• Animal Bioscience
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• v.36 no.2_spc
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• pp.364-373
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• 2023

The gastrointestinal (GI) tract of ruminants contains diverse microbes that ferment various feeds ingested by animals to produce various fermentation products, such as volatile fatty acids. Fermentation products can affect animal performance, health, and well-being. Within the GI microbes, the ruminal microbes are highly diverse, greatly contribute to fermentation, and are the most important in ruminant nutrition. Although traditional cultivation methods provided knowledge of the metabolism of GI microbes, most of the GI microbes could not be cultured on standard culture media. By contrast, amplicon sequencing of 16S rRNA genes can be used to detect unculturable microbes. Using this approach, ruminant nutritionists and microbiologists have conducted a plethora of nutritional studies, many including dietary interventions, to improve fermentation efficiency and nutrient utilization, which has greatly expanded knowledge of the GI microbiota. This review addresses the GI content sampling method, 16S rRNA gene amplicon sequencing, and bioinformatics analysis and then discusses recent studies on the various factors, such as diet, breed, gender, animal performance, and heat stress, that influence the GI microbiota and thereby ruminant nutrition.

• Genomics & Informatics
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• v.21 no.3
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• pp.40.1-40.11
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• 2023

Microbial community profiling using 16S rRNA amplicon sequencing allows for taxonomic characterization of diverse microorganisms. While amplicon sequence variant (ASV) methods are increasingly favored for their fine-grained resolution of sequence variants, they often discard substantial portions of sequencing reads during quality control, particularly in datasets with large number samples. We present a streamlined pipeline that integrates FastP for read trimming, HmmUFOtu for operational taxonomic units (OTU) clustering, Vsearch for chimera checking, and Kraken2 for taxonomic assignment. To assess the pipeline's performance, we reprocessed two published stool datasets of normal Korean populations: one with 890 and the other with 1,462 independent samples. In the first dataset, HmmUFOtu retained 93.2% of over 104 million read pairs after quality trimming, discarding chimeric or unclassifiable reads, while DADA2, a commonly used ASV method, retained only 44.6% of the reads. Nonetheless, both methods yielded qualitatively similar β-diversity plots. For the second dataset, HmmUFOtu retained 89.2% of read pairs, while DADA2 retained a mere 18.4% of the reads. HmmUFOtu, being a closed-reference clustering method, facilitates merging separately processed datasets, with shared OTUs between the two datasets exhibiting a correlation coefficient of 0.92 in total abundance (log scale). While the first two dimensions of the β-diversity plot exhibited a cohesive mixture of the two datasets, the third dimension revealed the presence of a batch effect. Our comparative evaluation of ASV and OTU methods within this streamlined pipeline provides valuable insights into their performance when processing large-scale microbial 16S rRNA amplicon sequencing data. The strengths of HmmUFOtu and its potential for dataset merging are highlighted.

• Journal of Microbiology and Biotechnology
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• v.33 no.9
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• pp.1162-1169
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• 2023

16S rRNA short amplicon sequencing-based microbiota profiling has been thought of and suggested as a feasible method to assess food safety. However, even if a comprehensive microbial information can be obtained by microbiota profiling, it would not be necessarily sufficient for all circumstances. To prove this, the feasibility of the most widely used V3-V4 amplicon sequencing method for food safety assessment was examined here. We designed a pathogen (Vibrio parahaemolyticus) contamination and/or V. parahaemolyticus-specific phage treatment model of raw oysters under improper storage temperature and monitored their microbial structure changes. The samples stored at refrigerator temperature (negative control, NC) and those that were stored at room temperature without any treatment (no treatment, NT) were included as control groups. The profiling results revealed that no statistical difference exists between the NT group and the pathogen spiked- and/or phage treated-groups even when the bacterial composition was compared at the possible lowest-rank taxa, family/genus level. In the beta-diversity analysis, all the samples except the NC group formed one distinct cluster. Notably, the samples with pathogen and/or phage addition did not form each cluster even though the enumerated number of V. parahaemolyticus in those samples were extremely different. These discrepant results indicate that the feasibility of 16S rRNA short amplicon sequencing should not be overgeneralized in microbiological safety assessment of food samples, such as raw oyster.

• Journal of Microbiology and Biotechnology
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• v.27 no.2
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• pp.321-334
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• 2017

Process surveillance within agricultural biogas plants (BGPs) was concurrently studied by high-throughput 16S rRNA gene amplicon sequencing and an optimized quantitative microscopic fingerprinting (QMF) technique. In contrast to 16S rRNA gene amplicons, digitalized microscopy is a rapid and cost-effective method that facilitates enumeration and morphological differentiation of the most significant groups of methanogens regarding their shape and characteristic autofluorescent factor 420. Moreover, the fluorescence signal mirrors cell vitality. In this study, four different BGPs were investigated. The results indicated stable process performance in the mesophilic BGPs and in the thermophilic reactor. Bacterial subcommunity characterization revealed significant differences between the four BGPs. Most remarkably, the genera Defluviitoga and Halocella dominated the thermophilic bacterial subcommunity, whereas members of another taxon, Syntrophaceticus, were found to be abundant in the mesophilic BGP. The domain Archaea was dominated by the genus Methanoculleus in all four BGPs, followed by Methanosaeta in BGP1 and BGP3. In contrast, Methanothermobacter members were highly abundant in the thermophilic BGP4. Furthermore, a high consistency between the sequencing approach and the QMF method was shown, especially for the thermophilic BGP. The differences elucidated that using this biphasic approach for mesophilic BGPs provided novel insights regarding disaggregated single cells of Methanosarcina and Methanosaeta species. Both dominated the archaeal subcommunity and replaced coccoid Methanoculleus members belonging to the same group of Methanomicrobiales that have been frequently observed in similar BGPs. This work demonstrates that combining QMF and 16S rRNA gene amplicon sequencing is a complementary strategy to describe archaeal community structures within biogas processes.

• Journal of Microbiology and Biotechnology
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• v.25 no.6
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• pp.765-770
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• 2015

Recent sequencing technology development has revolutionized fields of microbial ecology. MiSeq-based microbial community analysis allows us to sequence more than a few hundred samples at a time, which is far more cost-effective than pyrosequencing. The approach, however, has not been preferably used owing to computational difficulties of processing huge amounts of data as well as known Illumina-derived artefact problems with amplicon sequencing. The choice of assembly software to take advantage of paired-end sequencing and methods to remove Illumina artefacts sequences are discussed. The protocol we suggest not only removed erroneous reads, but also dramatically reduced computational workload, which allows even a typical desktop computer to process a huge amount of sequence data generated with Illumina sequencers. We also developed a Web interface (http://biotech.jejunu.ac.kr/ ~abl/16s/) that allows users to conduct fastq-merging and mothur batch creation. The study presented here should provide technical advantages and supports in applying MiSeq-based microbial community analysis.

• Korean Journal of Microbiology
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• v.39 no.1
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• pp.45-50
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• 2003

The diversity and change of microbial communities during kimchi fermentation at $4^{\circ}C$ were analyzed by denaturing gradient gel electrophoresis (DGGE). Kimchi samples were taken every 5 days over the fermentation periods (for 60 days) to extract total DNA for DGGE analysis. Touchdown polymerase chain reaction was performed to amplify the V3 region of 16S rRNA gene. Sequencing results of partial 16S rDNA amplicons from DGGE profiles revealed that lactic acid bacteria (LAB), especially Weissella koreensis, Lactobacillus sakei and Leuconostoc gelidum were dominants in kimchi fermentation at $4^{\circ}C$. And we knew that W. koreensis steadily existed throughout the whole fermentation period, also Lb. sakei and Leuc. gelidum appeared from 10th day and 30th day of fermentation time, respectively and then these species were to be dominant microorganisms.

• International Journal of Oral Biology
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• v.46 no.3
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• pp.146-153
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• 2021

Accurate identification of microbes facilitates the prediction, prevention, and treatment of human diseases. To increase the accuracy of microbiome data analysis, a long region of the 16S rRNA is commonly sequenced via paired-end sequencing. In paired-end sequencing, a sufficient length of overlapping region is required for effective joining of the reads, and high-quality sequencing reads are needed at the overlapping region. Trimming sequences at the reads distal to a point where sequencing quality drops below a specific threshold enhance the joining process. In this study, we examined the effect of trimming conditions on the number of reads that remained after quality control and chimera removal in the Illumina paired-end reads of the V3-V4 hypervariable region. We also examined the alpha diversity and taxa assigned by each trimming condition. Optimum quality trimming increased the number of good reads and assigned more number of operational taxonomy units. The pre-analysis trimming step has a great influence on further microbiome analysis, and optimized trimming conditions should be applied for Divisive Amplicon Denoising Algorithm 2 analysis in QIIME2 platform.

• Journal of Animal Science and Technology
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• v.63 no.3
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• pp.640-650
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• 2021

Particulate matter (PM) produced in pig houses may contain microbes which can spread by airborne transmission, and PM and microbes in PM adversely affect human and animal health. To investigate the microbiome in PM from pig houses, nine PM samples were collected in summer 2020 inside and outside of pig houses located in Jangseong-gun, Jeollanam-do Province, Korea, comprising three PM samples from within a nursery pig house (I-NPH), three samples from within a finishing pig house (I-FPH), and three samples from outside of the pig houses (O-PH). Microbiomes were analyzed using 16S rRNA gene amplicon sequencing. Firmicutes was the most dominant phylum and accounted for 64.8%-97.5% of total sequences in all the samples, followed by Proteobacteria (1.4%-21.8%) and Bacteroidetes (0.3%-13.7%). In total, 31 genera were represented by > 0.3% of all sequences, and only Lactobacillus, Turicibacter, and Aerococcus differed significantly among the three PM sample types. All three genera were more abundant in the I-FPH samples than in the O-PH samples. Alpha diversity indices did not differ significantly among the three PM types, and a principal coordinate analysis suggested that overall microbial communities were similar across PM types. The concentration of PM did not significantly differ among the three PM types, and no significant correlation of PM concentration with the abundance of any potential pathogen was observed. The present study demonstrates that microbial composition in PM inside and outside of pig houses is similar, indicating that most microbe-containing PM inside pig houses leaks to the outside from where it, along with microbe-containing PM on the outside, may re-enter the pig houses. Our results may provide useful insights regarding strategies to mitigate potential risk associated with pig farming PM and pathogens in PM.

• Journal of Animal Science and Technology
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• v.64 no.5
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• pp.897-910
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• 2022

Bovine fecal microbiota is important for host health and its composition can be affected by various factors, such as diet, age, species, breed, regions, and environments. The objective of this study was to evaluate the impact of diet and gender on fecal microbiota in Korean native Hanwoo cattle. The 16S rRNA gene amplicon sequencing of fecal microbiota was conducted from 44 Hanwoo cattle divided into four groups: (1) 11 heifers fed an oat hay plus total mixed ration (TMR) diet for breeding (HOTB), (2) 11 heifers fed an early fattening TMR diet (HEFT), (3) 11 steers fed the early fattening TMR diet (SEFT), and (4) 11 steers fed the late fattening TMR diet (SLFT). Firmicutes and Bacteroidota were the first and second most dominant phyla in all the samples, respectively. The Firmicutes/Bacteroidota (F/B) ratio associated with feed efficiency was significantly greater in the SLFT group than in the other groups. At the genus level, Romboutsia, Paeniclostridium, and Turicibacter were the most abundant in the SLFT while Akkermansia, Bacteroides, and Monoglobus were the most abundant in the HOTB group. Although the same early fattening TMR diet was fed to Hanwoo heifers and steers, Marvinbryantia and Coprococcus were the most abundant in the HEFT group while Alistipes and Ruminococcus were the most abundant in the SEFT group. Shannon and Simpson diversity indices were significantly lower in the SLFT group than in the other groups. Distribution of fecal microbiota and functional genetic profiles were significantly different among the four treatment groups. The present study demonstrates that different diets and genders can affect fecal microbiota and the F/B ratio may be associated with feed efficiency in Hanwoo cattle. Our results may help develop strategies to improve gut health and productivity through manipulation of fecal microbiota using the appropriate diet considering Hanwoo cattle gender.

• Korean Journal of Food Science and Technology
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• v.50 no.4
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• pp.400-406
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• 2018

Takju and yakju are traditional Korean alcoholic beverages that are prepared by fermentation of glutinous rice with nuruk, a cereal starter containing various bacteria, fungi, and yeast. In this study, physicochemical and microbial properties of a total of 12 commercial takju and yakju samples were analyzed; their pH, sweetness, and alcohol content were varied, depending on the type of alcohol, from pH 3.64-4.8, $5.1-24.8^{\circ}Bx$, and 4.6-18.5%, respectively. Microbial communities were analyzed with 16S rRNA amplicon sequencing using MiSeq system. At the phylum level, Firmicutes (86.2%) was the most dominant, followed by Proteobacteria (8.08%), Actinobacteria (2.56%), and Cyanobacteria (3.13%). Lactic acid bacteria, including Lactobacillus, Lactococcus, Leuconostoc, and Weissella were also frequently detected. Among eukaryotes, Saccharomyces cerevisiae was the most dominant in these samples.