Comparative Examination of the Olive Mill Wastewater Biodegradation Process by Various Wood-Rot Macrofungi

Olive mill wastewater (OMW) constitutes a major cause of environmental pollution in olive-oil producing regions. Sixty wood-rot macrofungi assigned in 43 species were evaluated for their efficacy to colonize solidified OMW media at initially established optimal growth temperatures. Subsequently eight strains of the following species were qualified: Abortiporus biennis, Ganoderma carnosum, Hapalopilus croceus, Hericium erinaceus, Irpex lacteus, Phanerochaete chrysosporium, Pleurotus djamor, and P. pulmonarius. Fungal growth in OMW (25%v/v in water) resulted in marked reduction of total phenolic content, which was significantly correlated with the effluent's decolorization. A. biennis was the best performing strain (it decreased phenolics by 92% and color by 64%) followed by P. djamor and I. lacteus. Increase of plant seeds germination was less pronounced evidencing that phenolics are only partly responsible for OMW's phytotoxicity. Laccase production was highly correlated with all three biodegradation parameters for H. croceus, Ph. chrysosporium, and Pleurotus spp., and so were manganese-independent and manganese dependent peroxidases for A. biennis and I. lacteus. Monitoring of enzymes with respect to biomass production indicated that Pleurotus spp., H. croceus, and Ph. chrysosporium shared common patterns for all three activities. Moreover, generation of enzymes at the early biodegradation stages enhanced the efficiency of OMW treatment

Bioconversion of agricultural by-products into detoxified derivatives and edible biomass through the use of selected strains of basidiomycetes

The agricultural industry produces every year a huge amount of by-products generated from the processing of primary products and raw materials. The effective management of the resulting residues constitutes a great environmental challenge for both the industry and scientific community.The use of saprotrophic fungi, which are able to decompose lignin, cellulose and hemicellulose through the production of oxidative enzymes, offers a potent solution in the management of lignocellulosic waste. Furthermore, the presence of carbon sources and nutrients in these materials provide suitable conditions for the development of microorganisms, and therefore permit the bio-transformation of agro-industrial and forest residues into edible biomass under solid-state fermentation (SSF) conditions. In particular, the SSF process is suitable for the production of edible or/and medicinal mushrooms of high organoleptic and nutritional characteristics. Additionally, mushrooms contain bioactive ingredients that have been associated with anticancer, antidiabetic, anti-inflammatory, immunomodulatory and other health-beneficial properties.In the frame of this Thesis, the cultivation process of several edible and medicinal mushrooms was evaluated, in terms of growth performance and yield, through the biotransformation of lignocellulosic residues. The intraspecific variability of Pleurotus osrteatus, one of the most-widely cultivated mushrooms, was initially assessed in respect to cultivation parameters and mushroom quality aspects by using 16 different strains under identical conditions (substrate, environmental conditions). P. ostreatus strains demonstrated a significant intra-variability/heterogeneity both in cultivation parameters (yield, biological efficiency, productivity etc.) and in bioactive compounds content (e.g. sterols, glucans, antioxidants etc.) by showing a strong dependence on intrinsic/genetic factors. Results also contributed at selecting P. ostreatus strains for use in subsequent experiments. Production of P. ostreatus and Cyclocybe cylindracea mushrooms on nine different lignocellulosic by-products was then investigated while the effect of substrates on mushrooms composition was also assessed. The highest biological efficiency was noted with substrates containing olive oil mill or winery by-products for both species examined. Furthermore, significant differences were observed in crude composition of mushrooms produced in different media.Consequently, this study focused on the evaluation of winery residues (grape marc) and olive oil by-products as substrates for the cultivation of eight species of the genera5Pleurotus, Cyclocybe, Hericium and Ganoderma. Results demonstrated that the use of these substrates (in mixtures with materials ordinarily used in conventional cultivation, e.g. wheat straw) not only improves mushroom productivity, but it also highly increase mushrooms content in bioactive components. Furthermore, the reduction of fibrous components in spent cultivation substrates facilitates their subsequent management without requiring additional processing.At a further stage, a large number of basidiomycetes (white-rot fungi) were comparatively examined as regards the detoxification of olive mill wastewater (OMW), and the most efficient were selected to study OMW’s degradation and the role of ligninolytic enzymes produced in the respective process. In addition to the high reduction of OMW phenolics content, colour and toxicity, encouraging results were also obtained when crude enzyme extracts were used from the cultivation of Abortiporus biennis under optimized conditions.Finally, preliminary studies were performed aiming at examining the effect of biotreated two-phase olive mill waste on vegetable crops; results demonstrated that the final product could be used both as substrate for the germination of tomato and lettuce seeds, and the growth of lettuce plants, contributing thus to the reduction in applications of chemical fertilizers and to the valorization of olive oil by-products.Η αξιοποίηση αγροβιομηχανικών αποβλήτων και υπολειμμάτων αποτελεί σημαντική πρόκληση για την επιστημονική κοινότητα, ιδιαίτερα αν ληφθούν υπόψιν η αφθονία και η εποχικότητα της παραγωγής καθώς και οι φυσικοχημικές ιδιότητες (και ως εκ τούτου η δυσκολία στην επεξεργασία) τους. Η χρήση μακρομυκήτων, οι οποίοι αναπτύσσονται στη φύση σαπροτροφικά εκκρίνοντας συγκεκριμένα οξειδωτικά ένζυμα για την αποδόμηση της λιγνίνης, των ημικυτταρινών και της κυτταρίνης, μπορεί να προσφέρει λύσεις για την ολοκληρωμένη διαχείριση τέτοιων υλικών. Αξίζει να σημειωθεί, ότι οι εν λόγω μικροοργανισμοί αποδεικνύονται ιδιαίτερα αποδοτικοί στη βιομετατροπή διαφόρων γεωργικών, αγροβιομηχανικών και δασικών παραπροϊόντων σε εδώδιμη βιομάζα. Η συγκεκριμένη διαδικασία συνιστά μια ελεγχόμενη ζύμωση στερεάς φάσης που οδηγεί στην παραγωγή εδώδιμων ή/και φαρμακευτικών μανιταριών, παρέχοντας κατ’ αυτό τον τρόπο, τρόφιμα υψηλής οργανοληπτικής και διαιτητικής αξίας. Επιπροσθέτως, τα βιοδραστικά συστατικά που περιέχουν έχει αποδειχθεί ότι σχετίζονται με αντικαρκινικές, αντιδιαβητικές, αντιφλεγμονώδεις, ανοσορυθμιστικές και άλλες ευεργετικές για την ανθρώπινη υγεία ιδιότητες.Στα πλαίσια της παρούσας διατριβής πραγματοποιήθηκαν εργασίες οι οποίες συνδέονται ευθέως με τους στόχους που προαναφέρθηκαν και μπορούν να ομαδοποιηθούν σε δύο επιμέρους ενότητες. Η πρώτη, αναφέρεται στη βιομετατροπή λιγνοκυτταρινούχων υπολειμμάτων με τη χρήση επιλεγμένων βασιδιομυκήτων για την παραγωγή εδώδιμων – φαρμακευτικών μανιταριών. Αρχικά μελετήθηκε η ενδοειδική ποικιλότητα που εμφανίζει το Pleurotus ostreatus, δηλαδή ένα από τα πιο ευρέως καλλιεργούμενα είδη μανιταριών, όσον αφορά στις καλλιεργητικές παραμέτρους και επιλεγμένα ποιοτικά χαρακτηριστικά των μανιταριών που παράγονται υπό όμοιες συνθήκες (υπόστρωμα, περιβαλλοντικές παράμετροι ανάπτυξης). Ανάμεσα στα 16 στελέχη που εξετάστηκαν διαπιστώθηκε μεγάλο εύρος στις τιμές παραμέτρων που σχετίζονται με την αποδοτικότητα της καλλιέργειας αλλά και την περιεκτικότητα των βιοδραστικών συστατικών. Τα εν λόγω αποτελέσματα συνέβαλαν και στην επιλογή συγκεκριμένων στελεχών P. ostreatus προς αξιολόγηση σε επόμενα πειράματα. Ακολούθως, διερευνήθηκε η παραγωγή μανιταριών σε μεγάλο εύρος διαφορετικών λιγνοκυτταρινούχων παραπροϊόντων χρησιμοποιώντας δύο είδη βασιδιομυκήτων (P. ostreatus και Cyclocybe cylindracea) όπως και η επίδραση που τα συγκεκριμένα υποστρώματα παρουσιάζουν στη σύσταση των καρποφοριών. Σχετικά με τη βιολογική αποδοτικότητα της καλλιεργητικής διαδικασίας, ξεχώρισαν τα υποστρώματα που2περιείχαν παραπροϊόντα οινοποιίας και ελαιοκομίας και για τα δύο είδη βασιδιομυκήτων, ενώ παρατηρήθηκαν σημαντικές διαφορές στη βασική σύσταση των μανιταριών που αναπτύχθηκαν σε διαφορετικά υλικά. Συν τοις άλλοις, η χρήση μεγάλου εύρους υποστρωμάτων έδωσε τη δυνατότητα για τη μελέτη της επίδρασης των επιμέρους συστατικών του υποστρώματος τόσο στις καλλιεργητικές παραμέτρους όσο και στη σύσταση των μανιταριών.Στη συνέχεια, η παρούσα μελέτη εστίασε στην αξιολόγηση υπολειμμάτων οινοποιίας και αποβλήτων-παραπροϊόντων ελαιουργίας και ελαιοκομίας ως προς την καταλληλότητα τους να υποστηρίξουν την καλλιέργεια διαφορετικών ειδών βασιδιομυκήτων (οκτώ είδη από τα γένη Pleurotus, Cyclocybe, Hericium και Ganoderma). Σε όλες τις περιπτώσεις, αποδείχθηκε ότι τα παραπάνω υπολείμματα μπορούν (σε κατάλληλες αναλογίες με υλικά που χρησιμοποιούνται στη συμβατική καλλιέργεια) όχι μόνο να βελτιώσουν την απόδοση σε παραγόμενα μανιτάρια, αλλά να αυξήσουν σημαντικά τη συγκέντρωση συγκεκριμένων βιοδραστικών συστατικών, σημαντικών για την ανθρώπινη υγεία. Επιπλέον, η μείωση των δύσκολα αποδομήσιμων συστατικών στα εξαντλημένα υποστρώματα της καλλιέργειας, επιτρέπει την εύκολη διαχείριση τους χωρίς να απαιτείται επιπλέον επεξεργασία τους.Η δεύτερη ενότητα περιλαμβάνει τη διαχείριση υγρών αποβλήτων ελαιοτριβείου (ΥΑΕ) με τη χρήση μυκήτων λευκής σήψης. Αρχικά, εξετάστηκε μεγάλο πλήθος διαφορετικών ειδών βασιδιομυκήτων όσον αφορά στην αποτοξικοποίηση ΥΑΕ και στη συνέχεια τα αποδοτικότερα από αυτά χρησιμοποιήθηκαν περαιτέρω για να αποσαφηνιστεί ο ρόλος των ενζύμων που εκκρίνουν στη διαδικασία βιοαποδόμησης. Εκτός από την υψηλή μείωση των ρυπαντικών παραμέτρων των ΥΑΕ μετά την ανάπτυξη των αποδοτικότερων μυκητιακών στελεχών, πολύ ενθαρρυντικά αποτελέσματα σημειώθηκαν και με την χρήση εκχυλισμάτων ενζύμων που προήλθαν από την καλλιέργεια του μύκητα Abortiporus biennis υπό βελτιστοποιημένες συνθήκες. Τέλος, πραγματοποιήθηκαν προκαταρκτικές δοκιμές αξιοποίησης βιοεπεξεργασμένου αποβλήτου ελαιοτριβείου στην παραγωγή οργανικών εδαφοβελτιωτικών και αξιολογήθηκε η εφαρμογή του σε καλλιέργεια κηπευτικών, αποδεικνύοντας ότι το τελικό προϊόν θα μπορούσε να εφαρμοστεί τόσο ως υπόστρωμα για τη βλάστηση σπόρων ντομάτας και μαρουλιού όσο και ανάπτυξης φυτών μαρουλιού, μειώνοντας τη χρήση χημικών λιπασμάτων και συμβάλλοντας στην περαιτέρω αξιοποίηση των ελαιουργικών παραπροϊόντων

Composted versus Raw Olive Mill Waste as Substrates for the Production of Medicinal Mushrooms: An Assessment of Selected Cultivation and Quality Parameters

Two-phase olive mill waste (TPOMW, “alperujo”) is a highly biotoxic sludge-like effluent of the olive-oil milling process with a huge seasonal production. One of the treatment approaches that has so far received little attention is the use of TPOMW as substrate for the cultivation of edible mushrooms. Fifteen fungal strains belonging to five species (Basidiomycota), that is, Agrocybe cylindracea, Pleurotus cystidiosus, P. eryngii, P. ostreatus, and P. pulmonarius, were evaluated for their efficacy to colonize media composed of TPOMW, which was used either raw or composted in mixtures with wheat straw in various ratios. Qualified strains exhibited high values of biological efficiency (e.g., 120–135% for Pleurotus spp. and 125% for A. cylindracea) and productivity in subsequent cultivation experiments on substrates supplemented with 20–40% composted TPOMW or 20% raw TPOMW. Only when supplementation exceeded 60% for raw TPOMW, a negative impact was noted on mushroom yields which could be attributed to the effluent's toxicity (otherwise alleviated in the respective composted TPOMW medium). Earliness and mushroom size as well as quality parameters such as total phenolic content and antioxidant activity did not demonstrate significant differences versus the control wheat-straw substrate. The substrates hemicellulose content was negatively correlated with mycelium growth rates and yields and positively with earliness; in addition, cellulose: lignin ratio presented a positive correlation with mycelium growth and mushroom weight for A. cylindracea and with earliness for all species examined. TPOMW-based media revealed a great potential for the substitution of traditional cultivation substrates by valorizing environmentally hazardous agricultural waste

Elemental Content in Pleurotus ostreatus and Cyclocybe cylindracea Mushrooms: Correlations with Concentrations in Cultivation Substrates and Effects on the Production Process

Few data exist about the effect of substrates’ elemental content on the respective concentrations in cultivated mushrooms, on the degradation of lignocellulosics or on production parameters. Sixteen elements (14 metals and 2 metalloids) were measured by inductively coupled plasma mass spectrometry (ICP-MS) in Pleurotus ostreatus and Cyclocybe cylindracea mushrooms, and in their seven cultivation substrates composed of various plant-based residues. Results revealed a high variability in elemental concentration among substrates which generally led to significant differences in the respective mushroom contents. High bioconcentration factors (BCFs) were noted for Cd, Cu, Mg and Zn for both species in all substrates. BCF of each element was variously affected by substrates’ pH, crude composition, and P and K content. Significant positive correlations were demonstrated for Cu, Fe, Mn and Li concentrations vs. a decrease of cellulose and hemicellulose in P. ostreatus substrates, and vs. mushrooms’ biological efficiency. In the case of C. cylindracea, Be, Mg and Mn concentrations were positively correlated with the decrease of hemicellulose in substrates, while a significant positive correlation was also recorded vs. mushroom productivity. Finally, it was found that 15% to 35% of the daily dietary needs in Mg, Se and Zn could be covered by mushroom consumption

Pleurotus Mushrooms Content in Glucans and Ergosterol Assessed by ATR-FTIR Spectroscopy and Multivariate Analysis

Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was used to monitor the infrared absorption spectra of 79 mushroom samples from 29 Pleurotus ostreatus, P. eryngii and P. nebrodensis strains cultivated on wheat straw, grape marc and/or by-products of the olive industry. The spectroscopic analysis provided a chemical insight into the mushrooms examined, while qualitative and quantitative differences in regions related to proteins, phenolic compounds and polysaccharides were revealed among the species and substrates studied. Moreover, by using advanced chemometrics, correlations of the recorded mushrooms’ spectra versus their content in glucans and ergosterol, commonly determined through traditional analytical techniques, allowed the development of models predicting such contents with a good predictive power (R2: 0.80–0.84) and accuracy (low root mean square error, low relative error and representative to the predicted compounds spectral regions used for the calibrations). Findings indicate that FTIR spectroscopy could be exploited as a potential process analytical technology tool in the mushroom industry to characterize mushrooms and to assess their content in bioactive compounds

Spent substrate from mushroom cultivation: exploitation potential toward various applications and value-added products

ABSTRACTSpent mushroom substrate (SMS) is the residual biomass generated after harvesting the fruitbodies of edible/medicinal fungi. Disposal of SMS, the main by-product of the mushroom cultivation process, often leads to serious environmental problems and is financially demanding. Efficient recycling and valorization of SMS are crucial for the sustainable development of the mushroom industry in the frame of the circular economy principles. The physical properties and chemical composition of SMS are a solid fundament for developing several applications, and recent literature shows an increasing research interest in exploiting that inherent potential. This review provides a thorough outlook on SMS exploitation possibilities and discusses critically recent findings related to specific applications in plant and mushroom cultivation, animal husbandry, and recovery of enzymes and bioactive compounds

On the Identification and Quantification of Ergothioneine and Lovastatin in Various Mushroom Species: Assets and Challenges of Different Analytical Approaches

In recent years, mushrooms have drawn the attention of agro-industries and food-industries as they were considered to be valuable natural sources of health promoting compounds such as β-glucans, ergothioneine, and lovastatin. The detection and quantification of such compounds by implementing reliable analytical approaches is of the utmost importance in order to adjust mushrooms’ cultivation conditions and maximize the production in different species. Toward this direction, the current study focuses on the comparison of ultraviolet–visible (UV–Vis) spectrometry and liquid chromatography–mass spectrometry (LC–MS) methods (a) by evaluating the content of ergothioneine and lovastatin in mushrooms and (b) by highlighting any possible substrate-based interferences that hinder the accurate determination of these two compounds in order to propose the technique-of-choice for a standardized bioactive compounds monitoring. For this purpose, mushrooms produced by three species (i.e., Agaricus bisporus, Pleurotus ostreatus, and P. citrinopileatus) on various cultivation substrates, namely wheat straw (WS), winery (grape marc (GM)), and olive oil (OL) by-products, were examined. Among the two applied techniques, the developed and validated LC–MS methods, exhibiting relatively short analysis time and higher resolution, emerge as the methods-of-choice for detecting ergothioneine and lovastatin in mushrooms. On the contrary, UV–Vis methods were hindered due to co-absorbance of different constituents, resulting in invalid results. Among the studied mushrooms, P. citrinopileatus contained the highest amount of ergothioneine (822.1 ± 20.6 mg kg−1 dry sample), whereas A. bisporus contained the highest amounts of lovastatin (1.39 ± 0.014 mg kg−1 dry sample). Regarding the effect of different cultivation substrates, mushrooms produced on OL and WS contained the highest amount of ergothioneine, while mushrooms deriving from GM-based substrates contained the highest amount of lovastatin

Toward an Increased Functionality in Oyster (<i>Pleurotus</i>) Mushrooms Produced on Grape Marc or Olive Mill Wastes Serving as Sources of Bioactive Compounds

<i>Pleurotus ostreatus</i>, <i>P. eryngii</i>, and <i>P. nebrodensis</i> were cultivated on nonconventional substrates containing grape marc (GMC) or olive mill byproducts (OMB); wheat straw (WHS) served as control. GMC-based media demonstrated equal/better mushroom productivity than WHS for <i>P. eryngii</i> and <i>P. nebrodensis</i>, while the cultivation performance of <i>P. eryngii</i> was improved in OMB-based media. Both GMC and OMB substrates led to large increase of fruit-bodies content in phenolic acids, resveratrol, triterpenic compounds, and ergosterol; in particular, <i>P. eryngii</i> mushrooms presented significantly more total phenolics and exhibited much higher antioxidant activity (2- to 8-fold increase). Furthermore, substrates containing GMC or OMB presented up to 27% increase in mushroom β-glucans. Overall, <i>Pleurotus</i> species responded in a different and mostly substrate-specific manner by selectively absorbing organic compounds. Phenolics and squalene content of substrates correlated very well with mushrooms antioxidant activity and ergosterol, respectively; the same was observed for triterpenics’ content of substrates and mushrooms

Fermentation Supernatants of Pleurotus eryngii Mushroom Ameliorate Intestinal Epithelial Barrier Dysfunction in Lipopolysaccharide-Induced Caco-2 Cells via Upregulation of Tight Junctions

In recent years, modulation of gut microbiota through prebiotics has garnered interest as a potential to ameliorate intestinal barrier dysfunction. The aim of the study was to examine the in vitro effect of fermentation supernatants (FSs) from rich in β-glucan Pleurotus eryngii mushrooms on the expression levels of tight junctions (TJs) genes in Caco-2 cells stimulated by bacterial lipopolysaccharides (LPS). Mushrooms were fermented using fecal inocula in an in vitro batch culture model. Caco-2 cells were subjected to LPS and FS treatment under three different conditions: pre-incubation with FS, co- and post-incubation. Reverse transcription PCR was applied to measure the expression levels of zonulin-1, occludin and claudin-1 genes. FSs from P. eryngii mushrooms led to a significant upregulation of the TJs gene expression in pre-incubation state, indicating potential preventive action. Down-regulation of all TJs gene expression levels was observed when the cells were challenged with LPS. The FS negative control (gut microbiota of each donor with no carbohydrate source) exhibited a significant upregulation of TJs expression levels compared to the cells that were challenged with LPS, for all three conditions. Overall, our data highlighted the positive and potential protective effects of P. eryngii mushrooms in upregulation of TJs’ genes.Funding agency:Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation under the call RESEARCH-CREATE-INNOVATE T1EDK-03404 </p

In Vitro Fermentation of Edible Mushrooms: Effects on Faecal Microbiota Characteristics of Autistic and Neurotypical Children

Children with autism spectrum disorder (ASD) often suffer gastrointestinal disturbances consistent with gut microbiota (GM) alterations. Treatment with pro/prebiotics may potentially alleviate gut symptoms, but the evidence for prebiotics is scarce. This study aims to evaluate the effects of edible mushrooms (Pleurotus, Basidiomycota) and prebiotic compounds on GM composition and metabolite production in vitro, using faecal samples from autistic and non-autistic children. Specific microbial populations were enumerated after 24 h of fermentation by quantitative PCR, and the metabolic production was determined by gas chromatography. Higher levels of Prevotella spp. and Bifidobacterium spp. were measured in neurotypical children compared to ASD children. A total of 24 h fermentation of Pleurotus eryngii and P. ostreatus mushroom powder increased the levels of Bifidobacterium, while known prebiotics increased the levels of total bacteria and Bacteroides in both groups. Only P. eryngii mushrooms resulted in significantly elevated levels of total bacteria Bacteroides and Feacalibacterium prausnitzii compared to the negative control (NC) in the ASD group. Both mushrooms induced elevated levels of butyrate after 24 h of fermentation, while short-chain fructooligosaccharides induced increased levels of acetate in the ASD group, compared to NC. Overall, this study highlights the positive effect of edible mushrooms on the GM and metabolic activity of children with ASD