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ARTICLE
Phosphorus-solubilizing bacteria improve the
growth of Nicotiana benthamiana on lunar regolith
simulant by dissociating insoluble inorganic
phosphorus
Yitong Xia1, Yu Yuan 2, Chenxi Li3& Zhencai Sun 1✉
In-situ utilization of lunar soil resources will effectively improve the self-sufficiency of bior-
egenerative life support systems for future lunar bases. Therefore, we have explored the
microbiological method to transform lunar soil into a substrate for plant cultivation. In this
study, five species of phosphorus-solubilizing bacteria are used as test strains, and a 21-day
bio-improving experiment with another 24-day Nicotiana benthamiana cultivation experiment
are carried out on lunar regolith simulant. We have observed that the phosphorus-solublizing
bacteria Bacillus mucilaginosus,Bacillus megaterium, and Pseudomonas fluorescens can tolerate
the lunar regolith simulant conditions and dissociate the insoluble phosphorus from the
regolith simulant. The phosphorus-solubilizing bacteria treatment improves the available
phosphorus content of the regolith simulant, promoting the growth of Nicotiana benthamiana.
Here we demonstrate that the phosphorus-solubilizing bacteria can effectively improve the
fertility of lunar regolith simulant, making it a good cultivation substrate for higher plants. The
results can lay a technical foundation for plant cultivation based on lunar regolith resources in
future lunar bases.
https://doi.org/10.1038/s42003-023-05391-z OPEN
1College of Agronomy and Biotechnology, China Agricultural University, Haidian District, Beijing, China. 2College of Engineering, China Agricultural
University, Haidian District, Beijing, China. 3College of Horticulture, China Agricultural University, Haidian District, Beijing, China. ✉email: zhencai_sun@cau.
edu.cn
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Series of successful unmanned lunar scientific research
programs1have aroused worldwide scientific interest in the
moon2. Considering the value of the moon in future deep
space exploration and the exploitation of lunar natural
resources3–6, it is necessary to establish a permanent crewed lunar
station. Transporting essential materials (e.g., food and water) for
human life support by cargo rockets has become a traditional
method in crewed space exploration. However, the tremendous
distance between the Earth and the moon makes it uneconomic
to use cargo rockets to transport these materials to the moon7,
and even economically unsustainable to provide terrestrial
resources to an extraterrestrial permanently crewed station from
the perspective of its expected lifespan8, because of the high cost
and resource/energy requirements and the difficulty and time
required to plan and execute a launch. A biological regenerative
life support system (BLSS) is the only advanced life support
system that human beings can rely on to carry out long-term,
long-distance, and multi-crewed space missions in the future9–11.
An efficient BLSS must be able to purify water, produce food, and
revitalize the atmosphere in a closed system12–14, which greatly
reduces the Earth-moon transportation frequency, and effectively
cut the economic cost of the initial construction and long-term
maintenance of the lunar base15.
Higher plants have the functions of providing food and O
2
,
absorbing CO
2
, and purifying wastewater. Therefore, they are
regarded as the core components of highly closed BLSS16,17.
However, among current BLSS simulation tests18–20, the culti-
vation of higher plants is highly dependent on hydroponic sys-
tems, or cultivation substrate based on earth soil21–25. Given the
vast amount of materials required to construct such systems, it is
not economically realistic to carry out these designs in extra-
terrestrial stations. A natural idea is: why not introduce the
unconsolidated fine granular weathering on the lunar surface, or
the lunar regolith, to BLSS as a cultivation substrate for higher
plants, in order to cut the cost of lunar base construction26. The
potential of this method, which is known as in-situ resource
utilization (ISRU), has been preliminarily studied. Analysis and
evaluation have been made on the elemental composition and
bioavailability of lunar soil27 and the ability of the regolith on
extraterrestrial planets to support the growth of
microorganisms8,28,29 and plants30–32. Studies have shown that
the composition of the elements essential for plant growth in
lunar regolith is very similar to those in the Earth’s soil27.
Unfortunately, the fertility of lunar regolith is greatly poorer than
that of the Earth soil, due to the influence of lunar environmental
conditions. It not only lacks carbon and nitrogen nutrients
necessary for plant growth but also holds other elements mainly
in an insoluble form, which is difficult to be absorbed by plants.
Because of this “inertness”, the lunar soil cannot provide the
absorbable soluble nutrients for plant growth as the Earth’s
soils do.
An interesting study conducted by Paul et al. 32. showed that
Arabidopsis thaliana could be seeded and grow directly on lunar
regolith with the support of exogenous nutrient solutions. How-
ever, compared with the volcanic rock control, Arabidopsis
thaliana grown on lunar regolith exhibits slower growth and a
severe stress phenotype, which is confirmed by transcriptome
data. This indicates that it is necessary to develop some technical
measures to improve the physical and chemical properties of
lunar regolith and increase the availability of essential plant
nutrients before the establishment of a practical extraterrestrial
higher plant cultivation system that effectively provides life sup-
port in the lunar station.
Reviewing the process of the earth’s terrestrial ecosystem
evolving hard rocks into porous and biologically active soils, it
has been believed that microbial groups (e.g., bacteria and fungi)
are one of the most important factors leading this process33,34.
Interactions between microbial metabolites and naked rock
resulted in the decomposition of silicate, phosphate, carbonate,
oxide, and sulfide minerals and the dissolution of some important
elements (Si, Al, Fe, Mg, Mn, P, Na, Ti, etc.) from minerals35–38,
which improves the fertility level of regolith and enables the
growth of plants. However, the feasibility study on microbial
weathering as an improvement measure for extraterrestrial
regolith has not been confirmed by academia yet. There are only a
few relevant studies, that mainly focus on two directions:
Exploration of the ability of cyanobacteria and other autotrophic
nitrogen-fixing microorganisms to improve lunar soil
fertility8,39–41, and the very preliminary verification of the
improving ability that artificial microorganisms coenosis have on
the growth of higher plants in a lunar regolith simulant30,42.
Unfortunately, considering phosphorus, a major element required
by plants and an important component of soil fertility, and stands
for about 1% of the total mass in the lunar regolith, current
studies either lack experimental evidence that microbial activity
directly decomposes mineral structures and leads to phosphorus
dissociation30, or even have obtained contrary experimental
evidence39 (in this study, cyanobacteria activity even led to a
decrease in phosphorus content in the simulated lunar soil).
Here, setting phosphorus as the object of the study, five kinds
of phosphorus-solubilizing bacteria (PSBs) were introduced to the
lunar surface regolith simulant (Fig. 1a). Through detailed
experimental design and analysis, a 21-day culture experiment
was carried out using a shaking flask method, measured the
dynamics of phosphorus content in different forms in the culture
medium on a time scale and screened out three strains with the
strong phosphorus-solubilizing ability to the lunar regolith
simulant. This study verified the potential of soil microorganisms
to sustain the growth of plants by dissociating insoluble phos-
phorus from the lunar regolith, which was again confirmed by
phenotypic data in a further Nicotiana benthamiana cultivation
experiment.
Results and discussion
The growth of five PSBs on the lunar regolith simulant. Con-
sidering that there is only a very small amount of lunar regolith
material brought back by the Apollo missions and a series of
unmanned probes to Earth, lunar regolith simulants or similar
minerals are used in place of true lunar regolith30,39,41,42 in most
studies related to the agricultural feasibility of lunar regolith.
These simulants, often made from volcanic scoria, used real lunar
samples for reference, to achieve good similarity in mineralogy,
physicochemical properties, and hydrological properties43. The
simulant used in our study is a copy of the CAS-1 lunar soil
simulant (Methods, Lunar surface regolith simulant materials),
which has a very similar elemental composition to Apollo
14 samples (Fig. 1b, c).
Five types of PSBs, namely Bacillus mucilaginosus,Bacillus
megaterium,Bacillus subtilis,Bacillus licheniformis, and Pseudo-
monas fluorescens were selected for the experiment. Their ability
to decompose insoluble inorganic phosphorus was verified in the
previous Ca
3
(PO
4
)
2
decomposition test (Fig. 1d). After seven days
of culture, the Ca
3
(PO
4
)
2
in the culture medium was decomposed,
and the concentration of soluble inorganic phosphorus in the
liquid medium was significantly increased by 212.7–519.7%
compared with that before culture (p=0.001 or less, n=6 for
each PSB). These results indicated that the five PSBs had a strong
potential to dissociate inorganic phosphorus elements from
Ca
3
(PO
4
)
2
. However, whether they can activate inorganic
phosphorus in the lunar soil simulants remains to be further
verified.
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The ability to reproduce and grow normally in lunar regolith
plays an important role in the function of PSBs to decompose
phosphate minerals in lunar regolith particles. Therefore, by
measuring the 600 nm light absorbance, or the OD
600
, of the
culture at different culture periods, we plotted the growth curve of
five PSBs and studied the impacts of lunar regolith simulant on
the growth of five PSBs in the shaking flask culture system
(Fig. 2a). The mass ratio of regolith simulant and LB medium was
1:2. In general, no matter whether the lunar regolith simulant was
added, the five PSBs showed typical growth characteristics, which
include an obvious logarithmic growth period followed by a
plateau period. This indicates that some components (e.g.,
peroxides and heavy metals) in the simulant as well as the real
lunar regolith that it referred to, do not reveal serious toxicity that
inhibits microbial growth.
By comparing the OD
600
of five PSB treatments in the culture
of simulant treatments with the control on a time scale., it was
found that the addition of the simulant had no obvious effect on
the growth and reproduction of five PSBs in the early stage of
culture. The growth curves of the five PSBs almost coincided with
their corresponding control, from the inoculation to the pre-
logarithmic and mid-logarithmic stages. The longest coincident
relationship could extend to 84 h after the inoculation (HAI) in
the B. subtilis treatment. This coincident relationship suggests
that the simulant has little impact on the growth of PSBs and
reproduction at the beginning of culture when it is considered to
be “inert”. This may be due to the very weak interaction between
the simulant particles and the PSBs in this stage. In contrast, from
the late logarithmic stage to the plateau stage of the culture, the
coincidence of growth curves between the simulant treatment and
the corresponding control gradually faded, and the different
reactions between the strains were shown. Specifically, some
strains had higher OD
600
in the culture at the plateau stage in
simulant treatments than control, such as B. mucilaginosus and B.
licheniformis treatments. On the contrary, the OD
600
of B.
megaterium,B. subtilis, and P. fluorescens was lower than that of
the corresponding control, indicating that the later growth of the
three PSBs was inhibited by simulants, and this inhibition impact
increased with time. This suggests that the plateau period is the
key period for the interactions between PSBs and simulants.
Therefore, it is necessary to study the dynamic changes of
medium composition on a time scale to further clarify the
Fig. 1 Preparations for the culture experiment. a Photography of lunar surface regolith simulant samples of different particle sizes. The simulants in the
plates are >5 mm, 0.5–0.9 mm, and 0.3–0.5 mm from up to down respectively. b,cThe determination of the elemental composition of the tested samples
of the regolith simulant and the comparison with CAS-1 lunar regolith simulant and the real lunar regolith samples brought back in the Apollo 14 mission.
Data of CAS-1 and Apollo 14 samples that used in this figure is provided by Zheng et al. 80. All error bars represent Standard Deviation. dThe identification
of all five PSBs’ability to decompose insoluble inorganic phosphorus compounds in the Ca
3
(PO
4
)
2
decomposition test. Different species of inoculated
bacteria contain different amounts of phosphorus, which leads to different levels of phosphorus at the beginning of the test. All error bars represent
Standard Deviation. *** indicates p< 0.001. Significance analyses are conducted using one-side one-way ANOVA tests. eThe comparison of the pH of the
culture medium at 7 DAI between the lunar regolith simulant treatments and their corresponding control in the PSBs culture experiment. All error bars
represent Standard Deviation. *** indicates p< 0.001. Significance analyses are conducted using one-side one-way ANOVA tests.
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inhibitory factors and mechanisms that simulants have on the
growth of PSBs.
Influential mechanisms of the lunar regolith simulant on
microbial growth. Two indexes including soluble salt content
and pH value of the culture medium were selected to measure
liquid samples at different culture times (Fig. 2b, c). All five PSBs
induced a rapid increase of soluble salt content at 4–10 days after
the inoculation (DAI), which reached 3.80–4.36% at 10 DAI and
gently increased to 4.36–5.11% at 21 DAI. However, there was
little difference among the five PSBs generally. Considering that
there was no significant change (p=0.933, n=6) in the soluble
salt content of the control group, we believe that the activity of
PSBs led to an increase in soluble salt content. Compared with
soluble salt content, the temporal dynamics of pH in the culture
medium of different PSBs showed an obvious difference. The
initial pH was determined by the medium itself, which was about
7.0. At 60 HAI, the pH of the culture medium of different strains
changed dramatically and reached stability (except for B. liche-
niformis) after 7 DAI. The stable pH values of B. mucilaginosus,B.
megaterium, and P. fluorescens are lower than 7 and are acidic,
while the stable pH values of B. subtilis and B. licheniformis are
higher than 7 and are alkaline. The pH of the culture medium in
lunar regolith simulant treatments with that of the corresponding
control at 7 DAI (Fig. 1e) was further compared. When the
simulant was not added, the culture medium of B. subtilis and B.
licheniformis were both weakly acidic, while when the simulant
was added, the two PSBs were strongly induced by the simulant,
Fig. 2 Growth curves of five PSBs and the dynamics of pH and the soluble salt content of the culture medium. a1–a5 The growth curves of five PSBs in
the regolith simulant treatment and corresponding control during the culture period of 21 days. a1 to a5 represent B. mucilaginosus, B. megaterium, B. subtilis,
B. licheniformis, and P. fluorescens, respectively. The mass ratio of regolith simulant and LB medium was 1:2. All error bars represent Standard Deviation.
bThe dynamics of the pH of the culture medium of five PSB treatments. All error bars represent Standard Deviation. cThe dynamics of the soluble salt
content in the culture medium of five PSB treatments. All error bars represent Standard Deviation.
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making the culture medium alkaline. The other three PSBs
showed little change under the two conditions. According to the
mineralogical properties of the simulant, it is unlikely that the rise
of pH values was caused by the decomposition of the simulant
minerals, and B. subtilis and B. licheniformis are believed to
secrete alkaline substances when treated with lunar regolith
simulant.
To evaluate the responses of different PSBs to salinity and pH
factors mathematically and statistically, the concept of growth
inhibition ratio (GIR, Supplementary Measurement Methods,
section 1) was applied. The higher the GIR is, the more serious
impact the simulant has on the inhibition of growth and
reproduction. Linear fitting analysis was conducted between the
GIR and the soluble salt content at 2, 4, 7, 10, 15, and 21 DAI, as
well between the GIR and the pH of the culture medium at 0, 10,
60 HAI, and 5, 8, 15 and 21 DAI. The results are as follows:
As can be seen from the Table 1, the coefficient of
determination, or the R2value, between the GIR of B.
mucilaginosus and P. fluorescens and its corresponding soluble
salt content was very low, showing that the growth inhibition of
these two PSBs did not respond to the change of soluble salt
content. On the contrary, the coefficient of determination
between the GIR of B. megaterium,B. subtilis, and B. licheniformis
and the corresponding soluble salt content ranged from 0.3566 to
0.6938. All of them were statistically significant (P=0.003, 0.000,
0.004, n=22, 18, 17, respectively), suggesting a certain positive
correlation between the two variables, which indicated that the
increase of soluble salt content inhibited the growth and
reproduction of these three PSBs to some degree.
The inhibitory impact caused by soluble salt concentration may
be a combination of high osmotic stress and ionic toxicity. At the
beginning of the culture, the medium provided a solution
environment with appropriate salt content for microorganisms,
which is conducive to maintaining the normal osmotic pressure
of cells. With the continuous decomposition of mineral particles
by PSBs, insoluble mineral elements were transformed into
soluble compounds, which increased the osmotic pressure of the
solution and may make PSBs cells unable to maintain
intracellular water potential, causing water outflow of cells44,45,
further leading to cell volume contraction. Meanwhile, consider-
ing the elemental composition of the lunar regolith simulant, the
salts dissolved in the culture medium may contain a large amount
of Fe, Al, Mg, K, and Na ions, which could replace the metal ions
in the active site of some proteases in the cells46,47. This
substitution reaction caused adverse changes in the structure of
the active sites and thus inhibited their activity. In addition, these
ions could induce the production of a large number of reactive
oxygen species (ROSs), which further led to severe damage to cell
membrane lipids and proteins that were essential to the normal
metabolic activities of cells48,49. Although microorganisms could
resist salt stress through initiative ion transportation50,51 and
synthesis of protective substances (e.g., glycerol52,53), growth and
reproduction would still be delayed. These factors could explain
the growth inhibition of B. subtilis and other PSBs when the
soluble salt content increased, which had been also reported by
transcriptome data analysis in the study conducted on the real
lunar regolith32.
The linear fitting analysis results of the GIR and the pH of the
culture medium showed that, within the range of pH changes
produced in the experiment, the coefficients of determination of
B. mucilaginosus,B. megaterium, and B. subtilis were very low,
which did not exceed 0.1, indicating that the change of pH was
not the main factor affecting the growth of these three PSBs. The
coefficient of determination of B. licheniformis and P. fluorescence
were 0.4207 and 0.6704 respectively, and both were statistically
significant (p=0.001, 0.000, and n=24, 28, respectively),
indicating that the decrease of pH inhibited the growth and
reproduction of the two PSBs to some degrees. What is more
interesting is B. mucilaginosus, whose growth and reproduction
were not affected by low pH stress or high salt stress. It even grew
better in the treatment with the addition of the lunar surface
regolith simulant. Based on the ability of B. mucilaginosus to
decompose silica-containing minerals, we speculated that B.
mucilaginosus might have a certain tendency to be silicophilic,
and the simulant provided silicon element.
In conclusion, under the activities of PSBs, the soluble salt
content of the lunar surface regolith simulant increased, which in
turn inhibited the growth and reproduction of all PSBs. In
addition, the metabolic products of PSBs also changed the pH of
the culture medium, which had a negative correlation to the
growth and reproduction of B. licheniformis and P. fluorescence
only. Meanwhile, the lunar surface regolith simulant also induced
B. licheniformis to increase the pH of the culture medium, thus
promoting its growth compared with its control after all. The
experiment we carried out is still a preliminary study, considering
only soluble salt content and the pH of the culture medium were
considered. If experimental conditions permit, detailed explora-
tion may further verify the conclusion of this experiment.
Dynamics of phosphorus content and forms in the lunar
regolith simulant treated by PSBs. The key to the microbial
regolith improvement program is to confirm the microbial dis-
sociation and activation of essential mineral elements for plants
(e.g., phosphorus) in the lunar surface regolith simulant. In this
study, the dynamics of dissolved inorganic phosphorus, dissolved
organic phosphorus, suspended microbial biomass phosphorus,
attached microbial biomass phosphorus, and adsorbed phos-
phorus were examined. The classification aimed to study the
differences in the spatial and temporal distribution of different
forms of phosphorus in more detail. At the beginning of the
culture, phosphorus in the medium and inoculation solution was
added to the culture system in the form of dissolved phosphorus
and microbial biomass phosphorus, and these exogenous phos-
phorus inputs would be a part of the start-up conditions of the
culture process.
Table 1 Results of linear fitting analysis between GIR and soluble salt content and pH of culture medium.
PSBs Linear fitting equation between GIR and salinity R2Linear fitting equation between GIR and pH R2
B. mucilaginosus y=9.1807x −54.269 0.1212 y =38.434x −264.56 0.0615
B. megaterium y=12.245x −8.4898 0.3566** y =12.4x −19.633 0.0096
B. subtilis y=20.721x −29.372 0.6938*** y =−1.4785x +42.075 0.0014**
B. licheniformis y=31.688x −124.44 0.541** y =−52.88x +328.52 0.4207**
P. fluorescens y=0.7443x +52.143 0.0034** y =−29.917x +172.23 0.6704**
Data used for these analyses is provided in Supplementary data, worksheets 18 and 19.
*** indicates p< 0.001, and ** indicates p< 0.01.
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Dissolved phosphorus is the most direct form of phosphorus
for plants and microorganisms to absorb, so it is considered as an
important indicator for the fertility of lunar regolith simulant. In
this study, with the progress of the culture, the overall trend of
dissolved phosphorus in regolith simulants with the treatments of
five PSBs was increasing. The dissolved phosphorus content at 21
DAI was significantly higher (p< 0.001, n=21) than that at the
beginning of the culture, ranging from 1.63 times to 2.15 times of
the initial value. The difference in dissolved phosphorus content
among all five PSBs at the end of culture (21 DAI) was not very
obvious compared with that at the middle of culture (4–15 DAI),
which may be a sign of sufficient processing. Dissolved
phosphorus in the five PSB treatments was mainly organic
(Fig. 3a) at 21 DAI, and dissolved organic phosphorus could
reach 2.54 times of dissolved inorganic phosphorus (B.
megaterium treatment) to 13.20 times of that (B. subtilis
treatment). This should be the result of the continuous absorption
and conversion of inorganic phosphorus into organic phosphorus
compounds by PSBs through intracellular biochemical activities.
The dissolved organic phosphorus compounds held by PSB cells
(e.g., nucleic acid molecules, phospholipid molecules, and
adenosine phosphate molecules) can be released back into the
culture medium through cell rupture after death.
Microbial biomass phosphorus is an important temporary
storage and transfer pool of phosphorus. Phosphorus in minerals
can be dissociated and stored in bacteria cells to prevent it from
Fig. 3 Dynamics of the phosphorus content in different forms during the culture. a The dynamics of dissolved inorganic phosphorus and dissolved
organic phosphorus of five PSB treatments. The corresponding control is presented in Supplementary Fig. 1. Each treatment included four flasks as
replicates. All error bars represent Standard Deviation. bThe dynamics of Suspended microbial biomass phosphorus and Attached microbial biomass
phosphorus content of five PSB treatments. Each treatment included four flasks as replicates. All error bars represent Standard Deviation. cThe dynamics
of Adsorbed phosphorus content of five PSB treatments. Each treatment included four flasks as replicates. All error bars represent Standard Deviation.
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being fixed again through absorption while the microbes are alive.
After they die, the phosphorus in the cells is released and can be
absorbed by higher plants or other microbes. In this study, total
microbial biomass phosphorus showed a large difference among 5
PSB treatments over the whole culture. For B. subtilis and B.
licheniformis, the microbial biomass phosphorus fluctuated at the
initial level and eventually was lower than the initial level at 21
DAI. Considering the obvious increase of dissolved organic
phosphorus in the treatment of these two PSBs from 10 to 21
DAI, it can be deduced that a large number of microorganisms
died at this stage, and cell rupture caused the transfer of microbial
biomass phosphorus to dissolved organic phosphorus. However,
the microbial biomass phosphorus of the simulant treated by B.
mucilaginosus,B. megaterium, and P. fluorescens maintained
higher than the initial level after 2 DAI, suggesting that these
three PSBs have a stronger ability to dissociate phosphorus
elements in the simulant through microbial metabolic activities,
in addition to using phosphorus compounds in the liquid
medium. Microbial phosphorus was divided into suspended
microbial biomass phosphorus and attached microbial biomass
phosphorus according to whether the PSB cells were attached on
the simulant particles. Suspended microbial biomass phosphorus
was always much higher than attached microbial biomass
phosphorus among all five PSB treatments (Fig. 3b), suggesting
that the major part of microorganisms in the culture system was
in a free state and did not have direct contact with mineral
particles. The attached microbial biomass phosphorus of five PSB
treatments was very small in 0–4 DAI, while that of B.
mucilaginosus,B. megaterium, and P. fluorescens treatments
began to increase substantially after 4 DAI. In particular, the
attached microbial biomass phosphorus of B. megaterium and P.
fluorescens treatments increased 29.6 times and 6.7 times
respectively at 15 DAI compared with that of 4 DAI, and still
only accounted for 15.63% to 43.93% of microbial biomass
phosphorus.
Adsorbed phosphorus is a dynamic regulating reservoir of
phosphorus elements. When the content of dissolved phosphorus
in the liquid medium increases, the equilibrium of adsorption-
desorption shifts backward and phosphorus tends to be adsorbed.
When the phosphorus in the liquid medium decreases with
biological absorption and utilization, the adsorbed phosphorus
dissolves again, maintaining the relative balance of dissolved
phosphorus concentration. In our study, the adsorbed phos-
phorus of all five PSBs treatments increased significantly
(p< 0.001, n=21) from the initial value to 21 DAI, ranging
from 1.50 times of B. subtilis treatment to 3.22 times of P.
fluorescens treatment (Fig. 3c). During the period of 7–10 DAI,
the five PSB treatments all experienced a relatively obvious
increase and maintained a relatively stable change in the
subsequent culture, which appeared to be similar to the dynamics
of soluble salt content, but we are yet insufficient with
experimental evidence to find out the relationship between two
indexes. After all, the adsorbed phosphorus content was relatively
very small in this study, with a maximum of 45.88 mg/kg (21DAI,
P. fluorescens).
In the control group, which was not treated with PSBs,
dissolved phosphorus content remained unchanged, while
dissolved inorganic phosphorus content only increased by
4.97 mg/kg after the culture. We believe that there was no
spontaneous phosphorus dissociation in the lunar regolith
simulants, and the liquid medium could not leach insoluble
inorganic phosphorus elements from the simulant, either.
To quantitatively measure the microbial dissociation of
phosphorus in the lunar regolith simulant, we defined the
concept of total available phosphorus which included all forms of
phosphorus, except insoluble phosphorus, representing the sum
of initial exogenous phosphorus input (dissolved phosphorus and
microbial biomass phosphorus from the medium and the
inoculant) and all phosphorus dissociated by PSBs. Statistical
analysis showed that the total available phosphorus capacity
could be significantly increased by B. mucilaginosus,B. mega-
terium, and P. fluorescens (p< 0.001, n=13), reaching the
maximum at 10 DAI, 21 DAI, and 21 DAI, which was
213.57%, 234.31%, and 247.09% of that of the initial level,
respectively (Fig. 4A–C). Considering that there was little
dissolved phosphorus in the lunar regolith simulant before the
culture, the initial total available phosphorus could be considered
to be entirely provided by liquid medium and inoculate solution.
Therefore, the extremely significant increase in the total available
phosphorus capacity during the 21-day culture process could be
completely regarded as the result of the dissociation of insoluble
inorganic phosphorus elements in the simulant by the three PSBs.
The amounts of insoluble inorganic phosphorus dissociated by B.
mucilaginosus,B. megaterium, and P. fluorescens at 21 DAI were
374.75 mg/kg, 887.46 mg/kg, and 904.03 mg/kg, respectively
(Fig. 5). In contrast, B. subtilis and B. licheniformis did not
significantly (p=0.141, n=8) increase the capacity of total
available phosphorus during the culture (Fig. 4D, E), and
even caused a relative decrease of that during 0-10 DAI. The
total available phosphorus of the two PSB treatments at the end of
the culture was only 1.11 times and 1.16 times of the initial level,
so it could be considered that the ability of the two PSBs to
dissociate insoluble inorganic phosphorus in the simulant was
very weak.
From the perspective of the forms of phosphorus, the
dissociation of phosphorus from the lunar surface regolith
simulant of B. mucilaginosus,B. megaterium,andP. fluorescens
treatments during the 21-day culture was analyzed (Fig. 4F). In
B. mucilaginosus treatment, dissolved organic phosphorus was
the most important factor for the increase of total available
phosphorus capacity, accounting for 45.70% of the total
increase. This indicated that B. mucilaginosus may have a
strong ability to excrete organic matter that contains phos-
phorus compounds, which is worth further investigation. The
main contributing factor for B. megaterium and P. fluorescens
treatments was microbial biomass phosphorus, accounting for
67.70% and 69.80% of the total increase, respectively. Among
the three PSB treatments, the contribution of adsorbed
phosphorus to the total increase was very small, ranging from
6.30% of B. mucilaginosus treatment to 3.39% of P. fluorescens
treatment, indicating that adsorbed phosphorus was not an
important factor.
The mechanisms of the microbial dissociation of phosphorus
in the lunar regolith simulant. In this study, the mechanisms of
the dissociation of insoluble inorganic phosphorus were explored
from the perspective of the pH of the culture medium and bac-
terial attachment on regolith simulant particles. Considering that
the pH of the culture medium of five PSB treatments remained
basically stable during 7–21 DAI, and the dissociation of inor-
ganic phosphorus is a continuous process, the average pH of 7
DAI, 15 DAI, and 21 DAI were chosen as the independent
variable, the amount of insoluble inorganic phosphorus dis-
sociated by PSBs (DIIP, mathematically equals to the difference
between the total available phosphorus and the initial phosphorus
input) at 21 DAI as the dependent variable, and carried out a
linear fitting analysis (Table 2). the coefficient of determination
was 0.8362 and reached an extreme statistical significance
(p< 0.001, n=16). We believed that the decrease of medium pH
is an important factor for the PSBs to dissociate insoluble inor-
ganic phosphorus in the simulant.
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The relationship between low pH and insoluble inorganic
phosphorus dissociation has been confirmed by a large number of
studies so far. Many studies believe that the acidity enhancement
of the culture medium is an important feature of microbial
phosphorus solubilization54–56. Lin et al. suggested that the
reduction of pH is an important condition for microbes to
dissociate inorganic phosphorus57, although not necessary. In the
culture system, protons come from two sources: one is from the
metabolic activities of cells, such as CO
2
produced by respiration
would form carbonic acid when dissolved in water, or the by-
product of absorption and utilization of NH
4
+58,59; The other is
organic acids produced and secreted by bacteria to the
extracellular environment60–63 (e.g., malic acid, oxalic acid, citric
acid, tartaric acid, etc.). Phosphoric acid ions are released when
protons exchange with mineral crystals. Meanwhile, according to
the research results based on the Earth soil, protons can promote
the decomposition of CaCO
3
in calcitic soil, while Fe, Al oxides,
and hydrating oxides can be decomposed in red soil, thus greatly
reducing the fixation of phosphorus in soil64–66. We believe a
similar process occurred on the lunar surface regolith simulant. In
addition, organic acid ions also facilitate the dissolution of
phosphate minerals through chelating with the metal ions in
Ca
3
(PO
4
)
2
, FePO
4
, AlPO
4
, and other insoluble phosphorus-
containing compounds, resulting in the release of phosphorus
acid ions. This mechanism has been confirmed by experimental
evidence67–70. The type and quantity of organic acids are believed
to be critical to the chelation ability with metal ions71,72, and this
may be the reason that different PSBs differ in their ability to
dissociate insoluble inorganic phosphate in regolith simulants.
In addition, linear fitting analysis was performed between the
amount of attached microbial biomass phosphorus and DIIP at 0,
2, 4, 7, 10, 15, and 21 DAI, with the coefficient of determination
of five PSB treatments ranging from 0.5262 to 0.8597, all reaching
extreme significance (p< 0.001, n=14, 24,19, 19, 23, respec-
tively). These results indicated that the attachment of PSBs to the
regolith simulant particles was also an important factor in the
dissociation of insoluble inorganic phosphorus. Considering that
B. mucilaginosus,B. megaterium, and P. fluorescens that showed
strong phosphorus dissociation ability in this study also had a
strong ability to secrete extracellular sticky substances such as
extracellular polysaccharides73 to form biofilms, we speculated
that these three PSBs may be able to bind themselves with
mineral particles through biological adsorption and developing
biofilms to tightly bind with mineral particles, forming a stable
bacteria-mineral complex74 and a relatively stable extracellular
microenvironment was formed in the biofilm, where bacteria
can better decompose and dissociate mineral particles through
the above-mentioned mechanisms, including protons and
organic acids.
In conclusion, we confirmed that B. mucilaginosus,B.
megaterium, and P. fluorescens have a strong ability to dissociate
insoluble inorganic phosphorus in the lunar regolith simulant due
to their ability to secrete acidic substances and their character-
istics to attach with simulant particles. On the contrary, B. subtilis
and B. licheniformis had poor performance in these two aspects,
so they had little dissociation activity on insoluble inorganic
phosphorus in the regolith simulant. It can be inferred that the
lunar regolith simulants reduced their ability to secrete acidic
Fig. 4 Dynamics of the content and forms of total available phosphorus in five PSB treatments. a–eRepresent B. mucilaginosus, B. megaterium, P.
fluorescens, B. subtilis, and B. licheniformis treatments respectively. fThe contribution ratio of different forms of phosphorus during the change of total available
phosphorus capacity. AII figures in Fig. 4 are plotted according to the average value of different forms of phosphorus, therefore no error bar is applicable.
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substances, thus making them unable to dissociate insoluble
inorganic phosphorus.
Plant growth in the lunar surface regolith simulant treated
with PSBs. Based on the results above, B. mucilaginosus,B.
megaterium, and P. fluorescens were selected for subsequent
experiments on plant growth. The cultivation experiment was
conducted on Nicotiana benthamiana. Most seeds had sprouted
two cotyledons at 6 days after sowing (DAS). At 21 DAS, there
were still 20–40 available plants for each treatment, which could
be used as samples for measurement and analysis.
During the cultivation, seedlings in two control groups showed
obvious growth inhibition and slower growth, compared with
Nicotiana benthamiana planted in horticultural soil. At 24 DAS,
the average fresh weight of whole plants (including root) in blank
and sterilized control groups was only 23.48% and 29.83% of that
in the horticulture soil group, respectively, and all reaching
extreme statistical significance (p< 0.0001, n=56, 33). The
regolith simulant in both control groups, which lacked microbial
treatment, is likely to have some negative effect on the growth and
development of higher plants, or it may just be delayed by the
poor fertility of the simulant.
Other results showed that this negative effect can be eliminated
by PSBs treatment. The rosette leaves diameter at 24 DAS, length
of hypocotyl and radicle of the seedlings at 6 DAS, fresh weight
per plant, and chlorophyll content of Nicotiana benthamiana
plants at 24 DAS (Fig. 6b, c) were measured, as important
indicating indexes of plant growth. It is worthwhile mentioning
ahead that we did not observe a significant difference in rosette
leaves diameter and chlorophyll content in the blank and
sterilized control groups (p=0.0668, 0.3454, n=56), while the
other two indexes had a small difference in their average values
with significance (p=0.0036, 0.0430, n=56). We may conclude
that the nutrients in dead microbes and broth medium of the
sterilized control group had little impact on plant growth in this
experiment, so it was needless to be considered in the subsequent
analysis.
Then, the differences in indexes in different treatments were
examined. Among all four indexes that we had measured, we
would firstly focus on the content of chlorophyll in Nicotiana
benthamiana plant leaves, as chlorophyll plays the most central
role in the light reaction of photosynthesis, and is widely
considered an important indicator of plant growth. It is obvious
in Fig. 6c that the chlorophyll content in the “Pre-cultured for 18
days”treatment is 104.08% higher than that in the sterilized
control, reaching an extreme statistical significance (p< 0.0001,
n=53). Meanwhile, the difference between the “Pre-cultured for
18 days”treatment with the horticultural soil group was not
significant (p=0.1580, n=44). This experimental evidence
shows that the growth of plants in the lunar regolith simulant
pre-cultured by PSBs for 18 days had been greatly improved,
reaching the level that was as good as horticultural soil on Earth.
The fresh weight of the plant in the “Pre-cultured for 18 days”
treatment, which was also significantly higher than that of the
sterilized control (p< 0.0001, n=53), also reflected the result of
the active and efficient photosynthesis.
We observed the overall trend that the growth of plants was
improved as the time of the pre-culture period extended, as
Figs. 6b, c, 7, and the Table 3represent.
The properties of the lunar regolith simulant samples at
different times during the culture process were examined to
confirm that the PSBs promoted plant growth. Similar to the flask
shaking experiment, the pH and OD
600
of the regolith extract, the
available phosphorus content, and the soluble salt content of the
simulant (Fig. 6d–g) was measured. The most noticeable changes
occurred within six days of starting the culture when the OD600
reached its peak and the regolith pH and available phosphorus
Table 2 Results of linear fitting analysis between the amount of DIIP and the pH of the culture medium and the attached
microbial biomass phosphorus.
PSBs Linear fitting equation between DIIP and
the pH
R2Linear fitting equation between DIIP and the attached
microbial biomass phosphorus
R2
B. mucilaginosus y=−220.27x +1838.1 0.8362*** y =1.8696x +139.79 0.8567***
B. megaterium y=1.6485x +12.589 0.8512***
B. subtilis y=4.9942x −148.97 0.5721***
B. licheniformis y=2.4674x −131.53 0.5262***
P. fluorescens y=2.1034x +15.736 0.8597***
Data used in these analyses are provided in Supplementary data, worksheets 20 and 21.
*** indicates p< 0.001.
Fig. 5 Dynamics of dissolved insoluble inorganic phosphorus (DIIP)
content of five PSB treatments. This figure is plotted according to the
average value of different forms of phosphorus, therefore no error bar is
applicable.
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content dropped to their lowest. Based on the results of the
shaking flask experiment, it is easy to understand that the PSBs
firstly used the nutrients in the medium to grow, consuming the
phosphorus in it. The low phosphorus content then promoted
PSBs to synthesize organic acids by using carbon sources in the
medium to reduce regolith pH, thus activating insoluble
inorganic phosphorus in lunar regolith simulant. After 6 days,
the OD
600
dropped and the regolith pH began to rise in all four
treatments, we suspect that this may be due to the consumption
of organic acids by PSBs as a carbon source for growth.
Meanwhile, we observed a steady increase of available phos-
phorus content before 6 DAS, with a regolith pH lower than 7 in
all four PSBs treatments. The phosphorus content was largely
determined by the addition of Murashige & Skoog medium after
6 DAS, which added 1.27 mg/kg phosphorus to the simulant
every 6 days. It could be concluded that microbial reactions in the
cultivation experiment followed the same pattern that we
concluded before in the shaking flask experiment, in which the
acidic substances were the key factor in dissolving inorganic
phosphorus from the simulant.
The result of the “Not pre-cultured”treatment was more
interesting. In this treatment, we observed an extremely
significant decrease in the length of seedlings at 6 DAS, compared
with sterilized control (p< 0.0001, n=22). The average was only
53.28% of that in sterilized control. However, plants in this
treatment did not show such extreme differences with the control
on other indexes, and we are yet unable to explain this decrease
with our data on the properties of soil samples. We may need
more careful examinations to explain this phenomenon.
The content of soluble salt in the regolith was also examined,
but we did not find any related growth inhibition in plants or
PSBs. The Nicotiana benthamiana plants grew better as the time
of the pre-culture period prolonged, while the content of the
soluble salt in the regolith also increased. We predicted that the
positive impact brought in by the pre-culture period, maybe extra
nutrients, or other microbial derived plant hormones,
Fig. 6 The Cultivation experiment of Nicotiana benthamiana.aThe workflow of the whole cultivation experiment, showing all sampling procedures on the
time axis of days before/after sowing. Negative numbers imply the number of days before sowing. bThe measurement results of the Diameter of rosette
leaves at 24 DAS, and the length of the seedlings at 6 DAS. All error bars in Fig. 6 represent Standard Deviation. Significance analyses are conducted using
one-side one-way ANOVA tests. *** indicates p< 0.0001. cThe content of fresh weight of whole plant (root included) and the chlorophyll content of
leaves at 24 DAS in different treatments. d–gThe dynamics of pH and the OD
600
of the regolith extract (regolith-water mass ratio was 1:2), the available
phosphorus content, and the soluble salt content in different treatments during the cultivation. The data points (small points) of different treatment groups
are offset horizontally, with the average value point (large points) as the center, to avoid overlap between data points.
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counteracted the negative impact that soluble salt content has on
plant growth. We look forward to explaining this in the next
phase of research.
The cultivation experiment of Nicotiana benthamiana further
confirmed the ability of PSBs to dissociate insoluble phosphorus
in lunar regolith simulant, making it a cultivation substrate for
higher plants and supporting their growth. The results of the
cultivation experiment showed that the growth of Nicotiana
benthamiana could be most improved by treating the regolith
simulant with PSBs 18 days before sowing. Meanwhile, the
experiment revealed the negative impacts caused by short
treatment of PSBs, which may be explained in further studies.
Application prospect of PSBs for lunar exploration and set-
tlement. Looking beyond the perspective of microbiology and
relooking our study from the perspective of lunar exploration and
settlement, it is not difficult to understand the strong potential of
PSBs to achieve sustainable space agriculture in the BLSS and to
improve the recycling capacity of manned closed ecosystems on
the moon in the further future, without bringing too much bur-
den in the transportation. A lunar base, as a closed, self-sustaining
artificial ecosystem, would require a constant exchange of matter
to keep it from collapsing and to support the survival and daily
activities of human crews. In the simplest model, the Earth
provides all the materials needed for the human crew to survive:
water, food, and air, just as we did in low-Earth orbit when space
exploration just began. As we have argued in the introduction, the
costs of these operations would be acceptable (though still very
high) for low-Earth orbit, but unaffordable for long-term
exploration plans towards more distant targets (e.g., the moon,
Mars, etc.) The introduction of photoautotrophs into an artificial
ecosystem is the first great step to try to resolve this problem. The
Fig. 7 The photography of partial plants in two control groups and four treatments. We selected 11 images for each group of plants, including plants at
the median leaf diameter of each treatment in the middle of the row, as well as five slightly larger and five slightly smaller plants on the left and the right.
Each row of images shares one ruler, which is given in the image on the far right. The identifier of each image is given at the bottom of each image. Other
images are available upon request.
Table 3 Comparison of four growth indexes between Sterilized control(setting the value as 100%) and other treatments.
Treatments Diameter of rosette leaves at
24 DAS
Length of seedling at 6
DAS
Fresh weight per plant at
24 DAS
Chlorophyll content at 24
DAS
Sterilized control 100% 100% 100% 100%
Not pre-cultured 120.86% 53.28% 134.52% 115.52%
Pre-cultured for 6 days 109.62% 126.62% 135.39% 125.80%
Pre-cultured for 12 days 139.49% 155.14% 230.83% 176.80%
Pre-cultured for 18 days 160.86% 147.53% 315.92% 204.08%
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great value of algae15,75,76, or higher plants77, has been repeatedly
demonstrated by studies in various countries for their ability to
purify the atmosphere, and water, and provide food. It has
become a consensus that a well-designed and carefully balanced
BLSS system supported by photoautotrophs is sufficient to meet
the oxygen requirements of human crews over long-term scales,
as well as to fix carbon and partially provide food through pho-
tosynthesis. By doing so, a material circulation system of two
components can be established within the closed artificial eco-
system, reducing the need for material exchange with the Earth.
But, looking at it another way, we are simply converting the
need to transport food, water, and air into the need to build an
extraterrestrial system of plant growth, including cultivation
substrate, nutrients, water, and equipment. Given the huge
requirements of existing designs on transportation capacity and
cost18–25, unless such a system can be self-sustaining over very
long periods, the net benefit remains very poor in the short term.
Introducing bacteria—micro decomposers of nature—will be the
key to solving this problem. Bacteria can transform the most
abundant resource of the moon—surface regolith or rocks, which
has undergone a lengthy space weathering process, into an active,
bio-friendly, and moderately fertile cultivation substrate, elim-
inating the need for complex extraterrestrial hydroponic systems,
and this possibility has been demonstrated by our experiments.
The even more exciting thing is that the applications of PSBs
also leave room for organic integration in another area of
microbial-mediated technology: soil-like substrate. The soil-like
substrate is a technology that uses organic waste including food
residues, human excrement, and plant residues to produce
cultivation substrate which is similar to earth horticultural soil
and requires the participation of microbial fermentation and soil
animals (e.g., earthworms78). Basically, it provides a method to
recycle organic wastes in the extraterrestrial closed ecosystem.
From the perspective of means of making cultivation substrates,
soil-like substrates still have a series of shortcomings. For
example, soil-like substrates often experience a large degree of
dry matter mass loss during the producing period78,79, making
them unable to cope with long-term plant cultivation needs.
However, in conjunction with the process by which PSBs treat the
lunar surface regolith, we can imagine like this: In a future lunar
base, all organic waste is composted in closed containers. The
compost products and the inoculation solution of PSBs will be
applied to untreated lunar surface regolith, which has been pre-
sifted to proper particle size. Here, PSBs use organic matter in
compost products as a nutrient source and decompose mineral
crystals in the regolith to further enhance the fertility level of the
regolith. Meanwhile, compost products, as an organic fertilizer,
increased the carbon content of lunar regolith, improving its
physical and chemical properties and avoiding its negative
influence of low output. By doing so, the demand for exogenous
nutrient solutions in culturing PSBs and cultivating higher plants
has been eliminated. It is reasonable to speculate that, in
combination with the PSBs process and the soil-like substrate
fermentation, the lunar regolith will become a cultivation
substrate that is very similar to horticultural soils on the Earth
and suitable for the growth of most agricultural higher plants.
This would greatly facilitate practices of space agricultural
systems in lunar bases without additional transportation
requirements.
However, considering the concerns on the potential biosafety
of microorganisms, it is necessary to conduct a thorough
pathogenic examination of any microorganisms introduced into
an extraterrestrial closed artificial ecosystem to ensure that their
biosafety risks to other organisms are fully controlled and do not
pose a threat to human crews. Synthetic biology may provide a
useful opportunity to create artificial life with efficient
phosphorous decomposition abilities by molecular biological
methods based on understanding the mechanism by which PSBs
and other microbial fertilizers improve the fertility of lunar
regolith, and ensure its existence being strictly restricted to
extraterrestrial closed artificial ecosystems.
In conclusion, our study shows that B. mucilaginosus,B.
megaterium, and P. fluorescens, can tolerate lunar regolith
simulant conditions and effectively dissociate insoluble inorganic
phosphorus in the simulant, improving the fertility of the
simulant making it a good cultivation substrate for higher plants.
We have therefore proved that it is feasible for PSBs to improve
lunar regolith, and PSBs have great application value and
prospects for future space exploration.
Methods
Lunar surface regolith simulant materials. The lunar surface
regolith simulant used in this study was modeled from the CAS-1
lunar soil simulant developed by Zheng et al. 80, and the reference
object was the lunar low-titanium basalt regolith. According to
the study of Zheng et al., the scoria of Jinlongding Volcano in the
Longwan volcanic Group at the western foot of Changbai
Mountain (located near Huinan County, Jilin Province, PRC) has
a very similar chemical composition and physical structure to the
lunar low-titanium basalt regolith and contain volcanic glass to a
degree of around 40%, which has a good simulation effect on the
lunar regolith. The initial material of the lunar regolith simulant
used in this study was collected from about 3 km east of the
Jinlongding volcanic cone, and the thickness of the scoria layer at
the sampling site was about 2 m and the sampling depth was
1.5 m. The surface of the sampling site was protected by thick
vegetation, thus preventing the scoria from the direct wash of
rain. The collected scoria is black porous trachyte basalt, about
1–2 cm in diameter. Scoria was then sufficiently broken up and
sifted to produce simulants with a range of particle sizes. In the
pre-study, it was found that: <0.3 mm simulation was easy to
cause turbidity in the shaking flask culture, which added difficulty
to analysis; In addition, the study of Paul et al. 33. showed that
when the soil with a large proportion of fine particles was used in
the cultivation of Arabidopsis, the soil showed a poor porosity
and water permeability, which could easily lead to heavy soil
viscosity and plant root dysplasia. Therefore, the particle size of
the simulant used in this study was 0.3–0.5 mm.
The samples were then digested and the mass ratio of Fe, Ca,
Mg, Na, K, P, and Mn was determined using an atomic flame
spectrophotometer. Three replicates of our samples were used for
measurement. This part of the determination was completed by
the Experimental Center of the College of Agriculture, China
Agricultural University.
Bacteria materials. The PSBs used in this study were selected
based on their ability to activate insoluble inorganic phosphorus
in Earth soils. According to the current research results81–88,five
PSBs were used in this study: B. megaterium (AS1.217), B. subtilis
(CMCC 63501), P. fluorescens (ATCC 13525), B. licheniformis
(ATCC 11946), and B. mucilaginosus (AS1.232), which are
commonly used as microbial fertilizers. All PSBs were provided
by the Shanghai Bioresource collection center. The PSBs were first
inoculated on agar medium plates for activation. After an incu-
bation of 24 h at 30 °C, single colonies with typical morphological
characteristics were picked and inoculated into 30 ml of liquid
medium for expanded culture. The strains were cultured over-
night in a shaking flask at 180 rpm and 30 °C. Subsequently, the
OD
600
value of the culture was adjusted to 0.6–0.8 with sterile
water (0.15 for B. mucilaginosus, because its cells were more
transparent), and the CFU of the solution was about 1 × 108,
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which could be used as the inoculation solution for subsequent
experiments. It should be noted that the culture medium for B.
mucilaginosus was silicate bacteria medium (glucose 5 g/L,
MgSO
4
0.5 g /L, CaSO
4
·2H
2
O 0.1 g/L, Na
2
HPO
4
2 g/L, FeCl
3
0.005 g/L, pH =7.0), as the provider of B. mucilaginosus suggest
that it would have maximum growth in silicate bacteria medium.
The medium for other PSBs was the LB medium. Solid medium
was made by adding a 2% mass ratio of agar to the liquid med-
ium. All mediums were sterilized at 115 °C for 15 min and tested
for sterility before use.
Plant materials. The seeds used in the cultivation experiments
were provided by Professor Ronghui Pan, Zhejiang University,
PRC. The seeds were collected in June 2022.
Standard test for the ability of microbes to dissolve insoluble
inorganic phosphorus. To ensure the validity of the experiment,
the ability of the five strains to dissolve insoluble inorganic phos-
phorus was tested using standard methods before inoculating them
with lunar regolith simulant. The inoculation solution of five
strains was inoculated to 30 ml liquid medium (glucose 10 g/L,
(NH
4
)
2
SO
4
0.5 g/L, Ca
3
(PO
4
)
2
5 g/L, NaCl 0.3 g/L, FeSO
4
0.03 g/L,
KCl 0.3 g/L, MgSO
4
0.3 g/L, MnSO
4
0.03 g/L, pH =7.0), using
sterile water as control, shaking flask culture at 30 °C, 180 rpm for
7 days. The culture medium was sampled right after the inoculation
and after the culture process. The soluble phosphorus content was
measured by the Mo-Sb colorimetric method.
Shaking flask experiment of PSBs. A suitable microbial culture
system was established after a series of preliminary experiments.
The culture medium included two parts: the solid phase and the
liquid phase. The solid phase consisted of 40 g of the lunar surface
regolith simulant (0.3–0.5 mm) which was pre-dried to constant
weight at 70 °C in a 150 ml flask. The liquid phase consisted of
80 ml glucose broth medium (peptone 10 g/L, yeast extract 3 g /L,
NaCl 5 g/L, glucose 5 g/L, pH =7.0). The preliminary test showed
that the growth of B. mucilaginosus was poor when the LB
medium was used (only the change of growth curve was con-
sidered). However, all five PSBs had better growth performance
when using a glucose broth medium. Therefore, to ensure the
uniformity of test conditions, 5 PSBs were cultured with glucose
broth medium. The preliminary experiment also found that
mixing the solid phase with the liquid phase before using steam
sterilization would cause obvious Browning of the medium, and
the sterilization effect was poor (the reason was unknown).
Therefore, we first sterilized the solid phase at 180 °C dry heat for
3 h, then steam-sterilized the liquid phase at 115 °C for 15 min,
and then mixed 80 ml of liquid phase with 40 g of solid phase
within an ultra-clean workbench, and sealed a sealing film of air-
permeability. Before inoculation, the mixed culture medium was
cultured at 30 °C and 180 rpm for 24 h to check whether it was
contaminated. The culture medium without contamination will
be inoculated with 0.1 ml inoculation solution, and continue
shaking culture under the above conditions. In the control groups
of the growth experiment, the simulant was not added to the
liquid phase. In the control group of the phosphorus measure-
ment, the medium mixed with simulant was not inoculated with
PSBs. Each treatment included four flasks as replicates.
We have conducted a series of pre-experiments with different
amount of regolith added to the culture, including half of the
weight of the culture to ten times of the weight of the culture. We
observed that: when the amount of regolith was more than the
culture, shaking the culture to maximum the microbial explosure
to the oxygen was impossible. Manual stirring of the culture may
introduce subjective errors, like the time or the degree of the
stirring procedure.
An extreme is adding culture to the regolith to a water ratio of
~20%. The moisture in the regolith allows the regolith to form a
relatively loose structure, which is conducive to microbial
respiration. However, fewer culture medium prevented micro-
organisms from further growing, and the regolith was treated less
adequately, as we have observed in the preparation of cultivation
substrate. The other extreme is adding a small amount of regolith
to the culture, which was described in reference 39 & 41. We
believed that such treatments are less practicle to be performed on
the moon, as water is predicted to be very rare and valuble on the
moon. Thus, we finally chose the ratio of 1 : 2, which enables the
microbe to be shaking-flask cultured and also a more adequate
treatment of regolith by PSBs.
We did not perform toxicity test, as we read from reference 39
& 41 that untreated lunar regolith simulant are “inert”and less
likely to affect biological activities, neither positive nor negative.
In fact, we observed from our experiment that the regolith
simulant treated with different PSBs may show different impact
on microbial growth with different mechanisms (salt content,
pH), at a addition ratio of 1 : 2. We may study this toxicity more
systematically in the future.
Sampling method of the shaking flask experiment. We devel-
oped and optimized a complex but meticulous sampling method
for a solid-liquid mixture shaking flask culture system. Corre-
sponding to the culture medium, the sample is divided into two
kinds including solid sample and liquid sample. Considering that
both solid and liquid phases have experienced sufficient shaking
during the culture process, we believe that the solid and liquid
phases have been fully mixed, and their chemical compositions
and properties are uniform in space. Before sampling, the culture
flask stopped shaking and was let still for about 1 minute to make
a full precipitation of the solid phase, separated from the liquid
phase completely. Then transfer the flask to the ultra-clean
workbench for sampling.
Liquid sampling. Transfer 500 μl of the culture to a marked and
sterilized centrifuge tube with a pipette; Dilute the sample if it is
necessary for a subsequent determination, and modify the effect
of dilution on concentration during analysis.
Solid sampling. Use a sterilized micro U-shaped medicine
spoon to scoop 2 g of the precipitated lunar regolith simulant at
the bottom of the flask and put it into a marked and sterilized
centrifuge tube.
Sampling schedule. Solid and liquid samples were sampled at 0,
2, 4, 7, 10, 15, and 21 DAI. Additional liquid samples were
sampled at 0, 10, 17, 26, 32, 48, 60, 72, 84, and 96 HAI, and 5, 6, 7,
8, 9, 10, 15, and 21 DAI for the determination of growth curves.
Sample storage. Both liquid and solid samples were stored at
4 °C and analyzed within 12 h after sampling.
Laboratory measurements of samples. The detailed methods of
laboratory measurements, including the measurement of dis-
solved inorganic phosphorus, dissolved organic phosphorus,
microbial biomass phosphorus, suspended microbial biomass
phosphorus, attached microbial biomass phosphorus, and
adsorbed phosphorus of five PSB treatments, are provided in
Supplementary Measurement Methods.
Preparation of cultivation substrate. In the cultivation experi-
ment, the substrates of experimental treatments were treated with
PSBs before sowing. According to the shaking flask culture
experiment results, B. mucilaginosus,B. megaterium, and P.
fluorescens were selected as treatment strains.
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COMMUNICATIONS BIOLOGY | ( 2023) 6:1039 | https://doi.org/10.1038/s42003-023-05391-z | www.nature.com/commsbio 13
Content courtesy of Springer Nature, terms of use apply. Rights reserved
We start the preparation from the inoculation solution. The three
strains mentioned above were cultured overnight in a shaking flask
at 180 rpm and 30 °C (described in Bacteria Materials). Subse-
quently, the CFU of the solution was adjusted with glucose broth
medium (described in the Shaking flask experiment of PSBs) to
about 1 × 108, and then the culture of three PSBs were mixed at a
volume ratio of 1:1:1. The mixture would be used as the inoculation
solution for subsequent experiments.
The experimental treatment was divided into four treatments
(Not pre-cultured, and pre-cultured for 6, 12, and 18 days) with
12 replicates each. Twelve 50 ml bottles were filled with 20 g of
sterilized lunar surface regolith simulant, 0.1 ml mixed inocula-
tion solution was added at a 0.5% mass ratio, and 1 ml glucose
broth medium was added at a 5% mass ratio. Then add sterilized
ddH
2
O to the water ratio of 20%. The medium was mixed
thoroughly and evenly, sealed with a sealing film of air-
permeability, and incubated at 30 °C away from light. The
simulant was remixed every 2-3 days so that the phosphorus
solubilizing bacteria were fully exposed to oxygen for normal
respiration. Three bottles were taken out at 0 DAI, 6 DAI, 12
DAI, and 18 DAI, respectively. They were then immediately filled
to a 24-well plate for further cultivation. Twelve of the 24 wells
are filled alternately. Each well was filled with 4.0 g of simulant
(wet weight).
The control group included blank control and sterilized control
with 12 replicates each. In the blank control, only sterilized water
was added. In sterile control, 1 ml glucose broth medium and
0.1 ml mixed steam-sterilized inoculation solution were added to
the lunar surface regolith simulant for every 20 g. Two control
groups were not pre-cultured.
Nicotiana benthamiana cultivation experiment. A 24-well cul-
ture plate was used as the cultivation vessel with a diameter of
17.5 mm and a depth of 17.5 mm for each well. The substrate is
saturated by wetting it with sterilized water until a very thin layer
of water can be maintained on the surface. Nicotiana benthami-
ana seeds were sterilized and 6-7 seeds were sown in each well.
Cover the culture plate with the cover, and promote germination
in the artificial climate room. The growth conditions were as
follows: temperature 24 °C, relative humidity 70%, light intensity
130 μmol/(m2·s), photoperiod 16/8 h. Nicotiana benthamiana
seedlings were artificially thinned after germination at 6 DAS,
remaining 2–4 plants for each well. The thinning was random, to
avoid the influence of subjective criteria on experimental results.
From 6 to 24 DAS, 30 μl Murashige & Skoog medium was added
to each well every 6 days. The plants were watered daily until the
substrate was basically saturated.
The sampling method of the cultivation experiment. The
samples of the cultivation experiment were divided into two parts:
regolith samples and plant samples. Regolith samples were taken
with a disposable single-use plastic sampling tube. When sam-
pling, the tube would be inserted directly into the bottom of the
soil, and ~0.3 g regolith substrate would remain in the tube when
the tube pulls out. The samples would be marked, stored at 4 °C,
and analyzed within 2 days.
Plant samples are taken at 6 and 24 DAS. When sampling, the
root of the plant would be carefully removed from the soil, and
washed with ddH
2
O.
The detailed methods of laboratory measurements are
provided in Supplementary Materials, Measurement Methods.
Software. In this study, all the original data determined by
Multiskan GO (Thermo Fisher Scientific) were outputted and
stored by SkanIt software. SPSS and Excel are used for data
sorting and simple statistical description. The correlation analysis,
linear regression analysis, and significance analysis of data are
completed in SPSS. OriginLab is used in data visualization.
Statistics and reproducibility. The numbers of data used in the
analyses (n) are shown together with the p-value.Inthisstudy,we
used four replicates for the culture of PSBs and twelve for the
cultivation of Nicotiana benthamiana. However, due to pollution or
other factors, some replicates were stopped from culture or culti-
vation. The decision was made on the status of each replicate,
including the color of the culture, spontaneous precipitation of cells,
growth of plants, or data from the measurement. The full
description of replicate numbers (and corresponding data numbers
used for analysis) at each time of sampling is provided in Supple-
mentary Table 1. In our study, the average value and the standard
deviation of the data were calculated. When comparing differences
between data groups, one-way ANOVA tests were performed.
Reporting summary. Further information on research design is
available in the Nature Portfolio Reporting Summary linked to
this article.
Data availability
Data supporting the findings of this work are available within the paper and in the
Supplementary files. Source data forfigures and tables is included in the Supplementary
Data. The data sets generated and analyzed for this study are available from the
corresponding author upon request.
Received: 18 April 2023; Accepted: 26 September 2023;
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Acknowledgements
This study is funded by the Undergraduate Research Program of China Agricultural
University (X2022100190381) and the Research Program of the National Key Laboratory
of Human Factors Engineering, Astronaut Center of China (HFNKL2023W08). We
thank Professor Qimei Lin, College of Environmental Sciences of China Agricultural
University for his support and recommendation, and his introduction of Associate-
Professor Zhencai Sun as our supervisor and responding author. We also thank Professor
Ronghui Pan and Hao Du, College of Agriculture and Biotechnology of Zhejiang Uni-
versity, for their permission to perform cultivation experiments in their lab. We thank
Yike Zhang, an undergraduate student at China Agricultural University, for her support
in the measurement of plant samples.
Author contributions
Y.X. conceptualized the experiment and took the lead in writing the manuscript as a team
leader. Y.Y. and C.L. developed the methodology and accomplished the culture
experiment of PSBs. Y.X. carried out the cultivation experiment of plants. Z.S. guided all
other authors to ensure the scientificity and strictness of the project and helped to
improve the design of experiments as a supervisor. All authors (Y.X., Y.Y., C.L., and Z.S.)
contributed to funding the project operations and writing the successful proposal. Y.X.
accomplished the statistics and data visualization of the project. All authors contributed
to the review and editing of the manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s42003-023-05391-z.
Correspondence and requests for materials should be addressed to Zhencai Sun.
Peer review information Communications Biology thanks Burcu Alptekin, Adnane
Bargaz and the other, anonymous, reviewer(s) for their contribution to the peer review of
this work. Primary Handling Editor: David Favero. A peer review file is available.
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