Use of mycelia as paths for the isolation of contaminant-degrading bacteria from soil.
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.
Affiliation: Helmholtz Centre for Environmental Research - UFZ, Department of Environmental Microbiology, 04318 Leipzig, Germany.Show MeSH
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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.
Affiliation: Helmholtz Centre for Environmental Research - UFZ, Department of Environmental Microbiology, 04318 Leipzig, Germany.