Collection and Molecular Ecology Analysis of Marine Microorganisms

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1		Lijuan Zhang	--	1771	2023-10-18 05:58:16	\|
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This entry is adapted from the peer-reviewed paper 10.3390/jmse11101981

The special characteristics of ocean ecosystems, such as the high salinity and pressure, low temperature, and nutrition, make 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.

marine microorganisms molecular ecology sample collection molecular analysis molecular markers

1. 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.

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

2. 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]. 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, 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), 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.

3. Molecular Ecology Analysis of Marine Microbial Samples

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 ^[26]. 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. 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 ^[27]. 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 ^[28]. 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 ^[29]. 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. 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 ^[30]. 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 ^[31]. 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 ^[32]^[33]^[34]. 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 ^[35]. 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 ^[36]. Additionally, the gyrB housekeeping gene was confirmed to be a valuable marker in distinguishing marine strains from the Bacillus pumilus clade ^[37]. 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 ^[38]^[39]. 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 δ¹³C-methane signatures, suggesting the potential roles of AOM in global methane budgets ^[40]. 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 ^[41]. 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.

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Subjects: Biochemistry & Molecular Biology

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Zhishan Huang

Fang Fang

Lingyun Ding

Ke Yu

Lijuan Zhang

Hailong Lu

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Update Date: 18 Oct 2023

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1. Introduction

2. Collection of Marine Microbial Samples

3. Molecular Ecology Analysis of Marine Microbial Samples

References