Characterization of Pleurotus ostreatus biofilms by using the calgary biofilm device

Abstract

The adequacy of the Calgary biofilm device, often referred to as the MBEC system, as a high-throughput approach to the production and subsequent characterization of Pleurotus ostreatus biofilms was assessed. The hydroxyapatite-coating of pegs was necessary to enable biofilm attachment, and the standardization of vegetative inocula ensured a uniform distribution of P. ostreatus biofilms, which is necessary for high-throughput evaluations of several antimicrobials and exposure conditions. Scanning electron microscopy showed surface-associated growth, the occurrence of a complex aggregated growth organized in multilayers or hyphal bundles, and the encasement of hyphae within an extracellular matrix (ECM), the extent of which increased with time. Chemical analyses showed that biofilms differed from free-floating cultures for their higher contents of total sugars (TS) and ECM, with the latter being mainly composed of TS and, to a lesser extent, protein. Confocal laser scanning microscopy analysis of 4-day-old biofilms showed the presence of interspersed interstitial voids and water channels in the mycelial network, the density and compactness of which increased after a 7-day incubation, with the novel occurrence of ECM aggregates with an α-glucan moiety. In 4- and 7-day-old biofilms, tolerance to cadmium was increased by factors of 3.2 and 11.1, respectively, compared to coeval free-floating counterparts.

Fig 1

Average P. ostreatus biofilm production, inferred by MTT reduction, on column pegs of the MBEC P&G device after 4 (■) and 7 days (□) growth on the LB medium at 30°C under orbital shaking (150 rpm). The data represent means ± the standard deviations of quadruplicate plates.

Fig 2

Total sugars (TS), lipid, protein, chitin, and ash contents and percent amounts of extracted ECM in free-floating (FF) and biofilm (B) biomasses after 4 and 7 days incubation in the MBEC P&G system at 30°C under orbital shaking (150 rpm). The inset shows the percent composition, referred to as the ECM dry weight, of protein and TS for each biomass type. With the sole exceptions of ECM and ash, data are the means ± the standard deviations of nine determinations (three replicates for three samples), and same lowercase letters above bars indicate the lack of statistically significant differences for each parameter among culture systems. ECM and ash determinations were performed in triplicate.

Fig 3

(A to D) SEM micrographs of P. ostreatus biofilms grown on the HA-P&G system and LB medium for 4 days (A and B) and 7 days (C and D) at 30°C (150 rpm). Insets show details at higher magnifications. (E and F) TEM micrographs of longitudinal and cross-view sections of a biofilm's constituent hypha, respectively. Abbreviations: DS, dolipore septum; ECM, extracellular matrix; HB, hyphal bundle; PS, parenthosome; SV, secretory vesicles; V, vacuoles.

Fig 4

CLSM images of 4-day-old (A to D) and 7-day-old (E to H) P. ostreatus biofilms grown on the HA-P&G device under orbital shaking (150 rpm) at 30°C and sequentially stained with concanavalin A conjugated with Texas Red (red emission) and Calcofluor White Stain M2R (green emission). Images A and E are horizontal (xy) and vertical side (xz and yz) views of three-dimensional reconstructed images of 4- and 7-day-old biofilms obtained by volume rendering, respectively, as described by Harrison et al. (32). Isosurfaces of 4- and 7-day-old biofilms are shown in panels B to D and F to H, respectively. Each image represents an area of 375 by 375 μm. The light blue and white arrows in panels A and E indicate a hydroxyapatite granule and ECM aggregates, respectively. The x, y, and z axes are coded in red, green, and blue, respectively. The direction of the blue arrow (z axis) is oriented toward the outermost biofilm layers.

Fig 5

(A to F) SEM micrographs of unperturbed 4- and 7-day-old P. ostreatus free-floating (A and C, respectively) and biofilm (E and G, respectively) cultures compared to their respective counterparts (B and D, respectively, and F and H, respectively) exposed to cadmium concentrations equal to their IC₉₀s (see Table 1) at 30°C for 48 h in the LB medium under orbital shaking (150 rpm).