Metagenomic Insights and Genomic Analysis of Phosphogypsum and Its Associated Plant Endophytic Microbiomes Reveals Valuable Actors for Waste Bioremediation

The phosphogypsum (PG) endogenous bacterial community and endophytic bacterial communities of four plants growing in phosphogypsum-contaminated sites, Suaeda fruticosa (SF), Suaeda mollis (SM), Mesembryanthmum nodiflorum (MN) and Arthrocnemum indicum (AI) were investigated by amplicon sequencing. Results highlight a more diverse community of phosphogypsum than plants associated endophytic communities. Additionally, the bacterial culturable communities of phosphogypsum and associated plant endophytes were isolated and their plant-growth promotion capabilities, bioremediation potential and stress tolerance studied. Most of plant endophytes were endowed with plant growth-promoting (PGP) activities and phosphogypsum communities and associated plants endophytes proved highly resistant to salt, metal and antibiotic stress. They also proved very active in bioremediation of phosphogypsum and other organic and inorganic environmental pollutants. Genome sequencing of five members of the phosphogypsum endogenous community showed that they belong to the recently described species Bacillus albus (BA). Genome mining of BA allowed the description of pollutant degradation and stress tolerance mechanisms. Prevalence of this tool box in the core, accessory and unique genome allowed to conclude that accessory and unique genomes are critical for the dynamics of strain acquisition of bioremediation abilities. Additionally, secondary metabolites (SM) active in bioremediation such as petrobactin have been characterized. Taken together, our results reveal hidden untapped valuable bacterial actors for waste remediation

Response Surface Methodology Optimization of an Acidic Protease Produced by Penicillium bilaiae Isolate TDPEF30, a Newly Recovered Endophytic Fungus from Healthy Roots of Date Palm Trees (Phoenix dactylifera L.)

To explore proteolytic activity of endophytic fungi inhabiting date palm roots, a Penicillium bilaiae isolate, displaying the highest level of protease production, has been recovered. Response surface methodology (RSM) was applied to optimize culture conditions for protease production by the fungus. Plackett-Burman design allowed for screening of variables effective in protease production. Results indicated that temperature, initial pH and glucose concentration dramatically affect protease yield. These factors were further optimized using a Box-Behnken design and RSM. A combination of initial pH (6.26), temperature (24.5 °C), glucose (13.75 g/L), NaNO3 (1.5 g/L), MgSO4 (0.2 g/L), KH2PO4 (0.5 g/L) and KCl (0.5 g/L) were optimum for maximum production of protease. A 1086-fold enhancement of protease production was gained after optimization. Biochemical properties of fungal protease including the effect of pH and temperature on the activity and the stability of proteolytic enzyme were determined. Moreover, the influence of carbon and nitrogen sources, metal ions, detergents as well as enzyme inhibitors was investigated. Our results highlighted that protease of Penicillium bilaiae isolate TDPEF30 could be considered as a promising candidate for industrial applications

Date Palm Trees Root-Derived Endophytes as Fungal Cell Factories for Diverse Bioactive Metabolites

Endophytic fungi of healthy and brittle leaf diseased (BLD) date palm trees (Phoenix dactylifera L.) represent a promising source of bioactive compounds with biomedical, industrial, and pharmaceutical applications. The fungal endophytes Penicillium citrinum isolate TDPEF34, and Geotrichum candidum isolate TDPEF20 from healthy and BLD date palm trees, respectively, proved very effective in confrontation assays against three pathogenic bacteria, including two Gram-positive bacteria Bacillus thuringiensis (Bt) and Enterococcus faecalis (Ef), and one Gram-negative bacterium Salmonella enterica (St). They also inhibited the growth of three fungi Trichoderma sp. (Ti), Fusarium sporotrichioides (Fs), Trichoderma sp. (Ts). Additionally, their volatile organic compounds (VOCs) were shown to be in part responsible for the inhibition of Ti and Ts and could account for the full inhibition of Fs. Therefore, we have explored their potential as fungal cell factories for bioactive metabolites production. Four extracts of each endophyte were prepared using different solvent polarities, ethanol (EtOH), ethyl acetate (EtOAc), hexane (Hex), and methanol (MetOH). Both endophyte species showed varying degrees of inhibition of the bacterial and fungal pathogens according to the solvent used. These results suggest a good relationship between fungal bioactivities and their produced secondary metabolites. Targeting the discovery of potential anti-diabetic, anti-hemolysis, anti-inflammatory, anti-obesity, and cytotoxic activities, endophytic extracts showed promising results. The EtOAc extract of G. candidum displayed IC50 value comparable to the positive control diclofenac sodium in the anti-inflammatory assays. Antioxidant activity was evaluated using α,α-diphenyl-β-picrylhydrazyl (DPPH), β-carotene bleaching, reducing power (RP), and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonique) (ABTS) radical scavenging assays. The findings revealed strong anti-oxidant power with an IC50 of 177.55 µg/mL for G. candidum EtOAc extract using DPPH assay, probably due to high polyphenol and flavonoid content in both fungal extracts. Finally, LC-HRMS (Liquid Chromatography–High Resolution Mass Spectrometry) and GC-MS (Gas Chromatography–Mass Spectrometry) analysis of G. candidum and P. citrinum extracts revealed an impressive arsenal of compounds with previously reported biological activities, partly explaining the obtained results. Finally, LC-HRMS analysis indicated the presence of new fungal metabolites that have never been reported, which represent good candidates to follow for the discovery of new bioactive molecules