Uncultured Microorganisms Research Articles

A Bioreactor Approach to Investigate the Linkage between Methane Oxidation and Nitrate/Nitrite Reduction in the Pelagic Oxic-Anoxic Transition Zone of the Central Baltic Sea

Evidence of aerobic methane oxidation coupled to denitrification has been provided for different freshwater environments, whereas the significance of this process for the marine realm has not been adequately investigated. The goal of this study was to investigate the methane-related reduction of nitrate/nitrite in a marine environment (salinity 8.5). A water sample was collected from the oxic-anoxic transition zone of the Gotland Deep (central Baltic Sea) and the microorganisms contained therein were cultivated in a bioreactor under hypoxic conditions (0.5 µM O2). To enrich the microorganisms involved in the coupled process the bioreactor was continuously sparged with methane as the sole energy and carbon source and simultaneously supplied with a nutrient solution rich in nitrate and nitrite. The bioreactor experiment showed a relationship between the turnover of methane and the concomitant concentration decrease of nitrite and nitrate at the early stage of the experiment. This relationship indicates the role of methanotrophs, which may support heterotrophic denitrifiers by the release of organic compounds as an energy source. Besides, a mixture of uncultured microorganisms, aerobic methanotrophic and heterotrophic denitrifying bacteria were identified in the enrichment culture. Microbial incorporation of nitrite and methane was proven on the cellular and gene levels via 15NO2- / 13CH4 incubation experiments and subsequent analyses with nano secondary ion mass spectrometry (NanoSIMS) and stable isotope probing (SIP). The NanoSIMS showed the incorporation of 15N in almost all the bacteria and in 9% of those there was a concomitant enrichment in 13C. The relatively low abundance of methane-consuming bacteria in the bioreactor was further reflected in specific fatty acids indicative for type I methanotrophic bacteria. Based on pmoA gene analyses, this bacterium is different from the one that was identified as the only key player of methane oxidation in previous studies in the Gotland Deep, indicating the existence of other subordinate methanotrophic bacteria at that site. The results provide the first indications for the predisposition of a methane-related reduction of nitrate/nitrite under hypoxic conditions in the marine realm, supporting the assumption of an interaction between methanotrophic and denitrifying bacteria which hitherto has only been described for fresh water environments.

Reflection on Molecular Approaches Influencing State-of-the-Art Bioremediation Design: Culturing to Microbial Community Fingerprinting to Omics.

Bioremediation is generally viewed as a cost effective and sustainable technology because it relies on microbes to transform pollutants into benign compounds. Advances in molecular biological analyses allow unprecedented microbial detection and are increasingly incorporated into bioremediation. Throughout history, state-of-the-art techniques have informed bioremediation strategies. However, the insights those techniques provided were not as in depth as those provided by recently developed omics tools. Advances in next generation sequencing (NGS) have now placed metagenomics and metatranscriptomics within reach of environmental engineers. As NGS costs decrease, metagenomics and metatranscriptomics have become increasingly feasible options to rapidly scan sites for specific degradative functions and identify microorganisms important in pollutant degradation. These omic techniques are capable of revolutionizing biological treatment in environmental engineering by allowing highly sensitive characterization of previously uncultured microorganisms. Omics enables the discovery of novel microorganisms for use in bioaugmentation and supports systematic optimization of biostimulation strategies. This review describes the omics journey from roots in biology and medicine to its current status in environmental engineering including potential future directions in commercial application.

Separating and characterizing functional alkane degraders from crude-oil-contaminated sites via magnetic nanoparticle-mediated isolation

Uncultivable microorganisms account for over 99% of all species on the planet, but their functions are yet not well characterized. Though many cultivable degraders for n-alkanes have been intensively investigated, the roles of functional n-alkane degraders remain hidden in the natural environment. This study introduces the novel magnetic nanoparticle-mediated isolation (MMI) technology in Nigerian soils and successfully separates functional microbes belonging to the families Oxalobacteraceae and Moraxellaceae, which are dominant and responsible for alkane metabolism in situ. The alkR-type n-alkane monooxygenase genes, instead of alkA- or alkP-type, were the key functional genes involved in the n-alkane degradation process. Further physiological investigation via a BIOLOG PM plate revealed some carbon (Tween 20, Tween 40 and Tween 80) and nitrogen (tyramine, l-glutamine and d-aspartic acid) sources promoting microbial respiration and n-alkane degradation. With further addition of promoter carbon or nitrogen sources, the separated functional alkane degraders significantly improved n-alkane biodegradation rates. This suggests that MMI is a promising technology for separating functional microbes from complex microbiota, with deeper insight into their ecological functions and influencing factors. The technique also broadens the application of the BIOLOG PM plate for physiological research on functional yet uncultivable microorganisms.

Dynamic functional characterization and phylogenetic changes due to Long Chain Fatty Acids pulses in biogas reactors.

The process stability of biogas plants is often deteriorated by the accumulation of Long Chain Fatty Acids (LCFA). The microbial community shifts due to LCFA disturbances have been poorly understood as the molecular techniques used were not able to identify the genome characteristics of uncultured microorganisms, and additionally, the presence of limited number of reference genomes in public databases prevented the comprehension of specific functional roles characterizing these microorganisms. The present study is the first research which deciphers by means of high throughput shotgun sequencing the dynamics of the microbial community during an inhibitory shock load induced by single pulses of unsaturated LCFA at two different concentrations (i.e. 2 g/L-reactor and 3 g/L-reactor). The metagenomic analysis showed that only the microbes associated with LCFA degradation could encode proteins related to “chemotaxis” and “flagellar assembly”, which promoted the ability to move towards the LCFA sources so as to degrade them. Moreover, the syntrophic interactions found between Syntrophomonas sp. together with Methanosarcina sp. were possibly assigned to the menaquinone-electron transfer. Finally, it was proven that a previously exposed to LCFA inoculum is more efficient in the degradation process of LCFA due to the specialization of the microbial consortium.

RAPID AND EFFICIENT METHOD FOR ENVIRONMENTAL DNA EXTRACTION AND PURIFICATION FROM SOIL

Large proportion of microbial population in the world is unculturable. Extraction of total DNA from soil is usually a crucial step considering to the difficulties of study the uncultivable microorganisms. Humic acid is considered as the main inhibitory agent in the environmental DNA studies. Here, we introduced a rapid and efficient method for DNA extraction and purification from soil. Yield of DNA extraction by the presented method was 130 ng/µl. Three conventional methods of DNA extraction including liquid nitrogen incursion, bead beating and sonication were performed as control methods. Yield of DNA extraction by these methods were 110, 90 and 50 ng/µl, respectively. A rapid and efficient one step DNA purification method was introduced instead of hazardous conventional phenol-chloroform methods. Humic acid removal percentage by the introduced method was 95.8 % that is comparable with 97 % gained by the conventional gel extraction method and yield of DNA after purification was 84 % and 73 %, respectively. This study could be useful in molecular ecology and metagenomics study as a fast and reliable method.

Diversity of methanogenic archaea from the 2012 terrestrial hot spring (Valley of Geysers, Kamchatka)

Archaeal diversity in the 2012 terrestrial hot spring (Valley of Geysers, Kronotsky Nature Reserve, Kamchatka, Russia) was investigated using molecular and cultivation-based approaches. Analysis of the 16S rRNA gene sequences revealed predominance among archaea of uncultured microorganisms of the pSL12 and THSCG clusters. Analysis of the mcrA genes revealed that members of the order Methanomassiliicoccales were predominant (68%) among methanogens; the latter constituted 0.15% of the total number of archaea. Five stable thermophilic methanogenic associations utilizing hydrogen, formate, acetate, or methanol as substrates were obtained from the sediments of spring 2012. The diversity of cultured methanogens was limited to members of the genera Methanothermobacter, Methanothrix, and Methanomethylovorans. The association growing at 65°C and producing methane from methanol contained two components, which probably formed a syntrophic relationship: a Methanothermobacter methanogenic archaeon and a bacterium representing an separate cluster within the Firmicutes phylum, which was phylogenetically related to the genera Thermacetogenium and Syntrophaceticus. These data indicate high diversity of methanogens, notwithstanding their low abundance among archaea. The group of thermophilic Methanomassiliicoccales, which predominated among methanogens, is of special interest.

Culture Independent Approaches for the Discovery of New Bacterial Metabolites

One of the key revelations to arise from the large‐scale sequencing of bacterial genomic DNA is that traditional approaches used for the discovery of natural products only provide functional access to a small fraction of the natural product biosynthetic gene clusters present in nature. The sequencing of DNA extracted directly from soil samples indicates that as yet uncultured soil microbes outnumber their cultured counterparts by at least two to three orders of magnitude. Environmental bacteria no doubt produces secondary metabolites that could serve as molecular probes of biological processes and therapeutic agents. Uncultivated microorganisms are a very attractive source of potentially new natural products, but they are not amenable to the traditional approaches used to characterize natural products from microbes grown in pure culture. I will discuss culture independent approaches we are using to access novel natural products.

Ultra-precise detection of mutations by droplet-based amplification of circularized DNA

BackgroundNGS (next generation sequencing) has been widely used in studies of biological processes, ranging from microbial evolution to cancer genomics. However, the error rate of NGS (0.1 % ~ 1 %) is still remaining a great challenge for comprehensively investigating the low frequency variations, and the current solution methods have suffered severe amplification bias or low efficiency.ResultsWe creatively developed Droplet-CirSeq for relatively efficient, low-bias and ultra-sensitive identification of variations by combining millions of picoliter uniform-sized droplets with Cir-seq. Droplet-CirSeq is entitled with an incredibly low error rate of 3 ~ 5 X 10-6. To systematically evaluate the performances of amplification uniformity and capability of mutation identification for Droplet-CirSeq, we took the mixtures of two E. coli strains as specific instances to simulate the circumstances of mutations with different frequencies. Compared with Cir-seq, the coefficient of variance of read depth for Droplet-CirSeq was 10 times less (p = 2.6 X 10-3), and the identified allele frequency presented more concentrated to the authentic frequency of mixtures (p = 4.8 X 10-3), illustrating a significant improvement of amplification bias and accuracy in allele frequency determination. Additionally, Droplet-CirSeq detected 2.5 times genuine SNPs (p < 0.001), achieved a 2.8 times lower false positive rate (p < 0.05) and a 1.5 times lower false negative rate (p < 0.001), in the case of a 3 pg DNA input. Intriguingly, the false positive sites predominantly represented in two types of base substitutions (G- > A, C- > T). Our findings indicated that 30 pg DNA input accommodated in 5 ~ 10 million droplets resulted in maximal detection of authentic mutations compared to 3 pg (p = 1.2 X 10-8) and 300 pg input (p = 2.2 X 10-3).ConclusionsWe developed a method namely Droplet-CirSeq to significantly improve the amplification bias, which presents obvious superiority over the currently prevalent methods in exploitation of ultra-low frequency mutations. Droplet-CirSeq would be promisingly used in the identification of low frequency mutations initiated from extremely low input DNA, such as DNA of uncultured microorganisms, captured DNA of target region, circulation DNA of plasma et al, and its creative conception of rolling circle amplification in droplets would also be used in other low input DNA amplification fields.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2480-1) contains supplementary material, which is available to authorized users.

Characterization of a Metagenome-Derived β-Glucosidase and Its Application in Conversion of Polydatin to Resveratrol

For the beneficial pharmacological properties of resveratrol, there is increasingly interest in enzymatic conversion of polydatin to resveratrol. The metagenomic technique provides an effective strategy for mining novel polydatin-hydrolysis enzymes from uncultured microorganisms. In this study, a metagenomic library of mangrove soil was constructed and a novel β-glucosidase gene MlBgl was isolated. The deduced amino acid sequences of MlBgl showed the highest identity of 64% with predicted β-glucosidase in the GenBank database. The gene was cloned and overexpressed in Escherichia coli BL21(DE3). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay demonstrated the purified recombinant β-glucosidase r-MlBgl with a molecular weight approximately of 71 kDa. The optimal pH and temperature of purified recombinant r-MlBgl were 7.0 and 40 °C, respectively. r-MlBgl could hydrolyze polydatin effectively. The kcat and kcat/Km values for polydatin were 989 s−1 and 1476 mM−1·s−1, respectively. These properties suggest that -r-MlBgl has potential application in the enzymatic conversion of polydatin to resveratrol for further study.

An overview of physico-chemical mechanisms of biogas production by microbial communities: a step towards sustainable waste management.

Biogas is a combination of methane, CO2, nitrogen, H2S and traces of few other gases. Almost any organic waste can be biologically transformed into biogas and other energy-rich organic compounds through the process of anaerobic digestion (AD) and thus helping in sustainable waste management. Although microbes are involved in each step of AD, knowledge about those microbial consortia is limited due to the lack of phylogenetic and metabolic data of predominantly unculturable microorganisms. However, culture-independent methods like PCR-based ribotyping has been successfully employed to get information about the microbial consortia involved in AD. Microbes identified have been found to belong mainly to the bacterial phyla of Proteobacteria, Chloroflexi, Firmicutes and Bacteroidetes. Among the archaeal population, the majority have been found to be methanogens (mainly unculturable), the remaining being thermophilic microbes. Thus, the AD process as a whole could be controlled by regulating the microbial consortia involved in it. Optimization in the feedstock, pH, temperature and other physical parameters would be beneficial for the microbial growth and viability and thus helpful for biogas production in AD. Besides, the biogas production is also dependent upon the activity of several key genes, ion-specific transporters and enzymes, like genes coding for methyl-CoM reductase, formylmethanofuran transferase, formate dehydrogenase present in the microbes. Fishing for these high-efficiency genes will ultimately increase the biogas production and sustain the production plant.

Exploration and isolation of novel thermophiles in frozen enrichment cultures derived from a terrestrial acidic hot spring.

An isolation strategy, exploring novel microorganisms in frozen enrichment cultures (ENFE), which uses a combination of enrichment culture and 16S rRNA gene clone analysis, was evaluated for isolating uncultured thermophiles from a terrestrial acidic hot spring. The procedure comprised (a) multiple enrichment cultures under various conditions, (b) cryostorage of all enrichments, (c) microbial community analyses of the enrichments using 16S rRNA gene sequences, and (d) purification of microorganisms from enrichments containing previously uncultured microorganisms. The enrichments were performed under a total of 36 conditions, and 16 of these enrichments yielded positive microbial growth with the detection of three previously uncultured archaea. Two of the three previously uncultured archaea, strains HS-1 and HS-3, were successfully isolated. Strain HS-1 and HS-3 represented a novel lineage of the order Sulfolobales and novel species of the genus Sulfolobus, respectively. Although innovative isolation methods play strategic roles in isolating previously uncultured microorganisms, the ENFE strategy showed potential for characterizing and isolating such microorganisms using conventional media and techniques.

ExoMeg1: a new exonuclease from metagenomic library.

DNA repair mechanisms are responsible for maintaining the integrity of DNA and are essential to life. However, our knowledge of DNA repair mechanisms is based on model organisms such as Escherichia coli, and little is known about free living and uncultured microorganisms. In this study, a functional screening was applied in a metagenomic library with the goal of discovering new genes involved in the maintenance of genomic integrity. One clone was identified and the sequence analysis showed an open reading frame homolog to a hypothetical protein annotated as a member of the Exo_Endo_Phos superfamily. This novel enzyme shows 3′-5′ exonuclease activity on single and double strand DNA substrates and it is divalent metal-dependent, EDTA-sensitive and salt resistant. The clone carrying the hypothetical ORF was able to complement strains deficient in recombination or base excision repair, suggesting that the new enzyme may be acting on the repair of single strand breaks with 3′ blockers, which are substrates for these repair pathways. Because this is the first report of an enzyme obtained from a metagenomic approach showing exonuclease activity, it was named ExoMeg1. The metagenomic approach has proved to be a useful tool for identifying new genes of uncultured microorganisms.

Metagenome-scale analysis yields insights into the structure and function of microbial communities in a copper bioleaching heap.

BackgroundMetagenomics allows us to acquire the potential resources from both cultivatable and uncultivable microorganisms in the environment. Here, shotgun metagenome sequencing was used to investigate microbial communities from the surface layer of low grade copper tailings that were industrially bioleached at the Dexing Copper Mine, China. A bioinformatics analysis was further performed to elucidate structural and functional properties of the microbial communities in a copper bioleaching heap.ResultsTaxonomic analysis revealed unexpectedly high microbial biodiversity of this extremely acidic environment, as most sequences were phylogenetically assigned to Proteobacteria, while Euryarchaeota-related sequences occupied little proportion in this system, assuming that Archaea probably played little role in the bioleaching systems. At the genus level, the microbial community in mineral surface-layer was dominated by the sulfur- and iron-oxidizing acidophiles such as Acidithiobacillus-like populations, most of which were A. ferrivorans-like and A. ferrooxidans-like groups. In addition, Caudovirales were the dominant viral type observed in this extremely environment. Functional analysis illustrated that the principal participants related to the key metabolic pathways (carbon fixation, nitrogen metabolism, Fe(II) oxidation and sulfur metabolism) were mainly identified to be Acidithiobacillus-like, Thiobacillus-like and Leptospirillum-like microorganisms, indicating their vital roles. Also, microbial community harbored certain adaptive mechanisms (heavy metal resistance, low pH adaption, organic solvents tolerance and detoxification of hydroxyl radicals) as they performed their functions in the bioleaching system.ConclusionOur study provides several valuable datasets for understanding the microbial community composition and function in the surface-layer of copper bioleaching heap.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-016-0330-4) contains supplementary material, which is available to authorized users.

Bacteria capable of degrading anthracene, phenanthrene, and fluoranthene as revealed by DNA based stable-isotope probing in a forest soil

Information on microorganisms possessing the ability to metabolize different polycyclic aromatic hydrocarbons (PAHs) in complex environments helps in understanding PAHs behavior in natural environment and developing bioremediation strategies. In the present study, stable-isotope probing (SIP) was applied to investigate degraders of PAHs in a forest soil with the addition of individually 13C-labeled phenanthrene, anthracene, and fluoranthene. Three distinct phylotypes were identified as the active phenanthrene-, anthracene- and fluoranthene-degrading bacteria. The putative phenanthrene degraders were classified as belonging to the genus Sphingomona. For anthracene, bacteria of the genus Rhodanobacter were the putative degraders, and in the microcosm amended with fluoranthene, the putative degraders were identified as belonging to the phylum Acidobacteria. Our results from DNA-SIP are the first to directly link Rhodanobacter- and Acidobacteria-related bacteria with anthracene and fluoranthene degradation, respectively. The results also illustrate the specificity and diversity of three- and four-ring PAHs degraders in forest soil, contributes to our understanding on natural PAHs biodegradation processes, and also proves the feasibility and practicality of DNA-based SIP for linking functions with identity especially uncultured microorganisms in complex microbial biota.

Bioprospecting of Uncultured Microorganisms: The Dawning of Antibiotic Discovery

Antibiotics are of great benefit for human health such as using in pathogenic infection, organ transplantation, cancer treatment and cholesterol control [1]. But a huge threat of global public health caused by multi-drug-resistance pathogens (e.g., gram-positive methicillin and vancomycin resistant Staphylococcus aureus and New Delhi metallo-beta-lactamase 1 gram-negative bacteria) has emerged extensively [2]. Despite this threat has ever-increasing public awareness regarding “superbug” infections, treatment options, unfortunately, continued to be limited [3]. The discovery of new antibiotics is one of direct action on the treat of drug resistant pathogenic infections for avoiding an epidemic [2]. However, the pace of antibiotic discovery and development with unique scaffold is dramatically declining after the past 60 years of intensive screening [4], especially the collapse of the antibiotic discovery pipeline that the last new class of antibiotics daptomycin discovered in 1987 has been successfully developed into a clinical therapeutic [5]. Currently, all microorganism derived commercial antibiotics come from cultivated species, especially five phyla Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and Proteobacteria that represent 95% of antibiotic-producing microbes [6]. Because uncultured microorganisms are approximately 99% of microbial species, a recent trend in antibiotic discovery from natural sources emphasizes the investigation of uncultured microorganisms (do not grow under laboratory conditions) to meet the urgent demand for novel drug.

Microbiology of shallow subsurface aquifer and carbonate rocks studied by culture-dependent and culture-independent methods. Preliminary results on an underground laboratory, the LSBB, Rustrel, France

We investigated the microbiology of a shallow subsurface site, the LSBB, located near Avignon. This site lies in carbonate rocks, belonging to the Urgonian facies. Rock, concrete and water samples were collected and directly transferred to the laboratory. Studies of microorganisms as pure cultures are the only way to get their real physiological properties. Nevertheless, microbiologists cannot cultivate and isolate the majority of microorganisms for several reasons, one being our lack of understanding of their minimal needs. Molecular studies, e.g. extraction and sequencing of the total nucleic acids present in an environment provide phylogenetic and metabolic information on uncultivated microorganisms. We performed aerobic and anaerobic culture with various electron acceptors and donors, searching for heterotrophic, methanogenic, sulphate- nitrate- and FeIII- reducing Prokaryotes. We also performed DNA extractions and PCR amplification of ribosomal RNA genes, to test if our protocols were adapted to this environment. Our results show that the LSBB galleries are colonized by a low diversity microbiote, with a strong influence of anthropogenic activities. Further studies will link the microorganisms biodiversity and the petrophysic properties of rocks.

A Simple Evaluation System for Microbial Property in Soil and Manure

Analyses of microbial properties in soil and manure had always included the problem that there was no available standard method to evaluate microbial property. The one of the major problems was the vast diversity and the enormous population of soil microorganisms [1], the other was an existence of numerically dominant unculturable microorganisms which comprise 99% of soil habitat [2]. We evaluated whether our newly developed method, by which taxonomies and their number of each bacterial groups were estimated, could be used as evaluation method of microbial properties of soils and manures. In the forest soil, β-Proteobacteria, which included Burkholderia sp., Ralstonia sp., and Alcaligenes sp., was numerically dominant bacteria (3.64 × 106 MPN g-1 dry soil), followed by γ-Proteobacteria (1.32 × 106 MPN), δ-Proteobacteria (0.006 × 106 MPN), and the other gram negative bacteria (0.006 × 106 MPN). In the commercial manure, Actinobacteria, which included Streptoverticillium salmonis, Mycrococcus sp., Streptomyces bikiniensis, and Microbacterium ulmi, was numerically dominant bacterial group (30.8 × 106 MPN), followed by α-Proteobacteria (26.0 × 106 MPN), β-Proteobacteria (17.1 × 106 MPN), δ-Proteobacteria (11.2 × 106 MPN), the other Firmicutes (1.71 × 106 MPN), γ-Proteobacteria (0.5 × 106 MPN), and the other gram negative bacteria (0.05 × 106 MPN). In the upland field, the other Firmicutes, which included Paenibacillus sp., was numerically dominant bacteria (4.41 × 106 MPN), followed by Actinobacteria (2.14 × 106 MPN), Bacillus sp. (2.14 × 106 MPN), and γ-Proteobacteria (0.35 × 106 MPN). Although the precision of the affiliations became lower because of higher diversity of samples and the number of some Antinobacteria and Firmicutes might be underestimated by the used PCR condition, the method was found suitable as a candidate of a new evaluation system of soil and manure.

Soil DNA Purification and Isolation

There is an increased interest in the extraction of nucleic acids from various environmental samples, since molecular techniques allow less biased access to a greater portion of uncultivable microorganisms. Two strategies have been developed to improve DNA recovery in terms of yield, purity and unbiased representation of the microbial diversity. Amplification of DNA from soil is often inhibited by co-purified contaminants. DNA is also suitable for PCR amplification using various DNA targets. This review presents an overview of the available methods to achieve this challenging objective. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol precipitation.

The underestimated diversity of phytoplasmas in Latin America.

Phytoplasmas ('Candidatus Phytoplasma') are insect-transmitted, cell-wall-less, plant-pathogenic bacteria that cause economically important crop diseases. Because phytoplasmas are difficult or impossible to culture in vitro, they are classified taxonomically according to the convention used for unculturable micro-organisms. The first coherent scheme of classification of phytoplasmas, based on the RFLP pattern of the 16S rRNA-encoding gene generated with 17 restriction endonucleases, was updated several times until the development of the iPhyClassifier. iPhyClassifier is an interactive online tool capable of determining the species, group and subgroup of 'Candidatus Phytoplasma' of unknown samples using the 16S F2nR2 sequence. Latin America, an important geographical area in relation to food production, has a high incidence of plant diseases caused by phytoplasmas. However, many phytoplasmas associated with these diseases have not been properly classified. An extensive literature review and the use of iPhyClassifier allowed us to identify two new tentative groups (16SrXXXIII-A and 16SrXXXIV-A) and the following tentative new subgroups among Latin American strains that were either previously unclassified or misclassified: six in 16SrI, six in 16SrII, one in 16SrIII, one in 16SrVII, one in 16SrIX, one in 16SrXII and two in 16SrXIII.

Preparing and characterizing Fe3O4@cellulose nanocomposites for effective isolation of cellulose-decomposing microorganisms

This study developed Fe3O4@cellulose nanocomposites by co-precipitation synthesis for bacteria capture and isolation. By surface modification with cellulose, the Fe3O4@cellulose nanocomposites have 20nm average particle size and 3.3–24.9emu/g saturation magnetization. Living bacteria could be captured by the Fe3O4@cellulose nanocomposites and harvested by magnetic field, with high efficiency (95.1%) and stability (>99.99%). By metabolizing cellulose and destroying the Fe3O4@cellulose@bacteria complex, cellulose-decomposing microorganisms lost the magnetism. They were therefore able to be isolated from the inert microbial community and the separation efficiency achieved over 99.2%. This research opened a door to cultivate the uncultivable cellulose-decomposing microorganisms in situ and further characterize their ecological functions in natural environment.