Micro/Nanofabrication, Assembly, and Actuation Based on Microorganisms: Recent Advances and Perspectives

Microorganisms become new sources for fabricating intricate micro/nano structures via bottom‐up approaches. Produced from nature, microorganism biotemplates exhibit unique structural and material features over thousands of years of revolution. They are considerably superior and cost‐effective for fabricating complex and heterogeneous structures when compared to other micro/nanofabrication techniques. Herein, the recent advances in biologically driven micro/nanofabrication, assembly, and actuation based on microorganisms are consolidated. Detailed development and recent advances of micro/nanoparticle fabrication, their ordered assembly, and controlled actuation properties, as well as certain typical engineering application examples, are mainly involved. The developing perspectives and challenges of microorganism‐based micro/nanofabrication are also discussed, aiming to inspire the relevant researches and promote the development in this field.

[36] They range in size from less than 100 nm to submillimeter, and most interestingly, lots of them exhibit diverse standard shapes (e.g., spherical, rodlike, threadlike, disklike, spiral), and some of them also owe exquisite uniform substructures such as multilevel pores of diatom frustules.These naturally grown features make microorganisms capable to act as ideal biotemplates to fabricate MNPs with controlled geometries, which can be further utilized to construct unique structures for functional materials and devices.[44][45][46] However, in recent years, a series of micro/nanofabrication strategies based on microorganisms have been proposed, and some novel promising researches have also been particularly explored including their assembly and actuation behaviors.To the best of our knowledge, no comprehensive review has been provided on the micro/nanofabrication, assembly, and actuation based on microorganisms up to now, especially from the perspectives of mechanical manufacturing.Moreover, the emerging scientific and engineering development of strategic significance of bio-manufacturing has also attracted extremely valuable attention.
To address the gap in this field, this review aims to present an informative literature survey of researches related to micro/nanofabrication based on microorganism, briefly explain the basic principles, review the advanced developments, discuss the key challenges, and further provide new insights and perspectives regarding its advances.To clearly demonstrate the contents of this review, we outlined the main researches into three parts, which includes the fabrication of mono MNPs with defined geometries, their ordered assembly/alignment, and also actuation for micro/nano actuator or robotic system (Figure 1).

Fabrication of MNPs Based on Microorganisms
The fabrication of MNPs based on microorganisms can be mainly classified into two types: biological synthesis and biotemplated forming methods.Generally, the biological synthesis method relies on the metabolic processes of natural living microorganisms, which can result in randomly distributed NPs inside or outside the microorganism cells.In addition, the biotemplated forming method directly take the substructure or the overall shape of microorganisms as binding sites or templates, to form ordered NPs aggregation or regular functional microparticles based on various chemical or physical deposition processes.

Biological Synthesis of MNPs
[52] The biologically synthesized magnetosomes can be organized in one or multiple magnetic chains, to endow the magnetotactic bacteria with unique capability to orient and even migrate along geomagnetic field lines.These biosynthesized magnetic MNPs exhibit fascinating advantages in terms of controlling crystal growth and structural properties.[60][61] According to the deposition position of the NPs, the synthesis of NPs can be classified as intracellular and extracellular types.For the intracellular synthesis, the forming mechanism of NPs involves biological trapping, reduction and capping.First, when the cell surfaces of microorganisms come into contact with the metal ions in the medium, positive charges of the metal ions can interact with negative charges of the cell walls electrostatically, resulting in transport of them into the cell.Subsequently, the enzymes existed in the cell walls can reduce the trapped metal ions into NPs.Finally, these synthesized NPs will get transmitted through the cell walls of microorganisms.
For the extracellular synthesis of NPs, the mechanism is mainly found to be nitrate reductase-mediated synthesis.In this case, nicotinamide adenine dinucleotide (NADH)-dependent nitrate reductase enzymes are secreted by microorganism cells.They play a vital role to convert metallic ions into NPs with different sizes, shapes, and compositions (Figure 2b-e). [54,56,62]So far, microbial synthesis of various metallic NPs (e.g., Ag, Au, Pt, Pd, Fe, Cd), as well as metal oxides (e.g., TiO 2 , ZnO), have been reported.These NPs are synthesized in a safe and eco-friendly manner, indicating significant potentials for applications in various fields such as biomedicine, cosmetic industry, and biochemical sensors.65][66][67] At present, a few successful examples have also been reported to fabricate mono-dispersed MNPs with high yield via optimizing various processing parameters (e.g., pH, temperature, time, chemical concentration).However, it still remains a major challenge to achieve stable production in bulk, and practical commercial applications of these biological synthesized NPs are still on its early state.This can be ascribed to two aspects: on the one hand, the yield efficiency of these NPs still needs to be enhanced; on the other hand, basic understanding of mechanisms and principles of biomineralization at molecular levels or in biochemical perspectives is still lacked.Thus, further researches in this field are still required to tackle with the aforementioned issues.For example, engineering of the biological synthesis process combing advanced genetic approaches and diverse novel functional materials is demanding; and efforts also need to be put into exploiting the metabolic potentials of the vast biological diversity of natural microorganisms.Reproduced with permission. [53]Copyright 1999, Horizon Scientific Press.b) Au nanoparticles (NPs).Reproduced with permission. [54]Copyright 2007, Wiley-VCH Verlag.c) Ag NPs.Reproduced with permission. [56]Copyright 2007, Wiley-VCH Verlag.d) Pt NPs.Reproduced with permission. [272]Copyright 2010, Elsevier.e) Au NPs.Reproduced with permission. [273]Copyright 2010, Wiley-VCH Verlag.

Biotemplated Forming of MNPs
Previously, biological synthesis of NPs is the earliest mode to utilize microorganisms for micro/nanofabrication, in which the distinguished advantages of microorganisms have been verified.However, it still remains constraint for microorganisms to achieve enzyme-based biomineralization, since the yield efficiency is relatively low.More importantly, the shape and structure diversity of microorganisms are neglected, resulting in random dispersion of the biosynthesized NPs in/on the cells.In contrast, biotemplated forming can directly utilize the microorganisms with various shapes/structures as templates for deposition of NPs, so as to fabricate MNPs with standard shapes/ structures.Thus, this method is capable of making full use of shape/structure diversity of microorganisms.Since the microorganisms can be artificially cultured in bulk easily, they are becoming superior choices for mass production of regular micro-/nanostructured particles, and the fabrication process are also facile and stable rather than using other biomaterials as scaffolds such as lipid and protein.According to the deposition sites of the synthesized NPs, biotemplated forming methods can be further classified into various types, including extracellular forming, intracellular forming, and also hybrid forming.

Microbial Extracellular Forming
The microbial extracellular forming strategy directly utilizes the outer surfaces of microorganism cells as substrate, to guide the deposition and assembly of inorganic materials onto them via various film deposition techniques.This method can be used to fabricate functional MNPs with different shapes and structures.Generally, natural microorganisms display an astonishing variety of sophisticated shapes which are hard to be fabricated using traditional fabrication methods.95][96][97] Based on microbial extracellular forming technique, a range of functional microparticles with various shapes and coating materials have been fabricated, including microspheres, microrods, microflakes, microcoils, etc. (Figure 3).
Twenty years ago, our group first pioneered the metallization as well as magnetic metallization of Citeromyces matritensis and Bacillus, to fabricate Ni-P microspheres and microrods via electroless plating technique. [98]Typically, activation of colloidal Pd-Sn was conducted to endow the bacteria surfaces with abundant catalytic sites.Due to the negative charges on bacteria cell walls, cations tended to be absorbed onto their surfaces to form the colloidal particles of {nSn 2þ •2(nÀx)Cl À }.As a result, the center was [Pd 0 ] m , surrounded by 2xCl À , forming a double-electric colloidal layer of {[Pd 0 ] m •nSn 2þ •2(n À x)Cl À }•2xCl À .After that, a peptization process was conducted to get rid of extra Sn around the colloidal Pd, which could expose the palladium NPs to activate for further electroless deposition.In the following coating process, chemical reactants of Ni 2þ and H 2 PO 2 À were slowly spread and adsorbed onto the activated cell surfaces.With the catalysis of core-like palladium NPs, redox reactions happened to deposit nickel and phosphorous on the cell surfaces.Since Ni atoms could serve as self-catalysis, the reactions continue to thicken the coating layer until the reactants were used up or jammed.As a result, nickel and phosphorous were co-deposited on the surfaces of bacteria cells to form a uniform-coating layer of Ni-P.
Similarly, Co-Ni-P film could also be coated onto the surfaces of rod-shaped Bacilli cereus to fabricate magnetic microrods. [99]he optimized bath formula and process conditions were obtained, which allowed enhanced magnetic performance of the coating film.The magnetic microrods were capable to be manipulated into parallel arrays in liquids under an externally applied magnetic field, indicating their sensitive magnetic response and motion potentials.After that, some other functional particles (solid or hollow) have also been fabricated via depositing specific nanomaterials onto microorganism cells including cocci, bacillus, and Chlorella.[105][106][107][108] For example, diatom frustules are nanostructured biomaterials synthesized by diatoms, which have hierarchical regular pores made of amorphous silica They have abundant shapes and standard morphologies, which can be mass collected from living diatom cells or purified diatom fossils (diatomite).Based on diatom frustules that own natural porous skeleton, lightweight functional particles can be fabricated.In our previous researches, flaky porous microparticles coated with magnetic materials (e.g., Co-Ni-P, Ni-Fe-P) were prepared using Coscinodiscus diatomite as templates via electroless plating. [93,109]hese electromagnetic microdisks exhibited superior microwaveabsorption properties, since the wheel-shaped structure was desired for enhancing electromagnetic susceptibility, and the porous substructure could also benefit scattering effect of electromagnetic wave. [94]oreover, the microorganism biotemplates can also be removed via acid/alkaline-based chemical etching, and further calcination after the surface coating can be conducted to obtain microparticles with porous/hollow features.For example, Yang et al. fabricated silver hollow microspheres using bacteria cocci as biotemplates via surface coating and a following sonication process to disrupt the bacteria template (Figure 3a). [110]Due to the porous hollow structure, the silver microspheres demonstrated superior surface enhanced Raman scattering (SERS) property.Using Spirulina cells as biotemplates, we fabricated hollow silver microsprings based on extracellular electroless silver plating and calcination treatment. [111]When the coating thickness was about 600 nm, and the annealing temperature was set at 300 °C, the average elongation of the as-prepared silver microsprings could reach up to %106.9%.In some cases, the shape or geometric size of microorganisms can be adjusted via tuning their growing conditions, such as nutrient solution formula, temperature, and illumination.For example, the geometric parameters of Spirulina cells (e.g., diameter, helical pitch, body length) can be adjusted via moderate regulation of the culturing conditions.Based on electroless plating onto the helical biotemplates, Kamata et al. fabricated metal microcoils with diverse structural parameters, and further demonstrated their active electromagnetic response in the terahertz-wave region due to structural resonance (Figure 3d). [90]

Microbial Intracellular Forming
As mentioned before, microbial extracellular forming mainly utilizes the outer morphology of microorganisms, yet the interior body structures are always neglected.Generally, microorganism cells are mainly composed of organic matters and water.For microbial intracellular forming, once the contained water is removed via cell dehydration, the porous cross-linked network of organic matters can form substructural cavity to work as loading sites for depositing NPs.By this way, the internal porous substructures can also ensure well dispersion of NPs without severe aggregation, so as to retain their excellent activity.
[114][115][116] The internal cavity of virus biotemplates can serve as nanoreactor for constrained growth of various nanomaterials, while the structural pores allow ion exchange between the outside and inside of virus.Thus, different NPs can be synthesized inside virus using this strategy, including linearly aligned NPs (e.g., Ag, Co-Pt, Pd, Au) or nanowires (e.g., Ni, Co, FePt 3 , CoPt, Au) (Figure 4a,b). [113,116,117] contrast to virus with natural open pores, it is more challenging to utilize the internal space of non-open microorganisms (e.g., bacteria, microalgae) for intracellular synthesis.Although biological synthesis based on enzymatic reduction is feasible for intracellular deposition of NPs, it is just confined to microorganisms with bioactive enzymes, and inherent defects also exist such as low yield efficiency and uncontrollable particle size.However, microbial intracellular deposition has ceased to advance over a long period of time, and the main obstacle is the poor permeability of microorganism biotemplates.Due to the compact structures of cell wall and membrane, the internal space of microorganism cells is difficult to access for external ions and even NPs in the medium.To solve this problem, our group took a typical algal microorganism (Spirulina) as example, explored the feasibility to enhance cell permeability, and further developed a series of general methods for intracellular deposition of functional NPs.Three diverse methods were conducted for permeability enhancement of Spirulina cells, including acid treatment, freezing-thawing method, and alcohol-based gradient dehydration.Based on the permeabilization process and electroless deposition, intracellular synthesis of highly dispersed NPs could be realized (Figure 4c). [118]In addition, we further demonstrated that a moderate permeabilization process could result in ordered assembly of Ag NPs intracellularly, and the morphology of Ag NPs could be tuned from nanospheres to nanosheets under the spatial confinement of cellular texture inside Spirulina cells (Figure 4d). [119]With the optimized concentration of NaOH, Ag NPs could be uniformly deposited inside the cells.By adjusting the deposition conditions, Ag NPs with size of %6.32, 17.85, and 38.98 nm could be synthesized intracellularly.This microbial intracellular deposition strategy indicated unique advantages such as high yield efficiency and loading amount, and most  [241] Copyright 2020, American Association for the Advancement of Science.Reproduced with permission. [274]Copyright 2010, Royal Society of Chemistry.b) Bacillus and the corresponding Co 3 O 4 microrods.Reproduced with permission. [270]opyright 2011, American Chemical Society.c) Coscinodiscus and the corresponding Ni-P microflakes.Reproduced with permission. [153]Copyright 2019, Elsevier.Reproduced with permission. [275]Copyright 2021, MDPI (Basel, Switzerland).d) Spirulina and the corresponding Ag microcoils.Reproduced with permission. [90]Copyright 2014, Springer Nature.
interestingly, firm confinement of the well-dispersed NPs inside the microorganism cells.These fascinating features could contribute to long-term stable functioning and easy recycling after uses.

Hybrid Forming Both External and Internal
Microorganisms have distinct advantages of standard shapes and interior multilevel structures.In this case, simultaneous utilization of the outer surface and inner space of microorganism cells make it possible to fabricate biomorphic functional MNPs with complex core-shell hierarchical structures.This hybrid strategy directly combines the unique advantages of microbial extracellular forming and intracellular deposition, so as to meet the demands of diverse potential applications spanning from catalysis, energy, electromagnetic materials, drug delivery, and environment remediation.
Except for the simple spherical capsules, functional MNPs with more complex shapes could also be fabricated via microbial  [116] Copyright 2010, American Chemical Society.b) Au nanowires.Reproduced with permission. [276]Copyright 2005, American Chemical Society.c) Optical, Scanning electron microscope (SEM), and TEM images (from left to right) of Spirulina with intracellularly synthesized Ag NPs.Reproduced with permission. [118]Copyright 2018, Wiley-VCH Verlag.d) Schematics of assembly process of Ag NPs spatially confined by the cellular texture of Spirulina, and TEM images of the synthesized Ag NPs with controllable size and structure.Reproduced with permission. [119]Copyright 2019, IOP Publishing Ltd.templated hybrid forming method.For example, Yan et al. fabricated magnetite porous hollow helical capsules based on Spirulina cells, which could be used for targeted delivery. [120]he fabrication process included the precursor deposition, annealing for template removal, and reduction treatment to form the magnetite.The porous hollow capsules possessed an outer shell aggregated by mesoporous spindle-like magnetite NPs and a helical inner cavity, indicating large specific surface area for further functionalization and cargo loading.Our group also developed a hybrid forming strategy for mass production of drugloaded magnetic helical microcapsules, and explored their synergistic chemo-photothermal therapeutic efficacy for cancer cells (Figure 5a). [121]They were fabricated by intracellular deposition of Pd@Au and extracellular deposition of Fe 3 O 4 simultaneously based on Spirulina cells.The Fe 3 O 4 layer enabled the microcapsules to be magnetically actuated precisely, and the intracellular Pd@Au core-shell NPs exhibited excellent photothermal conversion.Similarly, CuS nanodots were also intracellularly deposited within the scaffold of Spirulina cells, to construct magnetic helical microrobots with enhanced photothermal performance (Figure 5b). [122]After that, hybrid intracellular deposition of various functional NPs based on Spirulina was also achieved.We conducted intracellular deposition of Fe 3 O 4 and MnO 2 NPs in sequence to fabricate hybrid-deposited microrobots, and demonstrate their enhanced absorption performance to achieve efficient removal of Pb(II) (Figure 5c). [123]To the best of our knowledge, this was the first time to achieve hybrid intracellular loading of multiple functional NPs within microorganism cells.
Inspired by the selective transport and confined reaction of biological cells, Wu et al. fabricated a biomimetic bipolar microcapsule using Staphylococcus aureus as templates (Figure 5d). [124]he bipolar microcapsule processed a nonpolar core to load active materials and also a polar shell to selectively control the mass transport.They could act as a microreactor, which could adsorb sulfur with the porous carbon core and retard polysulfide migration with the polar TiO 2 shell.Since the resulting sulfur cathodes can interact with all sulfur species and effectively confine them, those intractable issues (e.g., S hosting, electron conducting, polysulfide migration, cycling capability) could be concurrently managed.
For microorganisms, the unique discrete biological structures can enable them to co-encapsulate diverse biomolecules spatially and precisely regulate hundreds of enzymatic reactions.Similarly, the construction of novel hybrid microcapsules with compartmental structures can be capable of achieving multiple complex functions, which are predictable and worth of deep study.

Summary and Perspectives
In recent years, the demands of micro/nanostructures with confined size, shape, and morphology are attracting increasing attention for various applications in the fields of electronics, environment, biotechnology, and healthcare.Fabrication of MNPs based on microorganisms is a significantly promising strategy, which exhibits great advantages of easy mass production, regular Figure 5. a) Optical and TEM images of original Spirulina, (Pd@Au)@Sp, and (Pd@Au)/Fe 3 O 4 @Sp.Reproduced with permission. [121]Copyright 2019, American Chemical Society.b) SEM image of CuS nanodots-loaded Spirulina.Reproduced with permission. [122]Copyright 2022, Elsevier.c) Crosssectional elemental mapping of Spirulina with intracellular loading of Fe 3 O 4 and MnO 2 NPs.Reproduced with permission. [123]Copyright 2021, American Chemical Society.d) Schematic illustration of fabricating Staphylococcus aureus-templated TiO 2 /S microcapsules, and SEM image of the final product.Reproduced with permission. [124]Copyright 2018, Wiley-VCH Verlag.
shape, tunable size, metal-binding capacity, and cost-effective feature.Despite considerable progress in this field, it is still in the early stage considering the abundant resource of microorganisms.
Regarding the development of microorganism-based fabrication of MNPs, the extracellular forming technique is relatively mature, and the as-prepared MNPs have also been explored for electromagnetic absorption and shielding materials in engineering applications.However, the intracellular forming and hybrid forming strategies are still in the starting stage, since the restriction of cell wall permeabilization was just broken with significant potentials of the intracellular space to be further explored.
Since the uniformity of shape, size, and composition are always dominant to the functions of MNPs, it is imperative to improve the yield efficiency of these biological nanofactories which may turn to genetic engineering.The precise control of shape, size, and micro/nanostructure is one key issue to address for the microorganism biotemplates.In addition, suitable fabrication processes need to be optimized to deposit diverse materials, and mass auto-fabrication facilities are also important for practical applications.The legend for key techniques and future developments are also shown, indicating various potential application fields to be further explored (Figure 6).

Assembly/Alignment of MNPs Based on Microorganisms
Alike biological cell populations that perform overall functions via ordered arrangement and differentiation into specific tissues or organs, microorganisms usually grow or assemble into ordered colony for variable environment adaption. [125,126]Inspired by this phenomenon, ordered assembly of functional MNPs has attracted much attention to construct advanced functional composites.[139] Compared with traditional particles, such as sphere-like, rodlike, and platelike types, the structures of biotemplated MNPs are more complicated, and it is more challenging to assemble them into ordered structures.To date, many researchers have tried various methods to fulfill this target, which are mainly based on external physical field-induced effects.In this part, we summarized the development on assembly/alignment of microorganism-templated functional MNPs, and also the ordered structures with special functions.

Magnetic Field-Induced Assembly/Alignment
[142][143] When magnetic MNPs are placed in an external magnetic field, they can be magnetized and orient their easy-axis along with the field direction.46][147]  Moreover, the alignment direction could be arbitrary in the free space and precisely controlled via tuning the direction of the magnetic field.First of all, to construct ordered structures via magnetic field-induced assembly/alignment, the microorganismbased MNPs should be magnetism responsive.Typically, magnetotactic bacteria (e.g., Magnetospirillum gryphiswaldense, MSR-1) can serve as excellent candidates to achieve ordered alignment due to their intrinsic magnetic properties.][150] Thus, it seems the magnetotactic bacteria are endowed with magnetic moments, which are capable to be driven under magnetic fields.For example, Bennet et al. achieved magnetic alignment of MSR-1 bacteria to fabricate a remotely tunable photonic device (Figure 7a). [151]The magnetotactic bacteria demonstrated distinct shape anisotropy, and rotation of its long axis could be controlled under magnetic actuation.When the bacteria were gradually aligned along with the sample plane, the light-transmission intensity could be effectively enhanced.
However, except for magnetotactic bacteria, most microorganisms in nature exhibit nonmagnetic property.Thus, it is imperative to realize magnetization of microorganisms, so as to enrich microorganism-based MNPs for fabrication of functional materials.Diverse magnetic MNPs have been fabricated, ranging from traditional 1D rodlike shape, 2D platelike shape to 3D helical structures.As mentioned in the previous chapter, early in 2002, our group deposited CoNiP on bacilli via electroless plating technique, and studied their response behavior under static magnetic fields, in which both arrays and chain-like clusters could be formed. [152]The bacilli-based particles were of 1D microrod structures, which could be magnetized and aligned under external static magnetic field.However, as for MNPs with more complicate structures (e.g., 2D or 3D shapes), alternating magnetic fields are desired to achieve effective assembly/alignment.Figure 7. Magnetic field-induced alignment of magnetotactic bacteria (living) and various microorganism-templated magnetic MNPs (nonliving): a) magnetotactic bacteria.Reproduced with permission. [151]Copyright 2017, Wiley-VCH Verlag.b) Diatomite-templated magnetic microplates.Reproduced with permission. [153]Copyright 2019, Elsevier.c) Spirulina-templated magnetic microcoils.Reproduced with permission. [134]Copyright 2019, Wiley-VCH Verlag.
Coscinodiscus diatomite with natural flaky microstructures can be utilized as biotemplates to fabricate magnetic microplates via electroless nickel deposition, which has also been demonstrated previously in Figure 3c. [153]In this case, easy-axes of the magnetic microplates were in-plane, thus they could be aligned via a rotating magnetic field. [154,155]The microplate could be driven to keep parallel with the plane of the rotating magnetic field and achieve highly ordered alignment, which opened doors for the fabrication of nacre-like biomimetic functional materials (Figure 7b).In addition, magnetic field-induced alignment of 3D biotemplated microcoils was also achieved in a large area.We used Spirulina cells to massively fabricate magnetic microcoils, which possessed an robust magnetic-coating layer and biotemplated core-shell structures (Figure 7c). [134]In a rotating magnetic field, the tight microcoil could rotate till its long axis was perpendicular to the rotating plane.In this case, orientation direction of the microcoils could be controlled via just tuning the rotating plane of the magnetic field, which made it feasible to realize programmable alignment of helical MNPs in arbitrary directions (e.g., vertical, horizontal) as well as hybrid types.To the best of our knowledge, this is the first time that mass alignment of complex 3D microcoils is achieved via a rotating magnetic field.Moreover, the as-prepared composite with vertical microcoils exhibited remarkable optical chirality, and those with horizontal microcoils showed high polarization conversion in terahertz frequencies, which could be promising to construct functional photonic devices. [156,157]2.Electric Field-Induced Assembly/Alignment Similar to magnetic field-induced assembly/alignment, electric field is also an effective approach to achieve highly ordered structures of conductive MNPs.When the MNPs were suspending in the medium under a nonuniform electric field, they could be electrically polarized.The resulting dielectrophoretic (DEP) force exerted on the MNPs could overcome other forces, such as gravity, stochastic force, and viscous force, resulting in effective actuation of them to form ordered assembly/alignment.[158][159][160][161][162] Consequently, the conductive MNPs could be either propelled toward the region of lager field intensity (so-called positive DEP) or repelled from it (so-called negative DEP), which was closely dependent on effective polarizability of the MNPs within the medium.[163,164] In general, AC fields are more desired for electric field-induced assembly/alignment other than DC fields.The voltage of DC fields is always limited, and a high DC voltage can result in water electrolysis and also cause damage to the micro-/nano objects.However, AC fields are applicable for various mediums such as water, organic solvent, and polymers, which can also typically avoid undesired electroosmosis and electrolysis.Thus, a higher voltage can be applied when using AC fields, to induce a larger field intensity and an electric force on the MNPs to achieve assembly/alignment.This strategy has been successfully applied to fabricate functional composites with ordered structures for various uses.Herein, constrained by this review subject, we just demonstrate typical examples related to microorganism-based MNPs.[165] Copyright 2017, Springer Nature.b) Spirulina-templated conductive microcoils.The electric field was set at 2 kV cm À1 , 1 kHz.Reproduced with permission. [137] Copight 2017, American Chemical Society.c) M13 bacteriophages.The electric field was set at 5-20 Vp-p, 15 kHz.Reproduced with permission.[167] Copyright 2019, MDPI (Basel, Switzerland). d) Escherchia coli bacteria.The electric field was set at 10 Vp-p, 4 MHz.Reproduced with permission.[168] Copyright 2011, American Institute of Physics.
In this case, Xu et al. prepared an electro-fluidic chip to achieve electrical manipulation of Euglena gracilis (Figure 8a). [165]An AC electric field (20 Vp-p, 0.5 MHz) was applied between two electrodes to stimulate an electric field intensity on the order of %2 Â 10 4 V m À1 , which could exert orientation torques to drive the microorganisms.As a result, the major axis of the elongated cells could be aligned along with the electric field direction.In addition, 2D and 3D electrical manipulation could also be conducted to investigate cell motion using cross-shaped and out-ofplane electrodes, respectively.This approach could be applied to align other elongated or anisotropic cells and microorganisms.Previously, our group used AC electric field to align helical Spirulina-templated metallic microcoils, and fabricate a highly anisotropic electrical conductive composite (Figure 8b). [137]Under an AC electric field (2 kV cm À1 , 1 kHz), the helical microcoils could be polarized, in which their long axis could be oriented along with the filed direction to align them into continuous long chains. [166]ore importantly, the adjacent microcoils could form unique endto-end physical contact in the alignment direction.In addition, electric field-induced assembly/alignment can also be applied to fabricate microorganism-based biosensors such as phage litmus, which is featured the characteristic colors of the aligned phage bundles and also color shifts based on structural changes to targeted molecules.Tronolone et al. applied AC electric fields (5-20 Vp-p, 15 kHz) to efficiently align phage filaments into colored thin films, which resulted in concentric color bands quantified with image analysis of red, green, and blue line profiles (Figure 8c). [167]Different from dipolar electric fields of one pair of electrodes, Chung et al. designed an integrated device of four electrodes, and used DEP force to manipulate the elongated E. coli bacteria for rapid antibiotic susceptibility tests (Figure 8d). [168]The AC field was set at 10 Vp-p and 4 MHz in this case.Moreover, except for DEP effects based on traditional electrodes, light-induced DEP is another effective method for electrical alignment.For example, Miccio et al. reported immobilization and orientation of E. coli bacteria using this approach based on photorefractive property of ferroelectric iron-doped lithium niobate crystals. [169]In this research, suitable light patterns were utilized to tune spatial distribution of charges within the substrate, thus adjust the resultant electric fields and DEP forces to immobilize and align the E. coli bacteria.This method allowed large-area manipulation and alignment of bacteria (areas of few square centimeters) without labeling, which was merely determined by modulation of the laser light.

Fluid Shear-Induced Assembly/Alignment
Fluid shear-induced method is a facile and cost-efficient strategy to align anisotropic MNPs in a large area, especially for MNPs of slender structures such as micro-/nanorods and micro-/nanowires.[172][173][174] The flow shear could adjust the nanowires to be orientated along with the bushing direction, and the fluidic surface tension which was always pointed to the normal direction at certain contacting points played dominant roles in this process.177] The unique features of fluid shear-induced method make it possible to align living microorganisms and various microorganismtemplated MNPs, such as original microbes, rodlike virus, and functional MNPs.Considering rheology property of the medium, previous researches have also verified applicability of alignment using shear flow in both Newtonian fluids (e.g., water, PDMS) and non-Newtonian fluids (e.g., hydrogels) (Figure 9).[180] It is generally easier to achieve alignment and chaining of MNPs in viscoelastic fluids under shear flow than in Newtonian fluids.For example, Marcos et al. found that elongated microorganisms displayed strong alignment with the flow streamlines when exposed to a vortex within a custom microfluidic device. [181]It was the shear force that adjusted orientation of the elongated microbes, resulting in light-scattering regulation of the microbial suspension.Similarly, microorganisms with smaller sizes, such as TMV and M13 bacteriophage, could also be effectively aligned in this way.In this case, the stiffness and aspect ratio of the microorganisms played a critical role in the alignment process.Due to the flow gradient, the fluidic shear force could drive the anisotropic MNPs to rotate till the long axis was oriented along with the flow direction.However, this method is still hard to realize robust physical connect between the adjacent MNPs compared with assembly/alignment process using magnetic or electric fields, which is more reliable due to adjacent dipolar interactions in these physical fields.In fact, it is significant to form robust physical connect between the adjacent MNPs for some specific applications, especially to form channels toward electrical conducting, yet it is not always demanding such as for optical uses and electromagnetic responses.Thus, there is no universal assembly/ alignment method, and the advantage and applicability of different strategies should be considered to achieve desired assembly/alignment for MNPs with various sizes and structures.
To date, effective assembly/alignment using fluid shear has been reported with unique advantages in convenience, applicability, and efficiency.Lee et al. reported large-scale unidirectional alignment of anisotropically functionalized M13 viruses in water (Figure 9a). [182]The virus ends were modified with a specially engineered peptide to form desired salt-bridge interactions with the graphene oxide substrates.Owing to the external shear force, the surface-bound viruses were aligned in one direction and stacked to form an ultrathin nanomesh membrane, which exhibited excellent permeability and size-selective exclusion efficiency.Using the facile alignment based on shear flow, Wu et al. realized alignment of rodlike TMV in hydrogel to afford a long-range order, which could be quickly fixed via fast sol-gel transition in situ (Figure 9b). [183]The orientation degree and distance between each other could be regulated via adjusting the concentration of hydrogels and TMVs.
In addition, based on controlling the flow rate and substrate surface properties, Zan et al. achieved alignment of various rodlike NPs in water, including TMV, gold nanorods, and M13 bacteriophage (Figure 9c). [184]These hierarchically ordered structures could be used to support cell growth and control cell orientation, indicating application potentials in tissue engineering and sensing fields.Furthermore, the shear flow method can also be used for microorganism-based MNPs with complex structures.Our group successfully aligned 3D helical microcoils in a large area using both flow shear and electrostatic interaction in PDMS medium (Figure 9d). [185]Due to the anisotropic structure of the microcoils, once they were anchored at one point, the posture could be adjusted to keep lowest resistance within the shearing flow.Such self-adjustment was mainly ascribed to the fluidic shearing torque, which allowed reorientation of the microcoils, yet robust contact and continuous chain-like structures could not be constructed.

Other Strategies for Assembly/Alignment
[188][189][190] For example, to align flaky diatom frustules (Coscinodiscus), Zhang et al. proposed a bubble-induced agitation method to achieve oriented assembly of the microplates.They simulated rising process of the frustules in water and their interactions between bubble-induced agitations.Finally, experiments were conducted to obtain close-packed frustule monolayers with up to nearly 90% of frustules achieving uniform orientation (Figure 10a). [191]In addition, as for MNPs of slender rods or other complex structures, space confinement is an effective method to realize highly ordered alignment/assembly.For example, Sheats et al. confined E. coli bacteria in a narrow slit to achieve regulation of their growth and nematic alignment (Figure 10b). [192]In this case, the assembly/alignment method based on space confinement should be specially designed considering inherent property of the microorganisms or microorganism-templated MNPs, especially their anisotropic structures.Using flow-induced alignment and torque balance, Basaran et al. achieved stable orientation equilibrium of rod-shaped bacteria in radial direction, and further revealed dynamics of the radially oriented structures (Figure 10c). [193]he results demonstrated the mechanism underlying structural order, and also helped to understand bacterial growth dynamics on complex surfaces.Moreover, our group utilized a facile and robust method of microgroove confinement to align and assemble Spirulina-templated microcoils densely in bulk (Figure 10d). [194]ased on the aligned conductive microcoils, a flexible radiofrequency antenna was further fabricated with excellent tunable and deformable performance, which could open new routes for Figure 9. Shear-induced alignment of anisotropic microorganisms and biotemplated MNPs: a) Unidirectional M13 viruses on the graphene-oxide surface.Reproduced with permission. [182]Copyright 2014, Wiley-VCH Verlag.b) Tobacco mosaic virus in the alginate hydrogel.Reproduced with permission. [183]Copyright 2017, Elsevier.c) Rodlike NPs (e.g., TMV, Au nanorods, and M13 bacteriophage), and their applications for control myoblast cells.Reproduced with permission. [184]Copyright 2013, American Chemical Society.d) Spirulina-templated conductive microcoils.Reproduced with permission. [185]Copyright 2019, Elsevier.
the development of wireless mobile communication systems, wearable electronics, and smart sensors.

Summary and Perspectives
Based on batch fabrication of various MNPs, it is demanding to apply collective MNPs to construct functional composites due to limited capabilities of individuals.For a vast number of MNPs, ordered assembly/alignment can further endow them with unique enhanced performance in many aspects including mechanical strength, electromagnetic property, and even anisotropic features (e.g., optical, electrical, and thermal conductivity).Considering the great significance in this field, we have briefly introduced typical strategies and applications of ordered assembly/alignment of MNPs based on microorganisms.In fact, there is no universal assembly/alignment method which can be Figure 10.Some other typical examples for assembly/alignment of microorganism and biotemplated MNPs.a) Simulated rising process of a concavedown diatom frustule and the velocity field of surrounding water.The results of diatom monolayer before and after bubble-induced agitation were also shown.Reproduced with permission. [191]Copyright 2016, Springer Nature.b) E. coli bacteria growth and orientation in wide and narrow slits.Reproduced with permission. [192]Copyright 2017, Royal Society of Chemistry.c) Inward growth of bacterial colonies and emergence of radial alignment.Reproduced under the terms of CC BY license. [193]Copyright 2022, The Authors, published by eLife Sciences.d) Space confinement-induced alignment of microcoils and its application as a tunable antenna.Reproduced with permission. [194]Copyright 2018, Royal Society of Chemistry.
applicable for all types of MNPs with various sizes and structures.Since the performance of most materials is not only related to inherent property of the embedded functional MNPs, but also related to distribution and organization of them.Thus, construction of well-ordered structures is dominated to further explore their practical applications.Considering MNPs with various intrinsic compositions and structures, electric/magnetic/shearing methods are still the most suitable and effective approaches for assembly/alignment.Moreover, some other methods including space confinement, fluid floating, and bubble agitation are also verified to be applicable in specialized cases to effectively accomplish highly ordered assembly of MNPs.
For the future development, not only massive assembly and alignment in one direction are required, but also complex structures made of functional MNPs with high-level or hierarchical assembly are demanding.For example, various types of functional MNPs can be prepared using novel and facile fabrication methods; hybrid programmable structures with function integration are also desired similarly to 3D/4D printing techniques.Based on integration of hybrid biotemplated fabrication and various elaborate assembly/alignment strategies, microorganismbased MNPs can be reorganized into highly ordered structures, and construct composite materials with hierarchical features and enhanced functions toward novel applications.Such promising technique is still on its early stage, and more efforts are needed to meet the growing demands of highly ordered assembly of functional MNPs (Figure 11).

Actuation of Micro/Nanorobots Based on Microorganisms
Most kinds of microorganisms in fluids are capable of swimming flexibly via diverse means during growing or living, so as to search nutrient and favorable habitat.[197][198][199] When considering the Reynolds (Re) number for propulsion in fluids, it can be defined as Re = ρLV/η.It is directly determined by the fluid (density ρ, viscosity η) and the moving object (characteristic length L, velocity V ).Due to the relatively small size and speed, both microorganisms and micro/nanorobots are moving in the fluids at small Re numbers (Re << 1), which means the viscous forces dominate compared with the inertial forces. [200][203][204] In general, there are mainly two types of research regarding this: one is to take inspiration from efficient motility of natural swimming microorganisms to design and fabricate micro/nanorobots with various propulsion strategies; the other is to directly apply microorganisms to construct micro/nanorobots, based on using the intact natural structures as biotemplates or applying the living microorganism cells as actuators to form biohybrid devices.

Propulsion Strategies of Micro/Nanorobots Based on Microorganisms
For small Reynolds numbers at micro/nanoscale, viscosity forces play a dominate role compared to inertial forces, which can form an obstacle for effective propulsion.207][208][209] Mostly, prokaryotic microorganisms can navigate via a corkscrewtype motion generated by helical flagella tails, such as E. coli bacteria. [210]In addition, eukaryotic flagellar propulsion is based on undulating waveform motion generated by one flexible flagellum, and the typical example was spermatozoa cell. [211]Another different locomotion type is ciliary propulsion which can swim forward based on nonreciprocal strokes performed by body-covered cilia, such as paramecium. [212]n turn, motile microorganisms with various propulsion mechanisms can provide researchers much inspiration to design and fabricate micro/nanorobots.[219][220][221][222] This strategy has been verified to be one most efficient propulsion method within low Reynolds number fluids.225][226][227] Moreover, artificial ciliary microrobots are also fabricated to be magnetically actuated based on nonreciprocal motion. [228]Due to the similarity in size scale and practical fluidic environment, mimicking swimming microorganisms at low Reynolds numbers significantly opens up new avenues to develop efficient robotic devices at micro/nanoscale.Novel propulsion strategies can also be further explored based on deep understanding of swimming mechanisms of motile microorganisms.

Fabrication of Micro/Nanorobots Based on Microorganisms
Microorganisms not only help a lot in designing micro/nanorobots with effective propulsion, but also can be utilized in the fabrication process to construct micro/nanorobots with delicate structures and special functions.This strategy can better take intrinsic advantages of biomaterials for microorganism-based micro/nanorobots.In this section, we focused on the main development in fabrication of microorganism-based micro/nanorobots, which can be classified into two diverse types, including biotemplated and biohybrid ones, in which typical microorganisms such as microalgae, fungi, and bacteria are covered.

Biotemplated Micro/Nanorobots Based on Microorganisms
Microorganisms can be directly utilized as biotemplates to fabricate micro/nanorobots based on various robotic designs, such as flagella tail structure, flaky structure, and helical structure.Based on integrating desired structures of modified microorganisms, it can provide much convenience to fabricate functional micro/ nanorobots.
Microalgae-Templated Micro/nanorobots: Microalgae are one species of microorganisms widely distributed in nature with diverse unique features as mentioned in previous sections.For example, Spirulina platensis that own natural grown helical structures can be used as ideal biotemplates to fabricate magnetic helical microrobots.In this field, Yan et al. fabricated magnetite helical microswimmers with porous hollow structures based on Spirulina for the first time (Figure 13a). [120]In the synthesis process, three key steps were involved including magnetite precursor deposition, annealing, and reduction.The microswimmers were endowed with a coating layer of mesoporous magnetite NPs and also helical inner cavity.They could be actuated via a rotating magnetic field and exhibited high loading capacity for targeted delivery.In addition, based on a facile magnetic dipcoating process, multifunctional helical microrobots were synthesized in batch using Spirulina (Figure 13b). [229]In this case, Fe 3 O 4 NPs in suspension were firmly bonded onto the cell surfaces of Spirulina, forming a uniform-coating layer.They could be flexibly propelled, and both fluorescence and magnetic resonance imaging were feasible in vivo to achieve imaging guided therapy.Such microrobots were further proved to be capable of loading, transporting, and releasing molecular cargos due to unique biological properties of microorganism cells (Figure 13c). [230]ur group previously also reported magnetic helical microswimmers made by nickel-plated Spirulina with an enhanced propulsion velocity. [231]We conducted electroless plating technique to coat a homogeneous and compact nickel layer onto helical biotemplates with good surface quality and magnetic property.The microswimmers exhibited superior swimming performance, which could reach a high forward velocity up to %12 body lengths per second under a low-strength rotating magnetic field.Furthermore, based on Spirulina, we fabricated various functional magnetic helical microrobots via optimized biotemplated forming.We first presented magnetic helical microrobots for synergistic Reproduced with permission. [277]Copyright 1988, Wiley-VCH Verlag.b) Undulating propulsion of eukaryotic flagella microorganisms, such as sperm cell.Reproduced with permission. [211]Copyright 2017, Springer Nature.c) Cilia propulsion of ciliary microorganisms, such as paramecium.Reproduced with permission. [212]Copyright 2011, Springer Nature.chemo-photothermal therapy via a facile fabrication process (Figure 13d). [121]Core-shell Pd@Au NPs were synthesized intracellularly via electroless deposition, and Fe 3 O 4 NPs were further coated on the cell surface via sol-gel method.Then, typical anticancer drug doxorubicin was also loaded using electrostatic interaction.The helical microrobots could be effectively actuated under rotating magnetic fields to achieve targeted drug delivery, and exhibited synergistic chemo-photothermal therapy.Similarly, we fabricated magnetic helical microrobots loaded with intracellularly deposited CuS nanodots, which exhibited enhanced photothermal conversion to effectively kill HeLa cells and E. coli bacteria (Figure 13e). [122]Based Spirulina cells, we also fabricated magnetic microrobots with hybrid intracellular deposition of both MnO 2 and Fe 3 O 4 NPs (Figure 13f ). [123]These helical microrobots could be spinning around to interact with the fluids for highly efficient adsorption-based removal of Pb 2þ in contaminated water, and could also be propelled forward for effective collection and recycling after uses.
Diatoms are one species of microorganisms widely distributed in all waters of the earth.[234][235][236][237] In 2009, Gordon et al. proposed a concept of diatom-based microrobot via attaching motile bacteria or depositing magnetic NPs onto the frustules, to introduce efficient mobility and realize flexible drug delivery. [238]To achieve effective actuation, Panda et al. presented self-propelled micromotors based on diatom frustules, in which strong oxidizing washing and pyrolysis treatment were conducted to embed iron oxide inside the frustules for catalytic propulsion. [239]The activated diatom micromotors could retain intact porous structure and achieve effective motion via facile catalytic decomposition of H 2 O 2 .In addition, our group used flaky diatomite as templates to fabricate magnetic wheel-shape microswimmers via electroless nickel plating (Figure 14a). [240]In this case, the as-prepared microrobots exhibited alternative locomotion modes as tumbling and rolling under vertical rotating magnetic fields of various frequencies.
Moreover, Chlorella is one genus of unicellular microalgae widely applied as biofuel and super food.[243] For example, Bi et al. fabricated rattle-type microspheres with multiple magnetite cores and porous shells based on Chlorella cells (Figure 14b). [71]ased on a controlled hydrothermal process, the precursor was reduced by initial biological substances to form distributed magnetite nanocores, and the cell wall further turned into a porous shell.These microspheres could not only be magnetically Reproduced with permission. [120]Copyright 2015, Wiley-VCH Verlag.b) Microrobots fabricated via a facile Fe 3 O 4 dip-coating process.Reproduced with permission. [229]Copyright 2017, American Association for the Advancement of Science.c) Microrobots based on dip-coated Spirulina platensis for molecular cargos loading and release.Reproduced with permission. [230]Copyright 2019, Elsevier.d) Microrobots loaded with Pd@Au photothermal NPs and anticancer drug.Reproduced with permission. [121]Copyright 2019, American Chemical Society.e) Microrobots loaded with CuS nanodots for enhanced photothermal therapy.Reproduced with permission. [122]Copyright 2022, Elsevier.f ) Microrobots loaded with MnO 2 and Fe 3 O 4 NPs for water treatment.Reproduced with permission. [123]Copyright 2021, American Chemical Society.
actuated, but also perform protein encapsulation and release via pH control, which were feasibility for promising biomedical applications.In addition, using Chlorella cells as biotemplates, our group fabricated novel ternary biohybrid magnetic microrobots with extracellular coatings of Fe 3 O 4 NPs and BiOCl nanosheets (Figure 14c). [244]These could be propelled under rotating magnetic fields, and perform enhanced photocatalytic degradation of Rhodamine B and inactivation of E. coli bacteria under visible light irradiation.Moreover, our group first proposed a novel strategy to fabricate Chlorella-based magnetic biohybrid microrobot multimers for targeted drug delivery (Figure 14d). [245]In this research, the permeabilized Chlorella cells were coated with Fe 3 O 4 NPs for magnetization, and then loaded anticancer drug with a high capacity.Based on controlled magnetic dipolar interaction, the microrobots could reconfigure into chain-like multimers and also disassemble reversibly.They exhibited diverse propulsion modes with high maneuverability under magnetic actuation, and could perform targeted drug delivery and chemotherapy toward HeLa cancer cells.
The micromotors exhibited chemotactic response to external chemical stimuli and could achieve bubble propulsion via catalytic decomposition of H 2 O 2 .Due to the mesoporous structures with negative zeta potentials, the iMushbots were capable of loading cationic anticancer drugs for effective targeted therapeutics.Similarly, yeast which were one common kind of fungi with ellipsoidal shapes have also been magnetically modified via optimized surface-coating techniques, indicating significant potentials to serve as biotemplates for fabricating micro/nanorobots with unique functions. [76,77,247]oreover, the widely existed spores produced by fungi can also be applied as biotemplates for microrobot fabrication due to their intricate 3D structures and excellent absorption properties.In this case, Zhang et al. developed magnetic microrobots via in situ growth of Fe 3 O 4 NPs on fungi spores, which possessed hierarchical porous structures as well as absorbing activity of biological matters (Figure 14f ). [248]The spore-based microrobots could be employed for fast and highly efficient removal of water pollutants when magnetically driven in controllable swarms.Furthermore, using the spore-based biohybrid magnetic microrobots, fluorescent probes could also be encapsulated via chemical conjugation to endow them with tracking capability, which could be applied for toxins detection in a motion-based method. [249]They could act as an efficient mobile sensing platform for selective fluorescence detection toward Clostridium difficile (C.diff ) toxins in clinical samples.

Biohybrid Micro/Nanorobots Based on Microorganisms
[252] Targeted for efficient actuation, powerful motility of microorganisms is desired to be employed for delivery and manipulation at small scale.Generally, living microorganisms are sensitive to the fluidic conditions (e.g., pH, chemical contents), thus their unique sensing capability can also be utilized.Biohybrid micro/nanorobots can take advantages of superior actuating and sensing properties of microorganisms, which can be combined with the artificial components to form an intact functional system.Taking motile microalgae for example, Sitti et al. used a kind of unicellular green microalga (Chlamydomonas reinhardtii) to fabricate biohybrid microrobots with biocompatibility and motility toward biomedical drug delivery.In this case, layer-by-layer polyelectrolyte deposition was conducted to coat various molecular layers onto magnetic polystyrene (PS) microparticles, which could be further  [253] Copyright 2018, Wiley-VCH Verlag.b) Bacteria-powered microrobots based on Salmonella typhimurium.Reproduced with permission. [254]Copyright 2013, Springer Nature.c-f ) Bacteriapowered microrobots based on E. coli with diverse designs.An E. coli bacterium was attached to c) Pt/PS Janus microsphere.Reproduced with permission. [257]Copyright 2016, Wiley-VCH Verlag.d) Polyelectrolyte-modified magnetic microparticle.Reproduced with permission. [258]Copyright 2017, American Chemical Society.e) Polymer microtube.Reproduced with permission. [259]Copyright 2017, Wiley-VCH Verlag.f ) Drug-loaded red blood cells, respectively.Reproduced with permission. [260]Copyright 2018, American Association for the Advancement of Science.g) Magnetotactic bacteria-based microrobots with drug-loaded nanoliposomes.Reproduced with permission. [262]Copyright 2014, American Chemical Society.h,i) Artificial magnetic bacteria with diverse designs: h) Internalizing magnetite NPs inside Tetrahymena pyriformis.Reproduced with permission. [266]Copyright 2010, American Institute of Physics.i) Self-assembly of maghemite NPs onto probiotic bacteria.Reproduced with permission. [267]Copyright 2014, Wiley-VCH Verlag.j) Biohybrid magnetic microrobot based on bacterial flagella.Reproduced with permission. [269]Copyright 2017, Springer Nature.
To fabricate biohybrid microrobots, living bacteria with superior motility have also been heavily utilized, such as Salmonella typhimurium (S. typhimurium), Serratia marcescens (S. marcescens), E. coli, as well as magnetotactic bacteria.Many researchers have tried various strategies to realize assembly and integration between living bacteria and artificial microstructures.Both nonspecific binding (e.g., polylysine binding) and specific binding (e.g., antibody binding, biotin-avidin binding) have been developed for reliable attachment.For example, Park et al. covalently bound attenuated S. typhimurium bacterium to one PS microparticle via strong biotin-streptavidin-binding affinity (Figure 15b). [254]ue to the biological chemotactic responses to tumor cell lysates, effective motility and tumor targeting capability of the bacteriabased microrobot were further realized.Based on this method, Sitti et al. also attached multiple S. marcescens bacteria onto the surface of a superparamagnetic microbead, to construct bacteria-driven biohybrid microrobot with magnetic steering capability. [255]n addition, living E. coli bacteria have also been applied to synthesize bacteria-driven biohybrid microrobot.Such microbes use a so-called "run-and-tumble" strategy based on the peritrichous flagella to navigate toward chemically favorable locations in fluids.Due to effective helical flagella propulsion, E. coli bacteria can attain a high speed over 30 μm s À1 , and the Reynolds number is at the magnitude of 10 À5 . [256]For example, Stanton et al. presented a simple and rapid fabrication method to attach E. coli bacteria with metal-capped PS Janus microsphere (Figure 15c). [257]The selective firm assembly could be ascribed to surface hydrophobicity as well as electron interaction, indicating efficient locomotion and biocompatibility features.Sitti et al. also performed electrostatic attachment between single E. coli bacterium and anticancer drug-loaded polyelectrolyte multilayer magnetic particles, which resulted in biohybrid microrobots for targeted drug delivery (Figure 15d). [258]To extend the limitation of cargo types and functions, single motile E. coli was also captured inside synthesized polypyrrole microtube to construct a biohybrid microrobot (Figure 15e). [259]In this case, the electropolymerized microtube was modified with polydopamine to trap single bacterium as a biological engine, and a layer of nickel was also deposited for magnetic directional guiding.Moreover, Sitti et al. also reported a deformable E. coli-driven microrobot using red blood cell as cargo carrier, which was loaded with anticancer drug molecules and superparamagnetic NPs via hypotonic/isotonic treatment (Figure 15f ). [260]The functionalized cell was attached to motile E. coli bacterium via biotin-avidin binding, forming erythrocyte-based biohybrid microrobots with superior biocompatibility and biodegradability.
Magnetotactic bacteria are a unique kind of bacteria with intracellularly synthesized magnetic nanocrystals (magnetosomes), which allow them to be directed along external magnetic fields.With sensitive response to oxygen gradients, most magnetotactic bacteria tend to localize toward oxygen-deprived regions.They can provide a high propulsion velocity of 200-300 μm s À1 at a body size of 1-2 μm, indicating great potentials to serve as biological actuators for cargo transport and drug delivery.For example, Khalil et al. proposed a closed-loop control approach to realize precise guiding and positioning of magnetotactic bacteria, which was desired for further targeted uses. [261]Felfoul et al. used Magnetococcus marinus to carry drug-loaded nanoliposomes, and transported them into hypoxic regions of mouse tumor in vivo under controlled magnetic guidance (Figure 15g). [262,263]However, considering the limitations of culturing and growing conditions for natural magnetotactic bacteria, it is imperative to develop artificially synthesized magnetic bacteria to serve as robust biohybrid microrobots.[266] The magnetite NPs were internalized within the bacteria cells via phagocytosis, and could be further magnetized to achieve directional motion guide.In addition, Martin et al. also used nonmagnetic probiotic bacteria as biological platforms, and assembled superparamagnetic NPs onto the surfaces to fabricate artificial magnetic bacteria with sensitive responses under external magnetic fields (Figure 15i). [267]o construct biohybrid microrobots, bacterial flagella which are crucial organelle for living microorganisms to swim in fluids can also be chosen as functional components.For example, Cheang et al. extracted the flagella filaments from S. typhimurium bacteria via ultracentrifugation, and used single flagellum to link PS microbead with magnetic NP via biotin-avidin interaction.This strategy could construct biohybrid microrobot with selfassembled structure, which could perform controlled propulsion under magnetic actuation. [268]Kim et al. also proposed biohybrid microrobots based on bacteria flagella.In this case, the sheared flagella of S. typhimurium were depolymerized into flagellin and further repolymerized into flagella fragments, which were utilized as polymerization seeds to form longer flagella.The regenerated flagella could also be magnetically modified or directly conjugated with magnetic microspheres to construct intact biohybrid microrobots (Figure 15j). [269,278]

Summary and Perspectives
To date, significant progress have been made in fabrication and application of microorganism-based micro/nanorobots, which represent an emerging and fascinating research field with distinct interdisciplinary features.The unique morphologies and structures provide possibility to understand propulsion mechanisms in low Reynolds number fluids, which further guide the specialized design and fabrication of micro/nanorobots.Except for the researches mentioned earlier, some certain microorganisms also have unique responsive properties, such as chemotaxis, geotaxis, phototaxis, magnetotaxis, and electrotaxis.Hence, these microorganisms are capable of being controlled by temperature change, food molecular concentration, surface softness, light, and magnetic and electrical stimulation, which can offer versatile strategies for actuation and control of microorganism-based micro/nanorobots.
Regarding the future development, there are still challenges should be focused on.First, it is demanding to optimize the design and fabrication of micro/nanorobots to achieve superior versatile functions considering motility, capacity, biocompatibility, and biodegradability.As for biotemplated micro/nanorobots, both intrinsic structure and geometric size should be taken into account, so as to meet the requirement for broader biomedical applications in vivo.In addition, as for biohybrid micro/nanorobots, the incubation process of motile living microorganisms should be optimized, and it is also desired to reduce the dependence on application environment (e.g., pH, temperature, fluids, chemicals).It is particularly imperative to keep motility and actuation activity of living microorganisms, which is extremely significant to increase the lifetime of such type of biohybrid robotic devices.Furthermore, drug resistance-induced invalidity and pathogenicity should be accounted for practical biomedical applications in vivo.It is always mandatory to guarantee material safety and limit cytotoxic effects.Another focus is to develop multimode actuation and manipulation control strategies, which may realize separated or swarming motion control precisely and efficiently within complex fluids.The key development strategies and challenges are also briefly illustrated in Figure 16.

Conclusion
Recent advances in biomedical and micro/nanofabrication technology have offered possible strategies to utilize microorganisms as building blocks for constructing functional MNPs, materials, and devices.Compared with the common concept that micro/ nanofabrication is a tool for microorganism research, using microorganisms as tools for micro/nanofabrication can also be one type of converse thought instead.As a novel strategy for micro/nanofabrication, bio-manufacturing based on microorganisms is still on its early stage compared with typical physical or chemical routes, and great potentials are also verified due to unique advantages of microorganisms as follows: 1) The natural grown size and structure diversity at micro/nanoscale provide possibility of complex biotemplates to fabricate various MNPs in a sustainable way.2) Based on biological synthesis or biotemplated forming process, microorganisms are applicable biotemplates with capability to meet the requirements of cost-efficiency, eco-friendliness, and reproducibility to fabricate functional MNPs.3) Both extracellular surface and inner substructure of microorganism cells can work as supporting scaffolds and loading sites for NP synthesis or assembly, which can endow them with significant stability for further modification and recycling utilization.4) The ordered assembly/alignment of microorganismbased MNPs can further enhance the performance of functional materials and composite structures, or even can endow them with some new properties or effects.5) Motile microorganisms of abundant species benefit the design and fabrication of micro/nanorobots for promising biomedical applications as the cornerstone of functional life, and distinct biological advantages could be highlighted based on (microorganism) life for (human) life.
Targeting practical scalable engineering applications, there are still obstacles need to conquer.For example, yield levels, reliability, as well as uniformity of nanostructures in biological synthesis need to be enhanced; potential applications of the as-fabricated MNPs need to be explored.Herein, the corresponding perspectives and research trends are summarized to be considered.1) Precise regulation of shape and hierarchical structure of microbial templates, which is one prerequisite to fabricate MNPs with designed parameters and functions, can be achieved with the interdisciplinary support of chemical and physical interventions, biological science, and genetic engineering technology.
2) It is demanding to guide and assemble microorganism-based MNPs into hybrid 2D/3D/4D microstructures with complex multiscale features toward diverse functional applications.In this case, various strategies may be combined such as biological selfgrowing, self-assembly, external physical field-aided arrangement, and also separated interfacial processing of the MNPs.
3) Using living microorganisms to guide design and fabrication of micro/nanorobots, so as to endow them with biohybrid activity, long-term stability, and biomedical versatility.Integrating bio-manufacturing strategies to construct microorganism-based micro/nanorobotic systems with intelligent features in individuals and swarms, which aim to construct independent and interactive "doctors" with versatile functions including biological detection, disease diagnosis, and therapy.
Herein, the blueprint and main directions of microorganismbased micro/nanofabrication are briefly summarized as shown in Figure 17.Despite those great advances in this field, the related fabrication technique and applications are still at the early state considering abundant diversity and complexity of microorganism resources.Based on deep understanding of biomolecule interactions within MNPs, mechanisms and principles of MNPs fabrication, assembly/alignment, as well as actuation, it will give rise to nextgeneration functional MNPs toward future advanced applications.

Figure 4 .
Figure 4. a,b) Transmission electron microscope (TEM) images of various synthesized MNPs inside virus biotemplates.a) Co-Pt nanowires.Reproduced with permission.[116]Copyright 2010, American Chemical Society.b) Au nanowires.Reproduced with permission.[276]Copyright 2005, American Chemical Society.c) Optical, Scanning electron microscope (SEM), and TEM images (from left to right) of Spirulina with intracellularly synthesized Ag NPs.Reproduced with permission.[118]Copyright 2018, Wiley-VCH Verlag.d) Schematics of assembly process of Ag NPs spatially confined by the cellular texture of Spirulina, and TEM images of the synthesized Ag NPs with controllable size and structure.Reproduced with permission.[119]Copyright 2019, IOP Publishing Ltd.

Figure 6 .
Figure 6.The legend for key technologies and development perspectives of MNPs fabrication based on microorganism.

Figure 11 .
Figure 11.The main strategies of assembly/alignment of functional particles based on microorganisms, and the perspectives in the future research.

Figure 16 .
Figure 16.The main development strategies and addressed challenges of microorganism-based micro/nanorobots.

Figure 17 .
Figure 17.The blueprint of micro/nanofabrication based on microorganism templates.