국립부경대학교 PKNU Lab of Microbial Ecology

● Culture-dependent and -independent Methods to Study Microbiomes

• ◇ All microorganisms and their genetic information in a specific environment is called microbiome and research on the microbiome is one of most important and exciting research topics and trends in microbiology in recent years. By analyzing the diversity of microorganisms, it is possible to reveal the influence of microbial communities on the surrounding environments and their interactions with other organisms.
• ◇ However, the number of microorganisms that can be isolated and cultured in the laboratory is extremely small, so there is a limit to microbial research through culture. Recently, research on microbiology is widely used to identify the types, amounts, and roles of various microorganisms that exist in the environment through research that combines culture-dependent and independent methods.
• ◇ In the microbial ecology laboratory in PKNU, we are conducting experiments to find out the characteristics and distribution of microorganisms living in various habitats, and their interactions, and to study how to apply useful microorganisms that are helpful to humans.

• ◇ For this purpose, research methods such as microbial community analysis, quantitative PCR, metagenomics, phylogenetics, genomics, bacterial systematics, next-generation sequencing, and bioinformatics are used to reveal the types and amounts of microorganisms in the ecosystem, to find new microorganisms and genes and understand the microbiome to use for application to industries.

• ◇ Recently, microbial community analysis is a method that can simultaneously analyze the diversity of hundreds of species of microorganisms in dozens of samples by obtaining tens to hundreds of millions of sequences using next-generation sequence analysis. To this end, tens of thousands to hundreds of thousands of sequences are compared with each other, divided into phylotypes (ASV, OTU, etc.), compared with the database, and the type and ratio of microorganisms can be identified by identifying which microorganisms they are. In addition, diversity indices such as Chao1 and Shannon index are analyzed for each sample, and samples can be divided into groups according to the type and ratio of microorganisms. Depending on the specific conditions, it is possible to confirm which types of microorganisms are more present, and statistical significance can be confirmed by performing statistical analysis.
• ◇ In the meantime, research has been conducted to reveal the diversity and role of microorganisms in various environments such as soil, ocean, marine life, and food, and has been published in a number of SCI journals. Recently, a paper analyzing the relationship between the factors affecting shrimp farming and the microbiome in shrimp farms was published in the SCI journal (Figure).
  

• Figure. Study to reveal microbiome changes according to environmental factors in the biofloc aquaculture system of the Pacific whiteleg shrimp, Litopenaeus vannamei.
  

• ◇ Isolating new bacterial strains from various environments (bacteria hunting), comparing them with existing strains (identification), setting the position of the new strains (classification), and making and proposing new scientific names (nomenclature) are the major topics of bacterial systematics, and what we are doing in this laboratory. So far, more than 30 new species and/or genera were proposed, accepted, and published. Among them, Pukyongia salina, Pukyongiella litopenaei, Salaquimonas pukyongi, and Pikeienuella piscinae are the cases in which the new scientific names followed the name of Pukyong National University, the university that the laboratory belongs to.
• ◇ For the analysis of new bacteria, chemical taxonomic analysis such as API test, Biolog test, fatty acid analysis, and polar lipid analysis are performed and students will learn how to do chemical analysis.
  

• ◇ According to the recent development of next-generation sequencing methods, the cost of analyzing the whole genome sequence of a bacterium has become cheaper, and accordingly, research on analyzing the entire genome of a bacterium is becoming common in microbiology. Also in bacterial systematics, such genome analysis research is essential to describe the new bacterial taxa. More than 20 bacterial genome analyses have been performed in this laboratory. 
• ◇ Comparative genomics is a useful tool to understand the group of bacteria and each member in the group. Pangenome analysis and comparative genomics are also being performed in this laboratory, some bioinformatics tools were used to analyze the bacterial genomes.

• ◇ In the case of general PCR, it is difficult to quantify the ratio of a gene in a sample. To solve this problem, qPCR using fluorescent dyes is widely used. In this laboratory, quantification of nitrifying and general microorganisms is performed through qPCR using amoA gene and 16S rRNA gene primers. For accurate quantification, standards with known concentrations or bacterial cultures are used as standard additions and internal standards, and a standard curve is drawn to perform accurate quantification. Regression curves are obtained using appropriate standards, PCR efficiency is calculated and accurate quantification is performed by confirming that qPCR is proceeding accurately.

• ◇ Recently, a lot of information and programs have been provided so that biologists can easily use biostatistics and bioinformatics tools, and this laboratory is also using these tools to perform various bioinformatics and statistical analyzes. In particular, in microbial ecology, various bioinformatic tools must be used to analyze tens to hundreds of millions of sequence information obtained through Next Generation Sequencing. For fast analysis, a server with a 64-core CPU and 256GB RAM is equipped in the laboratory to perform the analysis.
• ◇ In this laboratory, data obtained from amplicon sequencing, metagenomics, genomics, etc. are analyzed and visualized using programs and packages such as standalone BLAST, QIIME2, Picrust2, R project, Python, Conda, Matplotlib, Pandas, Seaborn, SciPy, Scikit-learn, etc. Statistical analyses such as ANOVA, PCoA, UniFrac, and PERMANOVA and ecological analyses such as alpha- and beta-diversity estimation are performed.

• ◇ The microbiomes of crustaceans, specially, shrimps and crabs are being analyzed as a project funded by the Ministry of Oceans and Fisheries. We are conducting tasks to isolate and identify new symbiotic microorganisms and to study the interaction between the host and the microbiome. The knowledge and bioresources will be applied to the industries such as aquaculture and fisheries. 
• ◇ We are carrying out a project funded by the National Research Foundation of Korea to isolate new microorganisms that have not been isolated before by combining non-culture and culture methods.