A Recent Overview of Microbes and Microbiome Preservation

Abstract

Microbes are mediators in almost all ecosystem processes and act as a pivotal game changer in various ecological activities, globally. Therefore, understanding of microbial community structure and related functions in different environmental and micro-environmental niches is not only critical, but also a matter of greatest importance. Due to our inability to cultivate and preserve all sorts of microorganisms, we are losing some ecologically and industrially relevant components of microbial community, due to extinction caused by environmental and climatic variations with time. Intact sample and microbiome preservation are crucial for future cultivation as well as to study the effects of ecological and climatic variations on community functionality and shift with time, using OMICS. Although, methods for pure culture preservation are almost optimized, the techniques of microbiome preservation still remain as an unsolved challenge for microbiologists due to technical and physiological constraints. Present article discusses, recent approaches of microbial preservation with special reference to intact sample, mixed culture and microbiome preservation. It also incorporates recent practices used to achieve the highest viability and metabolic activities in long-term preserved microbiome.

Introduction

Extensive research in microbiology has proven that, microbes are the key components of almost every process on earth and without them; there can be no operational ecosystem [1]. They are operators of all bio-geochemical cycles on the earth and are the providers of most of the ecosystem services. After the successful production of penicillin, microbial diversity has always remained in the spotlight for their bioactive and industrially important metabolites [2]. Microbes harbour valuable metabolite(s) producing genes of great industrial and clinical importance and form the backbone of modern biotechnology and bio-economic activities such as bio-pharmaceutical and bio-energy development [3–6]. Several activities like production of breathable oxygen, improved agricultural productivity by plant growth promotion and disease suppression, degradation of complex organics, solid waste management, generation of clean energy, global climate change and development of animal, plants and human diseases are directly or indirectly related to microbes [5, 6]. Several articles have already been published on industrial, clinical, environmental and agricultural application of microbes but their extensive description is out of the focus of current review article [5–7].

Current era of microbial science is mainly dependent on culture independent molecular approach using high throughput next generation sequencing platforms [8]. Past and recent advancement in metagenomics technology is opening a new insight about the glorious world of microbial diversity, inhibiting every part of the globe. It also revealed that a major part of microbial diversity (> 90%) of biotechnological importance is not yet cultured [1, 5, 6]. It is guessed; that remaining 90% not-yet-cultured microorganisms could play a very important role from ecological, clinical and industrial point of view [5]. Lack of optimized cultivation and preservation protocols might be one of the possible reasons behind non-culturabilty of these microorganisms. Unfortunately, culture independent molecular approach only gives structural details of microbiota but does not provide information about its biotechnological traits. Along with the development of OMICS, the importance of cultivation and preservation of microbes has been realized and several novel tools of cultivation have been developed recently [5, 7]. It is believed that, eventually with advancement in cultivation methods we will be able to cultivate and harness the biotechnological potential of not yet-cultured organisms in near future. Therefore, preservation of intact sample without losing the component of native microbiota is imperative for future cultivation. In addition, to conduct microbiome related comparative study on various samples and to study the temporal and spatial changes in community optimum tools of intact microbiome preservation is must. Furthermore, to achieve maximum and long-lasting benefits from preserved sample, pure cultures, or mixed microbiome, maintenance of viability, genetic stability and functions, are a matter of concern during preservation. Therefore; from biotechnology and bio-economy point of view, ex-situ as well as in-situ preservation of microbial biodiversity, along with their viability and cellular functionality is a vital task on the way to successfully exploit the beneficial microbial capacities [9]. Although, dozens of protocols and techniques are available on pure culture preservation, little work has been done on intact sample and microbiome preservation [6]. Intact microbiota and sample preservation is a much-needed exercise, from cultivation, OMICS and ecological point of view. Therefore, preservation and culturing of microbial resources with special reference to microbime is an emerging trend of current era [10, 11]. The aim of the present article is to provide a recent overview of intact microbiome and sample preservation. In addition, it also incorporates preservation techniques aimed at increasing microbial viability, functionality and stability during long-term storage and transport and discusses the current need of microbiome banking.

Need of Intact Sample and Microbiome Preservation

Microbial community or total microbiota of a specific habitat, living together constitutes microbiome of that particular ecosystem. Human-microbiome, animal-microbiome and plant-microbiome are some of the good examples to understand this concept. Microbiome of any particular ecosystem can be further divided into subsets, for example, human microbiome can be further divided into smaller subsets like oral microbiome, vaginal microbiome, gut microbiome and skin microbiome etc. These subsets can also be named based on the nature of microorganisms like bacterial-microbiome, fungal-microbiome etc. Recently, microbiome related studies are getting extra attention as it provides holistic view about microbial structure and function of any ecological and clinical samples. Emerging role of human-gut microbiome in human health and diseases is the best example of how microbiome related studies are important to understand the community structure and functions in a better way. Similarly, concept of soil, plant and environmental microbiome are fast emerging disciplines and are assisting to connect the role of native microbiota in soil, plant, and environmental health. Perturbation and disruption of intact structure of native microbiota of any microbiome is known as dysbiosis, which leads to diseases, disorders, and even causes extinction and endangerment of the host. It is also important to know how the missing component of native microbiome of any system affects its immunity, disease and survival potentials [11]. Detailed discussion about importance of intact sample and microbiome preservation is discussed below.

Initially, it was believed that unlike plants and animals; microbes do not face the problem of extinction and endangerment even after major change and environmental perturbations, because of their universal distribution and quick resilience quality. Due to this belief; despite the immense importance of microbial diversity they are not on biodiversity conservation agenda like birds, animal and plants [12]. Now it has been realized that; even after putting all the efforts and development of novel cultivation approaches, we cannot cultivate and preserve all the members of microbial community due to technical or physiological limitations. Inability to cultivate and preserve all the available microbial diversity of any ecosystem for long duration creates the possibility of shrinkage of core microbiota or extinction of certain valuable components of clinical, environmental, and industrial importance. The core microbiome is shrinking due to several factors [13, 14]. Rapid urbanization, industrialization, climate change, fast-growing civilization, changes in rate of precipitation and sudden change in carbon flux and overburden of pollutants are leading cause of habitat destruction and loss of valuable microbial niches and diversity [15]. Extinction of host plants and animals, caesarean birth, indiscriminate use of antibiotics, bottle or formula feeding is also contributing in shrinking of microbial diversity [16]. In addition, understanding the spatial organization and functionality of microbial community is very much needed to investigate the role of microbial biodiversity in various ecosystems. Rapid changes in microbial community structures are also related to micro-environmental changes in the atmosphere. Due to this, understanding of microbial dynamics and their habitat becomes a pivotal task in study of microbial ecology [17]. Preservation of intact sample or extracted DNA is one of the ways to study the effect of environmental and climatic variations on microbial communities in future.

Thus, the preservation of not-yet-cultured microbiota and intact microbiome is an important aspect of microbial research and as a microbiologist, protection of microbial biodiversity from such disruption should be our prime concern [18]. Intact sample and microbiome preservation is considered as one of the best approaches for future culturomics and to study the impact of ecological and environmental perturbation on community shift and ecological functionality. Preservation of intact microbiome is also required to reconstruct spatial organization of disturbed microbial community and dimensional structure of the sample. Loss of microbial biodiversity is considered pernicious to ecosystem functioning. Therefore, scientific communities are inclining their attention towards ecosystems conservation without disturbing the intact structure of their microbiome and restoration of natural microbial community for better ecosystem functioning. [6, 19, 20]. Restoration of native microbiota from unaffected individual and environment is considered as one of the remedies to restore the disturbed gut and environmental health respectively [15]. Intact microbial restoration is an urgent need with respect to health, environment and bio-economic perspective of the world. Therefore, preservation of intact sample for future OMICS studies and cultivation is a good idea as well as an important step towards preservation research. Any method, that preserves the maximum viability and functionality along with preservation of good quality nucleic acids from different ecological samples for future OMICS, single cell biology application and cultivation of valuable component for academic and industrial use, will be of great importance. Furthermore, there are certain applications, where purity of the culture is not mandatory and merely viability and functionality of the core-microbiota is required for quick establishment of ecosystem functionality again. Faecal Microbial Transplantation (FMT) and use of intact microbiome or enriched community as starter culture for biogas plant or in wastewater treatment plant are some of the good examples. Considering the above facts, concept of preservation of mixed culture, enriched consortium and whole microbiome is an emerging area in microbiology. Protection of maximum cell viability and functionality is the main aim of microbiome and mix culture preservation.

Importance of Sampling for Intact Microbiome Preservation

Exact representation of natural microbial communities by experimental technique is imperative for authentic microbiome related ecological studies associated with community shift and shrinkage with time and space. Method of sample collection, duration and temperature of sample transportation, sample archiving method, storage temperature, duration of storage and way of retrieving the sample for analysis purpose significantly affects the recovery of microbial community and metabolme from preserved samples (Fig. 1). Unfortunately, the quality control parameters are only taken seriously for analytical or instrumentation part, whereas the control procedures for the sampling and transportation of samples are often neglected or not taken very seriously. Improper sampling and storage of sample misleads the data creating variability in result of microbiome related studies and are also responsible for reduced viability of microbiota and loss of metabolite during storage and transportation. Thus, there arises a need for a detailed description of the sampling and preservation procedures. The objective of sampling is to collect a part of sample from an environmental niche, small enough in volume to be conveniently transported and handled in the laboratory, to maintain properties of the environment it is collected from. Samples for intact microbiome preservation should be carried at a temperature below 4 °C and in the dark as soon as possible, after sampling. Once in the laboratory, samples must be transferred a refrigerator as soon as possible. Delay in the sample processing may result in the alteration or shift of the microbiota of sample. It is always better to keep the relative proportions or concentrations of the components of interest, the same in samples as the original environment. This requires the samples to be handled and treated in such a way that no significant changes in composition occurs which may hamper proper analysis. During preservation, it is recommended to maintain the originality of the sample as much as possible and no gain, loss or deterioration must happen.

Fig. 1.

Concept of intact microbiome preservation. Figure depicts that sampling, transportation of samples, appropriate preservation and pre- and post-validation of viability and functionality of preserved samples are the essential components of preservation research

It is generally considered that the samples stored at room temperature for a longer period before extraction of DNA are unsuitable for downstream processes, due to changes in microbiome landscape of the samples. Earlier studies reported, the storage conditions do not strongly affect microbial community structure of the samples [21–23]. The study of gut-microbiome diversity, carried out by Roesch et al. (2009) has revealed that after 72 h storage of samples at room temperature results only in the modest changes. In contrast, other researchers reported that the storage temperature and conditions are very important to maintain the microbiome dynamics intact [24–26]. In comparison to environmental samples microbiome related studies are more prevalent on human microbiome. A number of research papers have been published recently on how to collect, transport, store and process faecal materials, oral washings, vaginal and skin swab for microbiome analysis [10, 27]. Effects of various parameters like sample homogenization, sudden freezing, storage at ambient temperature, storage in 95% ethanol, RNAlater and OMNIgene medium, and with different cryoprotectants has been studied. Detailed description of conditions and approaches of sampling and sample processing for microbiome studies have been discussed somewhere else [10, 27–35]. Researchers are still optimizing the effects of different storage conditions on microbial community fluctuation in different samples. In order to fill the knowledge gap, in this article, we also reviewed different methodologies and their significance for the long-term preservation of intact microbiome samples from different sources (Figs. 2,3).

Fig. 2.

Different preservation strategies used for intact sample and microbiome preservation

Fig. 3.

Overview of methods studied and recommended for preservation of intact sample and for further analysis of structure and functions of preserved microbiome [63, 67, 68, 70]

Methods of Intact Sample and Microbiome Preservation

Cell Alive System (CAS) Technique for Intact Microbiome Preservation

Conventional microbial preservation techniques have certain limitations and do not show promising result in terms of maintaining the viability and functionality of the microbiome during long-term preservation of intact samples. A novel technique, the Cell Alive System (CAS), which originated from Japan is nothing but a range of industrial freezers primarily developed to improve the preservation method in food industry, especially for preservation of raw seafood items. CAS-freezing technique is based on a very simple principle that the spinning motion of water during freezing prevents formation of crystal lattices. In addition, spinning motion also helps minimize the freezing point of water, which assists longer freezing effect and reduce the time required to achieve the super cool status of water [36]. In this system, presence of alternating or oscillating magnetic field and mechanical vibrations induce uniform cooling and minimum sized ice crystal formation in preserved sample. This process maintains the natural texture and prevents cellular and molecular damage in preserved materials and further claims to influence the tissue survival rate. Due to this feature and usefulness, in organ banking CAS has been attracting extra attention of life science researches in current era. Morono et al. (2015) carried out comparative study on effects of CAS and different freezing conditions on viability of microbial cells from preserved environmental samples and pure E. coli cells [37]. They found that samples preserved in CAS-freezer showed more viability than the samples preserved using other freezing techniques. Furthermore, CAS freezing technology has been approved as a well-optimized tool for long-term preservation of deep-sea sediment samples for geo-microbiological studies. Braun et al. (2016) used CAS freezing technique to preserve sub-seafloor sediment samples, for bio-molecule based microbial abundance studies and achieved satisfactory results [38]. Trembath-Reichert and co-authors also successfully used CAS freezing for deep-sea sediment preservation, for fluorescence-activated cell sorting (FACS) and 16S rRNA based microbial community analysis [39]. CAS gives better results as compared to other techniques after six- month storage of sub-seafloor sediment samples [37]. It is advisable here that CAS can be used as a tool for intact sample and microbiome preservation. With suitable modifications to this technique, researchers might be able to achieve increased storage period and viability [40].

Cryopreservation and Lyophilization in Microbiome Preservation

Cryopreservation and lyophilisation both are well-known methods for long-term preservation of microbial cultures. Use of ultra-low temperature with different cryoprotectants for preservation of microorganism is a traditional practice in every microbiological laboratory. Cryoprotectants are a group of chemicals, which prevent the formation of lattice from water molecules during preservation [41]. Prakash et al. (2013) and Kerckhof et al. (2014) [6, 42] discussed the chemistry, nature and mechanism of action of different groups of cryoprotectants in detail but it is not the subject matter of this review. Briefly, ultra-low temperature gives a high stability and viability to the microbial cells, [9, 43] while cryoprotectants reduce cellular lysis by preventing the formation of ice-crystal during the freezing process. Today, a range of good cryoprotectants is available in the market. Glycerol and Dimethyl Sulphoxide (DMSO) are well-known cryoprotectants which are currently available in market. Glycerol undergoes vitrification processes by forming hydrogen bond with water molecule present in the storage medium. There are no optimal methods, which can be equally applied for all types of microbes and cells. Different microbes behave differently in terms of viability and stability with different cryoprotectants and more work is needed to be done in this area. Different classes of polysaccharides are also used as cryoprotectants for long-term preservation and transport of the clinical samples from different groups [44]. In case of Lactobacillus, amino acids play an important role as cryoprotectants. They increase the mobility of fatty acid acyl of bacterial cell membrane and increases cell viability [45]. Nyanga et al. (2012) studied different storage conditions for preservation of yeast culture and discovered that preservation of yeast on rice cake gives better results [46]. It is believed; that starch present in rice reacts with yeast and forms glassing structure, which might protect the yeast cells from damage [9]. Galacto-oligosaccharides (GOS) composed of a number of Galactose units (2–9 glucose monomeric units) are an emerging cryoprotectant. As a cryoprotectant, GOS is getting more attention due to its prebiotic properties [47]. GOS is now in the limelight due to its unique properties like induction of vitrification process, ability to form glassing structure with embedded micro-molecule, replacement of water molecule and involvement in dehydration as well as rehydration processes [48]. Cryopreservation of intact environmental samples, mix culture and consortium with different cryoprotectants are still understudied and need extra attention.

Gelatine Disk Method: Preservation of Sample During Transportation

Lyophilisation is a widely accepted method for long-term storage of microbial samples and for transportation purpose. But due to excess cost of instrumentation many small laboratories and colleges cannot afford this method. Stamp et al. studied factors, which affect the bacterial survival ability after drying and suggested simple bacterial preservation technique using gelatine disc. This method gives good result for successful preservation of a number of pathogens but, did not perform well for prolonged preservation of Vibrio and Neisseria species. Obara et al. modified and standardized gelatine disc method given by Stamp for successful preservation of N. gonorrhoeae with stable morphology and antibiogram pattern for 3 years [49]. By using this technique, they maintained β-lactamase activity of penicillinase-producing N. Gonorrhoeae for 3 years. They also found that the gelatine disc method allows survival of fastidious organisms up to one year at − 20 °C storage. In another method, Kulkarni and Chitte successfully preserved thermophilic bacterial spores on filter paper [50]. According to their study, spores of thermophiles can be preserved at various temperature ranges from 4 to − 20 °C for almost one and a half year [50]. There is no direct report on use of gelatine disc method for preservation of intact microbiome but due to their capacity to maintain viability and functional stability of a number of bacterial strains, researchers might expect this technique as a possible alternative for long- term preservation of microbiome.

Cellular Immobilization or Entrapment

Cellular immobilization or entrapment in the gelling matrix is another alternative of long-term preservation of microbial viability and functionality. Formation of micro-droplets or beads with different gelling agents like alginate and Acacia-gum is well known methods of microbial cell entrapment. Several factors like size, texture, porosity, surface to volume ratio and chemical nature of gelling agents’ impacts cell viability and functionality during preservation. This method is getting good attention in probiotic industry due to high survivability and functionality of the entrapped cells compared to other preservation methods and sustained release in gut after intake. Several organisms of probiotic importance like Bifidobacterium and Enterococcus have been preserved using this method and giving good response. Encapsulation followed by drying is a good option but freeze drying did not give good response due to osmotic imbalance and oxidative stress created during freeze drying process. Fluidized bed drying gives better response in terms of viability and functionality. Furthermore, addition of cryoprotectants and antioxidant in gelling matrix provides long-term viability and functionality to entrapped cells. It is also noticed that use of biopolymers like Acacia-gum and Pullulan is better than inorganic polymers. Detailed description about these are available in Alanso 2016 [9]. Although these methods are used with pure culture of microbes however like other preservation methods it can be explored for consortium and mixed microbiome preservation and protocols needs to be optimized. Electrospinning and Electrospraying both are the advanced encapsulation method and their detail discussion is given below.

Electrospinning and Electrospraying (Microencapsulation) in Microbiome Preservation

Many microbial cells show sensitivity to micro-environmental changes. Their preservation using normal preservation techniques is quite difficult and lethal to their viability and functionality. Microencapsulation method of preservation works better for such type of microbes and is used to overcome such difficulties. High voltage Electro hydrodynamics processes like electro-spraying and electro-spinning are giving good results in preservation of microbes in recent times. Electrospinning and Electrospraying are novel microencapsulation techniques used to maintain the viability and functionality of microorganisms. Generally, both technologies are known as “sister technologies” because the way of working of both the technologies are almost similar with only difference in viscosity of the polymer solution. In Electrospinning polymer solution is highly viscous while the viscosity of polymer solution in Electrospraying is low which gives nano- or microscale fibres of polymer entrapped microbial cells and small droplets (beads) respectively. Typical diagrams of instrumentation of Electrospinning and Electrospraying are given in Fig. 4. Some studies in recent past have applied microencapsulation technique for preservation of probiotic bacteria. In their study López-Rubio et al. successfully used Electrospinning method for preservation of Bifidobacterium cells [51]. They used Poly-vinyl-alcohol (PVOH) as encapsulating material for bacterial cells due to their generally recognized as safe (GRAS) property, and easy recovery of bacterial cells during viability testing. Another study carried out by Liu et al. highlighted that electro-spinning mediated Pluronic F127 dimethacrylate (FDMA) fibrous hydro-gel material allows microbial cells to live for more than seven days at 4 °C and more than 2 months at − 70 °C along with retaining their metabolic activity [52].

Fig. 4.

A simple schematic outline of electro-spinning and electro-spraying methods use for long-term preservation of microbial cells. It consists a high voltage source operated in direct current (DC) mode, syringe pump, stainless steel needle with nozzle (Taylor cone) and ground collector plate. In electrospinning, high viscosity of solution and uneven charge distribution give bending motion to jet and form solid fibre (left) on collector plate while in Electrospraying, low viscosity of polymer solution forms fine droplets (right side)

Electrospinning and electrospraying are considered superior than other cell encapsulation or entrapment methods. Because, by changing the physical nature of polymers and applied voltage at jet-point fibres and droplets varying size and shapes can be obtained. In addition, both offers several other advantages like high surface to volume ratio and high porosity of produced beads and fibres that make nutrient and metabolite exchange easier. Unlike other methods, non-involvement of temperature in these processes reduced the protein- denaturation from harsh temperature conditions consequently increased the cellular viability and functionality during preservation. Efficient encapsulation potential with production of food grade polymers and use of varying nature of protein and polysaccharide based gelling materials makes the method more attractive. Although both the methods are used with limited groups of microbes and need to explore their potentials and preservation efficiency in futures using wide range of microbes giving difficulty with other preservation methods.

Human-Gut Microbiome, Faecal Bio-banking and Method of Assessment of Microbiome Preservation Efficiency

Human microbiota has coevolved with humans and confers benefits to the host and plays an important role in human health and physiology. It has been observed that shrinkage in the ancestral human microbiome or specific component of microbiome, is not entirely beneficial but leads to certain diseases and health consequences [13, 14]. Considering the role of fecal microbiota transplantation (FMT) in treatment of gut-dysbiosis, especially with encouraging result in Clostridium difficile infection (CDI), concept of establishment and running of stool banks is getting more attention. In 2015, the Netherlands Donor Feces Bank (NDFB) started optimizing protocols for donor selection, screening, sample collection, microbiota extraction and preservation along with pre- and post-preservation validation of viability and functionality of preserved samples [53]. A method to preserve intact microbiota which confers more viability and functionality as compared to others, needs to be optimized with different groups and sample type. The aim of any microbiome, mixed culture or enriched consortium preservation is to protect maximum cellular viability along with retaining the cellular functionality. In order to assess the efficiency of preservation methods, it is mandatory to evaluate the viability as well as the functionality of samples before preservation and after revival of the preserved cells. Although culture independent method like amplicon-based metagenomics is cheap, requires less labor and is quick, but is not extensively used as it cannot discriminate between live and dead cells. It is a good approach for assessment of suitability of storage conditions of samples for culture independent metagenomics approach [54]. Total viability count using selective media or universal media plate is a good approach for pre- and post-preservation viability assessment of pure culture, but due to limited cultivability of all the components of microbiome on solid rich medium plate, it is not applicable for assessment of viability from preserved microbiome. Most probable number (MPN) method of viability enumeration is relatively better and can be used even with anaerobic cultures in Hungate tubes with anoxic headspace. But MPN also has its own biases and is unable to assess the viability of all the component of microbiome due to growth limitation. Evaluation of membrane integrity using Propidium iodide (PI) and SYTO9, with the help of fluorescence activated cell sorter (FACS) is a good technique to measure live and dead cells present in a sample. It can be used for assessment of viability of cells from pre- and post- preserved samples [54–56]. Functionality testing is another concern. Some labs are using short chain fatty acid production as an indicator of functionality using gas chromatography, while others use rate of gas production after inoculation of post preserved micro biome.

Recent Development in Microbiome and Strict Anaerobes Preservation

Kerckhof worked on optimization of cryopreservation method of mixed microbial communities to retain the functionality and diversity during long term storage [32]. They used mixed microbiome of methanotrophic co-culture; oxygen limited autotrophic nitrification/ denitrification biofilm and human faecal material. They reported that addition of cryoprotectants preserved the mixed microbiome better than sample preserved without cryoprotectants. Addition of cryoprotectants also helped in quick recovery of functionality. Vandeputte et al. compiled different methods of faecal sample collection, preservation, transportation, and their suitability for various OMICS studies under the title “Practical considerations for large-scale gut microbiome studies”. They provided a detailed overview of different methods, protocol and techniques required for designing and execution of a large-scale microbiome cohort initiative [57, 58]. Aguirre et al. evaluated conditions of preparation of human faecal inoculum for in vitro fermentation studies in place of fresh sample. They preserved the faecal sample using different methods and studied the functionality and community recovery using differently preserved samples. They concluded that glycerol protects functionality and community structure better than others [59]. In all, the treatment diversity of Actinobacteria, Firmicutes, Fusobacteria, Verrucomicrobia and Proteobacteria preserved well whereas decline was observed in case of Bacteroidetes group. Yu et al. (2015) preserved the Switch grass enriched microbial community for its future use as starter culture for bio-ethanol production. They reported that glycerol and Dimethyl Sulfoxide (DMSO) both worked equally well but DMSO preserved community needed more time to get the same level of functionality after revival as compared to glycerol-preserved community [60].

Silkina et al. (2017) preserved the mixed algal consortium and evaluated its functionality in terms of bioremediation efficiency before and after cryopreservation [61]. They used 19% DMSO as cryoprotectant and slow cooling at the rate of 1 °C  min⁻¹ followed by storage in liquid nitrogen. They found that after thawing and inoculation, cryopreserved and non-cryopreserved community gave almost similar result in terms of removal of pollutants [61]. Due to recent interest in faecal microbiota transplantation (FMT) and probiotics related topics the concept of faecal bio-banking, intact stool preservation, preservation of mixed gut community and preservation of extracted microbiome is an emerging aspect in preservation research. In the same context, Bircher et al. (2018) tried to cryopreserve artificially produced colonic microbiota in 15% glycerol, 5% inulin and in combinations of both [62]. They also evaluated the structure and function of preserved community before and after preservation and used short chain fatty acid production as functional marker. They concluded that, beneficial butyrate producing microbial community could be best preserved using glycerol and inulin. Furthermore, Vekeman and Heylen (2015) published the protocol for preservation of mixed and pure culture of fastidious organisms and found that deep-freezer preservation with 5% DMSO works well with some modification for most of the tested community [63]. Fuertez et al. (2017) developed and successfully preserved the anaerobic methanogenic consortium for coal bio-gasification purpose and found good results post preservation [64]. Gaci et al. (2017) preserved the functionality of gut-microbiota using DMSO and other cryoprotective agents and found that the DMSO alone or in combination of other cryoprotectants gives better result [65]. Lee et al. (2019) preserved fungal microbial community using the DMSO, ethylene-diamine-tetra acetic acid, saturated salt (DESS), 15% glycerol, and phosphate buffered saline (PBS) and reported that DESS gives better protection than the other two and suggested to use DESS as preservative for microbial ecology research [66]. Optimization of cryoprotectants and preservation condition for strict anaerobic culture isolated from gut was also studied by Bircher et al. and concluded that protection effect of cryoprotectants are process and species specific [67]. Yarberry et al. (2019) preserved the intact inoculum from anaerobic digester and checked its post revival efficiency for methane production and found that lyophilisation with 10% skimmed milk gives better protection and retains 100% methane production efficiency [68]. Earlier studies have shown that, variation in sample storage condition impacts the assessment of microbial community structure qualitatively and quantitatively [68]. Use of electromagnetic field in combination with cryopreservation is not new for live cells and tissue preservation. Recently it is proven that, the electromagnetic fields can influence ice formation by non-thermal mechanisms [36]. Mapping of environmental microbiome requires efficient preservation methods. As discussed above; Morono et al. (2017) developed Cell Alive System (CAS) preservation technology to preserve intact microbiome of sub-seafloor sediment samples [37]. Tatangelo et al. (2014) studied effects of different storage conditions including freezing, adding two liquid-based preservatives or normal storage, on microbiome community structure of aliquots of organic-rich soil and water samples. They have also revealed that DMSO-EDTA-salt solution (DESS) is a good alternative to preserve organic content rich soil samples [69]. The preservation of intact microbiome with functionality is carried out by different researchers and is presented in brief in Table 1. Thus, it is clear from above mentioned data that works on microbiome preservation is going-on with different kinds of samples including human-faeces [70], water, soil, sediment and enriched microbial community and eventually get refined with time.

Table 1.

Various approaches of microbiome preservation from different types of samples

Sample type	Preservation methods and storage conditions	Duration of preservation	Techniques used to judge the efficiency of preservation	Observations	References
Sub-seafloor Sediment	Cell alive system (CAS)- freezing	6 months	Microbial cell count	CAS gives better viabilty	[37]
Garden Soil/ water	LifeGuard™ (MO BIO, Carlsbad, ca.)	15–30 days	TRFLP method	Lower than initial fresh sample	[69]
	DMSO–EDTA–salt solution (DESS)	15–30 days	TRFLP method	No significant changes
	Without preservative	15–46 days	TRFLP method	No significant changes
Soil	20 °C	90 days	16S rRNA gene-DGGE	31.4% variation from fresh sample	[24]
Soil	15 °C	120 days	16S rRNA gene-DGGE	20.9% variation from fresh sample
Human feces (Collected in RNAlater)	− 80 °C	6 months	16S rRNA sequencing (454 platform)	Minimum influence of length and storage temperature on microbiota composition	[71]
	− 80 °C	2 years	16S rRNA sequencing (MiSeq platform)	Change in abundance of some genera (increase Lactobacilli, decrease Staphylococci), Alpha-diversity for storage at RT decreases over time
	− 80 °C	5 years	16S rRNA sequencing (454 platform)	Minimum variability but some genera more affected than others
Compost of different age	4 °C, air dried, − 20 °C, lyophilized	3 and 14 days	16S rRNA gene based DGGE analysis	No to little change in extracted DNA quality and quantity	[22]
Anaerobic human gut microbes.	Lyophilization and cryopreservation	3 months	Cell viability by FACS	Cryoprotectant enhance viability	[67]
Switchgrass enriched communities	DMSO and glycerol as cryoprotectants	3 weeks	Viability	Both cryoprotectants were equally effective	[60]
Fecal microbiome	95% ethanol, OMNIgene Gut, FTA cards	8 weeks	16S-rRNA gene sequencing	Protect better community in comparison to 70% ethanol	[27]
Anaerobic digestion (AD) inoculum	Lyophilization cryopreservation		Functional assessment of CH4 recovery	Lyophilization with 10% skimmed milk protect better functionality	[68]
Human stool	+ 4 °C , − 80 °C, − 20 °C and RT	1 week	454 pyrosequencing	No significant changes	[70, 72]
Mixed microbiome	DMSO and DMSO plus trehalose	106 days	16S rRNA gene based community profiling	DMSO + trehalose and tryptic soy broth gave better protection	[32]

Perspective and Conclusion

In conclusion; pure culture and OMICS based studies has proven that microbes are the backbone of biotech industries and key player of various ecological processes. Culture independent metagenomics studies revealed that immense microbial diversity of clinical, environmental and industrial importance is untapped. Despite all the technological innovation only 1–10% microbial diversity has been cultured and rest 90–99% is not- yet- cultured. Due to technical and physiological limitation it is not possible to culture and preserve all the component of microbial community in a short period of time. It has been observed that due to ecological perturbations and climatic variations core microbiome of any ecological system is gradually shrinking and creating the possibility of extinction and loss of certain valuable components of microbiota with time. Therefore; concept of intact sample and microbiome preservation with retained viability and functionality for future OMICS, cultivation and application is fairly relevant in current scenario. Published data indicates that research work has been initiated in preservation of intact sample, microbiome, enriched culture, mix- culture and obligate anaerobes. Several papers have been published in last 5–6 years on this theme. It has been noticed that there is no any single method, process, preservation-protocol and cryoprotectant are available that works optimally for every kind of sample. Microbial heterogeneity in microbiome is the main factor that responds differently with different preservation methods. Due to this heterogeneity application of same protocol or preservation condition for all sorts of samples is difficult. Different samples and cells behave differently with different cryoprotectants and preservation conditions. Therefore, optimization of conditions of sampling, transportation, storage and cryoprotectants which provide maximum protection to different kinds of cell as well as cellular component is the need of current era. Validation of post preservation viability, functionality and health of preserved microbiota is also mandatory to develop better strategy for intact microbiome preservation. Samples from extreme environmental conditions and obligate anaerobic habitats are difficult to preserve for a long-period without alteration in the microbial community organization, viability and functionality. Due to these difficulties, it always remains as a challenge for the scientific communities and needs to be given extra attention for its successful preservation.