Time-Dependent Changes in Morphostructural Properties and Relative Abundances of Contributors in Pleurotus ostreatus/Pseudomonas alcaliphila Mixed Biofilms.

Silvia Crognale,Andrea Firrincieli,Alessandro D’Annibale,Silvia Rita Stazi,Maurizio Petruccioli,Lorena Pesciaroli,Stefano Fedi

doi:10.3389/fmicb.2019.01819

Silvia Crognale, Andrea Firrincieli + Show 5 more

Open Access

https://doi.org/10.3389/fmicb.2019.01819

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Abstract

Pleurotus ostreatus dual biofilms with bacteria are known to be involved in rock phosphate solubilization, endophytic colonization, and even in nitrogen fixation. Despite these relevant implications, no information is currently available on the architecture of P. ostreatus-based dual biofilms. In addition to this, there is a limited amount of information regarding the estimation of the temporal changes in the relative abundances of the partners in such binary systems. To address these issues, a dual biofilm model system with this fungus was prepared by using Pseudomonas alcaliphila 34 as the bacterial partner due to its very fast biofilm-forming ability. The application of the bacterial inoculum to already settled fungal biofilm on a polystyrene surface coated with hydroxyapatite was the most efficient approach to the production of the mixed system the ultrastructure of which was investigated by a multi-microscopy approach. Transmission electron microscopy analysis showed that the adhesion of bacterial cells onto the mycelial cell wall appeared to be mediated by the presence of an abundant layer of extracellular matrix (ECM). Scanning electron microscopy analysis showed that ECM filaments of bacterial origin formed initially a reticular structure that assumed a tabular semblance after 72 h, thus overshadowing the underlying mycelial network. Across the thickness of the mixed biofilms, the presence of an extensive network of channels with large aggregates of viable bacteria located on the edges of their lumina was found by confocal laser scanning microscopy; on the outermost biofilm layer, a significant fraction of dead bacterial cells was evident. Albeit with tangible differences, similar results regarding the estimation of the temporal shifts in the relative abundances of the two partners were obtained by two independent methods, the former relying on qPCR targeting of 16S and 18S rRNA genes and the latter on ester-linked fatty acid methyl esters analysis.

Highlights

Biofilms are microbial communities attached to a surface and encased within a self-produced matrix generally referred to as extracellular polymeric substances (EPSs)
Based on these results and in the attempt at simulating natural systems where the basidiomycetes offer a hyphal network for adhesion and movement of soil bacteria (Deveau et al, 2018; Guennoc et al, 2018), the application of bacterial inocula to already settled fungal biofilms was deemed to be the best approach to mixed biofilm formation
After 6 h from the application of the bacterial inoculum to the established fungal biofilm, the presence of mostly isolated cells or less frequently of aggregated cells P. alcaliphila adhering on the surface-attached mycelium was evident, as shown in Figure 2, and the adhesion appeared to be mediated by the presence of either filaments or aggregates of extracellular matrix (ECM)

Summary

Introduction

Biofilms are microbial communities attached to a surface and encased within a self-produced matrix generally referred to as extracellular polymeric substances (EPSs). One of the most recurring features of biofilm-associated cells is a high tolerance to antimicrobial agents, such as antibiotics, disinfectants, and biocides, which makes biofilms a major problem in clinical and industrial settings (Finkel and Mitchell, 2011; Wolfmeier et al, 2017) These properties have beneficial aspects in bioremediation, where biofilms were often more efficient than planktonic counterparts (Singh et al, 2006; Seneviratne et al, 2008; Pesciaroli et al, 2013a,c; Herath et al, 2014). One of the most widely used methods for testing the susceptibility of microbial biofilm to toxic compounds is TM the Calgary Biofilm Device, referred to as the MBEC device (Ceri et al, 1999) This system consists of a polystyrene lid with 96 downward-protruding pegs that can be adapted to either a standard 96-well microtiter plate or a multichannel trough tray. The large majority of studies dealing with dual fungal/bacterial biofilm systems have relied on the use of dimorphic fungi, such as Candida species (Peters et al, 2010; de Rossi et al, 2014; Sztajer et al, 2014), zygomycetes (Hover et al, 2016), or ectomycorrhizal species (Guennoc et al, 2017, 2018), as the fungal partners

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