In the last years, some progress in synthetic biology has been accomplished in this species; however, it is still limited compared to other NCYs such as *P. pastoris* since *K. marxianus* still lacks efficient genetic tools, there are also limited auxotrophic markers and very few constitutive and inducible promotes have been described [21]. Despite that, some examples and applications are discussed below.

**Codon Optimization.** This strategy is currently used to improve the expression of recombinant proteins in *K. marxianus*, especially vaccines. For instance, codon optimization has been used for the heterologous expression of the porcine circovirus type 2 (PCV2) Cap protein in *K. marxianus* as an alternative to produce PCV2 viruslike particle vaccines to treat porcine circovirus disease and help reduce economical losses in the swine industry. Duan et al. reported in their experiment higher yields compared with *E. coli* and *P. pastoris* as host vectors [76]. Other examples where codon optimization has been applied to *K. marxianus* include heterologous expression of single-chain antibodies, overproduction of inulinase, expression of the dengue virus type 1 nonstructural protein 1 and porcine parvovirus-like particles [77–80].

**Promoters.** In the last years, several promoters have been identified for this species which can be induced by heat, xylose, lactose or inulin [22]. In addition, several strong endogenous promoters of genes such as purine-cytosine permease, inulinase, enolase and glyceraldehyde 3-phosphate have been characterized [21, 69]. More recently, Kumar et al. identified two new strong promoters (PIMTCP1 and PIMTCP2) which are more efficient at different temperatures and carbon sources than previously known promoters in this species [21].

Interestingly, the relative strength of promoters can change depending on the carbon source provided [81]. For instance, Kumar et al. showed that the *K. marxianus* inulinase promoter has relatively higher activity in the presence of xylose than dextrose [21]. In addition to finding new promoters, already described promoters have been also engineered to improve their features. For example, Zhou et al. improved the expression of lignocellulolytic enzymes in *K. marxianus* by a mutation inside the inulinase promoter and a deletion of an A-T-rich region inside the 5'UTR [82].

Notably, if thermotolerance of this yeast is to be exploited when expressing heterologous proteins, it is not enough to only focus on utilizing thermostable proteins but also identifying thermotolerant promoters because promoter activity tends to decrease with elevated temperature as it was demonstrated by Yang et al. [81]. Despite the fact that lower promoter activity is observed when increasing the temperature, *K. marxianus* promoters have been found to be stronger than their corresponding promoters from *S. cerevisiae* at such temperatures. In fact, the *K. marxianus* constitutive promoter PPGK has been shown to retain relatively strong activity with an increase in temperature [81].

**Terminators.** Only a limited number of terminators have been examined in *K. marxianus* including terminators from *S. cerevisiae* such as TCYC1, TPGK1 and TADH1 [21, 22]. Additionally, new recently described *K. marxianus* terminators have widened the range of regulation of protein expression in this species. For instance, in a recent study researchers found an increase in EGFP expression (fourfold increase

of mRNA level) in *K. marxianus* when using the endogenous terminators TIMTT1 or TIMTT2 instead of *S. cerevisiae* TCYC1 [21].

**Signal Peptides.** Research about signal peptides in this species is still limited since only a few signal sequences have been characterized and employed in heterologous protein expression on *K. marxianus* including signal sequences from *K. marxianus* Inu1, *P. pastoris* Pho1, *S. cerevisiae* α-MF and *K. marxianus* α-MF [21]**.** Moreover, some experiments of signal sequence engineering conducted to improve its activity have been reported, for instance, Yarimizu et al. developed a synthetic signal sequence in the yeast *K. marxianus* by redesigning the hydrophobic core of *Gaussia princeps* secretory luciferase signal sequence. The hydrophobic sequence was replaced by a repeat of 16 methionine residues, resulting in 20-fold higher activity than that from the wild type [83].

**CRISPR-Cas9.** Cernak et al. first used a CRISPR-Cas9 system to inactivate genes responsible for spontaneous mating-type switching (common phenomenon in *K. marxianus*), enabling the production of stable heterothallic haploid strains which can mate. As a result, they combined three complex traits found in different strains (ability to take up exogenous DNA, thermotolerance capacity and higher lipid production) into single *K. marxianus* clones [71].

Li et al. developed a one-step multigene integration system based on CRISPR-Cas9, which is capable of integrating up to three cassettes in a single, targeted genomic locus in *K. marxianus.* It consists of the CRISPR plasmid (expression of the sgRNA and Cas9) and the homology donor plasmid (700 bp up- and down-stream homology to the targeted site). This system has been proven to have an efficiency comparable to single-gene integration and it can be performed within 4 days from transformation to confirmation [84].

In 2022 Bever et al. developed a highly efficient CRISPR-Cas9 system in *K. marxianus* that allows editing of multiple genes which can be used in both NHEJ-functional and -deficient strains showing nearly 100% efficiency of gene disruption in those two strains, whereas 100% efficiency of donor integration was observed only in NHEJdeficient strains*.* In addition, this system achieved a dual integration efficiency of 25.5% in an NHEJ-deficient strain [85].

### **4.3** *Kluyveromyces lactis*

*Kluyveromyces lactis* is an NCY known for its capacity to assimilate lactose and convert it into lactic acid. *K. lactis* is a respiratory Crabtree-negative yeast highly used in industries due to its ability to secrete the protein β-galactosidase, used for making lactose-free products [23]. Moreover, this yeast is also capable to produce methionol, which is a flavor-active compound important in the overall aroma of soy sauce and cheese [7].

Some advantages of working with *K. lactis* yeasts are the capacity of producing heterologous proteins in simple growth medium, complete knowledge of their genome and more importantly, they can be easily genetically manipulated [75]. Due to its similarity in biosynthetic capacities to *S. cerevisiae*, *K. lactis* toolkits for heterologous gene expression are mostly the same. However, *K. lactis* presents many attributes that make it more suitable for protein expression and extracellular secretion than *S. cerevisiae* [13]. In fact, *K. lactis* uses an inducible promoter PLAC4 which is commercially available due to its capacity to secrete recombinant proteins in the culture fluid under lactose presence, a very useful feature for protein purification [13].

**Promoters.** In general, the promoters used for heterologous protein production strategies in *K. lactis* are the same as *S. cerevisiae*, PGAL1 or PPGK, which, due to their

### *Upgrading Non-Conventional Yeasts into Valuable Biofactories DOI: http://dx.doi.org/10.5772/intechopen.109903*

high level of transferability, have shown the potential of promoter engineering in *S. cerevisiae* to be applied in the *K. lactis* expression system [6]. However, other engineering strategies involving *K. lactis* promoters have also been developed. For example, Sakhtah et al. have recently developed a novel auto-inducible promoter system in *K. lactis*. For this, portions of two promoters, the constitutive PGAP1 and the carbon source-sensitive PICL1, were combined to form a hybrid promoter called P350 [86]. This novel promoter is induced by the depletion of glucose or glycerol in the medium, making it auto-inducible as the carbon sources are consumed by the growing cells. The development of this hybrid promoter promises to be useful for the implementation of one-step protein expression methods for small- and large-scale bioprocesses [86]. Moreover, another hybrid promoter approach used in K*. lactis* involves the combination of core promoter elements of *Trichoderma reesei* PCBH1 and *K. lactis* PLAC4, which showed an increase in protein production in this yeast [13].

**CRISPR-Cas9.** For the implementation of CRISPR-Cas9 in *K. lactis*, Horwitz et al. adapted an *S. cerevisiae* system by exchanging the 2 μ element with the *K. lactis* specific pKD1 vector-stabilizing element and the constitutive promoter PFBA1 at a *GAL80* site [87–89]. Moreover, sgRNA expression was driven by the typical PSNR52 pol III promoter and a T*SUP*4 terminator, and the deletion of the *KU80* gene was performed to reduce NHEJ [6, 29, 87–89]. The implementation of this system resulted in the successful integration of three donor six-gene-DNA parts into three separate loci (*DIT1, ADH1 and NDT80*) with a triple integration efficiency of 2.1% [6, 29, 87, 88]. Despite this low efficiency, the speed and ability to screen strains reduced the design-builttest cycle for this non-conventional yeast, and further improvements in targeting this efficiency could enhance genome editing for wild-type or industrial strains [6].

On the other hand, CRISPR-Cas9 genome editing was used by Burghardt et al. in order to increase the enzymatic production of the prebiotic fructo-oligosaccharides (FOS) in *K. lactis*. For this, the fructosyltransferase gene (*FFT)*, needed for forward reactions, from *Aspergillus terreus* NIH2624 was integrated with a *K. lactis* GG799 production host. Furthermore, a CRISPR-Cas9 system was used to delete a native invertase gene, involved in reverse reactions. The results showed an increase in transferase activity by 66.9% when grown in a fed-batch process [90].

### **4.4** *Yarrowia lipolytica*

*Yarrowia lipolytica* belongs to the *Ascomycota*, *Dipodascaceae* family. It is naturally found in lipid and protein-rich substrates such as soil, sewage and oil-polluted environments due to their capacity to hydrolyse lipids, assimilate hydrocarbons and fatty acids and secrete extracellular proteases [91]. *Y. lipolytica* is a Crabtree-negative haploid, heterothallic yeast with mating types Mat A and Mat B, and low mating frequency in nature [92]. In a laboratory setting, cells appear spherical, ellipsoidal or elongated and arranged singly, in pairs or clustered in groups. Furthermore, colonies present a creamy texture and a convoluted pale white matte surface [91]. About the carbon sources, *Y. lipolytica* is capable to assimilate hydrophobic substrates like alkanes, alkenes, fatty acids, fatty acid methyl esters, triglycerides and hydrophilic substrates like glucose, fructose, some alcohols, many polyols and many organic acids [91]. Some important characteristics of this yeast are its efficient secretion pathway and lipid storage capacity, two qualities that have made it a research model for protein secretion and lipid metabolism [92]. Moreover, due to its lipogenic metabolism, *Y. lipolytica* has been studied for the biosynthesis of acetyl-CoA-derived molecules such as terpenes [93]. In the last years, because of its production capacity of industrial

interest compounds and the ability to grow at high cell densities, different synthetic biology tools have been developed and applied in *Y. lipolytica* [94]. As matter of fact, Wong and colleagues designed a collection of BioBricks for *Y. lipolytica* called YaliBricks, which contains compatible restriction enzyme sites that allows modular genetic engineering [95].

**Promoters.** In *Yarrowia lipolytica*, two important promoters have been isolated and characterized; the promoter from the *XRP2* gene, which codes for an alkaline extracellular protease and the constitutive promoter from the *TEF* gene, which codes for translational elongation factor-1 [96]. Furthermore, recent studies are focusing on the development of hybrid promoters that could increase the strength of the available ones. Early approaches to promoter hybridization led to the characterization of upstream activating sequences (UASs), native to *XPR2*, which resulted in an increase on promoter activity when hybridized in several tandem repeats [97]. Madzak et al. engineered four hybrid promoters (named hp1d, hp2d, hp3d and hp4d) containing up to four copies of one of its upstream activation sequences (UAS1XPR2) fused upstream from a PLEU2 promoter [98]. The resulting promoters showed an increase in their strength depending on the number of tandem UAS1XPR2 elements, with hp4d being the strongest hybrid promoter and therefore used widely for heterologous gene expression in *Y. lipolytica* [94, 99]. On the other hand, Schwarts et al. constructed a synthetic hybrid promoter, using *GAL1* UAS from *S. cerevisiae* and the *TEF* core promoter from *Y. lipolytica*, that achieved a slightly higher expression than P*UAS1B8*- *TEF*, hybrid promoter that has been widely used [93]. Moreover, other native promoters like PTDH1, PGPM1, PEXP1, PFBAINm, PGPAT, PGPD and PYAT have been characterized and used in expression of heterologous genes with promising results [94].

**Terminators.** The most commonly used terminators for expression of heterologous genes in *Y. lipolytica* are derived from the native *XPR2* and *LIP2* genes [99]. Moreover, some *S. cerevisiae* terminators have shown a high degree of transferability in *Y. lipolytica* [100]. Indeed, synthetic terminators designed for *S. cerevisiae* have been used in *Y. lipolytica* with an increase of 60% in expression level over some wild-type terminators [56]. Additionally, these synthetic terminators are commonly smaller than the natural ones, conferring them an advantage for transcription units (TU) and vector design since they show low risk of undesired HR between TU or with the genome, contributing high stability to genetically modified strains [50]. However, despite these advances, the number of studies of terminators in *Y. lipolytica*, in comparison to promoters, is still scarce [96].

**Golden Gate.** Larroude and collaborators have developed a modular toolkit based on the Golden Gate strategy that allows assembly in one step of three transcript units together with integration into *Y. lipolytica* genome. This approach comes with a collection of six selective markers and sequences for random or specific integration, nine promoters of variable strength and five terminators [100]. In such manner, the heterologous production of β-carotene is possible with the expression of three genes involved in the carotenoid pathway after a single transformation [101], making *Y. lipolytica* a competitive biotechnological producer of β-carotene.

**CRISPR-Cas9.** The use of CRISPR-Cas9 in *Y. lipolytica* has been widely studied. In recent years, a system from *Streptococcus pyogenes* has been adapted, with a synthetic RNA polymerase III promoter and an optimized Cas9 to perform a marker-free gene disruption and integration in *Y. lipolytica*. In fact, five loci have been recently identified that could serve as hotspots for targeting marker-free gene integration [97]. This system resulted in a single-gene disruption and HR with a 90 and 70% of efficiency

*Upgrading Non-Conventional Yeasts into Valuable Biofactories DOI: http://dx.doi.org/10.5772/intechopen.109903*

respectively when Cas9 and the sgRNA were co-transformed using donor DNA [94]. In addition, systems like CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) have been developed for controlling gene expression in *Y. lipolytica* [99]. Here, a deactivated Cas9 (dCas9) is fused to transcriptional repressors or activators, allowing binding to sgRNA-complementary DNA without cleavage that could result in DSBs [91]. An implementation of the CRISPRa system was performed by Schwarts et al. in order to upregulate *BGL1* and *BGL2* (β-glucosidase genes that are transcriptionally silent), and allow *Y. lipolytica* strains to use cellobiose as a carbon source. For this, a VPR activator was identified and fused to dCas9 to enable gene activation [93, 96].

Other CRISPR-Cas9 strategies have been developed, for example, a paired sgRNA, consisting of two vectors, each containing Cas9 gene and a sgRNA cassette, was used in order to target areas upstream and downstream the start and stop codon, respectively, and allow a complete gene knockout via gene excision with a 20% of efficiency [94].

**TALEN.** Used to direct DNA DSBs to occur at a specific target site, was applied in *Y. lipolytica* to produce structure-based mutagenesis of a fatty acid synthase (FAS) domain, and allow the synthesis of fatty acids with shorter chain lengths [99]. Moreover, site-directed mutagenesis improved in efficiency when homologous exogenous DNA was added to the targeted site, resulting in HR-mediated repair in 40% of clones [94].

### **5. Future perspectives**

To date, only about 1% of the yeast species found in nature have had their genomes fully characterized. Thus, it is not surprising, with the accessibility of new sequencing technologies, the complete genome analysis of many newly discovered yeast species with unique characteristics will be available. This will greatly expand the catalog of genetic parts allowing a more sensitive fine-tuning of desired economically relevant compounds and the discovery of new genes of interest. In addition, new yeast host vectors with desirable characteristics such as faster growth rates, stress-tolerance, efficient secretion systems and desired metabolic pathways will be engineered and domesticated to facilitate their use in industrial applications.

Moreover, in silico simulations will play a crucial role in the efficient design of new synthetic yeast biofactories since more accurate predictions will be made. Nevertheless, there are still limitations that have to be overcome such as the absence of gene regulatory information, lack of accurate metabolic models at genomic scale [102], or missing experimental design and testing of potential NCY biofactories. For instance, CRISPR-Cas9 still performs poorly regarding the adequate sgRNA production in NCYs. sgRNA expression is normally accomplished using RNA polymerase III (RNAP III) promoters (not well characterized in NCYs), implying more studies are needed for effective genetic engineering. Wagner and Alper suggest two approaches to overcome this issue: optimization of heterologous RNAP III promoters or the screening of native RNAP III promoters [6, 13] which are currently being tested in some NCYs such as *Y. lipolytica* [97].

Finally, a quite positive outcome of the use of NCYs as biofactories is the production of industrially relevant compounds in an economical manner. Synthetic biology helps to search and engineer strains capable of utilizing cheaper substrates, including "waste products" (e.g. whey and molasses), supporting a sustainable circular economy which in the future will certainly have a more relevant role.

### **6. Conclusions**

For several decades yeasts have proven to be of great importance for the development of modern society, contributing to industrial processes including food and pharmaceutics. In addition, the current application of synthetic biology techniques in these organisms has given them a greater potential to be used as substitutes for organisms commonly used in the industry, such as bacteria, given the benefits they present. The use of these techniques in unconventional yeasts such as *P. pastoris*, *K. marxianus*, *K. lactis* and *Y. lipolytica* has increased very rapidly in recent years. For example, the genome editing technique CRISPR-Cas9 has been developed in these four species to improve the production of compounds, such as the prebiotic fructooligosaccharides in the case of *K. lactis*. Other techniques, despite being recently applied, have shown promising results in improving the expression of genes and the production of compounds of interest. This is the case of the use of TALENs in *Y. lipolytica* and Golden Gate in *P. pastoris*.

On the other hand, the engineering of genetic parts has also been developed in these unconventional yeasts. Codon optimization in *P. pastoris* and *K. marxianus* has allowed the production of heterologous enzymes such as phytases, important in animal feed, and recombinant proteins such as PCV2 Cap protein, which can be used in vaccines against porcine circovirus disease. Modifications of promoters and terminators have also been investigated, with promoter studies being the most common, as shown by the recent literature existing on the four species described in this chapter. In sum, the advances shown here demonstrate the potential of non-conventional yeasts as alternatives to traditionally used organisms, or even for the discovery of new systems with potential industrial use, capable of improving the quality of people's life.

### **Author details**

Kevin Castillo-Mendieta1 , Jimmy Arias1 and Fernando Gonzales-Zubiate1,2\*

1 School of Biological Sciences and Engineering, Yachay Tech University, Urcuquí, Ecuador

2 MIND Research Group, Model Intelligent Networks Development, Urcuquí, Ecuador

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 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.

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Section 2


**4. Result**

After analyzing the data descriptively, the results are that the mean value of each question item is greater than the standard deviation number. Thus, it means that the study results have very low deviations so that the distribution of the data is normal and valid.

Based on each question item, it can be grouped according to the orientation of CSR practices and then analyzed the tendency of respondents' answers regarding

*CSR Practical Orientation in Small Medium Enterprises (SMEs): A Case Study in Solo City… DOI: http://dx.doi.org/10.5772/intechopen.99859*

whether or not the orientation is carried out in CSR practices. The grouping is as follows:


**Author details**

Bruno Vallespir\* and Anne Zouggar-Amrani Univ. Bordeaux, CNRS, IMS, UMR 5218, Talence, France

\*Address all correspondence to<EMAIL_ADDRESS>
© 2021 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.

*Model-Based Enterprise Continuous Improvement DOI: http://dx.doi.org/10.5772/intechopen.96856*


**5. Discussion and Conclusions**

B[e]SGs form a special class of evolved massive stars and are thought to represent a short-lived transition phase either in their red-ward, post-main sequence evolution, or in their blue-ward post-RSG evolution. The total number of currently known B[e]SGs is low, supporting the idea of a short transition phase. However, in which direction the stars evolve, and whether all B[e]SGs evolve in the same direction, is still an open issue. For the objects in M31 and M33, it has been argued that the B[e]SGs are more isolated than LBVs and hardly found in stellar associations, so that a post-RSG (or post-yellow supergiant) evolution was proposed to be more likely [142]. This assessment was based on the available numbers of putative B[e]SGs in these two galaxies at that time. However, after revision of the two samples (Tables 6 and 7), two of the four confirmed B[e]SGs in M31 and both confirmed B[e]SGs in M33 are associated with stellar groups, questioning the conclusion that B[e]SGs are isolated and thus post-RSGs.

The best age indicator for B[e]SGs we have to date is their surface abundance enrichment in 13C, as discussed in Section 2.3. If B[e]SGs were post-RSGs, their progenitors would all have started with a (very) low rotation speed. While such a scenario cannot be excluded, it may not be very likely considering that stars are born on average with a rotation rate of about 40% of their critical velocity [88].

Interaction within a (close) binary system, possibly even up to a binary merger, seems to be an alternative and popular scenario (e.g., [51,177]), but the number of currently confirmed binaries amongst B[e]SGs is still rather low to give preference to the binary channel as the sole possible way for the formation of B[e]SGs. Likewise, some B[e]SGs have been suggested as suitable supernova candidates. For example, the Galactic object MWC 137 appears similar to Sher 25 [178] and SBW1 [179], which both look like the progenitor of SN1987A. In addition, the SMC object LHA 115-S 18 has been proposed to be a viable SN1987A progenitor [27]. In this respect, it is vital to resolve B[e]SG populations and to study their properties.

In this review, a census of the currently known B[e]SG population in the Milky Way and in nearby star-forming galaxies within and beyond the Local Group is presented. The proposed candidates have been undertaken a critical examination, sorted into confirmed B[e]SGs and B[e]SG candidates, and unsuitable objects have been flagged as "misclassified"10.

During these investigations, a fundamental difference has been recognized between the identification issues for objects in our Galaxy compared to those in other galaxies. Extragalactic B[e]SGs bare the risk of being confused with LBVs in quiescence, which share very similar optical spectroscopic characteristics. To separate these two classes of objects, one can make use of clearly defined classification criteria based on certain sets of emission features identified in their optical and near-IR spectra, as outlined in Section 3. In addition, inspection of the location of possible B[e]SG candidates in

10 We would like to caution that, with insufficient knowledge of stellar properties, individual objects may easily be misclassified, as it happened in recently published catalogs [6,102,180,181], in which erroneously a number of (even confirmed) B[e]SGs are listed as LBV candidates.

the IR color–color diagrams is highly advisable, because B[e]SGs and LBVs populate clearly separate domains. This fact might also be used as starting point for future investigations of extragalactic samples. However, for such future studies, infrared photometry with higher spatial resolution than what is currently provided by 2MASS and WISE is desirable to prevent from contamination with neighboring sources in densely populated regions. Moreover, precise distances, as soon provided by GAIA, will help to separate foreground stars in the directions to other galaxies that might have been misclassified as luminous extragalactic stars.

In the Milky Way, an additional complication occurs due to the often uncertain luminosity estimates, and the overlap of low-luminosity B[e]SGs with the most luminous massive pre-main sequence objects (HAeBe). Here, special care needs to be taken, especially since both classes of objects occupy adjacent regions in the near-IR color–color diagram with a probable overlap. Without additional distinctive features for such low-luminosity, borderline B[e]SG candidates, their real nature remains elusive. One complementary classification criteria that was discussed, is provided by the enrichment of the circumstellar material of evolved objects with processed material that has been released from the stellar surface, as opposed to the non-processed material with interstellar abundance patterns found around HAeBes. The most ideal element to search for is 13C, which is bound in 13CO molecules in the circumstellar disks. Measuring the 13CO amount with respect to 12CO provides immediate insight into the nature of the object. The current HAeBe samples might hide such low-luminosity B[e]SG candidates, which can only be identified as such by careful and honest analysis.

The result from this census, after strict application of the classification criteria, is that we count nine confirmed and six candidate B[e]SGs in the Galaxy. Moving out to the MCs, the numbers amount to 13 (+2) in the LMC and 5 (+1) in the SMC. The situation in other members of the Local Group is not much better, where the numbers drop to four (+7) in M31 and two (+6) in M33. Even further away, only three candidates have been reported from M81. The total number of B[e]SGs found in the various galaxies are too small for statistical analyses with respect to a metallicity dependence of their number, but the comparable quantities within the Milky Way and the LMC suggest only a mild dependence of the amount of B[e]SGs on (i.e., a possible drop with decreasing) metallicity.

Further, we report that the Galactic sample of 15 B[e]SGs and candidates contains currently seven confirmed or suspected binaries (see Table 9), which is less than half of the population. In the MCs, this number is even lower, because thus far only three stars have been reported to be possible binaries, of which one, the SMC star LHA 115-S 6 (= RMC 4) has been proposed to be the remnant of a binary merger within an initially triple system [182,183]. The other two objects, the LMC star LHA 120-S 134 and the SMC object LHA 115-S 18, have been identified as optical counterparts of X-ray sources [27,29]. Nothing is known about possible binarity in the B[e]SG samples from the other galaxies, although photometric variability was seen in at least two M31 objects: the confirmed B[e]SG star J004417.10+411928.0 [137,184] and the candidate J004444.52+412804.0 [185,186]. Whether this variability is a sign of binarity or just of semi-regular variability which might be interpreted with pulsation activity such as reported from the *α* Cygni variables, needs to be studied in more detail.

For the sake of completeness, a special class of objects, which have not been discussed yet, should be briefly mentioned as well. These are the high-mass X-ray binaries (HMXB) with possible B[e] supergiant (candidate) companion. Some objects with an assigned B[e]SG status have been found to be too luminous in X-rays for being considered single stars. These objects have been proposed to be binary systems, in which the high energy emission is caused by either accretion onto a compact object, or by shocks in a colliding wind binary with a second massive star. Members of this group are the Galactic objects Cl\*Westerlund 1W 9 (=Wd1-9), which is considered a colliding wind system [85]; CI Cam (=MWC 84), which might be interpreted as supernova imposter [187]; and the high-mass X-ray binary (HMXB) IGR J16318-4848, in which the compact object was proposed to be a neutron star [188]. Two ultra-luminous X-ray sources (ULXs), Holmberg II X-1 and NGC 300 ULX1, the latter being also named as supernova imposter SN2010da, have also been proposed to be HMXBs including a B[e]SG [189–191]. Whether these objects indeed host a B[e]SG clearly needs to be investigated in more detail. However, since the behavior of these sources is considerably different from the confirmed B[e]SGs, I hesitate to include them into the census.

The presented populations represent the current knowledge of B[e]SGs and B[e]SG candidates in the closest star-forming galaxies. With the ever growing sensitivities of instruments and telescope sizes, the future in B[e]SG star research is bright, because many more candidates will be identified in even more distant galaxies and with metallicities spreading over a large range. With statistically meaningful samples, it will finally be possible to unveil the nature and fate of these fascinating objects.

**Funding:** This project received funding from the Grant Agency of the Czech Republic (GACR, grant number ˇ 17-02337S). The Astronomical Institute of the Czech Academy of Sciences, Ondˇrejov, is supported by the project RVO:67985815.

**Acknowledgments:** I wish to thank the editor, Roberta Humphreys, for the invitation to write this review. In addition, I am grateful to Michalis Kourniotis for inspiring discussions on searching for extragalactic B[e]SGs, and to Dieter Nickeler and Lydia Cidale for passionate discussions about B[e]SGs as well as for their proofreading of and suggestions on the draft versions. Moreover, I thank the anonymous referees for their careful reading and suggestions on the draft version. This research made use of the NASA Astrophysics Data System (ADS) and of the SIMBAD database, operated at CDS, Strasbourg, France. This publication makes use of data products from the 2MASS, which is a joint project of the University of Massachusetts and the Infrared Processing and Analysis Center/California Institute of Technology, funded by the National Aeronautics and Space Administration and the National Science Foundation. This publication makes use of data products from the Wide-field Infrared Survey Explorer, which is a joint project of the University of California, Los Angeles, and the Jet Propulsion Laboratory/California Institute of Technology, funded by the National Aeronautics and Space Administration.

**Conflicts of Interest:** The author declares no conflict of interest.


*4.11. Immunohistochemistry*

CAM sections were deparaffinized and rehydrated in an MICROM HMS740 automatic stainer (MICROM, Walldorf, Germany). Immunohistochemical analysis was performed using a streptavidin–biotin peroxidase complex system. Briefly, after rehydration, slides were subjected to heat-induced antigen-retrieval with 10 mM citrate buffer at pH = 6 for 2 min at 98 ◦C. The slides were washed with PBS and then incubated with 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidases. Another washing step was performed, and nonspecific binding was blocked with a 2.5% (*v/v*) horse serum (Vector Labs, Newark, CA, USA) for 30 min, before overnight incubation at 4 ◦C with mouse anti-human CD31 (1:30) (Dako, Cambridge, UK). Sections were washed with 0.1% tween in PBS and incubated with a secondary biotinylated antibody (Vector Labs, Newark, CA, USA) for 20 min. After thoroughly washed with 0.1% tween in PBS, samples were incubated with streptavidin-HRP (Vector Labs, Newark, CA, USA) for 20 min, followed by 3,3diaminobenzidine (DAB) incubation (Vector Labs, Newark, CA, USA). Finally, all sections were counterstained with Mayer's hematoxylin, dehydrated and mounted with resinous mounting medium Entellan®(Merck, Darmstadt, Germany).

### *4.12. In Situ Hybridization*

The presence of human cells within the implantation area was assessed using a human-specific DNA probe, according to the respective detection system BIO-AP REM-BRANDT ®Universal DISH & Detection kit (PanPath, Budel, The Netherlands). Briefly, after deparaffinization, proteolytic digestion was performed using a pepsin-HCL solution for 30 min at 37 ◦C, followed by dehydration in graded ethanol series. Sections were air-dried, and 1 drop of the probe was applied and covered with a coverslip. Samples were incubated at 95 ◦C for 5 min for DNA denaturation and then for 16 h at 37 ◦C in a moisturized environment for hybridization to occur. Samples were then washed in Tris-buffered saline (TBS) and incubated for 10 min with the stringency wash buffer. After rinsing with TBS, the detection was performed, and color was permitted to develop for 5 min at 37 ◦C in the dark. Samples were washed with water, counterstained with nuclear fast red, and observed under a Leica DM750 microscope (Leica, Wetzlar, Germany).


**4. Conclusions**

This work highlighted the valorisation of agro-industrial by-products for the efficient production of ethanol by yeast fermentation. The CCD-RSM methodology was employed as a powerful statistical tool to optimise ethanol productivity while considering the specific techno-economical process constraints such as the cost of *S. cerevisiae* yeast. Experimental results suggested that the raw sugar beet molasses should be diluted between 125–250 g/L of initial sugar and supplemented with a high concentration of nutrient (4 g/L) to achieve high ethanol productivity and yield responses. In addition, the quantity of yeast present in the fermentation media was significantly reduced from 0.70 to 0.27 g/L while achieving high ethanol production performances. Assays performed in 7.5 and 100 L bioreactors suggested that the fermentation process has the potential to be implemented at pilot and industrial scales for the production of bioethanol in the context of the circular economy. In addition, a detailed techno-economic evaluation of the whole fermentation process could provide valuable information for the potential commercialization of this integrated biorefinery approach.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/fermentation7020086/s1, Figure S1: Linear regression between experimental ethanol concentrations calculated by mass loss and quantified by HPLC, Figure S2: "Predicted versus obtained" analysis of the ethanol productivity values generated through the CCD-RSM, Table S1: Fermentation performances of the confirmation experiments designed during the process optimization.

**Author Contributions:** J.-B.B.: conceptualization, methodology, investigation, formal analysis, writing—review and editing; J.M.d.M.D.: conceptualization, methodology, investigation, formal analysis, writing—review and editing; J.-M.L.: supervision, writing—review and editing, funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Conseil de Recherches en Sciences Naturelles et en Génie du Canada (RDCPJ 531896-18) and the Consortium de Recherche et Innovations en Bioprocédés Industriels au Québec (2018-003-PR-C37).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to thank Jean Provencher and Philippe Robert (Gestion P.A.S Inc., Coaticook, QC, Canada) for proving the industrial sugar beet molasses and valuable information regarding its production. Further acknowledgements go to the Analytical Chemistry Laboratory of the Biomass Technology Laboratory for its support with various samples analysis.

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


Preface

Collagen is a ubiquitous protein, present in most tissues and a major contributor to their underlying architecture. Recent advances in molecular and structural biology have contributed to a better understanding of the structure and function of collagen. Collagen biomaterial is frequently employed for various applications such as a medium for delivery of drugs and genes, scaffold to support the growth of tissues, and much more. *Collagen Biomaterials* highlights some of the remarkable properties of collagen that make it one of the most widely studied and used biomaterials. With the advent of tissue engineering techniques, there has been a significant increase in the research interest in the development of novel applications of collagen as a biomaterial. The preference for collagen as biomaterials is also fueled by the fact that it is readily accessible, biocompatible with the human body, extremely malleable to one's requirements, and more importantly, biodegradable. Translational research on the various applications of collagen as a biomaterial is extremely important due to the diversity of roles that collagen and its various molecular types play in the body. Collagen is a major component of the extracellular matrix (ECM) of tissues and contributes to the stability, support, and growth of the tissue. Although there have been numerous books on collagen describing its structure, synthesis, and other properties, there have been only a few books that discuss collagen as a biomaterial. The current volume elucidates the various applications collagen biomaterials have found in the fields of medicine and tissue engineering.

*Collagen Biomaterials* is a collection of chapters that explore the various aspects of collagen that make it desirable as a biomaterial scaffold, its various applications, and the scope for further research. Chapter 1 by Silvestrini et al. discusses the involvement of collagen in health, disease, and medicine. The chapter elucidates the role of collagen in many functions of the body. The authors discuss the metabolism of collagen, factors affecting it, the disorders related to collagen deficiency, and its impact on the general health of individuals. Chapter 2 by Periasamy Srinivasan and Dar-Jen Hsieh discusses and compares the role of supercritical carbon dioxide extraction technology in the production of collagen scaffold biomaterials from various tissues and organs with the traditional decellularization techniques used in the production of collagen biomaterials, in the field of tissue engineering, and its implication in the field of regenerative medicine. The authors also elucidate the physicochemical properties, toxicity, biocompatibility, and bioactivity both in vitro and in vivo of the collagen scaffolds that are produced using carbon dioxide extraction technology. Chapter 3 by Makkithaya et al. discusses the application of collagen biomaterials for wound healing. The authors also elucidate the production of collagen in amalgamation with nanoparticles and its advantages for wound healing. The chapter also briefly describes the various applications of nano collagen in the biomedical field for wound healing. Chapter 4 by Bozsik et al. analyzes the various features of freeze-dried human collagen membranes consisting of cortical bone (SoftBone=SB) and folded platelet-rich fibrin (F-PRF) membranes after thermal modification and freeze-drying. The authors also compare the resistance properties

shown by collagen and PRF membranes. Detailed knowledge of the properties of collagen and PRF membranes is important during guided bone regeneration surgeries in dentistry. Chapter 5 by Amit Kumar Verma discusses the various methods of crosslinking collagen polymers and their implications in the field of pharmacology. The chapter also discusses the various formulations of collagen biomaterials that are used as a vehicle for the delivery of drugs. Chapter 6 by Ghosh et al. elucidates the application of 3D printing technologies to create collagen scaffolds. The authors discuss the various ways in which collagen in combination with other materials such as oxidized hyaluronic acid, bioinks, heparin sulfate, and many others can be used as scaffolds and implants in tissue engineering and regenerative medicine. The development of biomaterials with collagen as a structural protein is known to be beneficial in terms of the bioactivity and biocompatibility of said biomaterial because collagen is the structural component in the ECM of most tissues in the human body. Chapter 7 by Yunoki et al. elucidates the various mechanical methods employed for the fabrication of such biomaterials in considerable detail. The authors also summarize other well-known methods for producing collagen biomaterials.

The authors' contributions to this book as well as their timely responses to the reviewers' comments are greatly appreciated. We appreciate the reviewers for investing their time to make constructive suggestions and recommendations on the chapters, which helped us to improve the quality of content in the book. We also acknowledge the support and suggestions received from our colleagues. Our special thanks to Josip Knapić at IntechOpen for his contributions to the book. Most importantly, we wish our readers a pleasant and productive reading experience.

#### **Nirmal Mazumder**

Department of Biophysics, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India

#### **Sanjiban Chakrabarty**

**1**

Section 1

Health

Department of Cell and Molecular Biology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India

Section 1 Health

## **Chapter 1**


*Edited by Mohsen Mhadhbi*

This book describes advances in the development of smart and new ceramic materials for different applications. It also describes recent and expected challenges, along with their potential solutions, in advanced methods for the fabrication and characterization of nano-ceramics and their composites. This book provides readers with a comprehensive and updated review of the latest research in the field.

Published in London, UK © 2022 IntechOpen © MG1408 / Dollarphotoclub

Smart and Advanced Ceramics and Applications

Smart and Advanced

Ceramics and Applications

*Edited by Mohsen Mhadhbi*


**7. Conclusions**

In this chapter, the current scenario on the development of bentonite modified concrete was presented. It also reviews the literature about the physical & chemical properties of bentonite, bentonite blended cement mortar, bentonite modified cement concrete, and reinforced concrete. The history and development of Bentonite modified concrete were also briefly presented.


**2.6 Key technological breakthroughs of FOG**

Although FOG has many advantages over other gyroscopes, it still has some shortcomings because of the imperfect technology. Thus, we can employ some solution showed in **Table 3** to obtain better performance for FOG.