Rational Design and Development of Electrospun Nanofibrous

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Review

Rational Design and Development of Electrospun Nanofibrous Biohybrid Composites Brabu Balusamy, Omer Faruk Sarioglu, Anitha Senthamizhan, and Tamer Uyar ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00308 • Publication Date (Web): 22 Jul 2019 Downloaded from pubs.acs.org on July 25, 2019

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ACS Applied Bio Materials

Rational Design and Development of Electrospun Nanofibrous Biohybrid Composites Brabu Balusamy*†, Omer Faruk Sarioglu§, Anitha Senthamizhan† and Tamer Uyar*‡

†

Istituto Italiano di Tecnologia, Via Morego 30, 16163, Genova, Italy.

§

E-Kalite Software, METU Technopolis Twin Blocks, Middle East Technical University, 06800,

Ankara, Turkey. ‡Department

of Fiber Science & Apparel Design, College of Human Ecology, Cornell University,

Ithaca, NY 14853, USA.

ACS Paragon Plus Environment

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Keywords: Biohybrid composite, Electrospinning, Nanofibers, Core-shell, Encapsulation, Immobilization, Bacteria, Algae, Virus, Bioremediation, Wastewater treatment, Food, Agriculture

Abstract: Bio-encapsulation have gained substantial attention in wide spectrum of applications including bioremediation, sensing and catalysis over past few decades. However, such biohybrid systems suffer with many drawbacks in terms of less viability, low diffusion and loss of biological activity. Therefore, it is more important to preserve the pristine characteristics and activity of biological elements against various environmental factors. In the recent years, electrospinning has acknowledged as a feasible technique for fabricating biohybrid fibrous composites by incorporating various biological materials using several approaches including direct encapsulation, core-shell encapsulation and surface immobilization. In this review, the recent developments on the different methodologies in encapsulation and immobilization of microbial cells (i.e. bacteria, algae, viruses and yeast) in electrospun nanofibers and their potential applications in bioremediation, food, agriculture, biocatalysis, regenerative medicine and etc are briefly summarized. Further, ongoing challenges and future outlook in the electrospun nanofibrous biohybrid composites fabrication are concluded.


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1. Introduction Electrospinning has demonstrated as simple, versatile and cost-effective method that use numerous constituents like polymers, biomaterials and composites for producing fibers at nano and microscale dimensions.1-3 In electrospinning process, high electrostatic forces facilitate the production of ultrafine fibers in the form of nonwoven, or even aligned fibers or 3D structures can also be obtained by using various collector designs. Although the setup of electrospinning system remains simple, parameters including solution concentration, conductivity and viscosity, flow rate, work distance, intensity of electric field and humidity are playing governing role for uniform fiber production.1-3 In brief, upon high electric field is applied and which overcomes the droplet’s surface tension, a jet of a viscous solution is ejected. Further the released jet extends as longer and thinner spiralling loops, later results in solidification as solvent evaporates and finally collected on target in the form of fibers.1-3 Because of the exceptional features electrospun nanofibers possess including lightweight, high surface area, porosity, mechanical ﬂexibility, large scale production and structural diversity makes them potential candidate in numerous applications including environment remediation,4 sensing,5,6 catalysis,7 energy harvesting/conversion/storage,8 electronics,9 drug delivery,10 wound healing,11 regenerative medicine12 and tissue engineering.10,13 Electrospinning offers production of fibers with different characteristics and dimensions, moreover, incorporating various active agents through different approaches to enhance the functionalities of electrospun nanofibers for specific applications. Till now, several approaches have been adopted for the functionalization of electrospun fibers through blending the active agents in carrier matrix (mostly polymers) before electrospinning,14 encapsulation of active agents in nanostructures prior to their dispersion in carrier matrix,15 post treatment of electrospun


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composite membranes to convert precursor materials into their active form16,17 and attaching active agents onto surface of the fibers.18,19 Figure 1 depicts the basic electrospinning setup and various possible electrospinning approaches for functionalizing nanofibers by considering drug as model active agents, as well as different collectors presented for obtaining fibers with various morphologies.20,1-3 As evidenced with numerous studies, the multifunctional electrospun composite membranes showed enhanced performances from biomedicine to environmental applications. For instance, Peng et al. reviewed the improved performances of multifunctional electrospun nanofibers from water treatment to energy storage that are the important technological concern in the present scenario.21 Moreover, recent reviews demonstrate that the functional nanofibers can also be potentially extended for biosensor,22 cancer cell capture23 and many more clinical applications.24 Besides the tremendous efforts put forward in fabricating electrospun composite membranes using various active agents, a significant stride has also been made concerning fabrication of biocomposite membranes.25-27 As a consequence of industrial, agricultural and domestic activities, wide array of hazardous contaminants unceasingly released into the environment and possess detrimental effects to various organisms including humans. Therefore, decontamination of these substances is of significant importance. Among the several approaches available, physicochemical treatment methods attracted much concern in removing pollutants from water systems such as evaporation, burying, dispersion and washing, but generally such methods are labor intensive and highly expensive for use, and do not provide complete decomposition of organic pollutants.28 Therefore, more efficient, cost-effective and simple decontamination strategies are needed for complete detoxification of harmful pollutants.


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Figure 1. Schematic illustration of electrospinning process, various types of electrospinning techniques and collectors used for drug loading. Electrospinning techniques - co-electrospinning (A), side by side electrospinning (B), multi-jet electrospinning (C), co-axial electrospinning (D), emulsion electrospinning (E) and surface immobilization (F). Collectors - static plate (G), rotating mandrel (H), grid (I) and rotating disk (J). Reproduced with permission from reference 20. Copyright 2016 Royal Society of Chemistry.

Bioremediation of pollutants is considered a prominent process which use biological agents to remove or deactivate the contaminants in an environment. Bioremediation is considered to be harmless and more effective strategy for removal of various pollutants including heavy metals, agrochemicals, dyes, hydrocarbons and surfactants.29 While organic pollutants are generally degraded or removed from the contaminated site during bioremediation, some of the pollutants such as heavy metals are generally converted to non-toxic or less toxic forms to get rid of their harmful effects. Over several decades, microorganisms are used for differential purposes in the field of biotechnology and bioremediation. Microorganisms are generally preferred for bioremediation applications due to rapid growth of them, cost-effectiveness and ease of


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application. Microorganisms are generally applied by in planktonic form or as bio-integrated hybrid systems comprising specific microorganisms. Bacteria are the most preferred microorganism for bioremediation purposes. Use of bacteria has some advantages including metabolic diversity for metabolizing different types of contaminants, rapid growth of cells, formation of biofilms and higher resistance of cells in a biofilm to environmental extremes. Both alive and dead bacteria can be used, while alive bacteria have been used for biodegradation/bioremoval of contaminants, dead bacteria have been used for biosorption.30 Biosorption by dead bacterial cell biomasses can be a more advantageous strategy when biodegradation/bioremoval is not possible due to environmental restrictions. In biosorption, bacterial cells are unaffected by toxicity in the environment, they can be used without aeration and nutrient supply, and they can be reused in further applications.30 In addition, since some of the synthetic chemicals like textile dyes are intentionally manufactured as nonbiodegradable, biosorption can be a reasonable approach for remediation of such kinds of contaminants.30 Nevertheless, since living bacterial cells can perform both biodegradation/ bioremoval and biosorption simultaneously, better bioremediation performances can be achieved by living cells as previously reported.31 There are numerous instances for the use of bioremediative bacteria in both planktonic and biohybrid forms in literature. For example, the planktonic form of Acinetobacter calcoaceticus strain has potential in effective remediation of ammonium as reported by Zhao et al.32 and Sarioglu et al. reported that additional properties can be gained when a similar strain of Acinetobacter calcoaceticus is immobilized on a solid electrospun carrier system such as reusability and ease of application.33 Similarly, microalgae can also be explored for an ideal candidate in bioremediation and the application of microalgae for remediation of contaminants has several advantages; it is solar-power driven, sustainable,


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ecologically coherent and can stimulate production of microalgal biofuels for the biofuel industry.34 The microalgae and cyanobacteria are exceptionally employed in remediating nutrient waste and heavy metal contaminants from water system.35 Since microalgae are capable of transforming inorganic substances into organic via photosynthesis reactions using solar-power, they are promising candidates for removal of inorganic wastes (e.g. chloride).35 Like bacteria, microalgae can also be used in biohybrid systems36 and they can be included in constructing microbial consortia with other types of microorganisms for enhanced bioremoval performances.34 Food biotechnology applications have a great potential to improve human daily life. For instance, significant efforts have made to increase shelf life of nutraceuticals and development of antimicrobial food packages. Biocompatible or natural polymeric matrices can be used for storage of nutraceuticals or nutraceutical extracts with enhanced physical and chemical properties.37 Functional polymeric packages can comprise antimicrobial agents38 or antimicrobial probiotics39 to effectively avoid food spoilage. In order to develop functional biohybrid systems for food applications, use of electrospun polymeric systems is a rational approach.40, 41 Biotechnological applications on agriculture have being used for many years, and inclusion of beneficial bacteria in agricultural soils is an effective way to promote plant growth.42 These bacteria capable of phosphate solubilizing and fixing biological nitrogen using different mechanisms thus promoting the growth of plants and also protecting them from disease and abiotic stress.42 Furthermore, these bacteria can also provide reduced requirements of chemical fertilizers, thereby reducing environmental pollution and production costs, which eventually lead increased agronomic efficiency.42 Although these bacteria are generally included in agricultural soils in free form, development of integrated biohybrid systems have received more attention in


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recent years to make bacterial inoculants more resistant against environmental stress, and electrospun fibrous systems have been reported as potential carrier systems with high efficiency for those bacteria.43 Biocatalysis is use of bio-based systems such as enzymes or whole bacterial cells as catalysts for chemical reactions and it has broad application areas in the industry from food processing44 to production of active pharmaceutical ingredients (APIs).45 There is a tendency to use integrated biohybrid systems for biocatalysis applications for improved stability and recyclability,46 providing development of more economical and eco-friendly biocatalysis systems.47 Biointegrated electrospun fibrous systems are one of the potential hybrid systems for use in biocatalysis applications.48,49 Integrated biohybrid systems also have a great potential in biomedical field. Use of functional wound dressings comprising biological actives may enhance wound repair process considerably.50 In addition, by including other components such as antimicrobial agents51 or pain-killer medicines52 in their formulation, additional features might be gained for those materials. Since electrospinning allows preparation of highly tunable polymeric systems incorporating various active components, electrospun biohybrid systems have been tested for their potential application in biomedicine sector like regenerative medicine and tissue engineering.53-55 An enormous progress made in electrospinning has been disseminated in several reviews,6,12,19-24 but still an overview on the design and development of microbial cells incorporated electrospun nanofibrous biohybrid composites and their potential applications is lacking in the literature. In this review, we summarize the rational design and development of electrospun nanofibrous


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biohybrid composites using different microbial cells (i.e. bacteria, algae, viruses and yeast) through various functionalization approaches. Based on the different functionalization approaches, we organized this review into fabrication of electrospun nanofibrous biohybrid composites by encapsulation, core/shell and surface immobilization. In each section, we discuss the design and illustrate with several examples demonstrating their characteristic performances. Finally, we provide a conclusion on the review and future outlook of electrospun nanofibrous biohybrid composites are highlighted. 2. Fabrication of electrospun nanofibrous biohybrid composites and their applications 2.1. Direct encapsulation approach Besides surface functionalization of electrospun fibrous systems by attachment of microbes, encapsulation strategies by using direct or core-shell encapsulation methods have been received attention in recent years. These techniques provide integration of various microorganisms with fibrous systems in a single step, avoiding a requirement for further processing. Basically, in direct encapsulation method, living microorganisms including bacteria, algae, virus and yeast are integrated in the electrospinning solution as cell biomasses. No toxic and harmful solvent is used and biocompatible polymers soluble in non-hazardous solvents (e.g. water) are preferred for avoiding losses in microbial cell viabilities.56,57 Although electrospinning protocols may require applying lethally high voltages for microorganisms, desired amounts of living cells can be achieved after electrospinning by adding preservative chemicals (e.g. glycerol)58 or highly condensed amounts of living cells into the electrospinning solution.59 To date, several reports can be tracked from the literature for using electrospun fibrous systems in encapsulating microorganisms through direct encapsulation approach.49,56-70 These types of biohybrid systems can have application areas in tissue engineering,53-55 agriculture,43 food biotechnology40,41,60-62


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and environmental biotechnology.59,63 In the following paragraphs, some examples from the literature for use of direct encapsulation approach to generate biohybrid systems for different application areas will be mentioned. Cha et al. reported preparation of electrospun nanofibrous scaffold from silk fibroin (SF) and Spirulina extract for the purpose of application in tissue engineering.53 The scaffold was evaluated for performance and functionality, and found as a promising candidate for preventing blood clotting or antithrombogenicity. In addition, the scaffold has shown low cytotoxicity and very good cell adhesion capability with a positive impact for proliferation on different cell line models. These findings suggest that this scaffold can be used in tissue engineering applications in which high hemocompatibility is required. The studies of Kim et al. and de Morais et al. also generated novel electrospun biohybrid systems for potential use in tissue engineering applications, by incorporating Spirulina extract54 or biomass55 in polymeric matrices, respectively. Kim et al. reported that their newly generated PCL-Spirulina (PolycaprolactoneSpirulina) system has a potential to be used for treatment of CNS injured systems, based on positive experimental results of the biohybrid on primary astrocytes growth.54 The bacterial strains of Escherichia, Zymomonas and Pseudomonas which are industrially relevant has been efficiently encapsulated through electrospinning Pluronic F127 dimethacrylate [FDMA or PEO99-: polypropylene oxide (PPO)67-PEO99 DMA] as shown by Liu et al.49 A predominant concentration of bacteria growing in later stage of the log phase has been homogenously dispersed in FDMA/PEO solution and electrospun into fibers, further for the shake of obtaining 3D structure the fibers were collected using silicon wafer for a time period of 30 min. As a next step, the electrospun FDMA matrix was crosslinked using a catalytic system containing ferrous sulfate, AsA, and APS, further the electrospun membrane has been immersed


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in deionized water at the end of crosslinking process in order to remove PEO and swelling of scaffold. The fibers were characterized using SEM and TGA measurements. Consequently, encapsulated microbes were analyzed for their viability and morphology using laser-scanning confocal microscopy (LSCM) and SEM, respectively. Specifically, the bacterial viability was evaluated for a period of 7 days and two months at 4°C and −70°C, respectively. Further for the purpose of visualizing encapsulated Z. mobilis in confocal microscope, the FDMA cross-linked fibers with bacteria were stained with LIVE/DEAD bacterial viability kit. Figure 2A and B revealed 40% of the bacterial cells are alive following electrospinning and crosslinking and homogenously distributed. Further, Figure 2C and D confirms the encapsulation of bacteria in junctions of the fibers and also in single fibers. The findings of the study have potential implication in designing and application of biological thin-film catalysts and bio-hybrid materials.

Figure 2. Microscopical observation of cross-linked FDMA fibers encapsulated with Z. mobilis bacteria. The images of cross-linked FDMA fibers encapsulated with Z. mobilis bacteria captured using confocal microscope illustrate survival of about 40% encapsulated bacteria following electrospinning and cross-linking process. The scale bar in the image is corresponding to 10 μm (A). The SEM images of cross-linked FDMA fibers encapsulated with Z. mobilis bacteria depicts that the bacterial cells are encapsulated in the fiber junctions (B,C) and also in single fibers (B,D). The red arrows indicate location of encapsulated bacteria and the scale bars corresponds 20 μm in B and 1 μm in C and D. The figure inset in D shows the sketch of encapsulated bacteria in cross-linked FDMA fibers. Reproduced with permission from reference 49. Copyright 2009 National Academy of Sciences.


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Further in order to explore potential use in agricultural applications as an inoculant system, a novel biohybrid system generated by encapsulation of rhizobia within electrospun polyvinyl alcohol (PVA) nanofibers by direct encapsulation approach. Soybean seeds were either coated by electrospinning of PVA-rhizobia mixture or inoculated in a bacterial inoculum for use as a positive control. Rhizobia are a group of bacteria that have nitrogen fixation capability for use in agriculture.69 Encapsulation of rhizobia within PVA fibrous matrix system was intended to reduce the harmful effects coming from environmental stress on these bacteria. Rhizobia encapsulated biohybrid fibrous composites were evaluated for survival of bacteria within this system at different storage times and after exposure to fungicide, and their effects on the growth of soybean seeds. It was found that within 24 h, there was no significant change in the survival of bacteria, while a considerable change in bacterial numbers was observed after 48 h. It was also found that both free and encapsulated bacteria were negatively affected upon exposure to the fungicide. Inoculation of soybean seedlings with rhizobia incorporating PVA resulted in larger number of nodules compared to soybean seeds inoculated with rhizobia without PVA, suggesting that direct encapsulation of rhizobia within electrospun polymeric matrices might be a promising alternative to other protector inoculants. In order to fabricate functional food hydrocolloid, López-Rubio et al. generated a biohybrid system comprising a probiotic Bifidobacterium strain in a biopolymer matrix consisted of protein (whey protein concentrate, WPC) and carbohydrate (pullulan) by using electrospinning/ electrospraying.40 Briefly, ideal concentrations of these biopolymers were optimized to achieve functional biohybrid capsules with ease of handling as a food ingredient. The skimmed milk or PBS solutions were used for preparation of Bifidobacteriumanimalis subsp. lactis Bb12 incorporated microcapsules, which have differential morphologies and protection abilities for the


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biohybrid systems. Bacterial cell viability measurements were conducted at different relative humidity (75%, 53%, 11% and 0%) and different temperature including 20°C and 4°C. It was found, the skimmed milk use had a better effect on bacterial survival in comparison to use of PBS. In addition, WPC was found to be a better protective matrix for bacterial cells, which provided prolonged bacterial viability at higher relative humidity. Comparative cell viability tests demonstrated that bacterial cell viabilities considerably increased when encapsulated within the biopolymeric matrix in comparison to free cells especially at 20°C, suggesting that encapsulation of bacteria provides protection of bacterial cells from environmental conditions and prolonging their survival. There are similar studies in the literature showing electrospun fibrous systems can be promising systems for encapsulation, stabilization or storage of probiotic bacteria, by using polymeric60,61 or agrowaste-based nanofibers.62 In the case of preparing potential antibacterial food packaging materials, T4 bacteriophage was encapsulated in electrospun fibrous systems, and the results showed that encapsulated T4 bacteriophage had high viability, loading capacity, and storage time,41 suggesting the biohybrid systems are ideal candidates in application of antibacterial food packaging. Nevertheless, authors also commented that such virus containing materials need to overcome the negative perception of viruses by the public for real applications.41 For potential use in bioremediation of water systems, Sarioglu et al. generated two different biohybrid systems by encapsulating Pseudomonas aeruginosa strain which has commercial methylene blue (MB) removal capability within polyvinyl alcohol (PVA) and polyethylene oxide (PEO) electrospun nanofibers.59 A schematic representation for describing this process is illustrated in Figure 3. In order to achieve adequate numbers of viable bacteria in nanofibrous systems after the electrospinning process, highly condensed bacterial cell biomasses were


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included in the electrospinning solution. Bacterial cell viabilities were checked using staining of live/dead cells and viable cell counting assay (VCC). The nanofibrous webs of PVA and PEO encapsulated with bacteria were utilized for starting initial inoculum by dissolving these two water soluble systems in MB containing aqueous media. The MB removal performances of these two systems were evaluated and found as highly promising (about 70% removal of MB at 25 mg/L by both systems). Further, PVA and PEO fiber system encapsulated with bacteria were stored at room temperature and refrigerated condition (4°C) in order to evaluate their storability, the outcome revealed the storability of fibers for several months without losing cell viability when stored at refrigerated conditions, while the membranes can be storable for just several days at room temperature.

Figure 3. (a) Cartoon image showing electrospinning process for preparation of PVA/PEO webs containing bioremediative bacteria along with their representative photographs, (b) representative photograph and SEM micrograph of polymeric webs demonstrating bacterial encapsulation characteristics along with a cartoon drawing. Reproduced with permission from reference 59. Copyright 2017 Elsevier.


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Further, in a similar study by utilizing a different biopolymeric matrix, San Keskin et al. generated a biohybrid system by encapsulating a heavy metal and dye remediating bacterial strain, Lysinibacillus sp. NOSK into electrospun cyclodextrin (CD) nanofibers, as summarized in Figure 4.63 Cyclodextrins, which are cyclic oligosaccharides produced from starch, can be nutritional source for bacteria upon dissolving CD nanofibers in aqueous media. Optimization of viable bacterial numbers was done by testing inclusion of increasing concentrations of initial bacterial cells into the electrospinning solution. Although a higher concentration of bacterial concentration was also tested (2%, w/w), inclusion of 1% of bacterial cell biomass in the electrospinning solution was found to be optimal concentration to achieve homogenous nanofiber morphology with the desired amounts of viable bacteria. Bacterial cell viabilities

Figure 4. Cartoon image showing preparation of bacteria encapsulated electrospun nanofibers (CD: Cyclodextrin and CD-F: Cyclodextrin-Fiber). Reproduced with permission from reference 63. Copyright 2018 Elsevier.


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within electrospun CD nanofibers were checked by live/dead staining and VCC assay was performed for determining the viable bacterial cell numbers. The biohybrid system was also tested for storability at refrigerated condition (4°C), and encapsulated bacteria were found to be stable and viable for more than 7 days. Bioremediation capability of this system was evaluated using removal of contaminants including Reactive Black 5 dye, Chromium (VI) and Nickel (II) with 30 mg/L initial concentration, and the removal capabilities were found as about 70%, 58% and 82%, respectively. Removal performances of the biohybrid system were compared with removal performances of equal amounts of free bacteria without including CD in the system. It was found that the removal capabilities of the biohybrid system was higher for all of the contaminants, deducing bacterial cells can utilize CD in the system as an additional carbon source and improved growth rates can be achieved by using CD based fibrous systems.

Figure 5. SEM micrographs of 8 wt % PVA nanofibers (a) and 8 wt % of PVA nanofibers containing yeast cells (b). Illustration of optical microscopical images of PVA nanofibers (c) and nanofibers with yeast cells (d). SEM micrograph of nanofibers soaked in water for a period of 24 h (e) crosslinked PVA nanofibers and (f) crosslinked PVA/yeast nanofibers. Reproduced with permission from reference 70. Copyright 2013 American Chemical Society.


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Furthermore, several efforts made on encapsulating several bacteria, algae and bacteriophages in electrospun nanofibers, yeast cells are used for immobilization purpose. The EBY100 (Saccharomyces cerevisiae) cells functionalized with surface-display enhanced green fluorescent protein (eGFP) has been immobilized by electrospinning of PVA and yeast cell solution, followed by chemical vapor crosslinking.70 The cell suspensions of various ratios used for optimizing the condition with 8% PVA solution and electrospun into yeast cell PPVA polymer composite fibers, and then crosslinked using glutaraldehyde vapor. The Figure 5 depicts optical and scanning electron microscopical images of PVA nanofibers immobilized with yeast cells. The resultant membrane also displayed that immobilized yeast cells are viable and also possess the capability in expressing eGFP. Therefore, this approach expected to serve as potential tool in direct immobilization of agents in biocatalysis application. 2.2. Co-electrospinning approach Although electrospinning has been widely recognized for development of nanofibrous composite membranes encapsulating active agents, consequently many difficulties exist for encapsulating therapeutic agents and biologically active agents. Thus, several approaches have been adopted to prepare nanofibers with an inner core and outer shell of different composition, but among other approaches co-electrospinning and emulsion electrospinning has been renowned as viable choice for preparation of core-shell nanofibers efficiently and economically. The core-shell nanofibers offer encapsulated agents in the core remains biologically active since shell of the nanofibers favor protected environment for the inner core. Therefore, the core-shell electrospun nanofibers were mainly explored for their potency in targeted drug delivery and tissue engineering applications by encapsulating numerous active agents including antibiotics, proteins and growth


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factors.71-75 A typical schematic illustration representing coaxial electrospinning and emulsion electrospinning process are presented in Figure 6.76,77

Figure 6. Graphical representation of coaxial electrospinning technique (a), Reproduced with permission from reference 76. Copyright 2017 The American Chemical Society. (b) Emulsion electrospinning describing the formation of core-shell composite fibers. Reproduced with permission from reference 77. Copyright 2006 John Wiley and Sons.

Therefore, adopting the advantage of the core-shell nanofibers numerous attempts were made for encapsulation of microorganisms in these nanofibers. In this regard, whole microbial cells have been encapsulated in electrospun microtubes for the purpose of bioremediation application.78


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Briefly, polycaprolactone (PCL) with polyethylene glycol (PEG) has been used as shell polymer, whereas polyethylene oxide (PEO) was used as core polymeric solution with three different Pseudomonas ADP, Escherichia coli, and Pseudomonas putida S12::DsRed.78 The bacterial cells encapsulated microtubes with porous walls have been prepared by two coaxial spinnerets that use co-electrospinning approach for spinning core and shell polymeric solutions. Further, the microtubes support cell attachment, enzymatic activity and proliferation has been examined. In order to examine the location of bacterial cells in the microtubes, the bacterial strain Pseudomonas putida expressing red fluorescent protein (DsRed) has been encapsulated in microtubes and further fluorescence microscopy images revealed the encapsulation of the bacterial cells and their alignment in microtubes as depicted in Figure 7. The overall results shown well maintained β-galactosidase, phosphatase and denitrification activity of encapsulated cells in electrospun microtubes suggesting their suitability in various microbial cell immobilization.

Figure 7. Microtube morphology and bacterial cell viability analysis. HRSEM images of microtubes (a); fluorescent microscopy images of the bacterial cell P. putida S12::dsred encapsulated in microtubes (b,c). Reproduced with permission from reference 78. Copyright 2009 American Chemical Society.


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Similarly, Lopez-Rubio et al. reported living Bifidobacteria encapsulation in ultrathin PVA electrospun fibers.79 In brief, the Bifidobacterium animalis subsp. lactis Bb12 suspended in milk was used as core solution through pumping of inner needle, whereas the outer needle used for pumping the PVA solution that serves as shell thus resulted in bacterial cell encapsulated coreshell nanofibers. Further, encapsulation performances of obtained electrospun nanofibers were investigated through encapsulation of stained bacterial cells followed by immediate observation of the nanofibers in digital microscopy system which confirmed successful encapsulation of bacterial cells along fibers and the depth observation revealed certain agglomeration of bacterial cells in some areas of the nanofiber. Further, investigations on the nanofiber indicated incorporation on the electrospun nanofiber led to decrease the crystallinity and melting point of the PVA nanofibers and then polymer glass transition temperature increased. Further, viability results performed in 4°C, room temperature and -20°C confirms encapsulated B. animalis remains alive in room temperature condition for 40 days, whereas the refrigerated condition showed cell viability for 130 days, but viability of the not encapsulated bacteria significantly decreased in both cases. Bioremediation of atrazine has been demonstrated by Kleinn et al. by encapsulating the bacterium Pseudomonas sp. ADP that capable of atrazine degradation in electrospun microtubes.80 The bacterium Pseudomonas sp. ADP mixed with polyethylene oxide (PEO) used as core solution and the outer shell solution (type 1) was formulated by polycaprolactone (PCL)/ polyethylene glycol (PEG). In addition, another shell solution was also used to electrospun without PEG (type 2). The viability experiments indicated that polymeric solution of different composition shown to have negative effects on the viability of the bacterial cells. The type 2 composite had effects on the viability and as enzyme functionality and therefore conditions of


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the type 1 considered to be ideal scenario for the degradation of atrazine. The degradation studies indicated removal of 20.6±3 mg atrazine/g mat at day 3 and 47.6±5.9 mg atrazine/g mat at day 7 growth period. Therefore, prior to designing the electrospun nanofibrous biohybrid composite membranes through co-axial electrospinning, a significant attention need to be paid on the choice of ideal polymeric system in order to prepare core-shell fibers for the encapsulation of biologically active agents in that obviously have effects on the biological activity and lead to failure in the performances. In another approach, coaxial electrospinning with microfluidic timer was used for the preparation of PVA solution based bioreactive nanofibers through regulation of silica precursor reaction time for bioremediation application.81 In order to optimize the parameters responsible for producing highly reproducible quality core/shell nanofibers, a factorial design was used to evaluate effect of parameters including core solution flow rate, viscosity and protrusion. Figure 8 shows schematic illustration of designed electrospinning setup that used for the production of reactive membrane consists of PVA-based recombinant E. coli bacteria encapsulated biocompatible core and porous silica shell. The E. coli cells were mixed with PVA solution (8%, 18%, and 28%, w/v) for preparing core solution and loaded in syringe A, the syringe B was loaded with hydrolyzed tetramethyl orthosilicate (TMOS) and further connected to of the inlet in microfluidic timer and the other inlet was connected with syringe C containing 18% of PVA as this concentration had optimal viscosity upon mixing with TMOS. Moreover, electrospinning parameters were adjusted in order to have high encapsulation efficiency, less defect formation and uniform fiber diameter. The well optimized bioreactive membranes were further used for activity measurement by exposing to 150 µM (32.4 ppm) atrazine in potassium phosphate buffer (0.1 M, pH-7), following 20 min treatment the supernatant was collected and measured for atrazine concentration and


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Figure 8. Schematic illustration of designed electrospinning setup used for the preparation of core/shell nanofibers. Coaxial electrospinning setup and connected microfluidic system (a), brief description regarding the chemical reaction between silanol groups of silica and hydroxide groups of PVA in the electrospinning process (b), Effect of critical parameters (c): flow rate ratio between core and shell solution (Qc/Qs), viscocity ratio between core and shell solution (µc/µs) and ratio of core needle protrusion length to shell needle radius (Pc/Rs). Reproduced with permission from reference 81. Copyright 2014 John Wiley and Sons.


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hydroxyatrazine (metabolite of atrazine) through high‐performance liquid chromatography. The results indicate that the membrane encapsulated E. coli cells which express atrazine‐ dechlorinating enzyme AtzA had atrazine degradation rate as 0.24 µmol/g of E. coli/min that was relatively equal to degradation performance of free cells in solution. In continuation to the designing of core/shell electrospun nanofibers for microbial cell encapsulation, an approach using continuous reactor for high cell concentration maintenance under starvation conditions has been reported.82 In this approach, bacteria with polyvinylpyrrolidone (PVP) have been used as core solution and the shell was prepared using polyvinylidene fluoride-co-hexafluoropropylene (PVDF-HFP). The bacteria Pseudomonas sp. strain ADP (P. ADP) has been protected from drying conditions by adopting wet collection method. A schematic describing the electrospinning setup and core/shell microtube fabrication is illustrated in Figure 9. During the preparation process, the microtubes were directly dropped in a water bath followed by collection on a surface using rotating plastic carrier, where the surface has been partially submerged in sterile phosphate buffer solution. The atrazine degradation evaluation showed that the encapsulated bacterial cells had initial inhibition owing to the stress induced by electrospinning process and solvents used. But, the capability was recovered after several consecutive batches performed in reactor that operated for the degradation evaluation for a time period of 50 days and thus results in good degradation efficiency of 83.1±3.9% and ammonium recovery of 75.5±5.9% without substantial deterioration of microtubes. Besides the research on designing instrument for obtaining core/shell electrospun biocomposites, concerns were paid on selection of core/shell polymeric material that can be also evident from


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Figure 9. Preparation process of core/shell microtubes using electrospinning and wet collection method. The electrospinning spinneret was designed with two channels to move core (polyvinylpyrrolidone (PVP) and bacteria) and shell (polyvinylidene fluoride-co-hexafluoropropylene (PVDF-HFP)) polymeric solutions separately to electric field. The microtubes were gathered on a surface of carrier in collecting bath filled with sterile phosphate buffer using motor rotation. Reproduced with permission from reference 82. Copyright 2017 Elsevier.

the previous discussions. In order to produce coaxially electrospun core/shell fibers, processability features of pure hydrophilic glycerol core and starch has been assessed by Lancuški et al. for preparing suspension medium.83 The high-amylase starch formate shell serves as potential prebiotic substance and glycerol core can act as excellent cryo-protectant that enhances the bacterial cell viability, stability and also served as osmolality regulator. Further for the shake of demonstrating encapsulation of biotherapeutic products that are sensitive to environmental factors, the bacteria Lactobacillus paracasei used in the core solution as a model


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system. Then, the survival of bacteria was evaluated under different storage conditions including 4, 25 and 37°C for a period of 21 days. The HR-SEM images confirmed that the starch-formate fibers contain higher number of bacteria indicating their loading performance and further the cell viability performed on the fibers stored at 4°C and room temperature revealed excellent survival of bacteria up to 21 days (See Figure 10). Therefore, it should be noted that evaporation of solvents and porous nature of the shell layer need be consider in order to have better viability of the microbial cells.

Figure 10. Morphological analysis of L. paracasei bacteria, core-shell fibers and the survival measurements of encapsulated bacteria. (a) Optical microscope observation of bacteria dispersed in glycerol as indicated by white arrows. (b&c) HR-SEM images illustrating the appearance of SFG fiber core-shell interface. The white arrows indicate encapsulated bacteria. (d) diameter distribution graph of the fibers. (e) Bacterial shelf life encapsulated in electrospun mats of SGLP fibers stored at different temperatures for a period of 21 days. The results are presented as mean ± standard deviation. Reproduced with permission from reference 83. Copyright 2017 Elsevier.

Furthermore, accountable progress also made on encapsulating virus in electrospun fibers using co-axial electrospinning approach for sustained release. The phage therapy plays vital role in preservation of food and storage that effectively counteract bacterial growth on food surfaces.


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The T4 bacteriophage has been encapsulated in the fibers of PEO core and cellulose diacetate (CDA) shell through co-axial electrospinning.84 The encapsulation behavior of the nanofibers has been observed using transmission electron microscope and the images confirmed the incorporation of T4 phage inside fiber core. Further, the bacteriophage release has been analyzed through immersion of fibers in buffer and the results indicated that fibers with PEO shell shown immediate release, whereas the fibers prepared through blending CDA with PEO substantially decreased the phage release.84 Also, Korehei and Kadla used simple suspension, coaxial and emulsion electrospinning approaches for encapsulating T4 bacteriophage and further compared their activity upon encapsulation.41 In the emulsion electrospinning process, alginate nanoparticles reservoir dispersed in polyethylene oxide (PEO) and chloroform was preencapsulated with phages using emulsification approach and further electrospun as fibers. In another case, the suspension of T4 bacteriophage/buffer was served as core and whereas the PEO used to form shell by using co-axial electrospinning approach. Further, the fibers were undergone freeze drying and stored at different temperatures. The activity of encapsulated T4 bacteriophage has been evaluated using plaque assay and the results demonstrated that about five and two fold drop in their activity noted for simple suspension and emulsion electrospinning approaches, respectively. Interestingly, the encapsulated T4 bacteriophage in core/shell fiber exhibited complete cell viability after storing fibers at +4°C for several weeks.41 Another study demonstrated fabricating electrospun fibrous scaffold using adenovirus that encoded for the gene expressing green fluorescent protein in poly(ε-caprolactone) fibers as core component to achieve localized and sustained transduction.85 The effectiveness of virusencapsulated in cell transducing has been evaluated using critical parameters including cell transduction in scaffold and supernatant, cell proliferation in scaffold and adenovirus controlled


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release. Further, co-culture experiment and flow cytometry characterization were used to assess the performance of electrospun fibers for localizing cell transduction. The outcome of the experiment indicated that, the monolayer exhibit cell transduction of about 0% with 97% cells infected when the HEK 293 cells were pre-seeded in virus encapsulated scaffold and further cocultured for a time period of five days with monolayer of similar cell type separated by transwell membrane possessing 3 µm pore size. The study indicated higher transgene expression for a month in the scaffolds seeded with HEK 293 cells and furthermore virus encapsulated fibers seeded with RAW 264.7 cells activated macrophage cells as a result of lesser level production of IL-1 β, TNF-α and IFN-α. Therefore, the findings suggest an attractive approach for transferring viral gene in regenerative medicine.85 On the other area of living cells encapsulation using co-axial electrospinning, a try also given to encapsulate yeast cells in the electrospun nanofibers for bioremediation and ethanol production. In the study reported by Letnik et al. produced nanofibers with core containing yeast Candida tropicalis cells and nonbiodegradable polymer shell.86 The olive water waste was used to isolate yeast cells that holds the capability of degrading phenol and other natural polyphenols, further also accumulating ethanol. In order to comment on the encapsulation process, the polymer polyvinylpyrrolidone (PVP) of 15% with 20 vol% cell masses was used in core component production, whereas the copolymer comprising poly vinylidene fluoride-co-hexafluoropropylene (PVDF-HFP) of 15% in combination with PEG (2 wt %) has been used for preparation of shell. Finally, plastic carriers used to wound the electrospun fibers that have been collected on a surface earthed with bath containing PBS and then stored in PBS solution by which the fibers were not dried any time. Following the electrospinning process, the encapsulation and viability characteristics has been analyzed using microscopical techniques. Figure 11 shows the


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encapsulated yeast cells immediately following the electrospinning and 3 weeks of post process dictating the cell division. Also, the bioremediation experiments demonstrated significant effects in degradation of phenol and ethanol production postulating these composites have the capability for bioremediation application in water based system.86

Figure 11. Light microscopical images of electrospun microfibers containing C. tropicalis cells (A) immediately following process and (B) 3 weeks of post process illustrating yeast cells budding and division. Reproduced with permission from reference 86. Copyright 2015 American Chemical Society. 2.3. Surface immobilization approach Biofilm formation is a dynamic characteristic of the organisms belongs to archaea and bacteria lineages that considered to be structurally complex and ancient component of their life as evidences can be tracked on the appearance of biofilm in early fossil records (~3.25 billion years ago). The biofilm offers a protected mode of cell growth and further helps to survive against unfavorable or hostile environmental challenges including salinity, pH and metal toxicity.87,49 First and foremost, biofilms shown to have enhanced cell growth, nutrient availability, improved communication and genetic material exchange compared to their free-living planktonic


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forms.88,89 Therefore, embracing the advantage of the biofilms, there has been enormous efforts made with native or genetically modified microorganisms for remediation of several environmental pollutants including heavy metals, pharmaceuticals, dioxins, polycyclic aromatic hydrocarbons, polychlorinated ethenes and biphenyls, also personal care products that has been also listed as toxic compounds by United States Environmental Protection Agency.90,91 In this context, bacteria and algae have been exceptionally explored for their utilization in bioremediation technologies.92-94 Although there have been significant attempts made on encapsulating the microbial cells in electrospun nanofibers, still several challenges exist with electrospinning process including organic solvents and electric field that undoubtedly harm biological activity. Therefore, in recent years a substantial progress was fostered on functionalizing the electrospun nanofibers by immobilizing the microbial cells on the nanofiber surface for potential applications. Even if the electrospun nanofibers offer a higher surface for the viable cell adhesion, it has been noted that fiber diameter and surface chemistries of the nanofibers tends to have influence on the microbial cells attachment and proliferation.95,96 Arbigo et al. used confocal and scanning electron microscopy to investigate the interaction of different bacterial species including Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus with electrospun polystyrene nanofibers with different diameter.95 The experimental results indicated that the fiber diameter affects cell proliferation ability within fibrous network based on cell size and shape. The S. aureus with round shape preferentially proliferate in the fibers with diameter of about 500±200 nm, whereas other two rod shaped bacteria likely showed enhanced proliferation with the fiber diameter of 1000±100 nm revealing that the bacterial cells have enhanced growth in the fiber diameter possess nearly same size of the bacterial cells.95 Likewise, hydrophilic amine rich surface coating


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functionalization of polystyrene fibers demonstrated highest of E. coli, whereas the fiber surface containing higher carboxyl groups because of acrylic acid coating resulted in comparatively less interaction.96 The immobilization efficiency and viability of Escherichia coli strain Nissle 1917 (EcN) has been enhanced through the surface grafting than the coaxial process.97 Xie et al. compared three different methods for immobilizing bacterial cells including coaxial electrospinning, surface grafting with covalent binding and affinity adsorption with intended demonstration for drug delivery applications. The outcome of the study indicated that the bacteria entrapped in the core of nanofibers using coaxial electrospinning able to proliferate only 48% with 12 h passage period. On the other hand, Figure 12 clearly demonstrate the bacterial cells had higher immobilization through affinity adsorption on mannose-grafted fibers than the covalent binding. Therefore, to date, an accountable investigation paved towards attaching the bacterial and microalgal cells on the surface of electrospun nanofiber for functionalization and further used in different applications. For instance, Eroglu et al. demonstrated efficient removal of nitrate using chitosan electrospun nanofibers attached with green microalgae Chlorella vulgaris.98 Briefly, chitosan nanofibers were produced by electrospinning the solution of 6 wt% of chitosan and crosslinked by adding glutaraldehyde solution. Then the pieces of electrospun mats were used to add microalgae growing at exponential phase and kept in room temperature for two days to allow significant surface attachment of algal cells. The presence of negative charge on algal surface owing to uranic acid/sulfate group dissociation and primary amine groups with positive charge present in the chitosan might initiate electrostatic interaction, thus resulted in attachment of algal cells on the nanofiber mats. Further, bio-nanocomposite membrane was placed in nitrate containing artificial growth medium and nitrate level present in the medium was monitored. The


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study outcome resulted with the microalgae attached chitosan mats capable of removing 87±4% nitrate, whereas 32±3% of nitrate was removed by the chitosan mats without microalgae.

Figure 12. Representative (a) SEM and (b) fluorescence images of chitosan nanofibers loaded with bacterial cells through affinity absorption. (c) The graphical plot representing mannose density and bacterial cells absorbed on the fibers following amination of different time points (n = 3). Reproduced with permission from reference 97. Copyright 2016 Royal Society of Chemistry.

In recent years, Prof. Uyar research group extensively involved in development of various bacterial and microalgal cells attached functionalization of electrospun nanofibrous biohybrid composites for potential environmental remediation applications. To highlight, an efficient


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removal of ammonium from aquatic environments has been demonstrated by using a bionanocomposite immobilized with ammonia oxidizing Acinetobacter calcoaceticus STB1 bacteria.33 The bio-nanocomposite was prepared by electrospinning of porous cellulose acetate (CA) nanofibers followed by introducing the nanofibrous web into medium containing bacterial cells and incubated for a period of 35 days. The Figure 13 details the process of electrospinning and bacterial cell attachment on nanofiber surface. After incubation, removal experiments were performed using the basal medium introduced with various concentrations of ammonium (as NH4Cl) un-immobilized bacteria, pristine CA nanofibers or STB1 immobilized CA nanofibers (incubation parameters; agitation speed: 140 rpm, temperature: 30 °C, duration: 48 h). In order to evaluate ammonium removal performances of CA nanofibrous webs immobilized with STB1 strain, different ammonium concentrations were tested for representing low (50 mg L-1), medium (100 mg L-1) and high (200 mg L-1) concentrations. It was found that, CA webs with immobilized bacteria have successfully remediated ammonium within 48 h for each concentration, while their remediation performances in terms of percentile removal seems to be negatively correlated with the increase in initial ammonium concentration. The bacteria free fibrous web showed a negligible ammonium removal performance indicating that the removal was attributed to the bacterial metabolic activities. Interestingly bacteria immobilized CA fibrous web showed complete removal of ammonium from 50 mg L-1 initial concentration within 48 h similar to free bacteria. Further, the STB1 immobilized CA webs capable of removing 98.5% from the initial concentration of 100 mg L-1 and 72% from the initial concentration of 200 mg L1

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Figure 13. (a) Cartoon image showing electrospinning process for preparation of CA nanofibers along with a representative photograph and SEM micrograph. (b) Representative photograph and SEM image showing STB1 immobilized CA nanofibers along with a cartoon drawing. Reproduced with permission from reference 33. Copyright 2013 Royal Society of Chemistry.

Another study demonstrated that bacterial cells immobilized nanofibrous composite membrane was capable of efficient decolorization of a common dye pollutant (methylene blue) used in textile and paper industries.99 In this study, three different bacterial species namely Pseudomonas aeruginosa, Clavibacter michiganensis and Aeromonas eucrenophila were immobilized on surface of the electrospun cellulose acetate nanofibrous web (CA-NFW) following incubation period of 7 days in medium containing bacterial cells at a density of ∼107 CFU mL−1. Following the incubation, the SEM was employed in observation of bacterial attachment over the CANFW, the results revealed that a biofilm layer has been formed following the incubation and the webs were further used to decolorize methylene blue (MB) dye by means of effects based on


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various contact time and initial concentration in the range 0-48h and 20-500 mg/mL using UVvis spectrophotometer. In a typical experiment, to evaluate effect of contact time the bacteria immobilized CA-NFW (~20 mg) was introduced in the solution containing 20 mg/mL of MB dye and the decolorization efficiencies were monitored at 3, 12, 24 and 48 h. On the other hand, dyes at the initial concentration ranging from 20-500 mg L−1 that represent concentrations for low, medium and high were used to understand the effects due to initial concentration. The results indicated that the bacteria immobilized CA-NFW shown excellent decolorization performances (P. aeruginosa - 96.5±0.4%, A. eucrenophila - 96.1±0.4% and C. michiganensis 95.6±0.3%) which is almost comparable to the free bacterial cell decolorization efficiency. Interestingly, the decolorization performances of the CA-NFW immobilized with bacterial cells affected upon increasing the concentration of MB dye, as 20 mg/mL concentration dye solutions were decolorized above 90% after 48 h incubation, whereas comparatively less decolorization performances were noted for the dye solutions containing 100, 250 and 500 mg L−1. The reusability tests also indicated that about 45% decolorization was observed at fourth cycle indicating that the CA-NFW immobilized with bacteria possesses excellent decolorization capability even after several cycles of reuse. In a similar case, an attempt was made in removing Remazol Black 5 (RB5) and Reactive Blue 221 (RB221) using electrospun polysulfone nanofibrous web (PSU-NFW) immobilized with Chlamydomona reinhardtii microalgae.36 A detailed schematic demonstration on the electrospinning process, microalgae attachment and decolorization test are presented in Figure 14. The microalgae attachment on the PSU-NFW has been confirmed by SEM and Raman spectroscopy analysis following 10 days of incubation. Further, the microalgae immobilized membranes were used for removal of reactive dyes through evaluation of effects on contact time


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and dye concentration. The contact time experiment shown that, the microalgae immobilized electrospun nanofiber webs were capable of decolorizing RB5 (10 mg/mL) of 73.0 ± 0.3% within 14 days which is higher than the performance observed in free algal cells. But very less decolorization efficiency was observed for the RB221with a maximum decolorization performance of 30.2±0.2%. Consequently, the increase of initial dye concentrations has been dramatically affects the decolorization performance. Further, three cycles of reusability studies demonstrated significant decolorization performances that indicating microalgae/PSU-NFW can be used repeatedly in the purpose of decolorizing waste water from paint and textile industries.

Figure 14. Comprehensive schematic demonstration of preparing pristine PSU-NFW, attachment of microalgae decolorization test. The digital photographs and scanning electron microscopy images show before and after microalgae attachment after different days of incubation. Reproduced with permission from reference 36. Copyright 2015 American Chemical Society.

Further, the PSU-NFWs were extended in removing heavy metal and dye pollutants simultaneously through immobilizing Lysinibacillus sp. NOSK which is pollutant resistant and isolated from soil sample.30 The removal performances of PSU-NFWs towards Cr(VI) and Reactive Black 5 (RB5) was evaluated by monitoring effects of several parameters including temperature, initial pH values, static/shaking conditions, concentration of reactive dye and Cr(VI), based on the outcome their conditions have been optimized. Based on the optimized


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parameters, single and binary effects on removal of metal and dye pollutants have been performed. The overall results revealed that the bacteria attached PSU-NFWs shown excellent removal performances of RB5 (99.7±0.9%) and Cr(VI) (98.2±0.6%). The pristine membranes tend to have comparatively very less removal of these pollutants confirming the removal performances of the immobilized bacterial cells. Further, the adsorption isotherm studies estimated maximum adsorption capacities for RB5 and Cr(VI) was 35.17 and 5.67 mg g−1, respectively. In addition, the reusability studies also demonstrated a significant removal of these pollutants up to seven cycles. Consequently, a comparison on the bioremoval and biosorption of the pollutants have been investigated. Recently, electrospun nanofibrous membranes of polylactic acid (PLA) and polycaprolactone (PCL) immobilized with Clavibacter michiganensis tested against Setazol Blue BRF-X, a textile reactive dye and the outcome indicated a similar removal performances of dye pollutant.100 Besides the evaluation of potential bioremediation performances of the various pollutants using different bio-nanocomposites, a significant research has been also conducted to understand influence of fiber morphology on bacterial immobilization and their bioremediation performances. For instance, a novel electrospun biohybrid composites have been fabricated through immobilizing bacterial strains that capable of degrading sodium dodecyl sulfate (SDS) namely Achromobacter xylosoxidans STB4 and Serratia proteamaculans STB on electrospun nanofibrous webs of non-porous cellulose acetate (nCA) and porous cellulose acetate (pCA). The pCA nanofibers had initial interference on the bacterial attachment but the attachment became similar to nCA in 21-day time period. The SDS degradation experiments indicated that pCA nanofibrous bionanocomposites have higher degradation efficiency. The degradation efficiencies were mainly based on the bacterial immobilization properties based on the surface area offered


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by the nanofibrous webs.101 Another study highlighted the hydrophobic nature of the electrospun biohybrid composite plays a critical role in removal of Cr(VI) pollutant.102 Briefly, polystyrene (PS) and polysulfone (PSU) electrospun fibrous matrix were fabricated and characterized using appropriate techniques. The contact angle measurement showed PS webs found to have higher hydrophobic nature than the PSU web. The speculation arises in the higher removal performances of PS web could be attributed to the hydrophobic nature that provides hydrophobic interaction with Morganella morganii STB5 cells that results in better immobilization of bacteria than the less hydrophobic surface.102 Further, the fiber alignment tends to have influence on the bacterial immobilization and correspondingly influences the removal performances of pollutants. To claim the support of statement, the PSU nanofibers with different orientation (random and aligned) and diameter (thin and thick) were prepared and used for bacterial integration purpose as carrier matrix.103 The fibrous web immobilized with Acinetobacter calcoaceticus STB1 strain was characterized for determining the optimal fibrous type in bioremediation applications. Based on the cell viability experiments, the PSU fibers with random orientation and thinner diameter found to have highest bacterial immobilization than the other three fibrous types which was then employed for ammonium and methylene blue dye bioremediation. In summary, the electrospun biohybrid composite developed through the attachment of various microbial cells demonstrated for bioremediation of different environmental pollutants. In addition, a very recent study reported by Da Cesare et al. investigates the effects of nanofiber size on bacterial cell adhesion using 3D self-standing electrospun nanofibrous poly(ε-caprolactone) and Burkholderia terricola bacteria. The interaction studies revealed that the bacterial cells preferably attach on the nanofibers which are smaller than size of bacteria and films coated with bacterial origin organic substance, thus


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results in development of microcolonies which is an initial step in formation of biofilm.104 Based on the reports discussed, the development of electrospun nanofibrous biohybrid composites need to considered various factors including selection of polymer for electrospinning, morphology and alignment of the fiber, contact time of immobilization and concentration of the pollutants, pH of the solution, temperature and rotatory speed of the apparatus in order to have efficient removal performances of the biohybrid composites. 3. Conclusion and future outlook Ultimately, the pros and cons of each electrospun nanofibrous biohybrid composites judged based on the beneficial characteristics of fabrication approach and presence of individual components that offer potential for intended applications. Indeed, the present review summarize three different approaches in preparation of electrospun nanofibrous biohybrid composites, each approach demonstrated their potency with particular characteristics. For instance, the direct encapsulation and core-shell encapsulation approaches mainly offer the protection and control over unfavorable environment and physicomechanical properties thus improving the cell viability for longer time period. Correspondingly, the review presented many examples of encapsulated microbial cells retained their cell viability from several days to months that showed their potential in different application including tissue engineering and bioremediation. Particularly, encapsulation approaches always need special attention on the selection of polymeric system, solvent, humidity of the chamber and applied voltage in order to prevent the negative effects on the microbial cells during encapsulation process. In the case of surface immobilization, the electrospun nanofibrous membrane offer higher surface area which results in enhanced viable cell immobilization. Most importantly, in the surface immobilization approach the microbial cells can be immobilized using different surface functionalization binding


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strategies as described earlier. Furthermore, the electrospun nanofibrous biohybrid composites fabricated using surface immobilization approach can be used in several cycles of reuse especially in removal of toxic pollutants. But, the fiber diameter and alignment greatly influence the adhesion of microbial cells. In this review, rational design, development and potential applications of electrospun nanofibrous biohybrid composite systems based on various microorganisms (i.e. bacteria, algae, viruses and yeast) are summarized. Three different approaches are actively used for integration of microorganisms with electrospun fibrous systems. In the first approach, direct encapsulation of microbial cells within electrospun fibrous systems is provided by inclusion of microbial biomasses into electrospinning solutions. Non-hazardous solvents and biocompatible polymers are preferred for maximizing cell viabilities of microbial cells in this approach. In the second approach, core-shell electrospinning technique by utilizing a co-axial injection system is used for encapsulation of microbial cells within electrospun fibrous systems. Although direct encapsulation and core-shell electrospinning strategies share lack of necessity for further processing and enable fabrication of biohybrid systems in a single step, core-shell electrospinning differs from direct encapsulation approach by allowing preparation of polymeric mixture (shell) and microbial biomass (core) in two different solvent systems with reducing direct contact of electric field on microorganisms and cell viability losses. In the third approach, surfaces of electrospun nanofibrous materials are functionalized by attachment of microorganisms. The most preferred attachment method is natural adhesion of microorganisms, which may provide more stable and biochemically active cells as occurred by formation of bacterial biofilms. Various microorganisms integrated electrospun nanofibrous systems have great potentials for development of biohybrid composites systems for use in differential


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application areas including tissue engineering, food, agriculture, sensing and bioremediation. Nevertheless, these types of biohybrid nanofibrous systems are not yet plausible for use in situ applications. Optimizations are required for reducing manufacturing costs (e.g. using economical and more available natural polymer systems), reducing manufacturing time along with facilitating the methodologies for large scale industrial development, maximizing cell viability and biological activities, and enhancing performances (e.g. for bioremediation applications). Fortunately, there are recent progresses in the literature for such kind of optimizations as reported in the above sections and new start-up companies having a focus of this issue have been started to establish.105 The electrospun nanofiber based products has been tremendously commercially transformed through several companies around the world for various applications including air filtration, liquid filtration, molecular filtration, drug delivery, veterinary medicine, 3D cell culture products, regenerative medicine and functional apparels. Interestingly, the NanoSpun ‘Biology as Cassette’ line of products fabricated through effective encapsulation or immobilization of predetermined microorganisms aiming water and waste water treatment, health and medical and renewable chemical applications (http://www.nanospuntech.com). Corresponding Authors *Tamer Uyar: [email protected] *Brabu Balusamy: [email protected] ORCID ID Brabu Balusamy: 0000-0002-4103-566X Tamer Uyar: 0000-0002-3989-4481


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Author Contributions The manuscript was written through contributions of all the authors. All authors approved final version of the manuscript. Notes There are no conflicts to declare. References 1. Lin, T.; Fang, J. Fundamentals of Electrospinning & Electrospun Nanofibers, DEStech Publications, Inc: Pennsylvania, U.S.A., 2017. 2. Li, Z; Wang, C. One-Dimensional Nanostructures: Electrospinning Technique and Unique Nanofibers, Springer-Verlag Berlin Heidelberg, Germany, 2013. 3. Mitchell, G.R. Electrospinning: Principles, Practice and Possibilities, The Royal Society of Chemistry: Cambridge, UK, 2015. 4. Focarete, M.L.; Gualandi, C.; Ramakrishna, S. Filtering Media by Electrospinning: Next Generation Membranes for Separation Applications, 1st ed.; Springer International Publishing, Switzerland, 2018. 5. Macagnano, A.; Zampetti, E.; Kny, E. Electrospinning for High Performance Sensors, 1st ed.; Springer International Publishing, Switzerland, 2015. 6. Senthamizhan, A.; Balusamy, B; Uyar, T. Glucose sensors based on electrospun nanofibers: a review. Anal. Bioanal. Chem. 2016, 408, 1285–1306. 7. Ogunlaja, A.S.; Kleyi, P.E.; Walmsley, R.S.; Tshentu, Z.R. Nanofiber-supported metal-based catalysts. In Catalysis, Spivey, J.J., Han, Y.F., Dooley, K.M., Eds.; The Royal Society of Chemistry: Cambridge, UK, 2016; Volume 27, pp. 144-174. 8. Ding, B.; Yu, J.; Electrospun Nanofibers for Energy and Environmental Applications, 1st ed.; Springer-Verlag Berlin Heidelberg, Germany, 2014. 9. Bagherzadeh, R.; Gorji, M.; Sorayani Bafgi, M.S.; Saveh-Shemshaki, N. Electrospun conductive nanofibers for electronics. In Electrospun Nanofibers, Afshari, M., Eds.; Woodhead Publishing Series in Textiles, Elsevier, 2017; Volume 186, pp. 467-519. 10. Uyar, T.; Kny, E.; Electrospun Materials for Tissue Engineering and Biomedical Applications: Research, Design and Commercialization, 1st ed.; Woodhead Publishing Series in Biomaterials, Elsevier, United Kingdom, 2017. 11. Balusamy, B.; Senthamizhan, A.; Uyar, T. Electrospun nanofibrous materials for wound healing applications. In Electrospun Materials for Tissue Engineering and Biomedical Applications: Research, Design and Commercialization. Uyar, T.; Kny, E. Eds.; Woodhead Publishing Series in Biomaterials, Elsevier, United Kingdom, 2017; pp. 101-113.


41


Page 42 of 49

12. Braghirolli, D.I.; Steffens, D.; Pranke, P. Electrospinning for regenerative medicine: a review of the main topics. Drug Discov. Today 2014, 19, 743-753. 13. Almodovar, J.; Electrospun Biomaterials and Related Technologies, 1st ed.; Springer International Publishing, Switzerland, 2017. 14. Schneider, R.; Mercante, L.A.; Andre, R.S.; Brandão, H.M.; Mattoso, L.H.C.; Correa, D.S. Biocompatible electrospun nanofibers containing cloxacillin: Antibacterial activity and effect of pH on the release profile. React. Funct. Polym. 2018, 132, 26-35. 15. Ghalei, S.; Asadi, H.; Ghalei, B. Zein nanoparticle‐embedded electrospun PVA nanofibers as wound dressing for topical delivery of anti‐inflammatory diclofenac. J. Appl. Polym. Sci. 2018, 135, 46643. 16. Senthamizhan, A.; Balusamy, B.; Aytac, Z.; Uyar, T. Grain boundary engineering in electrospun ZnO nanostructures as promising photocatalysts. CrystEngComm 2016, 18, 63416351. 17. Anitha, S.; Brabu, B.; Thiruvadigal, D.J.; Gopalakrishnan, C.; Natarajan, T. S. Preparation of free-standing electrospun composite ZnO membrane for antibacterial applications. Adv. Sci. Lett. 2012, 5, 468-474. 18. Senthamizhan, A.; Balusamy, B.; Aytac, Z.; Uyar, T. Ultrasensitive electrospun fluorescent nanofibrous membrane for rapid visual colorimetric detection of H2O2. Anal. Bioanal. Chem. 2016, 408, 1347–1355. 19. Yoo, H.S.; Kim, T.G.; Park, T.G. Surface-functionalized electrospun nanofibers for tissue engineering and drug delivery. Adv Drug Deliv Rev. 2009, 61, 1033-1042. 20. Vellayappan, M.V.; Venugopal, J.R.; Ramakrishna, S.; Ray, S.; Ismail, A.F.; Mandal, M.; Manikandan, A.; Seal, S.; Jaganathan, S.K. Electrospinning applications from diagnosis to treatment of diabetes. RSC Adv. 2016, 6, 83638-83655. 21. Peng, S.; Jin, G.; Li, L.; Li, K.; Srinivasan, M.; Ramakrishna, S.; Chen, J.; Multi-functional electrospun nanofibres for advances in tissue regeneration, energy conversion & storage, and water treatment. Chem. Soc. Rev. 2016, 45, 1225-1241. 22. Zhang, M.; Zhao, X.; Zhang, G.; Wei, G.; Su, Z. Electrospinning design of functional nanostructures for biosensor applications. J. Mater. Chem. B 2017, 5, 1699-1711. 23. Xiao, Y.; Shen, M.; Shi, X. Design of functional electrospun nanofibers for cancer cell capture applications. J. Mater. Chem. B, 2018, 6, 1420-1432. 24. Stoddard, R.J.; Steger, A.L.; Blakney, A.K.; Woodrow, K.A. In pursuit of functional electrospun materials for clinical applications in humans. Ther Deliv. 2016, 7, 387-409. 25. Choi, J.; Yang, B.J.; Bae, G.N.; Jung, J.H. Herbal extract incorporated nanofiber fabricated by an electrospinning technique and its application to antimicrobial air filtration. ACS Appl Mater Interfaces 2015, 7, 25313-25320. 26. Yuan, T.T.; DiGeorge Foushee, A.M.; Johnson, M.C.; Jockheck-Clark, A.R.; Stahl, J.M. Development of electrospun chitosan-polyethylene oxide/fibrinogen biocomposite for potential wound healing applications. Nanoscale Res. Lett. 2018, 13, 88.


42

Page 43 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


27. Pilehvar-Soltanahmadi, Y.; Dadashpour, M.; Mohajeri, A.; Fattahi, A.; Sheervalilou, R.; Zarghami, N. An overview on application of natural substances incorporated with electrospun nanofibrous scaffolds to development of innovative wound dressings. Mini Rev Med Chem. 2018, 18, 414-427. 28. Banerjee, A.; Roy, A.; Dutta, S.; Mondal, S. Bioremediation of hydrocarbon – A Review. Int. J. Adv. Res. 2016, 4, 1303-1313. 29. Azubuike, C.C.; Chikere, C.B.; Okpokwasili, G.C. Bioremediation techniques–classification based on site of application: principles, advantages, limitations and prospects. World J. Microbiol. Biotechnol. 2016, 32, 180. 30. San Keskin, N.O.; Celebioglu, A.; Sarioglu, O.F.; Ozkan, A.D.; Uyar, T.; Tekinay, T. Removal of a reactive dye and hexavalent chromium by a reusable bacteria attached electrospun nanofibrous web. RSC Adv. 2015, 5, 86867-86874. 31. Malik, A. Metal bioremediation through growing cells. Environ. Int. 2004, 30, 261-278. 32. Zhao, B.; He, Y.L.; Hughes, J.; Zhang, X.F. Heterotrophic nitrogen removal by a newly isolated Acinetobacter calcoaceticus HNR. Bioresour. Technol. 2010, 101, 5194-5200. 33. Sarioglu, O.F.; Yasa, O.; Celebioglu, A.; Uyar, T.; Tekinay, T. Efficient ammonium removal from aquatic environments by Acinetobacter calcoaceticus STB1 immobilized on an electrospun cellulose acetate nanofibrous web. Green Chem. 2013, 15, 2566-2572. 34. Xiong, J.Q.; Kurade, M.B.; Jeon, B.H. Can Microalgae remove pharmaceutical contaminants from water? Trends Biotechnol. 2018, 36, 30-44. 35. Ramírez, M.E.; Vélez, Y.H.; Rendón, L.; Alzate, E. Potential of microalgae in the bioremediation of water with chloride content. Braz. J. Biol. 2018, 78, 472-476. 36. San Keskin, N.O.; Celebioglu, A.; Uyar, T.; Tekinay, T. Microalgae immobilized by nanofibrous web for removal of reactive dyes from wastewater. Ind. Eng. Chem. Res. 2015, 54, 5802-5809. 37. Sansone, F.; Mencherini, T.; Picerno, P.; d’Amore, M.; Aquino, R.P.; Lauro, M.R. Maltodextrin/pectin microparticles by spray drying as carrier for nutraceutical extracts. J. Food Eng. 2011, 105, 468–476. 38. Wang, X.; Yue, T.; Lee, T.C. Development of Pleurocidin-poly(vinyl alcohol) electrospun antimicrobial nanofibers to retain antimicrobial activity in food system application. Food Control 2015, 54, 150–157. 39. Espitia, P.J.P.; Batista, R.A.; Azeredo, H.M.C.; Otoni, C.G. Probiotics and their potential applications in active edible films and coatings. Food Res. Int. 2016, 90, 42–52. 40. López-Rubio, A.; Sanchez, E.; Wilkanowicz, S.; Sanz, Y.; Lagaron, J.M. Electrospinning as a useful technique for the encapsulation of living bifidobacteria in food hydrocolloids. Food Hydrocoll. 2012, 28, 159-167. 41. Korehei, R.; Kadla, J. Incorporation of T4 bacteriophage in electrospun fibres. J Appl Microbiol. 2013, 114, 1425-1434.


43


Page 44 of 49

42. de Souza R.; Ambrosini, A.; Passaglia, L.M.P. Plant growth-promoting bacteria as inoculants in agricultural soils. Genet Mol Biol. 2015, 38, 401–419. 43. Damasceno, R.; Roggia, I.; Pereira, C.; de Sá, E. Rhizobia survival in seeds coated with polyvinyl alcohol (PVA) electrospun nanofibres. Can. J. Microbiol. 2013, 59, 716-719. 44. Fernandes, P. Enzymes in Food Processing: A Condensed Overview on Strategies for Better Biocatalysts. Enzyme Res. 2010, 2010, 862537. 45. Arroyo, M.; la Mata, I.; García, J.L.; Barredo, J.L. Biocatalysis for Industrial Production of Active Pharmaceutical Ingredients (APIs). In Biotechnology of Microbial Enzymes: Production, Biocatalysis and Industrial Applications, 1st ed.; Brahmachari, G., Eds.; Academic Press, Cambridge, United States, 2017; pp. 451-473. 46. Sheldon R.A.; van Pelt, S. Enzyme immobilisation in biocatalysis: why, what and how. Chem. Soc. Rev. 2013, 42, 6223–6235. 47. Zajkoska, P.; Rebroš, M.; Rosenberg, M. Biocatalysis with immobilized Escherichia coli. Appl. Microbiol. Biotechnol. 2013, 97, 1441–1455. 48. Pinto, S.C.; Rodrigues, A.R.; Saraiva, J.A.; Lopes-da-Silva, J.A. Catalytic activity of trypsin entrapped in electrospun poly(ϵ-caprolactone) nanofibers. Enzyme Microb. Technol. 2015, 79–80, 8–18. 49. Liu, Y.; Rafailovich, M.H.; Malal, R.; Cohn, D.; Chidambaram, D. Engineering of bio-hybrid materials by electrospinning polymer-microbe fibers. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 14201-14206. 50. Shimizu, N.; Ishida, D.; Yamamoto, A.; Kuroyanagi, M.; Kuroyanagi, Y. Development of a functional wound dressing composed of hyaluronic acid spongy sheet containing bioactive components: evaluation of wound healing potential in animal tests. J. Biomater. Sci. Polym. Ed. 2014, 25, 1278–1291. 51. Simões, D.; Miguel, S.P.; Ribeiro, M.P.; Coutinho, P.; Mendonça, A.G.; Correia, I.J. Recent advances on antimicrobial wound dressing: A review. Eur. J. Pharm. Biopharm. 2018, 127, 130–141. 52. Maver, T.; Hribernik, S.; Mohan, T.; Smrke, D.M.; Maver, U.; Stana-Kleinschek, K. Functional wound dressing materials with highly tunable drug release properties. RSC Adv. 2015, 5, 77873–77884. 53. Cha, B.G.; Kwak, H.W.; Park, A.R.; Kim, S.H.; Park, S.Y.; Kim, H.J.; Kim, I.S.; Lee, K.H.; Park, Y.H. Structural Characteristics and Biological Performance of Silk Fibroin Nanofiber Containing Microalgae Spirulina Extract. Biopolymers 2014, 101, 307–318. 54. Kim, S.H.; Shin, C.; Min, S.K.; Jung, S.M.; Shin, H.S. In vitro evaluation of the effects of electrospun PCL nanofiber mats containing the microalgae Spirulina (Arthrospira) extract on primary astrocytes. Colloids Surf. B Biointerfaces 2012, 1, 113-118.


44

Page 45 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


55. de Morais, M.G.; Stillings, C.; Dersch, R.; Rudisile, M.; Pranke, P.; Costa, J.A.V.; Wendorff, J. Preparation of nanofibers containing the microalga Spirulina (Arthrospira). Bioresour Technol. 2010, 101, 2872-2876. 56. Greiner, A.; Wendorff, J.H.; Yarin, A.L.; Zussman, E. Biohybrid nanosystems with polymer nanofibers and nanotubes. Appl. Microbiol. Biotechnol. 2006, 71, 387-393. 57. Gensheimer, M.; Becker, M.; Brandis-Heep, A.; Wendorff, J.H.; Thaer, R.K.; Greiner, A. Novel biohybrid materials by electrospinning: Nanofibers of poly(ethylene oxide) and living bacteria. Adv. Mater. 2007, 19, 2480-2482. 58. Salalha, W.; Kuhn, J.; Dror, Y.; Zussman, E. Encapsulation of bacteria and viruses in electrospun nanofibres. Nanotechnology 2006, 17, 4675-4681. 59. Sarioglu, O.F.; San Keskin, N.O.; Celebioglu, A.; Tekinay, T.; Uyar, T. Bacteria encapsulated electrospun nanofibrous webs for remediation of methylene blue dye in water. Colloids Surf. B Biointerfaces 2017, 152, 245-251. 60. Heunis, T.D.; Botes, M.; Dicks, L.M. Encapsulation of Lactobacillus plantarum 423 and its Bacteriocin in Nanofibers. Probiotics Antimicrob. Proteins 2010, 2, 46-51. 61. Amna, T.; Hassan, M.S.; Pandeva, D.R.; Khil, M.S.; Hwang, I.H. Classy non-wovens based on animate L. gasseri-inanimate poly(vinyl alcohol): upstream application in food engineering. Appl. Microbiol. Biotechnol. 2013, 97, 4523-4531. 62. Fung, W.Y.; Yuen, K.H.; Liong, M.T. Agrowaste-based nanofibers as a probiotic encapsulant: fabrication and characterization. J. Agric. Food Chem. 2011, 10, 8140-8147. 63. San Keskin, N.O.; Celebioglu, A.; Sarioglu, O.F.; Uyar, T.; Tekinay, T. Encapsulation of living bacteria in electrospun cyclodextrin ultrathin fibers for bioremediation of heavy metals and reactive dye from wastewater. Colloids Surf. B Biointerfaces 2018, 161, 169-176. 64. Gensheimer, M.; Brandis-Heep, A.; Agarwal, S., Thauer, R.K.; Greiner, A. Polymer/bacteria composite nanofiber non-wovens by electrospinning of living bacteria protected by hydrogel microparticles. Macromol. Biosci. 2011, 11, 333-337. 65. Vajdai, A.; Szabó, B.; Süvegh, K.; Zelkó, R.; Ujhelyi, G. Tracking of the viability of Stenotrophomonas maltophilia bacteria population in polyvinylalcohol nanofiber webs by positron annihilation lifetime spectroscopy. Int. J. Pharm. 2012, 429, 135-137. 66. Tong, H.W.; Mutlu, B.R.; Wackett, L.P.; Aksan, A. Silica/PVA biocatalytic nanofibers. Mater. Lett. 2013, 111, 234-237. 67. Zussman, E. Encapsulation of cells within electrospun fibers. Polym. Adv. Technol. 2011, 22, 366-371. 68. Tromp, R.H.; Vink, C.; Stijnman, A.C. Encapsulation by Electrospinning of Live Bacteria used in the Food Industry. Gums and Stabilisers for the Food Industry 16, 1st ed.; Royal Society of Chemistry: London, UK, 2012, pp. 247-255. 69. Pagano, M.C.; Miransari, M. The importance of soybean production worldwide. In Abiotic and Biotic Stresses in Soybean Production, 1st ed.; Miransari, M., Eds.; Elsevier: Amsterdam, Netherlands, 2016; Volume 1, pp. 1-26.


45


Page 46 of 49

70. Canbolat, M.F.; Gera, N.; Tang, C.; Monian, B.; Rao, B.M.; Pourdeyhimi, B.; Khan, S.A. Preservation of cell viability and protein conformation on immobilization within nanofibers via electrospinning functionalized yeast. ACS Appl Mater Interfaces 2013, 5, 9349-9354. 71. Aytac, Z.; Uyar, T. Core-shell nanofibers of curcumin/cyclodextrin inclusion complex and polylactic acid: Enhanced water solubility and slow release of curcumin. Int. J. Pharm. 2017, 518, 177-184. 72. Romano, L.; Camposeo, A.; Manco, R.; Moffa, M.; Pisignano, D. Core-shell electrospun fibers encapsulating chromophores or luminescent proteins for microscopically controlled molecular release. Mol Pharm. 2016, 13, 729-736. 73. Moghe, A.K.; Gupta, B.S. Co‐axial electrospinning for nanofiber structures: preparation and applications. Polymer Reviews 2008, 48, 353-377. 74. Lu, Y.; Huang, J.; Yu, G.; Cardenas, R.; Wei, S.; Wujcik, E.K.; Guo, Z. Coaxial electrospun fibers: applications in drug delivery and tissue engineering. WIREs Nanomed. Nanobiotechnol. 2016, 8, 654–677. 75. McClellan, P.; Landis, W.J. Recent applications of coaxial and emulsion electrospinning methods in the field of tissue engineering. Biores Open Access 2016, 5, 212-227. 76. Wen, P.; Wen, Y.; Zong, M.H.; Linhardt, R.J.; Wu, H. Encapsulation of bioactive compound in electrospun fibers and its potential application. J. Agric. Food Chem. 2017, 65, 9161–9179. 77. Xu, X.; Zhuang, X.; Chen, X.; Wang, X.; Yang, L.; Jing, X. Preparation of core‐sheath composite nanofibers by emulsion electrospinning. Macromol Rapid Commun. 2006, 27, 1637-1642. 78. Klein, S.; Kuhn, J.; Avrahami, R.; Tarre, S.; Beliavski, M.; Green, M.; Zussman, E.; Encapsulation of bacterial cells in electrospun microtubes. Biomacromolecules 2009, 10, 1751-1756. 79. Lopez-Rubio, A.; Sanchez, E., Sanz, Y.; Lagaron, J.M. Encapsulation of living bifidobacteria in ultrathin PVOH electrospun fibers. Biomacromolecules 2009, 10, 2823-2829. 80. Klein, S.; Avrahami, R.; Zussman, E.; Beliavski, M.; Tarre, S.; Green, M. Encapsulation of Pseudomonas sp. ADP cells in electrospun microtubes for atrazine bioremediation. J. Ind. Microbiol. Biotechnol. 2012, 39, 1605–1613. 81. Tong, H.W.; Mutlu, B.R.; Wackett, L.P.; Aksan, A. Manufacturing of bioreactive nanofibers for bioremediation. Biotechnol. Bioeng. 2014, 111, 1483-1493. 82. Desitti, C.; Beliavski, M.; Tarre, S.; Avrahami, R.; Zussman, E.; Green, M. Durable electrospun microtubes for encapsulation of bacteria in atrazine bioremediation. J. Water Process Eng. 2017, 19, 205–211. 83. Lancuški, A.; Ammar, A.A.; Avrahami, R.; Vilensky, R.; Vasilyev, G.; Zussman, E. Design of starch-formate compound fibers as encapsulation platform for biotherapeutics. Carbohydr Polym. 2017, 158, 68-76. 84. Korehei, R.; Kadla, J.F. Encapsulation of T4 bacteriophage in electrospun poly(ethylene oxide)/cellulose diacetate fibers. Carbohydr Polym. 2014, 100, 150-157.


46

Page 47 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


85. Liao, I.C.; Chen, S.; Liu, J.B.; Leong, K.W. Sustained viral gene delivery through core-shell fibers. J Control Release 2009, 139, 48-55. 86. Letnik, I.; Avrahami, R.; Rokem, J.S.; Greiner, A.; Zussman, E.; Greenblatt, C. Living Composites of Electrospun Yeast Cells for Bioremediation and Ethanol Production. Biomacromolecules 2015, 16, 3322-3328. 87. Hall-Stoodley, J; Costerton, L. W.; Stoodley, P. Bacterial biofilms: from the Natural environment to infectious diseases. Nat. Rev. Microbiol. 2004, 2, 95-108. 88. Davies, D.G.; Parsek, M.R.; Pearson, J.P.; Iglewski, B.H.; Costerton, J.W.; Greenberg, E.P. The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 1998, 280, 295–298. 89. Costerton, J.W. Introduction to biofilm. Int. J. Antimicrob. Agents 1999, 11, 217–221. 90. Edwards, S.J.; Kjellerup, B.V. Applications of biofilms in bioremediation and biotransformation of persistent organic pollutants, pharmaceuticals/personal care products, and heavy metals. Appl. Microbiol. Biotechnol. 2013, 97, 9909-9921. 91. Mitra, A.; Mukhopadhyay, S. Biofilm mediated decontamination of pollutants from the environment. AIMS Bioengineering, 2016, 3, 44-59. 92. Abinandan, S.; Subashchandrabose, S.R.; Venkateswarlu, K.; Megharaj, M. Microalgaebacteria biofilms: a sustainable synergistic approach in remediation of acid mine drainage. Appl. Microbiol. Biotechnol. 2018, 102, 1131-1144. 93. Hayat, K.; Menhas, S.; Bundschuh, J.; Chaudhary, H.J. Microbial biotechnology as an emerging industrial wastewater treatment process for arsenic mitigation: A critical review. J Clean Prod. 2017, 151, 427-438. 94. Singh, R.; Paul, D.; Jain R.K. Biofilms: implications in bioremediation. Trends Microbiol. 2006, 14, 389-397. 95. Abrigo, M.; Kingshott, P.; McArthur, S.L. Electrospun polystyrene fiber diameter influencing bacterial attachment, proliferation, and growth. ACS Appl. Mater. Interfaces 2015, 7, 7644– 7652. 96. Abrigo, M.; Kingshott, P.; McArthur, S.L. Bacterial response to different surface chemistries fabricated by plasma polymerization on electrospun nanofibers. Biointerphases 2015, 10, 04A301. 97. Xie, S.; Tai, S.; Song, H.; Luo, X.; Zhang, H.; Li, X. Genetically engineering of Escherichia coli and immobilization on electrospun fibers for drug delivery purposes. J. Mater. Chem. B, 2016, 4, 6820-6829. 98. Eroglu, E.; Agarwal, V.; Bradshaw, M.; Chen, X.; Smith, S.M.; Raston, C.L.; Swaminathan Iyer, K. Nitrate removal from liquid effluents using microalgae immobilized on chitosan nanofiber mats. Green Chem. 2012, 14, 2682–2685. 99. San, N.O.; Celebioglu, A.; Tümtaş, Y.; Uyar, T.; Tekinay, T. Reusable bacteria immobilized electrospun nanofibrous webs for decolorization of methylene blue dye in wastewater treatment. RSC Adv. 2014, 4, 32249-32255.


47


Page 48 of 49

100. Sarioglu, O.F.; San Keskin, N.O.; Celebioglu, A.; Tekinay, T.; Uyar, T. Bacteria immobilized electrospun polycaprolactone and polylactic acid fibrous webs for remediation of textile dyes in water. Chemosphere 2017, 184, 393-399. 101. Sarioglu, O.F.; Celebioglu, A.; Tekinay, T.; Uyar, T. Evaluation of contact time and fiber morphology on bacterial immobilization for development of novel surfactant degrading nanofibrous webs. RSC Adv. 2015, 5, 102750-102758. 102. Sarioglu, O.F.; Celebioglu, A.; Tekinay, T.; Uyar, T. Bacteria-immobilized electrospun fibrous polymeric webs for hexavalent chromium remediation in water. Int. J. Environ. Sci. Technol. 2016, 13, 2057–2066. 103. Sarioglu, O.F.; Celebioglu, A.; Tekinay, T.; Uyar, T. Evaluation of fiber diameter and morphology differences for electrospun fibers on bacterial immobilization and bioremediation performance. Int. Biodeterior. Biodegradation 2017, 120, 66-70. 104. De Cesare, F.; Di Mattia, E.; Zussman, E.; Macagnano, A. A study on the dependence of bacteria adhesion on the polymer nanofiber diameter. Environ. Sci.: Nano 2019, 6, 778-797. 105. Persano, L.; Camposeo, A.; Tekmen, C.; Pisignano, D. Industrial upscaling of electrospinning and applications of polymer nanofibers: A review. Macromol. Mater. Eng. 2013, 298, 504–520.


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Rational Design and Development of Electrospun Nanofibrous

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