Field Of Environmental Microbiology Research Articles

FISH-TAMB, a Fixation-Free mRNA Fluorescent Labeling Technique to Target Transcriptionally Active Members in Microbial Communities

Keystone species or ecological engineers are vital to the health of an ecosystem; however, often, their low abundance or biomass present challenges for their discovery, identification, visualization and selection. We report the development of fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB), a fixation-free protocol that is applicable to archaea and bacteria. The FISH-TAMB method differs from existing FISH methods by the absence of fixatives or surfactants in buffers, the fast hybridization time of as short as 15 min at target cells’ growth temperature, and the omission of washing steps. Polyarginine cell-penetrating peptides are employed to deliver molecular beacons (MBs) across prokaryotic cell walls and membranes, fluorescently labeling cells when MBs hybridize to target mRNA sequences. Here, the detailed protocol of the preparation and application of FISH-TAMB is presented. To demonstrate FISH-TAMB’s ability to label intracellular mRNA targets, differentiate transcriptional states, detect active and rare taxa, and keep cell viability, labeling experiments were performed that targeted the messenger RNA (mRNA) of methyl-coenzyme M reductase A (mcrA) expressed in (1) Escherichia coli containing a plasmid with a partial mcrA gene of the methanogen Methanosarcina barkeri (E. coli mcrA+); (2) M. barkeri; and (3) an anaerobic methanotrophic (ANME) enrichment from a deep continental borehole. Although FISH-TAMB was initially envisioned for mRNA of any functional gene of interest without a requirement of prior knowledge of 16S ribosomal RNA (rRNA)-based taxonomy, FISH-TAMB has the potential for multiplexing and going beyond mRNA and thus is a versatile addition to the molecular ecologist’s toolkit, with potentially widespread application in the field of environmental microbiology.

MPN Drop Agar Method for Determination of Heterotrophic Microorganisms in Soil and Water Samples Using Tissue Plate as a Carrier

The most probable number (MPN) method is a culture-based method commonly used in the field of environmental microbiology to examine microbial populations in liquid substrates. The MPN tests require a wide range of special laboratory equipment, a lot of laboratory space, and skilled staff, which together limit their applicability. This paper presents a modified MPN method, which reduces the experimental requirements by applying tissue plate as a carrier. The modified MPN method introduces a fast-filled tissue plate with 5 × 5 squares as agar carrier, instead of the commonly used set of glass tubes. Further, self-refilling automatic syringe Socorex® was implemented to apply samples to the plate. The response of the modified MPN method was tested on eight selected bacterial strains as well as on soil and water samples. Simultaneously, all the strains and samples were tested by standard spread plate method. High linear correlation between the two methods was found, which makes a new modified MPN method a useful alternative within the field of environmental microbiology.

Discovery of an Inducible Toluene Monooxygenase That Cooxidizes 1,4-Dioxane and 1,1-Dichloroethylene in Propanotrophic Azoarcus sp. Strain DD4.

Cometabolic degradation plays a prominent role in bioremediation of commingled groundwater contamination (e.g., chlorinated solvents and the solvent stabilizer 1,4-dioxane [dioxane]). In this study, we untangled the diversity and catalytic functions of multicomponent monooxygenases in Azoarcus sp. strain DD4, a Gram-negative propanotroph that is effective in degrading dioxane and 1,1-dichloroethylene (1,1-DCE). Using a combination of knockout mutagenesis and heterologous expression, a toluene monooxygenase (MO) encoded by the tmoABCDEF gene cluster was unequivocally proved to be the key enzyme responsible for the cometabolism of both dioxane and 1,1-DCE. Interestingly, in addition to utilizing toluene as a primary substrate, this toluene MO can also oxidize propane into 1-propanol. Expression of this toluene MO in DD4 appears inducible by both substrates (toluene and propane) and their primary hydroxylation products (m-cresol, p-cresol, and 1-propanol). These findings coherently explain why DD4 can grow on propane and express toluene MO for active cooxidation of dioxane and 1,1-DCE. Furthermore, upregulation of tmo transcription by 1-propanol underlines the implication potential of using 1-propanol as an alternative auxiliary substrate for DD4 bioaugmentation. The discovery of this toluene MO in DD4 and its degradation and induction versatility can lead to broad applications, spanning from environmental remediation and water treatment to biocatalysis in green chemistry.IMPORTANCE Toluene MOs have been well recognized given their robust abilities to degrade a variety of environmental pollutants. Built upon previous research efforts, this study ascertained the untapped capability of a toluene MO in DD4 for effective cooxidation of dioxane and 1,1-DCE, two of the most prevailing yet challenging groundwater contaminants. This report also aligns the induction of a toluene MO with nontoxic and commercially accessible chemicals (e.g., propane and 1-propanol), extending its implications in the field of environmental microbiology and beyond.

On the Application of Statistical Analysis for Interpretation of Experimental Results in Environmental Microbiology

Statistical analysis is an integral part of every experiment, since it helps researchers to make conclusions out of the work done. Textbooks with detailed description of the algorithms for statistical analysis exist, as well as diverse software packages. Statistical methods are, however, often applied incorrectly, which leads to erroneous and inadequate conclusions. The present article is an attempt to determine the main problems emerging in the course of statistical analysis of experimental results in the field of environmental microbiology. For instance, the classical parametric tests, which are most often used in experimental articles (t-test, analysis of variance, Pearson correlation coefficient, etc.), are applicable only when a random variable of n independent observations has normal distribution. Importantly, the normality of the distribution must be proved, and it is possible to do only for minimal sample size of 20‒30 independent observations per group. The latter is crucial for the incubation, isotope, and molecular biological experiments. The family of normal distributions is not the only family of parameter-dependent (parametric) distributions of random variables. Moreover, real-life distributions usually differ from the normal ones, and normal distribution may be considered only as a certain approximation. High diversity of parametric families complicates the choice of criteria and statistical tests for analysis of a set of data collected from n independent observations. Nonparametric statistics may be of help, since it is free of sample size and distribution requirements, although, as any method of analysis, it also has limitations. An increasing number of experimental data, including those in the field of environmental microbiology, are nowadays analyzed by using nonparametric statistics, which indicates a certain tendency for substitution of parametric methods by nonparametric ones.

Environmental microbiology

Abstract For most people, microorganisms are out of sight and therefore out of mind but they are large, extremely diverse group of organisms, they are everywhere and are the dominant form of life on planet Earth. Almost every surface is colonized by microorganisms, including our skin; however most of them are harmless to humans. Some microorganisms can live in boiling hot springs, whereas others form microbial communities in frozen sea ice. Among their many roles, microorganisms are necessary for biogeochemical cycling, soil fertility, decomposition of dead plants and animals and biodegradation of many complex organic compounds present in the environment. Environmental microbiology is concerned with the study of microorganisms in the soil, water and air and their application in bioremediation to reduce environmental pollution through the biological degradation of pollutants into non-toxic or less toxic substances. Field of environmental microbiology also covers the topics such as microbially induced biocorrosion, biodeterioration of constructing materials and microbiological quality of outdoor and indoor air.

Enhanced bacterial affinity of PVDF membrane: its application as improved sea water sampling tool for environmental monitoring.

Isolation of diversified bacteria from seawater is a major challenge in the field of environmental microbiology. In the present study, an attempt has been made to select specific membrane with improved property of attaching diversified bacteria. Initially, different concentrations (15, 18, and 20% W/W) of polysulfone (PSF) were used to check their affinity for the attachment of selected gram-positive (Bacillus subtilis) and gram-negative (Escherichia coli) bacteria. Among these, 20% W/W PSF showed maximum attachment. Therefore, membrane prepared with other materials such as polyvinylidene fluoride (PVDF) and polyether sulfone (PES) were used with the same concentration (20% W/W) to check their improved bacterial attachment property. Comparative study of bacterial attachment on three different membranes revealed that PVDF possessed the highest affinity towards both the groups of bacteria. This property was confirmed by different analytical methods viz. contact angle, atomic force microscopy, zeta potential, and flux study and further validated with seawater samples collected from seven sites of western coast and Lakshadweep island of India, using Biolog EcoPlate™. All the samples showed that bacterial richness and diversity was high in PVDF membrane in comparison to surrounding seawater samples. Interestingly, affinity for more diversified bacteria was reported to be higher in water sample with less turbidity and low bacteria load. This finding can facilitate the development of PVDF (20% W/W) membrane as a simple, cheap, and less labor intensive environmental sampling tool for the isolation of diversified bacteria from seawater sample wih different physiochemical properties. Graphical abstract ᅟ.

Ring-Hydroxylating Oxygenase database: a database of bacterial aromatic ring-hydroxylating oxygenases in the management of bioremediation and biocatalysis of aromatic compounds.

Bacterial Rieske-type aromatic ring-hydroxylating oxygenases (RHOs) constitute a large family of enzymes, primarily involved in bioremediation of diverse aromatic compounds in the environment. In the present study, we have designed a manually curated database, Ring-Hydroxylating Oxygenase database (RHObase), which provides comprehensive information on all biochemically characterized bacterial RHOs. It consists of ∼ 1000 entries including 196 oxygenase α-subunits, 153 oxygenase β-subunits, 92 ferredoxins and 110 reductases, distributed among 131 different bacterial strains implementing a total of 318 oxygenation reactions. For each protein, users can get detailed information about its structure and conserved domain(s) with motif signature. RHObase allows users to search a query, based on organism, oxygenase, substrate, or protein structure. In addition, this resource provides analysis tools to perform blast search against RHObase for prediction of putative substrate(s) for the query oxygenase and its phylogenetic affiliation. Furthermore, there is an integrated cheminformatics tool to search for structurally similar compound(s) in the database vis-a-vis RHO(s) capable of transforming those compound(s). Resources in the RHObase and multiple search/display options therein are intended to provide oxygenase-related requisite information to researchers, especially working in the field of environmental microbiology and biocatalysis to attain difficult chemistry of biotechnological importance.

Integrating “omics” Technologies to Conceptualize Dynamic Antimicrobial Peptide Responses

Rarely in human disease can a single host factor be identified as the primary causal factor of a disease, syndrome, or disorder. Rather, the clinical manifestations that culminate in a symptomatic state are usually triggered by a perturbation of multiple factors derived from several distinct, but integrated, domains of cellular regulation. Host immune responses, such as antimicrobial peptide (AMP) production and regulation, are the result of interdependent dynamic interactions between host cells, extracellular milieu, and the external environment. In order to truly elucidate disease pathogenesis, it is imperative to consider the entire system at play from a global perspective, which is the promise of “omics” technology. The term “omics” was originally coined as a discipline in molecular biology that examines global sets of biological molecules (Micheel et al., 2012), which consequently stimulated an “Omics Revolution” throughout the scientific community. Genomics emerged following the sequencing of the human genome in the 1990s and early 2000s. This subsequently led to proteomics, which includes the entire complement of proteins and their related structure, modifications, and function. Pauling et al. (1971) proposed that estimating the abundance of metabolites in biological fluids may indicate the functional status of a specific biological system. Consequently, metabolomics has recently surfaced as an approach to comprehensively identify and quantify low molecular weight exogenous and endogenous metabolites and compounds in a biological system using high-throughput methods. Using 16S rRNA gene sequencing tools originally developed in the field of environmental microbiology, we can now generate a site-specific microbiome profile for any given patient. The Human Microbiome Project and numerous other projects aim to discern relationships between human-associated microbes and health and disease (Zoetendal et al., 2006). Such research has generated massive and complex data sets, which requires further advancement and refinement of bioinformatics and biostatistical applications. Often, the data is used to produce computational models to distinguish specific characteristics of a given population. Due to the unavoidable complexities and required statistical sophistication, few medical fields have developed clinically useful applications for the resulting data. Thus, AMPs are an ideal set of biological molecules to assess, since there biological role spans numerous areas of clinical research. Due to their diverse microbicidal and immunomodulatory functions (Steinstraesser et al., 2011), AMPs comprise a major aspect of the host innate immune response (Nakatsuji and Gallo, 2012). Alterations in AMP production and/or localization are associated with several cutaneous dermatoses. Patients diagnosed with atopic dermatitis and psoriasis ultimately develop epidermal barrier defects and microbial susceptibility, or a hyperproliferative/inflammatory epidermis, respectively (Schauber and Gallo, 2008; Schroder, 2011). The contributions of AMPs to tissue integrity and epithelial defense was also established in the brain (Schluesener et al., 2012; Williams et al., 2012), urinary tract (Saemann et al., 2007; Zasloff, 2007; Ali et al., 2009), gastrointestinal tract (Lisitsyn et al., 2012), and certain malignancies (Kesting et al., 2012; Scola et al., 2012). However, altered AMP regulation does not occur exclusively, but instead embodies dynamic transformations that emerge as a disturbed system. The integration of several different “omics” techniques may provide a more complete understanding of the diverse factors contributing to a specific disease state, responses to pharmacologic agents, or novel alternatives to current treatment modalities.

Toward Understanding, Managing, and Protecting Microbial Ecosystems

Microbial communities are at the very basis of life on earth, catalyzing biogeochemical reactions driving global nutrient cycles. However, unlike for plants and animals, microbial diversity is not on the biodiversity–conservation agenda. The latter, however, would imply that microbial diversity is not under any threat by anthropogenic disturbance or climate change. This maybe a misconception caused by the rudimentary knowledge we have concerning microbial diversity and its role in ecosystem functioning. This perspective paper identifies major areas with knowledge gaps within the field of environmental microbiology that preclude a comprehension of microbial ecosystems on the level we have for plants and animals. Opportunities and challenges are pointed out to open the microbial black box and to go from descriptive to predictive microbial ecology.

Identity and ecophysiology of filamentous bacteria in activated sludge

Excessive growth of filamentous bacteria in activated sludge wastewater treatment plants (WWTPs) can cause serious operational problems. With some filaments there may be the problem of bulking, where inadequate flocculation and settling of the biomass in the secondary clarifier results in a carryover of solids with the final treated liquid effluent. Their proliferation often encourages the development of stable foams on the surface of the reactors, and these foams may impact negatively on plant performance and operation. The availability of culture-independent molecular methods now allows us to identify many of the more common filamentous organisms encountered in WWTPs, which are phylogenetically diverse, affiliating to seven separate bacterial phyla. Furthermore, the extensive data published in the past decade on their in situ behaviour from the application of these culture-independent methods have not been summarized or reviewed critically. Hence, here, we attempt to discuss what we now know about their identity, ecophysiology and ecological niches and its practical value in better managing activated sludge processes. Some of this knowledge is already being applied to control and manage full-scale WWTPs better, and the hope is that this review will contribute towards further developments in this field of environmental microbiology.

Research progress in genomics of environmental and industrial microorganisms

Microbes contribute to geochemical cycles in the ecosystem. They also play important roles in biodegradation and bioremediation of contaminated environments, and have great potential in energy conversion and regeneration. Up to date, at least 150 genomes of non-pathogenic microbes have been sequenced, of which, the majority are bacteria from various environments or of industrial uses. The emerging field 'metagenomics' in combination with the high-throughput sequencing technology offers opportunities to discover new functions of microbes in the environment on a large scale, and has become the 'hot spot' in the field of environmental microbiology. Seven genomes of bacteria from various extreme environments, including high temperature, high and low pressure, and extreme acidic regions, have been sequenced by researchers in China, leading to the discovery of metabolic pathways, genetic functions and new enzymes, which are related to the niches those bacteria occupy. These results were published in Nature, PNAS, Genome Research and other top international journals. In the meantime, several groups in China have started 'metagenomics' programs. The outcomes of these researches are expected to generate a considerable number of novel findings, taking Chinese researchers to the frontier of genomics for environmental and industrial microorganisms.

Environmental Microbiology meets Microbial Biotechnology

<i>Environmental Microbiology</i> meets <i>Microbial Biotechnology</i>

Editorial

The rhizosphere of plants is one of the most fascinating microbial habitats for basic and applied studies in the field of environmental microbiology, as it is shaped by the soil, the plant and the microorganisms. The importance of this compartment for plant growth and soil microbiology had already been realized in the very pioneer times of microbiology in the late 19th century. In 1888, Hellriegel and Wilfarth proofed the special case of nitrogen nutrition of legumes through their root nodule bacteria, which Beijerinck finally isolated in 1889. Also Winogradskyi's studies on chemolithotrophy and nitrifying bacteria occurred in this time. As agricultural practice was in close contact to the new achievements of basic sciences, the challenge quickly arose whether at all, and how, these discoveries of the – at that time – very young science ‘soil bacteriology’ could be applied in the field. In 1885, the young scientist Lorenz Hiltner graduated from the University of Erlangen in Frankonia, Germany, and focused his scientific interests in plant sciences and soil microbiology. He joined the ‘Plant Physiological Research Station Tharandt’ near Dresden, Saxonia, headed by Professor Dr F. Nobbe. In his doctoral thesis, he worked on aspects of pathogen–root interactions. In 1887–88 Hiltner was certainly fascinated by the findings of Hellriegel and Wilfarth about the special case of nitrogen nutrition in legumes. Together with Professor Nobbe, intensive studies were conducted about the nature of the symbiotic interaction of nodule bacteria and legume roots. Apparently during these studies, Hiltner became aware of the importance of the ecological interactions in the root zone. Together with his teacher he developed the first inoculant based on root nodule bacteria for agricultural practice which they called ‘Nitragin’ in 1890. After several improvements and the discovery of the specificity of the Rhizobium–legume symbiosis, the inoculant worked very reliably and was successfully introduced into agricultural practice. In 1902, Dr Lorenz Hiltner (1862–1923) became the founding director of the Royal Bavarian Agriculture-Botanical Institute in Munich and also Professor of Soil Bacteriology at the Technical University of Munich. In his presentation at a meeting of the German Agricultural Society in 1904 in Eisenach, Thuringia, Germany, he clearly stated the general importance of plant–microbe interactions in the root zone for plant nutrition as well as for plant health. The international ‘Rhizosphere Congress 2004’ held in Munich, 12–17 September 2004, commemorated the 100 year anniversary of Lorenz Hiltner's definition of the Rhizosphere concept in 1904. As rhizosphere research is a true interdisciplinary field of biological and soil sciences, the presentations at the conference were dealing with new scientific findings and cutting-edge technologies in soil chemistry and physics as well as modelling and the molecular biological and functional genomic approaches of plant–microbe interactions. This special ‘Rhizosphere Issue’ of Environmental Microbiology presents some of the microbiological contributions at this conference but also research papers independent of this conference. The scope of the papers is very broad and concerns, for example, community studies of rhizosphere bacteria in different plant systems or detailed studies of the molecular interaction of microbes and plant roots, including signalling with quorum-sensing molecules or plant hormones. The special issue deals with phytopathogenic as well as symbiotic or plant growth-promoting bacteria and fungi. In addition, features of root endophytic bacteria as well as endofungal bacteria in ectomycorrhizae are presented. The understanding of the functional diversity and the underlying mechanisms of interactions between rhizosphere microbes and roots are the basis for further implementation and improvements of rhizosphere biotechnologies in agriculture and bioremediation.