Amplicon Sequencing Analysis Research Articles

Quantitative PCR assays as a monitoring tool for bacterial genera in fresh fish fillets

Fresh fish fillets are a valuable but highly perishable food, and their rapid microbial deterioration is a drawback for food safety and sustainability of aquaculture, food and retail industries. Quantitative PCR (qPCR) assays based on 16S rRNA gene (16S) sequences were developed for the most abundant bacteria genera detected by metagenomics in fresh or processed fish fillets. The efficiency and specificity of six qPCR assays (for 16S of all bacteria or genera Shewanella, Pseudomonas, Carnobacterium, Janthinobacterium and Massilia) was verified using in silico predictions, cloning, sequencing and phylogenetic analyses of amplicons obtained from refrigerated control or high-pressure processed (HPP) European sea bass (Dicentrarchus labrax) fillets. In HPP sea bass fillets, significant decreases in total bacteria 16S and of Shewanella, Pseudomonas, Carnobacterium and Janthinobacterium 16S compared to control fillets were confirmed by qPCR, after 11 days of refrigerated storage. The qPCR assays were successfully applied to monitor microbial contamination during refrigerated storage of fresh fillets from commercial (retail) sea bass and gilthead sea bream (Sparus aurata). Significant increases in total bacterial and Shewanella, Pseudomonas, Carnobacterium and Janthinobacterium contamination were detected after 7–14 days. 16S copy number for total bacteria and the four target genera positively correlated with total viable counts using culture enumeration. 16S of Massilia, that is abundant in fresh fish fillets, did not significantly change during storage. The six validated qPCR assays developed are proposed as specific, sensitive, culture-independent methods for monitoring quality or processing outcomes for fish fillets during cold chain storage.

Epimedium polysaccharides mitigates Porphyromonas gingivalis-exacerbated intestinal inflammation by suppressing the Th17 pathway and modulating the gut microbiota

This study aimed to investigate the potential alleviating effect of Epimedium polysaccharide (EP) on intestinal inflammation aggravated by Porphyromonas gingivalis (Pg). P. gingivalis, an oral pathogen, may play a role in intestinal inflammation, highlighting the necessity to explore substances capable of inhibiting its pathogenicity. Initially, in vitro screening experiments utilizing co-culturing and quantitative polymerase chain reaction revealed that EP significantly inhibited the growth of P. gingivalis and the levels of virulence genes, including Kgp and RgpA. Subsequent mouse experiments demonstrated that EP notably ameliorated Pg-aggravated weight loss, disease activity index, histopathological lesions, and disruption of intestinal barrier integrity, evidenced by a reduction in tight junction protein levels. Flow cytometry analysis further illustrated that EP attenuated Pg-induced Th17 differentiation and Th17-related cytokines, such as IL-17 and IL-6. Additionally, 16S rRNA amplicon sequencing analysis elucidated that EP significantly mitigated Pg-induced gut microbiota dysbiosis, enriching potentially beneficial microbes, including Akkermansia and Bifidobacterium. The metabolomic analysis provided further insight, indicating that EP intervention altered the accumulation of relevant intestinal metabolites and exhibited correlations with disease indicators. In conclusion, our research suggested that EP holds promise as a prospective therapeutic agent for alleviating P. gingivalis-aggravated intestinal inflammation.

Impact of Esophageal Motility on Microbiome Alterations in Symptomatic Gastroesophageal Reflux Disease Patients With Negative Endoscopy: Exploring the Role of Ineffective Esophageal Motility and Contraction Reserve.

Ineffective esophageal motility (IEM) is common in patients with gastroesophageal reflux disease (GERD) and can be associated with poor esophageal contraction reserve on multiple rapid swallows. Alterations in the esophageal microbiome have been reported in GERD, but the relationship to presence or absence of contraction reserve in IEM patients has not been evaluated. We aim to investigate whether contraction reserve influences esophageal microbiome alterations in patients with GERD and IEM. We prospectively enrolled GERD patients with normal endoscopy and evaluated esophageal motility and contraction reserve with multiple rapid swallows during high-resolution manometry. The esophageal mucosa was biopsied for DNA extraction and 16S ribosomal RNA gene V3-V4 (Illumina)/full-length (Pacbio) amplicon sequencing analysis. Among the 56 recruited patients, 20 had normal motility (NM), 19 had IEM with contraction reserve (IEM-R), and 17 had IEM without contraction reserve (IEM-NR). Esophageal microbiome analysis showed a significant decrease in microbial richness in patients with IEM-NR when compared to NM. The beta diversity revealed different microbiome profiles between patients with NM or IEM-R and IEM-NR (P = 0.037). Several esophageal bacterial taxa were characteristic in patients with IEM-NR, including reduced Prevotella spp. and Veillonella dispar, and enriched Fusobacterium nucleatum. In a microbiome-based random forest model for predicting IEM-NR, an area under the receiver operating characteristic curve of 0.81 was yielded. In symptomatic GERD patients with normal endoscopic findings, the esophageal microbiome differs based on contraction reserve among IEM. Absent contraction reserve appears to alter the physiology and microbiota of the esophagus.

Distinct changes in tomato-associated multi-kingdom microbiomes during Meloidogyne incognita parasitism

BackgroundThe interplay between root-knot nematode (RKN) parasitism and the complex web of host-associated microbiota has been recognized as pivotal for effective management of the pest. However, studies assessing this relationship have focussed on the bacterial and fungal communities, neglecting the unicellular eukaryotic members. Here, we employed amplicon sequencing analysis of the bacterial 16S rRNA, fungal ITS and eukaryotic 18S rRNA genes, and comprehensively examined how the microbiome composition, diversity and networking developed with time in the rhizospheres and roots of RKN-inoculated and non-inoculated tomato plants.ResultsAs expected, infection with the RKN Meloidogyne incognita decreased plant growth. At individual timepoints, we found distinct bacterial, fungal and eukaryote community structures in the RKN-inoculated and non-inoculated rhizospheres and roots, and RKN inoculation affected several taxa in the root-associated microbiome differentially. Correlation analysis revealed several bacterial and fungal and few protist taxa that correlated negatively or positively with M. incognita. Moreover, network analysis using bacterial, fungal and eukaryotic data revealed more dynamic networks with higher robustness to disturbances in the RKN-inoculated than in the non-inoculated rhizospheres/roots. Hub taxa displayed a noticeable successional pattern that coincided with different phases of M. incognita parasitism. We found that fungal hubs had strong negative correlations with bacteria and eukaryotes, while positive correlations characterized hub members within individual kingdoms.ConclusionOur results reveal dynamic tomato-associated microbiomes that develop along different trajectories in plants suffering M. incognita infestation and non-infested plants. Overall, the results identify stronger associations between RKN and bacterial and fungal taxa than between eukaryotic taxa and RKN, suggesting that fungal and bacterial communities could play a larger role in the regulation of RKN. The study identifies several putative RKN-antagonistic bacterial and fungal taxa and confirms the antagonistic potential previously identified in other taxa.

Ubiquity of methanogenic archaea in the trunk of coniferous and broadleaved tree species in a mountain forest.

Wetwood of living trees is a habitat of methanogenic archaea, but the ubiquity of methanogenic archaea in the trunk of various trees has not been revealed. The present study analysed methanogenic archaeal communities inside coniferous and broadleaved trees in a cold temperate mountain forest by culture-dependent or independent techniques. Heartwood and sapwood segments were obtained from the trunk of seven tree species, Cryptomeria japonica, Quercus crispula, Fraxinus mandshurica, Acer pictum, Aesculus turbinata, Magnolia obovata, and Populus tremula. Amplicon sequencing analysis of 16S rRNA genes showed that Methanobacteriaceae predominated the archaeal communities and Methanomassiliicoccaceae also inhabited some trees. Real-time PCR analysis detected methanogenic archaeal mcrA genes from all the tree species, with a maximum of 107copiesg-1 dry wood. Digital PCR analysis also detected mcrA genes derived from Methanobacterium spp. and Methanobrevibacter spp. from several samples, with a maximum of 105 and 104copiesg-1 dry wood. The enumeration by the most probable number method demonstrated the inhabitation of viable methanogenic archaea inside the trees; 106cellsg-1 dry wood was enumerated from a heartwood sample of C. japonica. Methanogenic archaea related to Methanobacterium beijingense were cultivated from a heartwood sample of Q. crispula and F. mandshurica. The present study demonstrated that the inside of various trees is a common habitat for methanogenic archaeal communities and a potential source of methane in forest ecosystems.

Phagotrophy in the nitrogen-fixing haptophyte Braarudosphaera bigelowii.

Biological nitrogen fixation provides fixed nitrogen for microbes living in the oligotrophic open ocean. UCYN-A2, the previously known symbiont of Braarudosphaera bigelowii, now believed to be an early-stage B. bigelowii organelle that exchanges fixed nitrogen for fixed carbon, is globally distributed. Indirect evidence suggested that B. bigelowii might be a mixotrophic (phagotrophic) phototrophic flagellate. The goal of this study was to determine if B. bigelowii can graze on bacteria using several independent approaches. The results showed that B. bigelowii grazed on co-occurring bacteria at a rate of 5-7 cells/h/B. bigelowii and that the overall grazing rate was significantly higher at nighttime than at daytime. Bacterial abundance changes, assessed with 16S rRNA gene amplicon sequencing analysis, may have indicated preferential grazing by B. bigelowii on specific bacterial genotypes. In addition, Lysotracker™ staining of B. bigelowii suggested digestive activity inside B. bigelowii. Carbon and nitrogen fixation measurements revealed that the carbon demand of B. bigelowii could not be fulfilled by photosynthesis alone, implying supplementation by heterotrophy. These independent lines of evidence together revealed that B. bigelowii engages in phagotrophy, which, beyond serving as a supplementary source of carbon and energy, may also facilitate the indirect assimilation of inorganic nutrients.

Coronal and Root Canal Microbiota in Apical Periodontitis with Different PAI.

Apical periodontitis is an inflammatory disease triggered by oral pathogens invading necrotic root canals. The aim of this study was to evaluate the coronal and root canal bacterial community profiles in primary endodontic infections with different periapical (PAI) indices in comparison to oral mucosa controls. A total of 31 patients with primary apical periodontitis, 14 with PAI-1 and 17 with PAI-3 were recruited. Microbial specimens from mucosa (control samples) and endodontic necrotic tissues were collected in each patient. Microbiota composition was studied through 16S ribosomal RNA gene amplicon sequencing analysis. Overall, 2953 taxa from 168 different genera of 451 various microbial species were retrieved in the controls and PAI-1 and PAI-3 groups. Firmicutes is the predominant phylum in the oral controls (34.5%) and PAI-1 (44.4%) groups, while Bacteroidetes is predominant in PAI-3 (38.6%). The Proteobacteria (21.5%) and Fusobacteria (12.5%) relative abundance is higher in oral controls while that of Synergistetes is higher in the PAI-1 (3.5%) and PAI-3 (2.5%) groups, being almost absent in controls (less than 0.1%). Most of the increased bacterial species found in the PAI groups were strict anaerobes. A diminished microbial diversity was found in apical periodontitis with higher PAI. These samples were also characterized by an increase in bacteria belonging to phyla and genera with an increased anaerobic character.

Combined effects of micropollutants and their degradation on prokaryotic communities at the sediment–water interface

Pesticides and pharmaceuticals enter aquatic ecosystems as complex mixtures. Various processes govern their dissipation and effect on the sediment and surface waters. These micropollutants often show persistence and can adversely affect microorganisms even at low concentrations. We investigated the dissipation and effects on procaryotic communities of metformin (antidiabetic drug), metolachlor (agricultural herbicide), and terbutryn (herbicide in building materials). These contaminants were introduced individually or as a mixture (17.6 µM per micropollutant) into laboratory microcosms mimicking the sediment–water interface. Metformin and metolachlor completely dissipated within 70 days, whereas terbutryn persisted. Dissipation did not differ whether the micropollutants were introduced individually or as part of a mixture. Sequence analysis of 16S rRNA gene amplicons evidenced distinct responses of prokaryotic communities in both sediment and water. Prokaryotic community variations were mainly driven by matrix composition and incubation time. Micropollutant exposure played a secondary but influential role, with pronounced effects of recalcitrant metolachlor and terbutryn within the micropollutant mixture. Antagonistic and synergistic non-additive effects were identified for specific taxa across taxonomic levels in response to the micropollutant mixture. This study underscores the importance of considering the diversity of interactions between micropollutants, prokaryotic communities, and their respective environments when examining sediment–water interfaces affected by multiple contaminants.

Elucidating Key Microbial Drivers for Methane Production during Cold Adaptation and Psychrophilic Anaerobic Digestion of Cattle Manure and Food Waste

At psychrophilic temperatures (<20 °C), anaerobic digestion produces less methane (CH4). For psychrophilic anaerobic digestion (PAD) to be successful, investigation of cold-adapted microbial consortia involved in methane production is critical. This study aimed to investigate the microbial community driving enhanced methane production from the cold-adaptation process and bioaugmentation of PAD with cold-adapted inoculum (BI). Microbial consortia in cattle manure (CM) and food waste (FW) were adapted and applied during batch PAD of CM and FW to bioaugment methane production at 15 °C. Cold adaptation and PAD with BI resulted in cumulative specific methane yields of 0.874 ± 0.231 and 0.552 ± 0.089 L CH4 g−1 volatile solids, respectively, after 14 weeks, while the absence of BI (control) led to acidification and no methane production during PAD. Following 16S rRNA V4–V5 amplicon sequencing and metagenomic analyses, Methanosarcina was revealed as a key driver of methanogenesis during cold adaptation and PAD bioaugmentation. Furthermore, based on the predictive functional and metabolic analysis of the communities, possible synergies were proposed in terms of substrate production and utilization by the dominant microbial groups. For instance, during methane production, Bacteroides and Methanobrevibacter were possibly involved in a syntrophic relationship, which promoted methanogenesis by Methanosarcina. These findings provide insight into the prospective microbial synergies that can be harnessed and/or regulated in cold-adapted inoculum for the improvement of methane production during PAD.

Elucidation of synbiotic diet comprising of Lactobacillus plantarum L20 and Sargassum polycystum on gastrointestinal microbiota, tissue structures and AHPND associated dysbiosis susceptibility in black tiger shrimp (Penaeus monodon)

This study investigates the effect of a synbiotic combination comprising of Lactobacillus plantarum L20 and Sargassum polycystum on gastrointestinal microbiota, tissue structures and AHPND infection in black tiger shrimp, P. monodon. The shrimp were randomly distributed into synbiotic (feed that was incorporated with 108 CFU/g of L. plantarum L20 and 2% S. polycystum) and control group (feed that was incorporated with PBS). After 35 days of feeding trial, all groups were subjected to an AHPND-causing V. parahaemolyticus S2–4 immersion challenge at 6 × 105 CFU/mL. The shrimp gastrointestinal tract from both groups were collected from the feeding trial and challenge test and subjected to high throughput 16S rRNA amplicon sequencing and histopathological analysis. Following 35th day of feeding trial, there were growth enhancement in shrimp, but no significant differences (p > 0.05) in alpha and beta diversities in gastrointestinal microbiota for both groups. However, synbiotic group exhibited a differential abundance of beneficial bacteria, including Lactobacillaceae and Lactiplantibacillus, which were not observed in the control group. Following 60 h post-challenge, there was improvement in shrimp survival in synbiotic group, with slightly significant differences (p < 0.05) were observed in alpha diversity and but no significant differences (p > 0.05) were observed in beta diversity. Synbiotic group demonstrated negative correlation between the gastrointestinal microbiota with Vibrionaceae, enriched potential metabolic pathway of energy generation and microbiota proliferation, and protected gastrointestinal tract structure during the study. The findings suggest that synbiotic diet comprising of L. plantarum L20 and S. polycystum improve growth performance, disease resistance and gastrointestinal structure protection through enhancement of few beneficial bacteria group abundance, despite insignificantly changes of gastrointestinal microbiota with control diet in the shrimp.

Nitrogen conversion performance of a polypropylene carrier designed to promote biofilm formation through foaming

The performance of plastic carriers in a moving bed biofilm reactor used for water treatment is strongly determined by the carrier material and its shape. Only a few reports have described the effect of foaming on biofilm formation and nitrogen conversion by polypropylene (PP) carriers. Here, we investigated a PP foam carrier for its biofilm forming ability and the resulting biofilm’s ability to remove nitrogen compounds. The results revealed that foaming increased the amounts of denitrifying bacterial biofilms attached to PP by approximately 44 times, indicating that unevenness by foaming promoted biofilm formation. Biofilms were also formed using activated sludges from nitrification and denitrification of a wastewater treatment facility. Amplicon sequencing analysis of the biofilms revealed that the families Xanthomonadaceae and Comamonadaceae, including nitrifying bacteria, were enriched in nitrifying sludge-derived biofilms, whereas Dechloromonas sp. and family Pseudomonadaceae, including denitrifying bacteria, were enriched in denitrifying sludge-derived biofilms. Furthermore, nitrifying sludge-derived biofilms completely removed ammonia, and its removal ability was superior to that of the original sludge. Denitrification by denitrifying sludge-derived biofilms was promoted by the addition of fumaric acid. Both nitrifying and denitrifying sludge-derived biofilms could remove nitrogen compounds from 1.4 mM ammonia-containing synthetic wastewater. Finally, the effect of biomass addition on the carrier was investigated. The addition of composted seaweed waste to PP foam carriers enhanced the formation of denitrifying sludge-derived biofilms by approximately 2.2 times, and the denitrification reaction by biofilms was promoted. These results revealed that waste biomass can be used to further enhance PP foam carrier performance.

Relationships between the gut microbiome and brain functional alterations in first-episode, drug-naïve patients with major depressive disorder

ObjectiveIncreasing evidence has shown that the microbiota-gut-brain axis (MGB) is involved in the mechanism of major depressive disorder (MDD). However, the relationship between the gut microbiome and brain function in MDD patients has not been determined. Here, we intend to identify specific changes in the gut microbiome and brain function in first-episode, drug-naïve MDD patients and then explore the associations between the two omics to elucidate how the MGB axis plays a role in MDD development. MethodsWe recruited 38 first-episode, drug-naïve MDD patients and 37 healthy controls (HC). The composition of the fecal microbiome and neural spontaneous activity alterations were examined using 16S rRNA gene amplicon sequencing analysis and regional homogeneity (ReHo). Spearman correlation analyses were conducted to assess the associations between the gut microbiome and brain function. ResultsCompared with HC, MDD patients exhibited distinct alterations in the gut microbiota and elevated ReHo in the frontal regions. In the MDD group, a positive relationship was noted between the relative abundance of Blautia and the HAMD-17 and HAMA scores, as well as between the relative abundance of Oxalobacteraceae and the HAMD-17 score. The relative abundances of Porphyromonadaceae and Parabacteroides were negatively correlated with the ReHo values of frontal regions. LimitationsOur study utilized a cross-sectional design, and the number of subjects was relatively small. ConclusionWe found that some specific gut microbiomes were associated with frontal function, and others were associated with clinical symptoms in MDD patients, which may support the role of the MGB axis underlying MDD.

Buccal Swab Samples from Japanese Brown Cattle Fed with Limonite Reveal Altered Rumen Microbiome.

The areas of the Mount Aso grasslands in Kumamoto, Japan, are the primary location for the breeding of the Kumamoto strain of Japanese Brown cattle (JBRK). Although Aso limonite, deposited by volcanic ash and magma, has been commonly fed to pregnant JBRK in this area, the mechanisms of its salutary effects on pregnant JBRK have not yet been elucidated. Approximately 100 days before the expected day of calf delivery, seven JBRK (four supplemented with limonite and three controls without limonite) were assigned to this study, from which a buccal swab was collected at the highest rumination every 30 days for 90 days. DNA extracted from these swabs was then analyzed using a 16S rRNA gene amplicon sequence analysis. Statistically significant differences between the two groups were discovered through beta-diversity analysis, though results from alpha-diversity analysis were inconclusive. The microbiota identified were classified into six clusters, and three of the main clusters were core-rumen bacteria, primarily cellulose digestion in cluster 1, oral bacteria in cluster 2, and non-core-rumen bacteria in cluster 3. In the limonite group, core-rumen bacteria decreased while non-core-rumen bacteria increased, suggesting that limonite feeding alters rumen microbiota, particularly activation of non-core-rumen microbiota.

Analyzing Cervical Microbiome Composition in HIV-Infected Women with Different HPV Infection Profiles: A Pilot Study in Thailand.

We conducted a pilot study to analyze the microbiome in cervical samples of women living with HIV with various profiles of HPV infections. The participants had an average age of 41.5 years. Sequence analysis of 16S rRNA V3 gene amplicons was performed using next-generation sequencing technology (Ion Torrent PGMTM). The bioinformatics pipeline was analyzed using the Find, Rapidly, OTUs with Galaxy Solution system (FROGS). Common genera were determined to identify Community State Types (CSTs). The cervical microbiome profiles showed a dominance of lactobacilli in 56% (five out of nine) of samples. All three women with normal cervical cells and high-risk HPV infection were classified as CST IV, characterized by anaerobic bacteria associated with bacterial vaginitis, such as Gardnerella, Prevotella, Atopobium, and Sneathia. Among the two women with abnormal cervical cells and high-risk HPV infection, one was classified as CST III, and the other had an unclassified profile dominated by L. helveticus. Four women with normal cervical cells and no HPV infection exhibited various CSTs. Our study demonstrated the feasibility of the protocol in analyzing the cervical microbiome. However, further analysis with a larger number of longitudinal samples is necessary to determine the role of cervical microbiota in HPV persistence, clearance, or the development of precancerous lesions.

Investigation of Mitochondrial Cytb Gene Region of Both Echinococcus granulosus Eggs from Dogs and Cystic Echinococcosis Isolates Obtained from Sheep and Cattle by Molecular Methods.

We aimed to determine the common Echinococcus granulosus genotypes in Ağrı, Türkiye and to obtain information on the transmission of this parasite. Cystic echinococcosis samples from 100 slaughtered cattle and 100 slaughtered sheep and faecal samples from 200 stray dogs were included in 2021. Collected cyst fluid samples and faces were examined microscopically. DNA was isolated from the germinal membrane of the cysts and from the parasite eggs in the stool samples. The mitochondrial cytb gene region of the parasite was amplified by PCR. Genotypes were determined using the Basic Local Alignment Search Tool (BLAST) after sequence analysis of PCR amplicons. The highest percentage of cysts was found in the lungs of sheep and the liver of cattle. In addition, 75% of sheep cysts and 25.6% of cattle cysts were fertile. Taenia spp./Echinococcus spp. eggs were found in 6% of the faeces of 200 dogs examined microscopically. E. granulosus eggs were detected in 4 out of 50 stool samples analysed by PCR. All samples analysed by sequence analysis were identified as E. granulosus s.s. G1 genotype. Sequence comparison revealed revealed one or more-point mutations in different regions of the five samples. E. granulosus s.s. G1 genotype, known as sheep strain, is common in the Ağrı, Türkiye. The controlled slaughter of livestock, especially sheep, and the avoidance of feeding hydatid cyst organs to dogs, together with public education, were necessary to prevent the spread of the disease.

Organophosphorus mineralizing-Streptomyces species underpins uranate immobilization and phosphorus availability in uranium tailings

Phosphate-solubilizing bacteria (PSB) are important but often overlooked regulators of uranium (U) cycling in soil. However, the impact of PSB on uranate fixation coupled with the decomposition of recalcitrant phosphorus (P) in mining land remains poorly understood. Here, we combined gene amplicon sequencing, metagenome and metatranscriptome sequencing analysis and strain isolation to explore the effects of PSB on the stabilization of uranate and P availability in U mining areas. We found that the content of available phosphorus (AP), carbonate-U and Fe-Mn-U oxides in tailings was significantly (P < 0.05) higher than their adjacent soils. Also, organic phosphate mineralizing (PhoD) bacteria (e.g., Streptomyces) and inorganic phosphate solubilizing (gcd) bacteria (e.g., Rhodococcus) were enriched in tailings and soils, but only organic phosphate mineralizing-bacteria substantially contributed to the AP. Notably, most genes involved in organophosphorus mineralization and uranate resistance were widely present in tailings rather than soil. Comparative genomics analyses supported that organophosphorus mineralizing-Streptomyces species could increase soil AP content and immobilize U(VI) through organophosphorus mineralization (e.g., PhoD, ugpBAEC) and U resistance related genes (e.g., petA). We further demonstrated that the isolated Streptomyces sp. PSBY1 could enhance the U(VI) immobilization mediated by the NADH-dependent ubiquinol-cytochrome c reductase (petA) through decomposing organophosphorous compounds. This study advances our understanding of the roles of PSB in regulating the fixation of uranate and P availability in U tailings.

Selective Elucidation of Living Microbial Communities in Fermented Grains of Chinese Baijiu: Development of a Technique Integrating Propidium Monoazide Probe Pretreatment and Amplicon Sequencing.

The fermentation process of Chinese Baijiu's fermented grains involves the intricate succession and metabolism of microbial communities, collectively shaping the Baijiu's quality. Understanding the composition and succession of these living microbial communities within fermented grains is crucial for comprehending fermentation and flavor formation mechanisms. However, conducting high-throughput analysis of living microbial communities within the complex microbial system of fermented grains poses significant challenges. Thus, this study addressed this challenge by devising a high-throughput analysis framework using light-flavor Baijiu as a model. This framework combined propidium monoazide (PMA) pretreatment technology with amplicon sequencing techniques. Optimal PMA treatment parameters, including a concentration of 50 μM and incubation in darkness for 5 min followed by an exposure incubation period of 5 min, were identified. Utilizing this protocol, viable microorganism biomass ranging from 8.71 × 106 to 1.47 × 108 copies/μL was successfully detected in fermented grain samples. Subsequent amplicon sequencing analysis revealed distinct microbial community structures between untreated and PMA-treated groups, with notable differences in relative abundance compositions, particularly in dominant species such as Lactobacillus, Bacillus, Pediococcus, Saccharomycopsis, Issatchenkia and Pichia, as identified by LEfSe analysis. The results of this study confirmed the efficacy of PMA-amplicon sequencing technology for analyzing living microbial communities in fermented grains and furnished a methodological framework for investigating living microbial communities in diverse traditional fermented foods. This technical framework holds considerable significance for advancing our understanding of the fermentation mechanisms intrinsic to traditional fermented foods.

The bacterial and viral communities associated with onion bacterial bulb rot

Microbes occur in complex communities within plants as endophytes and establish a network of interactions that can influence plant health positively or negatively. Positive interactions may be synergistic and confer abiotic and biotic stress tolerance. The aim of this study was to identify bacteria and viruses present in storage onion bulbs that were symptomatic and asymptomatic for bacterial bulb rot, from crops grown in Georgia and Washington states; and to assess their potential role in the bulbs based on functions of bacterial and viral genes detected. DNA was extracted from nine asymptomatic bulbs and nine bulbs displaying symptoms of bacterial bulb rot and subjected to 16S rRNA amplicon sequencing and Metagenomic Assembled Genome (MAG) analysis. The Illumina platform was used to sequence the hypervariable region (V3-V4) of the 16S rRNA gene. The 16S rRNA amplicon profiling revealed the presence of numerous bacteria including potential onion pathogens in the genera Panteoa and Burkholderia. MAG assembly identified P. agglomerans. B. gladioli and B. cepacia, known bulb rot pathogens, including genes linked to fitness and those involved in the Type II and III secretion systems. Eighty-nine unique viral genomes were identified, of which 67 could be classified taxonomically. The bacterial and viral genomes differed significantly in asymptomatic versus symptomatic bulbs. Viral genomes showed evidence of auxiliary metabolic genes (AMGs), including genes involved in fitness and pathogenicity to bacterial hosts. The bulbs hosted endophytic bacteria and viruses, some of which were potentially beneficial, and others potentially pathogenic to onion or as hosts to bacteriophages.

Metabolic Characterization and Geochemical Drivers of Active Hydrocarbon‐Degrading Microorganisms

AbstractUnderstanding the metabolic characteristics and controlled geochemical factors of functional microorganisms in petroleum‐contaminated areas at different locations is pivotal for enhancing pollutant removal strategies. To address the existing research gap in this domain, we employed stable‐isotope‐probing (SIP) with multi‐isotope labeling substrates, combined with 16S amplicon sequencing, metagenomic sequencing, and geochemical factor analysis. Utilizing n‐hexadecane and phenanthrene as model compounds, our study revealed location‐specific differences in the composition of functional microorganisms. Despite these variances, key players such as Pseudomonas, Marinobacter, Alcanivorax, Ochrobactrum, and Sphingomonas consistently emerged as active degraders of n‐hexadecane and/or phenanthrene. Several genera, including Pseudomonas, Ochrobactrum, Alcanivorax, Nitriliruptoraceae, and Sphingobacterium, demonstrated versatility by effectively degrading both contaminants. SIP‐metagenomic binning facilitated the acquisition of genomes from key active degraders, such as Pseudomonas sp., Ochrobactrum sp., Sphingomonas sp., and Shinella sp. This enabled a comprehensive analysis of petroleum hydrocarbon degradation pathways and genes, encompassing PAH dioxygenase genes, alkB genes, phthalate, and salicylate‐related pathways. Environmental factor and variation partitioning analysis revealed that oil pollution significantly influences the functional microbial community (12%), followed by available potassium and available nitrogen. Geochemical parameters and geographic location independently explained 14% and 21% of total variations, respectively. Intriguingly, more than half (51%) of the variation in functional microbial community structure remains unexplained, possibly due to unmeasured environmental variables. Our study contributes valuable insights into the in situ bioremediation mechanism for petroleum‐contaminated soil, elucidating factors influencing functional microbial structures across locations. These findings provide a vital theoretical reference for in situ regulation and bioremediation of petroleum hydrocarbon pollution in diverse environmental contexts.

Short-Term Response of Soil Microbial Community and Soil Biochemical Properties to Soybean Intercropping in a Cassava-Based Cropping System

A cassava-based cropping system study assessed the short-term response of soil chemical and biochemical properties and soil microbial communities to soybean intercropping. Two soybean varieties (‘Select Manchuria’ and ‘Tiwala 12’) were tested as intercrops. The treatments did not affect the soil’s chemical (pH, OM, N, P, K) and biochemical (basal respiration and dehydrogenase and urease enzyme activities) properties after one cropping period. Amplicon sequencing analysis found that intercropping promoted the abundance of bacterial orders Actinomycetales, Solibacterales, Sphingomonadales, and Rhodospirillales. These groups play active roles in organic matter decomposition and can potentially improve the soil quality in an area by enhancing the soil’s organic matter content. In terms of community analysis, principal coordinate analysis (PCoA) and redundancy analysis (RDA) showed separation between mono and intercropping systems, while ANOSIM and PEMANOVA did not detect any significance between varieties and cropping systems for both bacterial and fungal data. These results suggest that, in the short term, introduction of soybean in a cassava-based cropping system affects selected microbial groups, but the overall influence in the microbial community is not distinctly detected.