Culture-independent methods for studying environmental microorganisms: methods, application, and perspective

Since the application of molecular methods, culture-independent methods (CIMs) have been developed to study microbial communities from various environments. In the past 20 years, several methods based on the direct amplification and analyses of the small subunit ribosomal RNA gene have been developed to directly study environmental microorganisms. These methods include denaturing/temperature gradient gel electrophoresis, single-strand-conformation polymorphism, restriction fragment length polymorphism, terminal restriction fragment length polymorphism, and quantitative polymerase chain reaction (PCR). Similarly, non-PCR-based molecular techniques, such as microarray and fluorescence in situ hybridization have also been adopted. In recent years, several novel fields of investigation such as metagenomics, metatranscriptomics, metaproteomics, and single-cell genomics were developed, largely propelled by the innovation and application of next-generation sequencing methods. Several single-cell-based technologies such as Raman microspectroscopy and nano-scale secondary ion mass spectrometry are also increasingly used in the fields of microbial ecology and environmental microbiology. The application of these methods has revolutionized microbiology by allowing scientists to directly analyze natural microbial communities in situ, including their genes, transcripts, proteins, and metabolites and how their interactions impact their distribution patterns. In this review, we present an up-to-date review on different CIMs and their applications, our focuses are on the comparison of different CIMs and their application in the analyses of microbial diversities and communities.

[1]  J. Marchesi,et al.  Micro-eukaryotic diversity of the human distal gut microbiota: qualitative assessment using culture-dependent and -independent analysis of faeces , 2008, The ISME Journal.

[2]  Jinkui Yang,et al.  Bacterial diversities on unaged and aging flue-cured tobacco leaves estimated by 16S rRNA sequence analysis , 2010, Applied Microbiology and Biotechnology.

[3]  Y. Zo,et al.  Nonradioactive method to study genetic profiles of natural bacterial communities by PCR-single-strand-conformation polymorphism , 1996, Applied and environmental microbiology.

[4]  G. Casella,et al.  Pyrosequencing enumerates and contrasts soil microbial diversity , 2007, The ISME Journal.

[5]  Rob Knight,et al.  Short-Term Temporal Variability in Airborne Bacterial and Fungal Populations , 2007, Applied and Environmental Microbiology.

[6]  S. Hui,et al.  Phylogenetic analysis of the fecal flora of the wild pygmy loris , 2010, American journal of primatology.

[7]  J. Sibilia,et al.  Broad-range PCR, cloning and sequencing of the full 16S rRNA gene for detection of bacterial DNA in synovial fluid samples of Tunisian patients with reactive and undifferentiated arthritis , 2009, Arthritis research & therapy.

[8]  Eun Ju Lee,et al.  High Abundance and Role of Antifungal Bacteria in Compost-Treated Soils in a Wildfire Area , 2011, Microbial Ecology.

[9]  Å. Frostegård,et al.  Presence of Actinobacterial and Fungal Communities in Clean and Petroleum Hydrocarbon Contaminated Subsurface Soil , 2009, The Open Microbiology Journal.

[10]  S. Giovannoni,et al.  High-Throughput Methods for Culturing Microorganisms in Very-Low-Nutrient Media Yield Diverse New Marine Isolates , 2002, Applied and Environmental Microbiology.

[11]  Tao Zhang,et al.  The Airborne Metagenome in an Indoor Urban Environment , 2008, PloS one.

[12]  R. Daniel,et al.  Metagenomic Analyses: Past and Future Trends , 2010, Applied and Environmental Microbiology.

[13]  M. Kuypers Microbiology. Sizing up the uncultivated majority. , 2007, Science.

[14]  K. Schleifer,et al.  Linking phylogenetic and functional diversity to nutrient spiraling in microbial mats from Lower Kane Cave (USA) , 2010, The ISME Journal.

[15]  U. Pöschl,et al.  High diversity of fungi in air particulate matter , 2009, Proceedings of the National Academy of Sciences.

[16]  Richard D. Smith,et al.  Transport functions dominate the SAR11 metaproteome at low-nutrient extremes in the Sargasso Sea , 2009, The ISME Journal.

[17]  W. Stafford,et al.  Bacterial Diversity in Three Different Antarctic Cold Desert Mineral Soils , 2006, Microbial Ecology.

[18]  D. Benndorf,et al.  Functional metaproteome analysis of protein extracts from contaminated soil and groundwater , 2007, The ISME Journal.

[19]  S. Delgado,et al.  PCR-DGGE assessment of the bacterial diversity of breast milk in women with lactational infectious mastitis , 2008, BMC infectious diseases.

[20]  K. Márialigeti,et al.  Diversity of Sulfate-Reducing Bacteria Inhabiting the Rhizosphere of Phragmites australis in Lake Velencei (Hungary) Revealed by a Combined Cultivation-based and Molecular approach , 2008, Microbial Ecology.

[21]  David R. Herndon,et al.  Association of Mycoplasma ovipneumoniae Infection with Population-Limiting Respiratory Disease in Free-Ranging Rocky Mountain Bighorn Sheep (Ovis canadensis canadensis) , 2007, Journal of Clinical Microbiology.

[22]  Roger S Lasken,et al.  Single-cell genomic sequencing using Multiple Displacement Amplification. , 2007, Current opinion in microbiology.

[23]  Daniel H. Huson,et al.  Simultaneous Assessment of Soil Microbial Community Structure and Function through Analysis of the Meta-Transcriptome , 2008, PloS one.

[24]  Fabrice Armougom,et al.  Monitoring Bacterial Community of Human Gut Microbiota Reveals an Increase in Lactobacillus in Obese Patients and Methanogens in Anorexic Patients , 2009, PloS one.

[25]  D. Antonopoulos,et al.  16S rRNA gene-based analysis of fecal microbiota from preterm infants with and without necrotizing enterocolitis , 2009, The ISME Journal.

[26]  S. Tringe,et al.  Quantitative Phylogenetic Assessment of Microbial Communities in Diverse Environments , 2007, Science.

[27]  M. Bailey,et al.  Resolving Genetic Functions within Microbial Populations: In Situ Analyses Using rRNA and mRNA Stable Isotope Probing Coupled with Single-Cell Raman-Fluorescence In Situ Hybridization , 2008, Applied and Environmental Microbiology.

[28]  V. Orphan,et al.  Identification of diazotrophic microorganisms in marine sediment via fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS). , 2011, Methods in enzymology.

[29]  Jinkui Yang,et al.  Diversity and phylogeny of bacteria on Zimbabwe tobacco leaves estimated by 16S rRNA sequence analysis , 2011, Applied Microbiology and Biotechnology.

[30]  B. Jørgensen,et al.  The future of single-cell environmental microbiology. , 2007, Environmental microbiology.

[31]  A. Haffajee,et al.  Intra-oral microbial profiles of beagle dogs assessed by checkerboard DNA-DNA hybridization using human probes. , 2008, Veterinary microbiology.

[32]  B. Ahring,et al.  Specific single-cell isolation and genomic amplification of uncultured microorganisms , 2007, Applied Microbiology and Biotechnology.

[33]  Rob Knight,et al.  Human oral, gut, and plaque microbiota in patients with atherosclerosis , 2010, Proceedings of the National Academy of Sciences.

[34]  M. Polz,et al.  Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by 'reconditioning PCR'. , 2002, Nucleic acids research.

[35]  G. Laguerre,et al.  Rapid Identification of Rhizobia by Restriction Fragment Length Polymorphism Analysis of PCR-Amplified 16S rRNA Genes , 1994, Applied and environmental microbiology.

[36]  D. Relman,et al.  Incomplete recovery and individualized responses of the human distal gut microbiota to repeated antibiotic perturbation , 2010, Proceedings of the National Academy of Sciences.

[37]  M. Horn,et al.  Molecular analysis of bacteria in periodontitis: evaluation of clone libraries, novel phylotypes and putative pathogens. , 2003, Microbiology.

[38]  B. Peyton,et al.  Molecular analysis of prokaryotic diversity in the deep subsurface of the former Homestake gold mine, South Dakota, USA , 2009, The Journal of Microbiology.

[39]  François Robert,et al.  Diversity in the Archean biosphere: new insights from NanoSIMS. , 2010, Astrobiology.

[40]  R. Bhatnagar,et al.  Bacterial diversity analysis of larvae and adult midgut microflora using culture-dependent and culture-independent methods in lab-reared and field-collected Anopheles stephensi-an Asian malarial vector , 2009, BMC Microbiology.

[41]  M. Engelhard,et al.  Microbial and Mineralogical Characterizations of Soils Collected from the Deep Biosphere of the Former Homestake Gold Mine, South Dakota , 2010, Microbial Ecology.

[42]  Wendy S. Schackwitz,et al.  One Bacterial Cell, One Complete Genome , 2010, PloS one.

[43]  J. Lennon,et al.  Dormancy contributes to the maintenance of microbial diversity , 2010, Proceedings of the National Academy of Sciences.

[44]  Maureen L. Coleman,et al.  Microbial community gene expression in ocean surface waters , 2008, Proceedings of the National Academy of Sciences.

[45]  J. Chun,et al.  Diversity of bacterial community in freshwater of Woopo wetland , 2008, The Journal of Microbiology.

[46]  K. Nelson,et al.  Diversity of Cervicovaginal Microbiota Associated with Female Lower Genital Tract Infections , 2011, Microbial Ecology.

[47]  P. Visca,et al.  Microbial Community Structure and Dynamics of Dark Fire-Cured Tobacco Fermentation , 2006, Applied and Environmental Microbiology.

[48]  Tanja Woyke,et al.  Genomic sequencing of single microbial cells from environmental samples. , 2008, Current opinion in microbiology.

[49]  Vanja Klepac-Ceraj,et al.  Comparative Analyses of the Bacterial Microbiota of the Human Nostril and Oropharynx , 2010, mBio.

[50]  C. D. Long,et al.  Bacterial diversity in the oral cavity of 10 healthy individuals , 2010, The ISME Journal.

[51]  Ian P Thompson,et al.  Raman microscopic analysis of single microbial cells. , 2004, Analytical chemistry.

[52]  G. Andersen,et al.  Microbial Diversity in Uranium Mining-Impacted Soils as Revealed by High-Density 16S Microarray and Clone Library , 2009, Microbial Ecology.

[53]  R. Lasken,et al.  Genomic DNA Amplification from a Single Bacterium , 2005, Applied and Environmental Microbiology.

[54]  P. Pevzner,et al.  Efficient de novo assembly of single-cell bacterial genomes from short-read data sets , 2011, Nature Biotechnology.

[55]  W. Röling,et al.  Microbial Communities in the World's Largest Acidic Volcanic Lake, Kawah Ijen in Indonesia, and in the Banyupahit River Originating from It , 2006, Microbial Ecology.

[56]  P. Qian,et al.  Bacterial communities in PAH contaminated soils at an electronic-waste processing center in China , 2010, Ecotoxicology.

[57]  F. Thompson,et al.  Bacterial Community Diversity in the Brazilian Atlantic Forest Soils , 2010, Microbial Ecology.

[58]  K. Riedel,et al.  Environmental proteomics: Analysis of structure and function of microbial communities , 2010, Proteomics.

[59]  Mary Ann Moran,et al.  Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics , 2009, Journal of visualized experiments : JoVE.

[60]  H. Horz,et al.  T-RFLP-based mcrA gene analysis of methanogenic archaea in association with oral infections and evidence of a novel Methanobrevibacter phylotype. , 2009, Oral microbiology and immunology.

[61]  B. Díez,et al.  Epilithic Cyanobacterial Communities of a Marine Tropical Beach Rock (Heron Island, Great Barrier Reef): Diversity and Diazotrophy , 2007, Applied and Environmental Microbiology.

[62]  Anders F. Andersson,et al.  Comparative Analysis of Human Gut Microbiota by Barcoded Pyrosequencing , 2008, PloS one.

[63]  C. Lacroix,et al.  Population dynamics of two antilisterial cheese surface consortia revealed by temporal temperature gradient gel electrophoresis , 2010, BMC Microbiology.

[64]  K. Schleifer,et al.  Phylogenetic identification and in situ detection of individual microbial cells without cultivation. , 1995, Microbiological reviews.

[65]  A. Nocker,et al.  Genotypic Microbial Community Profiling: A Critical Technical Review , 2007, Microbial Ecology.

[66]  Peter F. Hallin,et al.  On the Origins of a Vibrio Species , 2009, Microbial Ecology.

[67]  R. Sorek,et al.  Prokaryotic transcriptomics: a new view on regulation, physiology and pathogenicity , 2010, Nature Reviews Genetics.

[68]  M. Kuypers Sizing Up the Uncultivated Majority , 2007, Science.

[69]  K. Horikoshi,et al.  Rapid Detection and Quantification of Members of the Archaeal Community by Quantitative PCR Using Fluorogenic Probes , 2000, Applied and Environmental Microbiology.

[70]  Danilo Ercolini,et al.  Application of FISH technology for microbiological analysis: current state and prospects , 2006, Applied Microbiology and Biotechnology.

[71]  F. Bäckhed,et al.  Host-Bacterial Mutualism in the Human Intestine , 2005, Science.

[72]  C. Kuske,et al.  Assessment of Microbial Diversity in Four Southwestern United States Soils by 16S rRNA Gene Terminal Restriction Fragment Analysis , 2000, Applied and Environmental Microbiology.

[73]  Les Dethlefsen,et al.  The Pervasive Effects of an Antibiotic on the Human Gut Microbiota, as Revealed by Deep 16S rRNA Sequencing , 2008, PLoS biology.

[74]  Y. Shouche,et al.  Molecular microbial diversity of a soil sample and detection of ammonia oxidizers from Cape Evans, Mcmurdo Dry Valley, Antarctica. , 2007, Microbiological research.

[75]  M. Nowrousian Next-Generation Sequencing Techniques for Eukaryotic Microorganisms: Sequencing-Based Solutions to Biological Problems , 2010, Eukaryotic Cell.

[76]  V. Kunin,et al.  Metatranscriptomic array analysis of 'Candidatus Accumulibacter phosphatis'-enriched enhanced biological phosphorus removal sludge. , 2010, Environmental microbiology.

[77]  T. Marsh,et al.  Fecal bacterial diversity of human‐habituated wild chimpanzees (Pan troglodytes schweinfurthii) at Mahale Mountains National Park, Western Tanzania , 2010, American journal of primatology.

[78]  P. Turnbaugh,et al.  Microbial ecology: Human gut microbes associated with obesity , 2006, Nature.

[79]  C. Gugliandolo,et al.  Distribution and Diversity of Bacteria in a Saline Meromictic Lake as Determined by PCR-DGGE of 16S rRNA Gene Fragments , 2010, Current Microbiology.

[80]  Eoin L. Brodie,et al.  Despite strong seasonal responses, soil microbial consortia are more resilient to long-term changes in rainfall than overlying grassland , 2009, The ISME Journal.

[81]  G. Muyzer DGGE/TGGE a method for identifying genes from natural ecosystems. , 1999, Current opinion in microbiology.

[82]  B. Zeng,et al.  The Development and Stability of the Genus Bacteriodes from Human Gut Microbiota in HFA Mice Model , 2011, Current Microbiology.

[83]  L. Bodrossy,et al.  Oligonucleotide microarrays in microbial diagnostics. , 2004, Current opinion in microbiology.

[84]  Adam Godzik,et al.  Shotgun metaproteomics of the human distal gut microbiota , 2008, The ISME Journal.

[85]  D. Bourne,et al.  Microbial diversity in sediments associated with a shallow methane seep in the tropical Timor Sea of Australia reveals a novel aerobic methanotroph diversity. , 2009, FEMS microbiology ecology.

[86]  R. Goodacre,et al.  Provided for Non-commercial Research and Educational Use Only. Not for Reproduction, Distribution or Commercial Use. Shining Light on the Microbial World: the Application of Raman Microspectroscopy , 2022 .

[87]  Byron F. Brehm-Stecher,et al.  Single-Cell Microbiology: Tools, Technologies, and Applications , 2004, Microbiology and Molecular Biology Reviews.

[88]  D. Relman,et al.  Linking Microbial Phylogeny to Metabolic Activity at the Single-Cell Level by Using Enhanced Element Labeling-Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (EL-FISH) and NanoSIMS , 2008, Applied and Environmental Microbiology.

[89]  Johannes Goll,et al.  Bioinformatics Applications Note Database and Ontologies Metarep: Jcvi Metagenomics Reports—an Open Source Tool for High-performance Comparative Metagenomics , 2022 .

[90]  P. Gemperline,et al.  Identification of single bacterial cells in aqueous solution using confocal laser tweezers Raman spectroscopy. , 2005, Analytical chemistry.

[91]  F. Bäckhed,et al.  Obesity alters gut microbial ecology. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[92]  Paul Wilmes,et al.  Metaproteomics Provides Functional Insight into Activated Sludge Wastewater Treatment , 2008, PloS one.

[93]  P. Wilmes,et al.  The application of two-dimensional polyacrylamide gel electrophoresis and downstream analyses to a mixed community of prokaryotic microorganisms. , 2004, Environmental microbiology.

[94]  G. Andersen,et al.  Bacterial Diversity Analysis of Huanglongbing Pathogen-Infected Citrus, Using PhyloChip Arrays and 16S rRNA Gene Clone Library Sequencing , 2009, Applied and Environmental Microbiology.

[95]  Paul C. Blainey,et al.  Genome of a Low-Salinity Ammonia-Oxidizing Archaeon Determined by Single-Cell and Metagenomic Analysis , 2011, PloS one.

[96]  Jianping Xu,et al.  INVITED REVIEW: Microbial ecology in the age of genomics and metagenomics: concepts, tools, and recent advances , 2006, Molecular ecology.

[97]  Yong-qing Li,et al.  Raman spectra and optical trapping of highly refractive and nontransparent particles , 2002 .

[98]  Yahai Lu,et al.  Life without light: microbial diversity and evidence of sulfur- and ammonium-based chemolithotrophy in Movile Cave , 2009, The ISME Journal.

[99]  R. Jónsdóttir,et al.  Bacterial composition and succession during storage of North-Atlantic cod (Gadus morhua) at superchilled temperatures , 2009, BMC Microbiology.