Volatiles Mediated Interactions Between Aspergillus oryzae Strains Modulate Morphological Transition and Exometabolomes

Abstract

Notwithstanding its mitosporic nature, an improbable morpho-transformation state i. e., sclerotial development (SD), is vaguely known in Aspergillus oryzae. Nevertheless an intriguing phenomenon governing mold's development and stress response, the effects of exogenous factors engendering SD, especially the volatile organic compounds (VOCs) mediated interactions (VMI) pervasive in microbial niches have largely remained unexplored. Herein, we examined the effects of intra-species VMI on SD in A. oryzae RIB 40, followed by comprehensive analyses of associated growth rates, pH alterations, biochemical phenotypes, and exometabolomes. We cultivated A. oryzae RIB 40 (S1VMI: KACC 44967) opposite a non-SD partner strain, A. oryzae (S2: KCCM 60345), conditioning VMI in a specially designed “twin plate assembly.” Notably, SD in S1VMI was delayed relative to its non-conditioned control (S1) cultivated without partner strain (S2) in twin plate. Selectively evaluating A. oryzae RIB 40 (S1VMI vs. S1) for altered phenotypes concomitant to SD, we observed a marked disparity for corresponding growth rates (S1VMI < S1)7days, media pH (S1VMI > S1)7days, and biochemical characteristics viz., protease (S1VMI > S1)7days, amylase (S1VMI > nS1)3–7days, and antioxidants (S1VMI > S1)7days levels. The partial least squares—discriminant analysis (PLS-DA) of gas chromatography—time of flight—mass spectrometry (GC-TOF-MS) datasets for primary metabolites exhibited a clustered pattern (PLS1, 22.04%; PLS2, 11.36%), with 7 days incubated S1VMI extracts showed higher abundance of amino acids, sugars, and sugar alcohols with lower organic acids and fatty acids levels, relative to S1. Intriguingly, the higher amino acid and sugar alcohol levels were positively correlated with antioxidant activity, likely impeding SD in S1VMI. Further, the PLS-DA (PLS1, 18.11%; PLS2, 15.02%) based on liquid chromatography—mass spectrometry (LC-MS) datasets exhibited a notable disparity for post-SD (9–11 days) sample extracts with higher oxylipins and 13-desoxypaxilline levels in S1VMI relative to S1, intertwining Aspergillus morphogenesis and secondary metabolism. The analysis of VOCs for the 7 days incubated samples displayed considerably higher accumulation of C-8 compounds in the headspace of twin-plate experimental sets (S1VMI:S2) compared to those in non-conditioned controls (S1 and S2—without respective partner strains), potentially triggering altered morpho-transformation and concurring biochemical as well as metabolic states in molds.

Non-obligate pairwise metabolite cross-feeding suggests ammensalic interactions between Bacillus amyloliquefaciens and Aspergillus oryzae

“…Aspergillus was cultured using freshly harvested conidia from WATM agar plates incubated for 216 h. Subsequently, 0.1% of the conidia suspension (~10 7 conidia/mL) was inoculated into CMM (100 mL) in 500 mL Erlenmeyer flasks (baffled type) and incubated at 30 °C and 200 rpm for 216 h. Cultures were harvested temporally at 0, 120, 168, and 216 h, and analyzed for conidial density (counts/mL), mycelial dry weight (mg/mL), and metabolite extraction. The growth rates of Aspergillus species were estimated based on conidiation and mycelial dry weight data using the method described by Singh and Lee 33 . Culture broth corresponding to the late-log growth phase was filtered using 0.2 μm bottle-top vacuum filter (Corning Inc. NY, USA).…”

Non-Obligate Pairwise Metabolite Cross-Feeding Suggests Ammensalic Interactions Between Bacillus and Aspergillus Species

“…Aspergillus was cultured using freshly harvested conidia from WATM agar plates incubated for 216 h. Subsequently, 0.1% of the conidia suspension (~10 7 conidia/mL) was inoculated into CMM (100 mL) in 500 mL Erlenmeyer asks (ba ed type) and incubated at 30 °C and 200 rpm for 216 h. Cultures were harvested temporally at 0, 120, 168, and 216 h, and analyzed for conidial density (counts/mL), mycelial dry weight (mg/mL), and metabolite extraction. The growth rates of Aspergillus species were estimated based on conidiation and mycelial dry weight data using the method described by Singh and Lee [27].…”

Effects of the Addition of Herbs on the Properties of Doenjang

“…The DPPH assay was performed with the method adapted from Dietz et al with some modifications [28,29]. For the DPPH assay, the doenjang extract (20 µL), which has been described in Section 2.2, was mixed with the DPPH solution (180 µL, 0.2 mM in ethanol) in a 96-well plate, followed by incubation for 20 min at room temperature in the dark.…”

“…Samples containing coriander, Korean mint, and peppermint were grouped on day 30 and day 150, except for control samples, indicating that each herb affected the primary metabolite as the fermentation progressed. To select the significant primary metabolites present in the doenjang samples using the PLS-DA model, the (VIP) value (>0.7) and p-value (<0.05) were determined from the GC-TOF-MS dataset [29]. These metabolites were subsequently identified using the standard compounds followed by comparing the resulting mass fragmentation patterns with the inhouse library (Supplementary Table S1).…”