Orientador: Eleni Gomes / Banca: Adalbeto Pessoa Júnior / Banca: Maria de Lourdes Teixeira de Moraes Polizeli / Banca: Márcia Regina Brochetto Braga / Banca: Gustavo Orlando Bonilla Rodriguez / Resumo: Pectinases termoestáveis apresentam características interessantes do ponto de vista da sua aplicação industrial, como alta estabilidade ao pH e à temperatura. Além disso, o tipo de processo fermentativo pode influenciar a produção e propriedades físico-químicas destas enzimas. A produção de poligalacturonase (PG) pelo fungo Thermoascus aurantiacus foi realizada em fermentação submersa (FSM) e em estado sólido (FES), usando substratos contendo pectina comercial ou subprodutos agro- industriais como fonte de carbono. A PG bruta obtida em FES apresentou atividade ótima a 65ºC e pH 5,0, com estabilidade na faixa de pH entre 4,0 e 5,0 e entre 7,5 e 8,5 e manteve 85% da atividade original quando incubada a 60ºC, por 1 hora. Em FSM, o melhor meio de cultivo foi a água amarela, com pH inicial de 5,5, após 5 dias de cultivo a 45ºC. A enzima em sua forma bruta apresentou temperatura ótima de 60ºC e pH ótimo de 5,0, maior estabilidade em pH ácido (3,0 a 4,5) e menor termoestabilidade, quando comparada com a obtida em FES, mantendo apenas 13% da atividade original quando incubada a 60ºC, por 1 hora. As enzimas foram purificadas utilizando-se cromatografias de filtração em gel e troca iônica. A PG purificada proveniente da FSM apresentou pH e temperatura ótimos de 5,5 e 60-65ºC, estabilidade em pH 5,0-5,5 e manteve, após 1 hora de incubação, 100% da atividade original até 50ºC. Resultados similares foram obtidos para a PG proveniente da FES. A PG de FES apresentou massa molar de 29,3 kDa, Km de 1,58 mg/mL e Vmáx de 1553,1 ? mol/min/mg, enquanto que a da FSM apresentou massa molar de 30,1 kDa, km de 1,46 mg/mL e Vmáx de 2433,3 ? mol/min/mg. Íons como Fe+3, Ca+2, e K+ praticamente não afetaram a atividade da enzima... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Thermostable pectinases present important characteristics under the view of their industrial application, as their high stability to pH and temperature. Besides, the type of fermentative process used can affect the ir production and physical-chemical properties. The polygalacturonase (PG) production by the thermophilic fungus Thermoascus aurantiacus was carried out by submerged fermentation (SMF) and solid state fermentation (SSF) using substrates containing commercial pectin or agro- industrial residues as carbon sources. The crude PG from SSF presented optimum activity at 65ºC and pH 5.0, with stability at pH 4.0-5.0 and 7.5-8.5 and maintained 85% of its original activity at 60º C for 1 hour. In SMF the best cultivation medium was the liquid waste from juice extraction, with initial pH of 5.5, after 5 days of cultivation at 45ºC. The crude enzyme showed an optimum activity at 60ºC and pH 5.0, higher stability in acid ic pH (3.0 to 4.5) and was less thermostable when compared to that obtained in SSF, wich maintained only 13% of its original activity at 60ºC, for 1 hour. Purification of enzymes was carried out using filtration and ion-exchange chromatographies. The purified PG, from SMF, showed optimum pH and temperature of 5.5 and 60-65ºC, stability at pH 5.0-5.5 and preserved, after 1 hour incubation, 100% of its original activity at 50ºC. Similar results were obtained to PG from SSF. The PG obtained by SSF presented molar mass of 29.3 kDa, Km of 1.58 mg/ml and Vmáx of 1553.1 ? mol/min/mg, while that the enzyme from SMF presented molar mass of 30.1 kDa, km of 1.46 mg/ml and Vmáx of 2433.3 ? mol/min/mg. Ions such as Fe3+, Ca2+ and K+ practically did not affect the enzyme activity, while Mg2+, Mn2+ and Zn2+ decreased 7%, 75% and 50%... (Complete abstract, click electronic address below) / Doutor

Orientador: Ranulfo Monte Alegre / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-03T21:39:56Z (GMT). No. of bitstreams: 1 Gonzales_TatianeAraujo_M.pdf: 808675 bytes, checksum: 754d8f090b92f6186edb2bc0bad0c5b2 (MD5) Previous issue date: 2004 / Resumo: O processo de batelada alimentada é útil para o estudo da cinética de processos fermentativos, pois permite a manutenção de baixos níveis de substrato por longo período de tempo, que é favorável à estimativa de parâmetros cinéticos; permite manter concentração celular constante e controlar a velocidade de crescimento em condições transientes. São as equações cinéticas que indicam como as variáveis de estado do processo em estudo interferem nas velocidades de crescimento e morte celular, de geração de produtos metabólicos e de consumo de substrato. Estudos de biorreatores de batelada alimentada têm sido freqüentemente relatados na literatura, entretanto para a modelagem destes processos necessita-se conhecer parâmetros cinéticos e informações sobre estes são escassas. A implantação do PROÁLCOOL no Brasil fez aumentar o número de destilarias. Atualmente, o processo de batelada alimentada responde por cerca de 60% do volume de etanol produzido. Outro processo fermentativo de alto valor comercial é a produção de ácido lático, uma vez que este tem grande aplicação no uso em alimentos, na área farmacêutica, de plásticos e indústria química. Desta forma, o objetivo deste trabalho foi estudar duas fermentações distintas com relação à cinética de produção e entender a fenomenologia do processo de batelada alimentada. Escolheram-se dois tipos de fermentações com cinética de produção diferentes, uma alcoólica e outra lática, o primeiro com produção associada ao crescimento e o outro de produção parcialmente associada ao crescimento. Realizaram-se ensaios de fermentações em batelada para obtenção dos parâmetros cinéticos e em batelada alimentada para aplicação dos parâmetros obtidos em modelos e estes em balanço de massa do processo batelada alimentada. Em seguida, foram conduzidas simulações do processo batelada alimentada com o auxílio do software AnaBio 1.0 e ajustados os parâmetros cinéticos através de análise visual, utilizando-se dois modelos encontrados na literatura: o modelo cinético de Monod e outro também baseado em Monod com inclusão de um termo de inibição pelo produto. Estes modelos representaram visualmente os dados experimentais de forma satisfatória para a fermentação alcoólica, no entanto, somente um ajuste regular dos dados experimentais em relação aos preditos foram obtidos para fermentação lática / Abstract: The fed-batch process is useful for studying of fermentation process kinetic, because it allows keeping low level of substrate for a long period, which is favorable to estimate kinetic parameters; it enables keeping a constant biomass concentration and controlling growing rate in transient conditions. The kinetic equations indicate how the process variables interfere in the cell growing rate and death, the metabolic product formation and substrate consumption. The bioreactors studies of fed-batch have been often found on specialized literature, however, its necessary to know the kinetic parameters that are not available. A very important program in Brazil, called PROALCOOL, leveraged a raise in the number of alcohol industries and 60% of ethanol assembled in this country, in the present time, use fed-batch process. Another interesting process is the production of lactic acid, which is important to food, pharmaceutical, plastic and chemical industry. Therefore the main objective of this project was to study two different fermentation processes to apply the material balance of the fed-batch reactor with determined parameters and known mathematical models. Two production kinetic fermentations were chosen, one alcoholic and another lactic. The first one is associated with the cell growth and the other is partially associated with cell growth. Some batch fermentations essays were carried out to obtain kinetics parameters and fed-batch processes were performed to study the kinetic process. Simulations of fed-batch process, running the software Anabio 1.0, were executed and the kinetic parameters fitted through visual analysis, using two known models, Monod kinetic model and Monod including inhibitory effect of product. Theses models were better visually represented by the alcoholic fermentation experimental data than lactic fermentation data. In the condition of the fed-batch assays the Monod or Monod with inhibitory effect of product inserted in the material balance do not describe these process / Mestrado / Mestre em Engenharia de Alimentos

Phenolics are critical to the sensory attributes and health benefits of hard cider due to their contribution to the flavor, mouthfeel, and antioxidant activity. With the increase in demand for cider, the use of dessert apple varieties has become more common leaving ciders lacking in phenolics. However, a promising method to increase their phenolic content is through maceration with apple pomace. This study evaluated the effect maceration with apple pomace during cider fermentation on the extraction of phenolic compounds, as well as its effect on the sensory properties of the final product. For this study, ciders were fermented with 0% (control), 20%, 35%, and 50% of the average pomace created during production of the apple must. After fermentation, the ciders were analyzed for acid content, total phenolic content (TPC), alcohol by volume (ABV), color, volatile profile, and sensory properties. The ciders fermented with apple pomace went through malolactic fermentation. During the maceration phenolics were extracted which resulted in an increase in phenolic content in cider. Compared to the control, the treatment ciders were also seen to have higher ABV and TPC, lower acidity, and a different aromatic profile: decreased acetaldehyde and increased ethyl acetate, isoamyl alcohol, and phenylethyl alcohol. The color measurements suggests that treatment ciders were lighter, with a higher red and yellow color compared to the control cider. The sensory analysis revealed the treatment ciders were perceived as less acidic and astringent, but more bitter than the controls. This study shows the addition of apple pomace in its native state increases total phenolic content and could be favorable to produce well rounded ciders. This study shows maceration is a promising technique for increasing phenolic content in ciders.

The research in this thesis was conducted at the Hampton Road Sanitation District's biological nutrient removal pilot, located at the Chesapeake-Elizabeth WWTP in Virginia Beach, VA. The pilot is operated in an A/B process with a high-rate, carbon-diverting A-stage, followed by a biological nitrogen removal B-stage containing four intermittently aerated CSTRs, followed by an anammox polishing MBBR. The goal of this research was to successfully combine short-cut nitrogen removal with sidestream enhanced biological nutrient removal (EBPR) in the most efficient way possible, specifically aiming to decrease cost and energy requirements, divert the most amount of carbon possible before B-stage, and to achieve low effluent nitrogen and phosphorus concentrations. A RAS fermenter (SBPR) and an A-stage WAS fermenter that feeds VFA into the SBPR (the supernatant of the fermenter is called fermentate) were implemented in order to enhance biological phosphorus removal. About 8 months after the RAS and WAS fermenter implementation, there was a 28 day consecutive period of low B-stage effluent OP <1 mg/L, with an average of 0.5 ± 0.1 mg/L OP. Following this low effluent OP period, bio-P became more unstable and there was high nitrite accumulation in the B-stage effluent for 106 days with concentrations ranging from 1.1-5.9 mg/L NO2. The nitrite accumulation was not due to NOB out-selection, confirmed by AOB and NOB maximum activity tests. It was determined that the nitrite accumulation was due to partial denitrification of nitrate to nitrite by bacteria using internally stored carbon, because profiles and activity tests showed anoxic nitrite accumulation at the end of the aerobic process. Post-anoxic denitrification using internally stored carbon compounds has been observed in other EBPR systems (Vocks, Adam, Lesjean, Gnirss, and Kraume, 2005). Fermentate addition was then halted, and nitrite accumulation and bio-P activity ceased all together, linking the fermentate addition to both bio-P activity and nitrite accumulation. Fermentate was then controlled to dose at 60% of the sCOD/OP (fermentate sCOD g/day / total OP- fermentate + influent - g/day) of the first low effluent OP period. During this fermentate dosing period where the average sCOD/OP was 15.6 ± 3.0 g/g, no nitrite accumulation was observed, but another consecutive low effluent OP period was observed with an average of 0.6 ± 0.2 mg/L OP. Linear correlation analysis shows that the highest r2 values relating the low effluent OP periods and the COD loads to the SBPR for both periods were between VFA g/day vs OP effluent mg/L, at r2=0.18 for the first low effluent OP period and r2=0.65 for the second. There were also high tCOD r2 values for the second low effluent OP period showing that COD hydrolysis in the SBPR could have impacted bio-P activity. However, the VFA r2 value was higher than any tCOD r2 value, concluding that the fermentate dosing mainly worked to enhance biological phosphorus removal by increasing the VFA load in g VFA as acetate/day. Since no nitrite was observed in a period with a lower VFA/OP dose, then the probable VFA load needed to provide enough internal storage to produce nitrite accumulation by partial denitrification is between 5-9 (g VFA as acetate/ g total OP). If sidestream EBPR systems could be studied further to promote nitrite accumulation and bio-P activity to produce low effluent OP, then short-cut nitrogen removal and EBPR could be successfully combined in an efficient way. / Master of Science / It is important to reduce nitrogen and phosphorus concentrations in wastewater treatment effluent in order to both protect the environment from eutrophication and to meet the increasingly stringent nutrient effluent discharge limits imposed by the EPA. Conventional biological nitrogen removal is achieved through nitrification and denitrification converting ammonia to nitrogen gas, where nitrogen gas is volatile and leaves the system naturally. Phosphorus removal can be achieved through either chemical addition or through biological phosphorus removal, where phosphorus is taken up in cells and removed from the system by the subsequent solids wasting of these cells. The combination of biological nitrogen and phosphorus removal can be improved to increase energy efficiency, reduce costs including aeration and chemical addition costs, increase system capacity and reduce tank sizes, and reduce biomass production, all while achieving low effluent N and P concentrations. Short-cut nitrogen removal can increase the efficiency of biological nitrogen removal. Deammonification, the combination of partial nitritation and anammox, has the potential to reduce wastewater treatment plant (WWTP) aeration costs by 63%, carbon requirements by 100%, and biomass production by 80% (Nifong, Nelson, Johnson, and B. Bott, 2013). Deammonification is the combination of partial nitritation and anammox. Anaerobic ammonia oxidation (anammox) is a useful class of bacteria that converts ammonia and nitrite straight to nitrogen gas in anaerobic conditions, which is a more direct pathway than the conventional nitrification-denitrification pathway. Anammox requires a nitrite supply, which can supplied by partial nitratation of ammonia to nitrite, performed by ammonia oxidizing bacteria (AOB) aerobically in the deammonification process. In order for partial nitratation to work, there needs to be nitrite oxidizing bacteria (NOB) out-selection so that the nitrite produced by AOB does not get oxidized to nitrate. Enhanced biological phosphorus removal (EBPR) is accomplished by the taking up and storing of orthophosphate (OP) by phosphorus accumulating organisms (PAOs). These organisms require an anaerobic carbon-storage phase followed by an aerobic growth phase where the internally stored carbon is used for growth. During the cell growth phase of PAOs in aerobic conditions, PAOs are able to take up more OP than they previously released in anaerobic conditions, creating a net OP removal from the system. There has been recent success in recycle activated sludge (McIlroy et al.) fermentation to enhance biological phosphorus removal, which works to promote hydrolysis, fermentation, and EBPR enhancement (Houweling, Dold, and Barnard, 2010). A portion of the RAS is introduced to an anaerobic zone before returning to the main process, allowing for extra VFA production and adsorption by PAOs. RAS fermentation solves the issue of carbon needed for EBPR in VFA/carbon limited systems without having to add too much additional carbon, creating a carbon efficient EBPR system. The research outlined in this study was done at the Hampton Road Sanitation District's (HRSD) pilot plant located within HRSD's Chesapeake-Elizabeth WWTP in Virginia Beach VA. The pilot is run in an A/B process that works in two separate steps: the A-stage is the first step that works to remove carbon by oxidation, and by adsorption so it can potentially be diverted, and the B-stage is the second step where biological nitrogen removal (BNR) is done. The BNR phase consists of an anaerobic selector followed by four completely stirred tank reactors (CSTRs) that are intermittently aerated to provide aerobic and anoxic phases. The pilot also has an anammox polishing step following B-stage. The nitrogen removal goal for this research was short-cut nitrogen removal via deammonification, by producing partial nitritation in B-stage and polishing with anammox. A B-stage RAS fermenter, along with an A-stage waste activated sludge (WAS) fermenter that feeds VFA into the RAS fermenter, was implemented to the existing pilot to enhance biological phosphorus removal. The overall goal of this study was to successfully combine short-cut nitrogen removal with sidestream EBPR to achieve low effluent N and P concentrations in the most energy and carbon efficient way possible. EBPR was achieved about eight months after the implementation of the RAS and WAS fermenter to the pilot. A period of B-stage effluent OP that was consistently below 1 mg/L OP was observed right before an unexpected period of high nitrite in the B-stage effluent. The high effluent nitrite lasted for 106 days and ranged from 1.1-5.9 mg/L of effluent nitrite during this time. The nitrite accumulation was unexpected because weekly maximum activity tests for AOB and NOB showed that NOB out-selection was not occurring. The first phase of this research investigates the cause of the nitrite accumulation. Based on profiles taken in the reactors in the aerobic and anoxic phases, and based on denitrification activity tests, it was determined that the nitrite accumulation was due to partial denitrification of nitrate to nitrite. Because this partial denitrification was happening in the reactor anoxic times where external should have been used up, it was determined that the source of the partial denitrification was from a bacteria using internally stored carbon during anoxic periods as the electron supply for partial denitrification. Research has showed that EBPR systems promote bacteria that are capable of storing carbon internally and keeping that carbon stored through an aerobic phase and then using that stored carbon for denitrification following an aerobic phase (Vocks et al., 2005), like observed in this research. The second phase of this research sought to link the nitrite accumulation and bio-P activity to the VFA added to the RAS fermenter. The VFA addition was decreased in phases, and with that a decrease in nitrite in the effluent was observed. The bio-P activity became more unstable after the nitrite accumulation occurred, but all bio-P activity ceased after VFA addition to the RAS fermenter ceased. It was concluded, unsurprisingly, that the VFA added to the RAS fermenter was the source of the internally stored carbon that caused the nitrite accumulation, and necessary for bio-P enhancement. The third phase of this research sought to recreate the low effluent OP period and the nitrite accumulation by controlling the VFA dose to the RAS fermenter. The average soluble chemical oxygen demand (sCOD) per OP (fermenter sCOD g/day / total OP-fermenter + influent- g/day) of the period of low effluent OP was calculated, and the dose from the WAS fermenter was controlled to meet 60% of the calculated value. The calculated dose was 13.6 gC/gP, but the actual average dose from controlling the load during this period was 15.6 ± 3.0 gC/gP. The average VFA/OP (g VFA as acetate/ g total OP) dose for the first low effluent OP period was 9.4 ± 3.6 g/g, and the average dose for the third phase of research was 5.5 ± 1.3 g/g. No nitrite accumulation occurred in this phase, but another consistent low effluent OP period did occur. From linear correlation analysis, the highest r2 values relating the low effluent OP periods and the COD loads to the RAS fermenter for both periods were between VFA g/day vs OP mg/L, at r2=0.18 for the first period and r2=0.65 for the second. This shows that effluent OP < 1 mg/L can be achieve at 5.5 or 9.4 (g VFA as acetate/ g total OP). Since no nitrite was observed in phase 3, than the probable VFA load needed to provide enough internal storage to produce nitrite accumulation by partial denitrification is probably between 5.5-9.4 (g VFA as acetate/ g total OP). This research was significant because the link between nitrite accumulation and bio-P enhancement with sidestream RAS and WAS fermentation was confirmed. Partial denitrification of nitrate to nitrite could be used as an alternative source of nitrite for anammox, instead of NOB out-selection and partial nitritation of ammonia to nitrite by AOB, in combined EBPR and short-cut nitrogen removal systems. If sidestream EBPR systems could be used to promote nitrite accumulation and bio-P activity to produce low effluent OP and nitrogen removal efficiently than short-cut nitrogen removal and EBPR could be successfully combined in an efficient way. Future work needs to be done on the organism that is capable of nitrite accumulation and if that organism can be enhanced in conjunction with EBPR organisms to promote both nitrite accumulation and low effluent OP simultaneously.

The US cider market has expanded in recent years, but limited research-based information is available on fermentation management. This study investigates how apple cultivar and yeast inoculation affect the chemical and sensory properties of cider. Four ciders were produced in triplicate using combinations of two different apple cultivars - Harrison, a cider cultivar and GoldRush, a dessert cultivar - and two fermentation strategies - inoculated with dry active yeast EC1118 or Pied de Cuvé ambient fermentation. Ciders were analyzed for alcohol content, free/total SO2, titratable acidity, volatile acidity, malic acid, pH, and residual sugar. Sensory evaluation was conducted using Descriptive Analysis with trained panelists. Results were analyzed via ANOVA and Principal Component Analysis. Apple cultivar and fermentation method resulted in significant differences for chemistry and sensory parameters. Malic acid concentration was greater in the control ciders while concentrations of both residual sugar and volatile acidity were higher in the PDC ciders. The interactions effect of cultivar*fermentation method influenced both malic acid and residual sugar concentrations, where concentration differences between control and ambient ciders is smaller for GoldRush than for Harrison, showing that fermentation style produces different results across cultivars. Volatile acidity produced opposite interaction effects as differences between fermentation styles was larger for GoldRush. For sensory attributes, Harrison ciders produced high intensities for multiple attributes, but also higher variability. Multiple sensory descriptors displayed interaction effects as the fermentation method produced different results in different cultivars. This study demonstrates that increasingly popular practices in the industry can produce significantly different ciders. / Master of Science in Life Sciences / The US cider market has grown rapidly in recent years, with many new products entering the market. However, there is limited research-based information available on cider fermentation management. This study investigates how production variables, namely apple cultivar and yeast inoculation, affect the chemical and sensory properties of the cider created. The overall goal of this project is to assess the chemical and sensory characteristics that come from cider production treatments. In this study, four experimental ciders were produced using combinations of two different apple cultivars – Harrison, a cider cultivar and GoldRush, a dessert/fresh market cultivar – and two fermentation management strategies – inoculated with dry active yeast strain EC1118 or indirect inoculation through a natural fermentation method. Ciders were analyzed for alcohol content, free and total SO2, titratable acidity, volatile acidity, malic acid, pH and residual sugar. Sensory evaluation was conducted by a trained panel providing descriptive terms and intensities for each sample. Both chemical and sensory results were analyzed to reveal significant differences in samples based on not only apple type and inoculation method, but also the interactions between those two variables. This study demonstrates that increasingly popular practices in the cider industry – like natural fermentation or the use of cider-apple varieties – can produce significantly different ciders. This highlights the idea that producers need to treat each apple cultivar differently, as they behave differently throughout production.

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Thesis (MSc)--Stellenbosch University, 1980. / Een kopie is mikrofiche. / ENGLISH ABSTRACT: No abstract available / AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar

published_or_final_version / abstract / toc / Botany / Master / Master of Philosophy

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