Microbes In Natural Environments Research Articles

Permeability decides the effect of antibiotics on sedimentary nitrogen removal in Jiulong River Estuary.

Sedimentary denitrification takes place beneath the oxic layer at the sediment-water interface, where nitrate and antibiotics need to diffuse through the overlying water. However, the antibiotics' effect on sedimentary N removal and associated N2O production has not been adequately investigated under in situ conditions. Here, isotope pairing techniques, including slurry incubations (potential) and intact core incubations (in situ), combined with metagenomic analysis were applied to investigate the impacts of two protein-inhibiting antibiotics (oxytetracycline and thiamphenicol) on sediment nitrogen removal in a subtropical estuary. Slurry incubations showed that the two antibiotics significantly inhibited denitrification (67-98%) and anammox (49-99%), while intact core incubations presented no antibiotic effect at upstream but significant inhibition (23%-52%) at downstream. Meanwhile, N2O yields were stimulated up to 20 folds in slurry incubations yet showing insignificant response in intact cores. Such contrasting results between up- and down-stream and between slurry and intact core incubations strongly indicated that permeability, which determines diffusion of antibiotics to microbes, is the key to exert the effect of antibiotics on in situ sedimentary nitrogen removal processes regardless the existence of antibiotics resistance genes. This diffusive obstruction may mitigate the toxic effect of antibiotics on nitrogen removal related microbes in natural environments.

Effects of Different Nanoparticles on Microbes.

Nanoparticles widely exist in nature and may be formed through inorganic or organic pathways, exhibiting unique physical and chemical properties different from those of bulk materials. However, little is known about the potential consequences of nanomaterials on microbes in natural environments. Herein, we investigated the interactions between microbes and nanoparticles by performing experiments on the inhibition effects of gold, ludox and laponite nanoparticles on Escherichia coli in liquid Luria-Bertani (LB) medium at different nanoparticle concentrations. These nanoparticles were shown to be effective bactericides. Scanning electron microscopy (SEM) images revealed the distinct aggregation of cells and nanoparticles. Transmission electron microscopy (TEM) images showed considerable cell membrane disruption due to nanoparticle accumulation on the cell surfaces, resulting in cell death. We hypothesized that this nanoparticle accumulation on the cell surfaces not only disrupted the cell membranes but also physically blocked the microbes from accessing nutrients. An iron-reducing bacterium, Shewanella putrefaciens, was tested for its ability to reduce the Fe (III) in solid ferrihydrite (HFO) or aqueous ferric citrate in the presence of laponite nanoparticles. It was found that the laponite nanoparticles inhibited the reduction of the Fe (III) in solid ferrihydrite. Moreover, direct contact between the cells and solid Fe (III) coated with the laponite nanoparticles was physically blocked, as confirmed by SEM images and particle size measurements. However, the laponite particles had an insignificant effect on the extent of aqueous Fe (III) bioreduction but slightly enhanced the rate of bioreduction of the Fe (III) in aqueous ferric citrate. The slightly increased rate of bioreduction by laponite nanoparticles may be due to the removal of inhibitory Fe (II) from the cell surface by its sorption onto the laponite nanoparticle surface. This result indicates that the scavenging of toxic heavy metals, such as Fe (II), by nanoparticles may be beneficial for microbes in the environment. On the other hand, microbial cells are also capable of detoxifying nanoparticles by coagulating nanoparticles with extracellular polymeric substances or by changing nanoparticle morphologies. Hence, the interactions between microbes and nanoparticles in natural environments should receive more attention.

Aspergillus Goes Viral: Ecological Insights from the Geographical Distribution of the Mycovirome within an Aspergillus flavus Population and Its Possible Correlation with Aflatoxin Biosynthesis.

Microbial multi-level interactions are essential to control the success of spreading and survival of most microbes in natural environments. Phytopathogenic mycotoxigenic fungal species, such as Aspergillus flavus, represent an important issue in food safety. Usually, non-toxigenic strains are exploited for biocontrol strategies to mitigate infections by toxigenic strains. To comprehend all the biological variables involved in the aflatoxin biosynthesis, and to possibly evaluate the interplay between A. flavus toxigenic and non-toxigenic strains during intraspecific biocompetition, the “virological” perspective should be considered. For these reasons, investigations on mycoviruses associated to A. flavus populations inhabiting specific agroecosystems are highly desirable. Here, we provide the first accurate characterization of the novel mycovirome identified within an A. flavus wild population colonizing the maize fields of northern Italy: a selection of A. flavus strains was biologically characterized and subjected to RNAseq analysis, revealing new mycoviruses and a peculiar geographic pattern distribution in addition to a 20% rate of infection. More interestingly, a negative correlation between viral infection and aflatoxin production was found. Results significantly expanded the limited existent data about mycoviruses in wild A. flavus, opening new and intriguing hypotheses about the ecological significance of mycoviruses.

A Simple Culture Method Enhances the Recovery of Culturable Actinobacteria From Coastal Sediments.

Molecular methods revealed that the majority of microbes in natural environments remains uncultivated. To fully understand the physiological and metabolic characteristics of microbes, however, culturing is still critical for microbial studies. Here, we used bacterial community analysis and four culture media, namely, traditional marine broth 2216 (MB), water extracted matter (WEM), methanol extracted matter (MEM), and starch casein agar (SCA), to investigate the diversity of cultivated bacteria in coastal sediments. A total of 1,036 isolates were obtained in pure culture, and they were classified into five groups, namely, Alphaproteobacteria (52.51%), Gammaproteobacteria (23.26%), Actinobacteria (13.32%), Firmicutes, and Bacteroidetes. Compared to other three media, WEM recovered a high diversity of actinobacteria (42 of 63 genotypes), with Micromonospora and Streptomyces as the most cultivated genera. Amplicon sequencing of the bacterial 16S ribosomal RNA (rRNA) gene V3–V4 fragment revealed eight dominant groups, Alphaproteobacteria (12.81%), Gammaproteobacteria (20.07%), Deltaproteobacteria (12.95%), Chloroflexi (13.09%), Bacteroidetes (8.28%), Actinobacteria (7.34%), Cyanobacteria (6.20%), and Acidobacteria (5.71%). The dominant members affiliated to Actinobacteria belonged to “Candidatus Actinomarinales,” “Candidatus Microtrichales,” and Nitriliruptorales. The cultivated actinobacteria accounted for a small proportion (<5%) compared to the actinobacterial community, which supported that the majority of actinobacteria are still waiting for cultivation. Our study concluded that WEM could be a useful and simple culture medium that enhanced the recovery of culturable actinobacteria from coastal sediments.

High-throughput sequencing clarifies the spatial structures of microbial communities in cadmium-polluted rice soils

Soil microbial communities are affected by environmental factors. Contamination with heavy metals such as cadmium (Cd) can decrease soil microbial species richness and substantially alter soil microbial species composition. Investigations of the microbial communities in Cd-contaminated soils are necessary to obtain data for soil bioremediation efforts. However, depth-associated variations in microbial community composition and structure in Cd-contaminated paddy soils are not well understood. Here, the effects of various degrees of long-term Cd pollution on soil microorganisms were investigated at different soil depths within the plough layer using 16S rRNA gene amplicon sequencing. We found that, in Cd-polluted soils, microbial communities were more similar between the surface soil and the underlying soil. In addition, microbial community richness and/or diversity were significantly reduced in the Cd-polluted underlying soil as compared with the non-polluted underlying soil. However, species richness in the surface layer was significantly greater in the mildly and severely Cd-polluted soils. The soil microbial communities in the same soil layer differed significantly between the non-polluted and polluted soils. Furthermore, Cd contamination affected the microbial communities of different soil layers differently. Soil pH had a synergistic effect on microbial community abundance and composition. The potential functions of the soil microbiota were mainly related to environmental processing, genetic processing, and metabolic pathways. Notably, our identification of the phyla that were differently abundant among sites with different levels of Cd pollution will provide experimental guidance for further explorations of the effects of Cd on soil microbes in natural environments. Our results not only demonstrate that long-term Cd pollution leads to a marked reduction in microbial richness and diversity in the underlying soil layer, but they also help to clarify how long-term heavy metal contamination affects the soil bacterial community.

Cultivation-independent and cultivation-dependent metagenomes reveal genetic and enzymatic potential of microbial community involved in the degradation of a complex microbial polymer

BackgroundCultivation-independent methods, including metagenomics, are tools for the exploration and discovery of biotechnological compounds produced by microbes in natural environments. Glycoside hydrolases (GHs) enzymes are extremely desired and important in the industry of production for goods and biofuel and removal of problematic biofilms and exopolysaccharide (EPS). Biofilms and EPS are complex, requiring a wide range of enzymes for a complete degradation. The aim of this study was to identify potential GH microbial producers and GH genes with biotechnological potential, using EPS-complex structure (WH15EPS) of Acidobacteria Granulicella sp. strain WH15 as an enrichment factor, in cultivation-independent and cultivation-dependent methods. We performed stable isotope probing (SIP) combined with metagenomics on topsoil litter amended with WH15EPS and coupled solid culture-EPS amended medium with metagenomics.ResultsSIP metagenome analysis of the soil litter demonstrated that phyla Proteobacteria, Actinobacteria, Acidobacteria, and Planctomycetes were the most abundant in WH15EPS amended and unamended treatments. The enrichment cultures in solid culture medium coupled to metagenomics demonstrated an enrichment in Proteobacteria, and the metagenome assembly of this enrichment cultures resulted in 4 metagenome-assembled genomes (MAGs) of microbes with low identity (42–86%) to known microorganisms. Among all carbohydrate-active enzymes (CAZymes) retrieved genes, glycoside transferase (GT) was the most abundant family, either in culture-independent or culture-based metagenome datasets. Within the glycoside hydrolases (GHs), GH13 was the most abundant family in both metagenome datasets. In the “heavy” fraction of the culture-independent metagenome SIP dataset, GH109 (α-N-acetylgalactosaminidases), GH117 (agarases), GH50 (agarases), GH32 (invertases and inulinases), GH17 (endoglucanases), and GH71 (mutanases) families were more abundant in comparison with the controls. Those GH families are affiliated to microorganism that are probably capable to degrade WH15EPS and potentially applicable for biofilm deconstruction. Subsequent in culture-based metagenome, the assembled 4 MAGs (unclassified Proteobacteria) also contained GH families of interest, involving mannosidases, lysozymes, galactosidases, and chitinases.ConclusionsWe demonstrated that functional diversity induced by the presence of WH15EPS in both culture-independent and culture-dependent approaches was enriched in GHs, such as amylases and endoglucanases that could be applied in chemical, pharmaceutical, and food industrial sectors. Furthermore, WH15EPS may be used for the investigation and isolation of yet unknown taxa, such as unclassified Proteobacteria and Planctomycetes, increasing the number of current cultured bacterial representatives with potential biotechnological traits.CTLt_7d-1ZnF5WvCrBnFSjVideo

Enumeration of Chemoorganotrophic Carbonyl Sulfide (COS)-degrading Microorganisms by the Most Probable Number Method.

Carbonyl sulfide (COS) is the most abundant sulfur compound in the atmosphere, and, thus, is important in the global sulfur cycle. Soil is a major sink of atmospheric COS and the numerical distribution of soil microorganisms that degrade COS is indispensable for estimating the COS-degrading potential of soil. However, difficulties are associated with counting COS-degrading microorganisms using culture-dependent approaches, such as the most probable number (MPN) method, because of the chemical hydrolysis of COS by water. We herein developed a two-step MPN method for COS-degrading microorganisms: the first step for chemoorganotrophic growth that supported a sufficient number of cells for COS degradation in the second step. Our new MPN analysis of various environmental samples revealed that the cell density of COS-degrading microorganisms in forest soils ranged between 106 and 108 MPN (g dry soil)–1, which was markedly higher than those in volcanic deposit and water samples, and strongly correlated with the rate of COS degradation in environmental samples. Numerically dominant COS degraders that were isolated from the MPN-positive culture were related to bacteria in the orders Bacillales and Actinomycetales. The present results provide numerical evidence for the ubiquity of COS-degrading microbes in natural environments.

Effect of motility on the transport of bacteria populations through a porous medium

The role of activity on the hydrodynamic dispersion of bacteria in a model porous medium is studied by tracking thousands of bacteria in a microfluidic chip containing randomly placed pillars. We first evaluate the spreading dynamics of two populations of motile and non-motile bacteria injected at different flow rates. In both cases, we observe that the mean and the variance of the distances covered by the bacteria vary linearly with time and flow velocity, a result qualitatively consistent with the standard geometric dispersion picture. However, quantitatively, the motiles bacteria display a systematic retardation effect when compared to the non-motile ones. Furthermore, the shape of the traveled distance distribution in the flow direction differs significantly for both the motile and the non-motile strain, hence probing a markedly different exploration process. For the non-motile bacteria, the distribution is Gaussian whereas for the motile ones, the distribution displays a positive skewness and spreads exponentially downstream akin to a Gamma distribution. The detailed microscopic study of the trajectories reveals two salient effects characterizing the exploration process of motile bacteria : (i) The emergence of an "active" retention effect due to an extended exploration of the pore surfaces, (ii) an enhanced spreading at the forefront due to the transport of bacteria along "fast-tracks" where they acquire a velocity larger than the local flow velocity. We finally discuss the practical applications of these effects on the large-scale macroscopic transfer and contamination processes caused by microbes in natural environments.

A Synthetic Ecology Perspective: How Well Does Behavior of Model Organisms in the Laboratory Predict Microbial Activities in Natural Habitats?

In this perspective article, we question how well model organisms, the ones that are easy to cultivate in the laboratory and that show robust growth and biomass accumulation, reflect the dynamics and interactions of microbial communities observed in nature. Today’s -omics toolbox allows assessing the genomic potential of microbes in natural environments in a high-throughput fashion and at a strain-level resolution. However, understanding of the details of microbial activities and of the mechanistic bases of community function still requires experimental validation in simplified and fully controlled systems such as synthetic communities. We have studied methane utilization in Lake Washington sediment for a few decades and have identified a number of species genetically equipped for this activity. We have also identified co-occurring satellite species that appear to form functional communities together with the methanotrophs. Here, we compare experimental findings from manipulation of natural communities involved in metabolism of methane in this niche with findings from manipulation of synthetic communities assembled in the laboratory of species originating from the same study site, from very simple (two-species) to rather complex (50-species) synthetic communities. We observe some common trends in community dynamics between the two types of communities, toward representation of specific functional guilds. However, we also identify strong discrepancies between the dominant methane oxidizers in synthetic communities compared to natural communities, under similar incubation conditions. These findings highlight the challenges that exist in using the synthetic community approach to modeling dynamics and species interactions in natural communities.

Survivability of Microbes in Natural Environments and Their Ecological Impacts.

Microbiology often focuses on the growth properties or physiological capabilities of microbes. However, most microbes in the natural environment are not actively growing. As has been proposed for plants, microbial life strategies should consider stress tolerance as well as competitive relationships and physiological and biological disturbance (19). Reports studying microbial communities in oligotrophic or nutrient-limited environments have increased recently, e.g. (10), (21), and (41). How do microbes live? Do they mostly simply do nothing? Recent reports on microbial survivability, introduced in this Research Highlight, are providing informative answers for these questions, and discovering new aspects of microbial ecosystems.

MaxBin: an automated binning method to recover individual genomes from metagenomes using an expectation-maximization algorithm.

BackgroundRecovering individual genomes from metagenomic datasets allows access to uncultivated microbial populations that may have important roles in natural and engineered ecosystems. Understanding the roles of these uncultivated populations has broad application in ecology, evolution, biotechnology and medicine. Accurate binning of assembled metagenomic sequences is an essential step in recovering the genomes and understanding microbial functions.ResultsWe have developed a binning algorithm, MaxBin, which automates the binning of assembled metagenomic scaffolds using an expectation-maximization algorithm after the assembly of metagenomic sequencing reads. Binning of simulated metagenomic datasets demonstrated that MaxBin had high levels of accuracy in binning microbial genomes. MaxBin was used to recover genomes from metagenomic data obtained through the Human Microbiome Project, which demonstrated its ability to recover genomes from real metagenomic datasets with variable sequencing coverages. Application of MaxBin to metagenomes obtained from microbial consortia adapted to grow on cellulose allowed genomic analysis of new, uncultivated, cellulolytic bacterial populations, including an abundant myxobacterial population distantly related to Sorangium cellulosum that possessed a much smaller genome (5 MB versus 13 to 14 MB) but has a more extensive set of genes for biomass deconstruction. For the cellulolytic consortia, the MaxBin results were compared to binning using emergent self-organizing maps (ESOMs) and differential coverage binning, demonstrating that it performed comparably to these methods but had distinct advantages in automation, resolution of related genomes and sensitivity.ConclusionsThe automatic binning software that we developed successfully classifies assembled sequences in metagenomic datasets into recovered individual genomes. The isolation of dozens of species in cellulolytic microbial consortia, including a novel species of myxobacteria that has the smallest genome among all sequenced aerobic myxobacteria, was easily achieved using the binning software. This work demonstrates that the processes required for recovering genomes from assembled metagenomic datasets can be readily automated, an important advance in understanding the metabolic potential of microbes in natural environments. MaxBin is available at https://sourceforge.net/projects/maxbin/.

Microbial Biofabrication for Nanomedicine: Biomaterials, Nanoparticles and Beyond

Author for correspondence: Institut de Biotecnologia i de Biomedicina, Universitat Autonoma de Barcelona, Bellaterra, 08193 Barcelona, Spain and Department de Genetica i de Microbiologia, Universitat Autonoma de Barcelona, Bellaterra, 08193 Barcelona, Spain and CIBER en Bioingenieria, Biomateriales y Nanomedicina, Bellaterra, 08193 Barcelona, Spain antoni.villaverde@uab.cat Microbial biofabrication for nanomedicine: biomaterials, nanoparticles and beyond

Biodegradation of glycerol using bacterial isolates from soil under aerobic conditions

Glycerol, a non-biodegradable by-product during biodiesel production is a major concern to the emerging biodiesel industry. Many microbes in natural environments have the ability to utilize glycerol as a sole carbon and energy source. The focus of this study was to screen for microorganisms from soil, capable of glycerol utilization and its conversion to value added products such as ethanol and 1,3-propanediol (1,3-PDO). Twelve bacterial isolates were screened for glycerol utilization ability in shake flask fermentations using M9 media supplemented with analytical grade glycerol (30 g/L) at various pH values (6, 7 and 8) and temperatures (30°C, 35°C and 40°C). Among these, six bacterial isolates (SM1, SM3, SM4, SM5, SM7 and SM8) with high glycerol degradation efficiency (>80%) were selected for further analysis. Highest level of 1,3-PDO production (15 g/L) was observed with isolate SM7 at pH 7 and 30°C, while superior ethanol production (14 g/L) was achieved by isolate SM9 at pH 8 and 35°C, at a glycerol concentration of 30 g/L. The selected strains were further evaluated for their bioconversion efficiency at elevated glycerol concentrations (50–110 g/L). Maximum 1,3-PDO production (46 g/L and 35 g/L) was achieved at a glycerol concentration of 70 g/L by isolates SM4 and SM7 respectively, with high glycerol degradation efficiency (>90). Three isolates (SM4, SM5 and SM7) also showed greater glycerol tolerance (up to 110 g/L). The isolates SM4 and SM7 were identified as Klebsiella pneumoniae and SM5 as Enterobacter aerogenes by 16S rDNA analysis. These novel isolates with greater glycerol tolerance could be used for the biodegradation of glycerol waste generated from the biodiesel industry into value-added commercial products.

Integrated proteomic and transcriptomic analysis reveals novel genes and regulatory mechanisms involved in salt stress responses in Synechocystis sp. PCC 6803

Salt stress is a common stress that limits growth and productivity of photosynthetic microbes in natural environments. Although cellular responses of a model cyanobacterium Synechocystis sp. PCC6803 to high and changing salt concentration have been studied, it remains undefined of the gene components and their regulation in the long-term salt acclimation networks. In this study, we performed an integrated study coupling a quantitative iTRAQ-LC-MS/MS proteomics and a next-generation sequencing-based RNA-seq transcriptomics on Synechocystis under salt stress for an extended period of time. Comparative quantification of protein abundances led to the identification of 68 and 108 proteins differentially regulated by salt treatment at 24 and 48h, respectively. RNA-seq transcriptomic analysis showed that genes involved in energy metabolism and protein synthesis, and genes encoding hypothetical proteins responded to salt stress in a phase-dependent pattern. Notably, a gene encoding CO2-uptake-related protein (CupA) and three genes encoding hypothetical proteins were induced significantly at either transcript or protein level after long-term salt stress. Gene knockout and comparative growth analysis demonstrated that these four genes were involved in salt tolerance in Synechocystis. In addition, a complementary proteome and transcriptome analysis showed that concordance between protein abundances and their corresponding mRNAs varied significantly between various gene-protein pairs, indicating divergent regulation of transcriptional and post-transcriptional processes during salt stress adaptation in Synechocystis. The study provided new insights on genes and regulatory mechanism involved in salt stress response in Synechocystis.

Editorial: ‘Disregarded’ microbial diversity and ecological potentials in aquatic systems: a new paradigm shift ahead

The introduction of molecular methods to the study of prokaryotes in the mid-1980s (Pace et al., 1986), and their expansion to small eukaryotes in the early 2000s (Lopez-Garcia et al., 2001; Moon-van der Staay et al., 2001) have allowed investigation of uncultured microbes in natural environments, and have greatly increased our knowledge of microbial biodiversity. The resulting major methodological, conceptual and empirical advances in aquatic microbial ecology are arguably the greatest advances in environmental sciences. Recent developments in sequencing technology and the use of SSU rRNA hypervariable tag sequencing are beginning to reveal the huge dimension of the microbial diversity hidden in natural aquatic ecosystems

Raman tweezers sorting of single microbial cells

We have selectively isolated microbial cells by identifying and then manipulating cells using a combination of Raman microspectroscopy and optical trapping. The criterion for cell discrimination is based on spectral peak shifts within the Raman spectrum of individual cells. A specific shift in the phenylalanine peak position from 1001 rel. cm(-1) to 965 rel. cm(-1) is utilized to indicate the uptake of (13) C within the cell that utilized (13) C-substrate. Cells were captured and manipulated using an infrared (1064 nm) laser while Raman spectra were acquired over shorter timescales (30 s) using a co-aligned 514.5 nm laser beam. Selected cells were manoeuvred to a clean part of a capillary tube and the tubes were cleaved to physically separate the cells. The technique was tested for cell viability and cross-contamination effects using 70 single yeast cells (Saccharomyces cerevisia). Following these tests, 58 single bacterial cells (Escherichia coli DH5α, and Pseudomonas fluorescens SBW25::Km-RFP) that exhibited (13) C uptake were sorted from bacterial populations. Among those isolated cells, 11 out of 18 yeast cells and 7 out of 18 single SBW25::Km-RFP cells were recovered by incubation; 2 out of 7 sorted yeast cells and 3 out of 8 sorted bacterial cells (single SBW25::Km-RFP) were genome amplified correctly. We show that the Raman tweezers approach has the potential to open a new frontier to study unculturable microorganisms, which account for more than 99% microbes in natural environment.

Amino acid variation in the GyrA subunit of bacteria potentially associated with natural resistance to fluoroquinolone antibiotics.

In studies of genetic diversity in natural microbial populations, we have analyzed nucleotide sequences in the quinolone resistance-determining region of the bacterial gyrA gene in ciprofloxacin-resistant and nonselected soil bacteria obtained from the environment. It is apparent that this sequence is highly variable, and resistance to fluoroquinolone antibiotics occurring in environmental populations of bacteria is due at least in part to natural sequence variation in this domain. We suggest that the development of new antimicrobial agents, including completely synthetic antimicrobials such as the fluoroquinolones, should incorporate the analysis of resistance mechanisms among microbes in natural environments; these studies could predict potential mechanisms of resistance to be encountered in subsequent clinical use of the agents and would guide chemical modification designed to evade resistance development.