Bacterial cellulose spheroids as building blocks for 2D and 3D engineered living materials

Engineered living materials (ELMs) based on bacterial cellulose (BC) offer a promising avenue for cheap-to-produce materials that can be programmed with genetically encoded functionalities. Here we explore how ELMs can be fabricated from millimetre-scale balls of cellulose occasionally produced by Acetobacteriacea species, which we call BC spheroids. We define a reproducible protocol to produce BC spheroids and demonstrate their potential for use as building blocks to grow ELMs in 2D and 3D shapes. These BC spheroids can be genetically functionalized and used as the method to make and grow patterned BC-based ELMs to design. We further demonstrate the use of BC spheroids for the repair and regeneration of BC materials, and measure the survival of the BC-producing bacteria in the material over time. This work forwards our understanding of BC spheroid formation and showcases their potential for creating and repairing engineered living materials.


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Engineered living materials (ELMs) are those containing cells on or within the material that play a 22 role in its functionalization or can produce the material itself 1,2 . Bacterial cellulose (BC) is a 23 carbohydrate polymer produced by many bacterial species as a structural element of their biofilm 24 and offers excellent opportunities for developing new ELMs 3 . The BC materials produced by several 25 Acetobacteriacea species are of particular interest as these are quickly and cheaply made as pellicles 26 -a large mass of thick BC -when the cells are grown in static rich media 4,5 . Bacterial cellulose 27 inherently has attractive mechanical properties and crystallinity, has a high water-retention capacity 28 and is ultra-pure compared to plant cellulose 6,7 . These outstanding properties of BC make it an 29 excellent candidate for developing new materials with improved technical and environmental 30 benefits. In the last decade, progress in understanding and producing BC has now led to its use in a 31 broad range of applications, including products used in textiles, cosmetics, healthcare, audio-visual 32 technology and architecture [8][9][10][11] . Most of these applications use sterile, purified BC as a bulk 33 specialised material, however bacterial cellulose has also shown promise as an ELM 12,13 . In one 34 recent example, incorporating Bacillus subtilis cells into BC-based wound dressings helped to 35 prevent wound infections by blocking the growth of several pathogenic bacteria 14 . 36 Two desirable features of ELMs not routinely seen in normal materials are regeneration in response 37 to damage, and modular design with patterned functionalities. Easy and cheap repair of damaged 38 materials (or their automatic regeneration) is an important consideration for the sustainability of all 39 new materials 15 . BC offers excellent opportunities in this regard, because the bacteria trapped in 40 the grown material have the potential to regenerate it by further growth and cellulose production 41 in the future. Just by providing nutrients, water and oxygen, the bacteria in theory can keep growing 42 and seal gaps and tears when they arise, so long as the material has not been sterilised after growth. 43 For patterned functionalities, this can also theoretically be achieved with BC-based materials by 44 growing these from genetically engineered cells programmed 3 . However, another possibility to 45 tackle this problem is to use modular ELM building blocks and pattern these physically to make 46 larger materials. Such a 'building block' approach to novel materials has been taken before in 47 nanotechnology to increase the complexity of materials and to facilitate industrial scaling of 48 complex pieces 16 . Modular BC-based building blocks have not been explored before in an ELM 49 context, but BC and in particular its rapid production from living cells within the material structure, 50 offer an excellent opportunity to tackle this challenge. Pre-cultures of K. rhaeticus were prepared by taking cells from -80°C stocks and growing in 50 ml 82 tubes with 10 ml of Hestrin and Schramm (HS) media (peptone 5 g/L, yeast extract 5 g/L, 2.7 g/L 83 Na2HPO4, 1.5 g/L citric acid, 2% glucose, 2% cellulase from Trichoderma reesei [Sigma-Aldrich]) in 84 shaking conditions at 250 rpm/min and at 30°C for 3 days. 2 x HS media was used for spheroid 85 production tests and was prepared by doubling the concentrations of peptone and yeast extract. 86 To grow pellicles, pre-cultures were centrifuged at 7000 g for 3 min, cells were then resuspended in 87 10 ml of HS without cellulase. This process was then repeated. Brighter 28 (Sigma-Aldrich). After 24 hours, growing cells were identified and a time-lapse was 101 started using the bright field and DAPI channels, and images were taken every 2 minutes for 2 102 hours. 103 104 3D structures and fluorescence 105 Spheroids from day 3 cultures of both wild-type and sfGFP-tagged strains were collected by filtering 106 the culture with filter paper in sterile conditions. The spheroids were seeded in the desired shape 107 with the help of a pipette tip and then incubated for 4 days at 30°C. To create patterns, spheroids 108 were taken one-by-one using sterile pipette tips and placed together in the desired positions. 109 Pellicles repaired with fluorescent spheroids and spheroid patterns made with fluorescently tagged 110 cells were imaged with an Amersham Typhoon Scanner, using 10 µm resolution. A far-blue light gel 111 transilluminator with amber filter was used to image spheroids produced by sfGFP-tagged cells. 112 113 Pellicle repair 114 A 0.5 cm hole puncture tool (Jenley hollow leather punch) was used to create holes in bacterial 115 cellulose pellicles. For repair using spheroids, spheres from day 3 cultures were filtered with filter 116 paper in sterile conditions to separate them from the liquid culture. The spheroids were placed in 117 the puncture holes and 50 µl of HS supplemented with 2% glucose was dropped over the spheroids. 118 2 ml of HS was then added into the surrounding container of the pellicle to maintain it in a hydrated 119 state. The pellicles were incubated in static conditions for 10 days at 30°C before imaging. In 120 unsuccessful attempts of pellicle repair the following were used i) fragments of biofilm found 121 adhered to the wall of a flask after 4 days in shaking conditions; ii) floating clumps formed in shaking 122 conditions from a initial culture set with high cell density (OD600 ~0.5); iii) cellulose aggregates 123 present in the culture medium under the pellicle; iv) a pellet of cells grown in shaking conditions 124 with cellulase, centrifuged, washed with HS and centrifuged again; v) cells from (iv) embedded in a 125 0.3% HS agar matrix at 40°C and immediately placed in the pellicle before it solidifies; vi) a cellulose 126 patch of slightly bigger dimensions than the hole produced in the pellicle, used to force the edges 127 of the patch and the hole to be in close contact. 128 129

Cell survival in pellicles 130
A homogeneous suspension to grow pellicles was prepared as described above. Pellicles were grown 131 in 96 squared deep well plates at 30°C. After 7 days, pellicles were stored in vacuum sealed plastic 132 bags at 4°C and 23°C and also in 2 ml tubes at 23°C. Samples in triplicate were collected at each time 133 point to assess survival. Pellicles were placed in 2 ml tubes, suspended in HS diluted 1 in 10 with 5% 134 cellulase and incubated at 37°C for 3 h in shaking conditions to degrade the cellulose. Serial dilutions 135 of each suspension were made, and these dilutions plated in four replicates on HS agar 136 supplemented with 2% of glucose. After 7 days of incubation at 30°C, colonies were counted from 137 the agar plates and colony forming units (CFUs) per cm 2 pellicle area was calculated. 138 139

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Bacterial cellulose spheroids 141 Bacterial cellulose (BC) spheroids have been reported in several previous works 20,22,23 , but how they 142 form and why has not been fully elucidated. It has been hypothesized that spheroid formation is 143 produced by the adhesion of bacteria to air bubbles produced in shaking media, with cellulose then 144 grown at the bubble air-liquid interphase to form a spheroid shape 24 . Past experiments in our lab 145 growing K. rhaeticus in shaking conditions occasionally produced spheroids. As with other BC-146 producing bacteria, we originally assumed that spheroid formation by K. rhaeticus occurred 147 randomly, either in response to stochastic processes during shaking growth or due to mutation or 148 another form of uncontrolled variation in cell behaviour that triggers their spontaneous production. 149 Here we set out to examine whether spheroid production by K. rhaeticus was indeed a random 150 event, or one that could be reproducibly triggered. To do this, we grew K. rhaeticus cells with shaking 151 at 30°C and tried combinations of more than 20 different growth variable (Supplementary Table 1). 152 During and after growth, we visually assessed the cultures for the presence of BC spheroids and 153 used this information to determine the key factors involved in spheroid formation and which 154 combination of growth variables leads to reproducible growth of BC spheroids. 155 Our results indicated that the main determining factor for BC spheroid formation is the initial optical 156 density of the culture, with more ideal spheroids seen when cultures begin at low optical densities 157 (OD600 = 0.001-0.0001) where bacteria are more likely to begin isolated from one another 158 (Supplementary Figure 1A). The second most important factor seen in our experiments was the 159 culture container. BC spheroids were only ever seen forming after shaking growth in 14 ml and 15 160 ml plastic culture tubes, and never grew in any of our attempts with 50 ml tubes or with larger With these three factors determined, it became possible for us to produce a protocol for reliable 176 spheroids production ( Figure 1A) in 14 ml culture tubes, yielding spheroids typically 0.2 to 1 mm in 177 diameter ( Figure 1B). We also tested if our protocol for spheroids cultivation would also work for 178 genetically modified strains of K. rhaeticus containing plasmids expressing transgenes. We observed 179 reliable growth of green fluorescent spheroids from K. rhaeticus expressing sfGFP and red 180 fluorescent protein (RFP) expressed from a plasmid ( Figure 1C). 181 In order to observe how BC is produced by our bacteria, we performed microfluidic time-lapse 182 culture experiments with calcofluor added to stain nascent cellulose production (Supplementary 183 Video 1). We observed band-like growth of BC chains coming from one side of the bacteria 184 longitudinal axis, producing branches of cellulose as the cells divide ( Figure 1C). In static culture, this 185 perturbed by shaking, the branches collapse on themselves, entangling the BC bands while the 187 chains continue growing and cells dividing. Although conditions in the microfluidic chamber do not 188 match those used for spheroid production in our protocol, we reason that the same processes lead 189 to the formation of spheroids when cultures are seeded at very low density ( Figure 1D). 190

Construction in 2D and 3D with BC spheroids 192
Given their mechanism of growth, we reasoned that spheroids would continue cellulose production 193 at their surfaces and thus when two spheroids interact for enough time they will grow together and 194 fuse. This property of our spheroids would allow them to act as millimetre-scale BC-based building 195 blocks that could then be used to produce 2D and 3D shapes (Figure 2A). To demonstrate this 196 application, we designed a simple 3D shape (a podium) and a more complex 3D shape (a serrated 197 ring). We then manually placed spheroids in these arrangements on sterile cotton pads, with the 198 help of a pipette tip. After 4 days of incubation at 30°C, the spheroids had visibly grown and fused 199 together to create a continuous BC-based shape roughly matching the seeded design ( Figure 2B). The use of cellulose spheroids as building blocks opens a new opportunity to create ELMs with 2D 210 and 3D patterns and multifunctional properties. As K. rhaeticus is a non-motile bacteria, the patterns 211 created from spheroid seeding will not get blurred and should remain conserved. To demonstrate, 212 we designed and created layer of BC seeded onto filter paper with both normal BC spheroids and 213 close to each other, setting a pattern to make diagonal lines of non-fluorescent spheroids within the 215 pattern. After 10 days of incubation at 30°C, we obtained a fused pellicle conserving the fluorescent 216 pattern of seeding, demonstrating the possibility of easily creating ELMs functionalized at the 217 millimetre scale -the diameter of a single spheroid ( Figure 2C). 218 We observed in these experiments that growth after seeding also led to the spheroid structure being 219 adhered to the sterile filter paper support. As paper is itself predominantly cellulose, we wondered 220 how the spheroids would behave if they were set to grow on a layer of bacterial cellulose. To 221 examine this, we seeded fluorescent spheroids on a normal K. rhaeticus pellicle produced from static 222 growth. After 4 days of incubation at 30°C, the spheroids were completely fused to the base pellicle, 223 revealing that a BC layer provides an excellent frame within which to set spheroids in patterns. 224 Fluorescence was localized exactly in the area of seeding ( Figure 2D). 225 226

Bacterial cellulose regeneration by BC spheroids 227
Given the strong interest in using BC as a basis for ELMs, there is a need to identify methods to 228 repair or regenerate a BC-based material when and if they are damaged. The efficient fusing of 229 spheroids into a pellicle as seen in pattern formation experiments ( Figure 2D) suggests that 230 spheroids could provide a method for pellicle damage repair ( Figure 3A). To investigate this, we 231 established a repair assay of BC pellicles BC using a hole punch to damage the material. We first 232 assessed whether just the addition of HS media and further incubation for 7 days in static aerated 233 conditions would result in regrowth of BC in the hole wound of a punctured fresh BC pellicle. 234 Although a thin BC layer did grow over the hole, the new thin pellicle layer was poorly adhered to 235 the original one below it (Supplementary Figure 2A). We considered that the poor adherence was a 236 result of adding too much liquid growth media, inducing the formation of a new BC layer only at the 237 air liquid interface and not well-adhered to the original pellicle. Thus, we decided to just add a few 238 drops of new HS media in the holes with and without also adding the circles of cellulose produced 239 by the hole puncture (Supplementary Figure 2B, 'Controls'). After 7 days incubation, this still failed 240 to show stable wound repair. 241 This failure to repair may be explained by the fact that BC pellicles grow anisotropically; producing 242 new cellulose predominantly in the horizontal axis at the top layer, building the pellicle from the 243 bottom up by stacking cellulose layers one over the other. In contrast, spheroids grow BC 244 isotropically, producing it in every radial direction ( Figure 3A). We have observed that the incubation 245 of two stacked pellicles does not produce regrowth and their fusion, so next we looked to see how 246 spheroids can behave as a repair material. After placing freshly-grown spheroids into the puncture 247 hole at high density ( Figure 3B) and incubating for 6 days to allow for cellulose growth, we saw 248 excellent repair that was not only stable but also restored the consistency and appearance of the 249 top layer of the material ( Figure 3C). We also compared this repair method to many other options 250 for repair where growing cellulose-producing bacteria or fragments of pellicles in different forms 251 are placed into the wound holes and allowed to grow (Methods and Supplementary Figure 2B). 252 Repair quality was assessed by holding the pellicle edges with tweezers and pulling. Only the pellicle 253 restored with cellulose spheroids maintained the continuity of the pellicle with high enough quality 254 to be stable upon further manipulation ( Figure 3C). We speculate that this is due the growth axis of 255 the materials used for restoration. 256 Notably, the BC layer repaired with spheroids did not look different to an unpunctured pellicle once 257 the repaired pellicle was lifted. This suggests that there is no change in the diffraction angle of the 258 light between the original BC and newly synthetized cellulose from the spheroids, perhaps due to 259 the spheroids producing similar cellulose that infiltrates and networks with the existing BC material 260 ( Figure 3C). To further investigate how spheroids interlink with a BC pellicle, we performed a further 261 repair assay experiment, but now refilling the wound holes with fluorescent spheroids produced by 262 GFP-tagged bacteria. After 10 days, the holes were repaired, and fluorescence could be observed 263 within the hole seeded with spheroids and also at a weaker level in the surrounding material of the 264 pellicle ( Figure 3D). This reveals that bacteria spread into the local region of the damaged cellulose 265 sheet into which they are placed. 266 to understand whether BC spheroids could be made in bulk in advance, stored and then used for 280 construction or repair when needed. Assessing cell survival within the spheroids proved difficult due 281 to the small size of spheroids and irregular shapes giving variable volumes and thus cell counts. 282 Therefore, we instead used small BC pellicles grown in 96 well microplates as an equivalent material. 283 After growth of this BC material, the small pellicle samples were immediately stored in either 284 vacuum-sealed bags (stored at 4°C and 23°C) or in 2 ml tubes at 23°C. Then over a period of time, 285 the samples were removed from storage, digested with cellulase and grown on solid agar plates in 286 order to determine cell number per sample by calculating colony forming units. This revealed that 287 the number of viable cells in the material decreased rapidly in the first 3 weeks for all three storage 288 methods tested ( Figure 3E) and that a by having a consistent low number of cells to begin the culture is critical. It may be the 300 case that all BC-producing bacteria can produce BC spheroids if seeded at the correct density. We 301 note that the protocol of the aforementioned work did not measure the cell density of the bacteria 302 inoculum before beginning culturing for BC spheroid production. 303 While previous work has hypothesised that attachment and growth around air bubbles triggers 304 spheroid production 25 , our data from microfluidic time-lapse imaging offer a new insight, showing 305 that there can be an entangling process of cells and cellulose chains during the early stages of culture 306 growth. As uncovered here, BC spheroid formation from K. rhaeticus iGEM strongly depends on the 307 bacteria concentration used to seed the culture, with low culture density being required. When the 308 shaking culture starts at high density, we always obtain amorphous aggregates of cellulose that 309 range from a very fibrous mass to single large piece of rounded cellulose. This tallies with previous 310 work with G. xylinus JCM 9730 that showed that differences in the number and size of spheroids 311 depended on the volume used to inoculate the culture 26 . The volume and shape of the container 312 also affected spheroid formation, likely due to how the motion of the growth media is affected and 313 how this changes the entangling process and air bubble formation. 314 Our work also demonstrated the potential for BC spheroids to be used as building blocks to create 315 2D and 3D shapes and create patterns that could find use as functionalized ELMs. The building block 316 approach opens a myriad of applications, especially when considering the possibility of using BC 317 spheroids grown from genetically reprogrammed cells that perform other tasks. Through synthetic 318 biology, bacteria can be made to sense a wide variety of biological, chemical and physical inputs and 319 also to signal to one another in ways analogous to electric circuitry 27-29 . Different functionalized 320 spheroids could be used to create 2D and 3D patterns that compute environmental information, or 321 even patterned materials that display anchor proteins to attract mammalian cells or cell 322 differentiation signals. Such a material could for example be used to seed and grow complex 323 mammalian tissues like skin or cartilage in defined patterns and layers. 324 BC spheroids also offered the best solution to BC material repair in the research shown here. 325 Regeneration of a damaged BC pellicle and restoration of its continuity was seen in only a few days 326 of growth with seeded spheroids. Our best results were obtained with thin pellicles. For thicker 327 pellicles, we observed that restoration was superficial, and we speculate that this is due to the poor 328 penetration of oxygen to the spheroids in deeper layers. To solve this problem, we now perform a 329 double incubation, turning over the pellicle after a few days to also let the material to heal on the 330 other surface. BC spheroids function as a successful vector to seed bacteria for BC repair and our 331 data suggest they may be able to be stored for several months prior to use, although likely with 332 reduced repair capacity over time. 333 For creating small 3D shapes, BC spheroids building blocks are limited by their millimetre size and 334 the low precision of fabrication by hand. For making smaller or more precise BC-based ELM shapes 335 it may be more convenient the use 3D printing methods developed for bacterial cultures, such as 336 the FLINK method where a non-living gel matrix that harbours the bacteria is printed into the desired 337 shape and the cells then grow and produce within this 19 . 3D printing allows the production of 338 functionalized ELMs using different bacteria, but its accuracy is limited by the width of the extrusion 339 printing head, which itself is limited by the high viscosity of the printed gel. A possible solution is a 340 hybrid approach where a 3D printer is programmed to precisely dispense BC spheroids, and the size 341 of these spheroids is reduced by harvesting them a day earlier than in the protocol given here. 342