Microbial induced calcite precipitation can consolidate martian and lunar regolith simulants

We demonstrate that Microbial Induced Calcite Precipitation (MICP) can be utilized for creation of consolidates of Martian Simulant Soil (MSS) and Lunar Simulant Soil (LSS) in the form of a ‘brick’. A urease producer bacterium, Sporosarcina pasteurii, was used to induce the MICP process for the both simulant soils. An admixture of guar gum as an organic polymer and NiCl2, as bio- catalyst to enhance urease activity, was introduced to increase the compressive strength of the biologically grown bricks. A casting method was utilized for a slurry consisting of the appropriate simulant soil and microbe; the slurry over a few days consolidated in the form of a ‘brick’ of the desired shape. In case of MSS, maximum strength of 3.3 MPa was obtained with 10mM NiCl2 and 1% guar gum supplementation whereas in case of LSS maximum strength of 5.65 Mpa was obtained with 1% guar gum supplementation and 10mM NiCl2. MICP mediated consolidation of the simulant soil was confirmed with field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD) and thermogravimetry (TG). Our work demonstrates a biological approach with an explicit casting method towards manufacturing of consolidated structures using extra-terrestrial regolith simulant; this is a promising route for in situ development of structural elements on the extra-terrestrial habitats.

the submission is accepted. Please make sure it is accurate. The authors have declared that no competing interests exist. NO   the European Space Agency (ESA) announced plans to restart manned missions for the exploration of outer space [2][3][4][5][6]. For feasible and sustainable space exploration, these habitats need to be built from available in situ resources on moon/Mars [7][8][9][10][11]. Within space lexicon, the term in situ resource utilization (ISRU) refers to any process that encourages processing of local resources found during exploration of extra terrestrial habitats in order to reduce dependency on materials chaperoned from Earth. Both martian and lunar surfaces have an abundance of fine soil on their surfaces, termed regolith, which can be utilized as building or raw construction material [1,3]. The primary challenge in this endeavour lies in the consolidation of the unbonded fine regolith particles into a structure of substantial mechanical integrity.
Towards this end, various methodologies for consolidating regolith have been proposed. Fusing of regolith particles though laser-sintering [11] use of microwave irradiation for bonding [12,13], production of cast basalt [9,[14][15][16], fabrication with lunar glass [17,18], creation of sulfur-based concrete [9,19], dry-mix/steam injection methods and various 3D printing methodologies [20][21][22] are examples of processes that have been proposed. These methods have their own advantages and disadvantages. For instance, laser based sintering of regolith has drawbacks such as high porosity, thermal cracking, and difficulty in casting long blocks. [13,23,24]. Given the importance and burgeoning interest in extra-terrestrial human settlements, it is necessary that other methods of creating structures from lunar/martian regolith be probed. As a step in this direction, we discuss here a casting method for fabrication of bricklike structures using regolith simulants and a biomineralization process called microbial induced calcite precipitation (MICP).
MICP is a biomineralization process that produces calcium carbonate by exploiting the metabolic activity of bacteria [25][26][27][28][29] via various pathways [30]. The urease pathway is widely explored [31][32][33][34] wherein the conditions for mineral precipitation are made favourable by controlled reactions involving hydrolysis of urea by ureolytic bacteria. Urease (E.C. 3.5.1.5), which is a nickel dependent and non-redox enzyme is primarily responsible for urea hydrolysis [35,36]. One possible route for enhanced activation of urease activity is by changing the concentration of Ni (II) ions. Crystal structures of microbial urease enyme studied from Klebsiella aerogenes [37] and Bacillus pasteurii [38] organisms suggest that it has divalent nickel ion at the centre, which aids in binding the substrate (urea), alleviates the catalytic transition state thus accelerating the ureolysis reaction rate [39].
In this work we explore this property to enhance the use of MICP based consolidation with martian and lunar regolith. Our study shows a significant increase in the compressive strength of the resulting consolidated brick-like structures with both martian and lunar regolith simulants. The present work also utilizes a naturally occurring and economically viable biopolymer guar gum, to further improve the strength of these bio-consolidated 'space bricks.' Guar gum acts as a stabilizer [26,40] with soil for improving mechanical strength, and is also stable with pH/temperature variation [41,42] thus making it an ideal additive for MICP mediated space brick formation. For our work, we used the microbe Sporosarcina pasteuriia much explored MICP capable bacteria and a gram positive non-pathogenic strain [29,[43][44][45].
We have also designed a modular self-contained lab-on-a-chip (LoC) device for the real time monitering of the MICP process and demonstrated the survival of this organism under miniaturized experimental environment [46]. We believe that such studies will help evaluate the possibilities of MICP as a sustained solution for building extra-terrestrial settlements utilizing in-situ resources.

Microorganisms and culture conditions
Bacterial-induced bio-consolidation was explored using ureolytic bacterial strain; namely, Flask condition experiments were performed in four different sets as follows: 1. Synthetic media (hereafter, SM); prepared with 0.1 g glucose, 0.1 g peptone, 0.5 g NaCl, 0.2 g mono-potassium phosphate and 3 g urea in 100 ml of distilled water.
Urea was filter sterilized (0.25µm) and added to the medium after autoclaving at 121ºC and 15 psi for 30 minutes to prevent degradation. Medium was inoculated with 5% culture of S. pasteurii grown upto log phase with 0.8 OD and further incubated at 30ºC. Parameters such as optical density (using UV/Vis spectrophotometer, Shimadzu, Japan) at 620 nm wavelength, pH (CyberScan pH meter, Eutech Instruments) and quantification of ammonium ion concentration using Nessler's reagent assay [33] were recorded at different time intervals. The data was recorded in triplicates and mean value was plotted. All chemicals were procured from Hi-Media, India and used without further purification.

Characterization of unconsolidated (raw) soil simulants
Martian soil simulant (MSS) procured from Class Exolith lab Florida [47], and lunar soil simulant (LSS) developed by the Indian Space Research Organisation (ISRO) [48] were used for the fabrication of the martian and lunar bricks. Particle size distributions for both soil simulants were measured by suspending 0.1g soil in 5ml ultrapure distilled water, followed by sonication to disperse the particles. After sonication, particle suspension was placed on a glass bottom petri dishes (ibid) and imaged using Leica DMI 8 optical microscope (Germany). The images were captured using a Leica DMi8 inverted microscope with Lecia DFC3000G camera and a 10x objective lens. The resulting image resolution was 1 micrometer per pixel. Particle size histograms were obtained using a 50 micrometer bin size. The images were processed using a standard MATLAB (Mathworks) routine (regionprops) to determine particle dimensions by fitting a horizontally aligned rectangle to each particle. The minimum equivalent diameter for LSS was 7.44 micrometers and for MSS particles was 6.62 micrometers. In order to avoid erroneous counting of small insignificant trace particles, the minimum major length of the fit rectangle was selected as 25 pixels (25 µm). Particle sizes were obtained from the corresponding rectangles as the radii of equivalent circles with the same area. Correspondingly, the minimum equivalent diameter for LSS was 7.44 m and for MSS particles was 6.62 m.

Casting process for repeatable and scalable sample preparation
In lieu of a conventional bioreactor, for the present work we designed and used aluminium molds to cast bricks of uniform size and shape, in a repeatable and scalable manner. The molds were made of aluminium alloy (Al6061-T6), machined using a vertical milling machine. Each mold was made in two mating parts, to enable easy casting and parting off, and consisted of five cuboidal cavities with a cross section of 32 x 32 mm 2 and a height of 35 mm. Thus a single two-part mould could be used to cast five samples simultaneously. A schematic representation of this casting process for MICP mediated consolidation is depicted in Figure 1 and representave image of space bricks are given in the Figure 2. The inner surface of the mold was covered with a thin transparent plastic (OHP) sheet to aid easy removal of consolidated samples from the mold. Fifty grams of autoclaved simulant soil (at 121℃ and 15 psi for 30 minutes) were mixed with media and various combinations of treatments as presented in Table 1, and discussed in Sec. 2.1. S. pasteurii with optical density at 620 nm (OD620) 1.5 in NH4-YE medium was used as inoculum in all the treatments. The soil-bacteria-medium mixture was tightly packed in the mold cavities and the upper part of the mold was sealed with parafilm strip, see Fig.1c. Incubation period was set for 5 days at 32ºC followed by drying in a hot air oven (BioBee, India) at 50ºC for a period of 24 h.

Mechanical and Materials Characterizations
Compression testing of consolidated MSS and LSS samples was carried out on a Universal Testing Machine (Instron-5697), witha a 5 kN capacity load cell and loading rate of 1 mm/min. To ensure uniform compression during the test, all surfaces of the cubical samples were ground and polished using a portable grinder (BOSCH GWS 600). The final specimen for testing was in the form of a cube with dimensions 25±2 mm. A minimum of three test specimens were made for each set of treatments, and the average value of the data was plotted.
All samples were tested in same condition except for MSS-SP-N and MSS-SP-GG-N. In the latter two cases, 2 mm rubber sheets were used between the sample surface and the UTM grips to uniformly distribute the load across the sample surface. This was because making perfectly flat samples for these two conditions proved to be extremely challenging, even after multiple grinding and polishing attempts. Furthermore, given the nonuniform nature of consolidated soil samples in general, the measured stress-strain curves contained a few small intermediate peaks.
Peaks with a maximum peak/valley distance of less than 6% of the overall load maximum were considered in the results reported and more than 6% of overall load maximum were considered as the failure of the samples.
The microstructure of bio-consolidated martian and lunar bricks was observed using field emission scanning electron microscopy (FESEM: Carl Zeiss AG -ULTRA 55, Germany).
Different calcium carbonate phases were identified using a X-ray diffractometer (XRD: PANalytical Philips diffractometer, Rigaku SmartLab, Rigaku Corporation) under Cu-Kα (λ= 1.54 Å) X-ray radiation and thoroughly indexed as per Inorganic Crystal Structure Database (ICSD) library using PANalytical X'Pert High Score Plus pattern analysis software.

RESULTS AND DISCUSSIONS
Lunar and martian soil/regolith simulants used in this experiment were drawn from two different sources and their mineral composition/particle size distribution was first established. Figure 3 depicts the size and morphological characterization for MSS and LSS particles. Figure   3a,b show the size histogram for MSS and LSS particles, respectively, with insets showing corresponding representative FESEM images. The mean particle diameter for LSS particles was approximately 34 µm with a standard deviation of 26 µm, while corresponding numbers for MSS were 31 µm and 33 µm, respectively. This indicates that that MSS typically contains a wider distribution of particle sizes than LSS. However, in both cases, particles were observed to be irregularly shaped. The particle aspect ratio, determined as discussed in Sec. 2.3, quantifies this irregularity; aspect ratios for MSS and LSS particles were around 0.5 and 0.56, respectively.

Lunar and martian soil simulants: Composition and particle properties
In order to identify the phases and the elements present in both soil simulants, X ray diffraction (XRD) and energy dispersive spectroscopy (EDS) were performed, see Fig. 4. In MSS, plagioclase, pyroxene and olivine ( Fig. 4 (a)) were identified as prime crystalline phases whereas in LSS, plagioclase ( Fig. 4 (b)) was the major crystalline phase. MSS possesses  [49]. Incidentally, iron appears in two oxidation states viz. Fe 2+ and Fe 3+ in the bacterial cells that can be transformed into each other. Though these ions are essential for bacterial growth and activity, their requirement is very low. It is reported that higher concentrations of iron may lead to toxic effects on bacterial cells due to generation of reactive oxygen compounds [50]. This may cause peroxidation of lipids cell membrane, protein and can also damage cellular DNA. [51]. The relevance of these observations will be discussed subsequently when consolidation results of MSS and LSS are discussed. In the case of LSS, the major mineral indentified was plagioclase, composed of NaAlSi3O8 and CaAl2Si2O8 and containing Si, Al, Na and Ca elements. These elements were, consequently, also detected in the EDS maps presented in Fig. 4(d).

MICP induced consolidation of martian regolith bricks
The molding/casting process adopted here allows us to make various shapes and even hollow structures that are simply not possible in a conventional bioreactor. For the purpose of determining the optimal process parameters, a mixture of microbial culture and regolith were pre-mixed in slurry form and incubated in the mold cavities for a period of about 5 days. At the end of the process, these cubical samples were retrieved, dried and subjected to quasi-static compression tests, as described in Sec. 2.3. Figure 5a shows the results of compressive strength measurement on various slurries containing different media combinations. MSS slurries mixed with only S. pasteurii or SP culture, when retrieved and dried, were not robust enough for uniaxial testing so data corresponding to this medium (MSS-SP) is not included in Fig. 5a.
Non-consolidation of MSS is probably due to the presence of approximate 12% Mg and 6.9% Structural changes arising in consolidated MSS due to MICP were by XRD analysis and supported by FESEM imaging presented in Figure 5  SP-GG treatment bacterial induced covering on the soil mass was observed (Fig. 5d).

MICP induced consolidation of lunar regolith bricks
An analogous procedure was followed the the lunar soil simulant (LSS) and the results are summarized in Fig. 6. Similar to the MSS slurry treatments, supplementation with both 1% guar gum and 10 mM NiCl2 resulted in bricks with the maximum compressive strength, see XRD analysis of lunar bricks showed multiple calcium carbonate peaks, as expected, thus confirming bio-consolidation ( Fig. 6 (b)). The major identified peaks in the treatment of LSS- Just as with the consolidated MSS bricks, aggreged soil masses were also observed in the case of the LSS bricks, see FESEM micrographs in Figs .6c and 6d. In the case of guar gum supplement, dense aggregated soil mass was observed (Fig 5c) while bacterial induced matrix and precipitates covering the LSS particle were observed in the treatment with 1% guar gum and 10 mM Nickel admixture (Fig. 6d).

Flask-condition evaluation of additives
The The biological origins of the increase in compressive strength with NiCl2, and guar gum separately, as well as together, were explored by performing physiological studies on the bacterial strain covering the primary parameters with respect to the ureolytic pathway. Figure   7 shows the change in medium pH, ammonium ion concentration and its effect on bacterial growth for these treatments. Typical lag, log, stationery and death phases of bacterial growth pattern can be seen clearly with and without guar gum and NiCl2 additives (Fig 7a). The lag phase (where bacteria is prepared for multiplication) and the log phase (where the bacteria actually multiply) of bacterial growth were observed to be comparatively longer with SM-GG, and SM-GG-N treatments where guar gum served as the sole carbon source. The lag phase for SM-GG lasted for 6 hours of incubation, as against 2 hours for SM and SM-N treatment.
Interestingly, the log phase was almost 1.5 times longer for treatments with guar gum supplementation in both cases with and without Ni supplementation. The stationary phase (i.e., where cell division and death rate become equal) was smaller and well defined for treatments without guar gum supplementation whereas with guar gum supplementation the stationary phase continued till the end of the experiment duration thus demonstrating sustainability of bacteria.
The supplementation of Ni in the media gave noteworthy results irrespective of the guar gum supplementation. There is a distinct shift seen with Nickel supplementation in the graph for ammonium ion concentration (in SM-N and SM-GG-N treatments as compared to SM and SM-GG treatments). The slope of the plot showed an increase with or without guar gum supplementation depicting an acceleration in the bacterial biochemical process. An increase in ammonium ion concentration was also recorded at the end of the log phase of bacterial growth in these treatments resulting in an incremental shift in the pH (inset image of Fig. 7b) of the medium. The supplementation of Nickel increased the ammonium ion concentration values approximately 1.7 times with increase in basicity of the media (Fig. 7b). However, the maximum urease activity was observed with the combination of both the additives (SM-GG-N treatment) exploiting their cumulative benefits and thus validating the results obtained for the compressive strength of both the simulants.

CONCLUSIONS
We have shown that bacteria-mediated MICP technique can efficiently consolidate lunar and martian regolith simulant into brick-like structures with promising structural strength. These brick-like structures have been christened 'space-bricks' [40]. Further, we have also presented a casting method, which can be ideal for making various shape structures as well as bricks with consistent strength. A natural polymer (guar gum) as an additive and appropriate concentration of NiCl2 tend to accelerates the overall MICP process thus contributing towards enhancing the strength of the space bricks. Hence this biological approach, coupled with a scalable casting method, towards manufacturing of bricks presents a promising and highly sustainable potential route for in situ utilization of structural elements on extra-terrestrial habitats.