38 research outputs found

    Recent advances in pretreatment of lignocellulosic wastes and production of value added products

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    This study highlights the recent advances in the treatment and value addition of lignocellulosic wastes (LCW) with main focus on domestic and agro-industrial residues. Mechanical, physical and biologicaltreatment systems are brought into perspective. The main value-added products from lignocellulosic wastes are summarized in a manner that pinpoints the most recent trends and the future directions. Physicochemical and biological treatment systems seem to be the most favored options while biofuels, biodegradable composites and biosorbents production paints a bright picture of the current and futurebio-based products. Engineered microbes seem to tackle the problem of bioconversion of substrates that are otherwise non convertible by conventional wild strains. Although the main challenge facing LCW utilization is the high costs involved in treatment and production processes, some recent affordable processes with promising results have been proposed. Future trends are being directed to nanobiotechnology and genetic engineering for improved processes and products. The paper presents state of the art review of the dual advantage of handling LCW for cleaner environment and productionof renewable bio-products

    Enzymes as catalysts in polymer chemistry

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    2009/2010The use of enzymes in synthetic chemistry is attracting the interest of many researchers thanks to their extraordinary efficiency under mild conditions, high stereo- regio- and chemoselectivity and low environmental impact. Their application in the field of polymer chemistry has provided new synthetic strategies for useful polymers. The advantages coming from the use of enzymes are mainly: i) the possibility to synthesize polymers with novel properties and difficult to produce by conventional chemical catalysts; ii) the improvement of the synthetic processes in terms of sustainability, for instance by avoiding toxic catalysts. In particular hydrolases have demonstrated to be efficient catalysts in the development of highly structured polyesters with low polydispersity characterized by the presence of different functionalisations. Although generally the enzymatic processes lead to the formation of oligomers of limited Mn, these can be successively chemically polymerized to synthesize new innovative products with high Mn. Hydrolases enzymes offer also a great opportunity for using non-petrochemical renewable resources as starting substrates thus contributing to global sustainability of polymerization processes. Also oxidases have proven to be efficient catalysts in polymer chemistry thanks to their capability to catalyze oxidation of phenolic groups. This radicalic reaction can be exploited either for: i) degrading lignin; ii) functionalizing lignin phenolic moieties thus improving their chemical and physical properties; iii) polymerize phenol derivatives or phenol based oligomers. Unfortunately, the use of enzymes in polymer chemistry on industrial scale is still hindered by many factors among which the most significant are: i) the instability of many commercial immobilized preparations under process conditions, ii) the high costs of both the biocatalyst and the monomers from renewable resources and iii) the limited availability of information concerning the main factors which affect the scalability and the industrialization of this kind of enzymatic processes. At this regard, the work described in Chapter 2 of the thesis exploits the catalytic potential of lipase B from Candida antarctica in polymerization processes. In collaboration with the group of Professor Karl Hult (KTH “Royal Institute of Technology”, Stockholm) a new synthetic route for the enzymatic synthesis of new innovative highly structured allyl functionalized polyester of industrial interest has been developed. Finally, products obtained have been efficiently employed in thiol-ene chemistry for films production. Chapter 3 deals with the synthesis of functionalized polyesters starting from renewable feedstock which can be employed as new starting materials for the production of surfactants for cosmetic and pharma industry. Continuing on the study of lipase catalyzed polyester synthesis, in Chapter 4 particular attention has been given to the feasibility of the biocatalysed technology at industrial level, namely addressing the problem of biocatalyst’s stability and formulation. In particular a highly stable covalently immobilized preparation of CALB, developed in collaboration with SPRIN Technologies, has been exploited for the synthesis of polyesters demonstrating the advantages coming from covalent immobilization over the other commercially available CALB adsorbed preparations, namely higher recyclability and absence of enzyme leaching. On this respect, one-step and multistep processes for the synthesis of polyesters employing adipic acid and 1,4-butanediol have been the subject of a deep investigation. A comparative analysis describes the effect of enzyme leaching from adsorbed preparations during polycondensation reactions evidencing meanwhile the stability of the covalent enzymatic preparation under harsh conditions. Within the context of this study characterization of the synthesized polymers has been feasible thanks to the collaboration with the group of Dr. Mario Malinconico at the “Istituto di Chimica e Tecnologia dei Polimeri” ICTP-CNR, Pozzuoli (NA). Moreover, a new methodology for polymer characterization which integrates DOSY NMR and GROMACS simulation techniques has been developed in collaboration with CBM (“Cluster in Biomedicine”, Area Science Park, Trieste). The obtained results open new perspectives for the study of polymer behavior in specific media. The final part of the work (Chapter 5) focuses on the potential applicability of laccases under non-conventional conditions, thus exploring new routes for more efficient lignin degradation. The most important obstacle to technological and commercial application of laccases is the limited number of enzymes readily available for industrial applications. At this regard, thanks to the collaboration with the group of Prof. Ludmila Golovleva (Institute of Biochemistry and Physiology of Microorganisms, “Russian Academy of Science”, Puschino, Russia), we have studied the stability and activity of three different fungal laccases in the conditions of interest for lignin processing, namely in organic/buffer mixtures and under microwaves radiation. The proof of concept for the application of the considered laccases in lignin valorization has been demonstrated assessing lignin over laccases mediated oxidation in organic/buffer mixtures media. Moreover, a laccase endowed with remarkable higher stability as compared to the other tested has been identified. The enzyme is currently under study for understanding the structural basis of its stability. In conclusion, the work of this thesis demonstrates that the lipase catalyzed synthesis of polyesters is a mature technology ready to be employed at industrial scale for those specific applications where the chemical properties of products or the sustainability of the process represent crucial issues in the classical chemical processes. Moreover, the new properties of the immobilized lipase employed in the present study induce to believe that the problem of biocatalyst stability and recyclability has been finally overcome. Concerning the application of laccases, the valorization of lignin in terms of production of bio-based chemicals is still a very new field of research, although it is expected to become one of the major topics for the future development of the “Sustainable Chemistry”.1 The present study intends to be a contribution for the identification of suitable biocatalysts and more efficient conditions for the degradation and modification of one of the most abundant biopolymers in Nature. The high stability of the laccase here reported induces to pursue in their characterization under non-conventional reaction conditions on different types of lignin. 1 J. E. Holladay, J. J. Bozell, J. F. White, D. Johnson, “Top Value-Added Chemicals from Biomass”, 2007, U.S. Department of Energy.(http://www1.eere.energy.gov/biomass/pdfs/pnnl-16983.pdf).L'impiego di enzimi nella chimica di sintesi sta attirando l'interesse di molti ricercatori, ciò è dovuto in particolare alla loro straordinaria efficienza in condizioni di reazione blande, alla loro elevata stereo-, regio- e chemoselettività e al loro basso impatto ambientale. L’ applicazione di biocatalizzatori nel campo della chimica dei polimeri ha fornito nuove strategie sintetiche per lo sviluppo di materiali innovativi. I principali vantaggi derivanti dall'impiego di enzimi sono: i) la possibilità di sintetizzare polimeri con nuove proprietà e difficili da ottenere mediante processi chimici convenzionali ed ii) il miglioramento dei processi di sintesi in termini di sostenibilità ambientale evitando l’impiego di catalizzatori tossici. Le idrolasi in particolare hanno dimostrato di essere catalizzatori efficienti per lo sviluppo di poliesteri altamente strutturati e a bassa polidispersità, caratterizzati inoltre dalla presenza di varie funzionalizzazioni chimiche terminali. Anche se generalmente i processi enzimatici portano alla formazione di oligomeri a basso peso molecolare, questi possono essere successivamente polimerizzati chimicamente per formare nuovi prodotti innovativi ad alto Mn. Le idrolasi offrono inoltre l’opportunità di impiegare monomeri provenienti da risorse rinnovabili contribuendo così alla sostenibilità ambientale dei processi di polimerizzazione. Anche le ossidasi hanno dimostrato di essere catalizzatori efficienti e facilmente impiegabili nella chimica dei polimeri grazie alla loro capacità di catalizzare l'ossidazione radicalica di gruppi fenolici. Queste reazioni radicaliche possono trovare diverse applicazioni come ad esempio: i) nella degradazione della lignina; ii) nella funzionalizzazione dei gruppi fenolici della lignina che consentano di migliorarne le proprietà chimiche e fisiche; iii) nella polimerizzazione di derivati fenolici o di oligomeri a base di fenolo. Sfortunatamente, l'impiego di enzimi su scala industriale è ancora ostacolato da una serie di fattori tra i quali i più significativi sono: i) l'instabilità delle preparazioni enzimatiche commercialmente disponibili nelle condizioni di processo, ii) gli alti costi del biocatalizzatore e dei monomeri provenienti da risorse rinnovabili e iii) la limitata disponibilità di informazioni riguardanti i principali fattori che influenzano la scalabilità e l'industrializzazione di questo tipo di processi enzimatici. A questo proposito, il lavoro descritto nel capitolo 2 di questa tesi sfrutta le potenzialità catalitiche della lipasi B da Candida antarctica nei processi di polimerizzazione. In particolare è stata sviluppata, in collaborazione con il gruppo del prof. Karl Hult (KTH “Royal Institute of Technology”, Stoccolma), una nuova via sintetica biocatalizzata per la produzione di poliesteri innovativi altamente strutturati recanti funzionalizzazione allilica, che trovano ampia applicabilità industriale. I prodotti così ottenuti sono stati successivamente impiegati con successo nella chimica dei tioleni per la produzione di films. Il capitolo 3 è invece dedicato alla sintesi di poliesteri funzionalizzati a partire da materie prime rinnovabili; i prodotti così sviluppati trovano potenziale applicazione come tensioattivi per l'industria cosmetica e farmaceutica. Proseguendo lo studio della sintesi biocatalizzata di poliesteri, nel capitolo 4 particolare attenzione è stata data alla fattibilità dei processi biocatalizzati a livello industriale affrontando il problema della stabilità del biocatalizzatore. In particolare, una preparazione di CALB immobilizzata covalentemente su supporti polimerici, sviluppata in collaborazione con SPRIN Technologies, è stata impiegata nella sintesi di poliesteri dimostrando i vantaggi dovuti all’ immobilizzazione covalente rispetto alle altre preparazioni adsorbite commercialmente disponibili, ovvero l’elevata riciclabilità del biocatalizzatore e la mancanza di rilascio di enzima nel prodotto finale. A tal proposito, la sintesi one-step e multistep di poliesteri a partire da acido adipico e 1,4-butandiolo è stata oggetto di una profonda indagine. Il lavoro riporta un' analisi comparativa che descrive l'effetto del rilascio dell’ enzima durante le reazioni di policondensazione evidenziando nel contempo la straordinaria stabilità della preparazione enzimatica covalente. La caratterizzazione dei polimeri così ottenuti è stata possibile grazie alla preziosa collaborazione con il gruppo del Dott. Mario Malinconico (Istituto di Chimica e Tecnologia dei Polimeri "ICTP-CNR, Pozzuoli (NA)). Al termine del capitolo 4 è stata sviluppata, in collaborazione con CBM ("Cluster in Biomedicine", Area Science Park, Trieste), una nuova metodologia per la caratterizzazione dei polimeri che integra DOSY NMR e tecniche di simulazione GROMACS. I risultati ottenuti da questo lavoro aprono nuove prospettive per lo studio del comportamento dei polimeri in solventi organici. La parte finale del lavoro di tesi (capitolo 5) si concentra sull’ applicabilità di laccasi in condizioni non convenzionali, esplorando nuove vie per i processi di degradazione della lignina. Il principale ostacolo nell’impiego tecnologico delle laccasi su scala industriale è il limitato numero di enzimi commercialmente disponibili per tali applicazioni. A questo proposito, grazie alla collaborazione con il gruppo della Prof.ssa Ludmila Golovleva (Istituto di Biochimica e Fisiologia dei Microorganismi, "Russian Academy of Science", Puschino, Russia), è stato possibile studiare la stabilità e l'attività di tre diverse laccasi fungine nelle condizioni di interesse per la trasformazione della lignina, vale a dire in miscele di solvente organico e sotto irradiazione di microonde. L’applicabilità delle laccasi considerate nella valorizzazione della lignina è stata dimostrata valutando l’ ossidazione della lignina nelle condizioni di interesse. E’stata quindi identificata una laccasi caratterizzata da una stabilità particolarmente elevata, l' enzima è attualmente in studio per la comprensione delle basi strutturali che gli conferiscono tali caratteristiche. In conclusione, questo lavoro di tesi dimostra che la sintesi di poliesteri CALB catalizzata è una tecnologia matura, pronta per essere impiegata su scala industriale laddove le proprietà chimiche dei prodotti o la sostenibilità ambientale del processo rappresentano ostacoli non sormontabili impiegando le calssiche metodologie sintetiche. Inoltre l’elevata stabilità della lipasi immobilizzata covalentemenete induce a credere che il problema della stabilità del biocatalizzatore e di conseguenza del suo riciclaggio è stato definitivamente superato. L'applicazione delle laccasi nella valorizzazione della lignina per lo sviluppo di prodotti chimici biobased è un campo di ricerca relativamente nuovo ma destinato a diventare uno dei settori più importanti per lo sviluppo della "chimica sostenibile".1 Il presente studio intende essere un contributo per l'identificazione del biocatalizzatore e delle condizioni di reazione più adatte per la degradazione e la trasformazione della lignina, uno dei biopolimeri più abbondanti presenti in natura. L’elevata stabilità della laccasi identificata in questo lavoro induce inoltre a proseguire con gli studi di applicabilità di questo enzima in mezzi non convenzionali. 1 J. E. Holladay, J. J. Bozell, J. F. White, D. Johnson, “Top Value-Added Chemicals from Biomass”, 2007, U.S. Department of Energy.)XXIII Ciclo198

    Enhanced large-scale production of laccases from Coriolopsis polyzona for use in dye bioremediation

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    Pollution from synthetic dyes, released by textile and paper pulping plants, draws major concern. Textile effluents have negative impact both on the environment and human health because they are toxic and some are carcinogenic. Apart from the textile industry, dyes are also widely used in manufacturing industries for leather products, cosmetics, pharmaceuticals, foods and beverages. It is known that white rot fungi can decolourise and detoxify various industrial effluents through the production of extracellular lignin modifying enzymes, a major class of which are laccases (EC 1.10.3.2). Considering the above, three strains of white rot fungi, Coriolopsis polyzona (MUCL 38443), Pleurotus ostreatus (ATCC no. MYA-2306) and Pycnoporus sanguineus (MUCL 41582) were studied for their ability to produce laccases in liquid media. The effects of mannan oligosaccharides (MO) as elicitor and ferulic acid as inducer were studied using central composite experimental design in liquid cultures of the three strains. The results showed that MO, either added alone or combined with ferulic acid, enhanced laccase activity in the three different cultures and the enhancement was species specific. The highest increase was in liquid cultures of P. sanguineus (88-fold) followed by P. ostreatus (3-fold) and C. polyzona (2-fold), among which C. polyzona resulted in the highest laccase activity. The combined addition of 150 mg/l of MO and 1 mM ferulic acid resulted in the optimal laccase activity by C. polyzona, whereas additions of 75 mg/l MO to the cultures of P. sanguineus and P. ostreatus led to the optimal activity. Extracellular laccase activity was considerably increased when C. polyzona was grown in glucose-bactopetone based culture medium induced by ferulic acid. The effect of inoculum conditions on laccase production was studied at reactor scales. Laccase activity achieved with conidia inoculation was higher compared with mycelium inoculation at the early stage of fermentation. However, the laccase levels were similar after 23 days of fermentation (110 U/ml and 100 U/ml for the conidia and mycelia pre-culture respectively). The conidia inoculation is preferred in scale-up when time-cost is considered. The maximal laccase activity with conidia inoculation in a 2 litre stirred tank reactor was 27% higher compared to that in shaken flasks. This showed that C. polyzona cultures have the potential to be scaled-up for increased laccase activity by applying conidia inoculum. The fermentation of C. polyzona was scaled-up to 20 litre and 150 litre stirred tank reactors applying fed-bath strategy. This resulted in 100 % enhancement of laccase activity. Addition of oak wood powder in the culture medium increased total laccase activity indicating the potential of lignocellulosic wastes as alternative substrates for enhanced laccase production with reducing cost. In order to investigate the application of laccases in dye decolourisation, two major laccase isozymes (Lac I & II) from C. polyzona were purified to apparent eletrophoretic homogenetity using hydrophobic interaction chromatography and ionexchange chromatography. Both enzymes were found to be monomeric proteins with the same molecular mass of 63 kDa, and isoelectronic point of 4.3. Their catalytic activities were studied under various substrates, pHs and temperatures. The highest enzyme affinity and efficiency were obtained with 2,2′-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Compared with other fungal laccases, the laccases from C. polyzona have very low Km values with ABTS as a susbtrate. The optimum pHs were 2.8, 3.0 and 5.0 on ABTS, 2, 6-dimethoxyphenol (DMP) and syringaldazine, respectively. Both isozymes had acidic optimal pH values. However, they were more stable in netural pH rather than at acidic pH. Moreover, mass spectrometry (MS) analysis of tryptic digestion products of the two isozymes was performed, which showed further similarity of these two isozymes. As common physical or chemical methods for dye removal are expensive, have low efficiency and sometimes generate other pollutants, the decolourisation of industrial effluents containing single and mixed dyes was investigated using purified laccase (Lac I) from C. polyzona as well as whole cell culture. The method appeared to be an attractive alternative for dye removal. Anthraquinone dyes were found to be more easily decolourised by Lac I compared to azo dyes. The addition of redox mediator ABTS and violuric acid (VA) improved considerably the catalytic efficiencies of azo dyes. Decolourisation, 40-50 %, was achieved for the reactive and the direct dye baths. Response surface technology (RSM) was applied to optimise the decolourisation of the diazo dye reactive black 5 (RB 5) by Lac I. Box-Behnken experimental design with three variables including laccase activity (100, 200, 300 U/l), pH (5, 7, 9) and VA concentration (0, 1.25, 2.5 mM) was studied to identify a significant correlation between the effect of these variables on decolourisation of RB5. The experimental values were in good agreement with the predicted values with the correlation coefficient of 97.4%

    Enhanced large-scale production of laccases from Coriolopsis polyzona for use in dye bioremediation

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    Pollution from synthetic dyes, released by textile and paper pulping plants, draws major concern. Textile effluents have negative impact both on the environment and human health because they are toxic and some are carcinogenic. Apart from the textile industry, dyes are also widely used in manufacturing industries for leather products, cosmetics, pharmaceuticals, foods and beverages. It is known that white rot fungi can decolourise and detoxify various industrial effluents through the production of extracellular lignin modifying enzymes, a major class of which are laccases (EC 1.10.3.2). Considering the above, three strains of white rot fungi, Coriolopsis polyzona (MUCL 38443), Pleurotus ostreatus (ATCC no. MYA-2306) and Pycnoporus sanguineus (MUCL 41582) were studied for their ability to produce laccases in liquid media. The effects of mannan oligosaccharides (MO) as elicitor and ferulic acid as inducer were studied using central composite experimental design in liquid cultures of the three strains. The results showed that MO, either added alone or combined with ferulic acid, enhanced laccase activity in the three different cultures and the enhancement was species specific. The highest increase was in liquid cultures of P. sanguineus (88-fold) followed by P. ostreatus (3-fold) and C. polyzona (2-fold), among which C. polyzona resulted in the highest laccase activity. The combined addition of 150 mg/l of MO and 1 mM ferulic acid resulted in the optimal laccase activity by C. polyzona, whereas additions of 75 mg/l MO to the cultures of P. sanguineus and P. ostreatus led to the optimal activity. Extracellular laccase activity was considerably increased when C. polyzona was grown in glucose-bactopetone based culture medium induced by ferulic acid. The effect of inoculum conditions on laccase production was studied at reactor scales. Laccase activity achieved with conidia inoculation was higher compared with mycelium inoculation at the early stage of fermentation. However, the laccase levels were similar after 23 days of fermentation (110 U/ml and 100 U/ml for the conidia and mycelia pre-culture respectively). The conidia inoculation is preferred in scale-up when time-cost is considered. The maximal laccase activity with conidia inoculation in a 2 litre stirred tank reactor was 27% higher compared to that in shaken flasks. This showed that C. polyzona cultures have the potential to be scaled-up for increased laccase activity by applying conidia inoculum. The fermentation of C. polyzona was scaled-up to 20 litre and 150 litre stirred tank reactors applying fed-bath strategy. This resulted in 100 % enhancement of laccase activity. Addition of oak wood powder in the culture medium increased total laccase activity indicating the potential of lignocellulosic wastes as alternative substrates for enhanced laccase production with reducing cost. In order to investigate the application of laccases in dye decolourisation, two major laccase isozymes (Lac I & II) from C. polyzona were purified to apparent eletrophoretic homogenetity using hydrophobic interaction chromatography and ionexchange chromatography. Both enzymes were found to be monomeric proteins with the same molecular mass of 63 kDa, and isoelectronic point of 4.3. Their catalytic activities were studied under various substrates, pHs and temperatures. The highest enzyme affinity and efficiency were obtained with 2,2′-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Compared with other fungal laccases, the laccases from C. polyzona have very low Km values with ABTS as a susbtrate. The optimum pHs were 2.8, 3.0 and 5.0 on ABTS, 2, 6-dimethoxyphenol (DMP) and syringaldazine, respectively. Both isozymes had acidic optimal pH values. However, they were more stable in netural pH rather than at acidic pH. Moreover, mass spectrometry (MS) analysis of tryptic digestion products of the two isozymes was performed, which showed further similarity of these two isozymes. As common physical or chemical methods for dye removal are expensive, have low efficiency and sometimes generate other pollutants, the decolourisation of industrial effluents containing single and mixed dyes was investigated using purified laccase (Lac I) from C. polyzona as well as whole cell culture. The method appeared to be an attractive alternative for dye removal. Anthraquinone dyes were found to be more easily decolourised by Lac I compared to azo dyes. The addition of redox mediator ABTS and violuric acid (VA) improved considerably the catalytic efficiencies of azo dyes. Decolourisation, 40-50 %, was achieved for the reactive and the direct dye baths. Response surface technology (RSM) was applied to optimise the decolourisation of the diazo dye reactive black 5 (RB 5) by Lac I. Box-Behnken experimental design with three variables including laccase activity (100, 200, 300 U/l), pH (5, 7, 9) and VA concentration (0, 1.25, 2.5 mM) was studied to identify a significant correlation between the effect of these variables on decolourisation of RB5. The experimental values were in good agreement with the predicted values with the correlation coefficient of 97.4%.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

    The potential of ligninolytic fungi in bioremediation of contaminated soils

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    Soils that are contaminated with the most recalcitrant organic contaminants, such as high molecular weight polyaromatic hydrocarbons (HMW PAH) and polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F), cannot be degraded efficiently by conventional composting. The only available treatment method for these soils, which destroys the contaminants, is combustion at high temperature. This thesis examines three alternative fungal methods to treat these soils: 1) treatment with fungal enzymes, 2) treatment with fungal inoculum, and 3) fungal treatment used as a pre-treatment to improve the energy efficacy in combustion. Manganese peroxidase (MnP), which belongs to lignin-modifying enzymes (LME), was produced and used to treat PCDD/F-contaminated soil in the laboratory scale. Nevertheless, no degradation with a MnP preparation was observed, although a substantial amount of MnP activity was found in the soil still after 10 days of incubation. Both PAH- and PCDD/F- contaminated soils were treated with fungal inoculum in the laboratory scale. HMW PAHs were degraded significantly more by the fungi than by the indigenous microbes alone in the laboratory experiments, where the PAH concentration of soil was 3500 mg kg -1 (sum of 16 PAH). Treatment with Phanerochaete velutina (inoculum) resulted to degradation of 96 % of 4- ring PAHs and 39 % of 5- and 6-ring PAHs in three months. With PCDD/F-contaminated soil, no degradation was observed in the control, but the degradation of PCDD/Fs with fungal treatments was significant ( P. velutina : 62 %, Stropharia rugosoannulata : 64 % of WHO-TEQ value). Fungal treatment of PAH-contaminated soil was also applied in the field scale (2 t). However, both P. velutina (inoculum) and control treatment resulted in equal degradation in soil with lower PAH concentration (1400 mg kg -1 , sum of 16 PAH): 94 % of the 16 PAHs were degraded in three months. Fungal treatment was even applied as a pre-treatment for contaminated soil with high organic matter content, and which will be later combusted. In the pilot-scale (300 kg), 13 % degradation of the original organic matter content was obtained in 6 months. To conclude, fungal treatment is reasonable to apply for soils with organic contaminants that cannot be bioremediated by composting. With soils contaminated by chlorinated dioxins, this is always the case, but also PAH-contaminated soils with high total concentration or high proportion of HMW-PAHs. In addition, with fungal treatment the amount of organic matter in the soil can be reduced and the efficacy of the combustion process is improved.Diss.: Aalto-yliopisto, 201

    Physiological roles and metabolism of fungal aryl alcohols

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    The major structural elements of wood and other vascular tissues are cellulose, hemicellulose and generally 20-30% lignin. Lignin gives the plant strength, it serves as a barrier against microbial attack and it acts as a water impermeable seal across cell walls of the xylem tissue. However, the presence of lignin has practical drawbacks for some of the applications of lignocellulosic materials. First, lignin has to be removed for the production of high quality pulps. Second, lignin reduces the digestibility of lignocellulosic materials. High quality pulps can be produced with chemical methods, however the abundant use of chemicals and energy, and the formation of an enormous waste stream has led scientists to investigate the possibilities of biodelignification. White-rot fungi give the most rapid and extensive degradation and have become subject of intensive research. Results obtained with the model organism Phanerochaete chrysosporium and other strains have revealed that lignin biodegradation is an extracellular, oxidative and non-specific process. This unique biodegradative potential has been considered for broader applications such as waste water treatment and the degradation of xenobiotic compounds. The research described in this thesis concentrates on the function of aryl alcohols in fungal physiology.Aryl alcohols In the physiology of white-rot fungi. White-rot fungi have a versatile machinery of enzymes, including peroxidases and oxidases, which work in harmony with secondary aryl alcohol metabolites to degrade the recalcitrant, aromatic biopolymer lignin. In chapter 2 literature concerning the important physiological roles of aryl (veratryl, anisyl and chlorinated anisyl) alcohols in the ligninolytic enzyme system has been reviewed. Their functions include stabilization of lignin peroxidase, charge-transfer reactions and as substrate for oxidases generating extracellular H 2 O 2 .The experimental research described in this thesis was initiated to evaluate the possibilities of white-rot fungi in the biopulping of hemp stem wood. Sixty-seven basidiomycetes were isolated and screened for high peroxidative activity (chapter 3). Several of the new isolates were promising manganese peroxidase-producing white-rot fungi. Enzyme assays indicated that for the production of H 2 O 2 either extracellular glyoxal or aryl alcohol oxidase were present. In contrast, lignin peroxidase was only detected in P. chrysosporium , despite attempts to induce this enzyme in other strains with oxygen and oxygen/veratryl alcohol additions. A highly significant correlation was found between two ligninolytic indicators: ethene formation from α-keto-γ- methylthiolbutyric acid and the decolorization of a polymeric dye, Poly R-478. Three of the new isolates had significantly higher Poly R decolorizing activities compared to P. chrysosporium .One of the best Poly R decolorizing strains, Bjerkandera sp. BOS55 was selected for further characterization. A novel enzyme activity (manganese independent peroxidase) was detected in the extracellular fluid of Bjerkandera sp. BOS55 (chapter 4). The purified enzyme could oxidize several compounds like phenol red, 2,6-dimethoxyphenol (DMP), Poly R-478, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and guaiacol with H 2 O 2 as an electron acceptor. In contrast, veratryl alcohol was not a substrate. This enzyme also had the capacity to oxidize DMP in the absence of H 2 O 2 . Bjerkandera sp. BOS55 also produced de novo several aromatic metabolites. Besides veratryl alcohol and veratraldehyde, compounds which are known to be involved in the ligninolytic system of several other white-rot fungi (chapter 2), other metabolites were formed. These included anisaldehyde, 3-chloro-anisaldehyde, 3,5-dichloro-anisaldehyde and small amounts of the corresponding anisyl, 3-chloro-anisyl and 3,5-dichloro-anisyl alcohol (chapters 5 and 6). This was the first report of de novo biosynthesis of simple chlorinated aromatic compounds by a white-rot fungus. These unexpected findings led us investigate the physiological role(s) of the denovo biosynthesized chlorinated anisyl alcohols (chapter 6). All metabolites were produced simultaneously with the extracellular ligninolytic enzymes. The monoand dichlorinated anisyl alcohols appeared to be excellent substrates for the extracellular aryl alcohol oxidases. The formed aldehydes were readily recycled via reduction by washed fungal mycelium, thus creating an extracellular H 2 O 2 production system regulated by intracellular enzymes. Lignin peroxidase does not oxidize the chlorinated anisyl alcohols both in the absence and in the presence of veratryl alcohol. It was therefore concluded that the chlorinated anisyl alcohols are well protected against the fungus's own aggressive ligninolytic enzymes. The relative amounts of veratryl alcohol and the chlorinated anisyl alcohols differ significantly depending on the growth conditions, indicating that the production of veratryl alcohol and the (chlorinated) anisyl metabolites are independently regulated.It was concluded that the chlorinated anisyl metabolites, biosynthesized by the white-rot fungus Bjerkandera sp. BOS55, are purposeful for ecologically significant processes such as lignin degradation. These results made us speculate if a significant biogenesis of chlorinated aromatics by fungi occurs in natural environments (chapter 7). Many common wood- and forest litter-degrading fungi were indeed detected that produced chlorinated anisyl metabolites (CAM). These compounds, which are structurally related to xenobiotic chloroaromatics, were present in the environment and occur at high concentrations of approximately 75 mg CAM kg -1wood or litter. The ubiquity among common fungi to produce large amounts of chlorinated aromatic compounds in the environment leads to the conclusion that these kind of compounds can no longer be considered to originate from anthropogenic sources only.Degradation of aryl alcohols by fungi. In chapter 2 the anabolic and catabolic routes of aryl alcohols by white-rot fungi has been reviewed. These fungi can not use veratryl alcohol as sole source of carbon and energy. However, several bacteria, yeasts and fungi were selectively isolated from paper mill waste water that grew on veratryl alcohol (chapter 8). Penicilliumsimplicissimum was selected for the characterization of the veratryl alcohol degradation route. P. simplicissimum oxidized veratryl alcohol via a NAD(P) +-dependent veratryl alcohol dehydrogenase to veratraldehyde which was further oxidized to veratric acid in a NAD(P) +-dependent reaction. Veratric acid-grown cells contained NAD(P)H-dependent O -demethylase activity for veratrate, vanillate and isovanillate. Ring-cleavage of protocatechuate was by a protocatechuate 3,4-dioxygenase. An interesting aspect of P. simplicissimum is the production of vanillyl alcohol oxidase with covalently bound FAD (chapter 9). The intracellular enzyme was purified 32-fold. SDS-PAGE of the purified enzyme revealed a single fluorescent band of 65 Kda. Gel filtration and sedimentation-velocity experiments indicated that the purified enzyme exists in solution as an octamer, containing 1 molecule flavin/subunit. The covalently bound prosthetic group of the enzyme was identified as 8α-(N 3-histidyl)FAD from pH dependent fluorescence quenching (p Ka = 4.85) and no decrease in fluorescence upon reduction with sodium borohydride. The enzyme showed a narrow and rather peculiar substrate specificity. In addition to vanillyl alcohol and 4-hydroxybenzyl alcohol, eugenol and chavicol are substrates for the enzyme (chapter 10). The formed products, coniferyl and coumaryl alcohol are the natural precursors of lignin in plants. This reaction has a potential application to produce coniferyl alcohol and subsequent synthetic lignin (DHP) from the inexpensive precursor eugenol

    The potential of ligninolytic fungi in bioremediation of contaminated soils

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    Diss. : Aalto University, 201

    Green degradation of mycotoxins by biotechnological application of enzymes from Pleurotus spp

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    Le micotossine sono metaboliti secondari tossici, prodotti da funghi filamentosi che appartengono principalmente ai generi Aspergillus, Penicillium, Fusarium e Alternaria. Sono comuni contaminanti alimentari di cereali, frutta, semi e spezie, prodotti a seguito di contaminazione fungina. La contaminazione da micotossine ha un impatto importante sulla salute mondiale e sulla sua economia. Alcune di esse sono classificate come sostanze cangerogene umane (aflatossine delle serie B e G) o possibili cancerogene umane (aflatossina M1, AFM1, fumonisina B1, FB1 e ocratossina A, OTA) dall’Agenzia Internazionale per la Ricerca sul Cancro (IARC). Inoltre, esplicano una azione tossica, sia acuta che cronica, su umani e animali. A causa della contaminazione da micotossine, milioni di dollari vengono persi ogni anno a causa di commodities invendute, problemi di salute e diminuzione della produttività degli animali e per la messa a punto di sistemi complessi e integrati di lotta e gestione di queste problematiche. Le strategie preventive non sono completamente efficaci e richiedono di essere implementate con nuove strategie di riduzione che agiscano nel post raccolta e che siano in grado di ridurre o di rimuovere le micotossine dai materiali contaminati. Lo scopo di questa tesi è stato quello di valutare e studiare la capacità delle laccasi di ridurre la contaminazione di micotossine, sia in vitro che in materiali contaminati, attraverso un approccio non invasivo sugli alimenti e sull’ambiente. Inoltre, fino a otto diversi mediatori redox sono stati utilizzati insieme alle laccasi nel cosiddetto Sistema Laccasi Mediatore (LMS) per massimizzare la degradazione delle micotossine. Per raggiungere questo scopo, l’attività di due diverse laccasi purificate, la Lac2 da Pleurotus pulmonarius e la ricombinante Ery4 da P. eryngii sono state testate contro le maggiori classi di micotossine. La Lac2 è stata identificata e valutata per la degradazione in vitro delle aflatossine, mentre la Ery4 è stata testata contro AFB1, AFM1, FB1, OTA, deossinivalenolo (DON), zearalenone (ZEN) e tossina T-2. iv Lo screening preliminare ha rivelato che l’inclusione di un mediatore redox nel mix di reazione è necessario per raggiungere un alto livello di degradazione con entrambi gli enzimi. Tuttavia, l’ LMS è risultato non completamente efficace per la degradazione dell’OTA e non efficace per la degradazione del DON. Attraverso l’uso di uno specifico LMS è stata ottenuta anche la degradazione simultanea di due diverse coppie di tossine, AFB1/ZEN e FB1/ tossina T-2. In aggiunta, il trattamento con LMS è stato utilizzato con successo in matrici artificialmente e naturalmente contaminate, latte e farina di mais, per la degradazione di AFM1 e ZEN, rispettivamente. Nonostante le grandi potenzialità mostrate da entrambi gli enzimi nel campo del biorisanamento, le laccasi restano dei biocatalizzatori versatili che possono essere utilizzati in una grande varietà di processi. L’applicazione dell’LMS è stato studiato in dettaglio nel latte per valutarne l’effetto sulle proteine del latte e la possibilità di produrre una cagliata con proprietà tecnologiche e nutrizionali migliorate. I risultati presentati in questa tesi pongono le basi per lo sviluppo di un metodo di biotrasformazione basato su un approccio enzimatico che apre nuove prospettive per l’utilizzo di un biocatalizzatore versatile e green, come la laccasi, nel campo della sicurezza e della qualità delle derrate alimentari contaminate da micotossine.Mycotoxins are toxic secondary metabolites produced by filamentous fungi mainly belonging to Aspergillus, Penicillium, Fusarium and Alternaria genera. They can be found as common contaminants of cereals, fruits, seeds and spices as a result of fungal spoilage. Mycotoxin contamination is an significant health and economic concern worldwide. Some of them were recognized by the International Agency of Research on Cancer (IARC) as carcinogenic (aflatoxin of the B and G series), possible carcinogenic (aflatoxin M1, AFM1; fumonisin B1, FB1; ochratoxin A, OTA) to humans. Moreover, they exert both acute and chronic toxic effects towards humans and animals. Because of mycotoxin contamination, billions of dollars are lost every year due to unsold commodities, decrease of animal health and productivity or to sustain a complex and integrated mycotoxin management system. Prevention strategies are not completely effective and require the implementation of novel post-harvest methods, able to mitigate or remove mycotoxins from contaminated materials. The aim of this thesis was to evaluate and study the capability of laccase enzymes to reduce mycotoxin contamination both in vitro and in contaminated materials through an environmental friendly and mild approach. In addition up to eight different redox mediators were used within the laccase mediator system (LMS) to maximize mycotoxin degradation. Within this purpose, the activity of two different purified LCs, native Lac2 from Pleurotus pulmonarius and the recombinant Ery4 from P. eryngii, was tested towards the main classes of mycotoxins. Lac2 was identified and evaluated for the in vitro degradation of aflatoxins, while Ery4 was tested towards AFB1, AFM1, FB1, OTA, deoxynivalenol (DON), zearalenone (ZEN) and T-2 toxin. The preliminary screening revealed that the inclusion of a toxin - specific redox mediator is required to achieve high levels of degradation with both enzymes. However, the use of the LMS resulted ineffective for DON and not efficient for OTA. i

    Optimisation of biopulping process by bacteria from rhynchophorus ferrugineus on empty fruit bunch for pulp industry

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    Lignin removal is an essential phase in pulping process. The conventional pulping process has many challenges such as high chemical and energy consumptions, as well as long period and sensitivity of fungal during fermentation process. A cleaner, cheaper, and more effective lignin removal method attracts the interest of industrialists. This research focuses on optimising lignin removal via biopulping process using several combinations of R. ferrugineus’s gut microbiome such as Klebsiella pneumoniae (K), Serratia marcescens (S), Pseudomonas citronellolis (P) and Enterobacter oryzae (E). The optimum conditions of biopulping process were determined through the design of experiment (DOE). DOE involves two phases: screening the significant parameter using Plackett-Burman design (PBD) and optimising conditions for biopulping process using Box-Behnken design (BBD). The chemical properties of EFB biopulp were characterised according to the Technical Association Pulp and Paper Industry (TAPPI), Chlorite and Kursher-Hoffner methods. On the application side, the handsheets produced were assessed on its physical and mechanical properties according to TAPPI methods. The results revealed that the quadruple culture (KSPE) combination having the highest capacity to degrade lignin by 61.86% (using alkali lignin) with the production of lignin degradation enzymes at 2230.10 U/mL (LiP), 314.84 U/mL (MnP) and 973.80 U/mL (Lac). The biopulping of EFB using KSPE combination identified the optimised conditions as follows; incubation time = 48 h, temperature = 35 °C and glucose load = 5 mL per 100 mL medium with 52.70% of lignin removal. Significantly, the cellulose, hemicellulose, lignin, and extractive contents of the biopulp were recovered at 47.37%, 31.36%, 12.70% and 1.77% respectively. The impact of percentage lignin being removed was reflected on the quality of pulp produced. In this case, the brightness (32.50%), tensile index (9.65 Nm/g), burst index (0.98 kPa.m2/g) and tear index (2.71 mN.m2/g) of handsheet produced were acceptable for the production of printing and writing paper grades. This study had successfully demonstrated the optimised biopulping process of KSPE microbes on EFB. The alternative approach of delignification could promote an effective and greener technology for the future of pulp and paper industries
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