Technological Advancements in a Field Investigation of Marine Microorganisms: From Sampling Strategies to a Molecular Analysis

: The special characteristics of ocean ecosystems, such as the high salinity and pressure, low temperature, and nutrition, makes marine microorganisms diverse in species, gene composition, and ecological functions. Recent advances in molecular biology techniques, together with the ongoing developments in bioinformatic and automatic technologies, have highlighted the scientiﬁc studies on marine microbial ecology, eliminating the total reliance on classical cultivation-based techniques. This review systematically summarizes the innovative aspects of a ﬁeld investigation of marine microorganisms. It covers various cutting-edge sampling approaches employed in the ﬁeld, highlighting the in situ high-ﬁdelity sampling strategies with practical applications. Particular emphasis is placed on the molecular analysis for marine ecology based on recently developed omics technologies. The key technical issues and substantial contribution of the contemporary instrumental analysis are discussed accordingly. It provides references for the in situ surveys of complex biogeochemical processes from marine microorganisms to ecosystems, relying on the development of new technical concepts and scientiﬁc methodologies in ﬁeld investigations.


Introduction
As one of the largest ecosystems, the ocean harbors vast amounts of microorganisms that account for most of the biodiversity on Earth.These marine microorganisms have attracted intensive research investigation for a long time and have been the research scope of the international ocean discovery program and many other comprehensive marine exploration programs more recently [1].With the implementation of advanced inter-disciplinary technologies aided by modern analytical equipment and tools, considerable achievements have been made in observing, mapping, and sampling marine microorganisms, as well as in the molecular analysis used to explore their biological and ecological functions [2,3].The great complexity of marine microbial communities and their diverse suite of interactions, however, pose a considerable challenge to contemporary biological oceanographers.
As the basic premise of marine resource exploration and development, the field investigation of marine microorganisms is of great significance (Figure 1).The first prerequisite for conducting marine microbial surveys is to adequately obtain microbial samples.The physiological status and sample quality of marine microorganisms is significantly affected by surrounding matrices and the in situ environmental variables of pressure, salinity, and temperature, etc., as well as the implementation of suitable collection and preservation apparatuses, which ultimately determine the accuracy and reliability of the analysis results [4,5].Identifying how to provide the most primitive microbial samples for scientific research has remained the focus for a long time.Beyond scientific marine biodiversity expeditions and sampling efforts, tremendous efforts have been made to isolate and culture marine microorganisms to investigate their diversity and functions, that is, to purify marine microorganisms from the natural environment, and then analyze the microbial communities in terms of their general biochemical properties or specific phenotypes [6][7][8].In recent years, alternative methods have been applied to avoid relying on pure culture approaches [9].The rapid development of microbial systematics and integrated meta-omics technologies, e.g., metagenomics, transcriptomics, proteomics, and metabolomics, has helped to conduct broad-scale diversity assessments of uncultured marine species [10].The cutting-edge analytical equipment, e.g., a fluorometer, DNA sequencer, and mass spectrometer [11][12][13], is more intensively used in scientific studies on marine microorganisms.Each of these technologies investigates a different aspect of marine communities, which provides us with comprehensive information about diverse biogeochemical cycles; however, most of them need to be adapted to the marine environment with special designs and fabrications in the field investigations.Therefore, for different marine habitats and research purposes, an increasing number of modern techniques have emerged for microbial studies, and the adoption of effective sampling and analytical strategies has become a priority.
salinity, and temperature, etc., as well as the implementation of suitable collection and preservation apparatuses, which ultimately determine the accuracy and reliability of the analysis results [4,5].Identifying how to provide the most primitive microbial samples for scientific research has remained the focus for a long time.Beyond scientific marine biodiversity expeditions and sampling efforts, tremendous efforts have been made to isolate and culture marine microorganisms to investigate their diversity and functions, that is, to purify marine microorganisms from the natural environment, and then analyze the microbial communities in terms of their general biochemical properties or specific phenotypes [6][7][8].In recent years, alternative methods have been applied to avoid relying on pure culture approaches [9].The rapid development of microbial systematics and integrated meta-omics technologies, e.g., metagenomics, transcriptomics, proteomics, and metabolomics, has helped to conduct broad-scale diversity assessments of uncultured marine species [10].The cutting-edge analytical equipment, e.g., a fluorometer, DNA sequencer, and mass spectrometer [11][12][13], is more intensively used in scientific studies on marine microorganisms.Each of these technologies investigates a different aspect of marine communities, which provides us with comprehensive information about diverse biogeochemical cycles; however, most of them need to be adapted to the marine environment with special designs and fabrications in the field investigations.Therefore, for different marine habitats and research purposes, an increasing number of modern techniques have emerged for microbial studies, and the adoption of effective sampling and analytical strategies has become a priority.This study reviews the development of sample collection and preservation techniques, and the advancements in the sample analysis strategies for marine microorganisms to date.Starting from enumerating different sampling and analytical strategies, a comparison between traditional sampling and in situ high-fidelity collection approaches is performed for marine microorganisms in different sample matrices, followed by an illustration of the advantages and limitations of different molecular analysis methods in a complex ecological niche.The research gaps, significances, and opportunities for current and future studies are also discussed to inspire more contemporary biological This study reviews the development of sample collection and preservation techniques, and the advancements in the sample analysis strategies for marine microorganisms to date.Starting from enumerating different sampling and analytical strategies, a comparison between traditional sampling and in situ high-fidelity collection approaches is performed for marine microorganisms in different sample matrices, followed by an illustration of the advantages and limitations of different molecular analysis methods in a complex ecological niche.The research gaps, significances, and opportunities for current and future studies are also discussed to inspire more contemporary biological oceanographers to further investigate the fundamental characteristics and biological complexity of marine microorganisms in field investigations.

Field Investigation of Marine Microorganisms
A typical workflow of the scientific studies on marine microorganisms includes sample collection, preservation, preparation, and analysis (Figure 2), which is described in more detail in the subsequent sections.The primary focus of the research was on the molecular biological techniques, particularly the in situ field investigations in the omics era.
oceanographers to further investigate the fundamental characteristics and biological complexity of marine microorganisms in field investigations.

Field Investigation of Marine Microorganisms
A typical workflow of the scientific studies on marine microorganisms includes sample collection, preservation, preparation, and analysis (Figure 2), which is described in more detail in the subsequent sections.The primary focus of the research was on the molecular biological techniques, particularly the in situ field investigations in the omics era.

Collection of Marine Microbial Samples
The marine environments that microorganisms inhabit are usually under extreme conditions.Appropriate microbial specimens should be sampled according to the local conditions of the seawater and seabed [14].According to the traditional sampling strategies, pre-sterilized bottles, bags, and/or corers, such as Niskin bottles and Kullenberg-type piston corers for shipboard water and sediment sample collections, should be accurately placed in predetermined column layers and seabed sites, strictly controlled at set stagnation durations, and returned in a timely manner to the deck for processing and a downstream analysis [15,16].Considering the short lifespans of prokaryotic transcripts averaged on the order of several minutes, however, traditional apparatuses are not appropriate for a nucleic acid-based analysis due to the time lapses, of up to hours, between the sample collection, preservation, and analysis [17].In addition, the possible changes in pressure, temperature, and redox conditions from anoxic deep-sea zones to marine surfaces may alter microbial cell and community structures [4,5].As a result, the gene expression and transcription profiles are likely to vary, caused by lost or interfered with fractions of DNA and rRNA as phylogenetic identifiers, making it difficult to obtain an accurate picture of marine microbial ecology.
In order to avoid the inaccuracy and variability of samples of a higher quality, the in situ collection, preservation, and detection of microbial samples was initiated using integrated sampling instruments in terms of deep-sea microbial sampling technology [18,19].As shown in Table 1, the in situ sampling technologies currently used mainly focus on five technical requirements, i.e., low disturbance, pressure retaining, temperature retaining, no pollution, and no pressure drop transfer, demonstrating a better fidelity effect on marine microorganisms than the traditional methods [20].With a pressure-retaining and thermal-insulation sampler (Figure 3a), the laboratory-measured rates of microbial

Collection of Marine Microbial Samples
The marine environments that microorganisms inhabit are usually under extreme conditions.Appropriate microbial specimens should be sampled according to the local conditions of the seawater and seabed [14].According to the traditional sampling strategies, pre-sterilized bottles, bags, and/or corers, such as Niskin bottles and Kullenberg-type piston corers for shipboard water and sediment sample collections, should be accurately placed in predetermined column layers and seabed sites, strictly controlled at set stagnation durations, and returned in a timely manner to the deck for processing and a downstream analysis [15,16].Considering the short lifespans of prokaryotic transcripts averaged on the order of several minutes, however, traditional apparatuses are not appropriate for a nucleic acid-based analysis due to the time lapses, of up to hours, between the sample collection, preservation, and analysis [17].In addition, the possible changes in pressure, temperature, and redox conditions from anoxic deep-sea zones to marine surfaces may alter microbial cell and community structures [4,5].As a result, the gene expression and transcription profiles are likely to vary, caused by lost or interfered with fractions of DNA and rRNA as phylogenetic identifiers, making it difficult to obtain an accurate picture of marine microbial ecology.
In order to avoid the inaccuracy and variability of samples of a higher quality, the in situ collection, preservation, and detection of microbial samples was initiated using integrated sampling instruments in terms of deep-sea microbial sampling technology [18,19].As shown in Table 1, the in situ sampling technologies currently used mainly focus on five technical requirements, i.e., low disturbance, pressure retaining, temperature retaining, no pollution, and no pressure drop transfer, demonstrating a better fidelity effect on marine microorganisms than the traditional methods [20].With a pressure-retaining and thermal-insulation sampler (Figure 3a), the laboratory-measured rates of microbial methane oxidation in the Joetsu Knoll cold seep in Japan, mediated by both aerobic and anaerobic methanotrophs of gammaproteobacterial Methylococcales and ANME archaea at 10 MPa and 4 • C for 45 d, were generally consistent with the methane oxidation rates reported in other oceanographic sites [21].By deploying a submersible-mounted sampler pressure-retaining and pressure-compensation unit, in-site sediment samples were successfully collected for the microbial communities investigation from hadal zones at a full ocean depth of 11,000 m in the West Philippine Basin, where the pressure change remained within ±6% [22].The advent of genomic technologies has expanded the studies of gene diversity and expression in situ.The microbial sampler submersible incubation device (MS-SID) (Figure 3b), allowing for in situ tracer incubations coupled with in situ sampling and preservation, was used for profiling the gene expressions of marine microbiota in a bathypelagic water column in the Eastern Mediterranean Sea [23].A higher percentage of MS-SID contigs were annotated (44%) than those in Niskin samples (29%), which might have contributed to the increased community complexity in the MS-SID samples with minimum environmental perturbations.More recent studies have also reported the dramatic differences in the microbial community characteristics between samples collected using multiple in situ nucleic acid collections (MISNACs), in situ microbial filtration and fixation (ISMIFF) apparatuses, and Niskin bottles [24,25], indicating the necessity for the in situ sampling and preservation of deep-sea marine samples.In the future, more high-fidelity and intelligent sampling methods must be proposed and practiced in research and development programs, and they will better meet the needs of expanding the exploration of complex marine ecosystems.

Analysis of Marine Microbial Samples 2.2.1. Molecular Analysis for Molecular Ecology
At present, marine microorganisms are phylogenetically studied according to the difference in their genetic structure and diversity, so as to explore their novel functions and, especially, uncultured microbial resources.Recent advances in studying the composition of marine microbial communities revealed several orders of magnitude of novel, uncultured species [9].Molecular ecology, a science that studies the structure and functions of biological molecules, such as nucleic acids, proteins, and metabolites, has rapidly expanded our knowledge of marine microbial abundance, diversity, and ecological functions.Certain molecular technologies are at present commonly applied to marine microbial samples, including clone libraries, fingerprinting tools of denaturing/temperature gradient gel electrophoresis (DGGE/TGGE), catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH), real-time qPCR (qRT-PCR), and oligonucleotide microarrays (Geochip), as well as the emerging omics approaches of metagenomics, transcriptomics and proteomics, and metabolomics [27].All of these technologies aim to understand the transmission of genetic and cellular information from marine microorganisms to marine ecosystems, each with its own advantages and limitations (Table 2).PCR-DGGE technology is one of the most commonly used methods in microbial molecular ecology, directly providing fingerprints of microbial communities in environmental samples after DNA extraction.However, there is no detailed classification and there are obvious shortcomings concerning the detection abundance and detection limit.qRT-PCR can determine the expression levels of functional genes; however, the key to its application is the design of functional gene primers [28].Different environmental samples require redesigning primers for various functional genes, and the emergence of rapid and accurate functional Geochip technology has solved this problem.The probe labeling and hybridization of numerous genes can be completed in one experimental process with a high degree of automation [29].CARD-FISH uses specific oligonucleotide probes to track and detect specific microorganisms; however, its sensitivity is not high, and sample processing requirements are high when dealing with complex communities [30].Hence, the application of different molecular analysis methods needs to be evaluated based on the specific field situations.In comparison to conventional phenotype-based markers, molecular markers can provide us with requisite landmarks for the elucidation of genetic variations in microbial ecology (Table 3).The 16S rRNA genes are the most frequently used phylogenetic markers in prokaryotic genomes.Based on 16S rRNA gene amplicon sequencing, the taxonomic identities of 26 tracer bacteria containing pathogenic and antimicrobial-resistant members were linked to coastal water pollution [34].By sequencing the 16S rRNA genes of the V3-V4 region, 25 bacterial genera were correlated with water depth, temperature, salinity, redox, as well as Pb, Al, and aliphatic and aromatic hydrocarbon contents, deciphering the influence of specific environmental variables on the benthic microbial communities and dynamics from shallow-to deep-sea sites (44-3573 m) on spatiotemporal scales [35].The housekeeping genes, e.g., rpoB and gyrB encoding ribosomal or DNA-linked proteins and amino-acyl synthetases, were recently highlighted as robust phylogenetic markers to precisely discriminate closely related species in microbial ecology [36][37][38].A single copy of rpoB simplified the ecological interpretation of PCR-DGGE profiles than that of 16S rRNA genes with multiple copies and sequence heterogeneity [39].A phylogenetic resolution of 4.5-times higher was recorded on the rpoB-based tree than on the 16S rRNA-based tree, providing more specific and sensitive DNA-sequencing subtypes for 13 Bacillus species from marine environments [40].Additionally, the gyrB housekeeping gene was confirmed to be a valuable marker in distinguishing marine strains from the Bacillus pumilus clade [41].As compared to 16S rRNA and housekeeping genes, functional gene amplicons are able to provide more accurate insights into the ecological potentials of highly diverse marine microorganisms in different and distant lineages.By analyzing genes for dissimilatory sulfite reductase and oxidase (dsrB and soxB) using combined high-through sequencing and qPCR, the distribution and composition of sulfur-oxidizing and sulphate-reducing bacteria in complex communities were depicted in marine sediment cores and surficial sediments along a bathymetric gradient [42,43].In deep submarine permafrost, anaerobic methanotrophic archaea (ANME-2a/b and ANME-2d) responsible for the anaerobic oxidation of methane (AOM) were identified by functional marker genes (mcrA), CARD-FISH, and δ 13 C-methane signatures, suggesting the potential roles of AOM in global methane budgets [44].With an isocitrate lyase (icl) gene maker and Geochip 2.0 targeting > 10,000 bacterial genes of ecological function with 50-mer probes, 25 bacterial isolates were determined with putative cold-adapted alleles in seacoast permafrost samples [45].There are numerous studies on molecular markers interpreting the ecological significance of marine microorganisms, and more information will be provided by biomolecular omics technologies, advancing our knowledge of marine resource exploration, biodiversity conservation, and global climate change mitigation.

Molecular Analysis by Omics Technologies Marine Metagenomics
Metagenomics is a discipline that takes the total genetic materials of all microorganisms in environmental samples as targets, sequences genomes, and screens functional genes to avoid the limitations of traditional cultivation-based microbiology [48].It makes it possible to study the microbial samples collected directly from marine environments, showing the potential to efficiently address the taxonomic and functional compositions of heterogeneous marine microorganisms [49].Metagenomics technologies for marine microbiota studies mainly adopt high-throughput sequencing approaches, represented by the next-or secondgeneration sequencing of Roche 454 and Illumina sequencing platforms [50,51].Among them, 454 pyrosequencing can generate large-scale DNA fragments of short read lengths, which is an ideal platform for the rapid sequencing of environmental genomes.On a hybrid platform of clone-dependent long-read Sanger and high-throughput short-read 454 pyrosequencing, high-quality draft assemblies were sequenced for 6 small marine microbial genomes in the most timely and cost-effective way [50].With complementary of 16 18S rDNA clone libraries corresponding to 454 pyrosequencing data, near full-length sequence information on the biodiversity and community compositions was obtained for micro-, nano-, and pico-planktons in marine environments, illustrating the potential for retrieving taxa from the rare biosphere [51].By providing millions of reads in a single run, Illumina HiSeq, MiSeq, and NovaSeq sequencers considerably contributed to the 16S rRNA gene libraries in the NCBI GenBank database with a greater sequencing depth and higher base-calling accuracy [52,53]; though, they generally they suffer from limitations regarding sequence assemblies with high biodiversity and the retrieval of adaptive genes in marine prokaryotes [54].Illumina MiniSeq, the latest benchtop Illumina sequencer, has been proved to rapidly and accurately predict antibiotic resistance by a fully automated bioinformatics system in clinical microbiology [2], which is also a potential candidate for field studies in marine microbiology.
The Pacific Biosciences (PacBio) sequencer, a third-generation platform for long-read metagenomics, allows for the direct retrieval of full-length genes to avoid the bias of sequence splicing, assembly, and annotation in bioinformatics analyses [55].For 23 bacterial and 3 archaeal species, the PacBio complete 16S rRNA gene sequences showed less ambiguous classifications than the Illumina V4 16S rRNA gene sequences [56], demonstrating it to be a powerful tool for detecting more marine microbial species at the genus and species levels [57].Oxford nanopore technology, another third-generation platform for singlemolecule real-time sequencing, depends on a single base of nucleic acids and a protein nanopore reader.It does not employ PCR amplification or chemical labeling, leaving no theoretical limit on the reading length of DNA or RNA fragments [58].It has been applied to decode the longer sequences of 16S rRNA, 18S rRNA, and internal transcribed spacer loci, achieving comprehensive taxonomic profiling and high-resolution mapping results for the genera and species of bacteria, protists, and fungi thriving on marine plastic debris and seawater [59,60].Taking advantage of its scalability and portability, a mobile Min-ION sequencer and nanopore flow cell were incorporated into an in situ field sequencing setup (Figure 4a), where 32 samples were sequenced simultaneously for DNA barcoding and species identification onboard [61].In a novel MICRObial life-detection platform (Figure 4b), 39 microbial isolates and an active L-serine metabolism community were identified by cryo-iPlate and a microbial activity microassay, which were further analyzed by Min-ION for biosignature characterizations and validated by Illumina MiSeq results.With the assistance of the base calling and uplink/downlink of nucleic acid sequences through satellite Internet, a metagenome was completed for viable microbes in the Canadian High Arctic environment [62].With an added advantage of low capital costs, nanopore sequencers are promising shipborne solutions for the on-site analysis of complex marine microorganisms.On the whole, the development and application of long-read, real-time, and highly flexible sequencing platforms allows for the achievement of more complete metagenomeassembled genomes from marine microorganisms containing many adaptive genes with ecological functions, which significantly contribute to the exploration and conservation of marine resources.

Marine Transcriptomics and Proteomics
Transcriptomics focuses on all RNA transcripts, including those of mRNA, non-coding RNA, and small RNA, to identify their transcriptional and post-transcriptional gene expressions and regulations, aiming to interpret the underlying biological processes in response to environmental perturbations [63].Current transcription technologies mainly include microarray or tiling arrays and transcriptome sequencing technology (RNA-Seq) [64][65][66].Microarray or tiling arrays utilize microchips with hybridization probes of known or entire DNA sequences to specifically survey global patterns in gene expressions and regulations [67], while RNA-Seq is the high-throughput sequencing of transcribed cDNA most frequently performed on Illumina HiSeq platforms [66].With a high-definition tiling array of marine diatom Phaeodactylum tricornutum, the first whole-genome methylome was obtained from a stramenopile to illustrate the adaptive evolutionary processes of gene-of 16 body methylation among eukaryotes, and the expression levels of methylated genes were quantified by RNA-seq data [68].The ever-growing transcriptomic evidence can reveal niche differentiations, account for certain metagenomic limitations, or identify potential functional genes in marine microorganisms.By exhibiting various co-occurring expression patterns of carbon, nitrogen, phosphorus, and sulfur elemental cycling genes, the transcriptomic activities of three primary marine Thaumarchaeota ecotypes were found to respond differently to chlorophyll α, salinity, and nutrient concentrations that were related to seawater depth and seasonal changes [69].With the advent of total RNA sequencing, 47 new viruses associated with vertebrate, host fish diet, or their microbiomes were identified by characterizing the viromes of 19 wild-caught fish species, exploring more of the marine virosphere [70].Combined with Illumina HiSeq mRNA-seq, comparative transcriptomic, and qRT-PCR analyses, a series of potential genes regulating lipid fraction migration, docosahexaenoic acid biosynthesis, signal transduction, and cell transport were analyzed in a heterotrophic marine microalga, Schizochytrium sp., which could promote the industrial production of single cell oil [71].Proteomics is an additional to metagenomics and transcriptomics by comparative or quantitative, structural, and functional analyses.After protein separation by electrophoresis or liquid/gas chromatography, traditional Edman sequencing has a low throughput result and requires a large sample size, and advances in mass spectrometry (MS) have made highly sensitive proteomics more cost effective [72].Tandem mass tag-based proteomics showed that, by screening and analyzing 437 proteins with differential expression levels, marine bacterium Halobacillus sp.P1 inhibited the nitrogen metabolism of diatom Skeletonema costatum during nutrient competition, hence revealing the algicidal mechanism in marine algae-bacteria interactions [73].Quantitative proteomics suggested that three marine bacterial lineages, i.e., Oceanospirillaceae, Roseobacter, and Flavobacteria, profoundly altered their proteomic profiles during growth transition, with characteristic patterns in the abundance of key enzymes in energy and central carbon metabolism processes [74].The cold and alkane-induced proteins, which were depicted by LC-MS/MS shotgun proteomics, were up to 21-fold higher in obligate hydrocarbon-degrading psychrophile Oleispira antarctica RB-8 grown on n-alkanes at 4 • C than in the non-hydrocarbon control group [75].These studies pointed out the scientific potential of proteomics to address the divergent ecological strategies of marine microorganisms in adapting to deteriorated marine environments with algal blooms, oil spills, and other contaminants.

Marine Metabolomics
Metabolomics is an emerging omics technology following transcriptomics and proteomics, which is the closest to the biological phenotype in various omics studies.Metabolite detection commonly relies on nuclear magnetic resonance (NMR) and mass spectrometry (MS) that couples with gas chromatography (GC), liquid chromatography (LC), and capillary electrophoresis (CE) (Table 4) [76,77].NMR can identify high concentrations of metabolites; however, it is limited to sensitivity and signal congestion in detecting metabolites at low levels [78].The GC/LC/CE-MS detection of metabolites is superior, with a lower analysis limit and higher repeatability, and is been applied to comprehensively and intensively analyze marine metabolomics [79].As a powerful tool to depict the internal metabolome of targeting cells, classical metabolic fingerprinting enables the identification of potential metabolite targets in marine drug discovery [80,81].Metabolic footprinting, a more recently developed approach for detecting secreted, excreted, or consumed metabolites, is of particular interest in investigating the interactions between environment/genetic and marine ecosystems [82].Marine metabolomics focuses on developing marine microbial resources and understanding the dynamics of marine ecosystems.In many natural product research programs, metabolomics technologies have been adopted for the separation, quantification, and elucidation of bioactive molecules.Based on LC-MS and molecular networking, five new pyrrole-derived alkaloids were isolated from marine Micromonospora sp.bacterium, where two of them exhibited antibacterial activity against methicillin-resistant strains [93].With the advent of metabolomics, the MS/MS data of an increasing number of natural products from microorganisms inhabiting marine environments have been archived in global natural products social molecular networking (GNPS, http://gnps.ucsd.edu(accessed on 12 May 2023)).Metabolomics has also taken a central role in profiling the ecological impacts of chemical residues/pollutants on the metabolome of various marine microorganisms, so as to illustrate their biological responses to environmental changes.Under stresses of heavy metals, the NMR-based metabolomics of microalgae Chlorella sp.revealed an increased yield of extracellular polymeric substances to perform Cu uptake [94].Felline, et al. [90] characterized the metabolome profile of the brown algae Fucus virsoides under the stress of glyphosate-based herbicides.The NMR result showed that herbicides down-regulated the metabolism of aromatic amino acids but up-regulated the shikimate content of exposed thalli.In marine microalgae Dunaliella salina exposed to polystyrene microplastics, the significant up-regulations of the amino acid biosynthesis, ATP-binding cassette transportation, and glycerophospholipid metabolism were speculated by LC-MS analysis [95].With up-/down-regulated metabolic pathways identified under environmental stressors, it is possible to perform the biomarker identification and ecological risk assessment and achieve the toxicant action elucidation and discovery of many other marine processes.

Conclusions
This review summarized the cutting-edge technologies for the collection of marine microbial samples and the applications of molecular ecology on marine microorganisms.
(1) With the expansion of marine resource explorations to deeper and wider sea areas, deep-sea sampling equipment tends to be diversified in structure, highly functional, and intelligent in operation.Traditional sampling methods are usually time consuming, while in situ collection, preservation, and detection have become the mainstream methods to maintain the integrity of marine microbial samples.
(2) Metagenomics, transcriptomics, and other omics technologies help to conduct broad-scale diversity assessments of both cultured and uncultured marine species.Based on the analyses of microbial diversity, population structure, and evolutionary relationships, the functional activity, mutual collaboration, and relationship with the environment of microbial populations can be further explored.

Challenges
The global marine resources investigation projects show the large groups of new species and gene resources in the ocean ecosystems.For example, the results of the Tara Oceans Expedition project indicate that the rich diversity of marine microorganisms in colony structure, metabolic pathways, physiological and biochemical reactions, metabolites, and other aspects are a abundant resource with great potential [96,97].At present, however, the research on marine microorganisms is still confronted by many challenges that need to be addressed urgently in the near future.
(1) Due to the extreme environmental conditions of ocean ecosystems, how to provide the most original marine microbial samples for scientific studies has become a worldwide research focus.The high-fidelity samples for scientific research are important indicators to measure the performance of deep-sea sampling equipment.Therefore, the development of low disturbance, pressure-retaining, and thermal-insulation deep-sea samples will better meet the needs of deep-sea resource explorations and developments, deep-sea biogeochemical cycles, and basic marine science research, and will certainly become an important development direction for marine microbial sampling strategies.
(2) How to stably preserve the collected high-fidelity samples, so as to provide reliable samples for omics research and ensure the accuracy of the research results, has become a key issue in the field investigation of marine microorganisms.The development of a stable preservation and in situ analysis platform for marine microbial samples in the future will greatly facilitate the mining of deep-sea resources.
(3) The continuous improvement of effective cultivation methods of marine microorganisms is an important aspect to promote the development of marine microbiome from descriptive to functional and applied research methods.The combination of multiple research methods further makes up for the shortcomings of single technology methods, identifies the diversity of marine microorganisms, and thoroughly explores the functions of the obtained microorganisms at the gene, protein, and metabolite levels.
To date, we achieved important information on marine biodiversity and the spatiotemporal distribution patterns of microbial communities.However, our understanding of the underlying mechanisms for maintaining the ecological functions of marine microorganisms is insufficient.With the continuous development of metagenomics, transcriptomics, proteomics, metabolomics, and other multi-omics techniques, the ecological functions of microorganisms can be revealed at molecular levels in marine ecosystems.The microbial roles can be better understood in biogeochemical processes, such as carbon cycling, nitrogen cycling, and sulfur cycling, in response to environmental changes.In addition, marine microbial research is not limited to scientific explorations and also applies to practical applications as bioenergy production, biomaterial manufacturing, and drug discovery, etc. Future research should focus more extensively on multi-scale research, from micro-to macro-scales, and from microbial single cells to complex ecosystems, in order to gain a more comprehensive understanding of the marine ecosystems driven by diverse microorganisms.

Figure 1 .
Figure 1.An overview of the field investigation into marine microorganisms with modern techniques.

Figure 1 .
Figure 1.An overview of the field investigation into marine microorganisms with modern techniques.

Figure 2 .
Figure 2. A typical workflow of the scientific studies on marine microorganisms.

Figure 2 .
Figure 2. A typical workflow of the scientific studies on marine microorganisms.

Figure 3 .
Figure 3. (a) The high-pressure (HP) core.(b) The microbial sampler submersible incubation device (MS-SID) equipped with a conductivity, temperature, and depth (CTD) instrument; two turbidity sensors; an oxygen optode; a synchronous digital subscriber line (SDSL); and a photosynthetically active radiation (PAR) sensor.Adapted with permission from references [21,23].Copyright 2017, the authors.Frontiers in Microbiology, published by Frontiers Media S.A and 2016 Elsevier Ltd.

Figure 3 .
Figure 3. (a) The high-pressure (HP) core.(b) The microbial sampler submersible incubation device (MS-SID) equipped with a conductivity, temperature, and depth (CTD) instrument; two turbidity sensors; an oxygen optode; a synchronous digital subscriber line (SDSL); and a photosynthetically active radiation (PAR) sensor.Adapted with permission from references [21,23].Copyright 2017, the authors.Frontiers in Microbiology, published by Frontiers Media S.A and 2016 Elsevier Ltd.

J 18 Figure 4 .
Figure 4. (a) A typical laboratory-based next-generation sequencing and in situ field sequencing workflow.(b) A MICRObial life-detection platform for microorganisms in remote and extreme environments.Adapted with permission from references [61,62].Copyright 2020, the authors.Genes published by MDPI, Basel, Switzerland, 2017, the authors.Frontiers in Microbiology, published by Frontiers Media SA, Switzerland.Marine Transcriptomics and Proteomics Transcriptomics focuses on all RNA transcripts, including those of mRNA, non-coding RNA, and small RNA, to identify their transcriptional and post-transcriptional gene expressions and regulations, aiming to interpret the underlying biological processes in response to environmental perturbations [63].Current transcription technologies mainly

Figure 4 .
Figure 4. (a) A typical laboratory-based next-generation sequencing and in situ field sequencing workflow.(b) A MICRObial life-detection platform for microorganisms in remote and extreme environments.Adapted with permission from references [61,62].Copyright 2020, the authors.Genes published by MDPI, Basel, Switzerland, 2017, the authors.Frontiers in Microbiology, published by Frontiers Media SA, Switzerland.

Author Contributions:
Conceptualization, H.L.; writing-original draft preparation, Z.H. and F.F.; writing-review and editing, L.D., K.Y., L.Z. and H.L.; visualization, Z.H. and L.Z.; supervision, L.Z. and H.L.; project administration, H.L.; funding acquisition, H.L. and L.D. All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by the Major Project for Basic and Applied Basic Research of Guangdong Province (grant number 2020B0301030003), the China Geological Survey Project (grant numbers DD20221703 and DD20230063), and the Natural Science Foundation of Top Talent of SZTU (grant number GDRC202115).Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.

Table 1 .
Advantages and disadvantages of different sampling strategies.

Table 1 .
Advantages and disadvantages of different sampling strategies.

Table 2 .
Advantages and limitations of different molecular analysis methods.

Table 3 .
The housekeeping and functional genes as molecular markers for marine microorganisms.

Table 4 .
Identification of marine microbial metabolites based on NMR and MS techniques.