Diversity, metabolism and cultivation of archaea in mangrove ecosystems

Abstract

Mangroves comprise a globally significant intertidal ecosystem that contains a high diversity of microorganisms, including fungi, bacteria and archaea. Archaea is a major domain of life that plays important roles in biogeochemical cycles in these ecosystems. In this review, the potential roles of archaea in mangroves are briefly highlighted. Then, the diversity and metabolism of archaeal community of mangrove ecosystems across the world are summarized and Bathyarchaeota, Euryarchaeota, Thaumarchaeota, Woesearchaeota, and Lokiarchaeota are confirmed as the most abundant and ubiquitous archaeal groups. The metabolic potential of these archaeal groups indicates their important ecological function in carbon, nitrogen and sulfur cycling. Finally, some cultivation strategies that could be applied to uncultivated archaeal lineages from mangrove wetlands are suggested, including refinements to traditional cultivation methods based on genomic and transcriptomic information, and numerous innovative cultivation techniques such as single-cell isolation and high-throughput culturing (HTC). These cultivation strategies provide more opportunities to obtain previously uncultured archaea.

Electronic supplementary material

The online version of this article (10.1007/s42995-020-00081-9) contains supplementary material, which is available to authorized users.

Introduction

Mangroves are located in the tropical and subtropical coastal areas of the world. They provide ecological services such as maintaining biodiversity, improving water quality, and protecting coastlines. As one of the world’s most productive ecosystems, mangroves are characterized as an important “blue carbon” reservoir (Alongi 2014). Mangroves are located in a buffer zone connecting land and ocean, supporting relatively high microbial diversity and complex microbial communities (Moopantakath et al. 2020; Zhang et al. 2019). Archaea, one of the most important microbial components, are widespread in mangrove ecosystems. In mangroves, the number of archaeal 16S rRNA genes ranges from 10⁷ to 10⁸ copies per gram of wet sediment (Li et al. 2012) and from 10⁷ to 10¹⁰ copies per gram of dry sediment (Zhou et al. 2017). The cultivation-independent approaches, such as 16S rRNA gene sequencing and metagenomics, have revealed a high diversity and range of metabolisms in the archaea of mangroves (Bhattacharyya et al. 2015; Pan et al. 2019; Zhang et al. 2019). Archaea have thus been proposed to play important roles in nutrient recycling in these ecosystems. However, only a few archaeal lineages have so far been isolated from mangroves and this impedes our understanding of the roles of archaea in these unique ecosystems.

Carl Woese proposed that archaea represented the third domain of life in parallel with bacteria and eukaryote more than 40 years ago (Woese 1990; Woese and Fox 1977). Since then, examinations of biochemical properties have found that archaea share some similarities with bacteria, such as lack of intracellular compartments (Londei 2005) but also have some shared traits with eukarya, such as the absence of peptidoglycan in the cell wall (Kandler and Hippe 1977) and the presence of multiple RNA polymerases (Zillig et al. 1985). Although the Woeseian three-domain tree hypothesis has been adopted as the major theory for the universal tree of life for many years, the debate has always been accompanied by the “eocyte tree hypothesis” (Lake 1988, 1990; Lake et al. 1984), especially because of the discovery of Asgard archaea in recent years, which are proposed to be the closest prokaryotic relatives of eukaryotes (Spang et al. 2015; Zaremba-Niedzwiedzka et al. 2017). Originally, archaea were discovered and described from extreme environments, e.g., hot spring (Hua et al. 2019), hydrothermal vents (Anantharaman et al. 2016), acid mine drainage (Kuang et al. 2013), and highly saline lakes (Sorokin et al. 2017). The development of high-throughput sequencing techniques facilitates the investigation of archaeal diversity. Now it has been recognized that various archaea are distributed globally, e.g., in freshwater, seawater, soils and sediments (Adam et al. 2017; Zhou et al. 2018, 2019).

Our understanding of archaeal diversity has been expanded in recent years due to the discovery of many novel archaeal lineages that have changed the shape of the phylogenetic tree of archaea (Spang et al. 2017). Since 1990, Euryarchaeota and Crenarchaeota had been recognized as the only two phyla. However, more recently, high-throughput sequencing, metagenomics assembly and binning have revealed many new archaeal phyla, based on the phylogenetic and genomic analyses. Until recently, with the exception of Euryarchaeota, three superphyla have been recognized: TACK, DPANN, and Asgard (Spang et al. 2017). The number of archaeal phyla has been expanded from the original two phyla to at least 27 phyla now (Baker et al. 2020), ushering in a new era of archaeal research.

In this review, we first briefly summarize the potential roles of archaea from mangrove ecosystems and then focus on the diversity, metabolism and cultivation of the archaea from these wetlands. We aim to enhance understanding of archaeal diversity and provide cultivation strategies for particular lineage of archaea from mangroves.

The potential roles of archaea in mangrove ecosystems

Mangroves are present in the tropical and subtropical coastal areas of 112 countries and territories. Together they comprise an area of 0.11–0.24 million km² that extend over a quarter of the world’s coastline (Nellemann et al. 2009). Mangrove ecosystems contain complex environments that have been formed under the influence of tides, the influx of fresh water, high temperature and high humidity (Sahoo and Dhal 2009). The sediments are characteristically anoxic, rich in organic matter and provide eutrophic and brackish environments for a number of archaeal communities (Zhang et al. 2019). Mangroves are also one of the most productive ecosystems in the world and are characterized by high nutrient turnover rates. The diverse archaeal communities living in mangroves are also likely to play crucial roles in global biogeochemical cycles (Sahoo and Dhal 2009). For example, Euryarchaeota are involved in the production and oxidation of methane, a potent greenhouse gas (Taketani et al. 2010b). Ammonia-oxidizing archaea (AOA) in Thaumarchaeota are responsible for ammonia oxidation (Li et al. 2011). Bathyarchaeota and Asgard archaea may be involved in nitrite and sulfur reduction (Cai et al. 2020; Pan et al. 2020). The high rates of organic matter input and low rates of decomposition also contribute to carbon accumulation in mangroves (McKee 2011). Bathyarchaeota and Asgard archaea may play an important role in the degradation of multiple types of organic matters in mangroves (Cai et al. 2020; Pan et al. 2020). Also, Thorarchaeota might be involved in detoxification of arsenic (Liu et al. 2018). Furthermore, archaea possibly have a relationship with the geochemical transformation of iron (Fe) and manganese (Mn) in mangrove sediments (Otero et al. 2014). According to function prediction, archaea are also likely to be important in thiosulfate respiration, sulfur compounds respiration and aerobic chemoheterotrophy in mangroves (Marie Booth et al. 2019).

Archaea is an important microbiological component of sediments in mangrove wetlands. They are generally more abundant in deeper sediment layers, with relative abundances ranging from 20.8% to 41.3% of the 16S rRNA gene sequences of prokaryotic communities in mangroves (Luis et al. 2019). The 16S rRNA gene sequence-based approaches and metagenomics have revealed the high diversity of archaea in mangrove sediments. To clearly illustrate the diversity and relative abundance of archaea in mangroves, we undertook a search of 24 studies to identify the archaeal community and metabolic potentials in mangroves across the world (Fig. 1, Supplementary Table S1). The detailed methods are listed in Supplementary Information.

Fig. 1.

Locations of the mangrove sites that obtained from the 24 studies. The sites with 16S sequence data are in red (26 sites), while the sites with Illumina HiSeq data are in blue (9 sites)

Diversity and distribution of archaea in mangrove wetlands

According to the 16S rRNA gene sequence data analysis, the most abundant archaeal groups in mangroves are Bathyarchaeota, Euryarchaeota, Thaumarchaeota, Woesearchaeota and Lokiarchaeota (Fig. 2). Bathyarchaeota, the most dominant archaeal phylum, is widely distributed in sediments of mangroves, accounting for an average of 39.8% of the total archaea (Fig. 2). Previous studies have shown that Bathyarchaeota comprised more than 70% of archaea in the bottom sediment layer of the Cardoso Island State Park, Brazil (Mendes et al. 2012; Otero et al. 2014). Bathyarchaeota occupied about 60% of the total archaea in the Jiulong River, China (Li et al. 2012). Kubo et al. (2012) conducted a comprehensive analysis of the biogeographical distribution of Bathyarchaeota and found that it was the dominant archaeal population in anoxic, low-activity subsurface sediments. So far, based on 16S rRNA gene analysis, 25 subgroups of Bathyarchaeota have been proposed; these subgroups are likely to have different strategies to adapt to the marine and freshwater environments (Zhou et al. 2018). For examples, subgroups 6, 8, 15, and 17 were the major Bathyarchaeotal subgroups in Shenzhen Futian Mangrove Nature Reserve, China (Pan et al. 2019).

Fig. 2.

Archaeal community composition in mangrove ecosystems of different sites. Only taxa that represent > 5% of the relative abundance in at least one site are represented. Minor phyla that represent < 5% of the relative abundance in all sites are not shown

In Euryarchaeota, Thermoprofundales (Marine Benthic Group D, MBG-D), Methanosarcinales, Marine Group III, and Methanofastidiosales were the most abundant orders, representing 13.4%, 3.7%, 2.8%, and 1.2% of the total archaea, respectively (Fig. 2). Thermoprofundales is one of the most frequently encountered archaeal lineages with a widespread distribution and high abundance (Zhou et al. 2019). For example, Thermoprofundales reached 5–53% of the total archaea in the sediment layer of Cardoso Island, Brazil (Mendes et al. 2012; Otero et al. 2014). Thermoprofundales encountered for 43% of the total archaea in sediments of New Caledonia (Luis et al. 2019). Methanogens are also one of the most important groups from the phylum Euryarchaeota. Multiple methanogenic lineages have been identified in mangrove sediments, including Methanobacteriales, Methanocellales, Methanofastidiosa, Methanosarcinales, Methanomicrobiales, and Methanomassiliicoccales (Zhang et al. 2020a). Methanobacteria, Methanococci, and Methanomicrobia represented 5%, 8%, and 35% of the total archaea in Kerala (India), respectively (Imchen et al. 2017). It has been reported that salinity was the major environmental factor regulating the methanogenic community assemblage and different methanogens occupied different niches (Zhang et al. 2020b). Methanococcoides, Methanoculleus, and Methanogenium preferentially existed in saline sediments, whereas Methanomethylovorans, Methanolinea, Methanoregula and Methanomassiliicoccales were more abundant in freshwater-oligohaline sediments (Zhang et al. 2020b).

Thaumarchaeota is one of the most abundant and cosmopolitan phyla in mangrove sediments, accounting for 1–74% of the total archaea (Fig. 2). It is the most abundant phylum (44–74%) on the west coast of India (Singh et al. 2010). Thaumarchaeota is also the dominant phylum in surface sediments of mangroves in Sundarbans (Bhattacharyya et al. 2015). Since Thaumarchaeota are aerobic archaea, they are considerably more abundant in oxic environments and oxic/anoxic interface zones than in the corresponding subsurface samples at the same sampling site (Zhou et al. 2017).

Woesearchaeota is abundant in mangrove sediments in some particular sites, e.g., in the mangrove sediments of Daya Bay (China), and Dongzhaigang (China), accounting for 10–27% of the total archaea (Li et al. 2016; Zhang et al. 2018). Lokiarchaeota, formerly named MBG-B (Marine Benthic Group B) and DSAG (Deep Sea Archaeal Group), are also important components of mangrove sediments, accounting for 2–15% of the total archaea (Fig. 2). It has been detected in sediments in the Gulf of Mexico, Bamenwan (China), Shenzhen Futian Natural Reserve (China) and Hong Kong Mai Po wetland (China) (Devereux et al. 2015; Liu et al. 2019; Zhang et al. 2019; Zhou et al. 2017). As with Bathyarchaeota, Lokiarchaeota are more abundant at greater depths within the sediments than in surface layers. Notably, Hydrothermarchaeota comprised 3–23% of total archaea in the Sundardans area (Bhattacharyya et al. 2015) and Odinarchaeota comprised 32% of total archaea in Ilha Grande, Brazil (Silveira et al. 2013), respectively.

The community composition of archaea showed distinct patterns at different sites. Mangrove archaeal communities were found to change with geographic location, which might be driven by a variety of environmental variables (etc., pH and carbon and nitrogen contents) (Li et al. 2019; Zhang et al. 2019). The community composition of archaea at the same site is also dependent on environmental properties such as silt–clay percentage, amount of organic matter and pH (Colares and Melo 2013; Zhou et al. 2017).

Mangrove archaeal communities might also be influenced by factors such as anthropogenic activities, including oil spills, municipal and industrial discharge and shrimp farming (Bhattacharyya et al. 2015; Dias et al. 2011; Taketani et al. 2010a). Mangrove sediments with these contaminants show an increase in the organic carbon content, which leads to alteration of the archaeal community composition. Halobacteriales from the Euryarchaeota was found to be the predominant group in hydrocarbon-polluted mangrove sediments (Mukherji et al. 2020). The release of municipal and aquaculture sewage upstream from estuaries might also promote the growth of Methanosarcinaceae and enhance methane production in mangroves (Zhao et al. 2019).

Depth is another important factor structuring archaeal communities in mangrove sediments, principally driven by oxic state of the sediments (Li et al. 2012; Zhou et al. 2017). Thaumarchaeota and Euryarchaeota predominate in surface sediments, while Bathyarchaeota and Lokiarchaeota dominate in subsurface sediments (Luis et al. 2019; Zhou et al. 2017). Previous study has shown that Bathyarchaeota accounted for 1.6% of total prokaryotic sequences in surface sediments, while it increased to 26% of total 16S rRNA gene sequences of prokaryotic communities in deep layers (Luis et al. 2019).

Mangrove trees may also affect archaeal diversity and composition, as there is extensive nutrient exchange between mangrove plants and various archaeal groups (Li et al. 2016; Pires et al. 2012). Previous studies have shown that the abundance of Methanobacteriales and Methanosarcinaceae was significantly higher in mangrove sediments than in the non-mangrove sediments (Zhao et al. 2019).

Metabolic potentials of archaea in mangrove wetlands

Recently, metagenomics techniques have obtained many metagenome-assembled genomes (MAGs) of archaea. The MAGs of archaea, recovered from mangrove ecosystems, revealed special metabolic potentials in carbon, nitrogen, and sulfur cycling, indicating important ecological functions of archaea in mangrove ecosystems (Fig. 3, Supplementary Table S2).

Fig. 3.

Archaeal metabolic potentials in mangrove ecosystems. Ecological roles of archaeal lineages in carbon, nitrogen and sulfur cycles based on physiological and genomic information. Black, grey and white circles indicate complete, incomplete and absent pathways, respectively

Multiple archaeal lineages have potential to fix inorganic carbon. Metabolic reconstruction revealed that Thermoprofundales, Bathyarchaeota, Lokiarchaeota, Thorarchaeota, Gerdarchaeota and Helarchaeota, identified from mangroves, have the genetic potential for inorganic carbon fixation via the archaeal Wood-Ljundahl (WL) pathway (Cai et al. 2020; He et al. 2016; Liu et al. 2018; Pan et al. 2020; Sousa et al. 2016; Zhou et al. 2019). Analysis of MAGs showed that Thermoprofundales might also encode an incomplete 3-hydroxy propionate/4-hydroxybutyrate cycle to fix CO₂ (Zhou et al. 2019). Recently, the presence of genes for rhodopsins, cobalamin biosynthesis, and the oxygen-dependent metabolic pathways in some Bathyarchaeota subgroup 6 genomes suggest a light-sensing and microoxic lifestyle within this subgroup (Pan et al. 2020).

Mangrove ecosystems contain a large number of organic carbon compounds, including carbohydrates, amino acids, and lipids (Alongi 2014). Previous researches have shown that almost all archaeal groups are predicted to degrade organic carbon into fermentation byproducts (Baker et al. 2020; Li et al. 2015). Thermoprofundales, Bathyarchaeota, Lokiarchaeota, and Thorarchaeota are hypothesized to have a heterotrophic lifestyle. Thermoprofundales are able to transport and assimilate peptides and generate acetate and ethanol through fermentation (Zhou et al. 2019). Several studies have suggested that Bathyarchaeota is capable of utilizing a variety of organic matter types, including cellulose, chitin, aromatic compounds, and fatty acids (He et al. 2016; Lazar et al. 2016; Lloyd et al. 2013; Meng et al. 2014). Lokiarchaeota, Thorarchaeota, and Gerdarchaeota in the Asgard superphylum are hypothesized to be not strictly autotrophic but might also participate in the degradation of organic carbon in mangroves (Cai et al. 2020). The prevalence and high relative abundance of Thermoprofundales, Bathyarchaeota, and Lokiarchaeota indicate that they may contribute to the turnover of organic matter in mangrove ecosystems.

Euryarchaeota undertake methane production, anaerobic methane oxidation and anaerobic oxidation of other short-chain alkanes (i.e., ethane and butane) (Borrel et al. 2019; Chen et al. 2019; Laso-Perez et al. 2019; Lyu et al. 2018). Mangrove sediments are usually muddy, anoxic, and have a high organic carbon content, which is a suitable habitat for methanogens. Sequencing of 16S rRNA genes, metagenomics, and metatranscriptomics has shown that hydrogenotrophic Methanomicrobiales, and H₂-dependent methylotrophic Methanomassiliicoccales were highly abundant and active, suggesting that these methanogenic pathways contribute the most to methane emission in mangroves (Xiao et al. 2017; Zhang et al. 2020a). MAGs annotations have revealed that methanogens contain genes encoding organic osmotic solute transporters to adapt to the high salinity of mangrove environments. Hydrogenotrophic Methanomicrobiales MAGs encoded multiple membrane-bound hydrogenases and a large number of electron transporters, which is favorable for their adaption to low substrate (H₂) environments (Zhang et al. 2020a). Methanomassiliicoccales MAGs contain genes encoding substrate-specific methyltransferases for multiple methylated compounds including methanethiol, methanol, and trimethylamine (TMA) (Zhang et al. 2020a). Methanol can be produced by degradation of lignin or pectin (Lyimo et al. 2009). Methylated amines can be formed from decomposition of choline and glycine betaine, which are osmolytes produced by many marine organisms to cope with osmotic stress. These methylated compounds are not easily utilized by sulfidogenic bacteria, but they can be rapidly fermented by methanogens to methane (Jones et al. 2019). In addition, methyl-coenzyme M reductase (McrA)-like transcripts have been found in Helarchaeota, indicating that Helarchaeota might be involved in short-chain alkane oxidation in mangroves, where ethane and butane originate from oil–gas seepage or human activities (Cai et al. 2020).

Multiple archaea are involved in the global nitrogen cycle. In mangrove ecosystems, AOA in Thaumarchaeota contain the ammonia monooxygenase gene (amoA) that can oxidize ammonia to nitrite (Li et al. 2011). Bathyarchaeotal and Thorarchaeotal genomes contain the nitrogenase iron protein gene (nifH) for nitrogen fixation, indicating that they can use nitrogen to synthesize ammonia (Liu et al. 2018; Pan et al. 2020). Thorarchaeota contain the nitrite reductase gene (nirB) that coverts nitrite to ammonia (Liu et al. 2018). There is genomic evidence that Bathyarchaeota contains the nitrate reductase gene (narH), the nitrite reductase gene (nirB) and mono/di/trimethylamine aminotransferase genes (mttB/mtbB/mtmB), suggesting Bathyarchaeota can utilize diverse nitrogen compounds to produce ammonium and then convert ammonium to urea (Pan et al. 2020). It has been reported that Thermoprofundales contains nitrate reductase genes (nar), suggesting they might participate in the initial step of denitrification (Zhou et al. 2019). The above results indicate that archaea may play an important role in the global nitrogen cycle.

Archaeal MAGs also contain sulfur cycle related genes. Sulfate concentration has previously been reported to range from 1.23 to 3.61 g/kg of dry sediment in mangroves (Wu et al. 2019). Sulfate reduction genes (sat) and phosphoadenosine phosphosulfate reductase (cysC), that participate in the first two steps of assimilatory sulfate reduction, reducing sulfate to sulfite through adenylyl-sulfate, can be identified in MAGs of Bathyarchaeota and Thermoprofundales, suggesting they probably depend on sulfate assimilation (Pan et al. 2020; Zhou et al. 2019, 2018). Furthermore, Bathyarchaeota and Thorarchaeota contain the hydrogenase/sulfur reductase gene (hydA) to reduce S to sulfide (Liu et al. 2018; Pan et al. 2020). All of these results indicate the role of archaea in the global sulfur cycle. In addition, annotation of Thorarchaeota MAGs reveals that they are involved in arsenic detoxification, suggesting that they could be applied to bioremediation of As-contaminated sediment or water (Liu et al. 2018).

Archaeal isolation from mangrove sediments

Cultivation-independent approaches provide a great many insights into the diversity, ecology, and metabolism of archaea. However, only seven phyla (i.e., Euryarchaeota, Crenarchaeota, Thaumarchaeota, Nanoarchaeota, Nanohaloarchaeota, Micrarchaeota, and Lokiarchaeota) have been isolated and cultured, while the other archaeal phyla have not yet been cultured (Baker et al. 2020). Euryarchaeota contains the most isolates, including members in the classes Archaeoglobi (Hartzell and Reed 2006), Halobacteria (Oren 2006), Thermococci (Bertoldo and Antranikian 2006; Zhao et al. 2015), Thermoplasmata (Huber and Stetter 2006a), as well as methanogens in the class Methanobacteria (Bonin and Boone 2006), Methanococci (Whitman and Jeanthon 2006), Methanopyri, Methanomicrobia (Garcia et al. 2006; Kendall and Boone 2006). In recent decades, the class Methanonatronarchaeia and the order Methanomassiliicoccales in the class Thermoplasmata have also been isolated (Dridi et al. 2012; Sorokin et al. 2017). There are pure cultures of three orders of the class Thermoprotei within the phylum Crenarchaeota, including Thermoproteales (Huber et al. 2006b), Sulfolobales (Huber and Prangishvili 2006c), and Desulfurococcales (Huber and Stetter 2006d). In Thaumarchaeota, Nitrosopumilus maritimus SCM1 has been isolated from a marine aquarium (Konneke et al. 2005). A few members of the Nanoarchaeota (Huber et al. 2002, 2006e; Wurch et al. 2016), Nanohaloarchaeota (Hamm et al. 2019), and Micrarchaeota (Krause et al. 2017) are co-cultured with their hosts. In the Asgard superphylum, a co-culture of a Lokiarchaeota, named as Prometheoarchaeum syntrophicum MK-D1, and one methanogen Methanogenium has recently been obtained from deep marine sediments (Imachi et al. 2020). No pure cultures of Bathyarchaeota have so far been successfully established. Recently, Bathyarchaeota subgroup 8 has been enriched from estuarine sediments in a consortium using lignin as an energy source (Yu et al. 2018). Here, we present an overview of the 16S rRNA gene-based phylogenetic tree of archaea with cultured archaeal lineages marked with red five-pointed stars (Fig. 4, Supplementary Table S3), which indicates the demand for isolates of the underexplored groups. Figure 4 was adapted from Sun et al. (2019) and supplemented with new published data (Cai et al. 2020; Jay et al. 2018; Probst et al. 2018; Wang et al. 2019).

Fig. 4.

Phylogenetic tree based on archaeal 16S rRNA gene sequences. Those with red five-pointed stars are groups that have cultivated members. Figure 4 was adapted from Sun et al. (2019) and supplemented with new published data (Cai et al. 2020; Jay et al. 2018; Probst et al. 2018; Wang et al. 2019). The phylogeny was generated with 1002 archaeal 16S rRNA gene sequences whose length longer than 660 bp using FastTree. Bootstraps are based on 1000 replicated trees. The alignment was generated with MUSCLE. Helarchaeota in the Asgard superphylum was not included in the phylogenetic tree, since there was no Helarchaeotal 16S rRNA gene sequences obtained from MAGs or references

Nitrogen-fixing bacteria, phosphate solubilizing bacteria, and sulfate-reducing bacteria (e.g., Bacillales, Actinomycetales, Vibrionales) and fungi (e.g., Pestalotipsis foedans, Fusarium solani) have been isolated from mangrove environments (Sahoo and Dhal 2009). However, there have been few isolations of archaea from mangroves (Supplementary Table S4). A few methylotrophic methanogens have been isolated and cultured from mangrove sediments. For example, Methanolacinia paynteri was isolated from the mangrove swamps located in the Cayman Islands (Rivard et al. 1983). Methanococcoides methylutens, which grew on trimethylamine (TMA) and methanol, was isolated and characterized from the mangrove sediments of Southeast India (Mobanraju et al. 1997). Methanosarcina semesiae was isolated from mangrove sediments in Tanzania (Lyimo et al. 2000). A mesophilic methylotrophic methanogenic archaeon Methanococcoides strain MM1 was isolated from mangrove sediments in Tanzania, which was capable of utilizing methanol and methylated amines as the only substrates (Lyimo et al. 2009). Mangroves at some sites are strongly affected by anthropogenic activities and disturbances. Haloarchaea are extremophiles surviving in extreme salinities and are involved in bioremediation of contaminations. Two major haloarchaeal genera Haloferax and Haladaptatus have recently been successfully isolated form hydrocarbon polluted mangrove sediments in Sundarban area, which could be used to reduce the chemical oxygen demand (COD) of polluted mangrove sediments (Mukherji et al. 2020).

Future cultivation strategies and suggestions for mangrove archaea

Obtaining pure cultures remains a priority for microbial ecological studies. For example, pure cultures are essential for a comprehensive understanding of the physiology and biochemistry of microbes and offer solid evidence for their ecological functions. Pure cultures enable the discovery of functionally important products such as new antibiotics and other secondary metabolites (Zhao et al. 2019). Nearly all existing archaeal strains were isolated and described more than ten years ago and only a few novel strains have been pure cultured using traditional enrichment and isolation methods, such as single colony picking and serial dilution-to-extinction, within the last ten years. There are several reasons why traditional archaeal cultivation methods have reached a plateau (Sun et al. 2019). Firstly, many archaea, such as the DPANN superphylum, may be symbionts or have interactions with other microbial species and so they cannot be isolated as single strains (Moissl-Eichinger et al. 2017). Secondly, the slow growth rates and low abundances of archaea also make them difficult to culture. Thirdly, limited knowledge of archaeal metabolisms impedes the establishment of suitable culture media and growth conditions for isolation.

In the recent decades, exciting progress has been made in cultivation techniques. There are two major strategies that could be taken advantage of for future archaeal isolation. The first strategy is the refinement of conventional cultivation processes based on genetic and transcriptomic information. When strains are enriched or cultivated, metagenomic and transcriptional data can be used as guidelines for selecting culture media and growth conditions (Laso-Perez et al. 2018). For example, a reconstruction of the metabolism of Bathyarchaeota, Thermoprofundales, and Asgard archaea predicts their substrate preferences for organic matter (Cai et al. 2020; Pan et al. 2020; Zhou et al. 2019). Mangrove sediments are typically rich in organic matter. Bathyarchaeota, Thermoprofundales and Lokiarchaeota are abundant in mangroves. Hence, a variety of organic compounds (such as amino acid, peptide, lipid, lignin) could be added as substrates to enrich and culture Bathyarchaeota, Thermoprofundales and Lokiarchaeota from mangroves. Genome-guided isolation could be also applied to the culture of methylotrophic methanogens. Methanogenesis is a dominant terminal processes in the degradation of organic matter in mangroves (Lyimo et al. 2009). According to genome annotation, Methanosarcinales, Methanofastidiosa, and Methanomassiliicoccales are able to utilize methylotrophic compounds (methanol, methanethiol, and trimethylamine) as substrates for methanogenesis (Zhang et al. 2020a). Therefore, a variety of methylated compounds could be added as substrates to enrich and culture methylotrophic methanogens. Sulfate is available and sulfate reduction predominates in mangroves. Archaea involved in sulfate reduction, such as Bathyarchaeota and Thermoprofundales, could be a target for isolation (Pan et al. 2020; Zhou et al. 2019). Furthermore, co-occurrence networks are used to analyze potential interactions and seek “key node” microbes in a microbial communities (Xian et al. 2020). Co-cultures of Rice Cluster I with propionate-oxidizing H₂-producing bacterium and Prometheoarchaeum syntrophicum MK-D1 with Methanogenium are two successful examples for microbial interaction (Imachi et al. 2020; Sakai et al. 2008). Co-occurrence networks in mangroves have shown that methanogens have significant non-random association with Woesearchaeota (Zhang et al. 2020a). Above all, specific substrate or co-culture microbes can be added to promote the growth of target archaea based on guidance from genomic information and co-occurrence analysis.

For the second strategy, numerous innovative culture techniques have been applied to microbial cultivation, such as single cell isolation using microfluidics (Boitard et al. 2015; Kaminski et al. 2016), capillary tube, encapsulation techniques, optical and raman tweezers (Park and Chiou 2011), and high-throughput cultivation (HTC) methods such as Microtite plates (MTP), a Million-Well Growth Chip (Hesselman et al. 2012; Ingham et al. 2007) and culturomics (Lagier et al. 2012). Single cell isolation, which is different from traditional “first culture then isolate” method, mainly includes three steps: (1) separate single cells, (2) detect the target cell, and (3) obtain pure culture of the cell. Optical tweezers, one of the single cell isolation techniques, has been successfully used for the isolation of the DPANN archaea Nanoarchaeota (Wurch et al. 2016). Raman-Activated Droplet Sorting (RADS), another single cell isolation technique, can achieve a higher throughput, preserve the vitality of cells and facilitate downstream single-cell cultivation (Wang et al. 2017). High-throughput cultivation means that microbes are indiscriminately isolated using a variety of culture media (Mu et al. 2018). Substrate concentrations in each well are three orders of magnitude lower than those in common laboratory media, which is suitable for growth of oligotrophic archaea (Welte 2018). These innovative methods show great potential for the isolation and culture of single archaeal cell from mix microbial community in mangroves.

Outlook

Mangroves are a unique coastal ecosystem that provides a wide range of microbial resources. Recent culture-independent and culture-dependent studies have revealed the large diversity and important potential functions of archaea in biogeochemical cycles in mangroves. Additional understanding of the taxonomy and metabolic potential of archaea will come from future genomic interpretation. However, there is still a need for more studies on archaea isolation and cultivation to verify their physiological and ecological roles. Using both refined traditional isolation methods and innovative isolation techniques, more previously uncultivable archaea will be successful cultivated or enriched in future. This will open the door for the enhancement of our understanding of archaeal ecological process and evolution.

Electronic supplementary material

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Author contributions

ML provided the idea and revised the manuscript. C-JZ collected and analyzed data with help from Y-LC, Y-HS, JP and M-WC. C-JZ contributed in preparing the manuscript with the help of all authors.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Animal and human rights statement

This article does not contain any studies with human participants or animals performed by any of the authors.