Engineered living building materials (LBMs) use biology to confer multiple functionalities to materials for the built environment. Microorganisms can be leveraged for multiple purposes in the design of LBMs, including increasing the rate of manufacturing, imparting mechanical benefit, and sustaining biological function. In this work, we used photosynthetic microorganisms to biomineralize inert sand-gelatin scaffolds to create LBMs. These materials are capable of exponential regeneration of the living component in response to physical switches. Thus, from one starting generation of material, multiple regenerations are produced on demand. In this study, microorganism-precipitated calcium carbonate conferred high fracture toughness to the LBMs. More broadly, LBMs represent a platform technology whereby biology can be leveraged to potentially deliver multiple functionalities to infrastructure materials by design.

Living building materials (LBMs) were engineered that are capable of both biological and structural functions. LBMs were created by inoculating an inert structural sand-hydrogel scaffold with Synechococcus sp. PCC 7002, a photosynthetic cyanobacterium. The scaffold provided structural support for Synechococcus, which toughened the hydrogel matrix via calcium carbonate biomineralization. Temperature and humidity switches were utilized to regulate the metabolic activity of the microorganisms and achieve three successive regenerations of viable LBMs from one parent generation. Microbial viability in LBMs maintained in at least 50% relative humidity for 30 days was 9%–14%, which far exceeded literature values of microorganisms encapsulated in cementitious materials for similar timeframes (0.1%–0.4%). While structural function was maximized at ultradesiccated conditions, prolonged dehydration compromised microbial viability. Despite this tradeoff in biological-structural function, LBMs represent a platform technology that leverages biology to impart novel sensing, responsive, and regenerative multifunctionality to structural materials for the built environment.

In this work, we engineered LBMs capable of successive regeneration in response to environmental switches. LBMs were created with a sand-hydrogel structural scaffold inoculated with Synechococcus sp. PCC 7002 (Synechococcus)—a robust photosynthetic cyanobacterium capable of MICP ( Figure 2 ). First, we inoculated sand with dissolved gelatin, media, and Synechococcus. Gelatin was chosen because its melting point (37°C) is compatible with bacterial viability and also because gelatin scaffolds gain strength through physical crosslinking during dehydration.The LBM was then cooled to form a three-dimensional hydrogel networkreinforced with biogenic CaCO. Synechococcus utilizes the enzyme Rubisco to convert COto sugars during photosynthesis. In low-COmedia, Ocompetitively binds to the Rubisco active site and diminishes COcarboxylation efficiency. Synechococcus surmounts this limitation by concentrating HCOfrom media to COwithin the cell and exporting OHoutside of the cell,thereby increasing local pH and promoting CaCOprecipitation. Cyanobacteria are a diverse phylum of microorganisms well known to survive extreme environmental conditions, including high and low temperature, salinity, and humidity.These characteristics make Synechococcus particularly suitable for LBMs, since in-service environmental conditions of building materials (i.e., fluctuating moisture and temperature) can impart physical stress to microorganisms. Using Synechococcus, LBMs were generated and successively regenerated from one parent inoculum using temperature and humidity switches. The mechanical properties, mineral characteristics, cell viability, and regeneration ability of LBMs are reported here.

If long-term viability were improved, microorganisms could be utilized to create or “grow” living building materials (LBMs) with structural and sustained biological functions. LBMs necessitate two principal components: (1) an inert scaffold that provides structural support for (2) a living component that, together with the structural scaffold, endows the LBM with structural and biological function. The living component must be robust to a range of environmental conditions and respond to physical switches (e.g., temperature, pH, light, moisture, pressure) with changes in metabolic activity. Microorganisms capable of MICP, for example, could be used to grow load-bearing building materials with self-sustaining functions.Control over microbial metabolism through environmental switches would enable on-demand growth, biomineralization, dormancy, and subsequent regeneration of LBMs. These environmental switches could enable regeneration of LBMs from one parent inoculum, which would enable new possibilities for infrastructure material manufacturing, use, and post-use remanufacturing ( Figure 1 ).

(1) LBMs are created by mixing Synechococcus sp. PCC 7002 cells with calcium-containing nutritional media, gelatin, and sand. (2) LBMs can be exponentially regenerated from an original LBM through use of temperature and humidity switches. (3) LBMs gain structural integrity through desiccation. After service as a load-bearing structural material, LBMs could be deconstructed and recycled as an aggregate source for new LBMs.

Today, microbially induced calcium carbonate (CaCO) precipitation (MICP) is utilized for soil stabilization,in situ concrete crack repair,fracture sealing of oil and gas wells,bioremediation of metals,and mitigating leakage from geologically sequestered carbon dioxide (CO).During MICP, the metabolic activity of microorganisms increases the saturation state local to the bacterial cell and promotes CaCOprecipitation.While ureolytic microorganisms have been the focus of most MICP applications,several alternative metabolic pathways also achieve CaCOprecipitation, such as carbonic anhydraseand the carbon-concentrating mechanism of cyanobacteria.Reparative (i.e., self-healing) applications of MICP require prolonged microorganism viability. However, ureolytic bacteria have only limited viability in the harsh, high-pH environment of cementitious materials.While viability is improved somewhat by utilizing spore-forming bacteria strains or by encapsulating microorganisms in a protective bead or matrix,long-term survival of the initial inoculum is still limited in these enhanced systems.

Jonkers, H.M., and Schlangen, E. (2007). Self-healing of cracked concrete: a bacterial approach Proceedings of the 6th International Conference on Fracture Mechanics of Concrete and Concrete Structures. 3, 1821–1826.

Compressive strength, assessed for mortar cubes, was not influenced by gelatin batch but was significantly affected by specimen type ( Table 1 ). Specifically, the abiotic high-pH control had significantly lower compressive strength than either the abiotic control (−29.3%, p < 0.05) or the LBMs (−28.4%, p < 0.05). Notably, both the LBMs and abiotic controls had strength similar to the minimum acceptable strength for ordinary Portland cement-based mortars (∼3.5 MPa).

Post hoc testing revealed that fracture energy was significantly higher for the LBMs than for the abiotic controls (+15.6%, p < 0.05) and abiotic high-pH controls (+17.0%, p < 0.05) ( Figure 7 ). From ANOVA, specimen type (i.e., LBM, abiotic control, abiotic high-pH control) and gelatin batch both had significant main effects on fracture energy. There was no interaction between these factors.

As observed in nature, biomineral deposits within polymer matrices can yield composites with high toughness.Thus, the fracture energy of LBMs was assessed and the results compared with the fracture energies of the abiotic and abiotic high-pH controls cured at room temperature. Rectangular prism LBMs and controls were desiccated to equilibrium mass at ambient temperature before testing ( Figure S4 ). While these desiccated samples do not retain microorganism viability, dehydration best enabled direct assessment of the mechanical benefit imparted by microbial biomineralization.

SEM-EDS revealed that the regenerated LBMs were able to biomineralize gelatin, similar to the parent generation. SEM-EDS confirmed the presence of CaCOin the parent, first, second, and third generations ( Figure 6 ). The abundance of CaCOappeared to increase over subsequent generations.

Mineralization was assessed with SEM-EDS for all viable generations of LBMs at each RH. CaCOmineralization qualitatively increased in abundance with each subsequent generation. All images are shown at 1,500×. Representative SEM-EDS spectra are provided in Supplemental Information

From X-ray diffraction (XRD), LBMs and the abiotic control each produced a mixture of calcite and gypsum minerals, which were detected along with halite introduced by the media. By contrast, some calcite and halite (but not gypsum) were precipitated in the abiotic high-pH control ( Figure S2 ). From scanning electron microscopy (SEM), larger minerals were precipitated by the abiotic control compared with the LBMs and high-pH control ( Figure 5 ). Energy-dispersive spectroscopy (SEM-EDS) showed that most of the sampled minerals in the abiotic controls were calcium sulfate (likely gypsum) ( Figure S3 ). Most of the minerals sampled by SEM-EDS for the abiotic high-pH control as well as the LBMs were CaCO

(D) The abiotic high-pH (pH 10) control forms CaCO, although these precipitates are smaller than when bacteria are present. Representative SEM-EDS spectra are provided in Supplemental Information

(A) For LBMs as well as controls, gelatin bridges sand particles and provides a substrate for mineralization.

Because CaCO 3 can spontaneously precipitate, we sought to understand whether the composition and morphology of mineral formation was influenced by the presence of the gelatin matrix and cyanobacteria. Two controls were used to assess whether mineral precipitated in the presence of cyanobacteria differed from mineralization spontaneously occurring in the absence of biotic influence. The “abiotic control” was created with the same medium as the cyanobacterial LBMs, but without cells. As with the initial LBM, the abiotic control was set to pH 7.6. The “abiotic high-pH control” was an abiotic control set to pH 10 to incite maximum precipitation of CaCO 3 . Mineral deposited from cyanobacteria and controls were compared for samples prepared without and with gelatin.

Next, we evaluated whether parent LBMs could be utilized to regenerate successive generations of viable LBMs. For the parent generation, viability was measured at 0 days and 7 days. For each generation (first, second, third), new abiotic media (i.e., sand and calcium-containing nutritional media) were added to form two new LBMs from one parent LBM. Thus, in three generations separated by 7 days each, eight specimens were formed from one initial parent LBM. Within each generation, viability decreased from day 0 to day 7. However, the addition of new medium at each regeneration and low-temperature storage sustained LBM viability through three generations for 50% RH and 100% RH ( Figure 4 ). As expected, greater viability was maintained for 100% RH compared with 50% RH. Viability measured at day 7 of the third generation was 20% and 40% of the viability of the initial inoculum (i.e., parent generation, day 0) for 50% and 100% RH, respectively.

To form the next generation of LBMs, the previous generation was subjected to a high-temperature switch and refreshed with abiotic medium (high-humidity switch). The new LBM was then gelled (low-temperature switch). Viability for each generation was measured at 0 days and again at 7 days of storage at 4°C. Greater RH (50%–100%) allows at least three viable regeneration events. Data indicate the mean and 95% confidence intervals.

We first assessed whether cyanobacteria would maintain long-term viability in the parent LBMs at ambient temperatures (20°C), refrigerated temperatures (4°C), or both ( Figure 3 ). At ambient temperature and relative humidity (RH), samples were viable at the time of demolding (9.5 h) but were not viable at 7 days. Because Synechococcus sp. PCC 7002 was expected to have slower metabolism at lower temperatures, viability was again assessed for refrigerated samples. When maintained at either 50% or 100% RH, ∼69% of the initial inoculum survived up to 14 days. When assessed at 30 days, ∼9% and 14% of the initial inoculum survived in the LBM stored at 50% RH and 100% RH, respectively. As expected, LBMs stored at 4°C with ambient RH (24%) exhibited reduced viability compared with those stored at higher humidity. At this lower humidity, ∼ 37% of the initial inoculum survived at 14 days while viability was not detectable at 30 days. This loss of viability closely aligned with the time to reach equilibrium mass via controlled dehydration ( Figure S1 ).

Discussion

We engineered LBMs with the capacity for regeneration and biomineralization ( Figure 1 ). Specifically, at least three successive generations of LBMs with viable Synechococcus were regenerated from one parent generation ( Figure 4 ). The regenerative ability of LBMs demonstrates a potential for exponential “growth” in material manufacturing. For each subsequent generation, one LBM from the previous generation was supplemented with new abiotic medium and sand to form two new LBMs. Thus, in three generations, one LBM formed eight new specimens from one parent microbial inoculum.

28 Pacheco-Torgal F.

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Schlangen E. Application of bacteria as self-healing agent for the development of sustainable concrete. , 18 Jonkers, H.M., and Schlangen, E. (2007). Self-healing of cracked concrete: a bacterial approach Proceedings of the 6th International Conference on Fracture Mechanics of Concrete and Concrete Structures. 3, 1821–1826. The viability of Synechococcus in the sand-hydrogel composite was, in general, much higher than other reported biomineralizing microorganisms in cementitious materials. Greater viability in our system may be attributed to the lack of harsh conditions that exist inside cement paste, including ultrahigh pH (>12), high ionic strength, elevated temperatures that occur during exothermic cement hydration, and nutrient depletion.Achal et al.reported that only 0.1% of vegetative (i.e., metabolically active) Bacillus megaterium cells remained viable in an aged cementitious mortar; similarly, Bundur et al.reported that 0.4% of vegetative Sporosarcina pasteurii cells remained viable in a cement paste at 28 days. Jonkers and coworkersexamined the viability of endospores (i.e., metabolically inactive) in bacterial mortars and reported that only 2% of the initial inoculum were detected in a mortar mixed with either Bacillus cohnii or Bacillus halodurans at 10 days, while 7% of the initial inoculum was detected in a mortar mixed with Bacillus pseudofirmus at 10 days.

The viability and regenerative potential of Synechococcus were enabled by the use of temperature and humidity (i.e., rehydration) switches. The first high-temperature switch corresponded to the incubation and growth temperature (37°C), which was sufficient to dissolve the gelatin matrix and encourage bacterial metabolic activity and mineral precipitation. The low-temperature switch corresponded to the storage temperature (4°C). At this temperature, the gelatin matrix effectively encapsulated the cyanobacteria and medium to form a solid LBM. The cyanobacteria remained viable at the storage temperature as long as humidity was sufficient to prevent excessive cell desiccation (50%–100% RH). Importantly, 50% RH was similarly effective in maintaining viability as 100% RH. This finding is of significance because many climates worldwide have at least 50% RH. Thus, during LBM regeneration, the addition of new liquid abiotic media and higher temperatures were the high-humidity and high-temperature switches that rekindled metabolic activity.

4 Wang J.Y.

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De Belie N. Self-healing concrete by use of microencapsulated bacterial spores. , 32 Zhang Z.

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Qian S. Influence of bacterial incorporation on mechanical properties of engineered cementitious composites (ECC). From an engineering perspective, the responsiveness of LBMs to these temperature and humidity switches is opportune. Material systems that protect ureolytic microorganisms with encapsulating gels or other solid media generally require physical damage to the encapsulant in order to trigger additional biomineralization.While this approach may be appropriate for in situ crack sealing, it is challenging to elicit a uniform metabolic response from embedded microorganisms. By contrast, environmental switches, such as those employed herein, can be applied uniformly to precisely control microbial activity.

CaCO 3 biomineralization increased with each regeneration event. Because biomineralization from Synechococcus is a consequence of metabolism, viable microorganisms were expected to precipitate additional CaCO 3 with each regeneration event. While SEM revealed a qualitative increase in mineralization with each regeneration, it is not known whether mineralization efficiency changed with regeneration. Likewise, the effect of additional biomineral content on LBM mechanics is not known and is a limitation of the present work. These questions would benefit from further study.

33 Wittmann F.H. Crack formation and fracture energy of normal and high strength concrete. 32 Zhang Z.

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Qian S. Influence of bacterial incorporation on mechanical properties of engineered cementitious composites (ECC). 3 precipitation compared with a wild-type control, suggesting that bacteria themselves increase crack-tip resistance. Alternatively, CaCO 3 precipitated in the presence of cyanobacteria may also have distinct mechanical behavior compared with abiotic calcite. CaCO 3 minerals from brachiopods, for example, exhibit high nanoindentation hardness compared with abiotic CaCO 3 , likely due to nanoscale polysaccharide inclusions. 24 Merkel C.

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et al. Tuning hardness in calcite by incorporation of amino acids. 3 can nucleate directly on this membrane, and biological macromolecules (e.g., polysaccharides, lipids, proteins) can incorporate into the crystal, 36 Obst M.

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Hitchcock A.P. Soft X-ray spectro-tomography study of cyanobacterial biomineral nucleation. The increased fracture energy of LBMs compared with controls likely relates to the characteristics of the biogenic mineral. LBMs, as well as both control materials, experienced a profound toughening effect from the mineralized gelatin matrix compared with cementitious mortars with similar aggregate size.Because the abiotic and abiotic high-pH controls had similar fracture energy despite precipitating predominantly gypsum and calcite, respectively, the specific phase of precipitated mineral may be less influential to material toughness than other factors. In particular, cyanobacteria themselves or their mineral precipitates may increase composite toughness. Zhang and coworkers reported that the inclusion of ureolytic bacteria conferred a toughness benefit to fiber-reinforced cement.Fracture toughness was similar for an engineered strain with higher urease activity and CaCOprecipitation compared with a wild-type control, suggesting that bacteria themselves increase crack-tip resistance. Alternatively, CaCOprecipitated in the presence of cyanobacteria may also have distinct mechanical behavior compared with abiotic calcite. CaCOminerals from brachiopods, for example, exhibit high nanoindentation hardness compared with abiotic CaCO, likely due to nanoscale polysaccharide inclusions.Calcite precipitated by ureolytic microorganisms also has high nanoindentation hardness,as does nacre.The cell membrane of Synechococcus, as with many other microorganisms, is substantially composed of polysaccharides and amino acids. CaCOcan nucleate directly on this membrane, and biological macromolecules (e.g., polysaccharides, lipids, proteins) can incorporate into the crystal,perhaps conferring a toughening effect. Although outside of the scope of the present work, identifying specific toughening mechanisms is an important topic for further investigation.

27 Rilem L.C. Functional classification of lightweight concrete. 3 crystals. The strength and toughness of LBMs was assessed at 7 days but are not expected to change with greater curing time. While cementitious materials gain strength with hydration and it is commonplace to assess mechanical properties at 28 days, LBMs instead dehydrate to impart strength to the gelatin scaffold. Because equilibrium mass for LBMs is obtained at ambient conditions in approximately 4 days, further changes to strength and fracture energy with curing time beyond 7 days are not anticipated. Nonetheless, if comparisons are made between the mechanical properties of LBMs and cementitious materials, both types of materials should be sufficiently cured. Compressive strength of the LBMs and control specimens in this study were similar to the minimum compressive strengths of cementitious mortars.As expected, compressive strength was not affected by cyanobacteria-mediated mineralization. Since compressive strength is predominantly influenced by the strength, size, and distribution of aggregate, strength would be minimally affected by the much smaller bacterial-precipitated CaCOcrystals. The strength and toughness of LBMs was assessed at 7 days but are not expected to change with greater curing time. While cementitious materials gain strength with hydration and it is commonplace to assess mechanical properties at 28 days, LBMs instead dehydrate to impart strength to the gelatin scaffold. Because equilibrium mass for LBMs is obtained at ambient conditions in approximately 4 days, further changes to strength and fracture energy with curing time beyond 7 days are not anticipated. Nonetheless, if comparisons are made between the mechanical properties of LBMs and cementitious materials, both types of materials should be sufficiently cured.

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Cohen Y. Accumulation of trehalose and sucrose in cyanobacteria exposed to matric water stress. , 38 Wingler A. The function of trehalose biosynthesis in plants. , 39 Hagemann M. Molecular biology of cyanobacterial salt acclimation. Our results illustrate a tradeoff between biological viability and mechanical performance for this class of LBMs. Given that gelatin gains strength with dehydration,peak mechanical performance of LBMs is obtained at maximum dehydration. By contrast, viability of cyanobacteria requires sufficient humidity, and minimum mechanical performance would be obtained at maximum viability conditions (i.e., 100% RH and 4°C). While viability was compromised for LBMs that were desiccated enough for peak mechanical performance, nonviable desiccated structures could be recycled as the abiotic component for new structures made from LBMs. The tradeoff between viability and mechanical performance could be mitigated by exploring molecular additives or other strategies to improve extreme desiccation tolerance of microorganisms (e.g., trehalose).

Engineered LBMs with a capacity to regenerate in response to controllable environmental switches represent a new frontier for exponential material manufacturing and end-of-life reuse. The high fracture energy of LBMs suggests that these materials may be particularly well suited for applications in which resistance to crack propagation is valued. Although this technology is in its nascence, potential applications of LBMs range from temporary civil and military structures to paving, façades, and other light-duty load-bearing materials. LBMs are not intended to broadly replace cementitious materials, but instead represent a new class of materials in which structural function is complemented by biological functionalities.

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Stankovic A.R. Biota as toxic metal indicators. Optimizing the biological and structural characteristics of LBMs (e.g., temperature, humidity, aggregate gradation, hydrogel chemistry, and inclusion of biological molecules) may extend the utility of LBMs to a myriad of advanced applications. More broadly, other microorganisms and physical switches could be deployed within the LBM framework for the design and fabrication of multifunctional building materials capable of sensing, actuation, and chemical response. For example, microorganisms could potentially sense—and respond to—toxic chemicals or reveal structural damage with fluorescence.