Earthworms and rice straw enhanced soil bacterial diversity and promoted the degradation of phenanthrene

Abstract Background Since the industrial revolution, the contamination of agricultural soils by polycyclic aromatic hydrocarbons (PAHs) has increasingly become of serious global environmental concern and poses a huge threat to human beings and natural ecosystems. Microbial degradation is a proved technology mostly used to depollute polycyclic aromatic hydrocarbon (PAH) in the environment. However, very limited information is available regarding the interaction of earthworms with rice straw on the soil microbial community and the degradation of phenanthrene. This study was performed to enlighten the rice straw and earthworms’ interaction on soil bacterial abundance and structure and phenanthrene removal. Results Result about functional gene information revealed that both rice straw and earthworm enhanced phenanthrene degradation. Subsequently, both Shannon diversity index (r2 = − 0.8807, p < 0.001) and bacterial 16S rRNA genes (r2 = − 0.7795, p < 0.001) negatively correlated with the remaining phenanthrene concentration in soil. The application of both rice straw and earthworms in soil had the lowest ratio of soil remaining phenanthrene concentration (0.16 ± 0.02), the highest Shannon diversity index (6.45 ± 0.2) and the highest bacterial 16S rRNA genes. This implied that both earthworms and rice straw might improve the phenanthrene metabolism by increasing soil bacteria diversity. The abundance of genera Pseudomonas, Luteimonas, Rhodanobacter, Sphingomonas, Gemmatimonas, Flavobacterium, and Leifsonia was significantly increased in the presence of both earthworms and rice straw and was found to negatively correlate with the remaining phenanthrene concentration in soil. Conclusion Based on these results, this study offers clear and strong evidences that the positive interaction between earthworms and rice straw could promote phenanthrene degradation in soil. These finding will improve our understanding on the importance of the natural resources forsaken and how they can interact with the soil macro- and microorganisms to change soil structure and enhance PAH degradation in soil.

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The spleen bacteriome of wild rodents and shrews from Marigat, Baringo County, Kenya

PeerJ ◽

10.7717/peerj.12067 ◽

2021 ◽

Vol 9 ◽

pp. e12067

Author(s):

Rehema Liyai ◽

Gathii Kimita ◽

Clement Masakhwe ◽

David Abuom ◽

Beth Mutai ◽

...

Keyword(s):

16S Rrna ◽

Bacterial Diversity ◽

Small Mammals ◽

Pathogenic Bacteria ◽

Diversity Index ◽

Shannon Diversity Index ◽

Gene Sequences ◽

16S Rrna Analysis ◽

Shannon Diversity ◽

Mitochondrial Cytochrome B

Background There is a global increase in reports of emerging diseases, some of which have emerged as spillover events from wild animals. The spleen is a major phagocytic organ and can therefore be probed for systemic microbiome. This study assessed bacterial diversity in the spleen of wild caught small mammals so as to evaluate their utility as surveillance tools for monitoring bacteria in an ecosystem shared with humans. Methods Fifty-four small mammals (rodents and shrews) were trapped from different sites in Marigat, Baringo County, Kenya. To characterize their bacteriome, DNA was extracted from their spleens and the V3–V4 regions of the 16S rRNA amplified and then sequenced on Illumina MiSeq. A non-target control sample was used to track laboratory contaminants. Sequence data was analyzed with Mothur v1.35, and taxomy determined using the SILVA database. The Shannon diversity index was used to estimate bacterial diversity in each animal and then aggregated to genus level before computing the means. Animal species within the rodents and shrews were identified by amplification of mitochondrial cytochrome b (cytb) gene followed by Sanger sequencing. CLC workbench was used to assemble the cytb gene sequences, after which their phylogenetic placements were determined by querying them against the GenBank nucleotide database. Results cytb gene sequences were generated for 49/54 mammalian samples: 38 rodents (Rodentia) and 11 shrews (Eulipotyphyla). Within the order Rodentia, 21 Acomys, eight Mastomys, six Arvicanthis and three Rattus were identified. In the order Eulipotyphyla, 11 Crucidura were identified. Bacteria characterization revealed 17 phyla that grouped into 182 genera. Of the phyla, Proteobacteria was the most abundant (67.9%). Other phyla included Actinobacteria (16.5%), Firmicutes (5.5%), Chlamydiae (3.8%), Chloroflexi (2.6%) and Bacteroidetes (1.3%) among others. Of the potentially pathogenic bacteria, Bartonella was the most abundant (45.6%), followed by Anaplasma (8.0%), Methylobacterium (3.5%), Delftia (3.8%), Coxiella (2.6%), Bradyrhizobium (1.6%) and Acinetobacter (1.1%). Other less abundant (<1%) and potentially pathogenic included Ehrlichia, Rickettsia, Leptospira, Borrelia, Brucella, Chlamydia and Streptococcus. By Shannon diversity index, Acomys spleens carried more diverse bacteria (mean Shannon diversity index of 2.86, p = 0.008) compared to 1.77 for Crocidura, 1.44 for Rattus, 1.40 for Arvicathis and 0.60 for Mastomys. Conclusion This study examined systemic bacteria that are filtered by the spleen and the findings underscore the utility of 16S rRNA deep sequencing in characterizing complex microbiota that are potentially relevant to one health issues. An inherent problem with the V3-V4 region of 16S rRNA is the inability to classify bacteria reliably beyond the genera. Future studies should utilize the newer long read methods of 16S rRNA analysis that can delimit the species composition.

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Bacterial Populations Colonizing and Degrading Rice Straw in Anoxic Paddy Soil

Applied and Environmental Microbiology ◽

10.1128/aem.67.3.1318-1327.2001 ◽

2001 ◽

Vol 67 (3) ◽

pp. 1318-1327 ◽

Cited By ~ 122

Author(s):

Sabine Weber ◽

Stephan Stubner ◽

Ralf Conrad

Keyword(s):

Bacterial Community ◽

16S Rrna ◽

Rice Straw ◽

Paddy Soil ◽

Denaturing Gradient Gel Electrophoresis ◽

Blot Hybridization ◽

16S Rrna Genes ◽

Rrna Genes ◽

Dot Blot ◽

Cell Counts

ABSTRACT Rice straw is a major substrate for the production of methane, a greenhouse gas, in flooded rice fields. The bacterial community degrading rice straw under anoxic conditions was investigated with molecular methods. Rice straw was incubated in paddy soil anaerobically for 71 days. Denaturing gradient gel electrophoresis (DGGE) of the amplified bacterial 16S rRNA genes showed that the composition of the bacterial community changed during the first 15 days but then was stable until the end of incubation. Fifteen DGGE bands with different signal intensities were excised, cloned, and sequenced. In addition, DNA was extracted from straw incubated for 1 and 29 days and the bacterial 16S rRNA genes were amplified and cloned. From these clone libraries 16 clones with different electrophoretic mobilities on a DGGE gel were sequenced. From a total of 31 clones, 20 belonged to different phylogenetic clusters of the clostridia, i.e., clostridial clusters I (14 clones), III (1 clone), IV (1 clone), and XIVa (4 clones). One clone fell also within the clostridia but could not be affiliated to one of the clostridial clusters. Ten clones grouped closely with the genera Bacillus (3 clones), Nitrosospira (1 clone), Fluoribacter (1 clones), andAcidobacterium (2 clones) and with clone sequences previously obtained from rice field soil (3 clones). The relative abundances of various phylogenetic groups in the rice straw-colonizing community were determined by fluorescence in situ hybridization (FISH). Bacteria were detached from the incubated rice straw with an efficiency of about 80 to 90%, as determined by dot blot hybridization of 16S rRNA in extract and residue. The number of active (i.e., a sufficient number of ribosomes) Bacteria detected with a general eubacterial probe (Eub338) after 8 days of incubation was 61% of the total cell counts. This percentage decreased to 17% after 29 days of incubation. Most (55%) of the active cells on day 8 belonged to the genus Clostridium, mainly to clostridial clusters I (24%), III (6%), and XIVa (24%). An additional 5% belonged to theCytophaga-Flavobacterium cluster of theCytophaga-Flavobacterium-Bacteroides phylum, 4% belonged to the α, β, and γ Proteobacteria, and 1.3% belonged to the Bacillus subbranch of the gram-positive bacteria with a low G+C content. The results show that the bacterial community colonizing and decomposing rice straw developed during the first 15 days of incubation and was dominated by members of different clostridial clusters, especially clusters I, III, and XIVa.

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Differentiation Of Clarias Batrachus, C. Gariepinus And Heteropneustes Fossilis By Pcr-Sequencing Of Mitochondrial 16s Rrna Gene

Journal of the Asiatic Society of Bangladesh Science ◽

10.3329/jasbs.v41i1.46190 ◽

2015 ◽

Vol 41 (1) ◽

pp. 51-58

Author(s):

Mohammad Shamimul Alam ◽

Hawa Jahan ◽

Rowshan Ara Begum ◽

Reza M Shahjahan

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Fish Larva ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Valuable Insight ◽

Multiple Sequence ◽

Pcr Rflp ◽

Alternative Means

Heteropneustesfossilis, Clariasbatrachus and C. gariepinus are three major catfishes ofecological and economic importance. Identification of these fish species becomes aproblem when the usual external morphological features of the fish are lost or removed,such as in canned fish. Also, newly hatched fish larva is often difficult to identify. PCRsequencingprovides accurate alternative means of identification of individuals at specieslevel. So, 16S rRNA genes of three locally collected catfishes were sequenced after PCRamplification and compared with the same gene sequences available from othergeographical regions. Multiple sequence alignment of the 16S rRNA gene fragments ofthe catfish species has revealed polymorphic sites which can be used to differentiate thesethree species from one another and will provide valuable insight in choosing appropriaterestriction enzymes for PCR-RFLP based identification in future. Asiat. Soc. Bangladesh, Sci. 41(1): 51-58, June 2015

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On a Variational Definition for the Jensen-Shannon Symmetrization of Distances Based on the Information Radius

Entropy ◽

10.3390/e23040464 ◽

2021 ◽

Vol 23 (4) ◽

pp. 464

Author(s):

Frank Nielsen

Keyword(s):

Diversity Index ◽

Diversity Indices ◽

Shannon Diversity Index ◽

Probability Measures ◽

Variational Optimization ◽

Shannon Diversity ◽

Concept Of Information ◽

Jensen Shannon Divergence

We generalize the Jensen-Shannon divergence and the Jensen-Shannon diversity index by considering a variational definition with respect to a generic mean, thereby extending the notion of Sibson’s information radius. The variational definition applies to any arbitrary distance and yields a new way to define a Jensen-Shannon symmetrization of distances. When the variational optimization is further constrained to belong to prescribed families of probability measures, we get relative Jensen-Shannon divergences and their equivalent Jensen-Shannon symmetrizations of distances that generalize the concept of information projections. Finally, we touch upon applications of these variational Jensen-Shannon divergences and diversity indices to clustering and quantization tasks of probability measures, including statistical mixtures.

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Effects of Supplement of Marichromatium gracile YL28 on Water Quality and Microbial Structures in Shrimp Mariculture Ecosystems

Genes ◽

10.3390/genes12010040 ◽

2020 ◽

Vol 12 (1) ◽

pp. 40

Author(s):

Liang Cui ◽

Bitong Zhu ◽

Xiaobo Zhang ◽

Zhuhua Chan ◽

Chungui Zhao ◽

...

Keyword(s):

Water Quality ◽

16S Rrna ◽

Relative Abundance ◽

Animal Health ◽

Denitrifying Bacteria ◽

Nitrite Reduction ◽

16S Rrna Genes ◽

Rrna Genes ◽

Sequencing Analysis ◽

Shrimp Culture

The elevated NH3-N and NO2-N pollution problems in mariculture have raised concerns because they pose threats to animal health and coastal and offshore environments. Supplement of Marichromatium gracile YL28 (YL28) into polluted shrimp rearing water and sediment significantly decreased ammonia and nitrite concentrations, showing that YL28 functioned as a novel safe marine probiotic in the shrimp culture industry. The diversity of aquatic bacteria in the shrimp mariculture ecosystems was studied by sequencing the V4 region of 16S rRNA genes, with respect to additions of YL28 at the low and high concentrations. It was revealed by 16S rRNA sequencing analysis that Proteobacteria, Planctomycete and Bacteroidetes dominated the community (>80% of operational taxonomic units (OTUs)). Up to 41.6% of the predominant bacterial members were placed in the classes Gammaproteobacteria (14%), Deltaproteobacteria (14%), Planctomycetacia (8%) and Alphaproteobacteria (5.6%) while 40% of OTUs belonged to unclassified ones or others, indicating that the considerable bacterial populations were novel in our shrimp mariculture. Bacterial communities were similar between YL28 supplements and control groups (without addition of YL28) revealed by the β-diversity using PCoA, demonstrating that the additions of YL28 did not disturb the microbiota in shrimp mariculture ecosystems. Instead, the addition of YL28 increased the relative abundance of ammonia-oxidizing and denitrifying bacteria. The quantitative PCR analysis further showed that key genes including nifH and amoA involved in nitrification and nitrate or nitrite reduction significantly increased with YL28 supplementation (p < 0.05). The supplement of YL28 decreased the relative abundance of potential pathogen Vibrio. Together, our studies showed that supplement of YL28 improved the water quality by increasing the relative abundance of ammonia-oxidizing and denitrifying bacteria while the microbial community structure persisted in shrimp mariculture ecosystems.

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Ultra-accurate microbial amplicon sequencing with synthetic long reads

Microbiome ◽

10.1186/s40168-021-01072-3 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Benjamin J. Callahan ◽

Dmitry Grinevich ◽

Siddhartha Thakur ◽

Michael A. Balamotis ◽

Tuval Ben Yehezkel

Keyword(s):

Microbial Community ◽

16S Rrna ◽

Amplicon Sequencing ◽

Species Level ◽

Full Length ◽

16S Rrna Genes ◽

Rrna Genes ◽

Strain Identification ◽

Long Reads ◽

Long Read

Abstract Background Out of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge. Methods Here, we describe and analytically validate LoopSeq, a commercially available synthetic long-read (SLR) sequencing technology that generates highly accurate long reads from standard short reads. Results LoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq perfectly recovered the full diversity of 16S rRNA genes from known strains in a synthetic microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kb in length. LoopSeq full-length 16S rRNA reads could accurately classify organisms down to the species level in rinsate from retail meat samples, and could differentiate strains within species identified by the CDC as potential foodborne pathogens. Conclusions The order-of-magnitude improvement in length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex- to low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics.

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Microdiversity and phylogeographic diversification of bacterioplankton in pelagic freshwater systems revealed through long-read amplicon sequencing

Microbiome ◽

10.1186/s40168-020-00974-y ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Yusuke Okazaki ◽

Shohei Fujinaga ◽

Michaela M. Salcher ◽

Cristiana Callieri ◽

Atsushi Tanaka ◽

...

Keyword(s):

16S Rrna ◽

Regional Scale ◽

Scale Up ◽

Amplicon Sequencing ◽

Freshwater Ecosystems ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Metagenomic Sequencing ◽

Long Read

Abstract Background Freshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on > 97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the eco-evolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe. Results Our single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7–101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages (e.g., Iluma-A2 and acI-A1) achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages (e.g., CL500-11) may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnion-specific lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion. Conclusions Our novel approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future.

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Microbiological and Geochemical Heterogeneity in an In Situ Uranium Bioremediation Field Site

Applied and Environmental Microbiology ◽

10.1128/aem.71.10.6308-6318.2005 ◽

2005 ◽

Vol 71 (10) ◽

pp. 6308-6318 ◽

Cited By ~ 172

Author(s):

Helen A. Vrionis ◽

Robert T. Anderson ◽

Irene Ortiz-Bernad ◽

Kathleen R. O'Neill ◽

Charles T. Resch ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

16S Rrna Genes ◽

Rrna Genes ◽

Acid Volatile Sulfide ◽

Rrna Gene ◽

Gene Sequences ◽

16S Rrna Gene Sequences ◽

Geochemical Heterogeneity

ABSTRACT The geochemistry and microbiology of a uranium-contaminated subsurface environment that had undergone two seasons of acetate addition to stimulate microbial U(VI) reduction was examined. There were distinct horizontal and vertical geochemical gradients that could be attributed in large part to the manner in which acetate was distributed in the aquifer, with more reduction of Fe(III) and sulfate occurring at greater depths and closer to the point of acetate injection. Clone libraries of 16S rRNA genes derived from sediments and groundwater indicated an enrichment of sulfate-reducing bacteria in the order Desulfobacterales in sediment and groundwater samples. These samples were collected nearest the injection gallery where microbially reducible Fe(III) oxides were highly depleted, groundwater sulfate concentrations were low, and increases in acid volatile sulfide were observed in the sediment. Further down-gradient, metal-reducing conditions were present as indicated by intermediate Fe(II)/Fe(total) ratios, lower acid volatile sulfide values, and increased abundance of 16S rRNA gene sequences belonging to the dissimilatory Fe(III)- and U(VI)-reducing family Geobacteraceae. Maximal Fe(III) and U(VI) reduction correlated with maximal recovery of Geobacteraceae 16S rRNA gene sequences in both groundwater and sediment; however, the sites at which these maxima occurred were spatially separated within the aquifer. The substantial microbial and geochemical heterogeneity at this site demonstrates that attempts should be made to deliver acetate in a more uniform manner and that closely spaced sampling intervals, horizontally and vertically, in both sediment and groundwater are necessary in order to obtain a more in-depth understanding of microbial processes and the relative contribution of attached and planktonic populations to in situ uranium bioremediation.

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