Isolation of Previously Uncultured Slow-Growing Bacteria by Using a Simple Modification in the Preparation of Agar Media (original) (raw)
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Laboratory Cultivation of Widespread and Previously Uncultured Soil Bacteria
Applied and Environmental Microbiology, 2003
Most soil bacteria belong to family-level phylogenetic groups with few or no known cultivated representatives. We cultured a collection of 350 isolates from soil by using simple solid media in petri dishes. These isolates were assigned to 60 family-level groupings in nine bacterial phyla on the basis of a comparative analysis of their 16S rRNA genes. Ninety-three (27%) of the isolates belonged to 20 as-yet-unnamed family-level groupings, many from poorly studied bacterial classes and phyla. They included members of subdivisions 1, 2, 3, and 4 of the phylum Acidobacteria, subdivision 3 of the phylum Verrucomicrobia, subdivision 1 of the phylum Gemmatimonadetes, and subclasses Acidimicrobidae and Rubrobacteridae of the phylum Actinobacteria. In addition, members of 10 new family-level groupings of subclass Actinobacteridae of the phylum Actinobacteria and classes Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria of the phylum Proteobacteria were obtained. The high degree of phylogenetic novelty and the number of isolates affiliated with so-called unculturable groups show that simple cultivation methods can still be developed further to obtain laboratory cultures of many phylogenetically novel soil bacteria.
New Strategies for Cultivation and Detection of Previously Uncultured Microbes
Applied and Environmental Microbiology, 2004
An integrative approach was used to obtain pure cultures of previously uncultivated members of the divisions Acidobacteria and Verrucomicrobia from agricultural soil and from the guts of wood-feeding termites. Some elements of the cultivation procedure included the following: the use of agar media with little or no added nutrients; relatively long periods of incubation (more than 30 days); protection of cells from exogenous peroxides; and inclusion of humic acids or a humic acid analogue (anthraquinone disulfonate) and quorumsignaling compounds (acyl homoserine lactones) in growth media. The bacteria were incubated in the presence of air and in hypoxic (1 to 2% O 2 [vol/vol]) and anoxic atmospheres. Some bacteria were incubated with elevated concentrations of CO 2 (5% [vol/vol]). Significantly more Acidobacteria were found on isolation plates that had been incubated with 5% CO 2 . A simple, high-throughput, PCR-based surveillance method (plate wash PCR) was developed. This method greatly facilitated detection and ultimate isolation of target bacteria from as many as 1,000 colonies of nontarget microbes growing on the same agar plates. Results illustrate the power of integrating culture methods with molecular techniques to isolate bacteria from phylogenetic groups underrepresented in culture.
Microbes and Environments, 2020
We previously demonstrated that a simple modification in the preparation of agar media, i.e., autoclaving phosphate and agar separately (termed the "PS protocol"), improved the culturability of aerobic microorganisms by reducing the generation of reactive oxygen species. We herein investigated the effects of the PS protocol on the cultivation of anaerobic microorganisms using sludge from a wastewater treatment system as a microbial source. The application of the PS protocol increased colony numbers and the frequency of phylogenetically novel isolates under aerobic, nitrate reduction, and fermentation conditions. The PS protocol is useful for isolating both aerobic and anaerobic microorganisms.
A novel method for cultivating soil bacteria
Http Dx Doi Org 10 1893 0005 3155 84 2 65, 2013
Ribosomal RNA analyses of soil microbial communities consistently indicate that the majority of microorganisms in soil have never been cultivated or described in the laboratory. It has been suggested that microorganisms fail to grow in traditional culture media because the media fails to simulate the organisms' natural environment. We designed a unique culture technique that can cultivate an average of 40% and as high as 90% of the bacteria present in a soil sample. The 16S rDNA of 50 of these isolates were PCR amplified, sequenced, and analyzed. Nineteen, 26, 2 and 3 of the isolates were affiliated with the phyla Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes, respectively. Ten isolates possessed less than 98% sequence similarity to any previously characterized organism.
Journal of microbiological methods, 2009
Traditional methods for bacterial cultivation recover only a small fraction of bacteria from all sorts of natural environments, and attempts have been made to improve the bacterial culturability. Here we describe the development of a cultivation method, based on the embedment of pure bacterial cultures in between two layers of agar. Plates containing either embedded Pseudomonas putida or Arthrobacter globiformis resulted in higher numbers of CFUs of soil bacteria (21% and 38%, respectively) after 833 h of incubation, compared to plates with no embedded strain. This indicates a stimulatory effect of the bacterial pure cultures on the cultivation of soil bacteria. Analysis of partial 16S rRNA gene sequences revealed a phylogenetical distribution of the soil isolates into 7 classes in 4 phyla. No difference was observed at the phylum or class level when comparing isolates grouped according to embedded strain. The number of isolates belonging to the same class as the embedded strain was...
Frontiers in microbiology, 2024
Phosphorus (P) deficiency is a common problem in croplands where phosphatebased fertilizers are regularly used to maintain bioavailable P for plants. However, due to their limited mobility in the soil, there has been an increased interest in microorganisms that can convert insoluble P into a bioavailable form, and their use to develop phosphate-solubilizing bioinoculants as an alternative to the conventional use of P fertilizers. In this study, we proposed two independent experiments and explored two entirely different habitats to trap phosphatesolubilizing bacteria (PSBs). In the first experiment, PSBs were isolated from the rhizoplane of native plant species grown in a rock-phosphate (RP) mining area. A subset of 24 bacterial isolates from 210 rhizoplane morphotypes was selected for the inorganic phosphate solubilizing activities using tricalcium phosphate (TCP) as the sole P source. In the second experiment, we proposed an innovative experimental setup to select mycohyphospheric bacteria associated to arbuscular mycorrhizal fungal hyphae, indigenous of soils where agronomic plant have been grown and trapped in membrane bag filled with RP. A subset of 25 bacterial isolates from 44 mycohyphospheric morphotypes was tested for P solubilizing activities. These two bacterial subsets were then screened for additional plant growth-promoting (PGP) traits, and 16S rDNA sequencing was performed for their identification. Overall, the two isolation experiments resulted in diverse phylogenetic affiliations of the PSB collection, showing only 4 genera (24%) and 5 species (17%) shared between the two communities, thus underlining the value of the two protocols, including the innovative mycohyphospheric isolate selection method, for selecting a greater biodiversity of cultivable PSB. All the rhizoplane and mycohyphospheric PSB were positive for ammonia production. Indol-3-acetic acid (IAA) production was observed for 13 and 20 isolates, respectively among rhizoplane and mycohyphospheric PSB, ranging, respectively, from 32.52 to 330.27 μg mL −1 and from 41.4 to 963.9 μg mL −1. Only five rhizoplane and 12 mycohyphospheric isolates were positively screened for N 2 fixation. Four rhizoplane PSB were identified as siderophore producers, while none of the mycohyphospheric isolates were. The phenotype of one PSB rhizoplane isolate, assigned to Pseudomonas, showed four additive PGP activities. Some bacterial strains belonging to the dominant genera Bacillus and Pseudomonas could be considered potential candidates for further formulation of biofertilizer in order to develop bioinoculant consortia that promote plant P nutrition and growth in RP-enriched soils.
Isolation and Physiology of Bacteria from Contaminated Subsurface Sediments
2010
The majority of environmental microorganisms cannot be grown by traditional techniques. Here we employed, and contrasted with conventional plating, an alternative approach based on cultivation of microorganisms inside diffusion chambers incubated within natural samples, followed by subculturing in petri dishes. Using this approach, we isolated microorganisms from subsurface sediments from the Field Research Center (FRC) in Oak Ridge, TN. The sediments were acidic and highly contaminated with uranium, heavy metals, nitrate, and organic pollutants. Phylogenetic analysis of 16S rRNA gene sequences revealed clear differences between diversity of isolates obtained by the diffusion chamber approach and those obtained by conventional plating. The latter approach led to isolation of members of the Alpha-and Gammaproteobacteria, Actinobacteria, and Verrucomicrobia. Isolates obtained via the diffusion chamber approach represented the Alpha-, Beta-, and Gammaproteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. Notably, one-third of the isolates obtained by the new method were closely related to species known from previous molecular surveys conducted in the FRC area. Since the initial growth of microorganisms inside diffusion chambers occurred in the presence of the environmental stress factors, we expected the isolates we obtained to be tolerant of these factors. We investigated the physiologies of selected isolates and discovered that the majority were indeed capable of growth under low pH and/or high concentrations of heavy metals and nitrate. This indicated that in contrast to conventional isolation, the diffusion chamber-based approach leads to isolation of species that are novel, exhibit tolerance to extant environmental conditions, and match some of the species previously discovered by molecular methods.
Bioprospecting of facultatively oligotrophic bacteria from non-rhizospheric soils
A B S T R A C T Conventional cultivation method relies on the use of nutrient-rich medium, which might limit the exploration of oligotrophic bacteria from soil ecosystems. In the present study attempts were made to isolate oligotrophic bacteria from non-rhizospheric samples collected from mountain roadside, beach, bay and wetland using 1000-fold diluted nutrient agar. Phylogenetic analysis and biogeographic studies of these isolates were performed based on their 16S rDNA sequences. Besides, tests for the utilization of methanol or agar as their carbon source (methylotrophic or agarolytic), light as their energy source (photoheterotrophic), fixation of nitrogen or solubilization of tricalcium phosphate were conducted. A total of 74 isolates with facultatively oligotrophic behavior were obtained, since they can proliferate on both 1000-fold diluted and conventional nutrient agar. 16S rDNA sequence analyses assigned them mainly to class Actinobacteria and a-Proteobacteria. All these isolates belonged to 24 genera which encompassed 35 species, demonstrating that these facultatively oligotrophic bacteria occupied a wide range of bacterial lineages. Fifty-seven out of 74 isolates formed colonies on methanol-containing agar, while only 21 showed better growth in methanol-containing medium. Nine isolates were verified to utilize agar instead of methanol for growth. A total of 4 isolates showed photoheterotrophic trait and the pufLM gene was successfully PCR amplified and sequenced. The methylotrophic or photoheterotrophic behaviors might provide advantages for them to inhabit oligotrophic environments. Members affiliated with many genera were first demonstrated to fix nitrogen or solubilize phosphate, and isolates with profound activities have potentials to be developed as bioinoculants used to promote plant growth. In addition, the use of diluted nutrient agar helped to explore several unrecognized species from non-rhizospheric soils. All these isolates may provide opportunities for studying their oligotrophic growth, metabolism and interaction with plants in a near future.
Journal of Advanced Research, 2015
Our previous publications and the data presented here provide evidences on the ability of plantbased culture media to optimize the cultivability of rhizobacteria and to support their recovery from plant-soil environments. Compared to the tested chemically-synthetic culture media (e.g. nutrient agar and N-deficient combined-carbon sources media), slurry homogenates, crude saps, juices and powders of cactus (Opuntia ficus-indica) and succulent plants (Aloe vera and Aloe arborescens) were rich enough to support growth of rhizobacteria. Representative isolates of Enterobacter spp., Klebsiella spp., Bacillus spp. and Azospirillum spp. exhibited good growth on agar plates of such plant-based culture media. Cell growth and biomass production in liquid batch cultures were comparable to those reported with the synthetic culture media. In addition, the tested plant-based culture media efficiently recovered populations of rhizobacteria associated to plant roots. Culturable populations of >10 6-10 8 cfu g À1 were recovered from the ecto-and endo-rhizospheres of tested host plants. More than 100 endophytic culturedependent isolates were secured and subjected to morphophysiological identification. Factor and cluster analyses indicated the unique community structure, on species, genera, class and phyla levels, of the culturable population recovered with plant-based culture media, being distinct from that obtained with the chemically-synthetic culture media. Proteobacteria were the dominant (78.8%) on plant-based agar culture medium compared to only 31% on nutrient agar,