scholarly journals Effect of Ag Nanoparticles on Denitrification and Microbial Community in a Paddy Soil

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiao Zhang ◽  
Di Dang ◽  
Lingsi Zheng ◽  
Lingyu Wu ◽  
Yu Wu ◽  
...  
Keyword(s):  
Microbial Community ◽  
Real Time ◽  
Quantitative Pcr ◽  
High Throughput ◽  
Paddy Soil ◽  
Ag Nanoparticles ◽  
High Throughput Sequencing ◽  
Emission Rate ◽  
Rrna Gene ◽  
Real Time Quantitative Pcr

The extensive application of Ag nanoparticles (AgNPs) in industry, agriculture, and food processing areas increases the possibility of its release and accumulation to agroecosystem, but the effects of AgNPs to denitrification and the microbial community in paddy ecosystems are still poorly studied. In this study, microcosmic simulation experiments were established to investigate the response of soil denitrification to different levels of AgNPs (i.e., 0.1, 1, 10, and 50 mg/kg) in a paddy soil. Real-time quantitative PCR and high-throughput sequencing were conducted to reveal the microbial mechanism of the nanometer effect. The results showed that, though 0.1–10 mg/kg AgNPs had no significant effects on denitrification rate and N2O emission rate compared to CK and bulk Ag treatments, 50 mg/kg AgNPs significantly stimulated more than 60% increase of denitrification rate and N2O emission rate on the 3rd day (P < 0.05). Real-time quantitative PCR revealed that 50 mg/kg AgNPs significantly decreased the abundance of 16S bacterial rRNA gene, nirS/nirK, cnorB, and nosZ genes, but it did not change the narG gene abundance. The correlation analysis further revealed that the cumulative N2O emission was positively correlated with the ratio of all the five tested denitrifying genes to bacterial 16S rRNA gene (P < 0.05), indicating that the tolerance of narG gene to AgNPs was the key factor of the increase in denitrification in the studied soil. High-throughput sequencing showed that only the 50-mg/kg-AgNP treatment significantly changed the microbial community composition compared to bulk Ag and CK treatments. The response of microbial phylotypes to AgNPs suggested that the most critical bacteria which drove the stimulation of 50 mg/kg AgNPs on N2O emission were Firmicutes and β-proteobacteria, such as Clotridiales, Burkholderiales, and Anaerolineales. This study revealed the effects of AgNPs to denitrification in a paddy ecosystem and could provide a scientific basis for understanding of the environmental and toxicological effects of Ag nanomaterials.

scholarly journals Flow Cytometry and Real-Time Quantitative PCR as Tools for Assessing Plasmid Persistence

2014 ◽  
Vol 80 (17) ◽  
pp. 5439-5446 ◽  
Author(s):  
Wesley Loftie-Eaton ◽  
Allison Tucker ◽  
Ann Norton ◽  
Eva M. Top
Keyword(s):  
Flow Cytometry ◽  
Real Time ◽  
Quantitative Pcr ◽  
High Throughput ◽  
Performance Criteria ◽  
Plate Count ◽  
Real Time Quantitative Pcr ◽  
Advantages And Disadvantages ◽  
Free Cells ◽  
Loss Rates

ABSTRACTThe maintenance of a plasmid in the absence of selection for plasmid-borne genes is not guaranteed. However, plasmid persistence can evolve under selective conditions. Studying the molecular mechanisms behind the evolution of plasmid persistence is key to understanding how plasmids are maintained under nonselective conditions. Given the current crisis of rapid antibiotic resistance spread by multidrug resistance plasmids, this insight is of high medical relevance. The conventional method for monitoring plasmid persistence (i.e., the fraction of plasmid-containing cells in a population over time) is based on cultivation and involves differentiating colonies of plasmid-containing and plasmid-free cells on agar plates. However, this technique is time-consuming and does not easily lend itself to high-throughput applications. Here, we present flow cytometry (FCM) and real-time quantitative PCR (qPCR) as alternative tools for monitoring plasmid persistence. For this, we measured the persistence of a model plasmid, pB10::gfp, in threePseudomonashosts and in known mixtures of plasmid-containing and -free cells. We also compared three performance criteria: dynamic range, resolution, and variance. Although not without exceptions, both techniques generated estimates of overall plasmid loss rates that were rather similar to those generated by the conventional plate count (PC) method. They also were able to resolve differences in loss rates between artificial plasmid persistence assays. Finally, we briefly discuss the advantages and disadvantages for each technique and conclude that, overall, both FCM and real-time qPCR are suitable alternatives to cultivation-based methods for routine measurement of plasmid persistence, thereby opening avenues for high-throughput analyses.

scholarly journals Composition and Origin of the Fermentation Microbiota of Mahewu, a Zimbabwean Fermented Cereal Beverage

2019 ◽  
Vol 85 (11) ◽  
Author(s):  
Felicitas Pswarayi ◽  
Michael G. Gänzle
Keyword(s):  
16S Rrna ◽  
Quantitative Pcr ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Starter Cultures ◽  
Rrna Gene ◽  
Culture Methods ◽  
Content Type ◽  
Cell Counts

ABSTRACTMahewu is a fermented cereal beverage produced in Zimbabwe. This study determined the composition and origin of mahewu microbiota. The microbiota of mahewu samples consisted of 3 to 7 dominant strains of lactobacilli and two strains of yeasts.Enterobacteriaceaewere not detected.Candida glabratawas present in high cell counts from samples collected in summer but not from samples collected in winter. Millet malt is the only raw ingredient used in the production of mahewu and is a likely source of fermentation microbiota; therefore, malt microbiota was also analyzed by culture-dependent and high-throughput 16S rRNA gene sequencing methodologies. Millet malt contained 8 to 19 strains ofEnterobacteriaceae, lactobacilli, bacilli, and very few yeasts. Strain-specific quantitative PCR assays were established on the basis of the genome sequences ofLactobacillus fermentumFUA3588 and FUA3589 andLactobacillus plantarumFUA3590 to obtain a direct assessment of the identity of strains from malt and mahewu.L. fermentumFUA3588 and FUA3589 were detected in millet malt, demonstrating that millet malt is a main source of mahewu microbiota. Strains which were detected in summer were not detected in samples produced at the same site in winter. Model mahewu fermentations conducted with a 5-strain inoculum consisting of lactobacilli,Klebsiella pneumoniae,andCronobacter sakazakiidemonstrated that lactobacilli outcompeteEnterobacteriaceae, which sharply decreased in the first 24 h. In conclusion, mahewu microbiota is mainly derived from millet malt microbiota, but minor components of malt microbiota rapidly outcompeteEnterobacteriaceaeandBacillusspecies during fermentation.IMPORTANCEThis study provides insight into the composition and origin of the microbiota of mahewu and the composition of millet malt microbiota. Fermentation microbiota are often hypothesized to be derived from the environment, but the evidence remains inconclusive. Our findings confirm that millet malt is the major source of mahewu microbiota. By complementing culture methods with high-throughput sequencing of 16S rRNA amplicons and strain-specific quantitative PCR, this study provides evidence about the source of mahewu microbiota, which can inform the development of starter cultures for mahewu production. The study also documents the fate ofEnterobacteriaceaeduring the fermentation of mahewu. There are concerns regarding the safety of traditionally prepared mahewu, and this requires in-depth knowledge of the fermentation process. Therefore, this study elucidated millet malt microbiota and identified cultures that are able to control the high numbers ofEnterobacteriaceaethat are initially present in mahewu fermentations.

scholarly journals Real-Time Quantitative PCR (QPCR) and Reverse Transcription-QPCR for Detection and Enumeration of Total Yeasts in Wine

2006 ◽  
Vol 72 (11) ◽  
pp. 7148-7155 ◽  
Author(s):  
Núria Hierro ◽  
Braulio Esteve-Zarzoso ◽  
Ángel González ◽  
Albert Mas ◽  
Jose M. Guillamón
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Reverse Transcription ◽  
Acetic Acid Bacteria ◽  
Rrna Gene ◽  
Real Time Quantitative Pcr ◽  
Yeast Extract Peptone Dextrose ◽  
Different Strains ◽  
26S Rrna ◽  
First Time

ABSTRACT Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of acetic acid bacteria and lactic acid bacteria. Numerous standard curves were constructed with different strains and species grown in yeast extract-peptone-dextrose medium or incubated in wine. The small standard errors with these replicas proved that the assay is reproducible and highly robust. This technique was validated with artificially contaminated and natural wine samples. We also performed a reverse transcription-QPCR (RT-QPCR) assay from rRNA for total viable yeast quantification. This technique had a low detection limit and was more accurate than QPCR because the dead cells were not quantified. As far as we know, this is the first time that RT-QPCR has been performed to quantify viable yeasts from rRNA. RT-QPCR is a rapid and accurate technique for enumerating yeasts during industrial wine fermentation and controlling the risk of wine spoilage.

scholarly journals Real-Time Quantitative PCR for Assessment of Abundance of Pseudoalteromonas Species in Marine Samples

2004 ◽  
Vol 70 (4) ◽  
pp. 2373-2382 ◽  
Author(s):  
Torben L. Skovhus ◽  
Niels B. Ramsing ◽  
Carola Holmström ◽  
Staffan Kjelleberg ◽  
Ingela Dahllöf
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Green Alga ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Rrna Gene ◽  
Marine Animals ◽  
Real Time Quantitative Pcr ◽  
Marine Samples ◽  
Marine Habitats ◽  
Pcr Method

ABSTRACT A real-time quantitative PCR (RTQ-PCR) method for measuring the abundance of Pseudoalteromonas species in marine samples is presented. PCR primers targeting a Pseudoalteromonas-specific region of the 16S rRNA gene were tested at three different levels using database searches (in silico), a selection of pure cultures (in vitro), and a combined denaturing gradient gel electrophoresis and cloning approach on environmental DNA (in situ). The RTQ-PCR method allowed for the detection of SYBR Green fluorescence from double-stranded DNA over a linear range spanning six orders of magnitude. The detection limit was determined as 1.4 fg of target DNA (1,000 gene copies) measured in the presence of 20 ng of nontarget DNA from salmon testes. In this study, we discuss the importance of robust post-PCR analyses to overcome pitfalls in RTQ-PCR when samples from different complex marine habitats are analyzed and compared on a nonroutine basis. Representatives of the genus Pseudoalteromonas were detected in samples from all investigated habitats, suggesting a widespread distribution of this genus across many marine habitats (e.g., seawater, rocks, macroalgae, and marine animals). Three sample types were analyzed by RTQ-PCR to determine the relative abundance of Pseudoalteromonas ribosomal DNA (rDNA) compared to the total abundance of eubacterial rDNA. The rDNA fractions of Pseudoalteromonas compared to all Eubacteria were 1.55% on the green alga Ulva lactuca, 0.10% on the tunicate Ciona intestinalis, and 0.06% on the green alga Ulvaria fusca.

scholarly journals Development of an Inexpensive and Rapid Operation Device for High-Throughput Real-Time Quantitative PCR-Based CYP2D6 CNV Genotyping

2019 ◽  
Vol 42 (10) ◽  
pp. 1761-1765 ◽  
Author(s):  
Miho Imai ◽  
Madoka Kisoi ◽  
Miwako Moritsugu ◽  
Shigenori Murata ◽  
Atsushi Ichikawa ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
High Throughput ◽  
Real Time Quantitative Pcr

Analysis of changes in the microbial community structure and physicochemical properties during the fermentation of sand crab juice

2021 ◽  
Author(s):  
Meiyan Luo ◽  
Leilei Chen ◽  
Huanming Liu ◽  
Jiahui Jiang ◽  
Chongxi Lai ◽  
...  
Keyword(s):  
Microbial Community ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Species Abundance ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Isolation Technique ◽  
Bacterial Phyla ◽  
Staphylococcus Equorum ◽  
Rrna Gene Sequencing

Abstract The structure of the microbial community during sand crab juice fermentation was analyzed using culture-based methods and high-throughput 16S rRNA gene sequencing. Additionally, the changes in amino acid nitrogen (AAN) and total volatile basic nitrogen (TVB-N) were evaluated. Staphylococcus equorum, Staphylococcus arlettae, Staphylococcus saprophyticus, Salinicoccus amylolyticus, and Bacillus cereus were isolated by traditional culture isolation technique. The Good's coverage obtained by high-throughput sequencing was over 99.5%, and the Chao1 and Simpson indices showed small fluctuations, indicating that the species abundance and diversity did not change significantly during the fermentation process, although the abundance decreased. Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria were the dominant bacterial phyla observed during fermentation, whereas Aquabacterium, Roseovarius, Muribaculaceae, and Silicimonas were the dominant bacterial genera. The AAN content increased from 0.15 to 0.43 g/100 mL during the 15-day fermentation, indicating the production of small peptides and amino acids during fermentation. The TVB-N content (25.2 mg/100 mL) on day 15 indicated slight spoilage of sand crab juice, although the freshness conformed to the production standard. These results provide a theoretical basis for improving the quality and optimizing the production process of sand crab juice.

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