Environmental Science and Pollution Research (2022) 29:3171–3183

https://doi.org/10.1007/s11356-021-17164-4

REVIEW ARTICLE

Storage of soil microbiome for application in sustainable agriculture:

prospects and challenges
Annapurna Bhattacharjee1 · Shubham Dubey1 · Shilpi Sharma1

Received: 2 September 2021 / Accepted: 19 October 2021 / Published online: 31 October 2021
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021

Abstract
Soil microbiome is a dynamic micro-ecosystem driving and fine-tuning several biological processes in the global macro-
ecosystems. Its tremendous potential towards mediating sustainability in the ecosystem necessitates the urgent need to store
it optimally and efficiently as “next-generation biologicals” for future applications via soil transplantation. The challenge,
therefore, is to devise a strategy for the storage of soil microbiome such that its “functionality” is preserved for later applica-
tion. This review discusses the current endeavours made towards storage of the soil microbiome. The methods for assessing
the integrity of soil microbiome by targeting the structural diversity and functional potential of the preserved microbiomes
have also been discussed. Further, the success stories related to the storage of fecal microbiome for application in trans-
plants have also been highlighted. This is done primarily with the objective of learning lessons, and parallel application of
the knowledge gained, in bringing about improvement in the research domain of soil microbiome storage. Subsequently,
the limitations of current techniques of preservation have also been delineated. Further, the open questions in the area have
been critically discussed. In conclusion, possible alternatives for storage, comprehensive analyses of the composition of the
stored microbiome and their potential have been presented.

Keywords Soil microbiome · Storage of microbiome · Fecal microbiome · Structural diversity · Sustainable agriculture

Introduction determinant of plant fitness. Involvement of the plant-

Soil microbiome as “next‑generation biologicals”
The next-generation sequencing tools have revolutionized
environmental microbiology by unravelling the microbi-
ome from various compartments on the earth and beyond.
In the current times, with an emphasis on sustainability, the
soil microbiome has emerged as a major driver of various
ecosystem processes like carbon sequestration, buffering
the adverse impact of climate change, and nutrient cycling
(Jacoby et al. 2017; Jansson and Hofmockel 2020; Song
et al. 2020). Soil is also the most important source of the
plant-associated microbiome, the latter being the major
Responsible Editor: Kitae Baek
* Shilpi Sharma
shilpi@dbeb.iitd.ac.in
1
Department of Biochemical Engineering and Biotechnology,
Indian Institute of Technology Delhi, Hauz Khas,
New Delhi 110016, India
associated microbiome, considered the second genome to
a plant (Berendsen et al. 2012), has been explored for vari-
ous aspects of plant development, stress management, and
disease control (Lakshmanan et al. 2014; De Corato 2019;
Arif et al. 2020). Correlation between soil health and human
health has also been drawn (Brevik et al. 2020), with the
microbiome being considered an efficient marker of health
in both systems. Interaction networks have been established
between diverse ecosystems like plant and rumen microbi-
omes as well (Attwood et al. 2019).
Traditionally, the potential of individual soil microbes,
and their consortia, has been harnessed for various bio-
technological applications. However, to circumvent the
major constraint of their application, that is their limited
survival and efficiency in real-life systems, there has been
a need to turn towards employing microbiomes instead
(Ray et al. 2020). The microbiome is expected to be more
robust and sturdy, with a tightly regulated web of interac-
tions contributing to its efficiency. The potential of soil
microbiomes in attaining sustainability in agriculture by
enhancing plant attributes and mitigation of stresses has

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been explored extensively in recent times by employing the
now, well-established approach of rhizospheric engineering
(Mueller and Sachs 2015; Mitter et al. 2019; Morella et al.
2020; Anand et al. 2021; Zhang et al. 2021). Transfer of
soil microbiome has also been successfully performed for
plant protection (Wei et al. 2019). Besides, soil microbiomes
have found application in other sectors like bioremediation
(Bell et al. 2016; Breister et al. 2020; Ying and Wei 2019)
and plant microbial fuel cells (Nitisoravut and Regmi 2017)
(Fig. 1). Hence, soil microbiomes have emerged as “next-
generation biologicals” with a huge potential to usher in
agricultural sustainability (Compant et al. 2019; Mitter et al.
2019).
Storage of soil microbiome
The promising potential of soil microbiome in agricultural
sustainability, besides other benefits, has necessitated the
urgency to explore and strategize suitable methods for its
preservation, which can ensure efficient future applica-
tions. Further, with current emphasis on United Nations’
sustainable development goals, recent initiatives for genera-
tion of national soil repositories, soil archives, microbiome
biobanks, and plant microbiome vaults, based on “minimal
effective microbiome set” (Manter et al. 2017; Gopal and
Gupta 2019; Benucci et al. 2020; Ryan et al. 2021), have
also emphasized the need to devise efficient means to store
microbiomes. The concept of soil transplantation has been
investigated in only a handful of studies, wherein the poten-
tial of fresh soil microbiome application has been explored
(Yergeau et al. 2015; Howard et al. 2017; Anand et al. 2021).
However, to the best of our knowledge, no study to date has
envisaged the potential of soil microbiome transplant from
preserved samples in the context of sustainable agriculture.
Fig. 1  Modes of application of soil microbiome for different ecosys-
tem services
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This is possibly because efforts to design an optimum strat-
egy for storage of soil microbiome to retain its functionality
are still in its infancy (Tatangelo et al. 2014; Pavlovska et al.
2021). Compared to the soil microbiome, the application
of which has been relatively recently realized, the practice
of fecal microbiome transplantation for improved human
health (Kim and Gluck 2019), has been under practice for
a relatively long time. Hence, parallels can be drawn from
the strategies adopted for the storage of fecal microbiomes,
on the common ground of preservation of microbiomes in a
matrix to retain its functionality.
Scope of the review
The present review is an attempt to critically compile the ini-
tiatives to date towards the preservation of soil microbiomes.
Storing of soil samples for assessing microbial community
diversity at a later time point (other than the sampling point),
as done routinely by environmental microbiologists, does not
fall within the review’s focus. It is targeted towards the stor-
age of soil microbiome to retain its functionality for future
soil transplantation. Next, it highlights the efforts made
towards devising storage methods for the preservation of
fecal microbiomes, from which lessons can be learnt towards
designing more potent soil microbiome storage strategies.
The approaches employed to comprehend the changes in
microbial community structure and functional potential of
both soil and fecal microbiomes have been critically pre-
sented. The impact of preservatives and amendments on the
storage of soil and fecal microbiome samples has also been
delineated. Eventually, the limitations of the present state
of the art, open questions, and future strategies for improve-
ment in the preservation approaches have been proposed.
Overall, this review underlines the importance of designing
robust storage strategies for microbiomes to enhance sustain-
ability, and eventually improve environmental and human
health.
State of the art in the storage of microbiome
Since the advent of microbial biotechnology, several
approaches have been successfully employed to preserve
various microbes for crucial biotechnological applications.
This has been exhaustively reviewed by Prakash et al. (2013,
2020). Scientists are now probing into the immense poten-
tial of the soil and plant microbiomes to facilitate the next
Green Revolution (Qiu et al. 2019; Hirt 2020). While syn-
thetic/artificial consortia have been designed using a limited
number of culturable microbes to mimic the functionality
of microbiomes, it is far from attaining the same efficiency.
A pre-requisite for widespread application of microbiome
transplant is to have a long-term method of storage, which
Environmental Science and Pollution Research (2022) 29:3171–3183
minimizes the loss of functionality of microbiomes. In the
following sections, the present state of art in the storage
of soil and fecal microbiomes, and subsequent analyses for
assessing the microbiome’s structure and functional poten-
tial have been discussed.
Status of storage of soil microbiome
When immediate analysis is not performed/required after
sampling, soil preservation at low temperatures has been
routinely practiced by molecular microbiologists to assess
the microbial community structure (Table 1). Compared to
storage at room temperature and at 4 °C, samples frozen
at − 20 °C and − 80 °C have been reported to reflect the
microbial community structure better (Rubin et al. 2013;
Weiβbecker et al. 2017). Such assessments of the structure
of a soil microbial community have been performed using
stable markers like fatty acids and nucleic acids, which origi-
nate from both dead and living cells in the stored samples.
Hence, freezing has been considered an efficient method to
answer majorly two critical questions in soil microbiome
research, viz. who are there, and how many are there. In an
attempt to extract DNA from only viable cells after storage
at 4 °C, − 80 °C, and upon air drying, Ouyang et al. (2021)
reported minimal deviations in samples stored for a short
duration at 4 °C. The strong physiological stress to microbes
when frozen, was proposed as the reason for significant
shifts in community composition in these samples. This
questions the potential of conventional methods of freezing
for storage of soil with the objective of microbiome trans-
plantation, the latter requiring a functional microbiome.
When answering the more pertinent questions determin-
ing the application of soil microbiome, viz. how many are
active, and what are they doing, it was realized that com-
pared to frozen soil, fresh soil is the best in terms of meta-
bolic activity (Cui et al. 2014). Till recently, the evaluation
of the functional potential of the microbiome has been per-
formed by relatively conventional methods like quantifi-
cation of enzyme activities in stored soil samples. While
estimation of enzyme activity has been indicative of the
metabolic status of soil samples, there are also contradic-
tory results in terms of the impact of storage conditions on
the metabolic markers. Abellan et al. (2011) demonstrated
that freezing at − 20 °C lowered the enzyme activity of soil
samples compared to storage at 4 °C. On the other hand,
DeForest (2009) did not find a significant difference between
enzyme activities of soil samples stored at 4 °C and − 20 °C.
However, he emphasized that such an observation is site-
and enzyme-specific. The impact on enzymatic activities has
been found to be stronger upon drying the soil samples as
compared to freezing (Peoples and Koide 2012).
Simultaneous analysis of microbial community structure
by fingerprinting and estimation of activity by respirometry
3173
to compare three storage conditions, i.e., air drying, 4 °C,
and − 20 °C for a period of 1 year, by Marti et al. (2012)
revealed alterations under all conditions. However, depend-
ing upon the soil type, the most appropriate storage method
differed; air drying proved to be a better approach for dry
soil, while freezing was found to be more efficient for clayey
soil type. Similarly, upon comparing a flooded and drained
paddy soil stored at 4 °C and − 20 °C, the total phospholipid
fatty acid (PLFA) was mostly unaffected, and so was the
microbial community structure analyzed by next-generation
sequencing (Wang et al. 2015). However, community level
physiological profiling (CLPP) by MicroResp™ was signifi-
cantly affected by the storage conditions, and the extent of
alterations was dependent on the soil type. With reports on
the effect of storage conditions on different forms of nitrogen
(Wu et al. 2018) and soil labile carbon (Sun et al. 2015),
such a dependence of efficiency of storage method on soil
type is expected (Cui et al. 2014; Benucci et al. 2020).
The use of various additives for better preservation of soil
microbiome has been tested. Storage of soil samples after
the addition of liquid-based preservatives, LifeGuard™, and
DMSO-EDTA-saturated salt (DESS) solution was compared
with samples stored without any additives (Tatangelo et al.
2014). Terminal restriction fragment length polymorphism
(T-RFLP) was employed to assess the changes in the
microbial community structure. DESS was found to be
effective in preserving the microbial community, with
profiles similar to that obtained after immediate processing
of samples. The proprietary liquid, LifeGuard™, on the
other hand, showed a reduction in the number of terminal
restriction fragments (TRFs). Interestingly, a similar number
of TRFs were detected in samples stored at 4 °C and 30 °C
without preservatives, and freshly analyzed samples. This
was postulated to be because of maintenance of temperature
identical to ambient, during storage and shipping. Even
Brandt et al. (2014) did not observe differences in microbial
community structure, which was assessed by T-RFLP and
quantitative PCR of markers (using both DNA and RNA
extracts) between soil samples stored without additives at
room temperature, 4 °C, − 22 °C, and − 80 °C for a short
duration of 11 days. Similarly, for a storage duration of
14 days, Lauber et al. (2010) reported similarity in the
relative abundance of most taxa analyzed by next-generation
sequencing, when soil samples were stored at 20 °C,
4 °C, − 20 °C, and − 80 °C without any cryoprotectant.
DESS proved to be more efficient than DNA/RNA Shield™
(Zymo Research), in preserving the microbial community
structure in terms of being closer to the optimal treatment
of snap freezing in liquid nitrogen (Pavlovska et al. 2021).
Another proprietary liquid, RNAlater®, has been shown
to adversely affect the microbial community composition
when used for storing soil samples (Schnecker et al. 2012;
Delavaux et al. 2020).
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13
Table 1  Methodological details of storage of soil microbiome and post-storage assessment
S. no Sample processing Additive Temperature of storage Duration of storage Assay for structural integ- Functional assay for Reference
rity of microbiome microbiome

Without additives
1 Sieved and homogenized None Dried at RT, − 20 °C 28 days None Enzymatic activity esti- Peoples and Koide 2012
mation
2 Sieved and homogenized None 25 °C, 4 °C, − 20 °C 30 days None Measurement of different Wu et al. 2018
forms of N, and micro-
bial biomass
3 Air dried, homogenised, None Air dried and kept at RT, 30 days None Enzymatic activity esti- Abellan et al. 2011
and sieved 4 °C, − 20 °C mation
4 Sieved and moistened None Air dried, 4 °C, − 20 °C 30 days PLFA estimation, Metabolism assessed by Wang et al. 2015
and 16S rRNA gene MicroResp™ technique
sequencing
5 Sieved and homogenized None Air dried, 4 °C, − 20 °C 30 days None Measurement of water Sun et al. 2015
soluble organic content
6 Homogenized and sieved None Air dried and kept at 2 months 16S/18S rRNA PCR- Biolog™ analysis Cui et al. 2014
4 °C), − 20 °C (with DGGE profiling
field moisture)
7 Sieved and homogenised None Air dried and kept at RT, 12 months 16S rRNA PCR-DGGE Heterotrophic aerobic Marti et al. 2012
4 °C, − 20 °C) profiling microbial activity esti-
mation by respirometry
8 None None Air dried 20 years 16S rRNA gene sequenc- None Benucci et al. 2020
ing

Environmental Science and Pollution Research (2022) 29:3171–3183

With additives
9 Sieved and homogenized Freeze dried samples kept 4 °C, − 80 °C, RT 7 days 16S and 18S rRNA gene None Weißbecker et al. 2017
with blue silica gel sequencing
10 Homogenized and filtered LifeGuard™, 4 °C, 20 °C, 30 °C 30 days Terminal restriction frag- None Tatangelo et al. 2014
on nitrocellulose mem- DMSO-EDTA-saturated ment length polymor-
brane salt (DESS) solution phism of 16S rRNA
gene fragments
11 Homogenized DNA Shield™, RNA 4 °C, − 20 °C, 23 °C 30 days 16S rRNA gene sequenc- None Pavlovska et al. 2021
Shield™, DESS solu- ing
tion
12 Homogenized and sieved Tween-20, sodium deoxy- 4 °C, − 80 °C, air dry 33–35 days 16S rRNA gene sequenc- Cell viability assessed Ouyang et al. 2021
cholate and Nycodenz® ing by flow cytometry and
fluorescence staining
13 Homogenized RNAlater® 4 °C, − 20 °C 40 days None Phospholipid fatty acid Schnecker et al. 2012
analysis
RT, 4 °C, liquid N
­ 2 60 days 16S and18S rRNA gene None Delavaux et al. 2020
sequencing and qPCR

RT room temperature
Environmental Science and Pollution Research (2022) 29:3171–3183
In a unique approach for preserving the microbiome,
Howard et al. (2017) mixed soil and soil wash with sterile
commercial potting mix. However, upon analysis of bacte-
rial and fungal communities after storage for 3 weeks, they
advised direct soil transfer to be better than storage, albeit
each method exhibited a distinct impact on microbial com-
munity diversity. A relatively novel approach of cell free
alive system (CAS) for samples from sub-sea floor sediments
has been attempted since it ensures better cell viability in
stored samples, but its feasibility for practical application
needs to be assessed (Morono et al. 2015).
Lessons to be learnt from approaches for storage
of fecal microbiome
In recent times, there has been an increasing acceptance of
fecal microbial transplantation (FMT) for the treatment of
different diseases (Giles et al. 2019; Gundling et al. 2019).
Hence, the protocols of processing and efficient storage of
fecal samples have received tremendous attention (Table 2).
Analogous to the requisite of retaining functionality after
storage of soil samples for its transfer, the process of FMT
also needs a metabolically active microbiome. Notably, the
storage of microbiomes from both systems is performed in
their respective matrices. So, for developing an efficient
strategy for storage of soil microbiome, lessons can be learnt
from the exhaustive recent literature available for storage of
fecal microbiome (Wu et al. 2019; Nicco et al. 2020).
For transporting the samples from the collection site to
the laboratory, and even for short-term storage, a cold envi-
ronment (4 °C) has been recommended (Tedjo et al. 2015;
Burz et al. 2019; Wu et al. 2019; Liang et al. 2020). For a
longer duration of storage, freezing at − 20 °C or − 80 °C
has been considered a gold standard for FMT (Vandeputte
et al. 2017; Ma et al. 2020; Nel Van Zyl et al. 2020). In a
large-scale study conducted by Allegretti et al. (2020), the
FMT was tagged as robust, with little impact of processing
time and storage duration, by testing its clinical effectiveness
in treating Clostridioides difficile infection after storing the
fecal microbiome at − 80 °C. However, the study did not
compare the use of preservatives, and different storage con-
ditions. Using equine feces, Gavriliuc et al. (2021) demon-
strated a similar efficiency of storage at − 20 °C and − 80 °C
for microbiome-based studies. Interestingly, when long-term
storage (18 months) of fecal samples at − 80 °C was assessed
by differentiating the microbial community composition of
total DNA extracted from the samples versus DNA from
only intact bacterial cells, the latter showed greater dissimi-
larities with the fresh samples (Dorsaz et al. 2020).
Additives like 95% ethanol, guanidine, dimethyl sul-
phoxide (DMSO)-EDTA, DMSO-EDTA-NaCl, eNAT®
medium, RNAlater®, and DNA/RNA Shield™ have been
shown to preserve the taxonomic diversity using DNA and/
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or RNA as markers (Vogtmann et al. 2017; Ribeiro et al.
2018; Byrd et al. 2019; Ezzy et al. 2019; Moossavi et al.
2019; Young et al. 2020; Kazantseva et al. 2021). However,
evaluation at the level of metabolome brings forth a different
picture. RNAlater® has been discouraged for use as a stor-
age medium, since it induces significant alterations in stored
samples, as observed using integrated omics approaches
(Hickl et al. 2019). Compared to the functional profiles of
proteins, taxonomic differences were reported to be higher
between flash freezing and RNAlater® amended samples,
with the former tagged as a superior method (Hickl et al.
2019). Using a similar approach for assessment of the effi-
cacy of storage conditions, and impact on microbiome and
metabolome of samples post-storage, Liang et al. (2020)
discouraged the use of RNAlater® as a stabilizer. This is
understandable as the product is recommended for use as
a stabilizing solution for RNA in unfrozen samples by the
manufacturers (Qiagen, Sigma-Aldrich, and Thermo Fisher
Scientific). Similarly, the principle of stabilization of DNA
by 95% ethanol is the latter’s strong protein denaturing abil-
ity, thereby inactivating DNases (Wu et al. 2019).
Earlier studies reported OMNIgene®-GUT solution
to be a promising preservative for retaining the structural
composition of fecal microbiomes (Choo et al. 2015; Song
et al. 2016). But, by employing a non-targeted metabo-
lomics approach, Wang et al. (2018) reported the solution
to detrimentally affect the number of metabolites detected,
when compared to immediate freezing at − 20 °C. Its use
was found to adversely affect the absolute abundance of
metabolites as compared to the control without any pre-
serving solution (Byrd et al. 2019; Lim et al. 2020). The
authors attributed this to the chemical nature of the pre-
serving solution that affected the process of analysis. Other
additives like maltodextrin-trehalose solution have been
shown to exhibit promising potency for the revival of fecal
samples when incubated at 37 °C, after storage at − 80 °C
for 3 months (Burz et al. 2019). Cryoprotective agents like
DMSO, DMSO + trehalose, and tryptic soy broth (TT) added
to the fecal samples did not affect the profile of single-chain
fatty acids (SCFAs), which was analyzed as a parameter
for microbial activity, upon resuscitation after 3 months of
freezing at − 80 °C (Kerckhof et al. 2014).
Alternative methods for sample storage have been tested
for preserving many biological samples collected from field
studies. Flinders Technology Associates (FTA®) matrix
cards and fecal immunochemical test (FIT) tubes have
emerged as encouraging modes of preservation of fecal sam-
ples to date (Song et al. 2016; Wang et al. 2018; Byrd et al.
2019). However, both were designed to stabilize and protect
DNA (Rajendram et al. 2006; Byrd et al. 2019). Moreover,
both the modes of preservation suffer from the limitation of
collection of a small amount of samples, and issues related
to determining the initial weight (Vandeputte et al. 2017).
13
 Table 2  Approaches adopted for storage of fecal microbiome and post-storage analysis
13
S. no Sample processing
Without additives
1 Homogenized and None
aliquoted, also fecal
swabs collected
With additives
2 Samples collected in RNAlater®-ICE and
MedAuxil stool collec- RNAlater®
tors, homogenised
3 Samples incubated with/
without specific pre-
servative
4 Homogenized and
swabbed in tubes
5 Homogenized
6 Homogenized
7 None
8 Homogenised
9 Homogenised in 0.05 M
NaCl (4 °C)/sterile
milli-Q water
3176
Additive Temperature of storage Duration of storage Assay for structural Functional assay for Reference
integrity of microbiome microbiome
4 °C, 24 h for all temperatures 16S rRNA gene None Tedjo et al. 2015
RT, − 20 °C, − 80 °C except for − 20 °C sequencing
and RT (1 week)
− 80 °C and 4 °C 16 h in RNAlaterICE 16S rRNA gene Metatranscriptomic Hickl et al. 2019
and 6 h in RNAlater sequencing analysis, Metaprot-
eomic analysis using
HPLC–MS
Swab on FTA® card, − 20 °C, RT 24 h 16S rRNA gene Untargeted and targeted Wang et al. 2018
95% ethanol and sequencing metabolomics (SCFA
RNAlater® with glass based)
beads, OMNIgene®-
Gut solution
95% ethanol, blood col- − 80 °C and RT Frozen for 2 months; RT 16S rRNA gene None Moossavi et al. 2019
lection card storage for 48 h sequencing
6 M Guanidine 4 °C, 48 h 16S rRNA gene None Ribeiro et al. 2018
RT, − 20 °C, − 80 °C sequencing
and RT
95% ethanol, RNAl- 4 °C, 48 h 16S rRNA gene None Vogtmann et al. 2017
ater®, or fecal col- RT, − 20 °C, − 80 °C sequencing
lection in fecal occult and RT
Environmental Science and Pollution Research (2022) 29:3171–3183
blood test (FOBT)
cards or FIT tubes
RNAlater® Flash-frozen in liquid 52 h (48 h in frozen 16S rRNA gene Metabolomic profiling Liang et al. 2020
­N2, − 16 °C followed state/RNALater/ sequencing using UPLC/MS–MS
by RT with ice bag RT + 4 h with/without
(gradual thawing), ice/RNALater/RT)
and − 16 °C followed
by RT without ice bag
(fast thawing), RT
7 × volume TE buffer, 4 °C, − 80 °C, RT 72 h 16S rRNA gene None Choo et al. 2015
7 × volume RNAlater®, sequencing
OMNIgene®-GUT
stabilisation kit
7 × volume 20% DMSO- − 80 °C, − 20 °C, 4 °C, 120 h at respective 16S rRNA gene None Ezzy et al. 2019
0.25 M EDTA, pH 8.0 RT (dry collection conditions followed by sequencing
(DETA), 7 × saline tube) freezing at − 80 °C
saturated DETA,
7 × volume of 95%
ethanol
 Table 2  (continued)

Environmental Science and Pollution Research (2022) 29:3171–3183

S. no Sample processing Additive Temperature of storage Duration of storage Assay for structural Functional assay for Reference
integrity of microbiome microbiome

10 None 70% and 95% ethanol, − 80 °C, RT 4 days (− 80 °C) and 16S rRNA gene None Byrd et al. 2019
RNAlater®, collection 7 days (RT) sequencing
in FTA® cards or FIT
tubes
11 Homogenised 10% glycerol, lyophili- − 80 °C 48 h followed by 9 days 16S metabarcoding, Measurement of pro- Deschamps et al. 2020
zation agents like treha- fermentation in bio- qPCR duction of gases and
lose and maltodextrins reactor SCFAs during fermen-
tation process
12 Samples diluted in Two different maltodex- 2–6 °C, 20 ± 2 °C 2 weeks 16S rRNA gene Metabolomic finger- Burz et al. 2019
cocktail under oxic and trin-trehalose cocktail or − 80 °C sequencing printing
anoxic conditions recipes in normal saline
medium
13 Homogenized DNA/RNA Shield™ 4 °C, RT, − 20 °C 3 weeks 16S rRNA gene None Kazantseva et al. 2021
sequencing
14 Homogenized OMNIgene®-GUT − 80 °C, RT 21 days 16S rRNA gene Metabolite profiling by Lim et al. 2020
solution sequencing LC–MS
15 Homogenised eNAT® medium, RNAl- 20 °C, − 80 °C 30 days 16S rRNA gene None Young et al. 2020
ater® sequencing
16 None 100 mM EDTA and 4 °C, RT, − 20 °C, 33 days at ambient 16S rRNA gene Metabolic analysis by Jenkins et al. 2018
Lysis buffer and − 80 °C oxygen level and under sequencing inferring the pan-
hypoxic conditions genome based func-
tional profiles
17 Homogenised 70% and 95% ethanol, 4 °C and − 20 °C 8 weeks 16S rRNA gene None Song et al. 2016
Whatman® FTA® sequencing
cards, RNAlater®, and
the OMNIgene®-Gut
kit
18 Stored in AnaeroGen bag Cryoprotective − 80 °C 3 months 16S rRNA gene Enzymatic activity meas- Kerckhof et al. 2014
agents, viz. DMSO, sequencing ured after resuscitation
DMSO + trehalose and by thawing at 37 °C
tryptic soy broth
19 Homogenised 70% ethanol, 4 °C, − 80 °C 6 months 16S rRNA gene Assessment of functional Ma et al. 2020
OMNIgene®-Gut, sequencing bacterial abundance
MGIEasy, Longsee kits related to bile acids
(BA) and short chain
fatty acids (SCFA)
metabolism
20 Homogenized in 0.9% 80% glycerol in 0.9% − 80 °C 18 months 16S rRNA gene None Dorsaz et al. 2020
NaCl solution NaCl sequencing
13

RT room temperature

3177
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Open questions related to storage of soil
microbiome, and proposed solutions
With the technological advancements, significant progress
has been made in terms of the storage and application of
microbiomes from diverse habitats (Kerckhof et al. 2014;
Wang et al. 2015; Song et al. 2016; Jenkins et al. 2018;
Pavlovska et al. 2021). However, there are still certain inherent
challenges related to the methodological details regarding
microbiome storage as well as its post-storage analysis and
application, which need to be overcome. The basic steps for
the preservation of soil microbiomes and associated critical
points have been illustrated in Fig. 2. Specific factors that
can affect the efficacy of methods for the preservation of
microbiomes have been discussed below.
Choice of cryoprotectant
The choice of cryoprotectant for storage of soil microbiome
depends on the objective of storage. The use of preservative
solutions or strategies for the protection of nucleic acid suf-
fices when the objective is to analyze microbial community
composition by next-generation sequencing (Tatangelo et al.
2014; Hickl et al. 2019; Young et al. 2020; Pavlovska et al.
2021). However, most of these solutions work by inactivat-
ing proteins and enzymes, which subsequently adversely
impact the viability of cells (Wu et al. 2019). Besides, DNA
extraction protocols mostly do not differentiate between
nucleic acids originating from live cells and the remnant
DNA. If, however, the objective is transplantation of the
microbiome to transfer any beneficial trait to the recipient
system, e.g., tolerance of stresses in plants (terHorst et al.
2014; Jochum et al. 2019; Anand et al. 2021), plant growth
promotion (Panke-Buisse et al. 2017; Lu et al. 2018), and
transformation of conducive soil to disease suppressive ones
(Taparia et al. 2021), any compromise with cell viability of
microbiomes is not acceptable.
Without any protective agent, cell viability for a com-
mon soil bacterium like Pseudomonas fluorescens has been
reported to decline by orders of magnitude (Stephan et al.
2016). On the other hand, survival of dried P. fluorescens
cells has been observed to be enhanced when supplemented
Fig. 2  Steps involved in the
preservation of soil microbi-
omes, and associated critical 1.
points Sampling
• Mode of
transfer to lab
• Duraon unl
processing
13
Environmental Science and Pollution Research (2022) 29:3171–3183
with osmoprotectants like fructose and trehalose (Schisler
et al. 2016). Even exopolysaccharide (EPS) has been shown
to exhibit shielding of bioinoculants when applied for plant
growth enhancement (Tewari and Sharma 2020). However,
these studies have been performed with only individual
bacterial strains.
Researchers need to elucidate the mechanism of action of
cryoprotective agents for making a rational decision regard-
ing the choice of preserving solution to be used during stor-
age. Further, a thorough assessment of any selective biases
that they offer to a particular taxon needs to be characterized.
Antioxidants have been shown to enhance the culturabil-
ity of bacteria from the intestinal microbiome by offering
an advantage to the anaerobes during storage (Bellali et al.
2019). Such an approach can aid in protecting the oxygen-
sensitive components of the soil microbiome as well.
Storage with or without matrix
Most of the studies until now have attempted microbiome
storage with the soil matrix (Marti et al. 2012; Rubin et al.
2013; Cui et al. 2014; Wang et al. 2015; Pavlovska et al.
2021). However, a limited number of studies have evalu-
ated the efficacy of soil washes in conferring the desired
property (Wagner et al. 2014; Howard et al. 2017). Albeit in
the preliminary stage, the approach of using washed micro-
biota for transplantation has yielded encouraging results
with feces (Ye et al. 2020; Zhang et al. 2020). Storage of
microbiome washes (without soil matrix) might prove to be
logistically convenient for transplants at a later time point.
Further studies assessing the biases introduced (microbiome
fraction loosely adhering to the soil will be favoured), if
any, by such a strategy, need to be performed before such an
approach can be brought to application.
Assessment of microbial community composition
of stored soil
When comparing different parameters related to stor-
age of soil microbiome, e.g., the choice of cryoprotec-
tive agent, the storage conditions, and its duration, the
assessment of the structure of microbial community post
2. 3. 4.
Processing Storage Revival
• Separaon from matrix • Temperature • Microbial community
• Homogenizaon • Duraon of composion
• Sieving storage • Tesng for
• Addives funcon/efficacy
Environmental Science and Pollution Research (2022) 29:3171–3183
storage is critical. The analysis of total DNA extracted
from the soil samples furnishes an over-representation of
the same as the extraction does not differentiate between
DNA from live vs dead cells, with the latter eventually
not contributing to the functionality of the stored micro-
biome. The metatranscriptomic approach of assessing the
microbial community composition can be an effective
means of surpassing this limitation to an extent, as RNA
is relatively less stable than DNA. An easier approach for
analysis is targeting the culturable fraction of the stored
microbiome. However, this can only offer an impression
of the impact on a fraction of the community, since char-
acterization of non-culturable fraction would still remain
elusive due to technical limitations. Ramirez et al. (2017)
studied the effect of freeze-storage of soil samples on
the viability of culturables, and concluded the method to
be efficient for storage up to 8 months. Another strategy
that can be adopted to target only the functionally active
component of the microbiome is to restrict to the extrac-
tion of nucleic acids from viable cells as performed by
Ouyang et al. (2021).
Assessment of stored microbiome’s functionality
A defined set of methods is required to evaluate whether
the functionality of the stored soil samples has been
retained, before its transplantation. Basic techniques of
respirometry and quantification of enzymatic activities
have been employed to determine the functionality of the
soil microbiome (DeForest 2009; Abellan et al. 2011;
Peoples and Koide 2012; Marti et al. 2012). However,
these are general methods for estimating the “live frac-
tion” of the microbiome, without offering any resolution
in terms of specific activity for which the microbiome
has been preserved. On lines similar to the assessment of
specific markers like SCFA production by fecal micro-
biome (Wang et al. 2018; Ma et al. 2020; Deschamps
et al. 2020), sensitive markers can also be selected for the
soil microbiome. This, of course, depends on the focused
objective for which the storage has to be done, like spe-
cific metabolites conferring properties like nutrient acqui-
sition, stress tolerance, biological control to the plant,
and soil health can be targeted. Besides, a non-targeted
approach of generating metabolic profiles can also help in
identifying deviations, if any, between the fresh samples
and the stored ones. The best approach, however, is to
assess the efficacy of preserved microbiomes after the
revival of the stored samples, in microcosms with model
plants, and monitoring the plants’ fitness. Most research-
ers have established the proof-of-concept for the process
of soil microbiome transplant by employing freshly sam-
pled rhizospheric soil (Panke-Buisse et al. 2015; Jochum
et al. 2019; Anand et al. 2021).
3179
Characterizing the microbiome components
to design minimal microbiome
Before attempting soil transplantation, it is crucial to delineate
the minimal active microbial community required for
imparting the desired trait. Using next-generation sequencing,
characterization of the functional core and satellite
microbiome (Lemanceau et al. 2017), and the keystone species
(Banerjee et al. 2018), will enable researchers to specifically
focus on the efficient storage of a rather simplistic but robust
“minimal microbiome” (Mendes et al. 2013; Raaijmaakers
2015; Gopal and Gupta 2019) without attempting to preserve
the total microbiome. This can also ease out the complexities
involved in the resuscitation of an otherwise complex
microbiome. Integration of the reductionist and ecological
approaches should be considered when selecting a minimal
microbiome (Fitzpatrick et al. 2020).
Continuous propagation for storage
The ideal condition for optimal storage of an active soil micro-
biome is to incubate it under conditions mimicking the real-life
scenario. There has been an attempt to continuously propagate
the microbiome by incubating it in a sterilized commercial pot-
ting mix (Howard et al. 2017). The incubation time required
for the microbiome in the potting mix to attain similarity to the
initial soil inoculum was found to depend on the size of the
inoculum. Such a propagation bypasses the step of revival of
the active community for transplantation. However, one can-
not ignore the action of evolutionary forces on the microbiome
upon its continuous propagation, thereby altering the original
microbiome. Continuous propagation of the rhizospheric micro-
biome can also be done by applying it successively during every
plant growth cycle (Jochum et al. 2019; Mueller et al. 2019;
Anand et al. 2021). This would ensure retention of the efficacy
of soil microbiome (adapted over multiple rounds of plant
growth) in soil. While this approach is tedious, it can maintain
the functionality of the soil microbiome, which is vital for future
applications.
Conclusions and future perspective
With increasing pressure on shifting to sustainable means of
agriculture, microbiome-based strategies have exhibited tre-
mendous potential as “next-generation biologicals.” Hence,
the emphasis has now shifted from storage of potent individual
strains for application, to preservation of the microbiomes, such
that its functionality is retained for transfer. Microbiome-based
strategies are believed to be more efficient, especially in compet-
ing with the resident soil microbiome, and in their performance,
considering the intact intricate web of interrelationships between
the members. However, this is still an area that requires further
13
3180
research towards devising an optimal, universal method of stor-
age as well as post-storage analysis of the soil microbiome’s
efficacy. Efforts till date have been majorly confined to evaluate
the impact of storage on a rather stable nucleic acid marker,
DNA. Conventional methods of stabilizing the nucleic acids
for assessment of microbial community structure cannot solely
meet this urgent requirement. Based on the lessons learnt from
the success of optimal storage and transplantation of the fecal
microbiome, further improvisations are the need of the hour for
soil microbiome storage and subsequent application. Besides,
a more critical analysis of the functionality of the stored soil
microbiome has to be done by integrating omics approaches, like
metatranscriptomics and metabolomics together with metagen-
omics. Testing the efficacy of the stored microbiome should be
performed in systems closer to real life, as performed for the
fecal microbiome (Deschamps et al. 2020). Also, markers for the
assessment of the functionality of the soil microbiome should be
well defined. Extensive, long-term (in years) studies adopting
a polyphasic approach will pave the way for wider applicability
and acceptance of soil microbiome transplantation for achiev-
ing agricultural sustainability. This will require scientists from
diverse area of expertise like environmental microbiologists,
plant agronomists, systems biologists, and chemical ecologists,
to come together for a sturdy outcome.
Acknowledgements A. B. acknowledges the award of National Post-
Doctoral Fellowship from the Science and Engineering Research
Board, Department of Science and Technology, Government of India
(PDF/2018/001905). S. D. received fellowship from Indian Institute
of Technology Delhi.
Author contribution S. S. conceptualized the idea. A. B., S. D., and S.
S. wrote the first draft, and S. S. reviewed the manuscript. All authors
approved the manuscript.
Funding The work was funded by a grant received from the Depart-
ment of Biotechnology, Government of India (BT/PR27680/
BCE/8/1434/2018).
Data availability Not applicable.
Declarations
Ethics approval and consent to participate Not applicable.
Consent for publication Not applicable.
Competing interests The authors declare no competing interests.
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