How the basidiomycetes respond to biogenic aspartate-bound metals(II) of variable valency in growth media

Current studies on artificial mushroom cultivation are aimed at optimizing mineral nutrition and the delivery of metals(II). Organically bound trace metals are superior to their inorganic precursors. Cu, Mn, Fe, Zn, and Co metal(II) complexation with essential amino acids seems to be a solution to the bioavailability problem, thus making amino acid chelates of biogenic metals relevant for study. In particular, aspartic acid salts potentially could improve cultivable mushroom growing due to use of bioavailable organic compounds of microelements. However, a comprehensive study on mineral nutrition of cultivated mushrooms using biogenic metal chelates has not been conducted previously. This paper is the first to discover and characterize the effect of metal(II) aspartates on growth, biochemical response, antibacterial activity of mycelium submerged cultures, and fruiting-body formation in basidiomycetes Ganoderma lucidum strain 1315, Grifola umbellate strain 1622, Laetiporus sulphureus strain 120707, Lentinula edodes strain F-249, and Pleurotus ostreatus strains 69, BK1702 and HK352. The work was aimed at elucidating and comparing action of the variable-valent metal(II) aspartates on the physiological and biochemical parameters of the basidiomycetes. Glucose- and wheat powder-based nutrient media supplemented with 1½10-4 mol/l Cu(II), Mn(II), Fe(II), Zn(II), and Co(II) aspartates were used to grow mycelia in submerged culture, comparing growth parameters and production of fruiting bodies. Media without any supplements or with 2½10-4 mol/l L-aspartic acid were control. Antimicrobial activity of the metal-containing biosamples against phytopathogenic bacteria Micrococcus luteus B-109, Pectobacterium carotovorum subsp. сarotovorum (strains 603 and MI), Pectobacterium atrosepticum 1043, Pseudomonas fluorescens EL-2.1, Xanthomonas campestris B-610 was determined by agar well diffusion method. A pool of secondary metabolites was analyzed by high performance liquid chromatography/high resolution time-of-flight mass-spectrometry method. Metal levels in specimens were quantified by atomic absorption spectroscopy technique. The fruiting body formation was tested in the lab and upon commercial growing. In lab tests, it was established that amino acid chelates of biogenic metals(II) intensify mycelium growth in liquid-submerged culture and fruiting body formation. Chelates of copper, manganese, zinc, and to a lesser extent iron, exhibited the significant growth-promoting effect on the basidiomycetes' mycelium under the submerged culture conditions, especially in respect to lacquered polypore, umbrella polypore, and sulfur-yellow polypore. The additives of Cu(Asp)2, Mn(Asp)2, Zn(Asp)2 showed only slight stimulation or even inhibition of P. ostreatus 69 growth. Aspartic acid caused a suppressing impact on mycelia accumulation, regardless of the basidiomycetes' taxonomic characteristics. At the oyster mushroom fermentation in the presence of biogenic metal aspartates, the interstrain distinctions occurred among rapidly and slower growing cultures in relation to the metal chelates' exogenic action. Thus, in assays with Cu(Asp)2 and Zn(Asp)2, the strain P. ostreatus BK1702 had an advantage over others in accumulating biomass. Manganese chelate exerted the most profound positive effect on the fast-growing strain P. ostreatus HK352. The latter, however, was suppressed in its development to the greatest extent compared with BK1702 or 69, when the cobalt organic salt appeared in the liquid medium. Earlier we discussed in detail the items related to these substances' increased level resulted from the exogenic action of some compounds. As a biochemical response of cultures to the above aspartates occurrence in the starting nutrient media, the organic substances with double bond, which were not detected in the absence of the same additives, appeared in the growth liquid. These substances were aromatic alcohol 2-phenylethanol, as well as para- hydroxyphenylacetic acid, the latter's maximal extracellular concentration evaluated by the analytical signal being observed at Mn(Asp)2 introduction. According to the data we gathered by physicochemical research, the metal(II) aspartates, notably Mn(Asp)2, Cu(Asp)2 in the growth liquid induced the increased level of 5-hydroxymethyl-2-furaldehyde, dihydropyrone (structurally similar to kojic acid), para- hydroxyphenylacetic acid, which antioxidative properties are important for mushroom culture. Positive impact of the certain combinations of Mn(II), Cu(II), Fe(II), Zn(II) chelate compounds on P. ostreatus vital functions could be efficiently used for elaborating upon scientific foundations and developing the technology of mushroom mineral nutrition, including wide-scale growing. Biogenic metal aspartates could serve as the active ingredient in biopreparations for commercial mushroom culture.