Aquesta tesi tracta l'estudi de la producció de la vitamina K3 des d'un punt de vista biotecnològic amb l'objectiu de plantejar noves alternatives a la síntesis química clàssica d'aquesta vitamina.La síntesi clàssica de la vitamina K3 es realitza per un procediment químic que comporta una oxidació del 2-metilnaftalè com a material de partida, mitjançant un agent oxidant molt fort, el dicromat sòdic, en un medi que conté àcid sulfúric, creant-se una reacció que genera una elevadíssima quantitat de residus de crom en aigües àcides.Així doncs es tracta d'una reacció molt contaminant que alhora té unes limitacions des del punt de vista químic molt important, com és la no utilització d'un catalitzador, la generació de grans quantitats de residus, així com la formació d'un isòmer de la vitamina K3, la 6-metil-1,4-naftoquinona, de manera que es produeix un compost no biològicament actiu, del qual cal desfer-se mitjançant dificultosos processos de purificació.Els experiments realitzats en aquest treball van estar dirigits cap a la solució d'aquests problemes, és a dir, la utilització de biocatalitzadors en unes condicions suaus per a minimitzar l'impacte ambiental del procediment de síntesi , així com la millora de la selectivitat de la reacció, eliminantse la formació de l'isòmer de la vitamina K3, punt clau per a l'èxit d'aquest nou sistema de síntesi.Amb aquests objectius es va treballar amb microorganismes salvatges, així com amb microorganismes de col.leccions internacionals, fins que es va poder determinar que dos microorganismes salvatges aïllats del producte de partida de la reacció, el 2-metilnaftalè, així com tres soques de les col.leccions internacionals, eren capaços de produir vitamina K3 a partir d'aquest substracte. Un cop obtingudes les soques productores de vitamina K3 es va poder determinar el mecanisme de la reacció, que indicava la formació dels intermediaris 2-metil-1-naftol i 2-metil-4-naftol, seguit del menadiol i finalment la formació de vitamina K3. També es va poder dur a terme la determinació de que els microorganismes productors de vitamina K3 tenien un 100% de selectivitat pel que fa a la formació de quinones, evitantse la formació de l'isòmer no desitjat, fet determinant per a l'interès del món químic-farmacèutic en una reacció d'aquest tipus. Es van poder mostrar diferents alternatives de síntesi d'aquesta vitamina utilitzant sistemes enzimàtics, cèl.lules senceres actives i cèl.lules senceres no proliferants, essent la producció amb Bacillus subtilis i Bacillus cereus biotip I en condicions de no proliferació en glucosa, les millor condicions de productivitat.Des del punt de vista dels estudis mol.leculars, es va poder determinar que la proteïna responsable de la síntesi de vitamina K3, utilitzant com a substracte el 2-metil-1-naftol, era la citocrom aa3-600quinol oxidasa de Bacillus subtilis , però no es va poder determinar la proteïna responsable del primer pas de la reacció, l'oxidació del 2-metilnaftalè a 2-metil-1-naftol.Els resultats obtinguts permeten veure un ventall de possibilitats que donen alternatives reals als processos clàssics de síntesi de la vitamina K3, tot i que són processos en els quals encara s'ha de treballar molt per augmentar-ne la productivitat fins a uns nivells òptims. / The classical synthesis of vitamin K3 is done by a chemical process consisting of an oxidation of the substrate, 2-methylnaphtalene, with a powerful oxidizing agent such as sodium dichromate in a sulphuric acid medium, which leads to a stechiometric reaction that generates an extremely high amount of chromium waste in acidic waters.This process is extremely polluting and at the same time, from a chemical point of view, its limitations are very important. The most important limitations are the low atom echonomy, because no catalysis is achieved as it is a stechiometric reaction that produces a great amount of waste, and the production of an isomer(6-methyl-1,4-naphtoquinone) of the vitamin, which is not a biologically active compound, so producing companies must get rid of this compound by a complex purification process.Experiments done in this study were performed with the aim of improving the reaction using biocatalysts in mild conditions to solve the environment impact , and in order to obtain a 100% selective oxidizing agent avoiding the production of the undesired isomer, which is the key point for the success of this new process.The strains used in this work were mainly isolated from the environment, except for some of them which were purchased from international collections, and two of the wild strains, which were isolated from the substrate of the reaction, and three of the purchased strains showed a positive results for the produciton of vitamin K3 using 2-methylnaphtalene as substrate.The mechanism of the reaction involved the sequential oxidation of 2-methylnaphtalene to 2-methyl-1-naphtol and 2-methyl-4-naphtol, leading to menadiol and finally leading to the formation of menadione as final product, with a 100% selective process for the production of quinones, avoiding the production of the undesired isomer.Different alternatives for the production of menadione were shown by using enzyme systems, active whole cells and whole resting cells of the 5 different menadione producing strains, from which the best results were achieved with Bacillus subtilis and Bacillus cereus biotype I resting cells in glucose.Molecular studies involving the cloning and expression of the cytochrome aa3 600 quinol oxidase of Bacillus subtilis in E.coli BL21, permitted us to determine that this protein is the responsible for the production of menadione using the substrate 2-methyl-1-naphtol, althoug no evidence of its activity against 2-methylnaphtalene was achieved.The results obtained in this work show a broad range of possibilities for alternative processes for the synthesis of vitamin K3, although this processes need to develope much further in order to compete with the stablished chemical process in terms of yield and production.

This work is included in a largest project focused on the improvement of transgenic rice plants resistance against pests and phytopathogens. Our goal is the obtention of an improvement of the rice resistance against the lepidopterean Chilo suppressalis, the most important rice pest. The chosen strategy was the expression of the mpi gene (Maize Proteinase Inhibitor, coding for a proteinase inhibitor) on rice, by both biolistic and Agrobacterium transformation methods. The proteinase inhibitors are part of the natural defence system of plants against insect predation. Expression of this gene, and accumulation of the MPI protein, is stable through successive generations of rice transgenic lines. We have demonstrated that constitutive expression of the mpi gene confers resistance towards Chilo suppressalis larvae infestation in transgenic rice. Bioassays performed with transgenic rice plants expressing the mpi gene constitutively that were infested with Chilo larvae, showed that ingestion of the MPI lead to a significant reduction of the larvae weight with respect to the control larvae group (as far as 60 %). It is also important to point out that larvae fed on transgenic MPI plants showed a significant delay in their development, preventing the achievement of the L4 larval stage in most of them.The mpi promoter is functional in rice and confers wound-inducibility of gus reporter gene. The 2K fragment of mpi promoter confers a more rapid and intense induction than shortest C1 fragment. Moreover, 2K fragment of mpi promoter doesn't confer expression of gus reporter gene in the rice seed endosperm.On the other hand, the maize mpi gene is correctly expressed in rice plants under control of his own regulatory regions. The observed expression levels allow obtaining similar effects on Chilo larvae than those obtained with constitutive mpi expression.Besides, we fixed the optimal pH of digestive proteinase activities of Chilo suppressalis and Cacyreus marshalli (Geranium pest) as alkaline (pH 10.5 and 10 respectively). In Chilo digestive system participate serine proteinases activities of trypsin and chimotryspsin type, and also carboxy and aspartic proteinases. We also observed an adaptation effect on Chilo digestive system of larvae feed on transgenic rice plants, with an increase of 40% of total proteolytic activities in comparison to the control larvae activities. Nevertheless, this increase is not effective to overcome the MPI inhibitor effect.The analysis of AFP (Antifungal Protein) allows to conclude that this protein shows an elevated antifungal activity against F. verticillioides and M. grisea at low nM concentrations. Besides, AFP shows activity against the oomycete Phytophthora infestans at low mM concentrations. The presence of AFP protein leads to important morphological abnormalities of these phytopathogens. We have not detect toxic effects of AFP protein towards rice protoplasts at high AFP concentrations. The topic application of AFP protein on rice plants confers protection to Magnaporthe grisea infection.

En aquest treball s'exposen dues línies de recerca centrades en el procés de producció d'una lipasa de Rhizopus oryzae (ROL) en Pichia pastoris: la utilització de diferents tècniques de monitoratge i l'aplicació d'estratègies de cultiu que permetin millorar la producció de la ROL en una soca Mut+ d'aquest sistema d'expressió. En els cultius discontinus alimentats amb P. pastoris cal disposar d'una mesura en línia i en temps real de la concentració de substrat, el metanol. Per això es va desenvolupar un analitzador d'injecció seqüencial. Es va comprovar que aquest té una freqüència d'anàlisi òptima per a cultius amb la soca Muts però baixa per a la soca Mut+. Amb aquesta última soca, es van utilitzar i comparar dos mesuradors comercials en fase gas. Es va avaluar també la fluorimetria com a tècnica de monitoratge per a la determinació de tres variables clau: la biomassa, el substrat (glicerol/metanol) i la proteïna recombinant.Inicialment es va fer un seguiment de l'evolució de la biomassa a partir del senyal de fluorescència off-line del triptòfan. Es pot predir la biomassa correctament però es van evidenciar diferents desavantatges: la necessitat de dilució de les mostres i d'un sistema de presa de mostra específic. Per això es va decidir aplicar i avaluar la fluorimetria multivariable in situ. Amb la utilització d'una sonda fluorimètrica in situ, combinada amb mètodes quimiomètrics de tractament de dades multivariables, es va aconseguir predir la biomassa i el substrat a partir de la determinació del senyal de diversos fluoròfors. No es va aconseguir una bona predicció de la producció de ROL. Per millorar el seguiment de la proteïna, es va fusionar a la GFP. Es van detectar dos inconvenients: els nivells de producció disminuïen respecte a la producció de ROL únicament i la riboflavina, que Pichia excreta al medi, interferia amb el senyal de fluorescència del mutant de GFP escollit. Tant amb la filtració de la mostra abans de la determinació del senyal de GFP com si s'hagués escollit un mutant de GFP amb emissió més allunyada de la riboflavina es podria haver evitat aquest inconvenient i fer un seguiment de la ROL excretada. Es pot seguir la ROL intracel·lular.Quant a la producció de la ROL, es va estudiar l'efecte del nivell de metanol residual en cultius discontinus alimentats amb la soca Mut+. Existeix una concentració òptima entorn els 2.5 g·l-1 de metanol al medi. A concentracions superiors es va apreciar inhibició per substrat. Es va observar un fenomen de limitació per transferència d'oxigen al final del cultiu i una important disminució de la viabilitat cel·lular.Per això, es van avaluar estratègies de cultiu alternatives. Primer es va aplicar una estratègia de metanol limitant (MLFB) per evitar la limitació d'oxigen al final d'un cultiu a 2.5 g·l-1 de metanol residual (MNLFB). Es va millorar la productivitat un 40%. En segon lloc, es va aplicar una estratègia de temperatura limitant per avaluar el seu efecte sobre la producció. No es van millorar els resultats. Finalment, es va aplicar una estratègia MNLFB a 2.5 g·l-1 de metanol però amb un medi amb una menor osmolaritat i una fase final amb limitació de temperatura per evitar la limitació d'oxigen. Es va aconseguir reduir la mortalitat cel·lular però la productivitat disminuïa respecte el cultiu on s'aplica una fase de MLFB al final de la inducció. Tot i això, es va obtenir un producte final un 30% més pur en quant a activitat lipolítica respecte la proteïna total. Posteriorment es va procedir a escalar la producció en planta pilot. Es va observar limitació en la transferència d'oxigen en fases inicials de la inducció. Això va originar l'aparició d'un subproducte, associat a una reducció en la producció de ROL extracel·lular. En millorar la transferència d'oxigen es va minimitzar aquesta limitació i va millorar la producció. No es van aconseguir els mateixos nivells de productivitat que a escala laboratori però es continua treballant en la millora de la transferència de matèria per assolir-los. / In this work two research lines, applied to the production of a Rhizopus oryzae lipase (ROL) in Pichia pastoris, are shown: the application of different monitoring and cultivation techniques to improve ROL production in a P. pastoris Mut+ strain.During P. pastoris fed-batch cultures, methanol needs to be on line measured and monitored in real time. For this purpose, a sequential injection analyzer was developed. Although it presented a suitable analysis frequency for a Muts strain it was too low for a Mut+ strain. When the later was used, two different methanol commercial sensors in the outlet gas steams were utilized and compared. Fluorometry was also evaluated as a monitoring technique for three key variables: biomass, substrate (glycerol/methanol) and recombinant protein. Initially, biomass was followed by the off-line determination of tryptophan's fluorescence. Biomass was correctly predicted with this system but different disadvantages appeared: the need of a sample dilution procedure and a specific sampling device. Therefore, multivariable in situ fluorometry was subsequently evaluated. By means of an in situ multivariable fluorimetric probe, combined with chemometric methods to data processing, biomass and substrate prediction was properly achieved. ROL production could not be satisfactorily estimated.To improve ROL monitoring, it was fusioned to the green fluorescent protein (GFP). Two main disadvantages were found: production levels were lower when compared to solely ROL expression and riboflavin, naturally excreted by the yeast, interfered to GFP's signal. This problem could be solved with sample filtration prior to GFP's measurement and also if a different GFP mutant had been chosen with emission signal further to riboflavin's.With respect to ROL production, the effect of methanol concentration was studied in Mut+ fed-batch cultures. There is an optimal methanol concentration about 2.5 g·l-1. Substrate inhibition was observed at higher methanol levels. Oxygen transfer limitation at the end of the induction phase and an important cell viability decrease were also found. Therefore, alternative culture techniques were evaluated. First a methanol limited fed-batch phase (MLFB) was applied when oxygen limitations appeared at the end of a 2.5 g·l-1 methanol fed-batch phase (MNLFB). Productivity increased up to 40% with this strategy. Secondly, a temperature limited fed-batch was applied. No better results were obtained compared to the reference MNLFB culture. Finally, a 2.5 g·l-1 methanol fed-batch was applied with a lower salt content medium and a final temperature limited phase when oxygen limitation appeared. Cell death was reduced but productivity decreased with respect to the reference MNLFB culture. However, a 30% purer lipase was obtained in terms of lipase activity to total protein.Thereafter, the scaling of the production process in a pilot plant was evaluated. Oxygen limitation was found in the early induction phase. This caused a byproduct secretion associated to a ROL production decrease. When oxygen transfer was enhanced, byproduct secretion was reduced and ROL production improved. Similar laboratory scale productivities were not achieved but oxygen mass transfer is being further enhanced to reach the objective levels.

Sp1 i Sp3 pertanyen a la família de factors de transcripció Sp que controla la transcripció de gens implicats en gairebé tots els processos cel.lulars. Estudis previs al nostre grup van estudiar la regulació del promotor del gen Sp1 i van demostrar que la transcripció de Sp1 estava regulada principalment de forma positiva per Sp1 i NF-Y, i que Sp3 era capaç de contrarrestar l'activació produïda per Sp1 sobre el seu propi promotor. Donat que la relació Sp1/Sp3 a la cèl·lula és important per a la regulació dels gens diana, en aquest treball ens vam proposar estudiar la regulació del promotor de Sp3.Vam clonar una regió de 546 bp que corresponia al promotor de Sp3 i vam procedir a la seva caracterització. Sp3 presenta múltiples inicis de transcripció localitzats entre les posicions -132 i -70 respecte de l'inici de traducció i la seva màxima activitat promotora es localitza a la regió fins a -281bp respecte de l'inici de traducció. Mitjançant les tècniques de retardament de la movilitat electroforètica (EMSA) i Immunoprecipitació de la Cromatina (ChIP) hem demostrat la unió dels factors Sp1, Sp3, NF-Y, NF-1, c-Myb, B-Myb i c-Jun al promotor de Sp3. D'altra banda hem estudiat l'efecte de la sobreexpressió i la inhibició d'aquestes proteïnes sobre l'activitat d'aquest promotor utilitzant assajos d'activitat luciferasa, i sobre els nivells endògens de mRNA utilitzant RT-Real Time-PCR. Sp3 activa la transcripció del seu propi promotor. El promotor de Sp3 també és activat de forma més potent per Sp1, Sp3 i NF-Y; tot i que NF-1, c-Myb, B-Myb, c-Jun i c-Fos també poden activar aquest promotor. Un altre fet remarcable és que E2F1 es comporta com a repressor del promotor de Sp3. Tots els resultats observats a nivell de l'activitat del promotor es van confirmar amb la mesura dels nivells endogens de mRNA per Sp3.Addicionalment, s'ha estudiat la interacció de Sp1 amb diferents proteïnes implicades en la regulació del cicle cel·lular i s'ha caracteritzat l'efecte de la seva sobreexpressió sobre l'activitat del promotor de Sp1, ja que està regulat per Sp1. Utilitzant un array d'anticossos, es va fer un cribatge de proteïnes que poguessin interaccionar amb Sp1, i algunes d'elles es van confirmar per co-immunoprecipitació. Això ens va permetre demostrar que Sp1 és capaç d'interaccionar amb CDK4, p21, SKP2 i BRCA2. Posteriorment, vam analitzar l'efecte d'aquestes i altres proteïnes que interaccionen amb Sp1 sobre el promotor de Sp1, i vam observar que el promotor de Sp1 és regulat de forma positiva per la sobreexpressió de CDK4, SKP2, BRCA2, Ciclina D1, E2F1/DP1 i Stat3; mentre que és reprimit per la sobreexpressió de p53 i NFB. Per tal d'analitzar si hi havia una correlació entre els efectes sobre el promotor de Sp1 i una alteració dels nivells endogens de Sp1, vam confirmar tots els efectes observats a nivell de l'activitat del promotor tot emprant la tècnica de RT-Real Time-PCR. A més, els efectes sobre el promotor de Sp1 es produeixen mentre aquestes proteïnes estan unides, directa o indirectament, al promotor tal com van demostrar els assajos de ChIP. També vam estudiar l'efecte de la sobreexpressió d'aquestes proteïnes sobre un promotor que només contenia caixes Sp1 i, en general, vam observar efectes equivalents als observats per al promotor de Sp1. La interacció entre Sp1-p21 va ser objecte d'estudi en més detall i vam determinar que l'expressió de p21 en cèl·lules de fibrosarcoma indueix el promotor de Sp1 així com els nivells de mRNA, però, al mateix temps, indueix la degradació de Sp1.Com a conclusió final, hem vist que el procés de transcripció és un mecanisme molt complex que involucra un gran número de factors de transcripció, així com d'altres proteïnes que puguin interaccionar amb aquests factors. / Sp1 and Sp3 belong to the Sp family of transcription factors that controls transcription of genes involved in almost all processes in the cell. We performed a detailed analysis of the promoter region of the Sp3 transcription factor, including the identification of its transcriptional start sites and the putative binding sites for transcription factors. Multiple transcriptional starts sites were located at position sranging from -70 to -132 relative to the translational start of the gene. We defined the minimal promoter region cooresponding to 281 bp relative to the translational start. Along the promoter sequence we demonstrated the binding of Sp1, Sp3, NF-Y, NF-1, c-Myb, B-Myb and c-Jun. Moreover, we studied the effect of the overexpression or knocking down of these factors on the Sp3 promoter activity and the endogenous mRNA levels. Sp3 is positively autoregulated and it is also activated by Sp1, NF-Y, Myb, AP-1 and NF-1. On the contrary, Sp3 transcription is repressed by E2F/DP1 overexpression.Additionally, we studied the interaction of Sp1 with other proteins involved in the cell cycle regulation and we characterized the effect the overexpression of these proteins on the Sp1promoter activity, given that this promoter is regulated by Sp1. Sp1 is able to interact with CDK4, p21, SKP2 and BRCA2. The Sp1 promoter is positively regulated by the overexpression of CDK4, SKP2, BRCA2, Ciclina D1, E2F1/DP1 and Stat3 whereas the overepression of p53 and NFB represses the promoter. The effects of all these proteins were also analyzed at the Sp1 mRNA level and by using an artificial promoter containing only Sp1 binding sites. The interaction between Sp1 and p21 was further analyzed and we demonstrated that, in fibrosarcoma cells, p21 induces the Sp1 promoter and its mRNA expression but, at the same time, it induces the degradation of Sp1 protein.The process of transcription is a very complex mechanism that involves a great number of transcription factors and other proteins interacting with these factors.

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No description available.

The bioproduction of materials and energy from renewable sources (industrial biotechnology) is getting more and more interest in order to improve environmental sustainability of chemical industrial processes and to decrease their dependence from oil. Anaerobic digestion of organic waste matrices (agricultural and industrial wastes, organic fraction of municipal wastes, sewage sludges etc.) may play an important role in the implementation of industrial biotechnology being a well developed strategy in the valorization of complex matrices, as it can mineralize them while producing bioenergy in the form of a biogas rich in methane. In this research the potential of anaerobic digestion in the treatment of polluted sewage sludge was studied by developing three set of anaerobic microcosms with sludges differently contaminated by xenobiotic compounds. The effect of different incubating temperatures and of exogenous carbon and vitamine sources was investigated along with the role of the occurring microbial populations in the pollutant degradation activity. So, while confirming the potential of anaerobic digestion for the biomethanization of sewage sludges, this work proved the effectiveness of this technology in the removal of pollutants too. Moreover, since the degradation of lignocellulose appears to be a limiting step in the anaerobic treatment of a wide range of biomass, the possibility of optimizing anaerobic digestion of lignocellulosic substrates was also studied. To this aim a research was carried out at the BOKUUniversity of Natural Resources and Applied Life Sciences, Department for Agrobiotechnology, IFA - Tulln, where mixed cellulolytic cultures were isolated from biogas plants while assessing the metabolic pathway leading to cellulose degradation and verifying their capability to grow on lignocellulose too, proving that on the long term such bacterial cultures could be used as inoculum in order to improve the hydrolysis of lignocellulose in anaerobic digestion plants.

No description available.

The ideal approach for the long term treatment of intestinal disorders, such as inflammatory bowel disease (IBD), is represented by a safe and well tolerated therapy able to reduce mucosal inflammation and maintain homeostasis of the intestinal microbiota. A combined therapy with antimicrobial agents, to reduce antigenic load, and immunomodulators, to ameliorate the dysregulated responses, followed by probiotic supplementation has been proposed. Because of the complementary mechanisms of action of antibiotics and probiotics, a combined therapeutic approach would give advantages in terms of enlargement of the antimicrobial spectrum, due to the barrier effect of probiotic bacteria, and limitation of some side effects of traditional chemiotherapy (i.e. indiscriminate decrease of aggressive and protective intestinal bacteria, altered absorption of nutrient elements, allergic and inflammatory reactions). Rifaximin (4-deoxy-4’-methylpyrido[1’,2’-1,2]imidazo[5,4-c]rifamycin SV) is a product of synthesis experiments designed to modify the parent compound, rifamycin, in order to achieve low gastrointestinal absorption while retaining good antibacterial activity. Both experimental and clinical pharmacology clearly show that this compound is a non systemic antibiotic with a broad spectrum of antibacterial action, covering Gram-positive and Gram-negative organisms, both aerobes and anaerobes. Being virtually non absorbed, its bioavailability within the gastrointestinal tract is rather high with intraluminal and faecal drug concentrations that largely exceed the MIC values observed in vitro against a wide range of pathogenic microorganisms. The gastrointestinal tract represents therefore the primary therapeutic target and gastrointestinal infections the main indication. The little value of rifaximin outside the enteric area minimizes both antimicrobial resistance and systemic adverse events. Fermented dairy products enriched with probiotic bacteria have developed into one of the most successful categories of functional foods. Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2002), and mainly include Lactobacillus and Bifidobacterium species. Probiotic bacteria exert a direct effect on the intestinal microbiota of the host and contribute to organoleptic, rheological and nutritional properties of food. Administration of pharmaceutical probiotic formula has been associated with therapeutic effects in treatment of diarrhoea, constipation, flatulence, enteropathogens colonization, gastroenteritis, hypercholesterolemia, IBD, such as ulcerative colitis (UC), Crohn’s disease, pouchitis and irritable bowel syndrome. Prerequisites for probiotics are to be effective and safe. The characteristics of an effective probiotic for gastrointestinal tract disorders are tolerance to upper gastrointestinal environment (resistance to digestion by enteric or pancreatic enzymes, gastric acid and bile), adhesion on intestinal surface to lengthen the retention time, ability to prevent the adherence, establishment and/or replication of pathogens, production of antimicrobial substances, degradation of toxic catabolites by bacterial detoxifying enzymatic activities, and modulation of the host immune responses. This study was carried out using a validated three-stage fermentative continuous system and it is aimed to investigate the effect of rifaximin on the colonic microbial flora of a healthy individual, in terms of bacterial composition and production of fermentative metabolic end products. Moreover, this is the first study that investigates in vitro the impact of the simultaneous administration of the antibiotic rifaximin and the probiotic B. lactis BI07 on the intestinal microbiota. Bacterial groups of interest were evaluated using culture-based methods and molecular culture-independent techniques (FISH, PCR-DGGE). Metabolic outputs in terms of SCFA profiles were determined by HPLC analysis. Collected data demonstrated that rifaximin as well as antibiotic and probiotic treatment did not change drastically the intestinal microflora, whereas bacteria belonging to Bifidobacterium and Lactobacillus significantly increase over the course of the treatment, suggesting a spontaneous upsurge of rifaximin resistance. These results are in agreement with a previous study, in which it has been demonstrated that rifaximin administration in patients with UC, affects the host with minor variations of the intestinal microflora, and that the microbiota is restored over a wash-out period. In particular, several Bifidobacterium rifaximin resistant mutants could be isolated during the antibiotic treatment, but they disappeared after the antibiotic suspension. Furthermore, bacteria belonging to Atopobium spp. and E. rectale/Clostridium cluster XIVa increased significantly after rifaximin and probiotic treatment. Atopobium genus and E. rectale/Clostridium cluster XIVa are saccharolytic, butyrate-producing bacteria, and for these characteristics they are widely considered health-promoting microorganisms. The absence of major variations in the intestinal microflora of a healthy individual and the significant increase in probiotic and health-promoting bacteria concentrations support the rationale of the administration of rifaximin as efficacious and non-dysbiosis promoting therapy and suggest the efficacy of an antibiotic/probiotic combined treatment in several gut pathologies, such as IBD. To assess the use of an antibiotic/probiotic combination for clinical management of intestinal disorders, genetic, proteomic and physiologic approaches were employed to elucidate molecular mechanisms determining rifaximin resistance in Bifidobacterium, and the expected interactions occurring in the gut between these bacteria and the drug. The ability of an antimicrobial agent to select resistance is a relevant factor that affects its usefulness and may diminish its useful life. Rifaximin resistance phenotype was easily acquired by all bifidobacteria analyzed [type strains of the most representative intestinal bifidobacterial species (B. infantis, B. breve, B. longum, B. adolescentis and B. bifidum) and three bifidobacteria included in a pharmaceutical probiotic preparation (B. lactis BI07, B. breve BBSF and B. longum BL04)] and persisted for more than 400 bacterial generations in the absence of selective pressure. Exclusion of any reversion phenomenon suggested two hypotheses: (i) stable and immobile genetic elements encode resistance; (ii) the drug moiety does not act as an inducer of the resistance phenotype, but enables selection of resistant mutants. Since point mutations in rpoB have been indicated as representing the principal factor determining rifampicin resistance in E. coli and M. tuberculosis, whether a similar mechanism also occurs in Bifidobacterium was verified. The analysis of a 129 bp rpoB core region of several wild-type and resistant bifidobacteria revealed five different types of miss-sense mutations in codons 513, 516, 522 and 529. Position 529 was a novel mutation site, not previously described, and position 522 appeared interesting for both the double point substitutions and the heterogeneous profile of nucleotide changes. The sequence heterogeneity of codon 522 in Bifidobacterium leads to hypothesize an indirect role of its encoded amino acid in the binding with the rifaximin moiety. These results demonstrated the chromosomal nature of rifaximin resistance in Bifidobacterium, minimizing risk factors for horizontal transmission of resistance elements between intestinal microbial species. Further proteomic and physiologic investigations were carried out using B. lactis BI07, component of a pharmaceutical probiotic preparation, as a model strain. The choice of this strain was determined based on the following elements: (i) B. lactis BI07 is able to survive and persist in the gut; (ii) a proteomic overview of this strain has been recently reported. The involvement of metabolic changes associated with rifaximin resistance was investigated by proteomic analysis performed with two-dimensional electrophoresis and mass spectrometry. Comparative proteomic mapping of BI07-wt and BI07-res revealed that most differences in protein expression patterns were genetically encoded rather than induced by antibiotic exposure. In particular, rifaximin resistance phenotype was characterized by increased expression levels of stress proteins. Overexpression of stress proteins was expected, as they represent a common non specific response by bacteria when stimulated by different shock conditions, including exposure to toxic agents like heavy metals, oxidants, acids, bile salts and antibiotics. Also, positive transcription regulators were found to be overexpressed in BI07-res, suggesting that bacteria could activate compensatory mechanisms to assist the transcription process in the presence of RNA polymerase inhibitors. Other differences in expression profiles were related to proteins involved in central metabolism; these modifications suggest metabolic disadvantages of resistant mutants in comparison with sensitive bifidobacteria in the gut environment, without selective pressure, explaining their disappearance from faeces of patients with UC after interruption of antibiotic treatment. The differences observed between BI07-wt e BI07-res proteomic patterns, as well as the high frequency of silent mutations reported for resistant mutants of Bifidobacterium could be the consequences of an increased mutation rate, mechanism which may lead to persistence of resistant bacteria in the population. However, the in vivo disappearance of resistant mutants in absence of selective pressure, allows excluding the upsurge of compensatory mutations without loss of resistance. Furthermore, the proteomic characterization of the resistant phenotype suggests that rifaximin resistance is associated with a reduced bacterial fitness in B. lactis BI07-res, supporting the hypothesis of a biological cost of antibiotic resistance in Bifidobacterium. The hypothesis of rifaximin inactivation by bacterial enzymatic activities was verified by using liquid chromatography coupled with tandem mass spectrometry. Neither chemical modifications nor degradation derivatives of the rifaximin moiety were detected. The exclusion of a biodegradation pattern for the drug was further supported by the quantitative recovery in BI07-res culture fractions of the total rifaximin amount (100 μg/ml) added to the culture medium. To confirm the main role of the mutation on the β chain of RNA polymerase in rifaximin resistance acquisition, transcription activity of crude enzymatic extracts of BI07-res cells was evaluated. Although the inhibition effects of rifaximin on in vitro transcription were definitely higher for BI07-wt than for BI07-res, a partial resistance of the mutated RNA polymerase at rifaximin concentrations > 10 μg/ml was supposed, on the basis of the calculated differences in inhibition percentages between BI07-wt and BI07-res. By considering the resistance of entire BI07-res cells to rifaximin concentrations > 100 μg/ml, supplementary resistance mechanisms may take place in vivo. A barrier for the rifaximin uptake in BI07-res cells was suggested in this study, on the basis of the major portion of the antibiotic found to be bound to the cellular pellet respect to the portion recovered in the cellular lysate. Related to this finding, a resistance mechanism involving changes of membrane permeability was supposed. A previous study supports this hypothesis, demonstrating the involvement of surface properties and permeability in natural resistance to rifampicin in mycobacteria, isolated from cases of human infection, which possessed a rifampicin-susceptible RNA polymerase. To understand the mechanism of membrane barrier, variations in percentage of saturated and unsaturated FAs and their methylation products in BI07-wt and BI07-res membranes were investigated. While saturated FAs confer rigidity to membrane and resistance to stress agents, such as antibiotics, a high level of lipid unsaturation is associated with high fluidity and susceptibility to stresses. Thus, the higher percentage of saturated FAs during the stationary phase of BI07-res could represent a defence mechanism of mutant cells to prevent the antibiotic uptake. Furthermore, the increase of CFAs such as dihydrosterculic acid during the stationary phase of BI07-res suggests that this CFA could be more suitable than its isomer lactobacillic acid to interact with and prevent the penetration of exogenous molecules including rifaximin. Finally, the impact of rifaximin on immune regulatory functions of the gut was evaluated. It has been suggested a potential anti-inflammatory effect of rifaximin, with reduced secretion of IFN-γ in a rodent model of colitis. Analogously, it has been reported a significant decrease in IL-8, MCP-1, MCP-3 e IL-10 levels in patients affected by pouchitis, treated with a combined therapy of rifaximin and ciprofloxacin. Since rifaximin enables in vivo and in vitro selection of Bifidobacterium resistant mutants with high frequency, the immunomodulation activities of rifaximin associated with a B. lactis resistant mutant were also taken into account. Data obtained from PBMC stimulation experiments suggest the following conclusions: (i) rifaximin does not exert any effect on production of IL-1β, IL-6 and IL-10, whereas it weakly stimulates production of TNF-α; (ii) B. lactis appears as a good inducer of IL-1β, IL-6 and TNF-α; (iii) combination of BI07-res and rifaximin exhibits a lower stimulation effect than BI07-res alone, especially for IL-6. These results confirm the potential anti-inflammatory effect of rifaximin, and are in agreement with several studies that report a transient pro-inflammatory response associated with probiotic administration. The understanding of the molecular factors determining rifaximin resistance in the genus Bifidobacterium assumes an applicative significance at pharmaceutical and medical level, as it represents the scientific basis to justify the simultaneous use of the antibiotic rifaximin and probiotic bifidobacteria in the clinical treatment of intestinal disorders.

Phenol and cresols represent a good example of primary chemical building blocks of which 2.8 million tons are currently produced in Europe each year. Currently, these primary phenolic building blocks are produced by refining processes from fossil hydrocarbons: 5% of the world-wide production comes from coal (which contains 0.2% of phenols) through the distillation of the tar residue after the production of coke, while 95% of current world production of phenol is produced by the distillation and cracking of crude oil. In nature phenolic compounds are present in terrestrial higher plants and ferns in several different chemical structures while they are essentially absent in lower organisms and in animals. Biomass (which contain 3-8% of phenols) represents a substantial source of secondary chemical building blocks presently underexploited. These phenolic derivatives are currently used in tens thousand of tons to produce high cost products such as food additives and flavours (i.e. vanillin), fine chemicals (i.e. non-steroidal anti-inflammatory drugs such as ibuprofen or flurbiprofen) and polymers (i.e. poly p-vinylphenol, a photosensitive polymer for electronic and optoelectronic applications). European agrifood waste represents a low cost abundant raw material (250 millions tons per year) which does not subtract land use and processing resources from necessary sustainable food production. The class of phenolic compounds is essentially constituted by simple phenols, phenolic acids, hydroxycinnamic acid derivatives, flavonoids and lignans. As in the case of coke production, the removal of the phenolic contents from biomass upgrades also the residual biomass. Focusing on the phenolic component of agrifood wastes, huge processing and marketing opportunities open since phenols are used as chemical intermediates for a large number of applications, ranging from pharmaceuticals, agricultural chemicals, food ingredients etc. Following this approach we developed a biorefining process to recover the phenolic fraction of wheat bran based on enzymatic commercial biocatalysts in completely water based process, and polymeric resins with the aim of substituting secondary chemical building blocks with the same compounds naturally present in biomass. We characterized several industrial enzymatic product for their ability to hydrolize the different molecular features that are present in wheat bran cell walls structures, focusing on the hydrolysis of polysaccharidic chains and phenolics cross links. This industrial biocatalysts were tested on wheat bran and the optimized process allowed to liquefy up to the 60 % of the treated matter. The enzymatic treatment was also able to solubilise up to the 30 % of the alkali extractable ferulic acid. An extraction process of the phenolic fraction of the hydrolyzed wheat bran based on an adsorbtion/desorption process on styrene-polyvinyl benzene weak cation-exchange resin Amberlite IRA 95 was developed. The efficiency of the resin was tested on different model system containing ferulic acid and the adsorption and desorption working parameters optimized for the crude enzymatic hydrolyzed wheat bran. The extraction process developed had an overall yield of the 82% and allowed to obtain concentrated extracts containing up to 3000 ppm of ferulic acid. The crude enzymatic hydrolyzed wheat bran and the concentrated extract were finally used as substrate in a bioconversion process of ferulic acid into vanillin through resting cells fermentation. The bioconversion process had a yields in vanillin of 60-70% within 5-6 hours of fermentation. Our findings are the first step on the way to demonstrating the economical feasibility for the recovery of biophenols from agrifood wastes through a whole crop approach in a sustainable biorefining process.