An External Loop Airlift Bioreactor with a small amount (99% porosity) of stainless steel mesh packing inserted in the riser section was used for bioremediation of a phenol polluted air stream. The packing enhanced VOC and oxygen mass transfer rates and provided a large surface area for cell immobilization. Using a pure strain of Pseudomonas putida, fed-batch and continuous runs at three different dilution rates were completed with phenol in the polluted air as the only source of growth substrate. 100% phenol removal was achieved at phenol loading rates up to 33120 mg/h.m3 using only one third of the column, superior to any previously reported biodegradation rates of phenol polluted air with 100% efficiency. A mathematical model has been developed and is shown to accurately predict the transient and steady state data.

This thesis presents the potential of integrating chemical and biological treatment technologies for the removal of high concentrations of phenol in a bioremediation medium. High concentrations of phenol in wastewater are difficult to remove by purely biological methods. Chemical oxidation is one way to treat high concentrations of phenol but complete oxidation is not always possible or will make the treatment process uneconomical. An experimental design approach, based on central composite rotatable design (CCRD) was used to evaluate the effects of process parameters on phenol oxidation by Fentons reagent and chlorine dioxide. Performance of the chemical oxidation was evaluated by determining the percentage of phenol oxidized at equilibrium. The reaction mechanism for the oxidation of phenol by Fentons reagent was proposed based on identification of the intermediate compounds.<p> The effects of H₂O₂ concentration (2000 to 5000 mg L^-1) and FeSO₄.7H₂O concentration (500 to 2000 mg L^-1) were investigated on phenol oxidation and optimal concentrations of H₂O₂ and FeSO₄.7H₂O for complete oxidation of 2000 mg L^-1 phenol in medium were found to be 4340 mg L^-1 and 1616 mg L^-1, respectively, at 25°C and pH 3. The main oxidation products were identified as catechol, hydroquinone and maleic acid.<p> In the case of phenol oxidation by chlorine dioxide, the effects of chlorine dioxide concentration (500 to 2000 mg L^-1), temperature (10 to 40°C) and pH (3 to 7) on the oxidation of 2000 mg L^-1 of phenol were determined. The optimal concentration of chlorine dioxide to completely oxidize 2000 mg L^-1 of phenol was 2000 mg L^-1. The other parameters did not significantly affect the oxidation over the ranges studied. The main oxidation products were identified as 1,4-benzoquinone and 2-chloro-1,4-benzoquinone.<p> Finally, the biodegradation of 1,4-benzoquinone, the main oxidation product of phenol oxidation by chlorine dioxide, was studied in batch and continuous systems using Pseudomonas putida 17484 in two dose McKinneys medium. The effects of 1,4-benzoquinone concentration and temperature were studied on biodegradation of 1,4-benzoquinone in batch reactors. Under optimal conditions, it was found that 150 mg L^-1 1,4-benzoquinone could be successfully biodegraded at 15°C. In a continuous reactor operating at 15°C the highest removal rate with 500 mg L^-1 of 1,4-benzoquinone was found to be 246 mg L^-1 h^-1. The values of µmax, Ks and yield were also determined as 0.74±0.03 h^-1 and 14.17±3.21 mg L^-1 and 2x10¹³ cell mg^-1, respectively.

Scytalidium thermophilum is a thermophilic fungus with an important role in the composting process of mushroom cultivation. An extracellular phenol oxidase of Scytalidium thermophilum (STEP) with novel features was previously studied in our laboratory. This enzyme later turned out to be a catalase having phenol oxidase activity. The aim of this study was to clone Scytalidium thermophilum bifunctional catalase/phenol oxidase encoding gene and express the gene in Aspergillus sojae for future site directed mutagenesis studies. Scytalidium thermophilum catalase gene was first cloned into E. coli XL1 Blue MRF&rsquo / and then heterologously expressed in Aspergillus sojae ATCC11906. For that aim, the catalase gene was amplified using specific primers, excluding the signal and pro-peptide coding regions and amplified fragment was then cloned into E.coli XL1 Blue MRF&rsquo / and sequenced. It was observed that the cloned gene, named as catpo, was 10 amino acids different from the amino acid sequence of the S.thermophilum catalase gene formerly cloned by Novo Nordisk. The catpo gene encoding a mature protein of 681 amino acids was then ligated onto expression vector pAN52-4 and the recombinant plasmid was transformed into Aspergillus sojae ATCC11906. Heterologous expression was observed under the control of the glyceraldehydes 3-phosphate dehydrogenese promoter of Aspergillus nidulans and the secretion signal of the glucoamylase gene of Aspergillus niger. Catalase activity of the transformants reached at a level of 13206 U/g at the end of the fourth day of cultivation. However, this is still lower than the catalase activity of the gene donor strain of Scytalidium thermophilum.

The main object of the presented study was investigation of the use of cross-linked enzyme (tyrosinase) aggregates (CLTA) obtained from crude mushroom extract for a rapid phenolic content analysis in wines. In addition, a comparison of phenolic characteristics of Turkish red wines was performed. Reproducible and reliable results in total phenolic measurement were obtained with CLTAs similar to pure tyrosinase and tyrosinase obtained from crude mushroom extract. Measurement of total phenolic content is possible both in standard solutions and in complex matrices, such as wine. In a very short time period, 10 seconds, phenolics content in red and white wines produced from grapes of Turkey were investigated by using CLTAs. Results were consistent when compared to a well known phenolic measurement method, Folin-Ciocalteau. CLTAs exhibited very high operational stability and retained more than 90% of its activity after 30th use. Moreover, it showed good shelf-life stability for about 2 months storage by maintaining 90% of its maximum activity. So, use of CLTAs prepared from crude mushroom extract is an effective, fast and cheap alternative in total phenolics measurements in wines. Moreover, a novel catalase phenoloxidase (CATPO) produced by a fungal microorganism, Scytalidium thermophilum, was studied to check its capabilities in phenolics measurements. This novel catalase phenol oxidase showed similarly good results, exhibiting widesubstrate selectivity.

Scytalidium thermophilum produces a novel phenol oxidase, which has turned out to be a bifunctional catalase-phenol oxidase (CATPO) during the course of this work, by other researchers of our group. Therefore, in the beginning of the studies, substrate specificity and inhibitor assays were conducted on the crude enzyme, followed by production, purification, cloning, expression, and mutagenesis and crystallography studies for further functional and structural analysis of CATPO. Accordingly, substrate specificity and inhibitory tests applied for crude enzyme characterisation presented the similarity of the phenol oxidase nature of CATPO essentially to catechol oxidase. Production studies were performed to investigate the effects of different factors including induction time, growth temperature, exogenous iron and hydrogen peroxide addition. In view of that, CATPO is constitutively produced in a growth associated manner. However, some phenolic compounds enhance its production. In this study, 15 phenolic compounds were tested for their ability to affect CATPO production. Of the phenolic compounds tested, catechol, resorcinol and vanillic acid caused repression of CATPO production. On the other hand, caffeic acid, myricetin and resveratrol enhanced CATPO production. As a biocatalyst, the efficiency of CATPO was examined and found to be a good candidate for getting pharmaceutically important drug intermediates. Its dual mechanism was analysed through side-directed mutagenesis. Two conserved residues (His101 and Val142) were mutated to discriminate catalase and phenol oxidase activities. Spectroscopic and mutagenesis studies exhibited the presence of heme d centre. Lastly, its structure was analysed by X-ray crystallography and found to have a tetrameric structure.

Staphylococcus aureus is one of the major causes of food-borne pathogenesis. Antibiotic consumption for these pathogens has been increasing year by year world-wide. In order to decrease the use of synthetic antibiotics, fresh fruits and dry herbs consumed as beverages in regular diets were examined as potential natural antibiotics for the treatment of food based infections against Staphylococcus aureus. Herbs consumed as tea infusions including Pimpinella anisum L. (anise), Anthemis arvensis L. (camomile), Rosa canina L. (rosehip), Salvia fruticosa Mill (sage) and fresh fruit juices including Vitis vinifera L. (grape), Citrus sinensis L. (orange), Prunus persica L. (peach) and Punica granatum L. (pomegranate) were selected as samples of hot or cold consumed beverages in our daily diets. Extracts of fresh fruit juices were prepared in methanol, on the other hand, tea infusions of herbs were filtered and lyophilized. Antioxidant capacities of the plant samples were investigated by radical scavenging methods, namely 2&rsquo / 2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) as well as determination of total phenolic and flavonoid contents. Furthermore, antimicrobial activities of plant samples were determined by minimum inhibitory concentration and minimum bactericidal concentration methods along with disc diffusion method. Trolox equivalent antioxidant capacities (TEAC) of the herbal tea infusions obtained by ABTS radical scavenging method were ranged between 48.38 &plusmn / 1.242 and 715.73 &plusmn / 4.265 (&mu / mol Trolox equivalent (TE)/g of extract) while, TEAC values of fresh fruits juices were between 26.86 &plusmn / 0.217 and 73.55 &plusmn / 0.973 (&mu / mol Trolox equivalent (TE)/g of extract). Moreover, EC50 values of the tea infusions obtained by DPPH radical scavenging method were ranged between 0.05 &plusmn / 0.001 and 1.53 &plusmn / 0.004 (mg/mL) while, EC50 values of the fruit juices were 1.6 &plusmn / 0.014 and 2 &plusmn / 0.093 (mg/mL). Total phenolic content of the plant samples tested in this study were varied from 1.383 (&mu / g gallic acid equivalent (GAE)/mg of extract) to 159.167 (&mu / g gallic acid equivalent (GAE)/mg of extract) and total flavonoid content varied from 0.111 (&mu / g quercetin equivalent (QE)/mg of extract) to 201.15 (&mu / g quercetin equivalent (QE)/mg of extract). Sage, orange and grape displayed higher antimicrobial activities with values of 1.5, 6 and 6 mg/mL minimum inhibitory concentrations and 1.5, 24 and 24 mg/mL minimum bactericidal concentrations, respectively. Inhibition zone diameters of sage, orange and grape were found to be 9, 9 and 11 mm.

The purpose of this study was to evaluate the treatment efficiency of phenol contaminated soils by electrokinetic (EK) process conducted in sand boxes (60 cm¡Ñ30 cm¡Ñ30 cm; L¡ÑW¡ÑH). The electric field strength, electrode polarity reverse, and Fenton reagent were employed as the experimental factors in this study to assess the variations of soil characteristics, potential difference, and residual phenol concentration distribution during a treatment period of 20 days and after the treatment. It was found that the anode reservoir pH decreased to around 2 and the cathode reservoir pH increased to approximately 12 after 2~3 days of treatment in the no electrode polarity reverse system. However, the variation of pH in the anode and cathode reservoirs was less obvious in the case with electrode polarity reverse. No matter a constant potential system or a constant current system was employed, a general trend of a lower pH at the anode reservoir and a higher pH at the cathode reservoir would be found. The acid front generated at the anode reservoir flushed across the soil specimen toward the cathode and the base front advanced toward the anode. However, in the central region of sand box, unsaturated and saturated soil specimen maintain neutral. For EK or EK-Fenton experiments, under the constant potential conditions, the potential difference relative to the cathode versus the distance from anode was found to have a linear relationship at the beginning of the electrical potential application. As the treatment time elapsed, the potential gradient became non-linear. Nevertheless, there was no remarked potential gradient change in the case with electrode polarity reverse. Although capillarity has resulted in an increase of the moisture content of unsaturated soil (from 25.34% to 30% after 20 days), electroosmotic (EO) flow was not obvious in the unsaturated zone. For the experiments with electrode polarity reverse, they had a much greater EO flow quantity, the electroosmotic permeability coefficients for constant potential and constant current systems were 6.42¡Ñ10-6 cm2/V¡Es and 9.47¡Ñ10-6 cm2/V¡Es, respectively. It was also found that the existence of contaminants did reduce the EO flow quantity. Regardless of the employment of a constant potential or constant current system, the maximum destruction and removal efficiency (DRE) of phenol was obtained for EK-Fenton process. The maximum DRE values of phenol for both constant potential and constant current systems were found to be 78.06% and 80.11%, respectively. However, the DRE of phenol was found to be much lower for the system with electrode polarity reverse. It was postulated that the destruction efficiency of phenol was less obvious than the removal efficiency in the electrode polarity reverse system. In addition, a frequent reverse of electrode polarity also resulted in a frequent change of EO flow direction. Thus, a flow hysteresis of phenol in the soil compartment was found.

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Το πρόβλημα της ρύπανσης του εδάφους και του υδροφόρου ορίζοντα έχει πάρει ανησυχητικές διαστάσεις τις τελευταίες δεκαετίες. Η βιομηχανική ανάπτυξη πέρα απο τις θετικές συνέπειες που έχει για την ζωή των ανθρώπων, έχει δυστυχώς επιφέρει προβλήματα ρύπανσης του υπεδάφους και του υδροφόρου ορίζοντα με επικίνδυνες οργανικές ενώσεις. Η αλόγιστη και ανεύθυνη διαχείριση και διάθεση των αποβλήτων έχει αρνητικές και συχνά μη αντιστρεπτές συνέπειες για το οικοσύστημα και την δημόσια υγεία. Ανάμεσα στις μεθόδους για την αντιμετώπιση του προβλήματος της ρύπανσης του εδάφους και των υπογείων υδάτων, οι βιολογικές μέθοδοι κερδίζουν ολοένα έδαφος λόγω του χαμηλού κόστους, της αποτελεσματικότητας και του ελάχιστου αριθμού παραπροϊόντων. Οι βιολογικές μέθοδοι αποσκοπούν στην αποκατάσταση των εδαφών με την χρήση γηγενών μικροοργανισμών του εδάφους. Η κινητική ανάπτυξης των μικροοργανισμών στο έδαφος διαφέρει σημαντικά από την κινητική ανάπτυξης όταν οι μικροοργανισμοί αιωρούνται σε καλά αναδευόμενα περιβάλλοντα. Η συγκεκριμένη εργασία έχει σαν στόχο να μελετηθεί η κινητική ανάπτυξης ενός γηγενούς βακτηριακού πληθυσμού, του Pseudomonas fluorescens κατά την βιοαποδόμηση της φαινόλης σε κατάλληλη πειραματική διάταξη, χρησιμοποιώντας ως πρότυπο πορώδες μέσο πυριτική άμμο (SiO2) η οποία αποτελεί ένα από τα βασικότερα συστατικά του εδάφους. Η φαινόλη είναι μία αρωματική ένωση η οποία χρησιμοποιείται συχνά στην βιομηχανία για την παρασκευή χρωμάτων, πλαστικών και φαρμάκων. Εξαιτίας της ευρείας χρήσης της, συναντάται συχνά στο έδαφος και τον υδροφόρο ορίζοντα. Για τον σκοπό αυτό έγιναν αρχικά πειράματα σε διαφορικές κλίνες άμμου με πηγή άνθρακα την γλυκόζη έτσι ώστε να αναπτυχθεί η κατάλληλη πειραματική διάταξη και διαδικασία για την περαιτέρω μελέτη της κινητικής ανάπτυξης του μικροοργανισμού Pseudomonas fluorescens. Στην συνέχεια μελετήθηκε η κινητική ανάπτυξης του μικροοργανισμού χρησιμοποιώντας ως πηγή άνθρακα την φαινόλη σε κλίνες άμμου. Παράλληλα έγιναν και πειράματα διαλείποντος έργου για την μελέτη της κινητικής ανάπτυξης του συγκεκριμένου μικροοργανισμού σε υγρές καλλιέργειες με θρεπτικό υπόστρωμα τόσο την γλυκόζη όσο και την φαινόλη. Σκοπός των πειραμάτων αυτών ήταν ο προσδιορισμός των κινητικών παραμέτρων. Τέλος αναπτύχθηκε κατάλληλο θεωρητικό μοντέλο για την προσομοίωση της κινητικής ανάπτυξης ενός βακτηριακού πληθυσμού στην μικροσκοπική κλίμακα. Απώτερος στόχος του θεωρητικού μοντέλου σε συνδυασμό με τα πειραματικά αποτελέσματα είναι να γίνει αποσαφήνιση των μηχανισμών αποδόμησης τοξικών ρύπων στην μικροσκοπική κλίμακα απο γηγενή βακτήρια και να αναπτυχθούν απλά κριτήρια για την πρόβλεψη και τον σχεδιασμό αποτελεσματικών μεθόδων αντιμετώπισης περιπτώσεων ρύπανσης του υπεδάφους και του υδροφόρου ορίζοντα. / The problem of soil and groundwater contamination has been increasing in the last few decades. Industrial growth is usually accompanied by pollution of groundwater with hazardous organic compounds. Irresponsible disposal of organic compounds into the soil has serious adverse consequences for the ecosystem and public health. Among methods that have been proposed for remediation of contaminated soils, biological methods using microorganisms which are indigenous in soil, are preferable because of their low cost, effectiveness and the low production of byproducts. Growth kinetics of microorganisms in soil differs significantly from growth kinetics of microorganisms suspended in a well-mixed stirred tank reactor. The aim of the present work was the experimental study of growth kinetics of a soilindigenous strain of the bacterium Pseudomonas fluorescens in sand packs (model soil) during the biodegradation of phenol. Phenol is an aromatic organic compound, which is widely used in industry, e.g. in paints, plastics, pharmaceuticals and many other products. Due to its extensive use, phenol is a common pollutant, especially in soil and groundwater. Experiments were initially conducted in sand packs, using glucose as a carbon source. The purpose of these experiments was the setup and test of the experimental procedure. Further experiments of growth kinetics in sand packs were conducted using phenol as a carbon source. In order to determine the growth kinetic parameters of Pseudomonas fluorescens for glucose and phenol biodegradation, batch experiments in liquid cultures were conducted. Finally, a hybrid simulator was developed for the theoretical investigation of growth kinetics of a bacterial population consisted a biofilm in microscale. The further aim of the theoretical simulator combined with the experimental results, was the elucidation of biodegradation mechanisms of toxic compounds by soil-indigenous bacteria in microscale, in order simple criteria for the prediction and remediation of polluted soils and groundwater to be developed.

La rápida industrialización y urbanización mundial ha creado un sin número de contaminantes para los medios acuosos tóxicos y peligrosos, los cuales en su gran mayoría son difícil de degradar de forma natural. Los fenoles son algunos de estos compuestos tóxicos que se encuentran con frecuencia en muchos efluentes industriales. Revisión literaria específica que la oxidación catalítica en aire húmedo utilizando carbón activado podría ser una solución prometedora para la destrucción de estos contaminantes fenólicos. Sin embargo, parece difícil lograr la estabilidad del catalizador dentro del reactor de lecho fijo, en estado estacionario, debido a la combustión lenta de carbón activado. Dentro de este contexto, el presente trabajo se centró en tres aspectos principales para extender los conocimientos actuales sobre la oxidación catalítica del aire húmedo, los cuales son: el establecimiento de las condiciones de arranque del reactor, el estudio de la cinética de reacción y la evaluación de la operación periódica de reactores de lecho fijo. Se ha demostrado que la cinética de la reacción puede llevarse a cabo en un reactor por goteo de lecho fijo sin limitaciones de transferencia de masa y calor. Y más importante aún, el catalizador pudo mantenerse estable durante la oxidación catalítica del aire húmedo de fenol sobre carbón activo en un reactor de lecho fijo, a través de la operación de un reactor dinámico mediante la optimización de los parámetros periódicos. / The fast world industrialization and urbanization creates a large number of water pollutants that are toxic and hazardous and in most cases too hard to amend naturally. Phenols are amongst those toxic compounds frequently found in many industrial effluents. A review of the specific literature points out that catalytic wet air oxidation using activated carbon can be a promising solution for the destruction of phenolic pollutants. However, it seems unlikely to achieve stable catalyst in fixed bed reactor under steady state operation due to the slow combustion of activated carbon. Within this context, the present work focus on three main aspects to extend the current state of art of catalytic wet air oxidation: establishing reactor start-up, kinetic measurements and periodic operation of fixed bed reactors. It has been shown that kinetic measurement can be conducted in trickle bed reactor without mass and heat transfer limitations. Most importantly, stable catalyst during catalytic wet air oxidation of phenol over active carbon was achieved in fixed bed reactor through dynamic reactor operation by optimizing periodic parameters.