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Advanced Composites and Hybrid Materials https://doi.org/10.1007/s42114-017-0018-x

REVIEW

Fabrication of nanocomposites and hybrid materials using microbial biotemplates Zhijun Shi 1 & Xudian Shi 1 & Muhammad Wajid Ullah 1 & Sixiang Li 1 & Victor V. Revin 2 & Guang Yang 1 Received: 19 September 2017 / Accepted: 27 November 2017 # Springer International Publishing AG, part of Springer Nature 2017

Abstract Microbes are important part of life that vary in sizes and shapes, diverse surface chemistry and biology, and porous nature of their cell walls. Besides their importance in industrial processes such as fermentation, these serve as biotemplates and provide a biomimetic approach for fabrication of multifarious complex constructs with predefined features, ordered composites and hybrid nanomaterials, microdevices, and micro/nanorobots through various strategies. The template or building blocks for such approaches can be bacterial, algal, and fungal cells or virus particles. Here, we have summarized recent advancements in biofabrication based on live microbes. Using engineering approaches and suitable methods, live microbes can be manipulated as functional Bmicro/nanodevices and -robots^ to further perform biological functions such as replication, distribution, motility, formation of colonies, and secretion of metabolites at will. Biofabrication based on microbes provides effective methods to control and manipulate microbes as functional live building blocks to create micro/nanodevices and -robots for biomedical and energy applications. Keywords Biofabrication . Microbes . Live building blocks . Artificial microbes . Synthetic biology

1 Introduction Biofabrication refers to a set of methodologies addressed to engineer complex constructs (both living and non-living) with predefined biological features using fragments or intact living cells, biological molecules, biomaterials, and extracellular matrices, etc. [1, 2]. Over the past few decades, there has been increasing demand for development of biomaterials for applications in organ transplant, regeneration of lost or damaged tissues, and treating injured and burned skin tissues, etc. However, due to shortage of donor cells, tissues, and organs and due to provoking of immunological response, the gap between supply and demand continues to widen. This encouraged the researchers to grow or Zhijun Shi and Xudian Shi contributed equally to this manuscript. * Guang Yang [email protected] 1

Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, People’s Republic of China

2

Federal State-Financed Academic Institution of Higher Education, National Research Ogarev Mordovia State University, Saransk 430005, Russia

fabricate tissues and organs using biomaterials-based scaffold and individual’s own cells to develop autografts. Although the success rate is very low and technology is limited by several factors, the development of recent fabrication strategies has made it more realistic. Living animal cells are important raw materials for biofabrication of animal-free meat [3], extracorporeal devices [4], and human tissues and organs [5, 6]. However, their usage has been overshadowed by limited availability, handling, controlled process development, and limited in vitro survival rate during extended incubation. In contrast, microbes such as bacterial, algae, and fungi and virus particles with a strong ability of adaptation, diversity in sizes and shapes, motility, presence of different chemical functional groups on their surfaces, surface biological features, intrinsic porosity of their cell walls, and ease of manipulation enable them to be used as biotemplates for biofabrication of different bionanomaterials [7]. Microbes are present in nature in all environments [8, 9] including air, rivers, sea, soil, body of plants and animals, and even in extreme conditions such as craters [10] or hot springs [11]. These exist in nature in different shapes such as spheres, rods, spirals, and icosahedrons, etc. and vary in size from nanoscale to mesoscopic dimensions. The intrinsically porous microstructure of microbial cell wall allows the deposition of different materials such as

Adv Compos Hybrid Mater

polyanions/cations, biopolymers, and magnetic (Fe3O4) nanoparticles without compromising their viability [12], thus supporting the development of core-shell hybrid structures and composites. Microbes not only can perform biological functions as individuals, but these also can replicate and be readily distributed and expanded to form functional colonies. These provide platform to understand the complexity of life and significantly improve the world around us and elucidate, map, and manipulate entangled networks of interaction among various organisms [13]. These are indispensable and play a key role on earth; even in human body, nine out of ten cells are microbial. Despite the pathogenic action of few types against humans, most are communal and use human body as their host [14]. These microbes cooperate with human cells to maintain body functions, and thus, their importance cannot be ignored. Microbes are readily viable and replicable, and because of their diversity, several useful functions can inspire us and be exploited. We can expect every individual microbe to behave as a living device, robot, or even as a factory if we can control and manipulate it. These features make microbes as ideal candidates for biofabrication practices. Thus, biofabrication based on microbes can serve as a promising approach to allow manipulation of living microbes as functional live building blocks to create micro/nanostructural units and devices. Inspired by the structure of microbes, various cost-effective and environmental-friendly biomaterials have been fabricated for potential applications in energy, nanotechnology, regenerative medicines, and biomedical fields [15]. This review focuses on different biofabrication methods and applications of microbe-based living biological systems and describes recent achievements in immobilization, patterning, movement control, and metabolites production by living microbes. Through immobilization and patterning methods, live microbes can be fixed on a chip to fabricate microstructured surfaces and arrays. It also describes the manipulation of microbial cell movement by controlling various factors such as chemotaxis, phototaxis, galvanotaxis, and magnetotaxis. It also provides insights for development of novel biofabrication strategies using microbes as a smart cell factory to manufacture functional metabolites. As an example, it describes the role of Gluconacetobacter xylinus as a smart textile microbiorobot for bacterial cellulose (BC) production. Specifically, inspired by the natural microbes, the development of synthetic biology and other methods provide several strategies to design various artificial microbes to perform different specialized functions.

2 Immobilization and patterning of live microbes: microbes on a chip fabricate microstructured surfaces and arrays Microorganisms, in the form of single cell, colony, or community, usually range in sizes from 102 nm to 102 μm.

Therefore, microstructural surfaces in this size range can be used for immobilization and patterning of live microbial cells. The live cells can be physically entrapped within micro-holes. For example, bacterial cells have been successfully entrapped in the pores of a nylon substrate [16] or microporous polymer [17] to be used in a microbioreactor. Bacteria immobilized in polyurethane [18, 19] have been used to repair concrete cracks. Silica gels can also be used to immobilize yeast spores and bacteria [20], where microbes can retain their enzymatic activity, but preserving the long-term viability of whole cells is a formidable challenge [21]. However, in all above examples, the living bacteria were randomly distributed in the substrates. Soft lithography, encompassing a number of different processes such as molding, printing, and embossing [22, 23], is a useful method to fabricate microstructured surfaces onto the surface of a substrate by utilizing an elastomeric stamp, with features typically having lateral dimensions of 1–1000 μm and vertical dimensions between 100 nm and hundreds of microns. This provides an effective method for immobilization and ordered patterning of live bacterial cells. In addition, live bacterial cells cannot only be immobilized in micro-holes through physical entrapment, but also by adhesion on different surfaces through specific interactions such as surface charges, hydrophilicity, covalent bonds, and antibody–antigen interactions [24] (Fig. 1). For example, motile Escherichia coli cells can be easily attached to a chemically modified and prefabricated surfaces through electrostatic interactions that exist between negatively charged groups on bacterial cell surface and positively charged poly(L-lysine) assemblies [25]. In another investigation, micro-holes were chemically modified through a soft lithographic process. The micro-holes had hydrophobic methyl-terminated n-alkanethiol groups at the bottom, which promoted bacterial adhesion and walls consisting of hydrophilic poly(acrylic acid)/poly(ethylene glycol) layered nanocomposites that inhibited adhesion [26]. Active molecules such as lectins can also be used to modify the surface and enhance microbial adhesion and aggregation. Besides, bacterial immobilization can also take place due to the specific antigen–antibody interaction. The fabrication of microarrays of motile E. coli on gold surfaces was achieved by that method [27]. Similarly, Salmonella enterica serovar typhimurium has also been successfully patterned on substrate through specific interaction of bacterial CFA/I fimbriae with corresponding antibody [28]. At present, several novel methods based on electric, optics, and magnetics have been proposed for immobilization and patterning of bacteria. For example, dielectrophoresis (DEP) is a phenomenon in which a force is exerted on a dielectric particle when it is subjected to a non-uniform electric field. The DEP force can be exerted on electrically neutral objects, such as cells and bacteria, when the objects have a different polarizability to the surrounding medium. For example, Kano et al. used a microfluidic device to immobilize microbes by


Fig. 1 Methods for immobilization and patterning of live bacterial cells. Reproduced with permission from ref. [25–27, 29–31]

DEP and found that the positive DEP was capable of selectively collecting live bacteria inside micropores. In addition, Miccio et al. [32] adopted electrode-free DEP for immobilization and orientation of bacteria. This electrode-free DEP was light induced, achieved through ferroelectric iron-doped lithium niobate crystals used as substrates. Due to photorefractive properties of such materials, suitable light patterns facilitate the writing of a conformational charge distribution inside its volume, and thus, the resulting electric field was able to immobilize E. coli on the surface. Another technique, called optical trapping, also known as optical tweezers, is based on a strongly focused laser beam. Small objects (such as viruses, bacteria, and eukaryotic cells) can be trapped in the focus by a

strong optical gradient force without any mechanical contact [33]. Similarly, other optic and magnetic techniques, such as optical nanofiber [34], silicon photonic crystal [35], rotating magnetic microrobot [36], and tapered optical fiber [37] have been designed and applied to trap microbes. All these noncontact trapping techniques are effective, offering easy fabrication and integration with microfluidic devices, and are promising for immobilization and patterning of live microbes. The immobilization and patterning techniques allow efficient manipulation and control of cells with an unprecedented degree of spatial and temporal precision; these allow the development of microbes on a chip device. This concept can be further developed to design and fabricate microdevices based


on bacterial cells to study the interactions between cells and their surroundings, biosensing and biodetection, smallmolecule screening, single-cell manipulation and analysis, biomaterials fabrication, and bioenergy synthesis [29]. For example, living bacteria were inserted into a microwell array formed at one end of an imaging fiber bundle, to be used as a biosensor for genotoxin monitoring. In this study, each microwell allowed only one cell to occupy; meanwhile, each fiber in the array possessed its own light pathway; thus, it enabled thousands of individual cell responses to be monitored simultaneously with both spatial and temporal resolution. As a result, this biosensor was capable of performing cell-based functional sensing with high sensitivity and short incubation times [38, 39]. Another interesting example is that immobilized human and microbial cells can be utilized to build an in vitro model that can support the co-culturing of human and microbial cells under conditions representative of the gastrointestinal human–microbe interface. Thus, it will provide tools to explore a range of fundamental research questions linking the gastrointestinal microbiome to human health and disease [40].

3 Manipulation of motile microbes: their exploitation as microswimmers Motility is the ability of entities to move spontaneously and actively, consuming energy in the process. Naturally, a large number of microorganisms are motile, since most of microorganisms possess one or more flagella or cilia on their surface allowing them to swim in the aqueous environment. The native environment for microorganism survival is dynamic and complex. Microorganisms can respond to changes in temperature [41], concentration of food molecules [42], surface softness [43], light [44], magnetic [45], or electric stimuli [46] (Fig. 2), by directing their motion to find a favorable environment. Therefore, control of environment is an effective method to manipulate motile microbes. Bacterial movement controls are under consideration from a variety of perspectives: for example, chemotactic, geotactic, phototactic, magnetotactic, and galvanotactic. A major challenge in microfabrication based on microorganisms is the independent manipulation of motile microbes and control the microenvironment at nanometer to micron scales. The magnitude and features of micro-channels can be customized to produce new environment or mimic the natural dynamic and complex environment of bacterial cells at these scales, or at picoliter to microliter level [47]. Microfluidic single-cell cultivation systems provide effective tools to study and manipulate motile microbes. For example, DiLuzio et al. [48] while studying the motility of E. coli cells in microchannels found that the cells swam preferentially along the right wall of the micro-channels and different surfaces were

influencing the movement of the cells, which indicates that the choice of materials can guide the motions of microbial cells in micro-channels. Herein, we describe different factors influencing the movement of microbial cells. Chemotaxis refers to the ability of microbial cells to respond to some chemical concentration gradient in the surrounding or microenvironment. Microfluidics can provide insights into bacterial chemotaxis, making it feasible to study the behavior of motile bacteria in concentration gradients of chemoattractants and chemo-repellents at as low as nanomolar levels [49]. By combining microfluidics with chemotaxis control, the motile microbes can be effectively manipulated. Besides chemotaxis, microbes can also respond to light stimuli, referred to as phototaxis, which is another adaptive response that greatly improves the competitive capacity of some microbes in diverse environments. A common example of phototaxis is the movement of Rhodospirillum centenum: a cell colony that can move in a linear direction toward and away from the source of infrared light (800–850 nm) and visible light (550–600 nm), respectively [50]. Steager et al. fabricated microstructures blotted with swarmer cells of Serratia marcescens into a monolayer. Without stimuli, the system moved randomly, but the phototactic response of the bacterial cell toward the source of UV light was exploited to induce the turn on/off control of the system. Compared to chemotactic control, the phototactic control is more useful and advantageous; for example, bacterial cell response is instant and consistent in the entire exposure region and the stimulus can be easily removed without disturbing the fluidic, as it is commonly produced by the refreshing chemicals in chemotactic control [44]. Galvanotaxis is another phenomenon influencing the microbial movement whereby cell migration is observed when they are subjected to a weak, direct current (DC) electric field. This effect can be exploited to induce directed microbial movement. For example, Tetrahymena pyriformis moved rapidly toward the cathode using cilia when an electric field was applied [46]. Due to the presence of charges on outer membrane of prokaryotic microbes, a Coulomb force is exerted on them when present in the electric field. As a result, individual microbial cell exhibit electrophoresis. Microstructures blotted with Serratia marcescens was responded immediately by seeking the positive electrode with direct movement when tested in DC electric filed. Interestingly, they immediately reversed their direction when polarity of electric field was switched [51]. Similarly, magnetotaxis is a motion of bacteria under the influence of magnetic field. During such movements, the microbial cells align themselves and swim in parallel along the lines of a magnetic field. Such microbes have the ability to synthesize nano-sized and membrane-bound magnetic particles within the single domain region and under mild conditions at a normal temperature and pressure. These

Adv Compos Hybrid Mater Fig. 2 Control of the environment is an effective method to manipulate motile microbes

nanoparticles possess regular morphology and size and are synthesized in aquatic environment; thus, this process is green and controllable and offers high technological potential since the microbes can be easily manipulated with magnetic forces. Live magnetotactic bacteria are natural nanorobots that can be controlled via magnetic forces. Practically, in order to orient bacteria and propel them to a predetermined position, a routing magnetic field was used that applied a torque to the magnetosome chain in bacteria [52]. Khalil et al. [53] thus used a magnetic manipulation system to control the motions of a magnetotactic bacterium within a microfabricated maze. Magnetotactic bacteria can prove to be a useful tool in microscale engineering tasks, for example, pathogen isolation [54], target transport [55], cell therapy [56], and in threedimensional micro-assemblies as microrobots [45]. For example, when bind to polyclonal antibodies, the magnetotactic bacteria can serve as microrobots for application in pathogenic separation. Pathogenic microorganism were loaded on microrobots due to the antigen–antibody reaction, and then carried away through magnetically controlled movement [54]. Another example is that Mokrani et al. [56] investigated potential of magnetotactic bacteria to penetrate 3D

Fig. 3 Bacteria-based microswimmers

multicellular tumor spheroids. They found that magnetotactic bacteria could be navigated within the tumor environment and function as microrobots for drug delivery when subjected to a computer-controlled magnetic field. Microbes are generally small in size, are easily acquired, and can survive in wide range of environmental conditions. These respond to an environmental stimulus; this property can be applied as a mean to steer the microbe-driven devices, such as microswimmers. Thus, a single bacterium can serve as microswimmer and can find potential applications in medicine and bioengineering, such as targeted drug, gene, and imaging agent delivery, biosensing, and cell transportation [57]. Besides, functional microparticles can be attached to a single bacterium; such biohybrid microswimmers can be used for targeted drug delivery [58]. In addition, several bacterial cells can get assemble to a whole structure, such as patterning onto a microstructure matrix or surface and thus, it can have a significant impact on motility enhancement of bacteriaactuated microswimmers [59–61]. Besides, bacterial cells can be attached onto the surface of microbeads where these selectively get attach on one side of microbead, which can control bacterial movement in same direction (Fig. 3). In


addition, these bacteria-based microswimmers have higher average velocities, and thus, microbeads can be used as drug carriers in drug delivery systems [62].

4 Artificial microbes: design an efficient microbe to achieve a specific desired output Microbes have relatively simple genome; in addition, complete genomes of several microbial strains have been sequenced and studied. As described above, microbes are relatively easy to manipulate; therefore, inspired by the natural microbes, artificial microbes have been fabricated by researchers to perform specialized functions, and meanwhile, similar to natural microbes, these artificial microbes can grow, reproduce, and develop without further human intervention. The dilemma of how to fabricate artificial microbes has been resolved by recent developments in synthetic biology which provide several promising approaches to achieve this goal. Synthetic biology is defined as the designing and construction of novel biologically based parts, devices, and systems or reprogramming the existing natural biological systems [24]. One approach of synthetic biology is the de novo creation of truly artificial life. This approach requires the construction of an artificial cell system that is able to self-maintain, selfreproduce, and potentially evolve. An artificial minimal cell at least needs to integrate three main components, including containment (e.g., liposome or polymersomes), cellular processes (e.g., energy conversion process), and information (e.g., genome). For containment, vesicle structures are derived from lipid molecular assembly and contain a thin bilayer membrane, 10-nm thick, that is used to encapsulate biomaterials such as DNA, RNA, proteins, or bacterial (e.g., Escherichia coli) cell-free expression system to build a cell-like bioreactor [63]. Polymersomes, obtained through self-assembly of amphiphilic block copolymers, also play a relevant role as cell mimics [64, 65]. In this encapsulation process, microfluidic methods playing a key role which offer unprecedented control over vesicle/polymersomes size, encapsulation efficiency, and membrane homogeneity [66]. The cellular processes involved during de novo construction of artificial cells include gene expression, protein expression, self-reproduction, nutrient synthesis, and energy conversion, etc. Although it is enormously challenging to mimic natural-like cell, several single steps of these processes have been performed within a celllike compartment such as mimicked protein synthesis [67], replication of genomic RNA [68], designing of cascade reactions [69], and translated chemical messages [70]. However, these different procedures have not been combined yet; the coordination of different cellular processes is still a major challenge. Information for development of artificial cells is provided by a genome. Synthetic biologists aim to produce a minimal organism based on a synthetic minimal genome. This

genome would be comprised of only absolutely essential genes required for survival under standardized laboratory conditions. Recently, the J. Craig Venter Institute (JCVI) and Synthetic Genomics, Inc. (SGI) used whole-genome design and complete chemical synthesis to minimize the 1079kilobase pair synthetic genome of Mycoplasma mycoides JCVI-syn1.0 and then produced JCVI-syn3.0 (531 kilobase pairs, 473 genes), which has a genome smaller than that of any autonomously replicating cell found in nature. JCVIsyn3.0 retained almost all genes involved in synthesis and processing of macromolecules. However, it also contains 149 genes with unknown biological functions [71]. At present, synthetic biology is enormously challenging, but surely, a promising discipline to creating truly artificial life de novo Fig. 4. Compared to de novo creation of truly artificial life, reprogramming an existing bacterial or eukaryotic cell is considered an easier and practical approach. Using a refined version of genetic engineering, bioengineers can design desired elaborated biochemical pathways and introduce to a host cell to perform required function. Using a host cell is more advantageous because the newly designed biochemical pathways can be assembled into the host cell and can use co-factors, metabolites, transcription pathways, and other components of the host cell [72]. For example, a synthetic gene regulatory circuit has been designed to permanently alter gene expression in response to the well-characterized inflammatory signal nitric oxide. The detection of nitric oxide initiates the expression of a DNA recombinase, causing permanent activation of a DNA switch. An engineered strain of E. coli as a host containing this synthetic circuit responded to nitric oxide from both chemical and biological sources, with permanent DNA recombination occurring in the presence of nitric oxide donor compounds or inflamed mouse ileum explants. This engineered microbe can be used to detect and respond to gut inflammation [73]. In another study, several Gram-negative pathogens encoding type-3 secretion, which can deliver proteins directly into the cytoplasm of mammalian cells, provided a potential application in therapeutic protein delivery. However, their utility has been limited by their inherent pathogenicity. To perform this specific function, reengineering E. coli as a host cell was performed with a tunable type-3 secretion system that can efficiently deliver heterologous proteins into mammalian cells, thereby circumventing the need for virulence attenuation. This reengineered system thus provided a highly flexible protein delivery platform with potential for future therapeutic applications [74]. Synthetic microbes, as a kind of cell therapy, have broad potential for future applications in human disease treatment. For example, bioengineers have been designing various engineered bacteria to perform different specialized functions, such as detection and diagnosis of diseases [75] and production of therapeutics [76] and their delivering [77], which can self-eliminate after therapy

Adv Compos Hybrid Mater Fig. 4 Two approaches to creating synthetic microbes: (1) creating life de novo, creating an artificial cell at least needs to integrate three main components: containment, cellular processes, and information and (2) bioengineering approach, using a refined version of genetic engineering, designing, and inserting elements inside the cells to perform specialized functions

[78]. With the development of synthetic biology, the future directions of bacterial cell therapy systems will set a variety of abilities in one and will be smarter as autonomous microbial Bphysicians^ [79]. Besides synthetic biology, other approaches are also used to fabricate artificial microbes. For examples, inspired by the natural magnetotactic bacteria, developing strategies (Fig. 5) to endow general microbes with magnetotaxis ability would be advantageous [80]. Genetic manipulation is an effective technique to achieve this goal [81], and there have been several reports regarding this. For example, selection of iron storage ferritin in yeast was carried out through specific mutagenesis and high-throughput screening that resulted in desirable ferritin biomineralization mutants being obtained. In these yeast mutants, iron storage ferritin in cells can enhance iron accumulation under physiological conditions [82]. Another technique is to modify Tetrahymena pyriformis such that it is capable of ingesting magnetite, which able to harness Tetrahymena pyriformisas as microrobots using magnetotaxis to steer the cells [83, 84]; in addition, they can steer the engineered motile cellular microrobots in two dimensions [85] and three dimensions [86], but this is limited to specific microbial cell types. Surface modification methods can also perform this specific task, including surface assembly, biomimetic mineralization, and surface hydrogel coating, etc. According to Miguel Martín et al., magnetic probiotic bacteria can be produced by using superparamagnetic maghemite nanoparticles assembled at the surfaces of non-magnetic probiotic bacteria Lactobacillus fermentum and Bifidobacteria

breve. These artificial magnetic bacteria present a collective ferromagnetic phase at room temperature [80]. Biomimetic mineralization is another method to create artificial magnetic cells. Through this method, an artificial mineral shell with magnetic particles in it was fabricated on the cell surface, which could offer the cell magnetic property. But the mineral shell between the cells and their surroundings reduces mass transport and biological communication [87]. In our previous study, we developed a strategy to fashion an artificial extracellular matrix (ECM) for the microbial cells based on Fe3O4 nanoparticle-doped hydrogels. With this method, various microbes including fungi (Saccharomyces cerevisiae) and bacteria (Flavobacterium heparinum) were modified to obtain the ability of magnetic and maintained cell activity and held the cell metabolic level [88].

5 Microbial cell factories: manufacturing functional metabolites Microbial cells use various materials and produce many metabolites for survival just like a factory. The metabolites from microbe have profound influence on the process of human civilization. Especially today, to achieve sustainable energy development, one good strategy is to use bio-based chemistry to alternative oil-based chemistry. The success of this strategy will require efficient, robust, and versatile cell factories. On the other hand, metabolites produced in microbial cell factories such as small


Fig. 5 Strategies to endow general microbes with magnetotaxis ability. Internalization: T. pyriformis can internalize spherical iron oxide particles through oral apparatus located at its anterior end [83]. Intracellular biomineralization: iron storage ferritin in yeast variants can enhance iron accumulation under physiological conditions [82]. Surface mineralization: in situ precipitation of calcium phosphates and Fe3O4 nanoparticles incorporated on an LbL-treated S. cerevisiae cell surface can encapsulate cells and be driven by a magnetic force [87]. Surface hydrogel coated: fashion an artificial extracellular matrix (ECM) for the

microbial cells based on Fe3O4 nanoparticles-doped hydrogels, various microbes including fungi (S. cerevisiae) and bacteria (Flavobacterium heparinum) can be modified to obtain the ability of magnetic and maintain cell activity and hold cell metabolic level [88]. Surface assembly: Magnetic probiotic bacteria can be produced by using superparamagnetic maghemite nanoparticles assembled at the surfaces of non-magnetic probiotic bacteria Lactobacillus fermentum and Bifidobacteria breve [80]. Reproduced with permission from ref. [80, 82, 83, 88]

molecule (e.g., ethanol [89], lactic acid [90, 91], glycerol [92]), antibiotic [93] (e.g., penicillin), pigment [94], protein [95] and glycoprotein [96], polysaccharide (e.g., hyaluronic acid [97], bacterial cellulose [98]), and even virus-like particles (as vaccine) [99] have been widely used in the production and life of human beings (Fig. 6). Therefore, the designing and fabrication of efficient, robust, versatile, and controllable microbe cell factories to produce functional metabolites will extend their range of applications in energy and biomedical fields.

The yeast S. cerevisiae is one of the oldest cell factory used in the production of biochemical, such as alcohol fermentations, baking processes, bioethanol production, and even for heterologous expression of proteins [100, 101]. Gram-positive bacterium Bacillus subtilis, an inhabitant of the upper layer of soil that has the capacity to secrete proteins in gram per liter range, is another promising cell factory for secreted enzyme production [102]. E. coli, Salmonella cerevisiae, Lactococcus lactis, and Bacillus subtilis, etc. are desirable and promising microbial cell factories because these possess several


Fig. 6 Employment of a microbe as designed cell factory for production of functional metabolites

important properties, including: (1) generally recognized as safe (GRAS) status, (2) probiotic properties, (3) absence of inclusion bodies and endotoxins, (4) surface display and extracellular secretion technology, and (5) a diverse selection of cloning and inducible expression vectors [103]. Beyond that, magnetotactic bacteria [104] act as a natural f a ct o r y f o r p r od u c t i o n of i n o rg a n i c m a t e r i a l s — magnetosomes: the intracellular, membrane-bound mineral crystals of magnetic iron oxide or iron sulfide [105]. These magnetosomes contain single magnetic domain crystals that possess useful magnetic and physical features. The magnetotactic bacteria produce these particles in natural aquatic environments, a green and controllable bioprocess.

Furthermore, in comparison with magnetic nanoparticles synthesized by chemical methods, the nanoparticles synthesized by magnetotactic bacteria have better properties (such as better size d istribution, biocompatibility, and easy functionalization over synthetic counterparts). Such nanoparticles find diverse applications, especially in biomedical field, such as in magnetic resonance imaging [106], cell tracking [107], drug delivery [108, 109], and gene delivery [110]. Gluconacetobacter xylinus (G. xylinus) is a microbial cell factory for production of bacterial cellulose (BC) [111]. BC is one of the most common fibrous materials in nature [112, 113]. Because of its unique properties such as a high mechanical strength, high permeability, high water-holding capacity,


considerable biocompatibility, and non-toxicity, BC can be used as scaffold for various tissue engineering applications such as the development of cartilage [114], skin [115, 116], dental implants [117], nerve conduits [118], and blood vessels [119, 120], etc. To further explore the applications of BC in biomedical field, one promising strategy is to make BC with similar ordered structure with natural tissue or organ, or guide the cell alignment to form ordered multicellular structures, and finally, make the artificial tissues or organs perform their functions. Fortunately, G. xylinus can serve as smart textile microbiorobot to produce BC with ordered microstructure (Fig. 7). During BC production by microbial cells, every bacterial cell secretes one cellulose fibril, which pushes the

bacterium forward and leads to cellulose aggregation. G. xylinus usually moves randomly in all directions; thus, BC can form multi-shaped pulps, filaments, and spheres under agitated conditions, and BC sheets are formed at the airmedium interface comprising of an irregular network of cellulose fibers when bacteria are cultured in a static liquid medium [121, 122]. Besides, series of BC tubes can be formed through culturing of G. xylinus in special tubular template [119, 120] and can also form an organ-like shape (such as outer ear) in a silicone mold [123]. However, in all of the abovementioned examples, cellulose fibers in BC are randomly distributed. If the motility of bacteria can be manipulated in an ordered fashion, ordered BC networks can be fabricated. Several methods have been reported to this end. For example,

Fig. 7 G. xylinus is a smart textile microbiorobot for BC production. BC have random microstructure to form multi-shaped pulps, filaments, spheres, and sheets; it can also form outer ear-shaped tube in special mold which have macro-order structure; in addition, several methods

can be used to guide G. xylinus synthesized BC with ordered microstructure. Reproduced with permission from ref. [123, 127, 128, 133–135]


Sano et al. [124] used electric fields to produce oxygen in culture medium. Since G. xylinus is an aerobic bacterium, oxygen induced their ordered motions, which made them biosynthesize ordered cellulose fibers. An ordered substrate was also obtained by Kondo et al. and formed a structure known as nematic ordered cellulose by dissolving the native cellulose and reprecipitating in a distinctive manner. In their study, they transferred G. xylinus cells to the oriented surface where cellulose ribbons were synthesized in parallel to the molecular orientation of the substrate [125]. Uniaxially oriented BC fibrils can also be obtained by culturing G. xylinus on poly-di-methyl-siloxane (PDMS) with a ridged topology [126]. When bacteria were cultured on an agarose film scaffold with honeycomb-patterned grooves, a honeycombpatterned BC was obtained [127]. In our research, we utilized ordered agarose or PDMS template as templates to produce micropatterns on BC surface during fermentation. The size of micropatterns on template surface was arranged from 5 to 50 μm (ridges and grooves of same width). The movement of G. xylinus on template was restricted by oxygen availability and by the groove pattern. BC films with grid and strip structures were also obtained through precise control of culture conditions [128, 129]. In addition, genetic manipulation is a promising technique to achieve this goal. Recently, Kubiak et al. reported the release of complete genome sequence of G. xylinus E25; the whole genome sequencing will allow us to regulate the BC production through genetic manipulation [130]. In addition, for cellulose biosynthesis in G. xylinus, oxygen is the first messenger, whereas 3′,5′-cyclic diguanylic acid (c-di-GMP, a naturally occurring small cyclic dinucleotide) is the second messenger. Since c-di-GMP provides allosteric regulation of cellulose synthase in G. xylinus, this provides bacterium with a mechanism to regulate cellulose synthesis based on environmental oxygen levels [131]. Another study reported the synthesis of c-di-GMP from guanosine triphosphate (GTP) in a light-dependent manner [132]. It can be a promising method in that we can engineer a bacterium which can regulate cellulose synthesis in response to light stimulus. Using this engineered bacterium, we can fabricate BC with diversified ordered microstructures by adjusting the pattern, intensity, and/or wavelength of light. The fine and ordered BC fibrils or hydrogels thus obtained are useful as tissue engineering scaffolds in the repair of nerves conduits, skeleton, and hamstrings. For example, in our previous study, ordered microstructures (grid and line structures) of BC led to a distinct neurite and glial guiding made the cells to show a well-organized strand-like formation on the line and grid structures, while at the same time, the cells showed a random cell distribution on random BC scaffolds and cover slips. Therefore, the ordered BC scaffolds can support the alignment of cells out growth, and furthermore, this method has the potential to fabricate large BC sheets with multilevel, microstructured surfaces with complex, variable

geometries at micrometer and even in nanometer range. Such multileveled microstructures could be modified with bioactive or functional molecules to several other advanced applications, such as fabrication of ordered conductive scaffolds to promote skin or neural regeneration under electrical stimulation [136]. In another study, we developed a strategy to rapidly fabricate multilayered tubular structures by employing the shape-memory property of BC membranes. The most interesting thing is that different types of cells were adhered in a flat state, and then BC membrane returned to its original shape as multilayered tubular structures. Patterning of human umbilical vein endothelial cells (HUVECs), human aortic smooth muscle cells (HASMC), and human skin fibroblasts (HSF) on its surface constituting multiple layers on the tubular wall can imitate blood vessels, and this BC tube showed good biocompatibility both in vitro and in vivo [135]. Further, this shapememory BC tube with ordered microstructures have the potential to fabricate artificial tissues by biomimetic method and to construct functional tissue-engineered artificial intestinal or intervertebral disc (IVD) [137], etc.

6 Conclusions and future perspective Recent developments in biotechnology and nanotechnology provide several possible pathways utilizing different microbes as functional live building blocks to create micro/nanodevices and -robots for biomedical and energy applications, etc. Soft lithography and microfluidic integration with electrical, optical, and magnetic techniques can effectively manipulate and control microbial cells with an unprecedented degree of spatial and temporal precision. Microbes, immobilized and patterned as microdevices, can be further used to study the interactions between the cells and their surroundings such as through biosensing, biodetection, small molecule screening, single-cell manipulation and analysis, and other applications such as biomaterials fabrication and bioenergy. Microbes are capable of moving spontaneously and actively in response to changes in their immediate extracellular environments, steerable environment can be regarded as a promising idea to operate motile microbes. Natural microbes owing to their unique properties, such as chemotactic, geotactic, phototactic, magnetotactic, and galvanotactic, can be controlled by the change of temperature, concentration of food molecules, surface softness, light, magnetic, or electrical stimuli. Inspired by natural microbes, developing a strategy to endow general microbes with special response ability would further explore their applications. Researchers have used different methods to immobilize and control the movement of single and group microbes and manipulate the microbes to be used as live building blocks to create micro/nanodevices and -robots. One of the major challenges moving forward is to increase the lifetime of these


micro/nanodevices and -robots based on microbes. There is also a need to develop multi-control strategies that can accurately and effectively guide their immobilization or motion and enable them to be used in more complicated environment. The development of synthetic biology and other methods provided several strategies to design various artificial microbes to perform different specialized functions; however, these are restricted by several challenges. At present, selfmaintaining, self-producing, and evolvable artificial cells have not been realized, but different preliminary constructions and procedures, such as containment, cellular processes, and minimal genome have been constructed or simulated. The coordination of different preliminary constructions and procedures are still major challenges. Bioengineering and other modification approaches can design systems based on known microbes to perform a specific task. The next challenge is how to make microbes become smarter and more complex, incorporating modules for sensing, diagnosis, signal integration and decision-making, compound production, delivery and control release, and safety, etc. Further, the development of biofabrication based on live microbes will provide effective methods to (1) utilize living microbes to guide and further to design micro/nanodevices and -robots for biomedical applications as fine, effective, long-term stable, and multi-functional live building blocks; (2) design microbe cell smarter as an autonomous Bphysicians^ used in cell therapy field, set a variety of abilities such as detecting and diagnosing disease and producing and delivering therapeutics, and self-eliminate after therapy, etc. integrated in one microbial cell; and (3) produce and guide microbe’s metabolic product to form 2D, 3D, or even 4D ordered microstructures to expand their applications. Acknowledgments This work was supported by the National Natural Science Foundation of China (31270150, 51603079, 21774039) and China Postdoctoral Science Foundation (2015M572132, 2016M602291).

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