The potential of flaxseed (Linum usitatissimum L.) protein to release bioactive peptides upon enzymatic hydrolysis was evaluated. Flaxseed protein released angiotensin I-converting enzyme inhibitory (ACEI) peptides during in vitro simulated gastrointestinal (GI) digestion in a static (no removal of digested products) and a dynamic model (removal of <1 kDa molecules in the intestinal phase). The ACEI activity of the gastric plus intestinal digest (absorbed fraction-IC50: 0.04 mg N/mL; retained fraction-IC50: 0.05 mg N/mL; degree of hydrolysis, DH: 46.78 %) of the dynamic model was significantly higher (P<0.05) than that of the static model (IC50: 0.39 mg N/mL; DH: 43.95 %). Polypeptides of 48, 41, 29 and 20 kDa could be releasing these ACEI peptides. Six peptides in the highest ACEI fraction (0.5-1 kDa) of the absorbable gastric plus intestinal digest were identified via de novo sequencing. Only digests of the static model exhibited hydroxyl radical (OH) scavenging activity (IC50: 0.40 mg N/mL), suggesting the inappropriateness of such models in this type of research. Presence of mucilage and oil interfered with the in vitro digestibility of flaxseed protein, which could limit the release of ACEI peptides during GI digestion. The protein digestibility of milled whole flaxseed (12.61 %) was significantly improved (P<0.05) with the removal of mucilage (51.00 %) and oil together with mucilage (66.79 %). The digestibility of isolated flaxseed protein was 68.00 %.<p> Flaxseed protein, hydrolyzed (DH: 11.94-70.62 %) with Flavourzyme® in a central composite rotatable design, possessed bioactivities with identified optimum enzyme/substrate and time of hydrolysis combinations, including ACEI activity (71.59-88.29 %, 83.7 LAPU/g protein, 19.9 h), scavenging of OH (12.48-22.08 %, 30.2 LAPU/ g protein, 1.5 h) and superoxide radical (O2-) (26.33-39.41 %, 4.9 LAPU/ g protein, 16.3 h) and inhibiting linoleic acid oxidation (0.71-94.33 %, 1.6 LAPU/ g protein, 12.6 h). The degradation pattern of polypeptides during enzymatic hydrolysis indicated that 48 and 13 kDa molecules could be releasing these bioactive peptides. De novo sequencing identified two ACEI and five OH scavenging peptides in the hydrolysate fractions (0.5-1.05 kDa) with the highest bioactivities. The findings suggest the importance of flaxseed protein as a source of cardioprotective bioactive peptides.

Anaerobic digestion of excess biological wastewater treatment sludge (WAS) from pulp mills has the potential to reduce disposal costs and to generate energy through biogas production. The organic matter in WAS is highly structured, which normally hinders biogas production. This study investigated three methods of pretreating WAS from two different pulp mills before anaerobic digestion to improve biogas yield and production rate. The three pretreatment methods tested were: i) thermal pretreatment at 170oC, ii) caustic pretreatment at 140oC and pH 12, and iii) sonication at 20 kHz and 1 W/mL. Thermal pretreatment proved to be the most effective, increasing biogas yield by 280% and 50% and increasing production rates 300-fold and 10-fold for the two samples, respectively. Caustic pretreatment showed similar results, but resulted in the formation of soluble non-biodegradable compounds. Sonication was the least effective pretreatment and did not substantially increase biogas yield, but increased biogas production rate.

The development and patenting of a new technology for the anaerobic digestion of solid waste is described. The design basis is explained and justified by extensive reference to the literature. The technology was specifically designed to be versatile, robust and affordable and is directly derived from other proven processes for organic waste management. The ways in which environmental regulations directly affect the development and commercialization of organic waste processing technologies are described. The great differences in regulations between Europe and North America are analyzed to explain why anaerobic digestion is common in Europe and rare in North America and why this is the result of waste management economics which are driven by these regulations. The new technology is shown to be competitive in the Province of Ontario in particular and North America in general; a detailed financial analysis and comparison with European technologies is provided in support of this conclusion.

To improve biogas production from lignocellulosics, methanogenic microbial enrichments were prepared from moose rumen fluid, beaver droppings, and internal circulation (IC) reactor granules amended with cellulose, pine needles, lignosulphonate, tannic acid, and poplar hydrolysate. Tannic acid delayed methanogenesis compared to cellulose only enrichments, both by reducing initial rates (up to 50% in beaver dropping cultures) and increasing lag times (up to 50 days in moose rumen cultures). Biogas yields from poplar hydrolysate were 56 % by beaver droppings, 51% % by IC granules, and 31 % by moose rumen enrichments. Bacterial community profiles, determined via denaturing gradient gel electrophoresis (DGGE) showed divergent populations between enrichments. Enrichment on pine needles or poplar hydrolysate promoted bioconversion of post extraction wash (PEW), and beaver dropping enrichments fed pine needles equaled the yield (about 23%) from IC granules. Together, the DGGE and PEW results provide evidence of acclimatization to previously recalcitrant feeds.

The development and patenting of a new technology for the anaerobic digestion of solid waste is described. The design basis is explained and justified by extensive reference to the literature. The technology was specifically designed to be versatile, robust and affordable and is directly derived from other proven processes for organic waste management. The ways in which environmental regulations directly affect the development and commercialization of organic waste processing technologies are described. The great differences in regulations between Europe and North America are analyzed to explain why anaerobic digestion is common in Europe and rare in North America and why this is the result of waste management economics which are driven by these regulations. The new technology is shown to be competitive in the Province of Ontario in particular and North America in general; a detailed financial analysis and comparison with European technologies is provided in support of this conclusion.

To improve biogas production from lignocellulosics, methanogenic microbial enrichments were prepared from moose rumen fluid, beaver droppings, and internal circulation (IC) reactor granules amended with cellulose, pine needles, lignosulphonate, tannic acid, and poplar hydrolysate. Tannic acid delayed methanogenesis compared to cellulose only enrichments, both by reducing initial rates (up to 50% in beaver dropping cultures) and increasing lag times (up to 50 days in moose rumen cultures). Biogas yields from poplar hydrolysate were 56 % by beaver droppings, 51% % by IC granules, and 31 % by moose rumen enrichments. Bacterial community profiles, determined via denaturing gradient gel electrophoresis (DGGE) showed divergent populations between enrichments. Enrichment on pine needles or poplar hydrolysate promoted bioconversion of post extraction wash (PEW), and beaver dropping enrichments fed pine needles equaled the yield (about 23%) from IC granules. Together, the DGGE and PEW results provide evidence of acclimatization to previously recalcitrant feeds.

Treatment of waste sludge has become an important issue in recent years around the world. However, the trend of waste sludge treatment has shifted from volume minimization and stabilization to reuse of the sludge and recover the energy potential of it. Therefore, anaerobic treatment of sludge is gaining popularity because of byproduct methane production and high percentage of VSS reduction. Pre-treatment of sludge before anaerobic digestion in order to increase methane production, and ozone pre-treatment in this context, is one such option. Domestic sludge also contains the recently recognized, so called, emerging compounds such as Endocrine Disrupting Compounds (EDCs). Therefore treatment of EDCs in sludge is another challenge in waste sludge treatment since direct discharge of such chemicals may harm the environment by causing gender shifts within the fauna. In this context two hormones (estrone and progesterone), three pharmaceuticals (acetaminophen, carbamazepine and diltiazem) and one plasticizer (benzyl-butyl phthalate) were routinely analyzed in sludge samples which were subjected to treatment during this study. Treatment of EDCs during anaerobic digestion and the effect of ozonation both on the performance of digestion and the treatability of EDCs were investigated in this study. Four 2.5L anaerobic jars were used for anaerobic digestion connected to four 1L plastic graduated cylinders immersed in salt-water to collect the off gas. Anaerobic sludge culture of the reactor and the sludge feed to the reactors were obtained from Ankara Tatlar Wastewater Treatment Plant anaerobic digester and return activated sludge (RAS) line, respectively. One of the anaerobic digesters was used as control (no ozonation) and the others were fed with sludge samples ozonated at three different ozone doses 0.65, 1.33 and 2.65 mg ozone/g biomass. Sludge ages of the reactors were initially set to 25 days and the reactors were fed once every 2 days. The TSS, VSS, total gas volume, COD, pH, CH4 percentage and EDCs were analyzed routinely. In the reactors, operated at 25 days, because of the observation of reduction of TSS, SRT was set to infinity / thus, sludge wastage was terminated. Following the startup it was seen that at 2.65 mg ozone/g biomass dose TSS and VSS did not stay constant in the reactor and dropped sharply in the course of operation, indicating that system was not steady at this SRT. However, upon stoppage of sludge wastage from the reactors, thereby setting SRT to infinity, a steady culture could be maintained in the reactors. Both total gas production and CH4 percentage increased with the increasing doses of ozone with respect to control reactor. For 2.65 mg/g ozonated reactor total gas volume doubled the amount produced in the control reactor. All the EDCs within the scope of this study were analyzed in sludge using ultrasound-aided sequential sludge extraction method twice a week and the results showed that ozonation affected treatment of EDCs for up to 96%. The highest removal rate was obtained with natural hormones. Rates of treatment of pharmaceuticals were the second best.

Anaerobic digestion of excess biological wastewater treatment sludge (WAS) from pulp mills has the potential to reduce disposal costs and to generate energy through biogas production. The organic matter in WAS is highly structured, which normally hinders biogas production. This study investigated three methods of pretreating WAS from two different pulp mills before anaerobic digestion to improve biogas yield and production rate. The three pretreatment methods tested were: i) thermal pretreatment at 170oC, ii) caustic pretreatment at 140oC and pH 12, and iii) sonication at 20 kHz and 1 W/mL. Thermal pretreatment proved to be the most effective, increasing biogas yield by 280% and 50% and increasing production rates 300-fold and 10-fold for the two samples, respectively. Caustic pretreatment showed similar results, but resulted in the formation of soluble non-biodegradable compounds. Sonication was the least effective pretreatment and did not substantially increase biogas yield, but increased biogas production rate.

Els residus carnis, o subproductes animals, són interessants per al procés de digestióanaeròbia i producció de biogàs, donat el seu elevat potencial energètic i l'actual marc legislatiu que prima la producció d'energia renovable. Tot i així, l'elevat contingut en lípids i proteïnes d'aquests residus pot limitar el seu tractament en introduir fenòmens d'inhibició, dels quals el més important és el produït pels àcids grassos de cadena llarga (AGCL), resultants de la hidròlisi dels lípids. L'objectiu de la present tesis és aprofundir en el coneixement d'aquest procés d'inhibició, en la capacitat d'adaptació dels microorganismes i en la recuperació o prevenció dels fenòmens d'inhibició. En una primera aproximació a la problemàtica, es caracteritzen residus orgànics d'escorxador, s'estudia la seva biodegradabilitat anaeròbia amb diferents relacions lípids/proteïnes i es realitzen assaigs discontinus seqüencials incrementant la concentració de substrat mitjançant pulsos consecutius. Es comprova que la hidròlisi i acidogènesi de proteïnes és molt ràpida i que la degradació dels lípids i AGCL limita la velocitat global del procés. Malgrat aquesta limitació, el sistema es recupera després dels pulsos aplicats, tot augmentant la taxa màxima de producció de metà. Per tal d'estudiar el fenòmen de recuperació, s'estudien i desenvolupen diferents estratègies en reactors sotmesos a processos d'inhibició per AGCL. L'increment dels ratis biomassa/AGCL o l'adició d'additius com la bentonita, per tal de reduir la biodisponibilitat o l'adsorció dels AGCL sobre la biomassa activa, es mostren com estratègies funcionals d'utilitat en l'operació de plantes industrials. Els resultats obtinguts reforcen la hipòtesi de que la inhibició és deguda a adsorció d'AGCL sobre la membrana cel·lular i que la recuperació es pot mesurar mitjançant un augment de l'activitat dels microorganismes. Per tal de dilucidar sobre la natura del augment de l'activitat en els processos de recuperació es caracteritza la inhibició-recuperació mitjançant tres tècniques: 1) estudi de les activitats dels microorganismes a diferents substrats 2) utilització de tècniques de biologia molecular per caracteritzar les poblacions, i 3) desenvolupant expressions cinètiques del procés d'inhibició, basades en l'adsorció, en el marc del model matemàtic ADM1 de la International Water Association. Mitjançant aquestes metodologies es comprova que els fenòmens d'inhibició i adaptació es poden explicar mitjançant un creixement poblacional específic i la inclusió dels fenòmens físic d'adsorció en el procés d'inhibició metabòlica. Finalment, s'avalua de forma més detallada el procés d'adsorció-inhibició mitjançant la determinació de les isotermes d'adsorció i monitoritzant mitjançant assaigs amb biomassa granular i tècniques de microscòpia de fluorescència. Aquesta caracterització ha permès obtenir estratègies de prevenció de la inhibició per AGCL, mitjançant competència amb adsorbents sintètics, i concloure que l'àcid palmític és el limitant en el procés de -oxidaciódels AGCL. Els resultats obtinguts constitueixen una base per al millor coneixement de les possibilitats de tractament anaerobi del residus carnis i dels processos d'inhibició per AGCL i adaptació de la biomassa. El procés físic d'adsorció ha estat directament relacionat amb el fenòmen d'inhibició metabòlica, obtenint-se una descripció matemàtica del mateix. Els resultats han permès plantejar estratègies operacionals, sent una eina a disposició d'operadors de plantes de biogàs per optimitzar la producció d'energia d'aquests residus >mitjançant la seva digestió anaeròbia. / Los residuos cárnicos, o subproductos animales, son interesantes para el proceso de digestión anaerobia y producción de biogás, dado su elevado potencial energético y el actual marco legal que prima la producción de energía renovable. A pesar de esto, el elevado contenido en lípidos y proteínas puede limitar su tratamiento, al introducir fenómenos de inhibición, de los cuales el más importante es el producido por ácidos grasos de cadena larga (AGCL), resultado de la hidrólisis de los lípidos. El objetivo de la presente tesis es profundizar en el conocimiento de este proceso de inhibición, en la capacidad de adaptación de los microorganismos t en la recuperación de sistemas inhibidos. En una primera aproximación a la problemática, se caracterizan los residuos orgánicos de matadero, se estudia su biodegradabilidad anaerobia con diferentes relaciones lípido/proteína y se realizan ensayos discontinuos secuenciales incrementando la concentración de substrato mediante pulsos consecutivos. Se comprueba que la hidrólisis y acidogénesis de las proteínas es muy rápido y que la degradación de lípidos y AGCL limita la velocidad global del proceso. A pesar de esta limitación, el sistema se recupera después de los pulsos aplicados aumentando la tasa máxima de producción de metano. A fin de estudiar el fenómeno de recuperación, se estudian y desarrollan diferentes estrategias en reactores inhibidos por AGCL. El incremento de los ratios biomasa/AGCL o la adición de aditivos como la bentonita, a fin de reducir la biodisponibilidad o la adsorción de los AGCL sobre la biomasa activa, se muestran estrategias funcionales de utilidad en la operación de plantas industriales. Los resultados obtenidos refuerzan la hipótesis de que la inhibición es debida a adsorción de AGCL sobre la membrana celular y que la recuperación se puede medir mediante un aumento de la actividad de los microorganismos. A fin de dilucidar sobre la naturaleza del aumento de la actividad en los procesos de recuperación se caracteriza la inhibición mediante tres técnicas: 1) estudio de las actividades de los microorganismos a diferentes substratos, 2) utilización de técnicas de biología molecular para caracterizar las poblaciones, y 3) desarrollando expresiones cinéticas del proceso de inhibición, basado en la adsorbió, en el marco del modelo ADM1 de la International Water Association. Mediante estas metodologías se comprueba que los fenómenos de inhibición y adaptación se pueden explicar mediante un crecimiento poblacional específico y la inclusión de la adsorción en el proceso de inhibición metabólica. Finalmente, se evalúa de forma detallada el proceso de adsorción-inhibición mediante la determinación de las isotermas de adsorción y monitorizando estos procesos mediante ensayos discontinuos con biomasa granular y técnicas de microscopia de fluorescencia. Esta caracterización ha permitido obtener estrategias de prevención de la inhibición por AGCL, mediante competencia con adsorbentes sintéticos, y concluir que el ácido palmítico es el limitante en el proceso de mutante -oxidación de los AGCL. Los resultados obtenidos constituyen una base para el mejor conocimiento de las posibilidades de tratamiento anaerobio de residuos cárnicos y de los procesos de inhibición por AGCL y adaptación de la biomasa. El proceso físico de adsorción se ha relacionado directamente con el fenómeno de inhibición metabólica, obteniéndose una descripción matemática del mismo. Los resultados han permitido plantear estrategias operacionales, siendo una herramienta a disposición de operadores de plantas de biogás para optimizar la producción de energía de estos residuos mediante su digestión anaerobia. / Slaughterhouse wastes are interesting for the anaerobic digestion process regarding its high biogas production potential and because the current legal scenario promotes renewable energy production. The high lipid and protein content of those residues limit its treatment due to inhibitory processes, in particular the inhibition caused by long chain fatty acids (LCFA). The objective of the present disertation is to obtain a deeper insight on the LCFA inhibition process, the microorganism adaptation ability and the prevention/recovery of inhibitory phenomena. In a preliminary approach, organic wastes generated in slaughterhouses are characterized, by studying the anaerobic biodegradability of waste mixtures containing diferents lipid/proteins concentrations. Anaerobic batch tests are performed at increasing substrate concentrations by sequential pulse feeding. From those experiments, the fast hydrolysis-acidogenesis of proteins is verified, being the lipids and LCFA degradation the main limiting step of the overall anaerobic process. Despite this limitation, the system is able to recover up to a higher methane production rate after each applied pulse. In order to elucidate on the mechanisms of the recovery process, several strategies to recover LCFA inhibited reactors are tested. The increase of the biomass/LCFA ratio and the adition of bentonite to reduce the biodisponibility or the adsorption of LCFA over microbial cell walls, are found to be effective approaches in the operation of fullscale biogas plants. The obtained results reinforce the hypothesis of the adsorptive nature of the LCFA inhibition, and that the recovery process can be followed as an increase in the microbial activity. The nature of the reported microbial activity improvement after subsequent sytem inhibition is characterized by three different techniques: 1) the study of specific microbial activities on different model substrates, 2) the application of molecular biology tools to monitor the microbial population structure and, 3) the development of kinetic expressions of the LCFA inhibition phenomena, based on the adsorption process, within the framework of ADM1 model of the International Water Association. The combined analysis of those confirmed that inhibition and adaptation phenomena are explained by a specific microbial growth, including adsorption in the metabolic LCFA inhibition process. The adsorption-inhibition process is evaluated in detail by determining LCFA adsorption isotherms on granular sludge, LCFA toxicity test, and fluorescence microscopy techniques. This multidisciplinary approach results in the definition of an inhibition preventing strategy based on the introduction of competitive adsorbents, and on stating the importance ofpalmitate during ß-oxidation of LCFA. This study contributes to the understanding of slaughterhouse wastes anaerobic treatment, the LCFA inhibition process, and the biomass adaptation phenomena. The physical adsorption process has been directly related with the LCFA metabolic inhibition, and a new mathematical kinetic expression is proposed. New strategies guiding the operation of anaerobic reactors are suggested in order to obtain high renewable energy yields from slaughterhouse wastes digestion.

Milk allergens were digested by allowing them to flow through a chromatography column, where pepsin was conjugated to the stationary phase of the column. The allergen fragments were then characterized both biochemically, by using SDS-PAGE and gel permeation chromatography, and immunologically, by examining their reactivity to IgE and monoclonal antibodies.