Thesis (MEng)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Bioconversion of hydrocarbons to value-added intermediates and products has significant industrial potential using both prokaryotic and eukaryotic organisms. In particular, alkanes can be converted to an expansive range of commercially important products using aerobic bioprocesses under mild process conditions. Coupled with the relative abundance of alkanes derived from gas to liquid (GTL) technologies, such as those employed by SASOL, South Africa, the commercial potential for bioconverison of alkanes is large. However, unlike carbohydrate substrates, alkane feedstocks are devoid of oxygen in their molecular structure. This means that the entire oxygen demand needs to be met by oxygen transfer. Furthermore, a decline in oxygen transfer in aqueous-hydrocarbon dispersions with increasing alkane concentration has been observed to result from depression of the overall volumetric oxygen transfer coefficient (KLa). Therefore, understanding KLa and the fundamental parameters underpinning its behaviour is critical to ensuring the bioprocess is kinetically, rather than transport, limited in terms of both operation and scale-up. Previous studies have examined KLa in aerated-alkane-aqueous systems. In light of the importance of oxygen transfer in bioprocesses, this study expands on the KLa understanding in 3-phase studies by including a fourth solid phase, thus more closely representing a hydrocarbonbased bioprocess. The project aimed to determine the impact of agitation, alkane concentration and solid loading on the Sauter mean bubble diameter (DSM), gas hold-up and specific interfacial area (a) and correlate these parameters to KLa. This ultimately determined which parameter was dominant over a range of process conditions. Furthermore, concurrent measurement of the KLa and interfacial area meant the behaviour of the liquid side oxygen transfer coefficient (KL) could be defined, providing further insight into how changes in the process conditions impact on KLa. Experiments were conducted in a 5 litre stirred tank bioreactor containing n-C14-20 straight chain alkane, sparged with air at 0.8 vvm. In line with process conditions typical of a hydrocarbonbased bioprocess, KLa and a were measured for agitation rates from 450 to 1000 RPM, alkane concentrations from 2 to 20% v/v and yeast solids from 1 to 10 g/l. KLa was measured using the gassing out procedure using a dissolved oxygen (DO) probe which measured the response of the system to a step change in the sparge gas oxygen pressure. The probe response lag ( P), equal to the time taken for the probe to reach 63.2% of the saturation DO concentration, was determined for every set of process conditions. The inverse of P, KP was taken into account when calculating KLa from the DO probe response. The area was calculated from DSM and gas hold-up. DSM was quantified using high speed photography and image analysis was performed in Matlab® using bespoke routines. Elimination of optical distortion and the development of an adequate light source was key to acquiring clear images. Both KLa and interfacial area were found to be affected by changes in agitation, alkane concentration and yeast loading. An increase in agitation increased the KLa over the entire range of alkane concentration and yeast loading. Similarly, an increase in agitation resulted in an increase in interfacial area, underpinned by a decrease in the DSM. It is therefore likely that the interfacial area plays a dominant role in defining KLa when considering an increase in agitation. Increases in alkane concentration resulted in a peak in KLa between 2.5 and 5% alkane concentration while further increases in alkane concentration depressed KLa. This peak was not observed in interfacial area, where an increase in alkane concentration resulted only in a decrease in interfacial area, thus indicating a positive influence of KL on KLa at low alkane concentrations. Further increases in alkane concentration beyond those creating the peak KLa resulted in KLa depression, suggesting that the increasing viscosity imparted by the alkane decreases both KL and interfacial area. Increased yeast loading had opposing effects at low and high agitation rates. At low agitation rates, increased loadings were observed to increase KLa, while increased loadings at high agitation rates caused a decrease in KLa. This behaviour was also evident in interfacial area, suggesting that in this regime KLa was defined by interfacial area behaviour. Increased yeast loading was observed to depress the KLa for all alkane concentrations when examined at a constant midpoint agitation rate. This trend was not evident in interfacial area, which increased with increasing yeast loading at the same agitation rate. The positive influence of yeast on interfacial area was likely caused by adhesion of the yeast particles to the bubble surface, lowering the DSM by preventing coalescence. The disagreement between the KLa and interfacial area results suggested that yeast loading impacted negatively on KL, which had an over-riding negative impact on KLa. The use of reliable methods for the determination of both interfacial area and KLa were demonstrated for application in model hydrocarbon-based bioprocesses. The combined results offer a unique insight into how changes in the process conditions impact independently on KL and interfacial area, which when combined ultimately defined the KLa behaviour. Quantification of the relative magnitude of the impact each parameter had on KLa contributed toward a fundamental understanding of oxygen transfer in model hydrocarbon-based bioprocesses. / AFRIKAANSE OPSOMMING: Biologiese omsetting van koolwaterstowwe na produkte met finansiële waarde het beduidende industriële potensiaal met behulp van beide prokariotiese en eukariotiese organismes. In die besonder, kan alkane omgeskakel word na ’n uitgebreide reeks van kommersieel belangrike produkte met behulp van aerobiese bioprosesse onder ligte proses voorwaardes. Tesame met die relatiewe oorvloed van alkane afgelei van GTL tegnologie, soos dié van Sasol, Suid-Afrika, die kommersiële potensiaal vir bioconverison van alkane is groot. Maar, in teenstelling koolhidrate substrate, alkaan voerstowwe is beroof van suurstof in hul molekulêre struktuur. Dit beteken dat die hele suurstof vereiste moet nagekom word deur suurstof oordrag. Verder het ’n afname in suurstof oordrag in waterige-koolwaterstof dispersies met toenemende alkaan konsentrasie waargeneem te lei van depressie van die algehele volumetriese suurstofoordragkoëffisiënt (KLa). Daarom verstaan KLa en die fundamentele parameters onderliggend sy gedrag is van kritieke belang om te verseker dat die bioprocess is kineties, eerder as vervoer, beperk in terme van beide werking en skaal-up van bioprosesse. Vorige studies het KLa in deurlug-alkaan-waterige stelsels ondersoek. In die lig van die belangrikheid van suurstof oordrag in bioprosesse hierdie studie brei uit op die KLa begrip in driefase studies deur die insluiting van ’n vierde soliede fase, dus meer nou wat ’n koolwaterstofgebaseerde bioprocess. Die doel van die projek is om die impak van vermengingstempo, alkaan konsentrasie en soliede inhout op die Sauter gemiddelde borrel deursnee (DSM), gas-vasvanging en spesifieke gas-vloistof oppervlakarea (a) te kwantifiseer en korreleer met KLa gedrag. Dit sou defineer die dominante parameter oor ’n verskeidenheid van proses voorwaardes. Verder, gelyktydige meting van die KLa en oppervlakarea kan die gedrag van die vloeistof-kant suurstofoordragkoëffisiënt (KL) gedefinieer. Dit sal verskaf verdere insig in hoe die veranderinge in die proses voorwaardes impak op KLa. Eksperimente was uitgevoer in ’n 5 liter belugte geroerde tenk bioreaktor bevat n - C14-20 reguitketting alkane, met lug met lug deurgeborrel by 0.8 VVM. In lyn met die proses voorwaardes tipies van ’n koolwaterstof-gebaseerde bioprocess, KLa en a was gemeet vir vermengignstempos van 450-1000 RPM, alkaan konsentrasies van 2-20 % v/v en gis vastestowwe van 1 tot 10 g / l. KLa is gemeet deur die vergassinguit prosedure met behulp van ’n suurstofmeter wat die reaksie van die stelsel na ’n stap verandering in die voer gas suurstof druk gemeet het. Die suurstofmeter reaksie vertraging ( P), gelyk aan die tyd wat dit neem vir die suurstofmeter 63.2 % van die versadiging DO konsentrasie te bereik, is bepaal vir elke procesopset. Die inverse van P, KP is in ag geneem by die berekening van KLa uit die suurstofmeter reaksie. Die gas-vloistof oppervlak is bereken vanaf DSM en gas hold-up. DSM is gekwantifiseer met behulp van hoë spoed fotografie en beeld analise is uitgevoer in Matlab ® roetines. Uitskakeling van optiese vervorming en die ontwikkeling van ’n voldoende ligbron was die sleutel tot die verkryging van helder beelde. Beide KLa en grens oppervlakarea gevind geraak word deur veranderinge in vermengignstempo, alkaan konsentrasie en gis laai. ’N toename in geroer het die KLa verbeter oor die hele reeks van alkaan konsentrasie en gis laai. Net so, ’n toename in geroer het gelei tot ’n toename in grens oppervlak, ondersteun deur ’n afname in die DSM. Dit is dus waarskynlik dat die grens oppervlak speel ’n dominante rol in die definisie van KLa by die oorweging van ’n toename in roering. Stygings in alkaan konsentrasie gelei tot ’n hoogtepunt in KLa tussen 2.5 en 5 % alkaan konsentrasie terwyl verdere verhogings in alkaan konsentrasie druk die KLa af. Die piek was nie in oppervlakarea duidelik, waar ’n toename in alkaan konsentrasie gelei net tot ’n afname in oppervlakarea, dus dui op ’n positiewe invloed van KL op KLa teen lae alkaan konsentrasies waargeneem. Verdere stygings in alkaan konsentrasie verder as die skep van die piek KLa gelei tot KLa depressie, wat daarop dui dat die toenemende viskositeit meegedeel deur die alkaan verminder beide KL en grens oppervlak. Verhoogde gis laai het opponerende effekte teen ’n lae en hoë vermengingstempo. By lae vermengingstempo, ’n verhoging in gis laai waargeneem KLa te verhoog, terwyl ’n verhoging in gis laai op ’n hoë vermengingstempo veroorsaak ’n afname in KLa . Hierdie gedrag was ook duidelik in grens oppervlak, wat daarop dui dat daar in hierdie regime KLa gedefinieer deur grens oppervlak gedrag. Verhoogde gis laai waargeneem die KLa te onderdruk vir alle alkaan konsentrasies wanneer ondersoek teen ’n konstante middelpunt vermengingstempo. Hierdie tendens was nie duidelik in tussenvlak gebied, wat verhoog met toenemende gis laai op dieselfde geroer koers. Die positiewe invloed van gis op grens oppervlak is waarskynlik veroorsaak deur adhesie van die gis deeltjies aan die borrel oppervlak, die verlaging van die DSM deur die voorkoming van die saamsmelting van gasborrels. Die meningsverskil tussen die KLa en grens oppervlakarea resultate voorgestel dat gis laai negatiewe uitwerking op KL, met ’n dominante negatiewe impak op KLa. Die gebruik van ’n betroubare metodes vir die bepaling van beide oppervlakarea en KLa gedemonstreer vir toepassing in model koolwaterstof-gebaseerde bioprosesse. Die gekombineerde resultate bied ’n unieke insig in hoe die veranderinge in die proses voorwaardes impak onafhanklik op KL en oppervlakarea, wat wanneer gekombineer gedefinieer die KLa gedrag. Kwantifisering van die relatiewe grootte van die impak elke parameter het op KLa bygedra tot ’n fundamentele begrip van suurstof oordrag in model koolwaterstof-gebaseerde bioprosesse.

No description available.

Thesis (MEng)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The expansion of the global fuels industry has caused an increase in the quantity of hydrocarbons produced as a by-product of refinery gas-to-liquid processes. Conversion of hydrocarbons to higher value products is possible using bioprocesses, which are sustainable and environmentally benign. Due to the deficiency of oxygen in the alkane molecule, the supply of sufficient oxygen through aeration is a major obstacle for the optimization of hydrocarbon bioprocesses. While the oxygen solubility is increased in the presence of hydrocarbons, under certain process conditions, the enhanced solubility is outweighed by an increase in viscosity, causing a depression in overall volumetric oxygen transfer coefficient (KLa). The rate at which oxygen is transferred is defined in terms of a concentration driving force (oxygen solubility) and the overall volumetric oxygen transfer coefficient (KLa). The KLa term comprises an oxygen transfer coefficient (KL) and the gas-liquid interfacial area (a), which are dependent on the uid properties and system hydrodynamics. This behaviour is not well understood for hydrocarbon bioprocesses and in a bubble column reactor (BCR). To provide an understanding of oxygen transfer behaviour, a model hydrocarbon bioprocess was developed using a BCR with a porous sparger. To evaluate the interfacial area, the Sauter mean bubble diameter (D32) was measured using an image analysis algorithm and gas holdup (ϵG) was measured by the change in liquid height in the column. Together the D32 and ϵG were used in the calculation of interfacial area in the column. The KLa was evaluated with incorporation of the probe response lag, allowing more accurate representation of the KLa behaviour. The probe response lag was measured at all experimental conditions to ensure accuracy and reliability of data. The model hydrocarbon bioprocess employed C14-20 alkane-aqueous dispersions (2.5 - 20 vol% hydrocarbon) with suspended solids (0.5 - 6 g/l) at discrete super ficial gas velocity (uG) (1 - 3 cm/s). For systems with inert solids (corn our, dp = 13.36 m), the interfacial area and KLa were measured and the behaviour of KLa was described by separation of the in uences of interfacial area and oxygen transfer coefficient (KL). To further the understanding of oxygen transfer behaviour, non-viable yeast cells (dp = 5.059 m) were used as the dispersed solid phase and interfacial area behaviour was determined. This interfacial area behaviour was compared with the behaviour of systems with inert solids to understand the differences with change in solids type. In systems using inert solids, a linear relationship was found between G and uG. An empirical correlation fo rthe prediction of this behaviour showed an accuracy of 83.34% across the experimental range. The interfacial area showed a similar relationship with uG and the empirical correlation provided an accuracy of 78.8% for prediction across the experimental range. In inert solids dispersions, the KLa increased with uG as the result of an increase in interfacial area as well as increases in KL. An increase in solids loading indicated an initial increase in KLa, due to the in uence of liquid-film penetration on KL, followed by a decrease in KL at solids loading greater than 2.5 g/l, due to diffusion blocking effects. In systems with yeast dispersions, the presence of surfactant molecules in the media inhibited coalescence up to a yeast loading of about 3.5 g/l, and resulted in a decrease in D32. Above this yeast loading, the fine yeast particles increased the apparent viscosity of the dispersion sufficiently to overcome the in uence of surfactant and increase the D32. The behaviour of G in yeast dispersions was similar to that found with inert solids and demonstrated a linear increase with uG. However, in yeast dispersions, the interaction between alkane concentration and yeast loading caused a slight increase in dispersion viscosity and therefore G. An empirical correlation to predict G behaviour with increased uG was developed with an accuracy of 72.55% for the experimental range considered. Comparison of yeast and inert solids dispersions indicated a 37.5% lower G in yeast dispersions compared to inert solids as a result of the apparent viscosity introduced by finer solid particles. This G and D32 data resulted in a linear increase in interfacial area with uG with no significant in uence of alkane concentration and yeast loading. This interfacial area was on average 6.7% lower than interfacial area found in inert solid dispersions as a likely consequence of the apparent viscosity with finer particles. This study provides a fundamental understanding of the parameters which underpin oxygen transfer in a model hydrocarbon bioprocess BCR under discrete hydrodynamic conditions. This fundamental understanding provides a basis for further investigation of hydrocarbon bioprocesses and the prediction of KLa behaviour in these systems. / AFRIKAANSE OPSOMMING: Die uitbreiding van die internasionale brandstofbedryf het 'n toename veroorsaak in die hoeveelheid koolwaterstowwe geproduseer as 'n deur-produk van raffinadery gas-tot-vloeistof prosesse. Omskakeling van koolwaterstowwe na hoër waarde produkte is moontlik met behulp van bioprosesse, wat volhoubaar en omgewingsvriendelik is. As gevolg van die tekort aan suurstof in die alkaan molekule, is die verskaffing van voldoende suurstof deur deurlugting 'n groot uitdaging vir die optimalisering van koolwaterstof bioprosesse. Terwyl die suurstof oplosbaarheid verhoog in die teenwoordigheid van koolwaterstowwe, onder sekere proses voorwaardes is die verhoogde oplosbaarheid oortref deur 'n toename in viskositeit, wat 'n depressive veroorsaak in die algehele volumetriese suurstofoordragkoëffisiënt (KLa). Die suurstof oordrag tempo word gedefinieer in terme van 'n konsentrasie dryfkrag (suurstof oplosbaarheid) en KLa. Die KLa term behels 'n suurstofoordragkoëffisiënt (KL) en die gas-vloeistof oppervlakarea (a), wat afhanklik is van die vloeistof eienskappe en stelsel hidrodinamika. Hierdie gedrag is nie goed verstaan vir koolwaterstof bioprosesse nie, asook in kolom reaktors (BCR). Om 'n begrip van suurstof oordrag gedrag te voorsien, is 'n model koolwaterstof bioproses ontwikkel met 'n BCR met 'n poreuse besproeier. Om die oppervlakarea te evalueer, is die gemiddelde Sauter deursnit (D32) gemeet deur 'n foto-analise algoritme en gas vasvanging ( G) is gemeet deur die verandering in vloeibare hoogte in die kolom. Saam is die D32 en G gebruik in die berekening van die oppervlakarea in die kolom. Die KLa is geëvalueer met insluiting van die meter se reaksie sloering, om n meer akkurate voorstelling van die KLa gedrag te bereken. Die meter reaksie sloering was gemeet op alle eksperimentele toestande om die akkuraatheid en betroubaarheid van data te verseker. Die model koolwaterstof bioproses gebruik n-C14-20 alkaan-water dispersies (2.5 - 20 vol% koolwaterstof) solide partikels (0.5 - 6 g/l) op diskrete oppervlakkige gas snelhede (1 - 3 cm/s). Vir stelsels met inerte solides (koring meel, dp = 13.36 m), is die oppervlakarea en KLa gemeet en die gedrag van KLa beskryf deur skeiding van die invloede van oppervlakarea en KL. Om die begrip van suurstof oordrag se gedrag te bevorder, is nie-lewensvatbare gisselle (dp = 5.059 m) gebruik as die verspreide solide fase en oppervlakarea is bepaal. Hierdie oppervlakarea gedrag is vergelyk met die van stelsels met inerte solides om die verskille met verandering in solide tipes te verstaan. In stelsels met inerte solides, is 'n line^ere verwantskap gevind tussen G en uG. 'n Empiriese korrelasie vir die voorspelling van hierdie gedrag is opgestel met 'n akkuraatheid van 83.34% in die eksperimentele reeks. Die oppervlakarea het 'n soortgelyke verhouding met uG en die empiriese korrelasie verskaf 'n akkuraatheid van 78,8% vir die voorspelling van oppervlakarea oor die eksperimentele reeks. In inerte solide dispersies, het die KLa toegeneem met uG as die gevolg van 'n toename in grens oppervlak asook stygings in KL. 'n Toename in solides belading het n aanvanklike styging in KLa aangedui, as gevolg van die invloed van die vloeistof-film penetrasie op KL, gevolg deur 'n afname in KL op vastestowwe ladings groter as 2.5 g/l, te danke aan diffusie blokkeer effekte. In stelsels met gis dispersies, het die teenwoordigheid van benattings molekules in die media samesmelting geïnhibeer tot 'n gis lading van ongeveer 3.5 g/l, en het gelei tot 'n afname in D32. Bo hierdie gis lading, het die fyn gis partikels die skynbare viskositeit van die verspreiding verhoog genoegsaam om die invloed van benattings molekules te oorkom en die D32 te verhoog. Die gedrag van G in gis dispersies was soortgelyk aan die van inerte solides en dui op 'n lineêre toename met uG. Maar in gis dispersies, het die interaksie tussen alkaan konsentrasie en gis lading 'n effense toename veroorsaak in die verstrooiing viskositeit en dus in G. 'n Empiriese korrelasie is ontwikkel om G gedrag te voorspel en het 'n akkuraatheid van 72,55% vir die eksperimentele verskeidenheid beskou. Vergelyking van gis en inerte patrikel dispersies wys 'n 37.5% laer G in gis dispersies in vergelyking met inerte vaste stowwe as 'n gevolg van die skynbare viskositeit bekendgestel deur fyner vastestowwe partikels. Hierdie G en D32 data het gelei tot 'n linere toename in grens oppervlak met uG met geen beduidende invloed van alkaan konsentrasie en gis lading nie. Die oppervlakarea was gemiddeld 6.7% laer as oppervlakarea gevind in inerte partikel dispersies as 'n waarskynlike gevolg van die skynbare viskositeit met fyner partikels. Hierdie studie bied 'n fundamentele begrip van die veranderlikes wat die suurstof oordrag definieer in 'n model koolwaterstof bioproses BCR onder diskrete hidrodinamiese voorwaardes. Hierdie fundamentele begrip bied n basis vir verdere ondersoek van koolwaterstof bioprosesse en en die voorspelling van KLa gedrag in hierdie stelsels.

Peptidase pode ser considerada, como uma subclasse das enzimas hidrolíticas que ocupam uma posição central em relação às suas aplicações na área fisiológica e também na área comercial. As peptidases representam um dos três maiores grupos de enzimas industriais e são responsáveis por cerca de 60% da venda mundial de enzimas. O presente trabalho visa avaliar a produção de peptidase em fermentação submersa (FSm) pelo fungo Scopulariopsis koningii, utilizando como substrato farinha de pena (FP), a caracterização bioquímica parcial, secagem do extrato bruto, purificação da peptidase e determinação de especificidade da peptidase isolada. Para avaliar a influência da farinha de pena (FP), na produção de peptidases, foram adicionadas às porcentagens de 0,2; 0,4 e 0,8% no meio de cultura. Os melhores níveis de produção de peptidases pelo fungo Scopulariopsis koningii foram obtidos nas concentrações de 0,4% e 0,8% de FP em 48h de fermentação com 1.427 U/mL. A caracterização bioquímica parcial do extrato bruto foi realizada com azocaseína 1% preparada em tampão com pH adequado. A peptidase presente no extrato enzimático apresentou atividade ótima em pH 6,5 e temperatura ótima de 55°C. A peptidase foi inibida por PMSF, indicando a presença de resíduo de aminoácido serino no sítio catalítco, e desta forma sendo classificada como serino peptidase. Entretanto, também observamos uma inibição por EDTA, sugerindo a presença de uma metalo peptidase presente no extrato bruto, desta forma podemos sugerir que o fungo S. koningii na presença do meio contendo FP secreta duas subclasses; serino e metalo peptidase, ou secreta uma serino peptidase dependente de íon. Zimograma constatou a presença de duas enzimas. A atividade enzimática do extrato bruto diminuiu significativamente quando exposta os íons Al+3, Ni2+ e Cu2+ e aumentada quando adicionados os íons Ba2+, Ca2+ e Mg2+. A purificação da metalo peptidase presente no extrato enzimático envolveu sete etapas de purificação, sendo cromatografia de troca-iônica e gel filtração determinantes, com recuperação de 6% e purificação de 3,4 vezes. Utilizando a peptidase pura (metalo) realizouse a caracterização bioquímica funcional e a determinação dos parâmetros cinéticos, ambos utilizando o substrato peptídico de supressão intramolecular de fluorescência. A peptidase pura apresentou atividade ótima em pH 6,0 e temperatura ótima de 40°C e mostrou-se estável em ampla faixa de pH e temperatura. Foi modulada positivamente pelos íons Na+ e K+ e negativamente por Al3+ e Cu2+. A análise dos parâmetros cinéticos revelou uma grande influência de aminoácidos apolares do lado \"linha\" do substrato sintético na eficiência catalítica e no lado não \"linha\" grande influência de aminoácidos polares neutros e apolares. A secagem foi realizada por liofilização foram utilizados três tipos de adjuvantes: maltodextrina, manitol e glicina, em diferentes concentrações. Após a secagem foi realizado estudo da estabilidade nas temperaturas 4, 25 e 60°C por 32 dias para avaliar o desempenho dos adjuvantes na manutenção da atividade do extrato enzimático liofilizado. O adjuvante considerado mais eficaz foi a maltodextrina na concentração de 4,5% que manteve cerca de 93% da atividade do extrato enzimático liofilizado por 32 dias. / Peptidase can be considered as a subclass of hydrolytic enzymes which occupy a central position in relation to their applications in physiological area and also in the commercial area. Peptidases represent one of the three largest groups of industrial enzymes and account for about 60% of worldwide sales of enzymes. This study aims to evaluate the production of peptidase in submerged fermentation (FSm) by the fungus Scopulariopsis koningii, using as substrate feather meal (FP), the partial biochemical characterization, drying the crude extract, purification and determination of specific peptidase peptidase isolated. To evaluate the influence of feather meal (FM), in the production of peptidases, were added to the percentages of 0.2, 0.4 and 0.8% in the culture medium. The best levels of production of peptidases by the fungus Scopulariopsis koningii were obtained at concentrations of 0.4% and 0.8% of FP 48h fermentation with 1,427 U/mL. Partial biochemical characterization of the crude extract was performed with 1% azocasein prepared in buffer with appropriate pH. This in peptidase enzyme extract showed optimal activity at pH 6.5 and optimum temperature of 55°C. The peptidase was inhibited by PMSF, indicating the presence of a serine residue at amino acid catalytic site, and thus being classified as serine peptidase. However, we also observed an inhibition by EDTA, suggesting the presence of a metallo peptidase present in the crude extract, thus we suggest that the fungus S. koningii in the presence of medium containing secret FP two subclasses; serine peptidase and metal, or secretes a serine peptidase dependent ion. Zymogram found the presence of two enzymes. The enzymatic activity of the crude extract decreased significantly when exposed the Al 3+, Ni 2+ and Cu2+ ions and increased when added to Ba2+, Ca2+ and Mg2+ ions. The purification of metallo peptidase present in the enzyme extract involved seven stages of purification, and ion-exchange chromatography and gel filtration determinants, with recovery and purification of 6 % from 3.4 times. Using pure peptidase (metallo) held functional biochemical characterization and determination of kinetic parameters using both the intramolecular peptide substrate fluorescence suppression. Pure peptidase showed optimal activity at pH 6.0 and optimum temperature of 40°C and was stable in a wide range of pH and temperature. The positively modulated by Na+ and K+ and negatively by Al3+ and Cu2+. Analysis of kinetic parameters revealed a strong influence of nonpolar amino acid side \"line\" of the synthetic substrate in the catalytic efficiency and not on the side \"line\" great influence of nonpolar and polar neutral amino acids. The freeze drying was performed by three types of additives were used: maltodextrin, mannitol and glycine in different concentrations. After drying stability study was conducted at the temperatures 4, 25 and 60°C for 32 days to evaluate the performance of additives in maintaining the activity of the enzyme extract lyophilized. The adjuvant was found more efficacious maltodextrin in a concentration of 4.5%, which retained about 93% of the extract lyophilized enzyme activity for 32 days.

Peptidase pode ser considerada, como uma subclasse das enzimas hidrolíticas que ocupam uma posição central em relação às suas aplicações na área fisiológica e também na área comercial. As peptidases representam um dos três maiores grupos de enzimas industriais e são responsáveis por cerca de 60% da venda mundial de enzimas. O presente trabalho visa avaliar a produção de peptidase em fermentação submersa (FSm) pelo fungo Scopulariopsis koningii, utilizando como substrato farinha de pena (FP), a caracterização bioquímica parcial, secagem do extrato bruto, purificação da peptidase e determinação de especificidade da peptidase isolada. Para avaliar a influência da farinha de pena (FP), na produção de peptidases, foram adicionadas às porcentagens de 0,2; 0,4 e 0,8% no meio de cultura. Os melhores níveis de produção de peptidases pelo fungo Scopulariopsis koningii foram obtidos nas concentrações de 0,4% e 0,8% de FP em 48h de fermentação com 1.427 U/mL. A caracterização bioquímica parcial do extrato bruto foi realizada com azocaseína 1% preparada em tampão com pH adequado. A peptidase presente no extrato enzimático apresentou atividade ótima em pH 6,5 e temperatura ótima de 55°C. A peptidase foi inibida por PMSF, indicando a presença de resíduo de aminoácido serino no sítio catalítco, e desta forma sendo classificada como serino peptidase. Entretanto, também observamos uma inibição por EDTA, sugerindo a presença de uma metalo peptidase presente no extrato bruto, desta forma podemos sugerir que o fungo S. koningii na presença do meio contendo FP secreta duas subclasses; serino e metalo peptidase, ou secreta uma serino peptidase dependente de íon. Zimograma constatou a presença de duas enzimas. A atividade enzimática do extrato bruto diminuiu significativamente quando exposta os íons Al+3, Ni2+ e Cu2+ e aumentada quando adicionados os íons Ba2+, Ca2+ e Mg2+. A purificação da metalo peptidase presente no extrato enzimático envolveu sete etapas de purificação, sendo cromatografia de troca-iônica e gel filtração determinantes, com recuperação de 6% e purificação de 3,4 vezes. Utilizando a peptidase pura (metalo) realizouse a caracterização bioquímica funcional e a determinação dos parâmetros cinéticos, ambos utilizando o substrato peptídico de supressão intramolecular de fluorescência. A peptidase pura apresentou atividade ótima em pH 6,0 e temperatura ótima de 40°C e mostrou-se estável em ampla faixa de pH e temperatura. Foi modulada positivamente pelos íons Na+ e K+ e negativamente por Al3+ e Cu2+. A análise dos parâmetros cinéticos revelou uma grande influência de aminoácidos apolares do lado \"linha\" do substrato sintético na eficiência catalítica e no lado não \"linha\" grande influência de aminoácidos polares neutros e apolares. A secagem foi realizada por liofilização foram utilizados três tipos de adjuvantes: maltodextrina, manitol e glicina, em diferentes concentrações. Após a secagem foi realizado estudo da estabilidade nas temperaturas 4, 25 e 60°C por 32 dias para avaliar o desempenho dos adjuvantes na manutenção da atividade do extrato enzimático liofilizado. O adjuvante considerado mais eficaz foi a maltodextrina na concentração de 4,5% que manteve cerca de 93% da atividade do extrato enzimático liofilizado por 32 dias. / Peptidase can be considered as a subclass of hydrolytic enzymes which occupy a central position in relation to their applications in physiological area and also in the commercial area. Peptidases represent one of the three largest groups of industrial enzymes and account for about 60% of worldwide sales of enzymes. This study aims to evaluate the production of peptidase in submerged fermentation (FSm) by the fungus Scopulariopsis koningii, using as substrate feather meal (FP), the partial biochemical characterization, drying the crude extract, purification and determination of specific peptidase peptidase isolated. To evaluate the influence of feather meal (FM), in the production of peptidases, were added to the percentages of 0.2, 0.4 and 0.8% in the culture medium. The best levels of production of peptidases by the fungus Scopulariopsis koningii were obtained at concentrations of 0.4% and 0.8% of FP 48h fermentation with 1,427 U/mL. Partial biochemical characterization of the crude extract was performed with 1% azocasein prepared in buffer with appropriate pH. This in peptidase enzyme extract showed optimal activity at pH 6.5 and optimum temperature of 55°C. The peptidase was inhibited by PMSF, indicating the presence of a serine residue at amino acid catalytic site, and thus being classified as serine peptidase. However, we also observed an inhibition by EDTA, suggesting the presence of a metallo peptidase present in the crude extract, thus we suggest that the fungus S. koningii in the presence of medium containing secret FP two subclasses; serine peptidase and metal, or secretes a serine peptidase dependent ion. Zymogram found the presence of two enzymes. The enzymatic activity of the crude extract decreased significantly when exposed the Al 3+, Ni 2+ and Cu2+ ions and increased when added to Ba2+, Ca2+ and Mg2+ ions. The purification of metallo peptidase present in the enzyme extract involved seven stages of purification, and ion-exchange chromatography and gel filtration determinants, with recovery and purification of 6 % from 3.4 times. Using pure peptidase (metallo) held functional biochemical characterization and determination of kinetic parameters using both the intramolecular peptide substrate fluorescence suppression. Pure peptidase showed optimal activity at pH 6.0 and optimum temperature of 40°C and was stable in a wide range of pH and temperature. The positively modulated by Na+ and K+ and negatively by Al3+ and Cu2+. Analysis of kinetic parameters revealed a strong influence of nonpolar amino acid side \"line\" of the synthetic substrate in the catalytic efficiency and not on the side \"line\" great influence of nonpolar and polar neutral amino acids. The freeze drying was performed by three types of additives were used: maltodextrin, mannitol and glycine in different concentrations. After drying stability study was conducted at the temperatures 4, 25 and 60°C for 32 days to evaluate the performance of additives in maintaining the activity of the enzyme extract lyophilized. The adjuvant was found more efficacious maltodextrin in a concentration of 4.5%, which retained about 93% of the extract lyophilized enzyme activity for 32 days.

O etanol é um dos biocombustíveis oriundos de fontes renováveis mais promissor e ambientalmente amigável. Visando-se ao atendimento de uma demanda crescente por este álcool, o aproveitamento integral dos materiais lignocelulósicos é uma alternativa interessante e, desta forma, trabalhos de pesquisa têm sido desenvolvidos visando-se à viabilização de biorrefinarias de produção integrada de etanol 1G e 2G. O presente trabalho teve como objetivo principal desenvolver e analisar economicamente modelos de biorrefinaria integrada 1G2G que empregam pré-tratamento ácido diluído, avaliando-se o impacto econômico do destino da hemicelulose para produção de etanol. Para isso, foram definidos cenários que incluíssem todas as etapas do processo de produção de etanol 1G e 2G, sendo avaliados dois teores de sólidos no reator de pré-tratamento (5% e 10%) e, para cada caso, três relações tempo e conversão da hidrólise enzimática, sendo estas de 45%, 51% e 55% para tempos de reação de 24h, 48h e 72h, respectivamente. Para cada condição, considerou-se a fermentação de pentose (C5) ou seu envio para tratamento de efluentes, constituindo-se, assim, 12 cenários. Foram realizadas as simulações destes cenários auxiliadas pelo pacote computacional Aspen Plus®, obtendo-se os dados de balanço de massa e energia, permitindo assim a realização da análise de viabilidade econômica empregando a plataforma Biorrefinaria Virtual de Cana - BVC. Foi possível observar que a produção de etanol 2G empregando pré-tratamento ácido diluído foi responsável por um aumento na produção de etanol de até 35%. Avaliando-se os cenários, observou-se que quando o reator de pré-tratamento foi operado com maior teor de sólidos, menor consumo de energia térmica foi necessário, requerendo menor quantidade de bagaço na caldeira e aumentando o rendimento final de etanol. Com relação ao custo de investimento, este correspondeu a R$799,2 milhões para a biorrefinaria 1G, enquanto que os cenários integrados 1G2G apresentaram valores cerca de 40% superiores. Dentre as áreas mais expressivas no custo de investimento, estão a de pré-tratamento e geração e distribuição de vapor. No que concerne ao custo de produção de etanol, os cenários que apresentaram menor valor para etanol 2G corresponderam ao uso de 10% de sólidos no reator de pré-tratamento com fermentação de C5. As áreas mais expressivas para esse custo foram os custos com insumos e capital. Todavia, os cenários com 5% de sólidos no reator de pré-tratamento e sem fermentação de C5 resultaram em menor custo de produção global de etanol (1G2G), sendo o custo com cana, insumos e capital os mais expressivos. Com base na taxa interna de retorno, esta se apresentou abaixo da taxa mínima de atratividade para todos os cenários, ocasionando um valor presente líquido negativo, inferindo-se que, nas condições avaliadas, a metodologia empregada nos cenários está inviável do ponto de vista de um investidor. No entanto, a análise econômica auxiliou a identificação dos gargalos tecnológicos do processo de produção integrada de etanol 1G2G quando estes empregam pré-tratamento ácido diluído, favorecendo a definição de metas operacionais a serem solucionadas em trabalhos de pesquisa. / Ethanol is one of the most promising and environmentally friendly biofuels obtained from renewable energy sources. Aiming at meeting a growing demand for this fuel, the integral use of lignocellulosic materials is an interesting alternative and, in this way, research Works have been performed to make viable biorefineries for integrated production of 1G and 2G ethanol. The present work had as main objective to develop and to analyze economically models of 1G2G integrated biorefineries using dilute acid pretreatment, evaluating the economic impact of using hemicellulose for ethanol production. Alternative scenarios were defined including all steps of the 1G and 2G ethanol production process. Two different solids contents were evaluated in the pre-treatment reactor (5% and 10%) and, for each case, three values of time and conversion for the enzymatic hydrolysis step were considered, namely 45%, 51% and 55% for reaction times of 24h, 48h and 72h, respectively. For each condition, the fermentation of pentose (C5) or its sending for effluents treatment was supposed, constituting, thus, 12 scenarios. Simulations of these scenarios were carried out using the software Aspen Plus®, generating mass and energy balance data, thus allowing carrying out economic viability analysis using the Virtual Sugarcane Biorefinery (VSB) platform. Production of 2G ethanol using dilute acid pretreatment was responsible for an increase in ethanol production of up to 35%. When the pretreatment reactor was operated with higher solids content, lower thermal energy consumption was necessary, requiring less bagasse in the boiler and thus increasing the final ethanol yield. Regarding to investment cost, it was of R$ 799.2 million for 1G biorefinery, compared to a value 40% higher observed for integrated 1G2G scenarios. Among the most significant areas of investment cost are the pretreatment and the generation and distribution of steam. With relation to the cost of etanol production, scenarios that presented the lowest value for 2G ethanol corresponded to the use of 10% solids in the pre-treatment reactor associated to C5 fermentation. The most significant areas for this cost were the input and capital costs. However, the scenarios with 5% solids in the pre-treatment reactor without C5 fermentation resulted in a lower global ethanol production cost (1G2G), with sugarcane, chemical inputs and capital cost representing the most significant fraction. Based on the internal rate of return, this was lower than the minimum attractiveness rate of return for all scenarios, resulting in a negative net present value, allowing inferring that, under the evaluated conditions, the methodology used in the scenarios would be unviable from an investor viewpoint. However, the economic analysis helped to identify technological bottlenecks of the integrated 1G2G etanol production process using dilute acid pretreatment, favoring the definition of operational goals to be solved in research works.

Os fungos filamentosos são utilizados em larga escala na produção de produtos biotecnológicos na indústria devido a sua versatilidade e um dos exemplos são as peptidases que representam uma das principais classes de enzimas hidrolíticas. As peptidases são hidrolases que catalisam a quebra das ligações peptídicas das proteínas e que nos microrganismos são responsáveis por funções fisiológicas e patológicas, além de ter muitas aplicações em diversos campos industriais. Neste estudo foram analisados diferentes bioprocessos fermentativos para produção de peptidases pelo fungo Aspergillus terreus. Este microrganismo foi capaz de produzir peptidases em ambos os bioprocessos, sólido ou submerso, obtendo melhor performance e o pico de produção no bioprocesso fermentativo sólido de valor 677U/mL, nas condições 5g de farelo de trigo, 72 horas, 30°C e 75% de umidade. Utilizando o bioprocesso fermentativo submerso também obtivemos resultados satisfatórios com pico de atividade de 360U/mL, nas condições de meio padrão suplementado com Caseína 0,5%, 72 horas e 35°C. A caracterização bioquímica parcial dos extratos dos dois bioprocessos mostrou semelhanças entre algumas características das enzimas produzidas como a faixa extensa de pH ótimo abrangendo regiões ácidas, neutra e alcalinas, temperatura ótima pontual de 55°C e perfil de inibição pelo PMSF e EDTA. Contudo, os perfis de estabilidade (pH e temperatura) e comportamento frente a adição de íons apresentaram respostas diferentes entre si, o que sugere a produção de enzimas diferentes produzidas pelo mesmo fungo em meios distintos. A microencapsulação por Spray Drying como processo de estabilização foi satisfatória obtendo rendimentos de 37,5-58,2% e com níveis acima de 50% de atividade enzimática. Em contrapartida, a imobilização enzimática demonstrou ser eficaz na etapa de ligação ao suporte, mas não foi capaz de estabilizar a enzima presente no extrato, o que ficou caracterizado pela perda de atividade proteolítica. / Filamentous fungi are extensively used in the production of biotechnological products in industry because of their versatility and one of the examples are peptidases which constitute a major class of hydrolytic enzymes. The peptidases are hydrolases which catalyze the cleavage of peptide bonds of proteins and microorganisms that are responsible for the physiological and pathological roles, in addition to having many applications in various industrial fields. This study evaluated various bioprocesses for fermentative production of peptidases by the fungus Aspergillus terreus. This microorganism was able to produce peptidase in both bioprocess, solid or submerged, achieving better performance in the solid bioprocess with peak of production of 677U/mL under the conditions 5g wheat bran, 72 hours, 30°C and 75% humidity . Using submerged fermentation bioprocess we also obtained satisfactory results with peak of activity of 360U/mL with conditions of standard medium supplemented with 0.5% casein, 72 hours and 35°C. Biochemical characterization of the two partial purified extracts showed similarities between some characteristics of the enzymes produced, as large optimum pH range spanning regions acidic, neutral and alkaline point temperature optimum of 55 ° C and the inhibition profile of PMSF and EDTA. However, the stability profiles (pH and temperature) and behavior in addition ions showed different responses to each extract, which suggests the production of different enzymes in different ways. Microencapsulation by Spray Drying and stabilization process was obtaining satisfactory yields of 37.5 to 58.2%, with levels above 50% of enzyme activity. In contrast, the enzyme immobilization step was effective in bonding the support, but was not able to stabilize the enzyme present in the extract, which was characterized by the loss of proteolytic activity

Submitted by Marilene Donadel (marilene.donadel@unioeste.br) on 2019-03-13T00:48:12Z No. of bitstreams: 1 Camila_Hinterholz_2015.pdf: 2996844 bytes, checksum: f54562c9458e8a2460e3978c1be500c2 (MD5) / Made available in DSpace on 2019-03-13T00:48:12Z (GMT). No. of bitstreams: 1 Camila_Hinterholz_2015.pdf: 2996844 bytes, checksum: f54562c9458e8a2460e3978c1be500c2 (MD5) Previous issue date: 2015-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Biopolymers, especially Poly(3-hydroxybutyrate) (PHB), have been receiving big attention in order to minimize environmental damage caused by plastics from petrochemical industries. In this context, the aim of this paper was to formulate a mathematical model to describe the PHB production by Cupriavidus necator, from a detailed theoretical study. To this end, 6 models were evaluated, being 5 for cell growth and 1 for product formation, all from the literature. The ordinary differential equations system was solved numerically by Rosenbrock method. To the parameters estimation, an algorithm based on the Genetic Algorithms was developed and implemented in the software Maple®. To validate the models, experimental data at 30, 32,5, 35 and 37,5 °C were obtained from the literature. From the data analysis, it was observed that the best temperature, for both cell growth and product formation was 32,5 °C, and that the PHB production in partially associated with cell growth. To the parameters estimation, the ordinary differential equations system, obtained from the phenomenological modelling of non-structured and non-segregated models, was evaluated together with the models from the literature. The results for the objective function and correlation coefficient indicated that all the studied models adjusted well to the experimental data at all temperatures. Thus, some statistical tests were applied in order to better evaluate the models fitting, and the results indicated the Andrews’s (1968) model as the one that best represents the data from 32,5 °C, and Heinzle e Lafferty’s (1980) model for 35 °C. For the data at 30 and 37,5 °C there was no statistically valid models found. In conclusion, the statistical methodology applied for the models discrimination and fitting evaluation made it possible to say which model best represents data at each temperature. / Os biopolímeros, em especial o Poli(3-hidroxibutirato) (PHB), têm recebido grande atenção na tentativa de minimizar danos ambientais causados pelo acúmulo de plásticos de origem petroquímica. Neste âmbito, o objetivo deste trabalho foi formular um modelo matemático para o processo de produção do PHB por Cupriavidus necator, a partir de um estudo teórico detalhado. Para tanto, foram avalliados 5 modelos de crescimento celular e 1 modelo para a formação do produto, todos obtidos da literatura. O sistema de equações diferenciais ordinárias foi resolvido numericamente pelo método de Rosenbrock. Para a estimação dos parâmetros dos modelos foi desenvolvido um algoritmo baseado nos Algoritmos Genéticos, o qual foi implementado no software Maple®. Para a validação dos modelos, dados experimentais de ensaios a 30, 32,5, 35 e 37,5 °C foram obtidos da literatura. A partir da análise dos dados foi verificado que a melhor temperatura, tanto para o crescimento da biomassa quanto para a formação do produto, foi de 32,5 °C, e que a produção de PHB está parcialmente associada ao crescimento celular. Para a estimação dos parâmetros, o sistema de equações diferenciais ordinárias obtidas a partir da modelagem fenomenológica de modelos não estruturados e não seguregados foi avaliado juntamente com os modelos da literatura. Os resultados obtidos para a função objetivo e coeficiente de correlação indicaram que todos os modelos estudados se ajustaram bem aos dados experimentais em todas as temperaturas. Assim, para uma melhor avaliação dos ajustes foram aplicados alguns testes estatísticos, cujos resultados indicaram o modelo de Andrews (1968) como sendo o que melhor representa os dados à temperatura de 32,5 °C, e o modelo de Heinzle e Lafferty (1980) para os dados à 35 °C. Para as temperaturas de 30 e 37,5 °C não foi encontrado um modelo estatisticamente válido. Logo, a metodologia estatística aplicada para a discriminação de modelos e avaliação da qualidade dos ajustes tornou possível a identificação do modelo que melhor representa os dados em cada temperatura.

O presente projeto analisou a expressão gênica de Amblyomin -X, uma proteína com potencial terapêutico expressa em Escherichia coli BL21(DE3), por intermédio de um vetor de expressão plasmideal induzido por IPTG ( Isopropyl α-D-1-thiogalactopyranoside). A quantificação da expressão gênica foi realizada utilizando a técnica de RT-qPCR absoluta. A molécula Amblyomin-X corresponde a um inibidor de serino protease do tipo Kunitz, que apresenta atividade antitumoral e anticoagulante. Devido a sua importância terapêutica, fez-se necessário implementar um protocolo de escalonamento de produção, utilizando biorreatores. A informação sobre expressão gênica pode ajudar a interpretar o funcionamento do microrganismo durante os cultivos em frascos agitados e biorreatores, auxiliando na obtenção de protocolos de cultivo. A partir do momento da indução do gene Amblyomin-X, a bactéria Escherichia coli recombinante direciona grande parte de seu metabolismo para a expressão e produção dessa proteína, que é expressa em forma de corpos de inclusão insolúveis. As proteínas em forma de corpos de inclusão insolúveis permitem uma fácil extração do produto, porém, dificulta na quantificação por técnicas cromatográficas, por esse motivo, buscou-se também uma correlação entre a expressão gênica e a síntese protéica de Amblyomin-X, com o objetivo de validar a quantificação por RT-qPCR como uma forma indireta de quantificação desse tipo de produto protéico. Poucos estudos abordam a correlação existente entre a expressão gênica e a síntese protéica de um gene e sua proteína, e essa comparação poderia, também, revelar aspectos importantes entre a transcrição e a tradução de proteínas heterólogas em microrganismo. Foram realizados experimentos em frascos agitados em duas diferentes temperaturas indução (37°C e 30°C), com a concentração do indutor IPTG de 1 mM e concentração da biomassa (X) de 0,2 g/L. Para os experimentos em biorreatores utilizou-se o ponto central do planejamento experimental com concentração de IPTG 1mM e mais duas condições com concentração de IPTG 0,1 mM e 2mM. Foi encontrada uma grande similaridade entre os gráficos das análises cinéticas referentes a expressão gênica e a velocidade específica de formação do produto (qP). Não foi possível Identificar uma semelhança entre o perfil referente a análise cinética da expressão gênica comparada com a síntese protéica. Esses resultados sugerem que a expressão gênica apresenta uma cinética semelhante a velocidade específica de formação do produto e não necessariamente a concentração de proteína. Não foram encontradas correlações significativas nas análises de cada momento após a indução, exceto pelo ponto t4 do biorreator 14, quando foram correlacionadas a expressão gênica e a velocidade de formação do produto (r=0,998 e valor P=0,03). / The present work analysed the gene expression of Amblyomin-X, a potential therapeutic protein expressed in a recombinant Escherichia coli with a plasmid vector inducible by IPTG ( Isopropyl β-D-1- thiogalactopyranoside). The gene expression was quantified by absolute RT-qPCR method. Amblyomin-X is a Kunitz-type serine protease inhibitor which demonstrates antitumor and anticoagulant activity. Due to Amblyomin therapeutic importance, it was necessary to implement a production protocol using bioreactors. The information on gene expression can helps to understand the microorganism behavior during the cultures in shaken flasks and bioreactors. From the moment of the induction of the gene Amblyomin-X, the recombinant Escherichia coli directs its metabolism for expression and production of this protein. The Amblyomin-X protein is expressed as insoluble inclusion bodies. Insoluble inclusion bodies proteins can be easyly extracted from the cell, however it difficults its quantification by chromatographic techniques. Because of that, besides analysis of the gene expression, by RT-qPCR method, we tried to correlate the gene expression with their protein levels concentration, whose objective was implement the RT-qPCR as an indirect method to quantify these type of protein product. Few studies correlate the gene expression in their protein, and this comparison can contribute to reveal important aspects between transcription and translation of heterologous proteins in Escherichia coli, for the achievement of scale up process in biorreactors. Therefore, the quantification by RT-qPCR, represents a most secure way to understand the expression of heterologous proteins in recombinant systems. Experiments were performed in shaken flasks at two different induction temperatures (37 °C and 30 °C), 0,2 g/L of biomass (X) at the induction moment with IPTG 1mM. Biorreactors are performed with three different IPTG concentrations (0,1 mM, 1 mM and 2 mM). A great similarity was found between the graphs of the kinetic analyzes concerning gene expression and the specific rate of product formation (qP). It was not possible to identify a similarity between the profile concerning kinetic analysis of gene expression compared to protein synthesis. These results suggest that gene expression exhibits similar kinetics to the specific rate of product formation and not necessarily to the protein concentration. were found no significant correlations in the analyzes of each moment after induction, except for point t4 of the bioreactor 14, when the gene expression and the rate of the product formation were positively correlated (r = 0.998 and P value = 0.03).

O etanol é um dos biocombustíveis oriundos de fontes renováveis mais promissor e ambientalmente amigável. Visando-se ao atendimento de uma demanda crescente por este álcool, o aproveitamento integral dos materiais lignocelulósicos é uma alternativa interessante e, desta forma, trabalhos de pesquisa têm sido desenvolvidos visando-se à viabilização de biorrefinarias de produção integrada de etanol 1G e 2G. O presente trabalho teve como objetivo principal desenvolver e analisar economicamente modelos de biorrefinaria integrada 1G2G que empregam pré-tratamento ácido diluído, avaliando-se o impacto econômico do destino da hemicelulose para produção de etanol. Para isso, foram definidos cenários que incluíssem todas as etapas do processo de produção de etanol 1G e 2G, sendo avaliados dois teores de sólidos no reator de pré-tratamento (5% e 10%) e, para cada caso, três relações tempo e conversão da hidrólise enzimática, sendo estas de 45%, 51% e 55% para tempos de reação de 24h, 48h e 72h, respectivamente. Para cada condição, considerou-se a fermentação de pentose (C5) ou seu envio para tratamento de efluentes, constituindo-se, assim, 12 cenários. Foram realizadas as simulações destes cenários auxiliadas pelo pacote computacional Aspen Plus®, obtendo-se os dados de balanço de massa e energia, permitindo assim a realização da análise de viabilidade econômica empregando a plataforma Biorrefinaria Virtual de Cana - BVC. Foi possível observar que a produção de etanol 2G empregando pré-tratamento ácido diluído foi responsável por um aumento na produção de etanol de até 35%. Avaliando-se os cenários, observou-se que quando o reator de pré-tratamento foi operado com maior teor de sólidos, menor consumo de energia térmica foi necessário, requerendo menor quantidade de bagaço na caldeira e aumentando o rendimento final de etanol. Com relação ao custo de investimento, este correspondeu a R$799,2 milhões para a biorrefinaria 1G, enquanto que os cenários integrados 1G2G apresentaram valores cerca de 40% superiores. Dentre as áreas mais expressivas no custo de investimento, estão a de pré-tratamento e geração e distribuição de vapor. No que concerne ao custo de produção de etanol, os cenários que apresentaram menor valor para etanol 2G corresponderam ao uso de 10% de sólidos no reator de pré-tratamento com fermentação de C5. As áreas mais expressivas para esse custo foram os custos com insumos e capital. Todavia, os cenários com 5% de sólidos no reator de pré-tratamento e sem fermentação de C5 resultaram em menor custo de produção global de etanol (1G2G), sendo o custo com cana, insumos e capital os mais expressivos. Com base na taxa interna de retorno, esta se apresentou abaixo da taxa mínima de atratividade para todos os cenários, ocasionando um valor presente líquido negativo, inferindo-se que, nas condições avaliadas, a metodologia empregada nos cenários está inviável do ponto de vista de um investidor. No entanto, a análise econômica auxiliou a identificação dos gargalos tecnológicos do processo de produção integrada de etanol 1G2G quando estes empregam pré-tratamento ácido diluído, favorecendo a definição de metas operacionais a serem solucionadas em trabalhos de pesquisa. / Ethanol is one of the most promising and environmentally friendly biofuels obtained from renewable energy sources. Aiming at meeting a growing demand for this fuel, the integral use of lignocellulosic materials is an interesting alternative and, in this way, research Works have been performed to make viable biorefineries for integrated production of 1G and 2G ethanol. The present work had as main objective to develop and to analyze economically models of 1G2G integrated biorefineries using dilute acid pretreatment, evaluating the economic impact of using hemicellulose for ethanol production. Alternative scenarios were defined including all steps of the 1G and 2G ethanol production process. Two different solids contents were evaluated in the pre-treatment reactor (5% and 10%) and, for each case, three values of time and conversion for the enzymatic hydrolysis step were considered, namely 45%, 51% and 55% for reaction times of 24h, 48h and 72h, respectively. For each condition, the fermentation of pentose (C5) or its sending for effluents treatment was supposed, constituting, thus, 12 scenarios. Simulations of these scenarios were carried out using the software Aspen Plus®, generating mass and energy balance data, thus allowing carrying out economic viability analysis using the Virtual Sugarcane Biorefinery (VSB) platform. Production of 2G ethanol using dilute acid pretreatment was responsible for an increase in ethanol production of up to 35%. When the pretreatment reactor was operated with higher solids content, lower thermal energy consumption was necessary, requiring less bagasse in the boiler and thus increasing the final ethanol yield. Regarding to investment cost, it was of R$ 799.2 million for 1G biorefinery, compared to a value 40% higher observed for integrated 1G2G scenarios. Among the most significant areas of investment cost are the pretreatment and the generation and distribution of steam. With relation to the cost of etanol production, scenarios that presented the lowest value for 2G ethanol corresponded to the use of 10% solids in the pre-treatment reactor associated to C5 fermentation. The most significant areas for this cost were the input and capital costs. However, the scenarios with 5% solids in the pre-treatment reactor without C5 fermentation resulted in a lower global ethanol production cost (1G2G), with sugarcane, chemical inputs and capital cost representing the most significant fraction. Based on the internal rate of return, this was lower than the minimum attractiveness rate of return for all scenarios, resulting in a negative net present value, allowing inferring that, under the evaluated conditions, the methodology used in the scenarios would be unviable from an investor viewpoint. However, the economic analysis helped to identify technological bottlenecks of the integrated 1G2G etanol production process using dilute acid pretreatment, favoring the definition of operational goals to be solved in research works.