Microorganisms In Environmental Samples Research Articles

Unveiling novel pathways and key contributors in the nitrogen cycle: Validation of enrichment and taxonomic characterization of oxygenic denitrifying microorganisms in environmental samples

Microorganisms play a crucial role in both the nitrogen cycle and greenhouse gas emissions. A recent discovery has unveiled a new denitrification pathway called oxygenic denitrification, entailing the enzymatic reduction of nitrite to nitric oxide (NO) by a putative nitric oxide dismutase (nod) enzyme. In this study, the presence of the nod gene was detected and subsequently enriched in anaerobic-activated sludge, farmland soil, and paddy soil samples. After 150 days, the enriched samples exhibited significant denitrification, and concomitant oxygen production. The removal efficiency of nitrite ranged from 64.6 % to 79.0 %, while the oxygen production rate was between 15.4 μL/min and 18.6 μL/min when exposed to a sole nitrogen source of 80 mg/L sodium nitrite. Additionally, batch experiments and kinetic analyses revealed the intricate pathways and underlying mechanisms governing the oxygenic denitrification reaction by using CARBOXY-PTIO, 18O-labelled water, and acetylene to unravel the intricacies of the reaction. The quantitative polymerase chain reaction (qPCR) results indicated a significant surge in the abundance of nod genes, escalating from 7.59 to 10.12-fold. Moreover, analysis of 16S ribosomal DNA (rDNA) amplicons revealed Proteobacteria as the dominant phylum and Thauera as the main genus, with the presumed affiliation. In this study, a new nitrogen conversion pathway, oxygenic denitrification, was discovered in environmental samples. This process provides the possibility for the control of nitrous oxide in the treatment of nitrogenous wastewater.

Fundamentals of genomics and its applications

Genomics is the discipline that studies the structure, function, and evolution of genomes and addresses the methodological processes used tosequence and assemble the genome. Analyzing sequencing data requires state-of-the-art computational resources and specialized mathematicalalgorithms and software, which are together known as bioinformatics. The history of genome sequencing can be divided into three stages: 1)first-generation, which is based on the sequencing of a single fragment using capillary electrophoresis; 2) second generation, characterized by themass parallelization of sequencing reactions, resulting in an increase in the amount of DNA fragments sequenced with a length of 50 to 300 basepairs; and 3) third generation, which also includes the mass sequencing, but of much longer fragments (> 10000 base pairs), which facilitatesgenome assembly. Advances in massive sequencing have allowed for the sequencing of a large number of genomes, which has had broadapplications in medicine, the improvement of economically important plant and animal species, and phylogenetic studies, among many others.One of the main branches of genomics is metagenomics, which has been highly important in generating knowledge of the composition anddiversity of microorganisms in environmental samples of water, air, and other materials, facilitating the recognition of new taxa. Another branchof genomics is phylogenomics, which is used to infer the evolutionary relationships among species. Therefore, the advances in sequencingplatforms as well as advances in the area of bioinformatics have resulted in a revolution of knowledge of genome complexity

The "Molecular Biodiversity of Bacteria Isolated from Medicago sativa Rhizosphere in Hail District, Saudi Arabia.

Global biodiversity is affected remarkably by global climate change, which in turn its effect reflected on all life aspects. Identifying microorganisms in environmental samples, particularly soil could be a valuable interest to study their effect on soil quality and plant growth. Through this study, we conducted a molecular characterization of bacteria found in the rhizosphere of Medico sativa plants grown in Hail soil and we highlighted their main properties. The sequences analyses revealed that the main bacterial isolates Pseudarthrobacter, Metabacillus, Priestia, and Massilia species. According to the sequences analysis and the phylogeny tree results, some of the identified bacteria were classified at the species level: Pseudarthrobacter was identified clearly as Pseudarthrobacter phenanthrenivorans; Metabacillus isolates grouped with Metabacillus sediminilitoris and the two Priestia isolates closely related to Priestia aryabhattai. We concluded that Hail soil is a niche of diverse bacteria with a high interest in soil environment and ecosystems. Further studies are required for further classification of all identified bacteria and to define their specific role in the environment.

Predicting the occurrence of monooxygenases and their associated phylotypes in soil microcosms

Cometabolic oxidation involves the oxidation of chemicals often by monooxygenases or dioxygenases and can be a removal process for environmental contaminants such as trichloroethene (TCE) or 1,4-dioxane. Information on the occurrence of these genes and their associated microorganisms in environmental samples has the potential to enhance our understanding of contaminant removal. The overall aims were to 1) ascertain which genes encoding for monooxygenases (from methanotrophs, ammonia oxidizing bacteria and toluene/phenol oxidizers) and other key enzymes are present in soil microcosms and 2) determine which phylotypes are associated with those genes.The approach involved a predictive tool called PICRUSt2 and 16S rRNA gene amplicon datasets from two previous soil microcosm studies. The following targets from the KEGG database were examined: pmo/amo, mmo, dmp/pox/tomA, tmo/tbu/tou, bssABC (and downstream genes), tod, xylM, xylA, gst, dhaA, catE, dbfA1, dbfA2 and phenol 2-monooxygenase.A large number of phylotypes were associated with pmo/amo, while mmo was linked to only five. Several phylotypes were associated with both pmo/amo and mmo. The most dominant microorganism predicted for mmoX was Mycobacterium (also predicted for pmo/amo). A large number of phylotypes were associated with all six genes from the dmp/pox/tomA KEGG group. The taxonomic associations predicted for the tmo/tbu/tou KEGG group were more limited. In both datasets, Geobacter was a key phylotype for benzylsuccinate synthase. The dioxygenase-mediated toluene degradation pathway encoded by todC1C2BA was largely absent, as were the genes (xylM, xylA) encoding for xylene monooxygenase. All other genes investigated were predicted to be present and were associated with a number of microorganisms. Overall, the analysis predicted the genes encoding for sMMO (mmo), T3MO/T3MO/ToMO (tmo/tbu/tou) and benzylsuccinate synthase (bssABC) are present for a limited number of phylotypes compared to those encoding for pMMO/AMO (pmo/amo) and phenol monooxygenase/T2MO (dmp/poxA/tomA). These findings suggest in soils contaminant removal via pMMO/AMO or phenol monooxygenase/T2MO may be common because of the occurrence of these enzymes with a large number of phylotypes.

Function-Oriented Graphene Quantum Dots Probe for Single Cell in situ Sorting of Active Microorganisms in Environmental Samples.

Functional microorganisms play a vital role in removing environmental pollutants because of their diverse metabolic capability. Herein, a function-oriented fluorescence resonance energy transfer (FRET)-based graphene quantum dots (GQDs-M) probe was developed for the specific identification and accurate sorting of azo-degrading functional bacteria in the original location of environmental samples for large-scale culturing. First, nitrogen-doped GQDs (GQDs-N) were synthesized using a bottom-up strategy. Then, a GQDs-M probe was synthesized based on bonding FRET-based GQDs-N to an azo dye, methyl red, and the quenched fluorescence was recovered upon cleavage of the azo bond. Bioimaging confirmed the specific recognition capability of GQDs-M upon incubation with the target bacteria or environmental samples. It is suggested that the estimation of environmental functional microbial populations based on bioimaging will be a new method for rapid preliminary assessment of environmental pollution levels. In combination with a visual single-cell sorter, the target bacteria in the environmental samples could be intuitively screened at the single-cell level in 17 bacterial strains, including the positive control Shewanella decolorationis S12, and were isolated from environmental samples. All of these showed an azo degradation function, indicating the high accuracy of the single-cell sorting strategy using the GQDs-M. Furthermore, among the bacteria isolated, two strains of Bacillus pacificus and Bacillus wiedmannii showed double and triple degradation efficiency for methyl red compared to the positive control (strain S12). This strategy will have good application prospects for finding new species or high-activity species of specific functional bacteria.

Next Generation High Throughput Sequencing to Assess Microbial Communities: An Application Based on Water Quality.

Traditional techniques to identify different contaminants (biological or chemical) in the waters are slow, laborious, and can require specialized expertise. Hence, the rapid determination of water quality using more sensitive and reliable metagenomic based approaches attains special importance. Metagenomics deals with the study of genetic material that is recovered from microbial communities present in environmental samples. In traditional techniques cultivation-based methodologieswere used to describe the diversity of microorganisms in environmental samples. It has failed to function as a robust marker because of limited taxonomic and phylogenetic implications. In this backdrop, high-throughput DNA sequencing approaches have proven very powerful in microbial source tracking because of investigating thefull variety of genome-based analysis such as microbial genetic diversity and population structure played by them. Next generation sequencing technologies can reveal a greater proportion of microbial communities that have not been reported earlier by traditional techniques. The present review highlights the shift from traditional techniquesfor the basic study of community composition to next-generation sequencing (NGS) platforms and their potential applications to the biomonitoring of water quality in relation to human health.

Assigning Function to Phylogeny: FISH-nanoSIMS.

Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) is an imaging method used to identify microorganisms in environmental samples based on their phylogeny. CARD-FISH can be combined with nano-scale secondary ion mass spectrometry (nanoSIMS) to directly link the cell identity to their activity, measured as the incorporation of stable isotopes into hybridized cells after stable isotope probing. In environmental microbiology, a combination of these methods has been used to determine the identity and growth of uncultured microorganisms, and to explore the factors controlling their activity. Additionally, FISH-nanoSIMS has been widely used to directly visualize microbial interactions in situ. Here, we describe a step-by-step protocol for a combination of CARD-FISH, laser marking, and nanoSIMS analysis on samples from aquatic environments.

Low Turnover of Soil Bacterial rRNA at Low Temperatures

Ribosomal RNA (rRNA) is used widely to investigate potentially active microorganisms in environmental samples, including soil microorganisms and other microbial communities that are subjected to pronounced seasonal variation in temperature. This raises a question about the turnover of intracellular microbial rRNA at environmentally relevant temperatures. We analyzed the turnover at four temperatures of RNA isolated from soil bacteria amended with 14C-labeled uridine. We found that the half-life of recently produced RNA increased from 4.0 days at 20°C to 15.8 days at 4°C, and 215 days at −4°C, while no degradation was detected at −18°C during a 1-year period. We discuss the implications of the strong temperature dependency of rRNA turnover for interpretation of microbiome data based on rRNA isolated from environmental samples.

TAMA: improved metagenomic sequence classification through meta-analysis

BackgroundMicroorganisms are important occupants of many different environments. Identifying the composition of microbes and estimating their abundance promote understanding of interactions of microbes in environmental samples. To understand their environments more deeply, the composition of microorganisms in environmental samples has been studied using metagenomes, which are the collections of genomes of the microorganisms. Although many tools have been developed for taxonomy analysis based on different algorithms, variability of analysis outputs of existing tools from the same input metagenome datasets is the main obstacle for many researchers in this field.ResultsHere, we present a novel meta-analysis tool for metagenome taxonomy analysis, called TAMA, by intelligently integrating outputs from three different taxonomy analysis tools. Using an integrated reference database, TAMA performs taxonomy assignment for input metagenome reads based on a meta-score by integrating scores of taxonomy assignment from different taxonomy classification tools. TAMA outperformed existing tools when evaluated using various benchmark datasets. It was also successfully applied to obtain relative species abundance profiles and difference in composition of microorganisms in two types of cheese metagenome and human gut metagenome.ConclusionTAMA can be easily installed and used for metagenome read classification and the prediction of relative species abundance from multiple numbers and types of metagenome read samples. TAMA can be used to more accurately uncover the composition of microorganisms in metagenome samples collected from various environments, especially when the use of a single taxonomy analysis tool is unreliable. TAMA is an open source tool, and can be downloaded at https://github.com/jkimlab/TAMA.

Stable Isotope Probing of Microorganisms in Environmental Samples with H218O.

We describe a protocol for investigating microbial growth in environmental samples via stable isotope probing (SIP) with H218O. Water is a universal substrate for all microorganisms and replication is required for DNA to become labeled with 18O. By measuring how much the DNA of each taxon becomes enriched with 18O when an environmental sample is incubated with H218O, it is feasible to quantify that population's DNA replication rate, which is a proxy for growth.

Can enzymatic stoichiometry be used to determine growth-limiting nutrients for microorganisms? - A critical assessment in two subtropical soils

The measurement of potential enzymatic activities has been proposed as an efficient method to infer nutrient limitations for microorganisms in environmental samples. To validate this use, confirmation with direct methods of microbial growth responses to resource additions are required. We experimentally manipulated nutrient-poor soils from the afromontane subtropics with relatively low (grassland soils, ca. 4% soil carbon (C)) or high organic matter content (forest soils, ca. 13% soil C) with nutrient additions (plant material added at 8 mg C g−1 soil combined with mineral N and/or P to reach C:N:P mass-ratios of 10:1:1) in a multifactorial design for one month in order to shift the microbial community towards C-, N- or P-limitation. We then measured the responses of the most commonly measured indicator enzymes used to infer growth limiting nutrients, using ß-1,4-glucosidase, ß-1,4-N-acetylglucosaminidase and leucine aminopeptidase, and acid phosphatase as indicators for C-, N- and P-acquiring enzymatic activities, respectively. In the same soil samples, we also determined the responses in bacterial (3H-leucine incorporation) and fungal growth rates (14C-acetate incorporation into ergosterol) to nutrient supplements, and also verified these with biomass responses (microbial PLFA and ergosterol concentrations) to the factorial nutrient loading amendments. Ratios of C-, N-, and P-acquiring enzymes indicated that the grassland soils were primarily P-limited, and secondarily co-limited by C and N, while the forest soils were co-limited by C and P. However, short-term responses in growth rates and respiration to nutrient additions, along with long-term growth rate, respiration and biomass responses to nutrient loading treatments all indicated that bacterial growth, fungal growth and respiration were primarily limited by C in both grassland and forest soils. We conclude that enzymatic ratios do not capture the growth-limiting factors for bacterial growth, fungal growth, or respiration in soil. Furthermore, the addition of C-rich plant material could shift the fungal community into N-limitation, while bacteria were shifted into co-limitation by both C and N, revealing that bacteria and fungi can be limited by different nutrients within the same soil environment.

Non-Specific Indication of Microorganisms in Environmental Samples

The review presents an analysis of modern methods and instruments for performing nonspecific detection of pathogenic biological agents in environmental objects. Discussed are technological characteristics of application of these methods for the detection of biological substances of protein nature in samples. The spectrum of means for non-specific PBA detection includes home-produced and foreign field devices based on protein contamination indication using various colorimetry variants. Technologies for remote and direct monitoring of environment for the presence of aerosols of biological nature are represented by hybrid lidar systems (biolidars) and biodetectors. For PBA nucleic acids tracing, the complexes based on DNA molecule binding with fluorophore with further fluorescence detection are described. Given are the examples of chemiluminescent analysis application in the developed automatic impurity detectors, as well as systems using bioluminescence. Based on the literature data analysis, put forward is a possible algorithm for indication of pathogenic biological agents when carrying out monitoring of the environment in zones of possible emergency situation occurrence and mass events holding.

DACE: a scalable DP-means algorithm for clustering extremely large sequence data.

Advancements in next-generation sequencing technology have produced large amounts of reads at low cost in a short time. In metagenomics, 16S and 18S rRNA gene have been widely used as marker genes to profile diversity of microorganisms in environmental samples. Through clustering of sequencing reads we can determine both number of OTUs and their relative abundance. In many applications, clustering of very large sequencing data with high efficiency and accuracy is essential for downstream analysis. Here, we report a scalable D irichlet Process Means (DP-means) a lgorithm for c lustering e xtremely large sequencing data, termed . With an efficient random projection partition strategy for parallel clustering, DACE can cluster billions of sequences within a couple of hours. Experimental results show that DACE runs between 6 and 80 times faster than state-of-the-art programs, while maintaining overall better clustering accuracy. Using 80 cores, DACE clustered the Lake Taihu 16S rRNA gene sequencing data (∼316M reads, 30 GB) in 25 min, and the Ocean TARA Eukaryotic 18S rRNA gene sequencing data (∼500M reads, 88 GB) into ∼100 000 clusters within an hour. When applied to the IGC gene catalogs in human gut microbiome (∼10M genes), DACE produced 9.8M clusters with 52K redundant genes in 1.5 hours of running time. DACE is available at https://github.com/tinglab/DACE . tingchen@mail.tsinghua.edu.cn or ningchen@mail.tsinghua.edu.cn. Supplementary data are available at Bioinformatics online.

Ecological connectivity shapes quasispecies structure of RNA viruses in an Antarctic lake.

RNA viruses exist as complex mixtures of genotypes, known as quasispecies, where the evolution potential resides in the whole community of related genotypes. Quasispecies structure and dynamics have been studied in detail for virus infecting animals and plants but remain unexplored for those infecting micro-organisms in environmental samples. We report the first metagenomic study of RNA viruses in an Antarctic lake (Lake Limnopolar, Livingston Island). Similar to low-latitude aquatic environments, this lake harbours an RNA virome dominated by positive single-strand RNA viruses from the order Picornavirales probably infecting micro-organisms. Antarctic picorna-like virus 1 (APLV1), one of the most abundant viruses in the lake, does not incorporate any mutation in the consensus sequence from 2006 to 2010 and shows stable quasispecies with low-complexity indexes. By contrast, APLV2-APLV3 are detected in the lake water exclusively in summer samples and are major constituents of surrounding cyanobacterial mats. Their quasispecies exhibit low complexity in cyanobacterial mat, but their run-off-mediated transfer to the lake results in a remarkable increase of complexity that may reflect the convergence of different viral quasispecies from the catchment area or replication in a more diverse host community. This is the first example of viral quasispecies from natural aquatic ecosystems and points to ecological connectivity as a modulating factor of quasispecies complexity.

Taxonomic and functional diversity of microbial community from a mining environment

Background Biomining uses acidophilic and chemolithotrophic microbes capable of oxidizing iron and sulfur to recover metals of interest from complex minerals. One of the most critical issues in mining environments is the generation of acid mine drainage (AMD) that pollutes water and sediments with acids and metals. The Sulfate-reducing bacteria (SBR) are an alternative to bioremediate contamination of AMD. The current knowledge on the microbial diversity and the metabolic pathways involved in bioming and bioremediation is still limited. In this context, metagenomics has become a valuable tool to investigate previously uncultured microorganisms in environmental samples. The aim of this study was to assess the taxonomic and functional microbial diversity in a mining area in the Brazilian Amazon.

Determination of specific types and relative levels of QPCR inhibitors in environmental water samples using excitation–emission matrix spectroscopy and PARAFAC

Assays that utilize PCR offer powerful tools to detect pathogens and other microorganisms in environmental samples. However, PCR inhibitors present in nucleic acid extractions can increase a sample's limit of detection, skew calculated marker concentrations, or cause false-negative results. It would be advantageous to predict which samples contain various types and levels of PCR inhibitors, especially the humic and fulvic acids that are frequently cited as PCR inhibitors in natural water samples. This study investigated the relationships between quantitative PCR (qPCR) inhibition and the humic and fulvic content of dissolved organic matter (DOM), as well as several other measures of DOM quantity and quality, in water samples. QPCR inhibition was also compared to water quality parameters, precipitation levels, and land use adjacent to the sampling location. Results indicate that qPCR inhibition in the tested water samples was correlated to several humic substance-like, DOM components, most notably terrestrially-derived, humic-like DOM and microbially-derived, fulvic-like DOM. No correlation was found between qPCR inhibition and water quality parameters or land use, but a relationship was noted between inhibition and antecedent rainfall. This study suggests that certain fractions of humic substances are responsible for PCR inhibition from temperate, freshwater systems. PARAFAC modeling of excitation–emission matrix spectroscopy provides insight on the components of the DOM pool that impact qPCR success and may be useful in evaluating methods to remove PCR inhibitors present in samples.

Portable self-contained cultures for phage and bacteria made of paper and tape

In this paper, we demonstrate that a functional, portable device for the growth of bacteria or amplification of bacteriophage can be created using simple materials. These devices are comprised of packing tape, sheets of paper patterned by hydrophobic printer ink, and a polydimethyl siloxane (PDMS) membrane, which is selectively permeable to oxygen but non-permeable to water. These devices supply bacteria with oxygen and prevent the evaporation of media for a period over 48 h. The division time of E. coli and the amplification of the phage M13 in this device are similar to the rates measured on agar plates and in shaking cultures. The growth of bacteria with a fluorescent mCherry reporter can be quantified using a flatbed scanner or a cell phone camera. Permeating devices with commercial viability dye (PrestoBlue) can be used to detect low copy number of E. coli (1-10 CFU in 100 μL) and visualize microorganisms in environmental samples. The platform, equipped with bacteria that carry inducible mCherry reporter could also be used to quantify the concentration of the inducer (here, arabinose). Identical culture platforms can, potentially, be used to quantify the induction of gene expression by an engineered phage or by synthetic transcriptional regulators that respond to clinically relevant molecules. The majority of measurement and fabrication procedures presented in this report have been replicated by low-skilled personnel (high-school students) in a low-resource environment (high-school classroom). The fabrication and performance of the device have also been tested in a low-resource laboratory setting by researchers in Nairobi, Kenya. Accordingly, this platform can be used as both an educational tool and as a diagnostic tool in low-resource environments worldwide.

Influence of DNA isolation on Q-PCR-based quantification of methanogenic Archaea in biogas fermenters

Quantitative real-time PCR (Q-PCR) is commonly applied for the detection of certain microorganisms in environmental samples. However, some environments, like biomass-degrading biogas fermenters, are enriched with PCR-interfering substances. To study the impact of the DNA extraction protocol on the results of Q-PCR-based analysis of the methane-producing archaeal community in biogas fermenters, nine different protocols with varying cell disruption and DNA purification approaches were tested. Differences in the quantities of the isolated DNA and the purity parameters were found, with the best cell lysis efficiencies being obtained by a combined lysozyme/SDS-based lysis. When DNA was purified by sephacryl columns, the amount of DNA decreased by one log cycle but PCR inhibitors were eliminated sufficiently. In the case of detection of methanogenic Archaea, the chosen DNA isolation protocol strongly influenced the Q-PCR-based determination of 16S rDNA copy numbers. For example, with protocols including mechanical cell disruption, the 16S rDNA of Methanobacteriales were predominantly amplified (81–90% of the total 16S rDNA copy numbers), followed by the 16S rDNA of Methanomicrobiales (9–18%). In contrast, when a lysozyme/SDS-based cell lysis was applied, the 16S rDNA copy numbers determined for these two orders were the opposite (Methanomicrobiales 82–95%, Methanobacteriales 4–18%). In extreme cases, the DNA isolation method led to discrimination of some groups of methanogens (e.g. members of the Methanosaetaceae). In conclusion, for extraction of high amounts of microbial DNA with high purity from samples of biogas plants, a combined lysozyme/SDS-based cell lysis followed by a purification step with sephacryl columns is recommended.

Fluorescence In Situ Hybridization for the Identification of Environmental Microbes

INTRODUCTIONThis protocol presents methods for the phylogenetic identification of microorganisms in environmental samples (e.g., water and sediments) by means of fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes, followed by signal amplification with catalyzed reporter deposition (CARD). In this procedure, FISH probes are conjugated with the enzyme horseradish peroxidase (HRP). After hybridization, the subsequent deposition of fluorescently labeled tyramides results in substantially higher signal intensities on target cells than after FISH with probes labeled directly with fluorochromes. This protocol describes the custom labeling of tyramides with different fluorochromes, as well as sample preparation and cell permeabilization strategies for various microbial cell wall types. A protocol for sequential multicolor CARD-FISH for the simultaneous detection of different phylogenetic groups is also presented.

Analyzing Microorganisms in Environmental Samples Using Stable Isotope Probing with H218O

INTRODUCTIONWhen analyzing environmental samples of microorganisms by stable isotope probing (SIP), labeling the DNA with H218O, instead of organic or nitrogenous compounds, offers important advantages because water cannot be used as an energy, carbon, or nitrogen source. As a result, addition of the label is unlikely to influence microbial growth rates in soil directly, and microbial communities can be exposed to the label for long periods of time because they are not exposed to abnormally high substrate concentrations. Because all organisms incorporate water into their DNA, performing SIP with H218O is a method for identifying microorganisms that have grown during incubation with H218O, as well as microorganisms that have not grown (i.e., did not incorporate the label) but survived the incubation. This protocol describes the use of SIP with H218O to study soil microorganisms.