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Use of mycelia as paths for the isolation of contaminant-degrading bacteria from soil.

Furuno S, Remer R, Chatzinotas A, Harms H, Wick LY - Microb Biotechnol (2011)

Bottom Line: Mycelia of fungi and soil oomycetes have recently been found to act as effective paths boosting bacterial mobility and bioaccessibility of contaminants in vadose environments.Except for Rhodococcus the NAPH-degrading isolates exhibited significant motility as observed in standard swarming and swimming motility assays.Interestingly, a high similarity (63%) between both the cultivable NAPH-degrading migrant and the cultivable parent soil bacterial community profiles was observed.

View Article: PubMed Central - PubMed

Affiliation: Helmholtz Centre for Environmental Research - UFZ, Department of Environmental Microbiology, 04318 Leipzig, Germany.

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Related in: MedlinePlus

Schematic diagram of the work flow of the procedure used in this study. The sketch of the reversed minimal medium agar (MMA) plate depicts the microcosm employing mycelia as paths for the separation and enrichment of NAPH‐degrading bacteria from soil: a potato dextrose agar (PDA) piece inoculated with filamentous P. ultimum was positioned in the lid of an upside‐down placed minimal medium agar (MMA) plate and covered with 1.5 g of NAPH‐contaminated urban soil (containing the bacterial community A prior to addition to the microcosm) allowing for > 0.5 cm headspace to the agar. The oomycete was subsequently allowed to penetrate the headspace and to serve as path for the movement of bacteria to the MMA. Air‐borne NAPH emanating from solid NAPH deposited at > 1 cm distance to the soil served as carbon source. Capital letters indicate the sample label of the T‐RFLP community analysis of bacterial 16S rRNA genes; i.e. enrichment B denominates the wash‐off of the migrant community from the agar surface 5 days after first contact of the hyphae with the agar, which gave rise to isolated colonies on MMA/NAPH (enrichment D) and R2A agar (enrichment C). Simultaneously, bacteria from soil overlying the P. ultimum‐inoculated patch (community E) were enriched on MMA/NAPH (enrichment F). Please refer to Supporting information for detailed description of the microcosms, the cultivation, sample handling techniques, the T‐RFLP analysis and the identification and phylogenetic characterization of the bacterial strains.
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f1: Schematic diagram of the work flow of the procedure used in this study. The sketch of the reversed minimal medium agar (MMA) plate depicts the microcosm employing mycelia as paths for the separation and enrichment of NAPH‐degrading bacteria from soil: a potato dextrose agar (PDA) piece inoculated with filamentous P. ultimum was positioned in the lid of an upside‐down placed minimal medium agar (MMA) plate and covered with 1.5 g of NAPH‐contaminated urban soil (containing the bacterial community A prior to addition to the microcosm) allowing for > 0.5 cm headspace to the agar. The oomycete was subsequently allowed to penetrate the headspace and to serve as path for the movement of bacteria to the MMA. Air‐borne NAPH emanating from solid NAPH deposited at > 1 cm distance to the soil served as carbon source. Capital letters indicate the sample label of the T‐RFLP community analysis of bacterial 16S rRNA genes; i.e. enrichment B denominates the wash‐off of the migrant community from the agar surface 5 days after first contact of the hyphae with the agar, which gave rise to isolated colonies on MMA/NAPH (enrichment D) and R2A agar (enrichment C). Simultaneously, bacteria from soil overlying the P. ultimum‐inoculated patch (community E) were enriched on MMA/NAPH (enrichment F). Please refer to Supporting information for detailed description of the microcosms, the cultivation, sample handling techniques, the T‐RFLP analysis and the identification and phylogenetic characterization of the bacterial strains.

Mentions: This study was motivated by earlier observations that mycelial growth of soil microorganisms enables the dispersal of defined bacterial populations in air‐filled porous media (Leben, 1984; Kohlmeier et al., 2005; Warmink and van Elsas, 2009) suggesting the idea of using ‘fungal highways’ (Kohlmeier et al., 2005), i.e. mycelia as paths for the separation and isolation of contaminant‐degrading bacteria. Figure 1 depicts the experimental set‐up and the work flow for the separation and enrichment of NAPH‐degrading bacteria by migration along the dense mycelial network of P. ultimum (Furuno et al., 2010): bacterial suspensions obtained from the mineral medium agar (MMA) positioned opposite to the soil 5 days after first contact with the hyphae (enrichment B) gave rise to isolated colonies on MMA/NAPH (enrichment D) and R2A‐agar (enrichment C). Simultaneously, bacteria from soil overlying the P. ultimum‐inoculated patch were isolated on MMA/NAPH (enrichment F). Amplified 16S ribosomal DNA restriction analysis (ARDRA) of the 57 isolates picked (based on visually different morphotypes) revealed five distinct operational taxonomic units (OTUs). Three of them were found in both the soil‐community and the migrant communities. 16S rRNA gene sequence analysis of several representatives of each OTU identified the five OTUs (sequence homology within one OTU: 98–100% similarity) as Arthrobacter sp. (n = 3 colonies), Pseudomonas sp. (n = 20), Stenotrophomonas sp. (n = 2), Rhodococcus sp. (n = 18) and Xanthomonas sp. (n = 14) (Table 1). Except for Arthrobacter sp., all OTUs were found in the migrant communities. NAPH‐degrading isolates were further tested for their ability to utilize selected polycyclic aromatic hydrocarbons (PAHs) (i.e. phenanthrene, fluorene, pyrene and anthracene). Except for Stenotrophomonas sp. and Xanthomonas sp., the isolates grew on most of the PAHs tested (Table 1). The mycelia‐based discrimination seems to be driven by the inherent motility of the bacteria. A recent study for instance revealed that mycelia allow for chemotactic movement of PAH‐degrading bacteria to substrate hotspots even in water‐unsaturated systems (Furuno et al., 2010). Such studies support the relevance of mycelial networks for successful colonization of new microhabitats in soil (Wick et al., 2007; Nazir et al., 2010) especially in vadose environments. The results of this study suggest that a majority of the cultivable NAPH‐degrading bacterial consortium (Table 1) may have been capable of using the hyphal network for dispersal. Both enrichments shared three out of the five NAPH‐degrading isolates (Pseudomonas sp., Rhodococcus sp. and Xanthomonas sp.). In contrast, Arthrobacter sp. and Stenotrophomonas sp. were detected only in enrichment F or C, respectively (Fig. 1). Most isolates exhibited either swarming or swimming motility on standard agar plate assays with average colony diameters ≤ of motile soil bacterium Pseudomonas putida PpG7 (NAH7) but ≥ of poorly motile Mycobacterium frederiksbergense LB501T (Table 1). It is remarkable that the poorly motile Arthrobacter sp. appears to be fully retained in the soil, whereas the likewise poorly motile Rhodococcus sp. was found to move along the hydrophilic (Smits et al., 2003) hyphae of P. ultimum. Due to the suspected effect of physicochemical cell surface properties of the bacteria on mycelia‐mediated bacterial transport (Kohlmeier et al., 2005), the water contact angles (θw) and ζ‐potentials of the migrating bacteria were further measured as descriptors for the hydrophobicity and charge of the isolates' cell surfaces. No significant differences, however, of θw were observed with all strains being moderately hydrophobic [θW of 30°–70° (Table 1)] according to the classification by others (Bastiaens et al., 2000). The ζ‐potentials of the isolates ranged from −3 to −46 mV and exhibited no significant difference between isolates derived from enrichments C, D (migrant communities) and F (soil bacterial community) as did the results from motility tests of the same strains.


Use of mycelia as paths for the isolation of contaminant-degrading bacteria from soil.

Furuno S, Remer R, Chatzinotas A, Harms H, Wick LY - Microb Biotechnol (2011)

Schematic diagram of the work flow of the procedure used in this study. The sketch of the reversed minimal medium agar (MMA) plate depicts the microcosm employing mycelia as paths for the separation and enrichment of NAPH‐degrading bacteria from soil: a potato dextrose agar (PDA) piece inoculated with filamentous P. ultimum was positioned in the lid of an upside‐down placed minimal medium agar (MMA) plate and covered with 1.5 g of NAPH‐contaminated urban soil (containing the bacterial community A prior to addition to the microcosm) allowing for > 0.5 cm headspace to the agar. The oomycete was subsequently allowed to penetrate the headspace and to serve as path for the movement of bacteria to the MMA. Air‐borne NAPH emanating from solid NAPH deposited at > 1 cm distance to the soil served as carbon source. Capital letters indicate the sample label of the T‐RFLP community analysis of bacterial 16S rRNA genes; i.e. enrichment B denominates the wash‐off of the migrant community from the agar surface 5 days after first contact of the hyphae with the agar, which gave rise to isolated colonies on MMA/NAPH (enrichment D) and R2A agar (enrichment C). Simultaneously, bacteria from soil overlying the P. ultimum‐inoculated patch (community E) were enriched on MMA/NAPH (enrichment F). Please refer to Supporting information for detailed description of the microcosms, the cultivation, sample handling techniques, the T‐RFLP analysis and the identification and phylogenetic characterization of the bacterial strains.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815281&req=5

f1: Schematic diagram of the work flow of the procedure used in this study. The sketch of the reversed minimal medium agar (MMA) plate depicts the microcosm employing mycelia as paths for the separation and enrichment of NAPH‐degrading bacteria from soil: a potato dextrose agar (PDA) piece inoculated with filamentous P. ultimum was positioned in the lid of an upside‐down placed minimal medium agar (MMA) plate and covered with 1.5 g of NAPH‐contaminated urban soil (containing the bacterial community A prior to addition to the microcosm) allowing for > 0.5 cm headspace to the agar. The oomycete was subsequently allowed to penetrate the headspace and to serve as path for the movement of bacteria to the MMA. Air‐borne NAPH emanating from solid NAPH deposited at > 1 cm distance to the soil served as carbon source. Capital letters indicate the sample label of the T‐RFLP community analysis of bacterial 16S rRNA genes; i.e. enrichment B denominates the wash‐off of the migrant community from the agar surface 5 days after first contact of the hyphae with the agar, which gave rise to isolated colonies on MMA/NAPH (enrichment D) and R2A agar (enrichment C). Simultaneously, bacteria from soil overlying the P. ultimum‐inoculated patch (community E) were enriched on MMA/NAPH (enrichment F). Please refer to Supporting information for detailed description of the microcosms, the cultivation, sample handling techniques, the T‐RFLP analysis and the identification and phylogenetic characterization of the bacterial strains.
Mentions: This study was motivated by earlier observations that mycelial growth of soil microorganisms enables the dispersal of defined bacterial populations in air‐filled porous media (Leben, 1984; Kohlmeier et al., 2005; Warmink and van Elsas, 2009) suggesting the idea of using ‘fungal highways’ (Kohlmeier et al., 2005), i.e. mycelia as paths for the separation and isolation of contaminant‐degrading bacteria. Figure 1 depicts the experimental set‐up and the work flow for the separation and enrichment of NAPH‐degrading bacteria by migration along the dense mycelial network of P. ultimum (Furuno et al., 2010): bacterial suspensions obtained from the mineral medium agar (MMA) positioned opposite to the soil 5 days after first contact with the hyphae (enrichment B) gave rise to isolated colonies on MMA/NAPH (enrichment D) and R2A‐agar (enrichment C). Simultaneously, bacteria from soil overlying the P. ultimum‐inoculated patch were isolated on MMA/NAPH (enrichment F). Amplified 16S ribosomal DNA restriction analysis (ARDRA) of the 57 isolates picked (based on visually different morphotypes) revealed five distinct operational taxonomic units (OTUs). Three of them were found in both the soil‐community and the migrant communities. 16S rRNA gene sequence analysis of several representatives of each OTU identified the five OTUs (sequence homology within one OTU: 98–100% similarity) as Arthrobacter sp. (n = 3 colonies), Pseudomonas sp. (n = 20), Stenotrophomonas sp. (n = 2), Rhodococcus sp. (n = 18) and Xanthomonas sp. (n = 14) (Table 1). Except for Arthrobacter sp., all OTUs were found in the migrant communities. NAPH‐degrading isolates were further tested for their ability to utilize selected polycyclic aromatic hydrocarbons (PAHs) (i.e. phenanthrene, fluorene, pyrene and anthracene). Except for Stenotrophomonas sp. and Xanthomonas sp., the isolates grew on most of the PAHs tested (Table 1). The mycelia‐based discrimination seems to be driven by the inherent motility of the bacteria. A recent study for instance revealed that mycelia allow for chemotactic movement of PAH‐degrading bacteria to substrate hotspots even in water‐unsaturated systems (Furuno et al., 2010). Such studies support the relevance of mycelial networks for successful colonization of new microhabitats in soil (Wick et al., 2007; Nazir et al., 2010) especially in vadose environments. The results of this study suggest that a majority of the cultivable NAPH‐degrading bacterial consortium (Table 1) may have been capable of using the hyphal network for dispersal. Both enrichments shared three out of the five NAPH‐degrading isolates (Pseudomonas sp., Rhodococcus sp. and Xanthomonas sp.). In contrast, Arthrobacter sp. and Stenotrophomonas sp. were detected only in enrichment F or C, respectively (Fig. 1). Most isolates exhibited either swarming or swimming motility on standard agar plate assays with average colony diameters ≤ of motile soil bacterium Pseudomonas putida PpG7 (NAH7) but ≥ of poorly motile Mycobacterium frederiksbergense LB501T (Table 1). It is remarkable that the poorly motile Arthrobacter sp. appears to be fully retained in the soil, whereas the likewise poorly motile Rhodococcus sp. was found to move along the hydrophilic (Smits et al., 2003) hyphae of P. ultimum. Due to the suspected effect of physicochemical cell surface properties of the bacteria on mycelia‐mediated bacterial transport (Kohlmeier et al., 2005), the water contact angles (θw) and ζ‐potentials of the migrating bacteria were further measured as descriptors for the hydrophobicity and charge of the isolates' cell surfaces. No significant differences, however, of θw were observed with all strains being moderately hydrophobic [θW of 30°–70° (Table 1)] according to the classification by others (Bastiaens et al., 2000). The ζ‐potentials of the isolates ranged from −3 to −46 mV and exhibited no significant difference between isolates derived from enrichments C, D (migrant communities) and F (soil bacterial community) as did the results from motility tests of the same strains.

Bottom Line: Mycelia of fungi and soil oomycetes have recently been found to act as effective paths boosting bacterial mobility and bioaccessibility of contaminants in vadose environments.Except for Rhodococcus the NAPH-degrading isolates exhibited significant motility as observed in standard swarming and swimming motility assays.Interestingly, a high similarity (63%) between both the cultivable NAPH-degrading migrant and the cultivable parent soil bacterial community profiles was observed.

View Article: PubMed Central - PubMed

Affiliation: Helmholtz Centre for Environmental Research - UFZ, Department of Environmental Microbiology, 04318 Leipzig, Germany.

Show MeSH
Related in: MedlinePlus