The focus of this study is on the use of bioremediation, as the primary method of decontamination for a soil contaminated with industrial waste oils. The area from which the samples were taken was used as a disposal site for oily wastewater for a period of more than 20 years. During this time the soil became severely contaminated. The site is approximately 1 acre in area and consists of three distinct soil strata: a solidified petroleum layer, a peat layer and a layer of muck and mud. This soil is approximately 96% organic matter. The purpose of this study is to determine if: given these site characteristics, is bioremediation a feasible option. Three phases were conducted to determine the usefulness of bioremediation in this situation. Phase one focused on the removal of total petroleum hydrocarbons (TPH) through nutrient addition and aeration. Phase two focused on quantifying and characterizing the reductions observed in phase one. Phase three again focused on quantifying and characterizing the reductions observed in phase one. The three phases of the study provided strong evidence that bioremediation was occurring in the soil and therefore, would be a viable means of remediation for a site with similar characteristics.

Pentachlorophenol (PCP) is a chlorophenol with a pronounced biocidal activity that has led to its use in a number of applications. It was introduced in the 1930s as a preservative for timber and lumber and since then has found wide use as a biocide in agricultural and industrial applications. Many different physical, chemical and biological methods have been tried for the removal of PCP from wastewater. However, using microalgae for the removal of PCP and other organochlorine compounds from water may prove to be a cheaper alternative and give complete degradation of the compounds. The aim of this project was to study the efficiency of microalgae to degrade PCP. An algal strain named VT -1 and a bacterial strain named AT -14 were isolated from PCP containing conditions in the laboratory. The growth of VT -1 in the presence of PCP was compared with Chlorella emersonii and Chlorella vulgaris in two different autotrophic media. It was observed that VT-1 had the highest IC50 value of 25-26mg }-l PCP and EC50 value of 11.3mg }-1 PCP in S&K medium. With glucose as an additional carbon source the IC50 value for VT-1 in S&K medium was 29-30mg t 1 PCP. Bacterium AT-14 could grow in the presence of PCP, only with glucose as a carbon source. Mineralization of PCP by VT -1 and the two Chlorella strains was compared by using 14C_PCp. With all the three algae exposed to 14C_PCp, only VT-1 showed release of 14C02, which was evidence of mineralization of PCP by VT-1 which occurred only in the presence of light. Bacterium AT-14 did not produce 14C02. However, the consortium of VT-1 and AT-14 showed enhanced 14C02 evolution in the presence of glucose. The release of chloride ions from PCP can also indicate PCP dehalogenation and degradation. The evolution of 14C02 lagged behind chloride release (90 %) indicating that dechlorination of PCP could be the first step in its biodegradation. Breakdown of PCP was also followed by its extraction from the cells and medium. Normally dichloromethane (DCM) was used to extract PCP. The changes in the label extracted in DCM and iso-butanol were studied under different light condjtions, which showed that the 14C counts in DCM reduced and those in iso-butanol extract increased with time. The 14C counts in the iso-butanol extract could be a metabolite of PCP which is more hydrophilic. VT-1 appeared not to degrade PCP completely, since only 15% of 14C was recovered as 14C02. It appears that intermediates are formed which are distributed in the growth medium and in the biomass. It can thus be concluded that VT -1 is tolerant of PCP, appears to dechlorinate PCP and then releases some part of it as CO2.

No description available.

The focus of this thesis is the bioremediation of oil impacted sites. Chapter 3 considered “Oil Lakes” in Kuwait, a consequence of the First Gulf War, which pose a considerable human and environmental hazard.  The data revealed that across even a small “Oil Lake”, the hydrocarbon concentration varied significantly and that hydrocarbon concentration was the most important descriptor for determining the rate of remediation.  Effective and sustainable bioremediation strategy must be led by the focused use of underpinning empirical data and its application in a predictive capacity. Chapter 4 considered a similar integrated approach to assess the bioremediation potential of historically contaminated soils from Kuwait.  The analyses reveal that an optimised combination of chemical and biological characterisation were necessary to monitor processes involved in remediation of heavily weathered oil contaminated soils.  A predictive equation derived from empirical chemical and biological data could enhance certainty in the adoption of remedial strategies. Chapter 5 focussed on the toxicity of fractionated crude oils undergoing remediation. Chapter 6 compared a full scale trial of two remediation strategies done on the same site contaminated with bunker-fuel.  This trial reveals the benefit of developing risk and hazard-based approaches in defining endpoint bioremediation of heavy hydrocarbons when engineered biopile or window are proposed as treatment option. Bioremediation, to be effectively applied requires a considerably intense monitoring regime.  For freshly contaminated soils, there is sound justification for applying a predictive capacity but as the samples age, this becomes less certain.  New technologies are evolving which when properly applied and interpreted in the correct context may enhance our ability to sustainably manage hydrocarbon bioremediation.

Many sites world-wide are contaminated with a range of pollutants of environmental concern. Bioremediation has the potential to be a cost-effective and efficient alternative clean-up technology. The success of bioremediation is influenced by both biological and environmental factors. A site, therefore, needs extensive characterisation to determine the extent of contamination and to evaluate the potential for bioremediation. Chemical analysis has traditionally been used to determine pollutant concentrations, but it provides no information about the bioavailability of the pollutants. Bioassays are able to complement chemical analyses by showing the bioavailability and ecotoxico logical effects of pollutants. Bioluminescent bacteria have been adapted as biosensors where the response to environmental stresses is monitored by a reduction in light output. Only metabolically active cells produce light and any substance or environmental condition which impairs cell metabolism and, thus, compromises cellular activity and viability will lead to a reduction in light output. Naturally bioluminescent marine bacteria (e.g. Vibrio fischeri) have been used for ecotoxicity testing, but soil and freshwater bacteria that have been marked with lux genes have several advantages. These genetically modified biosensors do not require high salinity and a neutral pH, and they have environmental relevance. A suite of biosensors has been developed at the University of Aberdeen that responds to a wide range of pollutants. These biosensors have been successfully used for acute ecotoxicity measurements. The research carried out in this study was part of a larger ICI project for the assessment and management of bioremediation of a BTEX contaminated site. The aim of this study was to develop a lux-marked biosensor based on a BTEX-degrader. None of the existing biosensors are known to be degraders. Environmental isolates from the ICI site that were able to degrade BTEX were characterised and assessed for their suitability for lux-marking. An appropriate isolate was selected for lux-marking, but the marking was not successful. It was concluded that there are several problems associated with obtaining suitable isolates in pure culture from a site. The enrichment, isolation, identification and characterisation of isolates is laborious and time-consuming, and the lack of characterisation of the isolates can complicate the Iwc-marking attempts. Selecting a well-characterised bacterium for lux-marking avoids these problems. Therefore, Pseudomonas putida FI was selected as the bacterium for lux-marking as a biosensor in this study. It was selected because it is a toluene-degrader and the degradative genes are located on the chromosome. P. putida FI also has environmental relevance for the ICI site. P. putida FI and P. putida FI06 (an isogenic mutant of P. putida FI) were lux-marked with the plasmid pUCD607, and P. putida FI and FI06 pUCD607 were characterised. Characterisation of P. putida FI and FI06 pUCD607 suggested that pUCD607 was not stable even under selective conditions due to segregational instability. This study, therefore, concluded that the plasmid pUCD607 is not appropriate for lux-marking bacteria as biosensors. P. putida FI was lux-marked with the mini-Tn5 luxCDABE transposon and P. putida FI Tn5 luxCDABE was characterised. The integration of the mini-Tn5 luxCDABE cassette did not affect growth of P. putida FI Tn5 luxCDABE and luminescence levels were higher than in P. putida FI pUCD607. P. putida FI Tn5 luxCDABE was also stable in the absence of selective pressure over time. This study, therefore, concluded that the mini-TnJ luxCDABE transposon is appropriate for lux-marking bacteria as biosensors.

Spring water bottled and sold for human consumption can only be subjected to certain treatment processes such as separation from unstable constituents by decantation, filtration and aeration, ultraviolet irradiation and ozonation. A spring water distribution system in the Western Cape, South Africa was experiencing microbiological problems. The aim of the study was to investigate bacterial contamination in the spring water distribution system and the application of bioremediation as treatment technology. Sampling at various points in the spring water distribution bottling system started in February 2004 and continued until November 2004. The acceptable microbiological limits for bottled spring water clearly states that the total viable colony count should be < 100 organisms per ml of water. Analysis of samples by the heterotrophic plate count (HPC) technique indicated significantly (p < 0.05) high counts which did not conform to the microbiological limit. The heterotrophic plate counts recorded for weeks one, four, eight & 46 in the final bottled water (Site J) were 3.66 x 107 cfu/ml, 9.0 x 106cfu/ml, 2.35 x 107 cfu/ml and 5.00 x 104 cfu/ml, respectively. The total cell counts [Flow cytometry analyses (FCM)] recorded for week one, four, eight & 46 in the final bottled water (Site J) were 5.44 x 107 microorganisms/ml, 8.36 x 107 microorganisms/ml, 9.09 x 107 microorganisms/ml and 5.70 x 107 microorganisms/ml, respectively. The higher viable total cell counts(FCM) indicate that flow cytometry was able to detect cells in the water sample that enter a viable but not culturable state and that the heterotrophic plate count technique only allowed for the growth of the viable and culturable cells present in the water samples. This indicated that the HPC is not a clear indication of the actual microbial population in the water samples. It could be concluded that FCM technique was a more reliable technique for the enumeration of microbial populations in bottled water samples. Various organisms were identified by means of the Polymerase Chain Reaction (PCR) using 16S rRNA specific primers. Purified PCR amplicons were sequenced and Phylogenetic trees were constructed. Neighbour-joining phylogenetic tree analysis of the bacterial species present in the water samples was performed. The dominant bacterial isolates that were sequenced from the various water samples throughout weeks one, four, eight and 46 were Bacillus sp. and Enterobacteriaceae. The pathogenic species isolated throughout the sampling period included Escherichia sp., Pseudomonas sp., Shigella boydii, Bacillus and Staphylococcus sp. A laboratory-scale bioreactor was constructed and water samples were analysed over a period of two weeks. Water samples were analysed using FCM and Direct Acridine Orange Count (DAOC) in conjunction with epiflourescence microscopy (EM). The FCM counts ranged from 1.53 x 107 microorganisms/ml in the initial sample (Day 0) to 1.16 x 107 microorganisms/mℓ in the final sample (Day 13). The results indicated a 24% decrease in the microbial numbers however, it was still above the limit of < 100 organisms/ml as set out by the South African Standards of Bottled Water, (2003). The total cell counts obtained by the DAOC method ranged from 1.43 x 106 microorganisms/ml to 9.54 x 105 microorganism/ml on day 13 (final). The results indicated a 33% decrease in microbial numbers. The total cell counts analysed by flow cytometry fluctuated throughout the sampling period. The total cell counts obtained from the DAOC method were lower in all the water samples when compared to the total counts obtained by flow cytometric analyses. Even though the FCM counts fluctuated throughout the sampling period, results clearly show that the FCM method yielded more accurate data for total cell counts than the DAOC method. Due to external environmental conditions such as changes in the weather conditions the results fluctuated and the final results clearly indicated that further studies are required to optimise the bioreactor system for its application in the spring water industry.

by Ching Mei Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 137-145). / List of Tables --- p.I / List of Figures --- p.III / Abbreviations --- p.VII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Present situation of municipal solid wastes in Hong Kong --- p.1 / Chapter 1.2 --- Landfill in Hong Kong --- p.1 / Chapter 1.2.1 --- Landfill leachate --- p.9 / Chapter 1.2.1.1 --- Generation --- p.9 / Chapter 1.2.1.2 --- Quality --- p.10 / Chapter 1.2.1.3 --- Environmental hazard --- p.17 / Chapter 1.2.1.4 --- Treatment --- p.18 / Chapter 1.2.1.5 --- Other alternatives --- p.24 / Chapter 1.3 --- Spent mushroom compost --- p.27 / Chapter 1.3.1 --- Production and nature --- p.27 / Chapter 1.3.2 --- Availability --- p.29 / Chapter 1.3.3 --- Physical and chemical properties --- p.31 / Chapter 1.3.4 --- Capabilities to degrade phenolic compounds --- p.31 / Chapter 1.3.5 --- Potential uses --- p.37 / Chapter 1.4 --- Proposal and exp erimental plan --- p.38 / Chapter Chapter 2 --- Materials and Methods --- p.41 / Chapter 2.1 --- Materials --- p.41 / Chapter 2.2 --- Physical and chemical analyses of pollutants --- p.41 / Chapter 2.3 --- Basic studies on removal capacities on potential pollutants --- p.48 / Chapter 2.3.1 --- "Removal of dyes, metals and ammonia" --- p.48 / Chapter 2.3.2 --- Removal of pentachlorophenol --- p.53 / Chapter 2.4 --- Applied studies on removal of pollutants --- p.58 / Chapter 2.4.1 --- Treatment of landfill leachate --- p.58 / Chapter 2.4.2 --- Microcosm to examine the decomposition of refuse --- p.60 / Chapter 2.4.3 --- Phytotoxicity --- p.65 / Chapter 2.5 --- Statistical analysis --- p.65 / Chapter Chapter 3 --- Results --- p.67 / Chapter 3.1 --- Characterization of spent mushroom compost and landfill leachate --- p.67 / Chapter 3.2 --- Removal capacities of spent mushroom compost --- p.67 / Chapter 3.2.1 --- Biosorption of dyes --- p.67 / Chapter 3.2.1.1 --- Evercion yellow --- p.67 / Chapter 3.2.1.2 --- Evercion navy H-ER blue --- p.73 / Chapter 3.2.1.3 --- Congo red --- p.74 / Chapter 3.2.1.4 --- Adsorption isotherm --- p.75 / Chapter 3.2.2 --- Biosorption of metals --- p.75 / Chapter 3.2.2.1 --- Lead --- p.75 / Chapter 3.2.2.2 --- Iron --- p.81 / Chapter 3.2.2.3 --- Cadmium --- p.82 / Chapter 3.2.2.4 --- Adsorption isotherm --- p.82 / Chapter 3.2.3 --- Removal of ammonia --- p.85 / Chapter 3.2.3.1 --- Basic study --- p.85 / Chapter 3.2.3.2 --- Applied removal of ammonia from landfill leachate --- p.85 / Chapter 3.2.3.2.1 --- Effect of indigenous micro-organims in landfill leachate --- p.85 / Chapter 3.2.3.2.2 --- Effect of spent mushroom compost and glucose --- p.85 / Chapter 3.2.3.2.3 --- Effect of sugar cane waste extract --- p.89 / Chapter 3.2.3.2.4 --- Effect of sugar cane waste and concentration of glucose --- p.89 / Chapter 3.2.4 --- Removal of pentachlorophenol --- p.91 / Chapter 3.2.4.1 --- Removal by spent mushroom compost --- p.91 / Chapter 3.2.4.2 --- Identification of two spent mushroom compost micro-organisms --- p.91 / Chapter 3.2.4.3 --- Pentachlorophenol-degrading abilities of the two micro-organisms --- p.99 / Chapter 3.2.5 --- A microcosm to examine the decomposition of refuse --- p.99 / Chapter 3.2.5.1 --- pH --- p.99 / Chapter 3.2.5.2 --- Salinity --- p.99 / Chapter 3.2.5.3 --- Turbidity --- p.103 / Chapter 3.2.5.4 --- Ammonia content --- p.103 / Chapter 3.2.5.5 --- Orthophosphate content --- p.106 / Chapter 3.2.5.6 --- "Inorganic, organic and total carbon contents" --- p.106 / Chapter 3.2.5.7 --- Metals --- p.106 / Chapter 3.2.5.8 --- Gases production --- p.112 / Chapter 3.2.6 --- Phytotoxicity --- p.112 / Chapter Chapter 4 --- Discussion --- p.117 / Chapter 4.1 --- Characterization of the spent mushroom compost --- p.117 / Chapter 4.2 --- Removal abilities of pollutants by the spent mushroom compost --- p.119 / Chapter 4.2.1 --- Metals and dyes --- p.119 / Chapter 4.2.1.1 --- Adsorption --- p.119 / Chapter 4.2.1.2 --- Adsorption specificity --- p.123 / Chapter 4.2.1.3 --- Adsorption isotherm --- p.125 / Chapter 4.2.2 --- Pentachlorophenol --- p.127 / Chapter 4.3 --- Decomposition of refuse --- p.129 / Chapter 4.4 --- Removal of ammonia in landfill leachate --- p.132 / Chapter 4.5 --- Phytotoxicity --- p.133 / Chapter Chapter 5 --- Conclusion --- p.135 / Chapter Chapter 6 --- Reference --- p.137 / Chapter Chapter 7 --- Appendix --- p.146

Coumaphos is an organophosphate insecticide used predominantly by the US Department of Agriculture, Animal and Plant Health Inspection Services for its tick eradication program. Bioremediation of the hydrolysis products of coumaphos, chlorferon and diethylthiophosphate (DETP), using Ca-alginate immobilized cells was the focus of this research. Consortia of indigenous microorganisms capable of degrading chlorferon and DETP were isolated separately. Since chlorferon inhibited both chlorferon-degrading and DETP-degrading organisms, it was not possible to enrich a consortium of organisms for simultaneous degradation of chlorferon and DETP. A two-step growth procedure was developed for degradation studies to provide biomass acclimated to the target compound and reaction medium since cells lost their degradation activity during the growth in a rich medium. Without acclimation, approximately a week-long lag period was required before degradation was initiated. Optimum reaction conditions were found for the degradation of chlorferon and DETP using free cells. Reaction kinetics of chlorferon and DETP were determined using enzyme kinetics because cell growth was not observed during the degradation. Chlorferon degradation followed substrate inhibition kinetics and DETP degradation followed simple Michaelis-Menten kinetics. A calcium-alginate immobilized cell system was developed, and the optimum bead loadings in the reactor were determined. Degradation rates for immobilized cells were enhanced up to five times that for free cells in untreated cattle dip (UCD) solution. The enhanced degradation of immobilized cells was due to protection of the cells from inhibitory substances present in the UCD solution. In addition, physiological changes of cells caused by Ca-alginate immobilization may have contributed to a slightly increased reaction rate in pure solution. Diffusion coefficients of chlorferon and DETP into Ca-alginate gel beads were studied to assist in designing and operating bioreactor systems. Diffusion coefficients of chlorferon and DETP increased with increasing agitation speed and decreasing substrate concentration. Increased cell concentration in gel beads caused lower diffusivity. Calcium-alginate gel beads used in this study were not subject to diffusional limitations. Both external and internal mass transfer resistances were negligible, and the degradation rate inside Ca-alginate gel beads was reaction-limited.

Phosphotriesters, also known as organophosphates (OP), represent a class of toxic compounds first synthesized in Germany. Enzymatic removal of harmful insecticides and breakdown products is a promising alternative to skimming or dredging. Wild type bacterial phosphotriesterase (PTE) was screened against 7 agricultural organophosphates: coumaphos, chlorpyrifos, fenitrothion, temephos, profenofos, pirimiphosmethyl and diazinon. The initial results laid the groundwork for a mutagenesis study to investigate the determining factors in enzyme reactivity. Coumaphos is hydrolyzed more efficiently than any other target by the wild type cobalt enzyme (kcat/Km = 2 x 10^7 M^-1s^-1). Coumaphos, fenitrothion and chlorpyrifos had the lowest Km values from the initial screen and were targets for steady state kinetic characterization of active site mutants. Site directed mutagenesis of binding sites was conducted and the most reactive point mutants, F132G, F132V and S308G, were used as backgrounds for subsequent mutation. Seven active site double mutants: F132G/S308G, F132G/S308T, F132V/S308G, F132V/S308T, F132G/I106T, F132V/I106T and G308/W309 were purified to homogeneity for kinetic characterization. The double mutant G308/F132V enhanced chlorpyrifos reactivity relative to the wild type enzyme. This enhancement of reactivity is proposed to result from conformational rearrangement following substrate bond hydrolysis.

Com a resultat de les activitats humanes, la contaminació deguda a productes químics alliberats al medi s’ha convertit en un problema global, essent una amenaça real per a l’activitat humana. La contaminació pot afectar qualsevol compartiment de l’ecosistema. Els hidrocarburs derivats del petroli, on s’inclouen els hidrocarburs policíclics aromàtics (HAPs), són contaminants que afecten de manera particular el sòl. Actualment existeixen diverses tècniques per a la restauració d’emplaçaments contaminats, inclosa la bioremediació. La micorremediació, àrea de coneixement en què s’emmarca la tesi, ha guanyat atenció en els últims anys ja que és una tècnica ambientalment respectuosa. Aquest treball presenta els resultats de les investigacions prèvies al desenvolupament d’un tractament de bioremediació de sols contaminats per hidrocarburs policíclics aromàtics mitjançant el fong ligninolític Trametes versicolor. Els resultats de l’esmentada investigació es presenten en tres apartats diferenciats. El primer es centra en la producció de biomassa del fong per a posteriors aplicacions en processos de bioremediació. Es divideix en dues seccions: • La primera es basa en la producció de biomassa en cultiu submergit. Els experiments es van centrar en formular un medi definit de cultiu de baix cost que permetés obtenir nivells elevats de biomassa, en la morfologia desitjada (pellets). El reactor fluïditzat per polsos d’aire amb control de pH va resultar ser el més adequat. La producció s’escalà a un bioreactor de 10 litres. • La segona secció analitza la colonització del fong sobre suports lignocel·lulòsics provinents de residus agrícoles per a posterior aplicació en el sòl. La selecció dels millors substrats per a la colonització es va basar en el nivell de biomassa (ergosterol), la producció de lacasa i la capacitat de degradar naproxè en 24 hores. Es va demostrar que el fong era capaç de colonitzar el sòl tant en condicions estèrils com en no estèrils mantenint, en tot cas, la capacitat degradativa. El segon apartat es centra en la degradació d’hidrocarburs policíclics aromàtics pel fong. Es divideix en tres seccions: • La primera es basa en la selecció d’un surfactant per a la degradació de HAPs en medi líquid, essent el millor el surfactant no iònic Tween 80. Es va poder demostrar la capacitat degradativa de diversos HAPs en medi líquid; tant en experiment per separat com en mescles. Així mateix, també es va demostrar que en les condicions de cultiu l’enzim lacasa podia degradar alguns dels compostos. • La segona secció fa referència a la identificació de productes intermediàris de degradació d’HAPs. També es va estudiar la capacitat de degradació d’aquests intermediaris per part del fong. • En la tercera secció es van provar diferents sistemes de degradació en sòl, on el bioslurry es presenta com el més efectiu en termes d’eficàcia de degradació. Es va poder comprovar que en les biopiles airejades el fong va tenir problemes derivats del rang termofílic assolit. El darrer apartat es basa en la degradació dels HAPs de la creosota. Es divideix en dues seccions: • La primera es centra en la degradació dels HAPs de la fracció aromàtica de la creosota. Es van estudiar diferents sistemes i es va determinar que el fong era més eficient en degradació en biopiles. En cultius submergits, medi líquid i slurry, el fong era efectiu en la degradació d’HAPs de baix pes molecular però no en aquells d’alt pes molecular. Es va observar l’efecte inhibitori sobre Trametes tant dels compostos addicionals afegits junt amb la creosota com en augmentar la concentració de HAPs. ! • La segona secció és fruit de la col·laboració amb el Laboratori de Biotecnologia Ambiental de l’Institut de Microbiologia de l’Acadèmia de les Ciències de la República Txeca de Praga. S’estudià l’efecte de la interacció del fong amb la població microbiana del sòl durant processos de bioremediació d’HAPs en sòl.! El treball experimental s’ha dut a terme en el “Grup de degradació de contaminants industrials i valorització de residus” del Departament d’Enginyeria Química de la UAB. L’objectiu general de recerca del grup és el desenvolupament de processos biotecnològics per a degradar compostos xenobiòtics difícilment degradables per tractaments convencionals. / Como consecuencia de las actividades humanas, la contaminación debida a productos químicos liberados en el medio se ha convertido en un problema global, siendo una amenaza real para la actividad y la salud de los seres vivos. La contaminación puede afectar a cualquier compartimento del ecosistema. Concretamente, los hidrocarburos derivados del petróleo son contaminantes que afectan de manera particular al suelo, entre los cuales destacan los hidrocarburos policíclicos aromáticos (HPAs). Actualmente existen diversas técnicas para la restauración de emplazamientos contaminados por estos compuestos. Cabe destacar que en los últimos años la comunidad científica ha centrado especialmente los esfuerzos en el campo de la micorremediación (área de conocimiento donde se enmarca la presente tesis) dado que se trata de una técnica ambientalmente respetuosa. El trabajo presenta los resultados de las investigaciones previas al desarrollo de un tratamiento de bioremediación de suelos contaminados por hidrocarburos policíclicos aromáticos mediante el hongo ligninolítico Trametes versicolor. Los resultados de la mencionada investigación se presentan en tres apartados diferenciados. El primero se centra en la producción de biomasa del hongo para posteriores aplicaciones en procesos de bioremediación. Se divide en dos secciones: • La primera se basa en la producción de biomasa en cultivo sumergido. Los experimentos se centraron en formular un medio de cultivo definido de bajo coste que permitiera obtener niveles elevados de biomasa, en la morfología deseada (pellets). El reactor fluidizado por pulsos de aire con control de pH resultó ser el más adecuado. La producción se escaló a un bioreactor de 10 litros. • La segunda sección analiza la colonización del hongo sobre soportes lignocelulósicos provenientes de residuos agrícolas para posterior aplicación en el suelo. La selección de los mejores sustratos para la colonización se basó en el nivel de biomasa (ergosterol), la producción de lacasa y la capacidad de degradar naproxeno en 24 horas. Se demostró que el hongo era capaz de colonizar el suelo tanto en condiciones estériles como no estériles manteniendo, en todo caso, la capacidad degradativa. El segundo apartado se centra en la degradación de hidrocarburos policíclicos aromáticos por el hongo. Se divide en tres secciones: • La primera se basa en la selección de un surfactante para la degradación de HPAs en medio líquido, siendo el surfactante no iónico Tween 80 aquel que dio mejores resultados. Se pudo demostrar la capacidad degradativa de diversos HPAs en medio líquido; tanto en experimentos por separado como en mezclas. Asimismo, también se demostró que en las condiciones de cultivo la enzima lacasa podía degradar algunos de los compuestos. • La segunda sección hace referencia a la identificación de productos intermediarios de degradación de HPAs. También se estudió la capacidad de degradación de estos intermediarios por parte del hongo. • En la tercera sección se probaron diferentes sistemas de degradación en suelos, donde el bioslurry resultó el más efectivo en términos de eficacia de degradación. Se pudo comprobar que en las biopilas aireadas el hongo tuvo problemas derivados del rango termofílico alcanzado. El último apartado se basa en la degradación de los HPAs de la creosota. Se divide en dos secciones: • La primera se centra en la degradación de los HPAs de la fracción aromática de la creosota. Se estudiaron diferentes sistemas y se determinó que el hongo era más eficiente en degradación en las biopilas. En cultivos sumergidos, medio líquido y slurry, el hongo era efectivo en la degradación de HPAs de bajo peso molecular pero no en aquellos de alto peso molecular. Se observó el efecto inhibitorio sobre Trametes versicolor tanto de los compuestos adicionales añadidos junto con la creosota como al aumentar la concentración de HPAs. • La segunda sección es fruto de la colaboración con el Laboratorio de Biotecnología Ambiental del Instituto de Microbiología de la Academia de las Ciencias de la República Checa en Praga. Se estudió el efecto de la interacción del hongo con la población microbiana del suelo durante procesos de bioremediación de HPAs en suelos. El trabajo experimental se ha llevado a cabo en el "Grupo de degradación de contaminantes industriales y valorización de residuos" del Departamento de Ingeniería Química de la UAB. El objetivo general de investigación del grupo es el desarrollo de procesos biotecnológicos para degradar compuestos xenobióticos difícilmente degradables mediante tratamientos convencionales. / As a result of human activities pollution aroused as a global concern due to improper release of chemicals into the environment. Contamination represents a real threat to humans and can affect any ecosystem compartment. Petroleum hydrocarbons affect typically soil, including polycyclic aromatic hydrocarbons (PAHs). Several conventional clean-up techniques are available for site restoration, including bioremediation. Mycoremediation, attained interest in the last decades as it is assumed to be an environmental-friendly technique. This work shows the results of the basic research previous to the development of a polycyclic aromatic hydrocarbons polluted soil bioremediation treatment by means of the white rot fungus Trametes versicolor. The results of the mentioned research are presented in three differentiated sections. The first section focuses on the fungal biomass production for posterior applications in bioremediation processes. It is divided into two subsections: • The first part focuses on biomass production of in submerged cultures. The experiments were aimed at formulating a low-cost defined medium to obtain high amounts of biomass, in the preferred morphology (pellets). The air-pulsed fluidized bioreactors equipped with pH control were the most appropriate. The production was scaled-up to a 10 liters bioreactor. • The second part analyzes the fungal colonization of lignocellulosic supports for further application in soil. The selection of the optimal substrate for colonization based on active biomass amounts, laccase production and the capacity to degrade naproxene in 24 hours. It was demonstrated that the fungus was capable of colonizing soil both under sterile and non-sterile conditions maintaining, in any case, the degradative capacity. The second section focuses on the fungal ability to degrade polycyclic aromatic hydrocarbons. It is ddivided into three subsections: • The first part focuses on the selection of an optimal surfactant for PAHs degradation in liquid medium, obtaining the best results with the non-ionic surfactant Tween 80. The degradation of several PAHs by T. versicolor in liquid medium was demonstrated; both in individual-PAHs experiment as well as in PAHs-mixtures. Likewise, it was also demonstrated that under culture conditions laccase might degrade some of the studied compounds. • The second section includes the identification of PAHs metabolites arising from degradation. The fungal degradation capacity of these intermediates was also examined. • In the third section different degradation systems of were tested for soil treatment, the bioslurry resulted as the most effective in terms of degradation efficiency. It was checked out that in aerated biopiles, the fungus had problems derived from attaining termophilic ranges. The last section deals with the creosote-PAHs degradation. It is divided into two subsections: • The first part focuses on the fungal PAHs-degradation of the creosote aromatic fraction. Different systems were studied and it was determined that the fungus was more efficient at degrading PAHs in biopiles approach. In submerged cultures, liquid medium and slurry, the fungus was effective at degrading low-molecular-weight PAHs but not those of high-molecularweight. It was also observed inhibitory effects on Trametes due to the additional compounds present in creosote as well as when increasing the total PAHs concentration. • The second section is a result of the collaboration with the Laboratory of Environmental Biotechnology in the Institute of Microbiology, which belongs to the Academy of Sciences of the Czech Republic (Prague). The effect on PAHs removal during the interaction between soil microbial population and white-rot fungi during soil bioremediation processes was studied. The experiments have been carried out in the "Group of degradation of industrial pollutants and valuation of waste" from the Department of Chemical Engineering in the UAB. The main research motivation of the group is to develop specific biotechnological processes to degrade xenobiotic compounds that are scarcely degraded by conventional treatments.