Biofilm formation by zygomycetes: quantification, structure and matrix composition

Abstract

Most studies on fungal biofilms have focused on Candida in yeasts and Aspergillus in mycelial fungi. To the authors’ knowledge, biofilm formation by zygomycetes has not been reported previously. In this study, the biofilm-forming capacity of Rhizopus oryzae, Lichtheimia corymbifera, Rhizomucor pusillus and Apophysomyces elegans was evaluated. At appropriate seeding spore densities, Rhp . oryzae (105 c.f.u. ml−1), L. corymbifera (104 c.f.u. ml−1) and Rhm. pusillus (104 c.f.u. ml−1) produced highly intertwined, adherent structures on flat-bottomed polystyrene microtitre plates after 24 h at 37 °C. The adhered fungal hyphae were encased in an extracellular matrix, as confirmed by phase-contrast and confocal microscopy. The thickness of Rhp. oryzae, L. corymbifera and Rhm. pusillus biofilms was 109.67±10.02, 242±23.07 and 197±9.0 µm (mean±sd), respectively. Biochemical characterization of the biofilm matrix indicated the presence of glucosamine, constituting 74.54–82.22 % of its dry weight, N-acetylglucosamine, glucose and proteins. Adherence and biofilm formation were not observed in A. elegans. Although A. elegans spores germinated at all three seeding densities tested (1×107, 1×106 and 1×105 c.f.u. ml−1), no significant difference was observed (P>0.05) between the A 490 of wells inoculated with A. elegans and the cut-off A 490 for biofilm detection. This study highlights the potential for biofilm formation by at least three medically important species of zygomycetes.

Filamentous fungal biofilm for production of human drug metabolites

“…These data strongly suggest that C. elegans biofilm matrix consists mainly of polysaccharides while the presence of proteins and DNA is scarce. The results are in accordance with the previous finding reporting that EPS of selected zygomycetes was primarily composed of amino sugars with proteins present in small quantities (Singh et al 2011). …”

“…These conditions are very similar to those that resulted in optimum biofilm growth of other zygomycete fungi (Singh et al, 2011).…”

“…It was recently reported that selected zygomycete strains showed biofilm-forming capacity on flat-bottomed polystyrene microtiter plates (Singh et al 2011). The starting inoculum concentration, presence of nutrients and charged surfaces may strongly influence biofilm formation and adhesion (Djordjevic et al 2002;Cerca et al 2005;Pierce et al 2008), thus these factors were investigated in relation to C. elegans.…”

“…Singh et al (2011) determined that the main monomeric constituent of the biofilm matrix in other zygomycete fungi was glucosamine and concluded that the matrix was mainly chitosan. It is likely that the matrix of C. elegans has a similar composition, since chitosan is a major cell wall component of this fungus (Stamford et al 2007).…”

“…Recently, Singh et al (2011) reported the first observations of zygomycete fungi growing as biofilms, by studying the morphology and matrix characteristics of the medically relevant fungi Rhizopus oryzae, Lichtheimia corymbifera and Rhizomucor pusillus. However, as far as we are aware, no work has been reported of a filamentous fungus growing as a biofilm being applied as a biocatalyst for fine chemicals production.…”