Encapsulating Microorganisms inside Electrospun ... - ACS Publications

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Biological and Medical Applications of Materials and Interfaces

Encapsulating microorganisms inside electrospun microfibers as a living material enables room-temperature storage of microorganisms Jinpeng Han, Chenyu Liang, Yuchen Cui, Likun Xiong, Xiaocui Guo, Xiaoyan Yuan, and Dayong Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b14978 • Publication Date (Web): 19 Oct 2018 Downloaded from http://pubs.acs.org on October 21, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26 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

ACS Applied Materials & Interfaces

Encapsulating Microorganisms inside Electrospun Microfibers as a Living Material Enables Room-temperature Storage of Microorganisms Jinpeng Han1#, Chenyu Liang1#, Yuchen Cui1, Likun Xiong1, Xiaocui Guo1, Xiaoyan Yuan2 and Dayong Yang*,1 1: School of Chemical Engineering and Technology, Key Laboratory of Systems Bioengineering (Ministry of Education), Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300072, P. R. China 2: School of Materials Science and Engineering, Tianjin Key Laboratory of Composite and Functional Materials, Tianjin University, Tianjin 300072, P.R. China # Jinpeng Han and Chenyu Liang contributed to this work equally. * E-mail: [email protected], [email protected]

Keywords: living materials, microorganism storage, electrospinning, microfiber, bioactive membrane

1

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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

Abstract Room-temperature storage and transportation of microorganisms maximize the power of microorganisms in healthcare, energy and environment. Recently, paper-based biotechnologies have been developed to enable room-temperature storage of a variety of non-living biosystems such as diagnostic devices and cell-free systems. Herein, room-temperature storage of living microorganisms is realized by an electrospun nonwoven paper containing convex region, which is composed of coiled microfibers with dense distribution of microorganisms. Microorganisms are encapsulated into the microfibers and remain intact after electrospinning. Poly(ethylene oxide) is used as polymer matrix, and glycerol and dextran are used as additives. When the contents of glycerol and dextran are optimized as 5% and 0.4%, the room-temperature time is prolonged to 2 days, more than 8 folds as compared with the control group. Upon demand, the microorganisms can be activated by adding water, and used for culturing microorganisms directly. Further, mechanisms which account for microbial activity and storage are studied. Our microfiber-based strategy is universal for the room-temperature storage of prokaryotic and eukaryotic microorganisms in the solid formulation. Besides, our microorganism/polymer complex structures represent novel living materials via bottom-up strategy, which are of great potential for new biomedical applications.

2


Page 2 of 26

Page 3 of 26 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


Introduction Storage and transportation of microorganisms maximize the power of biotechnology, for example facilitating the development of drug discovery and production, personalized therapies for diseases, ‘green’ means to fuel cars, and environmental sensing and protection.1-5 Recently, paper-based biotechnologies provide potential for the storage and transportation of non-living biosystems.6-12 For example, Whitesides and colleagues developed paper-based microfluidic devices for low-cost and point-of-care diagnostics.7 Collins and colleagues embedded DNA and cell-free machineries into the paper matrix, realizing long-term storage of cell-free systems and a diverse range of functional genetic bio-systems.8 Very recently, Jiang and colleagues presented a paper-based barcode assay system, in which biological reagents were patternedly immobilized within stacked sheets of paper.11 Inspired by these pioneer works, we herein report a paper-based strategy for room-temperature storage and transportation of microorganisms. The paper that we used is microfibrous non-woven membrane, structurally similar to that used in those pioneer works. Differently, they immobilized nonliving biological reagents on commercially available paper; and in contrast, we encapsulate living microorganisms inside microfibers. To achieve this goal, we fabricate the microfiber/microorganism complex structure by a technology named electrospinning. Electrospinning is a simple, cost-effective, and highly versatile technique to fabricate microfibrous non-woven membrane.13-16 Briefly, electrospinning applies electric field on polymer solution, in which polymer jet is split into microfibers upon electrostatic force and then assembled into non-woven membrane. Notably, in comparison with commercial paper such as filter paper, electrospinning processes polymeric materials de novel, and consequently could versatilely tune and program the formation process 3



and the morphology of microfibers. In particular, functional components could be conveniently encapsulated into electrospun microfibers, such as molecules, nanoparticles and even living cells.17-22 We encapsulate microorganisms within microfibers by electrospinning, and microorganisms remain intact inside microfibers. Remarkably, microorganisms can be stored at room temperature for a relatively long time. As a result, microorganisms can be conveniently transported as paper format to any place at room temperature. Furthermore, microorganisms can be on-site activated and cultured on-demand by only adding water. We further systemically study the mechanisms for the storage of microorganisms inside microfibers, and figure out that three factors account for microbial activity, including microfiber topologies, drastic change of osmotic environment during electrospinning and trace water content inside microfibers. Traditional storage and transportation of microorganisms usually requires cold chains, special equipment, and complex processing methods, which are generally expensive, technically demanding, manpower intensive or restricted by biosafety regulations.23,24 In contrast, our proposed microfiber-based strategy has the following major advantages: 1) all the processes including fabrication, storage and transportation are conducted at room temperature, thus avoiding cold chains; 2) microorganisms are encapsulated inside microfibers as paper format, thus avoiding liquid formulation which causes potential biosafety problems; 3) only electrospinning (simple instrument and process) is involved, thus avoiding massive equipment, and complex processing.

4


Page 4 of 26

Page 5 of 26 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


Results and discussion a)

PEO Glycerol Dextran

electrospinning

+

assembling

instantaneous drying

b)

room temperature storage and transportation

c)

water

water activate and culture

Scheme 1. Schematic diagram of the microfiber-based strategy for room-temperature storage and transportation of microorganisms. (a) Scheme for the fabrication of electrospun microfibers in which microorganisms are encapsulated through the instantaneous drying process. (b) Microorganisms are stored in paper formulation, and can be stored and transported at room temperature, such as to any places worldwide by air. (c) Upon demand, microorganisms are activated and further cultured through addition of water. Microfiber-based strategy for room-temperature storage and transportation of

microorganisms.

Scheme

1

describes

the

overall

procedure

of

the

microfiber-based strategy for room-temperature storage and transportation of 5



microorganisms.

Microfibrous

membrane

was

Page 6 of 26

fabricated

from

polymer/microorganism dispersions using electrospinning. The polymer matrix was poly(ethylene oxide) (PEO); glycerol (GL) and dextran were used as additives. In detail, microorganisms were mixed with PEO/GL/Dextran to yield bioactive dispersions first, and afterwards the dispersions were transferred into the electrospinning syringe. By applying electric field (generally as high as kilovolts), droplet formed at the tip of the syringe orifice (Figure 1a). Upon the strong electrostatic repulsion, the droplet overcame surface tension to form a thin stream and split into microfibers. During the process, water evaporated quickly and microfibers instantaneously dried inside which microorganisms were encapsulated. Electrospun microfibers further assembled into non-woven membrane as a sheet of paper format (Scheme 1a). Unlike some traditional methods by which microorganisms were stored as liquid formulation24 or attached on solid paper,25 in our strategy microorganisms were encapsulated inside microfibers as a solid formulation, avoiding microorganisms escaping to environment which would cause potential biosafety problems. In addition, the microorganisms encapsulated microfibrous membrane (MEM) could be easily detached from the substrate and flexible enough to be packed into any shapes and stored in containers such as test tubes or boxes. As a proof-of-application, MEM could be packed in a suitcase and transported to the destination by air (Scheme 1b). Afterwards, microorganisms could be activated by adding water and after which microorganisms could be cultured as usual (Scheme 1c). We used Escherichia coli (E. coli) as the demonstration microorganism to test the feasibility of our strategy given that E. coli is one of the most frequently used engineered strains. Moreover, to achieve better visualization results, E. coli was encoded with green fluorescent protein (GFP) gene. To obtain microfibers with 6


Page 7 of 26 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


appropriate diameter and maintain microorganisms with relatively high activity, we first optimized the electrospinning parameters, including the applied voltages, types and concentration of polymers, and electrospinning time (Discussions S1-S3). When PEO concentration was 20 wt. % and the applied voltage was 13 kV, the average fiber diameter was approximately 375 nm which was slightly smaller than that of E. coli (500 nm) (Figure S1b). As the applied voltage decreased or PEO concentrations increased, the fiber diameter increased slightly (Figure S1 &S2). However, when the applied voltage was less than 13 kV, it was difficult to fabricate continuous microfibers. Similarly, when PEO concentration was higher than 20 wt. %, homogeneous polymer/microorganism dispersions could not be prepared. As for polymer types, polyvinylpyrrolidone (PVP) and PEO were tested to encapsulate E. coli. After room-temperature storage for 3 hours, the colony forming units (CFU) number (the number of viable E. coli) in PVP MEM was 300; in contrast, the CFU number was 407 in PEO MEM (Figure S3&S4c). So we used PEO as the matrix material. Besides, when the electrospinning time was 1 hour, a circular and self-standing MEM could be fabricated and the thickness of MEM was approximately 160 m.

7



Figure 1. The morphology of MEM and activity of E. coli in MEM. MEM was fabricated from 20% PEO/5%Gl/0.4%Dextran/E. coli dispersions. (a-c) Digital photos of the electrospinning process. (d-f) Fluorescence and SEM images of the smooth region. Rhodamine B (red) was utilized to stain microfibers, and E. coli cells expressed GFP (green). (g-i) Fluorescence, SEM and 3D scanning images of particle aggregates at convex region. (j) Growth curve of E. coli in PEO/E. coli dispersions with different components. (k) Digital photos of agar plates seeded by the whole MEM diluted by 100 times immediately after electrospinning. Fabrication of the novel MEM. With deliberate optimization of electrospinning 8


Page 8 of 26

Page 9 of 26 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


parameters, a self-standing MEM was fabricated by utilizing PEO as polymer matrix, and GL and dextran as additives. MEM contained two different regions, named smooth and convex region, respectively (Figure 1b). In smooth region, the surface morphology was similar to that of traditional electrospun membrane. E. coli cells were successfully encapsulated inside microfibers as indicated by the fluorescence images that E. coli cells encoded with GFP were evenly distributed in microfibers and no aggregates were observed (Figure 1d-e). SEM images gave more details that microfibers were uniform and E. coli were encapsulated inside microfibers (Figure 1f). In contrast, convex region showed completely different morphologies. In the view of macroscopic, convex region was visible in the form of particle aggregates (Figure 1b), which was observed in previous work.26 The aggregates were interlaced with each other to form the whole convex region (Figure S5a-c). In the view of microscopic, the coiled microfibers were highly entangled to form 3D porous network, in which E. coli cells were densely distributed (Figure 1g). SEM images showed the surface morphology of particle aggregates, which was locally compact (Figure 1h&S5d). The height difference of particle aggregate was approximately 200 m. And the aggregated structure was porous, which indicated the formation of locally loose structure inside the aggregates (Figure 1i & S6). This structure was supposed to be favor to cell metabolism and survival as compared with dense fiber structure.27 As a result, E. coli were not evenly encapsulated in the two different regions of MEM. In addition, the effect of material components of MEM on microbial activity was tested (Figure 1j). PEO, GL and dextran had no effect on the growth of E. coli. The high initial optical density (OD 600) of PEO/Gl/Dextran/E. coli was due to the phase separation of PEO and dextran.28 After electrospinning, high microbial activity was obtained, which indicated that electrospinning process did not hurt cells severely, 9



even being subject to high voltages (Figure 1k).

Figure 2. The room-temperature storage of MEM fabricated by different PEO/E. coli systems. (a-d) Plot of CFU as a function of storage time in different PEO/E. coli systems. (e-h) Digital photos of agar plates seeded by different MEMs at different storage times. (a,e) 20% PEO/E. coli, (b,f) 20% PEO/5%Gl/E. coli, (c,g) 20% PEO/0.4%Dextran/E. coli, (d,h) 20% PEO/5%Gl/0.4%Dextran/E. coli. 10


Page 10 of 26

Page 11 of 26 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


Room-temperature storage of MEM. We studied the effect of electrospinning material recipe on the room-temperature storage of E. coli. As for MEM fabricated from PEO/E. coli, the room-temperature storage time was only 6 h (Figure 2a&2e). Significantly, the storage time could be extended to 24 h after introducing appropriate amount of glycerol or dextran (Figure 2f&2g). In comparison with PEO/E. coli MEM, the introduction of glycerol and dextran individually gave rise to much higher initial CFU value (160000 and 40000 vs. 1800), indicating that glycerol and dextran could protect E. coli cells during electrospinning (Figure 2b&2c). In detail, the incorporation of glycerol mainly increased the number of viable E. coli during electrospinning due to the highest initial CFU value (Figure 2b). The introduction of dextran mainly reduced the death rate during the storage process, due to the lower descending slope after room-temperature storage for 6 hours (Figure S7). By incorporating glycerol and dextran simultaneously, synergetic effects were achieved and the room-temperature storage time was extended to 48 h (Figure 2d&2h). The storage time was prolonged more than 8 folds as compared with PEO/E. coli MEM. As a traditional method, E. coli storage on filter paper was tested as a control. The storage time was only 24 h (Figure S8). Growth curve was measured to monitor the growth process of E. coli after room-temperature storage. MEM fabricated from 20% PEO/5%Gl/0.4%Dextran/E. coli had the highest microbial activity, as indicated by the fastest growth rate reaching the logarithmic growth period (Figure S9). In order to confirm whether E. coil mutated with subject to electrospinning and room-temperature storage process, gene sequencing of E. coil was conducted and the sequence identity was 100.00 % (Data S1). Rod-shaped E. coli could be obtained again after room-temperature storage for 48 h, and the GFP was successfully expressed (Figure S10). We emphasized that 11



low-temperature storage (such as 4 and -20 oC) of microorganisms was relatively easy to achieve;17,29 nevertheless, room-temperature storage was still a challenge. We achieved room-temperature storage of E. coli for 48 hours using electrospinning, which was the longest time to date as we know. More importantly, we supposed that transportation by air in 48 hours could reach any place on earth.

12


Page 12 of 26

Page 13 of 26 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


Figure

3.

The

mechanisms

accounting for

room-temperature

storage

of

microorganisms. (a) Scheme for the balance among microfiber topologies, osmotic environment, water content inside microfibers and microbial activity. (b) Scheme for the formation process of particle aggregates by introducing glycerol. (c) Fluorescence images of denser coiled microfibers. (d) Scheme for the formation process of microfibers with two-phase structure by introducing dextran. (e) Fluorescence images 13



Page 14 of 26

of microorganisms encapsulated microfibers with two-phase structure. (f) Plot of CFU and survival rate as a function of different PEO/E. coli systems. (g) Plot of CFU and water content as a function of different glycerol contents based on PEO/Gl/E. coli MEMs. The room-temperature storage time was 6 h. (h) Plot of CFU and water content as a function of different PEO/E. coli MEMs. The room-temperature storage time was 6 h. The

mechanisms

accounting

for

room-temperature

storage

of

microorganisms. We proposed that the balance among microfiber topologies, drastic change of osmotic environment during electrospinning, and trace water content inside microfibers were three key parameters to affect microbial activity and storage (Figure 3a). In particular, the topologies of MEM not only affected the distribution and microenvironment of microorganisms, but also changed the osmotic environment and water content, and finally influenced the metabolism level of microorganisms. The introduction of glycerol was vital for the formation of particle aggregates (Figure 3b &S11). The content of glycerol was optimized as 5 wt. % (Figure S12). Before forming the final aggregates, coiled microfibers with dense distribution of E. coli formed as the intermediate state (Figure 3c). SEM images show more detailed micromorphology of the coiled microfibers (Figure S13a). It was proposed that the coiled fibers intertwined with each other to form denser fibers first, and which were further clustered together to form aggregates via electrostatic interactions. The surface tension of polymer solutions and the charge accumulation during electrospinning were considered to account for the aggregates formation.30,31 Glycerol was liquid with low surface tension to water, which endowed fibers with better crimping properties.30,32 In addition, glycerol was nonconductive compound and the static-charge accumulation on the surface of MEM induced the coiled microfibers to cluster together.31,33 14


Page 15 of 26 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


The incorporation of dextran was important to fabricate microfibers with two-phase structure, in which E. coli were densely distributed inside (Figure 3d). E. coli were discontinuously distributed inside microfibers and the diameter of microfibers was increased to a few microns (Figure 3e). This structure was favor to maintaining appropriate microbial metabolic activity by providing ingestible nutritional ingredients.34 Regarding the formation mechanism, aqueous two-phase systems (ATPS) played a critical role. Due to the mutual influence of interfacial tension and intermolecular repulsion, PEO and dextran formed ATPS.28 E. coli cells were uneven distributed in two phases and the dextran phase contained more E. coli cells (Figure S13b-e). The CFU number in dextran phase was approximately 9 times higher than that of PEO phase (Figure S14). As a result, when the polymer/E. coli droplets were formed at the tip of syringe orifice during electrospinning, phase separation formed and the two-phase structures could be fabricated. In addition to microfiber topologies, drastic change of osmotic environment during electrospinning and trace water content inside microfibers were further studied. Because solvent volatilization during electrospinning was extremely fast (was estimated 10 ms),35 the osmotic environment drastically changed, which could influence the survival rate of microorganisms. In comparison with PEO/E. coli MEM, the introduction of glycerol could significantly increase the survival rate (2.18% and 2.10% vs. 0.09%) (Figure 3f). Similar effect was observed in previous study.29 Glycerol could enter E. coli, which protected cells from rapid dehydration.36 In addition, the presence of glycerol could reduce mortality of E. coli during the process of preparing PEO/E. coli dispersions, and thus leading to the higher CFU value before electrospinning (Figure 3f) Furthermore, the mechanical stresses could be generated during electrospinning, which was approximately 5 × 104 g·cm−1·s−2, and much lower 15



than that E. coli could withstand (3 × 106 g·cm−1·s−2).29 We therefore proposed that cell death during electrospinning was mainly due to the drastic change of osmotic environment rather than the mechanical stresses. Besides, glycerol was hydrophilic and could form hydrogen bond with water and bioactive molecules, which thus improved the stability and water-retaining property of bioactive molecules.37 As a result, glycerol was conducive to maintaining the amount of water inside microfibers. Water content inside microfibers increased with the increment of glycerol (Figure 3g). When glycerol was 5 wt. %, MEM had the highest microbial activity (the maximum CFU value) and the water content was approximately 14.5% (Figure 3g). Appropriate water content was critical to maintain the metabolic balance of E. coli (Figure S15&S16). The presence of dextran could improve preservation effect of cells.38 Microfibers with discontinuous distribution of E. coli provided a congregate and protective environment for microbial metabolism, as a result of the uneven distribution of E. coli cells in polymer dispersions. The dextran phase contained high E. coli concentrations. In contrast, high cell concentrations without forming ATPS for directly electrospinning were not able to obtain MEM. In comparison with 20% PEO/0.4%Dextran/E. coli MEM, the incorporation of glycerol gave rise to much higher initial CFU value (8000 vs. 2000), indicating that microbial metabolism was enhanced with the increase of water content (Figure 3h). Correspondingly, the water content was increased from 5.9% to 13.1% (Table. S4).

16


Page 16 of 26

Page 17 of 26 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


Figure 4. The universality of MEM for room-temperature storage of microorganisms. (a-c) The storage of multiple homologous E. coli using 20%PEO/5%Gl/0.4%Dextran/ E. coli MEM. The storage time was 48 h. (a) Digital photos of agar plates seeded by MEM. (b) Fluorescence images of MEM. (c) Laser scanning confocal microscopy images of MEM. (d-f) Room-temperature storage of S. cerevisiae. (d) Plot of CFU as a function of storage time in different MEMs. (e) Digital photos of agar plates seeded by 20%PEO/S. cerevisiae MEM at different storage times. (f) Digital photos of agar plates seeded by 20%PEO/5%Gl/0.4%Dextran/S. cerevisiae MEM at different storage times. The universality of MEM for room-temperature storage of microorganisms. In order to prove the universality of microfiber-based strategy for microorganism storage, the storage of multiple homologous microbes and eukaryotes were further 17



tested. Two kinds of homologous E. coli cells (CFP and GFP gene were transformed into E. coli cells) could be stored for more than 2 days at room temperature (Figure 4a). GFP and CFP were successfully expressed in MEM (Figure 4b). In order to identify the three-dimensional distributions of three kinds of homologous E. coli in MEM, laser scanning confocal microscopy was utilized and E. coli transfected with CFP, GFP and YFP genes were all independently distributed in MEM (Figure 4c). Moreover, the room-temperature storage time of MEM containing three kinds of homologous E. coli was more than 2 days. S. cerevisiae was chosen as an example to demonstrate the storage of eukaryotic cells. In PEO/S. cerevisiae MEM, the number of viable S. cerevisiae cells decreased quickly along with prolonged storage time, and the storage time was approximately 1 day (Figure 4d&4e). After introducing appropriate amount of glycerol and dextran, the storage time was remarkably extended to longer than 7 days without a significant decline in CFU number (Figure 4d&4f). Conclusion In summary, we developed a microfiber based strategy for room-temperature storage and transportation of microorganisms. Microorganisms were encapsulated inside electrospun microfibers. The polymer matrix was PEO; GL and dextran were used as additives. Microorganisms could remain intact inside microfibers. E. coli and S. cerevisiae cells could be stored for longer than 2 and 7 days at room temperature, respectively, which were the longest room-temperature storage time. Upon demand, the microorganisms could be activated by adding water, and used for culturing directly. The balance among microfiber topologies, drastic change of osmotic environment during electrospinning, and water content inside microfibers was the key factor to affect microbial activity. Only all of the three factors were optimized to an 18


Page 18 of 26

Page 19 of 26 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


optimal level, the balance of microbial metabolism could be achieved. Our microfiber-based strategy avoided cold chains and potential biosafety problems in process of microbial storage and transportation. In future, we envision that microfiber/microorganism complex structure will provide new ideas for the design of living materials and devices.39-41

19



Methods Electrospinning with microorganisms. Polymer/microorganisms dispersions were prepared by adding the desired amount of microbial culture to a sterile solution of polymer. Take 20%PEO/5%Gl/0.4%Dextran/ E. coli as an example, a mixture of 2g of PEO, 0.5g of Gl, 4.5g of LB and 1g of 4 wt. % dextran solution was prepared and autoclaved first. The obtained sterile PEO solution and 2g of 12 h E. coli culture were mixed at 250 rpm for 1 day at 37 oC to get homogeneous dispersions. Then the dispersions were transferred into the syringe for electrospinning process. The microfibers were collected on sterile aluminum foil. And the sample collector was placed at 15 cm from the needle tip. Contamination of fibers by unwanted microorganisms was prevented by intensively cleaning the whole apparatus with a 75vol% ethanol-water mixture. Afterwards, the microorganisms encapsulated microfibrous membrane (MEM) could be peeled off and stored at room temperature. All the experiments were performed at room temperature (∼25 oC), and a humidity of about 20%. Sample preparation and characterization. As-spun microfibers were collected on the sterile aluminum foil for 1 h and formed a macroscopic MEM. MEM could be detached from the aluminum foil and distributed into the vials. Then MEM was stored at room temperature for appropriate time and taken from the vials for viability tests. The water content of MEM could be determined. Specifically, MEM was put into vacuum drying oven for 5 days at 40 oC without any further weight loss. MEM was 20


Page 20 of 26

Page 21 of 26 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


weighed before and after desiccation. The samples for scanning electron microscopy (SEM, Hitachi S4800), fluorescence microscopy (PerkingElmer, UltraView Vox), atomic force microscope (AFM, NTEGRA Spectra) and laser scanning confocal microscopy (LSCM) (PerkingElmer, UltraView Vox) were prepared by direct deposition of microfibers onto pieces of slide glasses. Visual inspection of MEM was characterized by digital camera (Nikon) and 3D optical profiler (Sensofar, S neox). The growth curve of E. coli was obtained by using microplate reader (BioTek, Synergy H1). And the growth curve was obtained by measuring the optical density 600 (OD 600) of different polymer/microorganism dispersions at the appropriate concentration. Gene sequencing was conducted by GENEWIZ, Inc. Viability testing. For analysis of survival rate, the CFU (Colony Forming Units, CFU/mL) method was used. 100 L of sterile water was used to dissolve MEM. Also dilution series were made. Then 100 L of the dilutions were poured on agar plates. After incubation at 37 °C (E. coli) or 30 °C (S. cerevisiae) for 24 h, the number of surviving microorganisms was analyzed by counting the formed colonies. In order to determine the number of viable cells of MEM at different storage times, the CFU method was also utilized. At given times, MEM was dissolved in the tube (2 mL) with 100 L of sterile water, then the obtained solution was exposed on an agar plate giving the number of colony forming units. Abbreviations MEM: microorganisms encapsulated microfibrous membrane; E. coli: Escherichia 21



coli; ATPS: aqueous two-phase systems; GFP: green fluorescent protein; CFP: cyan fluorescent protein; YFP: yellow fluorescent protein. Supporting Information Discussion of fabricating microfibers with higher microbial activity and appropriate diameter; reagents and materials; and attached data, analysis and discussion of the project. Acknowledgements This work was supported in part by National Natural Science Foundation of China (grant no. 21621004, 21575101, and 21622404). We thank Ms. Wenfei Song, Mr. Xinpeng Han and Ms. Yang Liu at Tianjin University for their kind help on experiments and discussion. We thank Prof. Yingjin Yuan at Tianjin University for providing S. cerevisiae.

22


Page 22 of 26

Page 23 of 26 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


References (1) Quigley, E. M. M. Gut Microbiome as a Clinical Tool in Gastrointestinal Disease Management: Are We There Yet? Nat. Rev. Gastroenterol. Hepatol. 2017, 14, 315-320. (2) Bourdeau, R. W.; Leegosselin, A.; Lakshmanan, A.; Farhadi, A.; Kumar, S. R.; Nety, S. P.; Shapiro, M. G. Acoustic Reporter Genes for Noninvasive Imaging of Microorganisms in Mammalian Hosts. Nature 2018, 553, 86-90. (3) Sousa, D. Z.; Visser, M.; Gelder, A. H. V.; Boeren, S.; Pieterse, M. M.; Pinkse, M. W. H.; Verhaert, P. D. E. M.; Vogt, C.; Franke, S.; Kümmel, S. The Deep-subsurface Sulfate Reducer Desulfotomaculum Kuznetsovii Employs Two Methanol-degrading Pathways. Nat. Commun. 2018, 9, 239-247. (4) Berezamalcolm, L.; Mann, G.; Franks, A. E. Environmental Sensing of Heavy Metals Through Whole Cell Microbial Biosensors: A Synthetic Biology Approach. Acs Synth Biol 2015, 4, 535–546. (5) Yu, T.; Zhou, Y. J.; Wenning, L.; Liu, Q.; Krivoruchko, A.; Siewers, V.; Nielsen, J.; David, F. Metabolic Engineering of Saccharomyces Cerevisiae for Production of Very Long Chain Fatty Acid-derived Chemicals. Nat. Commun. 2017, 8, 15587-15596. (6) K, P.; AA, G.; T, F.; DE, C.; A, D.; P, Y.; JJ, C. Paper-based Synthetic Gene Networks. Cell 2014, 159, 940-954. (7) Pollock, N. R.; Rolland, J. P.; Kumar, S.; Beattie, P. D.; Jain, S.; Noubary, F.; Wong, V. L.; Pohlmann, R. A.; Ryan, U. S.; Whitesides, G. M. A Paper-based Multiplexed Transaminase Test for Low-cost, Point-of-care Liver Function Testing. Sci. Transl. Med. 2012, 4, 129-148. (8) Pardee, K.; Slomovic, S.; Nguyen, P. Q.; Lee, J. W.; Donghia, N.; Burrill, D.; Ferrante, T.; Mcsorley, F. R.; Furuta, Y.; Vernet, A. Portable, On-Demand Biomolecular Manufacturing. Cell 2016, 167, 248-259. (9) Guo, H.; Yeh, M. H.; Zi, Y.; Zhen, W.; Jie, C.; Liu, G.; Hu, C.; Zhong, L. W. Ultralight Cut-Paper-Based Self-Charging Power Unit for Self-Powered Portable Electronic and Medical Systems. ACS Nano 2017, 11, 4475-4482. (10) Gong, M. M.; Sinton, D. Turning the Page: Advancing Paper-Based Microfluidics for Broad Diagnostic Application. Chem. Rev. 2017, 117, 8447-8480. (11) Yang, M.; Zhang, W.; Yang, J.; Hu, B.; Cao, F.; Zheng, W.; Chen, Y.; Jiang, X. Skiving Stacked Sheets of Paper into Test Paper for Rapid and Multiplexed Assay. Sci. Adv. 2017, 3, 4862-4867. (12) Ma, L.; Yang, G.; Wang, N.; Zhang, P.; Guo, F.; Meng, J.; Zhang, F.; Hu, Z.; Wang, S.; Zhao, Y. Trap Effect of Three‐Dimensional Fibers Network for High Efficient Cancer‐Cell Capture. Adv. Healthc. Mater. 2015, 4, 838-843. (13) Jia, C.; Luo, B.; Wang, H.; Bian, Y.; Li, X.; Li, S.; Wang, H. Precise and Arbitrary Deposition of Biomolecules onto Biomimetic Fibrous Matrices for Spatially Controlled Cell Distribution and Functions. Adv. Mater. 2017, 1701154-1701163. 23



(14) Li, D.; Xia, Y. Electrospinning of Nanofibres: Reinventing the Wheel. Adv. Mater. 2004, 16, 1151-1170. (15) Cheng, S.; Jin, Y.; Wang, N.; Cao, F.; Zhang, W.; Bai, W.; Zheng, W.; Jiang, X. Self-Adjusting, Polymeric Multilayered Roll that can Keep the Shapes of the Blood Vessel Scaffolds during Biodegradation. Adv. Mater. 2017, 29, 1700171-1700178. (16) Yang, D.; Lu, B.; Zhao, Y.; Jiang, X. Fabrication of Aligned Fibrous Arrays by Magnetic Electrospinning. Adv. Mater. 2007, 19, 3702-3706. (17) Gensheimer, M.; Becker, M.; Brandis-Heep, A.; Wendorff, J. H.; Thauer, R. K.; Greiner, A. Novel Biohybrid Materials by Electrospinning: Nanofibers of Poly(ethylene oxide) and Living Bacteria. Adv. Mater. 2007, 19, 2480-2482. (18) Zhang, Y.; Shuai, Y.; Xiao, F.; Li, H.; Zhou, J.; Bo, W. Preparation of Nanofibrous Metal–Organic Framework Filters for Efficient Air Pollution Control. J. Am. Chem. Soc. 2016, 138, 5785-5788. (19) Baker, B. M.; Britta, T.; Wang, W. Y.; Sakar, M. S.; Kim, I. L.; Shenoy, V. B.; Burdick, J. A.; Chen, C. S. Cell-mediated Fiber Recruitment Drives Extracellular Matrix Mechanosensing in Engineered Fibrillar Microenvironments. Nat. Mater. 2015, 14, 1262-1268. (20) Han, F.; Jia, X.; Dai, D.; Yang, X.; Zhao, J.; Zhao, Y.; Fan, Y.; Yuan, X. Performance of a Multilayered Small-diameter Vascular Scaffold Dual-loaded with VEGF and PDGF. Biomaterials 2013, 34, 7302-7313. (21) Jiang, K.; Long, Y. Z.; Chen, Z. J.; Liu, S. L.; Huang, Y. Y.; Jiang, X.; Huang, Z. Q. Airflow-directed in situ Electrospinning of a Medical Glue of Cyanoacrylate for Rapid Hemostasis in Liver Resection. Nanoscale 2014, 6, 7792-7798. (22) Yang, D.; Niu, X.; Liu, Y.; Wang, Y.; Gu, X.; Song, L.; Zhao, R.; Ma, L.; Shao, Y.; Jiang, X. Electrospun Nanofibrous Membranes: A Novel Solid Substrate for Microfluidic Immunoassays for HIV. Adv. Mater. 2008, 20, 4770-4775. (23) Bjerketorp, J.; Håkansson, S.; Belkin, S.; Jansson, J. K. Advances in Preservation Methods: Keeping Biosensor Microorganisms Alive and Active. Curr. Opin. Biotechnol. 2006, 17, 43-49. (24) Cox, C. S. Method for the Routine Preservation of Micro-organisms. Nature 1968, 220, 1139-1139. (25) Hays, H. C.; Millner, P. A.; Jones, J. K.; Rayner-Brandes, M. H. A Novel and Convenient Self-drying System for Bacterial Preservation. J. Microbiol. Methods 2005, 63, 29-35. (26) Taskin, M. B.; Xu, R.; Gregersen, H.; Nygaard, J. V.; Besenbacher, F.; Chen, M. Three-Dimensional Polydopamine Functionalized Coiled Microfibrous Scaffolds Enhance Human Mesenchymal Stem Cells Colonization and Mild Myofibroblastic Differentiation. ACS Appl. Mater. Inter. 2016, 8, 15864-15873.

24


Page 24 of 26

Page 25 of 26 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) Vaquette, C.; Cooper-White, J. J. Increasing Electrospun Scaffold Pore Size with Tailored Collectors for Improved Cell Penetration. Acta Biomater. 2011, 7, 2544-2557. (28) Ryden, J.; Albertsson, P. A. Interfacial tension of dextran—polyethylene glycol—water two—phase systems. J. Colloid Interf. Sci. 1971, 37, 219-222. (29) Salalha, W.; Kuhn, J.; Dror, Y.; Zussman, E. Encapsulation of Bacteria and Viruses in Electrospun Nanofibres. Nanotechnology 2006, 17, 4675-4681. (30) Liu, W.; Lipner, J.; Moran, C. H.; Feng, L.; Li, X.; Thomopoulos, S.; Xia, Y. Generation of Electrospun Nanofibers with Controllable Degrees of Crimping through a Simple, Plasticizer-based Treatment. Adv. Mater. 2015, 27, 2583-2588. (31) Lu, X.; Zhou, J.; Zhao, Y.; Qiu, Y.; Li, J. Room Temperature Ionic Liquid Based Polystyrene Nanofibers with Superhydrophobicity and Conductivity Produced by Electrospinning. Chem. Mater. 2008, 20, 3420-3424. (32) Alkindi, A. S.; Alwahaibi, Y. M.; Muggeridge, A. H. Physical Properties (Density, Excess Molar Volume, Viscosity, Surface Tension, and Refractive Index) of Ethanol + Glycerol. J. Chem. Eng. Data 2015, 53, 2793-2796. (33) Uecker, J. C.; Tepper, G. C.; Rosell-Llompart, J. Ion-assisted Collection of Nylon-4,6 Electrospun Nanofibers. Polymer 2010, 51, 5221-5228. (34) 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. (35) Reneker, D. H.; Yarin, A. L.; Fong, H.; Koombhongse, S. Bending Instability of Electrically Charged Liquid Jets of Polymer Solutions in Electrospinning. J. Appl. Phys. 2000, 87, 4531-4547. (36) Richey, D. P.; Lin, E. C. Importance of Facilitated Diffusion for Effective Utilization of Glycerol by Escherichia Coli. J. Bacteriol. 1972, 112, 784-790. (37) Han, L.; Liu, K.; Wang, M.; Wang, K.; Fang, L.; Chen, H.; Zhou, J.; Lu, X. Mussel‐Inspired Adhesive and Conductive Hydrogel with Long‐Lasting Moisture and Extreme Temperature Tolerance. Adv. Funct. Mater. 2017, 28 1704195-1704206. (38) Wang, X.; Xu, H. Incorporation of DMSO and Dextran-40 into a Gelatin/alginate Hydrogel for Controlled Assembled Cell Cryopreservation. Cryobiology 2010, 61, 345-351. (39) Liu, X.; Tang, T.; Tham, E.; Yuk, H.; Lin, S.; Lu, T.; Zhao, X. Stretchable Living Materials and Devices with Hydrogel-elastomer Hybrids Hosting Programmed Cells. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 2200-2205. (40) Liu, X.; Yuk, H.; Lin, S.; Parada, G. A.; Tang, T.-C.; Tham, E.; de la Fuente-Nunez, C.; Lu, T. K.; Zhao, X. 3D Printing of Living Responsive Materials and Devices. Adv. Mater. 2018, 30, 1704821-1704829. (41) Schaffner, M.; Rühs, P. A.; Coulter, F.; Kilcher, S.; Studart, A. R. 3D Printing of Bacteria Into Functional Complex Materials. Sci. Adv. 2017, 3, 6804-6812.

25



Table of Contents Graphic

instantaneous drying

water activate and culture

26


Page 26 of 26

Encapsulating Microorganisms inside Electrospun ... - ACS Publications

Recommend Documents