minute intervals represent discrete changes in the degree of alertness. According to the response ratings point values are assigned for each time interval, which are then summated.

### **2.4 Multiple Sleep Latency Test (MSLT)**

The Multiple Sleep Latency Test by Carskadon and Dement (1977) recorded the sleep latency lying down and is recommended for the investigation of daytime sleepiness in OSAS patients in the ICSD-2. The MSLT is based on the assumption that a strong physiological sleepiness can reduce the sleep latency (Arand et al. 2005).

For a long time the MSLT has been considered a gold standard for the investigation of daytime sleepiness (Carskadon et al. 1986). The MSLT (as well as the Maintenance of Wakefulness Test (MWT)) is often used to determine the alertness with expert's investigations, e.g. to assess the driving ability (Poceta et al. 1992). Five times a day electrophysiological recordings (C3/A2, C4/A1, EOG, EMG) are performed in 2-hour intervals. The first time of measurement should be from 1.5 to 3 hours after waking. The patient lies in a darkened room and is asked to fall asleep. During the test procedure, the patient is monitored with a video recording.

A pathological fall asleep exists, when the medium sleep latency is < 5 minutes (Richardson et al. 1982). The gray area is between 5-10 minutes and > 10-20 minutes is a normal finding. But are also divergent standard values of 5-8 minutes; thereby establishing of normal values is equivalent to a kind of "rule of thumb" (Guilleminault et al. 1994, van den Hoed et al. 1981, Johns 2000). Although the MSLT perform and should be evaluated strictly according to objective criteria and standardized, it seems to have low implementation objectivity, because the results of individual tests vary greatly (Danker-Hopfe et al. 2006). As other reasons for the inconsistent individual test results Thorpy (1992) describes the different day times and measuring times and not objectified sleep deprivation and sedative or stimulating effects of drugs. In spite of these influences, however satisfactory test-retest reliabilities of rtt = .65 to .97 (van den Hoed et al. 1981, Zwyghuizen-Doorenbos et al. 1998) have been found.

Another problem of MSLT is the limited external generalization of daytime sleepiness in everyday situations (Johns 1994). The assumption that the MSLT describe daytime sleepiness - as reflection of everyday life - Johns (2000) keeps being wrong. As a predictor of MSLT is therefore not own, regardless how strict standards and criteria were met. In considering of the relationship between ESS and MSLT are unsatisfactory correlation of r = .27 (p <.001) or on those that are not significant (Mitler et al 1998.). Reasons for the inconsistent correlations are different: Either there are satisfactory (significant) correlations when all patients fell asleep in all MSLT times or when the patients rarely slept or not fell asleep (Chua et al. 1998).

### **2.5 Maintenance of Wakefulness Test (MWT)**

The Maintenance of Wakefulness Test of Poceta et al. (1992) examines the ability to stay awake in a sleep-inducing situation. The patient sits in a darkened room on a comfortable

The Effects of Sleep-Related Breathing Disorders on Waking Performance 125

daytime sleepiness in (Table 2). In a normal population (n = 349) between 20 and 60 years, was found a mean value for ln PUI of 1.50 ± 0.39 mm/min. Thus, abnormal values are obtained from ln PUI > 1.89 and pathological values from ln PUI > 2.28. The cut-off value of > 6.64 was found for 84.1% of a healthy sample (Wilhelm et al 2001), which was established that this is independent of gender and age (r = .85 to .94). The PUI correlated low, but significantly with the subjective estimates of daytime sleepiness in SSS (r = .29, p < .010). The implementation objectivity and evaluation objectivity seem to be sufficiently given, because the change in pupil size can be deliberately manipulated. The reliability was tested in healthy control subjects and is satisfactory (r = .64, p < .001) (Weeß et al. 2000).

**Value range Mean-2SD Mean-SD Mean Mean+SD Mean+2SD**  ln PUI (mm/min) 0.73 1.11 1.50 1.89 2.28 Percentile 2.3% 15.9% 50.0% 84.1% 97.7% PUI (mm/min) 2.07 3.05 4.50 6.64 9.80

In the first version of the *Reading test*, it was up to the patients and healthy controls, to select a passage according to their interests. Therefore, it was possible that the individual level of activation of OSAS patients may have influenced the excitement level of the books. For this reason, the story "One day, maybe one night" by Arnold Stadler (2003) was selected. This is a retrospective narrative. Due to the low excitement level of the narrative it was assumed

A B Fig. 2. In 2A is seen as the patient reads in a semi-recumbent position, the modified form of the story "One day, maybe one night" by Arnold Stadler (Fischer paperback 2003). In the face of the electrodes are glued EOG, EEG and the EMG and its right to recognize a

The text was justified, typed in the font "Times New Roman" and the size 12. The pages were not numbered and included 36 lines with 11 cm length. A lamp (40 watts) was used for lighting, placed at a distance of one meter above the patient's head. At the beginning of the *Reading test*, the patient was informed by a verbal instruction, to read the text as possible in the normal reading speed and without interruptions. Patients were asked to keep the book at a distance of 40 cm. Lack of vision and of reading ability has been excluded by

polysomnography. In **2B**, the patient is asleep and the book has resigned.

Table 2. Percentile of the normal reference range for ln PUI and PUI

that the degree of tonic activation would remain constant.

**2.7 Reading test (Fig. 2)**

chair or on the bed and will be asked to refrain movements (e.g., grimacing, shaking), which may prevent falling asleep to refrain (Hartse et al. 1982, Mitler et al. 1982). Three to four times a day electrophysiological recordings (C3/A2, C4/A1, EOG and EMG) are recorded in 2-hour intervals of 20 minutes. The earliest start of the first test procedure should be scheduled two hours after waking. As with the MSLT test history is filmed with a video camera. Evaluated will be the sleep latency from the moment "light off" until the onset of the first two epochs of sleep stage 1 or 2.

In various standardization studies, inconsistent cut-off values were found from 13.5 to 18 minutes (Banks et al. 2004, Rühle 2005). Reasons for the different standard values according to Shreter et al. (2006) are that the test exercises have a significant influence on occasion staying awake in the test situation. So they provided proof that the sleep latency on the MWT was deliberately suppressed because the OSAS patients were afraid to get the license revoked. In considering the relationship between the MWT and ESS were calculated a satisfactory correlation of r = .48 (p < .001), with the common variance of the two devices was only 23% (Sangal et al. 1997b).

## **2.6 Pupillography (Fig. 1)**

The Pupillograph Sleepiness Test (PST) from Amtech (Weinheim) reflects the fatigue waves of the pupil described by Löwenstein. Normally, the pupil size will be constant in normal central nervous system activation in the dark for a long time. However, occur with increased daytime sleepiness after a few minutes spontaneous fluctuations (oscillations) on the pupil, which are recorded with infrared videography. Cause of fluctuations in pupil size is a mechanism of the autonomic nervous system. With reduced central nervous system activating two divisions acting simultaneously, which inhibit the Edinger-Westphal nucleus. This leads to instability of the central sympathetic activation and consequently fluctuating in an inhibition of parasympathetic activity and the Edinger-Westphal nucleus (Löwenstein et al. 1963, Yoss et al. 1970).

Fig. 1. Experimental setup for the pupillography. The patient wears an infrared protective goggle, has propped his chin on a device and looks toward the infrared camera.

#### **Evaluation**

The average Pupil Unrest Index (PUI) is the average pupil size fluctuations in millimetres per second over a period of 11 minutes. Higher PUI values indicate a clinically significant daytime sleepiness in (Table 2). In a normal population (n = 349) between 20 and 60 years, was found a mean value for ln PUI of 1.50 ± 0.39 mm/min. Thus, abnormal values are obtained from ln PUI > 1.89 and pathological values from ln PUI > 2.28. The cut-off value of > 6.64 was found for 84.1% of a healthy sample (Wilhelm et al 2001), which was established that this is independent of gender and age (r = .85 to .94). The PUI correlated low, but significantly with the subjective estimates of daytime sleepiness in SSS (r = .29, p < .010). The implementation objectivity and evaluation objectivity seem to be sufficiently given, because the change in pupil size can be deliberately manipulated. The reliability was tested in healthy control subjects and is satisfactory (r = .64, p < .001) (Weeß et al. 2000).

Table 2. Percentile of the normal reference range for ln PUI and PUI


Contents

## **Chapter 8 185**

Recent Megafires Provide a Tipping Point for Desertification of Conifer Ecosystems *by Daniel G. Neary*

## **Chapter 9 203**

Respiratory Allergy to Conifers *by Denis Charpin, Hélène Sénéchal and Pascal Poncet*


**1. Introduction**

Lithium-ion batteries (LIBs) are extensively used in various applications, including from small-scale portable electronics to large-scale application in electric vehicles, along with other applications in the field of drones, airplanes, and robots [1]. LIBs have shown efficient and practical applications in domestic as well as hand-held devices [2]. The LIBs are preferred over other chemistries owing to their strong advantages of high-energy density, low self-discharge, fast charging ability, enhanced cycle life, among many other advantages [3].

However, LIBs are likely to fail in case of over-charging and over-discharging with the possibility of a severe accident in case of overheating or internal-external short circuit [4]. The thermal abuse of LIBs can be caused by penetration, external-short, overheating, or over-charge [5]. In the case of a thermal abuse incident, the available chemical energy in LIBs rapidly converts to heat energy, generating a large amount of heat with the possibility of thermal runaway—fire accompanied by the occasional explosion [6]. Owing to high-energy density and flammable components, LIBs are prone to safety issues. Physical abuse of LIBs could lead to a temperature rise to the levels of 100 to 150 ◦C,

which could trigger cascading exothermic electrochemical reactions along with chemical disintegration, resulting in the rise of temperature to the levels of 500 ◦C in a few seconds. Although many safety methods, including effective cooling, have been installed, several cases of hazards have been reported over the years [7,8]. The recent events of LIB failure events leading to fire and explosion accidents have focused research attention on LIB safety [9]. For example: iPod caught fire due to overheated LIB [10] and three fire accidents of Boeing 747 at different locations [11]. Although, recently, LIB safety issues have received attention due to increased instances of hazards in electrical vehicle application, the safety concerns were admitted for many years [12,13].

In the case of continuous increase of temperature, rapid reactions start to occur. At around 110 ◦C, the graphite anode reacts with the electrolyte solvent. At around 165 ◦C, cathode material decomposes and releases oxygen. At around 180 ◦C, electrolyte solvent decomposes, and flammable components undergo combustion leading to thermal runaway [14]. The mechanical abuse in terms of penetration, or crush leading to thermal abuse, is an important safety concern and has received increased attention in recent years [15]. In many applications, the overheating and overcharge is controlled as a safe temperature and cut-off voltage are maintained. However, mechanical abuse leading to a short circuit is a challenging threat, as LIB can be subjected to crushing or metal penetration in unpredictable situations, such as accidents. The penetration provides a low-resistance path, drawing extremely high levels of currents with a rapid discharge of available chemical energy and simultaneously converting it into thermal energy. In the absence of resistive load, with the short circuit providing a low-resistance path, the energy is dissipated as joule heat, resulting in a rapid temperature increase [16].

Chen et al. developed an electro-thermal model to investigate the thermal performance during normal discharge and internal short circuit. The authors investigated the effect of an internal short circuit at the center of the battery on the temperature rise of the battery, and suggested that the increasing thermal conductivity of a separator can be an effective method to reduce the heat accumulation at the location of the internal short circuit [17]. Shi et al. conducted an experimental study on the exothermic behavior of LIB under mechanical abuse and suggested that dibenzylamine (DBA) could be used to prevent thermal runaway. The authors suggested that under normal conditions DBA does not affect the battery performance, and during mechanical abuse, DBA is released, which increases electrolyte resistivity preventing thermal runaway [18]. Vyroubal et al. presented a finite element model of nail penetration into the lithium-ion battery and showed that shorting resistance has a significant influence on the cell electrochemical-thermal process of batteries [19]. Noelle investigated internal short circuit on LIB by conducting direct current internal resistance, extremal shorting, and nail penetration experiment, and presented an electrolyte resistance model with experimental validation [20]. Fang et al. developed a 3D electrochemical-thermal model for internal short in Li-ion cell, and predicted that discharge rate was higher in Anode-Aluminum short as compared to Anode-Cathode owing to lower short resistance [21]. Mao et al. investigated the failure mechanism of the lithium-ion battery during nail penetration tests and presented the influence of penetration position and depth on temperature of the jelly-roll-type cylindrical cell. The authors pointed out that maximum temperature was observed when the nail was penetrated at the center of the cylindrical cell with the region of thermal runaway covering the entire cell. According to the micro short-circuit cell model presented by authors, if the temperature reaches between 90–120 ◦C, heat can spread to whole battery. However, reactions cannot lead to thermal runaway due to the unavailability of oxygen. But if the temperature reaches up to more than 233 ◦C, then the separator shrinks, thus leading to a larger short-circuit area, with the start of a reaction between cathode and electrolyte releasing a large amount heat and combustion byproducts in terms of gases [22]. Zao et al. conducted external and internal short circuit tests on batteries with different capacities. The authors showed that for external short test, with lower internal resistance, heat was accumulated between clamps and battery tabs, whereas, in case of nail penetration, heat accumulation increases with battery capacity and may catch fire. The authors suggested hydrogel-based thermal management to prevent thermal runaway [23]. There are few studies on how to mitigate the effect of internal short circuit and prevent thermal runaway. For example, Wang et al. suggested a

modified current collector with surface-notch, instead of a flat current collector, leading to a negligible temperature increase associated with internal sorting [24]. However, safety concerns still remain for the accumulation of large amounts of heat leading to thermal runaway in LIB during internal short circuit, and more understanding is still needed of the thermal behavior of LIB during internal shorting. temperature increase associated with internal sorting [24]. However, safety concerns still remain for the accumulation of large amounts of heat leading to thermal runaway in LIB during internal short circuit, and more understanding is still needed of the thermal behavior of LIB during internal shorting.

current collector with surface-notch, instead of a flat current collector, leading to a negligible

*Symmetry* **2020**, *12*, x FOR PEER REVIEW 3 of 23

The goal of the present study is to investigate the thermal abuse behavior of the LIR2450 coin cell with internal short circuit by penetrating element. The experimental coin cell discharge study is conducted and validated with numerical study within ±5.0%. The effect of penetrating element size, location of penetrating element, state of charge (SOC), discharge rate, short-circuit resistance, and heat transfer coefficient on maximum temperature and heat generation rate are presented. The study provides comprehensive insights on the thermal behavior of LIBs during thermal abuse condition with internal short circuit by penetrating element. The goal of the present study is to investigate the thermal abuse behavior of the LIR2450 coin cell with internal short circuit by penetrating element. The experimental coin cell discharge study is conducted and validated with numerical study within ±5.0%. The effect of penetrating element size, location of penetrating element, state of charge (SOC), discharge rate, short-circuit resistance, and heat transfer coefficient on maximum temperature and heat generation rate are presented. The study provides comprehensive insights on the thermal behavior of LIBs during thermal abuse condition with internal short circuit by penetrating element.

#### **2. Experimental Study 2. Experimental Study**

Figure 1 shows the schematic for the experimental study. The LIB under consideration is the LIR2450 coin cell with 120 mAh capacity, and the specifications of the coin cell are presented in Table 1. The coin cell was connected to KIKUSUI electronic load PLU-150 for evaluating and maintaining current, voltage, and power. The discharge test of the coin cell was conducted under constant current condition. The coin cell was fully charged to 4.2 V and discharge tests were conducted with a cut-off voltage of 2.75 V. The thermocouple was attached to a coin cell positive tab for measuring the surface temperature. The second thermocouple was attached to measure the ambient temperature. All the experiments were conducted in a room set at a constant temperature. The temperature of the room was controlled and kept constant at 25 ◦C. The details of the equipment used in the experimental study are presented in Table 2. Figure 1 shows the schematic for the experimental study. The LIB under consideration is the LIR2450 coin cell with 120 mAh capacity, and the specifications of the coin cell are presented in Table 1. The coin cell was connected to KIKUSUI electronic load PLU-150 for evaluating and maintaining current, voltage, and power. The discharge test of the coin cell was conducted under constant current condition. The coin cell was fully charged to 4.2 V and discharge tests were conducted with a cut-off voltage of 2.75 V. The thermocouple was attached to a coin cell positive tab for measuring the surface temperature. The second thermocouple was attached to measure the ambient temperature. All the experiments were conducted in a room set at a constant temperature. The temperature of the room was controlled and kept constant at 25 °C. The details of the equipment used in the experimental study are presented in Table 2.

**Figure 1. Figure 1.** Experimental setup. Experimental setup.

The measured parameter uncertainty is presented based on the accuracy of the instrument. The measured parameters were voltage, current, and temperature. The accuracy for current measurement was calculated based on Equation (1). The maximum uncertainty in measuring current was 0.45%. The accuracy of the voltage measurement was calculated based on Equation (2). The maximum uncertainty in voltage measurement was 0.08%. The accuracy in temperature measurement was calculated based on Equation (3). The maximum uncertainty in temperature measurement was 1.25%.

$$I\_{\rm U} = \pm (0.2\% \text{ of set} + 0.2\% \text{ of full scale}) \, + \,\text{V}\_{\rm in}/500 \,\text{k}\Omega \tag{1}$$

$$V\_{U} = \pm (0.2\% \text{ of set} + 0.2\% \text{ of full scale}) \tag{2}$$

$$T\_{\rm II} = \pm (0.1\% \text{ of reading} + 0.5^{\circ} \text{C}) \tag{3}$$