Microbial induced calcite precipitation can consolidate martian and lunar regolith simulants

Abstract

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 bacteria, 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) and X-ray diffraction (XRD). 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.

Full text

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Microbial induced calcite precipitation can consolidate martian and lunar
regolith simulants
Rashmi Dikshit1, Nitin Gupta1, Arjun Dey2, Koushik Viswanathan1, Aloke Kumar1*
1Department of Mechanical Engineering, Indian Institute of Science, Bangalore 560012
2!Thermal Systems Group, U. R. Rao Satellite Centre (Formerly ISRO Satellite Centre),
Indian Space Research Organisation, Bangalore, 560017, India
*Corresponding author: alokekumar@iisc.ac.in
Abstract
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 bacteria, 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) and X-ray diffraction (XRD). 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.
Keywords: Microbially induced calcite precipitation (MICP), Martian Simulant Soil (MSS),
Lunar Simulant Soil (LSS), Biocementation, Sporosarcina pasteurii
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1. INTRODUCTION
Human curiosity to explore the deep mysteries of space may end up eventually demanding
the development of extra-terrestrial settlements. Mars and moon, being close to Earth, have
been identified as unprecedented sources of processable materials and have thus become the
preferred choices for space organizations around the world to build temporary structures and
observatories [1]. On the occasion of the 50th anniversary of manned mission landing on lunar
surface, space agencies like the National Aeronautics and Space Administration (NASA) and
the European Space Agency (ESA) announced plans to restart manned missions for the
exploration of outer space [2–6]. For feasible and sustainable space exploration, these habitats
need to be built from available in situ resources on moon/Mars [7–11]. 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 through laser-sintering [11], use of microwave irradiation
for bonding [12,13] cast basalt production [9,14–16], lunar glass preparation [17,18], sulphur-
based concrete preparation [9,19], dry-mix/steam injection methods and various 3D printing
methodologies [20–22] 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.
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[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 brick-
like 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 [25–29] via various pathways [30]. The urease pathway is widely
explored [31–34] 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 enzyme 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), and 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 bio-
polymer 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 pasteurii -
a much explored MICP capable bacteria and a gram positive non-pathogenic strain [29,43–45].
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In a separate work, we have also designed a modular self-contained lab-on-a-chip (LoC)
device for the real time monitoring 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.
2. MATERIALS AND METHODS
2.1 Microorganisms and culture conditions
Bacterial-induced bio-consolidation was explored using ureolytic bacterial strain; namely,
Sporosarcina pasteurii (Miquel) Yoon et al. ATCC®11859™, procured from American Type
Culture Collection (ATCC) and revived using ATCC recommended media (NH4-YE liquid
medium).
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.
2. Synthetic media-guar gum (hereafter, SM-GG); prepared by replacing glucose in SM
medium with 1% (w/v) guar gum (Urban Platter, India).
3. Synthetic media-NiCl2 (hereafter, SM-N); prepared by adding 10 mM NiCl2 in SM
medium.
4. Synthetic media-GG-NiCl2 (hereafter, SM-GG-N); prepared by adding 10 mM NiCl2
in SM-GG medium.
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 up to log phase with 0.8 optical density (OD) and further incubated at 30ºC.
Parameters such as OD (using UV/Vis spectrophotometer, Shimadzu, Japan) at 620 nm
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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.
2.2 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 approximately 1 µm per pixel.
Particle size histograms were obtained using a 50 µm bin size. The images were processed
using a standard MATLABÒ (Mathworks) routine (regionprops) to determine particle
dimensions by fitting a horizontally aligned rectangle to each particle [49]. 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 µm and for MSS particles was 6.62 µm.
2.3 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
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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 mm2 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
representative image of these consolidated samples, which we have termed ‘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.
Table 1. Different treatments used in experiment for bio-consolidation of lunar (LSS)
and martian (MSS) soil simulants
Name
Bacterial
Strain
Precipitation
Media
Calcium
Lactate
50 mM
Guar Gum
(% W/W)
NiCl2
(mM)
MSS-SP
S. pasteurii
SMU
Yes
0
0
MSS-SP-GG
S. pasteurii
SMU
Yes
1
0
MSS-SP-N
S. pasteurii
SMU
Yes
0
10
MSS-SP-GG-N
LSS-SP
LSS-SP-GG
LSS-SP-N
LSS-SP-GG-N
S. pasteurii
S.pasteurii
S. pasteurii
S. pasteurii
S. pasteurii
SMU
SMU
SMU
SMU
SMU
Yes
Yes
Yes
Yes
Yes
1
0
1
0
1
10
0
0
10
10
2.3 Mechanical and Materials Characterizations
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Compression testing of consolidated MSS and LSS samples was carried out on a
Universal Testing Machine (Instron-5697), with 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
not considered to points of failure, whereas the first peak larger than this 6% threshold was
taken to be the point of failure.
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.
3 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.
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3.1 Lunar and martian soil simulants: Composition and particle properties
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.
In order to identify the phases and the elements present in both soil simulants, 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 significant amount of pyroxene (XY
(Si,Al)2O6 ), that contains Mg and Fe along with Si and aluminium oxide. Correspondingly,
major elements observed in MSS from EDS mapping were O, Si, Al, Mg and Fe as shown in
Figure 4 (c). Fe and Mg are essential metal ions required in limited concentration for bacterial
growth. It is well known that Mg is important for cell division of the rod-shaped bacteria
whereas iron contributes in biological activity such as electron transfer and enzymatic activity
[50]. Incidentally, iron appears in two oxidation states viz. Fe2+ and Fe3+ 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 [51].
This may cause peroxidation of lipids cell membrane, protein and can also damage cellular
DNA. [52]. The relevance of these observations will be discussed subsequently when
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consolidation results of MSS and LSS are discussed. In the case of LSS, the major mineral
identified 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).
3.2 MICP induced consolidation of martian regolith
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%
iron causing inhibition of urease activity of S. pasteurii in absence of additives. Supplementing
the MSS slurry with 1% guar gum resulted in mean compressive strength of approximately 1.1
MPa, see MSS-SP-GG bar in Fig. 5a.
Since the urease enzyme is a metalloenzyme containing nickel at the centre [39,53], potential
enhancement of urease activity via Ni supplementation was evaluated next. Flask growth data
suggested that 10 mM of Ni supplementation can enhance urease activity significantly and
thereby accelerate the overall MICP process (data not shown). The mean compressive strength
of the consolidated brick sample obtained by 10 mM NiCl2 supplementation (MSS-SP-N) was
found approximately 2.67 MPa. Hence, the strength was consistently found to be twice as large
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as that of the MSS-SP-GG consolidated samples. Subsequently it was deemed natural to
evaluate the compressive strength of consolidated samples with both guar gum (GG) as well
as Nickel supplementation. The MSS supplemented with 1% guar gum and 10 mM Nickel
(MSS-SP-GG -N) showed a significantly larger mean compressive strength, of 3.3 MPa, which
was the largest among all the treatments, see Fig. 5(a). It is hence clear that the addition of guar
gum and Ni results in significantly stronger consolidates, approaching the compressive strength
of ice.
Structural changes arising in consolidated MSS due to MICP were by XRD analysis and
supported by FESEM imaging presented in Figure 5 (b, c & d). The XRD patterns for the three
different treatments (MSS-SP-N, MSS-SP-GG and MSS-SP-GG-N) discussed above are
presented in Fig. 5b. It is clear that several calcium carbonate phases (e.g., calcite, aragonite
and vaterite) of precipitates are observed in all three cases. In case of MSS-SP-GG-N treatment,
the peaks correspond to the calcite phase at 2θ values of 23.01°, 35.50°, 62.7°, 65.1° (hkl value
012, 111, 212, 122) matched with ICSD File nos. 98-000-5337, 98-001-442 respectively
whereas peaks at 2θ values of 20.8°, 52.32° (ICSD File no. 98-010-9796) were observed for
vaterite phase. Aragonite phase matching with ICSD File no. – 98-011-4648 was also observed
at 2θ values of 67.42°. In MSS-SP-N treatment major peaks at 2θ values 29.3°, 42.5° for calcite
phase with hkl (101, 002) matched with ICSD File no. 98-001-4421. Major identified peaks in
MSS-SP-GG treatments were at 2θ values 62.7° matching with ICSD File no. 98-001-4421 for
calcite phase of calcium carbonate. As expected, treatment without any additives (MSS-SP)
did not show any significant calcium carbonate peaks indicating inhibition of MICP activity
and, consequently, poor strength of the consolidated sample.
The FESEM micrograph of bio-consolidated MSS bricks also clearly showdepicted bacterial
mediated consolidation. An aggregated soil mass with appreciable bacterial induced
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precipitates were seen in MSS-SP-GG-N treatment (Figure 5c) whereas in the case of MSS-
SP-GG treatment bacterial induced covering on the soil mass was observed (Fig. 5d).
3.3 MICP induced consolidation of lunar regolith
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
Fig. 6. Furthermore, guar gum and Ni supplemented LSS samples (LSS-SP-GG-N) showed
mean compressive strength of 5.65 MPa, which was significantly higher than the corresponding
value for MSS-SP-GG-N. Additionally, LSS consolidation using a slurry with just the
microbial medium (LSS-SP) was found to be robust enough to be subjected to uniaxial testing,
with a resulting mean compressive strength of 0.75 MPa. When using only guar gum
supplement (LSS-SP-GG), mean compressive strength of 3.41MPa was obtained.
These results seem to suggest that MSS provides a less ideal environment for bacterial
growth as compared to LSS primarily due to the existence of metals such as Fe and Mg in
significantly higher quantities. An additional difference between the consolidated LSS and
MSS bricks is that LSS-SP-GG samples showed higher mean compressive strength compared
to LSS-SP-N samples. The reason for this difference is at present not well understood and
requires further investigation.
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-
SP-N corresponding to the calcite phases were found at 2θ values 23.02° (hkl 012), 29.40° (hkl
104), 35.9° (hkl 110) and 47.0° (hkl 018) and matched with ICSD File nos. 00-005-0586. In
the case of LSS-SP-GG-N, identified phases of calcite were at 2θ values 29.3° (hkl104), 35.40°
(hkl 110), 42.01° (hkl 200), matched with ICSD File nos. 00-005-0586,980005339 and 98-011-
4421 respectively, whereas for vaterite phase at 2θ values 45.6° (301), 49.6° (hkl 412), 53.7°
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(hkl 222) and matched with ICSD File nos. 98-010-9797.The peaks correspond to the calcite
phase at 2θ values of 24.60° (hkl 111), 31.50° (hkl 202) matched with ICSD File nos. 98-007-
8903, 98-009-6175 whereas peaks at 2θ values of 10.36° (hkl 002), 13.5°, 65.49° (hkl 420)
(ICSD File nos. 98-000-6092, 98-010-9797, 98-010-9796) was observed for vaterite phase.
Aragonite phase matched with ICSD File nos. – 98-011-4648, 98-011-4649 was also observed
at 2θ values of 57.71° with hkl 122 in case of LSS-SP-GG treatment.
Just as with the consolidated MSS bricks, aggregard 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).
3.4 Flask-condition evaluation of additives
The XRD and microstructural data of consolidated LSS and MSS bricks clearly confirm
bacterial induced consolidation in both soil simulants. Peaks representing different phases of
calcium carbonate (calcite, aragonite and vaterite) in MSS-SP-N and MSS-SP-GG-N for MSS
and in LSS-SP-GG and LSS-SP-GG-N for LSS clearly show the occurrence of bacterial
mediated bio-consolidation of martian and lunar bricks. These results serve to demonstrate that
supplementation with guar gum and Ni can significantly enhance urease activity in both soil
simulants, thereby accelerating the MICP process and leading to considerable increase in
compressive strength.
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
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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 suplementation. 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.
4. CONCLUSIONS
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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 christended ‘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.
Acknowledgments
RD acknowledges funding from Department of Biotechnology, Ministry of Science and
Technology, GOI for their grant under BioCare scheme (BT/PR31844/BIC/101/1206/2019).
Authors acknowledge thank Indian Space Research Organisation for providing the lunar soil
simulant. We thank Dr. Amrit Ambirajan, Research Professor, IISc and former Scientist, ISRO
for stimulating discussions and for suggesting the name ‘space bricks’.
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Figure and captions
Fig:1: Schematic representation of casting of aluminium mould and process involved in MICP
mediated consolidation of simulant soils.
Fig: 2 Representative image of consolidated samples using MSS. Such structures have been
termed space bricks.
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Fig:3: Characterization of raw MSS and LSS: Size distribution of a) MSS particles with an
inset FESEM image of MSS and b). LSS particles with an inset FESEM image of LSS c)
Aspect ratio for MSS particles and d) Aspect ratio for LSS particles.
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Fig:4: XRD pattern and EDS of raw soil simulants a) identified crystalline phases of MSS b)
identified crystalline phases of LSS c) elemental mapping of selected location of MSS d)
elemental mapping of selected location of LSS
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Fig: 5 a) Compressive strengths of martian bricks for different treatments with an inset image
of cubical biconsolidated martian brick. b) XRD pattern of bioconsolidated martian bricks
with different treatments (V – vaterite; C- calcite; A- argonite) c) showing SEM micrograph
of martian bricks for MSS-SP-GG-N treatments. d) SEM micrograph for MSS-SP-GG
treatment.
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Fig: 6 a) Compressive strengths of martian bricks for different treatments with an inset image
of cubical biconsolidated lunar brick b) XRD spectrum of consolidated lunar bricks with
different treatments. (V – vaterite; C- calcite; A- argonite) c) showing SEM micrograph of
lunar bricks with guar gum supplementation d) with guar gum and nickel chloride
supplementation.
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Fig: 7 Exploration of microbial physiology under flask condition with and without guar
gum additive: (a) microbial growth curve (b) temporal evolution of pH (inset) and
ammonium ion concentration in growth medium. Legend shows SM; media without
any supplementation, SM-GG; SM media supplemented with 1% guar gum, SM-N; SM
media with only 10 mM Nickel chloride supplementation, SM-GG-N; SM media
supplemented with 1% guar gum and 10 mM Nickel chloride. S. pasteurii was used as
inoculum in all cases. (Error bars represents the standard deviation of the data of three
independent experiments).
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