Dissertations / Theses on the topic 'Separate Hydrolysis and Fermentation'

Bioethanol from lignocellulose is expected to be the most likely fuel alternative in the near future. SEKAB E-Technology in Örnsköldsvik, Sweden develops the technology of the 2nd generation ethanol production; to produce ethanol from lignocellulosic raw material. The objective of this master’s thesis was to achieve a better knowledge of the potential and limitations of separate hydrolysis and fermentation (SHF) as a process concept for the 2nd generation ethanol production. The effects of enzyme concentration, temperature and pH on the glucose concentration in the enzymatic hydrolysis were investigated for pretreated spruce at 10% DM using a multiple factor design. Enzyme concentration and temperature showed significant effects on the glucose concentration, while pH had no significant effect on the concentration in the tested interval of pH 4.5-5.5. To obtain the maximum glucose concentration (46.4 g/l) for a residence time of 48 h, the optimal settings within the studied parameter window are a temperature of 45.7⁰C and enzyme concentration of 15 FPU/g substrate. However, a higher enzyme concentration would probably further increase the glucose concentration. If enzymatic hydrolysis should be performed for very short residence times, e.g. 6 h, the temperature should be 48.1⁰C to obtain maximum glucose concentration. The efficiency of the enzymes was inhibited when additional glucose was supplied to the slurry prior to enzymatic hydrolysis. It could be concluded that end product inhibition by glucose occurs and results in a distinct decrease in glucose conversion. No clear conclusions could be drawn according to different techniques for slurry and enzymes, i.e. batch and fed-batch, in the enzymatic hydrolysis process. Investigations of the fermentability of the hydrolysate revealed that the fermentation step in SHF is problematic. Inhibition of the yeast decrease the fermentation efficiency and it is therefore difficult to achieve the 4% ethanol limit. Residence time for enzymatic hydrolysis (48 h) and fermentation (24 h) need to be prolonged to achieve a sufficient SHF process. However, short processing times are a key parameter to an economically viable industrial process and to prolong the residence times should therefore not be seen as a desirable alternative. SHF as a process alternative in an industrial bioethanol plant has both potential and limitations. The main advantage is the possibility to separately optimize the process steps, especially to be able to run the enzymatic hydrolysis at an optimal temperature. Although, it is important to include all the process steps in the optimization work. The fermentation difficulties together with the end product inhibition are two limitations of the SHF process that have to be improved before SHF is a preferable alternative in a large scale bioethanol plant.

AgÃncia Nacional do PetrÃleo
CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior
In this work, the ethanol production from cashew bagasse was studied after acid followed by alkali pretreatment (CAB-OH) using the Separate Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF) processes. In SHF process, the hydrolysate obtained from enzymatic hydrolysis of CAB-OH was used as carbon source for fermentation with different strains of Saccharomyces (S. cerevisiae CCA008, S. cerevisiae 01, S. cerevisiae 02 and Saccharomyces sp. 1238), Kluyveromyces (K. marxianus CCA510, CE025 and ATCC36907) and Hanseniaspora sp. GPBio03. The bioprocess was conducted at 30 ÂC and 50 g.L-1 initial glucose concentration. The K. marxianus ATCC36907 achieved ethanol concentration of 20 g.L-1 with consumption of all glucose in the hydrolysate. Similar results were obtained with Saccharomyces strains and higher ethanol concentration (23.43 g.L-1) was obtained by Saccharomyces sp. 1238. The maximum ethanol concentration of 24.54 g.L-1 was achieved by Hanseniaspora sp. GPBio03. Focused on further studies using SSF process, it was evaluated the temperature influence of thermotolerant yeast K. marxianus ATCC36907 in glucose and enzymatic hydrolysate from CAB-OH. The results showed that the temperature (30, 35, 40, 45 and 50 ÂC) did not affect the values of YE/G (0.45 to 0.46 gethanol/gglucose) using glucose as substrate. Moreover, the ethanol yields obtained with enzymatic hydrolysate were slightly influenced by temperature, 0.39 and 0.43 gethanol/gglucose were obtained at 30 and 40 ÂC, respectively. Based on this, the SSF of CAB-OH and K. marxianus ATCC36907 was conducted at 40 ÂC with cellulases from Celluclast 1.5L at 15 FPU/gcellulose. The highest ethanol concentration (24.90 Â 0.89 g.L-1) was obtained with 76h of fermentation with 0.33 g.L-1.h-1, 0.34 gethanol/gglucose and 66.3% of productivity, YʹE/G and of ethanol efficiency, respectively. In enzymatic hydrolysis studies, the cellulase NS 22074 at 30 FPU/gcellulose without cellobiases supplementation resulted in glucose yield of 93.77 Â 2.72% which is promising for studies of SSF with this enzyme complex. The temperature (40, 42 , 45 and 50 ÂC) influence in SSF process using microcrystalline cellulose, in contrast with SHF results, higher ethanol concentration, 19.86 Â 0.32 g.L-1, was obtained at 40 ÂC. The SSF using CAB-OH, 30 FPU/gcellulose cellulases NS 22074 at 40 ÂC showed higher ethanol concentration of 37.35 Â 0.64 g.L-1 at 80h, with productivity of 0.46 g.L-1.h-1. In this condition, there was an increase of YʹE/G from 0.34 to 0.49 gethanol/gglucose and the ethanol efficiency from 66.3% to 95.59% when compared to results obtained with SSF using Celluclast 1.5L. Based on the results of efficiency and ethanol yield (YʹE/G), the cashew apple bagasse showed as lignocelulose feedstock promising material for second generation ethanol production by SSF process using the yeast K. marxianus ATCC36907 and NS 22074 cellulases complex.
Nesse trabalho, estudou-se a produÃÃo de etanol de bagaÃo de caju apÃs prÃ-tratamento Ãcido seguido de Ãlcali (CAB-OH) atravÃs dos processos de FermentaÃÃo e HidrÃlise Separadas (SHF) e FermentaÃÃo e HidrÃlise SimultÃneas (SSF). No processo SHF, o hidrolisado obtido da hidrÃlise enzimÃtica de CAB-OH foi submetido à etapa de fermentaÃÃo com diferentes linhagens de Saccharomyces (S. cerevisiae CCA008, Saccharomyces sp. 1238, S. cerevisiae 01, S. cerevisiae 02), Kluyveromyces (K. marxianus CCA510, CE025 e ATCC36907) e Hanseniaspora sp. GPBio03. A fermentaÃÃo do hidrolisado foi conduzida a 30 ÂC com concentraÃÃo inicial de glicose de 50 g.L-1. ApÃs o screening de leveduras, a linhagem de K. marxianus ATCC36907 destacou-se com maior concentraÃÃo de etanol de 20 g.L-1 com consumo de toda glicose no hidrolisado. Resultados similares foram obtidos com Saccharomyces sp. 1238 e com a levedura isolada do caju (Hanseniaspora sp. GPBio03) com maiores concentraÃÃes de etanol de 22,41 g.L-1 e 24,54 g.L-1, respectivamente. Com o propÃsito de estudos posteriores de SSF, avaliou-se a influÃncia da temperatura da levedura termotolerante K. marxianus ATCC36907 em glicose PA e hidrolisado enzimÃtico de CAB-OH. Os resultados mostraram que para a glicose PA, a variaÃÃo da temperatura (30, 35, 40, 45 e 50 ÂC) nÃo influenciou nos valores de conversÃo de glicose em etanol (YE/G) obtendo-se valores na faixa de 0,45-0,46 getanol/gglicose. Por outro lado, os resultados de YE/G em hidrolisado enzimÃtico foram ligeiramente influenciados pela temperatura, obtendo-se 0,39 getanol/gglicose a 30ÂC e 0,43 getanol/gglicose a 40 ÂC. Em seguida, realizou-se a SSF de CAB-OH com K. marxianus ATCC36907 a 40 ÂC e celulases de Celluclast 1.5L a 15 FPU/gcelulose. A maior concentraÃÃo de etanol (24,90  0,89 g.L-1) foi obtida em 76h de fermentaÃÃo com produtividade de 0,33 g.L-1.h-1, conversÃo de glicose em etanol (YʹE/G) de 0,34 e eficiÃncia de produÃÃo de etanol de 66,3%. Contudo, visando aumentar a produÃÃo de etanol em estudos posteriores de SSF, realizou-se o estudo de hidrÃlise enzimÃtica com outros complexos de celulases (NS 22074) e celobiases (NS 50010). Os resultados de hidrÃlise enzimÃtica mostraram que a atividade de celulases NS 22074 a 30 FPU/gcelulose sem suplementaÃÃo de celobiase resultou no rendimento de glicose de 93,77  2,72% sendo resultado promissor para estudos de SSF com esse complexo enzimÃtico. Nos ensaios de SSF com celulases do complexo NS 22074, inicialmente realizou-se o estudo da temperatura (40, 42, 45 e 50 ÂC) com K. marxianus ATCC36907 utilizando celulose microcristalina; e, em contrapartida com os resultados SHF, na temperatura de 40 ÂC foi obtida a maior concentraÃÃo de etanol de 19,86  0,32 g.L-1, em 72h de fermentaÃÃo. Diante desses resultados, realizou-se o processo de SSF de CAB-OH nas seguintes condiÃÃes: 40 ÂC de temperatura e 30 FPU/gcelulose do complexo de celulases NS 22074. A maior concentraÃÃo de etanol (37,35  0,64 g.L-1) foi obtida em 80h de fermentaÃÃo, com produtividade de 0,46 g.L-1.h-1. Diante desses resultados, observa-se que a mudanÃa do complexo enzimÃtico de Celluclast 1.5L para NS 22074 proporcionou o aumento no valor de YʹE/G de 0,34 getanol/gglicose para 0,49 getanol/gglicose e no rendimento de etanol de 66,3% para 95,59%, o que torna o bagaÃo de caju prÃ-tratado promissor como matÃria-prima para produÃÃo de etanol de segunda geraÃÃo por processo SSF utilizando a levedura K. marxianus ATCC36907.

The diploma thesis is focused on production possibilities of bioethanol from waste paper by yeast Kluyveromyces marxianus. Waste cardboard was used as a potential substrate for bioethanol production. Several methods for cardboard preparation were introduced and compared as well as methods of fermentation. Simultaneous sacharification and fermentation and separate hydrolysis and fermentation of preprepared cardboard paper were performed in different pH buffer (4,8-7). Simultaneous sacharification and fermentation was held at a temperature of 45°C. Hydrolysis in separate hydrolysis and fermentation was performed at 50°C and fermentation at 25°C. Procedures outputs were obtained by sampling in specific time intervals and samples were analyzed by HPLC for presence and concentration glucose and ethanol. The results of the analysis have shown that the highest concentration of glucose produced by enzymatic hydrolysis was achieved by using microwaves, 2% H2SO4 and 2% NaOH pretreated paperboard at pH 4,8. The highest yield of ethanol was obtained by separate hydrolysis and fermentation of pulp pretreated by microwaves, 2% H2SO4 and 2% NaOH in pH 5,4 buffer. The method SHF proved to be more effective for the production of ethanol than SSF.

Thesis (MScEng)--Stellenbosch University, 2012
ENGLISH ABSTRACT: Through many years of research, a number of production schemes have been developed for converting lignocellulosic biomass into transport fuels. These technologies have been assessed through a number of techno-economic studies for application in a particular context in terms of the technical and economic feasibility. However, previous studies using these methods have tended to lack vigour in various aspects. Either the energy efficiency of the processes were not maximised through adequate heat integration, or a competing technology which existed was not considered. From an economic perspective, the financial models would often lack the vigour to account for the risk and uncertainty that is inherent in the market prices of the commodities. This phenomenon is especially relevant for the biofuel industry that faces the full fledge of uncertainties experienced by the agricultural sector and the energy sector. Furthermore, from an environmental perspective, the techno-economic studies had often ignored the environmental impacts that are associated with biofuel production. Thus, a comparative study could have favoured an option due to its economic feasibility, while it could have had serious environmental consequences. The aim of this study was to address these issues in a South African context, where biofuels could be produced from sugarcane bagasse. The first step would be to modify an existing simulation model for a bioethanol scenario that operates with a Separate Hydrolysis and Fermentation (SHF process) configuration into a second processing scenario that operates with a Simultaneous Saccharification and Fermentation (SSF process) configuration using reliable experimental data. The second step was to ensure that the maximum energy efficiency of each scenario was realised by carrying out pinch point analysis as a heat integration step. In contrast to these biological models is the thermochemical model that converts bagasse to gasoline and diesel via gasification, Fischer-Tropsch synthesis and refining (GFT process). While there were no significant advances in technology concerning this type of process, the energy efficiency was to be maximised with pinch point analysis. The GFT process obtained the highest energy efficiency of 50.6%. Without the affects of pinch point technology, the efficiency dropped to 46%, which thus emphasises the importance of heat integration. The SSF had an efficiency of 42.8%, which was superior to that of the SHF at 39.3%. This resulted from a higher conversion of biomass to ethanol in the SSF scenario. Comparing the SHF model to an identical model found in literature that did not have pinch point retrofits, this study showed lower efficiency. This arose because the previous study did not account for the energy demands of the cold utility systems such as the cooling tower operation, which has been shown in this study to account for 40% of the electrical energy needs. The economic viability of all three processes was assessed with Monte Carlo Simulations to account for the risks that the fluctuations in commodity prices and financial indices pose. This was accomplished by projecting the fluctuations of these parameters from samples of a historical database that has been transformed into a probability distribution function. The consequences were measured in terms of the Net Present Value (NPV) and Internal Rate of Return (IRR) for a large number of simulations. The results of these variables were aggregated and were then assessed by testing the probability that the NPV<0, and that the IRR recedes below the interest rate of 12.64%. The investment was thus deemed unfeasible if these probabilities were greater than 20%. Both biological models were deemed profitable in terms of this standard. The probabilities were 13% for the SSF and 14% for the SHF. The GFT process however was deemed completely unfeasible because the probability that the NPV<0 was 78%. Given that the GFT process had the highest energy efficiency, this result arises mainly because the capital investment of 140,000USD/MWHHV of biomass energy input is to enormous for any payback to be expected. The environmental footprint of each process was measured using Life Cycle Assessments (LCAs). LCAs are a scientifically intricate way of quantifying and qualifying the effects of a product or process within a specified boundary. The impacts are assessed on a range of environmental issues, such as Global Warming, Acidification, Eutrophication and Human toxicity. Furthermore, if the project under concern has multiple output products, then the impacts are distributed between the output products in proportion to the revenue that each generates. The impacts were either relative to the flow of feedstock, which was 600MW of bagasse, or to the functional unit, which was the amount of fuel required to power a standard vehicle for a distance of 1 kilometre. In either case, the GFT scenario was the least burdening on the environmental. This was expected because the GFT process had the highest energy efficiency and the process itself lacked the use of processing chemicals. Relative to the feedstock flow, the SSF was the most environmentally burdening scenario due to the intensive use of processing chemicals. Relative to the functional unit, the SHF was the most severe due to its low energy efficiency. Thus, the following conclusions were drawn from the study:  The GFT is the most energy and environmentally efficient process, but it showed no sign of economic feasibility. iv  There is no significant difference in the economic and environmental evaluation of the SSF and SHF process, even though the SSF is considered to be a newer and more efficient process. The major cause of this is because the setup of the SSF model was not optimised.
AFRIKAANSE OPSOMMING: Deur baie jare van navorsing is ‘n aantal produksie-skemas vir die omskakeling van lignosellulose biomassa na vloeibarebrandstof ontwikkel. Hierdie tegnologië is geassesseer ten opsigte van die tegniese en ekonomiese haalbaarheid deur middel van tegno-ekonomiese studies in bepaalde tekste. Tog het hierdie vorige studies besliste beperkings gehad. Of die energie-doeltreffendheid van die proses is nie gemaksimeer deur voldoende hitte-integrasie nie, of 'n mededingende tegnologie wat bestaan is nie oorweeg nie. Vanuit 'n ekonomiese perspektief, was die finansiële modelle dikwels nie die omvattend genoeg om rekening te hou met die risiko en onsekerheid wat inherent is in die markpryse van die kommoditeite nie. Hierdie verskynsel is veral relevant vir die biobrandstof bedryf wat die volle omvang van onsekerhede ervaar waaraan die landbousektor en die energiesektoronderhewig is. Verder het die tegno-ekonomiese studies dikwels die omgewingsimpakte wat verband hou met biobrandstofproduksie geïgnoreer. Dus kon ‘n opsie deur die ekonomiese haalbaarheid bevoordeel word, ten spyte van die ernstige omgewingsimpakte wat dit kon inhou. Die doel van hierdie studie was om hierdie kwessies aan te spreek in 'n Suid-Afrikaanse konteks, waar biobrandstof uit suikerriet bagasse geproduseer kan word. Die eerste stap was om 'n bestaande simulasiemodel vir 'n bio-scenario wat met Afsonderlike Hidroliese en Fermentasie (SHF proses) stappe werk, te modifiseer vir 'n tweede verwerking scenario wat met 'n gelyktydige Versuikering en Fermentasie (SSF proses) konfigurasie werk. Die verandering is gedoen deur die gebruik van betroubare eksperimentele data. Die tweede stap was om te verseker dat elke scenario die maksimum energie-doeltreffendheid het, deur 'n hitte-integrasie stap, wat gebruik maak van “pinch-point” analise. In teenstelling met hierdie biologiese modelle, is daar die thermochemiese roete waar petrol en diesel van bagasse vervaardig word via vergassing, Fischer-Tropsch-sintese en rafinering (GFT proses). Daar was geen betekenisvolle vooruitgang in tegnologie vir hierdie proses nie, maar die energie-doeltreffendheid is gemaksimeer word deur energie-integrasie. Die GFT proses toon die hoogste energie-doeltreffendheid van 50,6%. Sonder die invloed van energie-integrasie het die doeltreffendheid gedaal tot 46%, wat dus die belangrikheid van hitte-integrasie beklemtoon. Die SSF het 'n effektiwiteit van 42,8% gehad, wat beter was as dié 39,3% van die SHF opsie. Hierdie hoër effektiwiteit wasas gevolg van die hoër omskakeling van biomassa na etanol in die SSF scenario. Die energie doeltreffendheid vir die SHF-model was laer as met 'n identiese model (sonder energie-integrasie) wat in die literatuur gevind wat is. Dit het ontstaan omdat die vorige studie nie 'n volledig voorsiening gemaak het met die energie-eise van die verkillingstelselsnie, wat tot 40% van die elektriese energie behoeftes kan uitmaak. Die ekonomiese lewensvatbaarheid van al drie prosesse is bepaal met Monte Carlo simulasies om die risiko's wat die fluktuasies in kommoditeitspryse en finansiële indekse inhou, in berekening te bring. Hierdie is bereik deur die projeksie van die fluktuasies van hierdie parameters aan die hand van 'n historiese databasis wat omskep is in 'n waarskynlikheid verspreiding funksie. Die gevolge is gemeet in terme van die netto huidige waarde (NHW) en Interne Opbrengskoers (IOK) vir 'n groot aantal simulasies. Die resultate van hierdie veranderlikes is saamgevoeg en daarna, deur die toets van die waarskynlikheid dat die NPV <0, en dat die IRR laer as die rentekoers van 12,64% daal, beoordeel. Die belegging is dus nie realiseerbaar geag as die waarskynlikhede meer as 20% was nie. Beide biologieseprosesse kan as winsgewend beskou word in terme van bostaande norme. Die waarskynlikhede was 13% vir die SSF en 14% vir die SHF. Aangesien die NHW van die GFT-proses onder 0 met ‘n waarskynlikheid van 78% is, is die opsie as nie-winsgewend beskou. Gegewe dat die GFT-proses die hoogste energie-doeltreffendheid het, is die resultaat hoofsaaklik omdat die kapitale belegging van 140,000 USD / MWHHV-biomassa energie-inset te groot is, om enige terugbetaling te verwag. Die omgewingsvoetspoor van elke proses is bepaal deur die gebruik van Lewens Siklus Analises (“Life Cycle Assessments”) (LCAS). LCAS is 'n wetenskaplike metodeom die effek van ‘n produk of proses binne bepaalde grense beide kwalitatief en kwantitatief te bepaal. Die impakte word beoordeel vir 'n verskeidenheid van omgewingskwessies, soos aardverwarming, versuring, eutrofikasie en menslike toksisiteit. Voorts, indien die projek onder die saak verskeie afvoer produkte het, word die impakte tussen die afvoer produkte verdeel, in verhouding tot die inkomste wat elkeen genereer. Die impak was met of relatief tot die vloei van roumateriaal (600MW van bagasse), of tot die funksionele eenheid, wat die hoeveelheid van brandstof is om 'n standaard voertuig aan te dryf oor 'n afstand van 1 kilometer. In al die gevalle het die GFT scenario die laagste belading op die omgewing geplaas. Hierdie is te verwagte omdat die GFT proses die hoogste energie-doeltreffendheid het en die proses self nie enige addisionele chemikalieë vereis nie. Relatief tot die roumateriaal vloei, het die SSF die grootse belading op die omgewing geplaas as gevolg van die intensiewe gebruik van verwerkte chemikalieë. Relatief tot die funksionele eenheid, was die SHF die swakste as gevolg van sy lae energie-doeltreffendheid.

Research about resource recovery from complex waste streams is getting an increased scientific attention since valuable resources can be produced by sustainable biological means. In anaerobic degradation processes, resources such as volatile fatty acids (VFAs) and biogas are highly coveted. One of the key parameters affecting the yield of resources is the hydrolytic efficiency in the waste stream by hydrolytic bacteria. The aim of this study was to examine how bioaugmentation can be implemented as a strategy to enhance hydrolysis in complex waste streams. In pursuit of this aim, three selected species of hydrolytic bacteria, Bacteroides thetaiotaomicron, Bacteroides amylophilus and Bacteroides ruminicola were inoculated both in pure culture combinations and bioaugmented with granular sludge as mixed culture in reactors. The studied waste stream was food waste mixed with primary sludge collected from Henriksdals wastewater treatment plant at Stockholm, Sweden.  The highest hydrolytic efficiency (90%) was reached by the pure culture fermented reactor inoculated with Bacteroides thetaiotaomicron and Bacteroides ruminicola. This efficiency was measured at day 10 after reactor set-up. Among the bioaugmented reactors, highest hydrolytic activity (66%) was achieved by the reactor inoculated with Bacteroides thetaiotaomicron and it was measured at day 10. The increase in hydrolytic efficiency for bioaugmented reactors was slower compared to pure culture fermented reactors and the most probable reason to that is due to competition amongst introduced species and pre-existing mixed culture in granular seed sludge.
Mer uppmärksamhet riktas till forskning kring resursåtervinning från komplexa avfallsströmmar eftersom värdefulla resurser kan produceras genom mer hållbara biologiska tillvägagångssätt. I anaeroba nedbrytningsprocesser är produkter såsom flyktiga fettsyror (VFAs) och biogas mycket eftertraktade. En av huvudparametrarna som påverkar utbytet av återvunna resurser är den hydrolytiska effektiviteten i avfallsströmmen av hydrolytiska bakterier. Syftet med studien var att undersöka hur bioaugmentering kan implementeras som strategi för att förstärka hydrolys i komplexa avfallsströmmar. Därav utfördes fermentering med tre valda hydrolytiska bakterier, Bacteroides thetaiotaomicron, Bacteroides amylophilus och Bacteroides ruminicola både i renkultur och bioaugmenterat med granulärt slam som mixad kultur i reaktorer. Avfallsströmmen som studerades var matavfall mixat med primärt slam hämtat från Henriksdals vattenreningsverk i Stockholm, Sverige.  Högsta hydrolytiska effektivitet (90%) uppnåddes för reaktorn inokulerat med Bacteroides thetaiotaomicron och Bacteroides ruminicola i renkultur. Denna effektivitet uppmättes dag 10 efter reaktorerna sattes upp. För de bioaugmenterade reaktorerna så uppnåddes högsta hydrolytiska effektivitet (66%) dag 10 av reaktorn inokulerat med Bacteroides thetaiotaomicron. Ökningen i hydrolytisk effektivitet var långsammare för de bioaugmenterade reaktorerna jämfört med reaktorerna med renkultur. Den mest sannolika förklaringen till det är tävling om näringsämnen och vitaminer mellan introducerade bakterier och de bakterier som redan existerar i det granulära slammet.

Due to the depletion of petroleum reserves and environmental concerns, bioethanol has been identified as an alternative fuel to petrol. Bioethanol is a fuel of bio-origin derived from renewable biomass. Starch and sugar containing materials are the primary sources of carbon for bioethanol production. Starch is firstly hydrolysed into simple sugars which are later fermented to bioethanol using Saccharomyces cerevisiae (S. cerevisiae). The fermentation of sugars to bioethanol is however limited by inhibition of S. cerevisiae by the major product of the process, bioethanol. The challenge is thus in keeping the bioethanol concentration at levels which are not harmful to the fermenting organism. Keeping bioethanol concentration low in the broth will provide a suitable environment for yeast to grow and thus increase the overall production. Currently bioethanol producers use high water dilution rates to keep the bioethanol concentrations in the broth low enough so that yeast is not harmed. This excess water has to be removed in the downstream process, which is expensive. The use of excessive amounts of water in the fermentation can be avoided by continual removal of bioethanol from the broth. During this investigation the experimental conditions for the hydrolysis process were determined. A pH of 5.5 was determined as the best pH for Termamyl SC at 95°C with a pH of 5.0 for Spirizyme Fuel at 55°C during the liquefaction and the saccharification step, respectively. During the fermentation process the influence of yeast concentration on bioethanol production was investigated by varying the yeast concentration between 2 g.L-1 and 7 g.L-1. A yeast concentration of 5 g.L-1 produced the highest bioethanol yield of 0.48 g.g-1 after 48 hours of fermentation using S. cerevisiae. Later during the investigation a coupled fermentation/pervaporation system was employed in a batch system for continual removal of bioethanol in the fermentation broth in a process called simultaneous fermentation and separation (SFS). Through the continuous removal of bioethanol from the fermentation broth, the bioethanol concentration in the broth was kept low enough so that it was not harmful to the fermenting organism but the overall fermentation yield was not improved. Pervaporation is a membrane separation process used to separate azeotropic mixtures such as bioethanol and water. It is highly efficient, cost effective and uses less energy than distillation. During the SFS process a bioethanol yield of 0.22 g.g-1 was obtained. The SFS process yield for bioethanol was low compared to 0.45 g.g-1 of the traditional batch fermentation process. The lower overall bioethanol yield obtained in the SFS process could be attributed to only the supernatant being used in the SFS process and not the entire fermentation broth as in the traditional process. The results from this study proved that the SFS process was less efficient compared to the traditional batch fermentation process with respect to the bioethanol yield, but that the fermentation could be carried out without the necessity for additional process water.
Thesis (M.Sc. Engineering Sciences (Chemical and Minerals Engineering))--North-West University, Potchefstroom Campus, 2010.

Efficient processing of cassava roots by wet milling requires overcoming challenges associated with disaggregation of the starch-containing parenchyma cells. These cells entrap starch granules and hinder their release during wet milling. Steeping of ground cassava in 0.75% (w/v) NaOH in combination with wet milling was investigated to determine whether and how dilute NaOH modifies cassava cell walls. Gas chromatography (GC) data of cell wall constituent sugar composition and Fourier transform infrared (FTIR) data showed that NaOH steeping caused solubilisation of the cell wall pectin fraction. FTIR and wide-angle x-ray scattering (WAXS) spectroscopy indicated that NaOH steeping combined with fine (500 ?m opening screen size) wet milling reduced cellulose crystallinity. Dilute NaOH steeping also produced pits (micropores) through the cell wall structure as shown by scanning electron microscopy (SEM). The micropores seemed to have weakened the cell walls, as revealed by increased cellular disaggregation as viewed by light microscopy. Disaggregation of cassava root cells was associated with a reduction in large (diameter > 250 ?m) residue particle size in the bagasse and consequently more starch yield. Thus, it seems that mechanistically, dilute NaOH solubilisation of alkaline-soluble pectin weakens the cell walls of starch-containing cassava root parenchyma cells. Weakening of cassava cell walls with a combination of biological (14 day endogenous fermentation under microaerophilic conditions) and dilute alkaline pre-treatment (0.75% NaOH steeping) was investigated in an attempt to further increase starch yield by wet milling. However, the combined pre-treatment resulted in approx. 11.8% more starch yield, slightly less than the 12.3% increase obtained by using endogenous fermentation alone. The absence of an additive effect was probably because although endogenous fermentation (retting) and dilute NaOH steeping weakened cassava cell walls through different mechanisms (hydrolysis/solubilisation of pectin), the resultant loss in pectin cohesiveness was similar. Solid state fermentation of ground cassava using various alkaliphilic Bacillus spp. starter cultures separately and in combination was also investigated to determine their extracellular hydrolytic enzyme induced weakening effects on parenchyma cell walls. GC and FTIR data indicated that fermentation with Bacillus akibai + endogenous microflora (EM), B. cellulosilyticus + EM, B. hemicellulosilyticus + EM and B. spp. in combination + EM caused reduction in cell wall pectin, xyloglucan and cellulose contents. Cell wall solubilisation/hydrolysis seemed to have primarily involved the amorphous constituents, as indicated by an increase in cellulose crystallinity by WAXS spectroscopy. Enzyme assay and SEM indicated that Bacillus spp. extracellular cellulase and polygalacturonase weakened the cell walls through formation of micropores and possible rupturing of cellulose microfibril structures. These modifications seemed to have aided disaggregation of the cassava parenchyma cells and consequently liberation of more starch granules as indicated by light microscopy. Fermentation with B. akibai + EM, B. cellulosilyticus + EM, B. hemicellulosilyticus + EM and B. spp. in combination + EM also resulted in less large (diameter > 250 ?m) residue particle size in the bagasse and consequently higher starch yield. Thus, dilute NaOH steeping and fermentation with alkaliphilic Bacillus spp. starter cultures are techniques capable of improving the effectiveness of wet milling in disintegrating cassava cell walls. However, with regard to the demand for environmentally cleaner production, potential utilisation of alkaliphilic Bacillus spp. and more specifically Bacillus cellulosilyticus appears more promising.
Thesis (PhD)--University of Pretoria, 2017.
Food Science
PhD
Unrestricted

Thesis (PhD)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: The development of ―energycane‖ varieties of sugarcane for ethanol production is underway, targeting the use of both sugar juice (first generation ethanol) and bagasse (second generation ethanol). Nevertheless, identification of the preferred varieties represents the biggest challenge to the development of energycane due to large number of samples produced during breeding. In the present study, dilute acid pretreatment, enzymatic hydrolysis and fermentation processes were used to evaluate the processability of bagasse (fibrous residue generated after juice sugar extraction) from different varieties of sugarcane to select preferred varieties with the properties of improving combined ethanol yield (ethanol from juice and bagasse) per hectare. The impact of variety selection on combined ethanol yield (ethanol from juice and bagasse) per hectare was also assessed. In the first part of this study, 115 varieties of sugarcane originated from classical breeding and precision breeding (genetic engineering) were screened based on agronomic data and experimental data from biochemical processes (dilute acid pretreatment and enzymatic hydrolysis) applied to the bagasse fraction of each variety. The results showed wide variations in the chemical composition of bagasse between the varieties. Structural carbohydrates and lignin content ranged from 66.6 to 77.6% dry matter (DM) and 14.4 to 23.1% DM, respectively. The majority of precision breeding varieties showed higher arabinoxylan, lower lignin and lower ash content than most of classical breeding varieties. Combined sugar yield from the bagasse after pretreatment and enzymatic hydrolysis also varied significantly among the varieties. Up to 27.9 g/100g (dry bagasse) difference in combined sugar yield was observed. Combined sugar yield was inversely correlated with lignin as well as ash content, but it correlated positively with structural carbohydrates content. Total potential ethanol yields per hectare, calculated based on cane yield, soluble and non-soluble sugar content also differed significantly among the varieties (8,602−18,244 L/ha). Potential ethanol from bagasse contributed approximately one third of the total potential ethanol yield. Interestingly, some of the varieties had combined properties of high potential ethanol yield per hectare and improved bagasse convertibility. Thus, six varieties (3 from each breeding technology) were selected as preferred varieties for further investigation. To enhance sugar yield from bagasse, optimisation of pretreatment was conducted on the selected varieties. Industrial bagasse was included for comparison purposes. The pretreatment optimisation was based on maximising combined sugar yield from the combined pretreatment-hydrolysis process. A central composite design (CCD) was applied to investigate the effects of temperature, acid concentration and residence time on the responses and was later used to determine the maximum combined sugar yield. Pretreatment optimisation was conducted at gram scale (22.9 ml reactor) and at bench scale (1000 ml reactor). Significant differences in sugar yields (xylose, glucose, and combined sugar) between the varieties were observed. The combined sugar yields from the best performing varieties and industrial bagasse at optimal pretreatment-hydrolysis conditions differed by up to 34.1% and 33% at gram and bench scale, respectively. A high ratio of carbohydrates to lignin and low ash contents increased the release of sugar from the substrates. At mild pretreatment conditions, the differences in bioconversion efficiency between varieties were greater than at severe conditions. This observation suggests that under less severe conditions the conversion efficiency was largely determined by the properties of the biomass. Furthermore, it was demonstrated that the pretreatment conditions with temperature ranged from 184 to 200 °C and varying residence time to provide a severity factor between 3.51 and 3.96 was observed to be the area in common where 95% of maximum combined sugar yield could be obtained. Simultaneous Saccharification and Fermentation (SSF) was performed on the unwashed pressed-slurry from bagasse pretreatment at conditions for maximum combined sugar yield at bench scale. Batch and fed-batch SSF feeding strategy at different solid loadings and enzyme dosages were used aiming to reach an ethanol concentration of at least 40 g/L. The results revealed significant improvement in overall ethanol yield after SSF for the selected varieties (84.5–85.6%) compared to industrial bagasse (74.8%). The maximum ethanol concentration from the best performing varieties was 48.6−51.3 g/l and for poor performing varieties was 37.1−38.3 g/l. Ethanol concentration in the fermentation broth was inversely correlated with lignin content and the ratio of xylose to arabinose, but it showed positive correlation with glucose yield from pretreatment-enzymatic hydrolysis. The overall assessment of the varieties showed greater improvement in combined ethanol yields per hectare (71.1–90.7%) for the best performing varieties with respect to industrial sugarcane. The performance in terms of ethanol yields of selected varieties from a number harvest years was evaluated. The results showed considerable variations in ethanol yields across harvests. The results showed that the best variety in terms combined ethanol yield was not maintained across harvests. The differences in ethanol yields were greater among the varieties than across the harvests. Prolonged severe drought significantly affected the ethnol yields of all varieties represented by lower and intermediate lignin content for cane yield compared to that which had highest lignin content. However, carbohydrates content in the bagasse and sugar yield/recovery between the harvest years did not change for the most of the varieties. In summary, the present study provides evidence of the impact of cultivar selection and pretreatment optimisation in increasing conversion efficiency of bagasse. The results demonstrate that varieties with lower lignin and ash content, as well as highly substituted xylan resulted in higher sugar and ethanol yields. These results suggest that lower process requirements can be achieved without adversely affecting juice ethanol and cane yield per hectare. Nonetheless, an attempt to reduce lignin content in the bagasse, to reduce processing requirements for ethanol production, can also target the improvement of crop tolerance toward severe drought conditions.
AFRIKAANSE OPSOMMING: Die ontwikkeling van ―energie-riet‖ rasse vir etanol produksie is goed op dreef, waar beide die sap (eerste generasie etanol) en die bagasse (tweede generasie etanol) geteiken word. Die groot aantal monsters wat tydens teling geproduseer word, bied egter die grootste uitdaging vir die identifisering van nuwe rasse ten einde energie-riet te ontwikkel. In die huidige studie is verdunde suurvoorbehandeling, ensiematiese hidrolise en fermentasie-prosesse gebruik om die verwerkbaarheid van bagasse (veselagtige residu gegenereer na sap suiker ekstraksie) van verskillende suikerrietrasse te evalueer om nuwe variëteite te selekteer wat eienskappe van verbeterde gekombineerde etanolopbrengs (etanol van sap en bagasse) per hektaar toon. Die impak van variëteit-seleksie op gekombineerde etanol opbrengs (etanol van sap en bagasse) per hektaar is ook beoordeel. In die eerste deel van hierdie studie het uit ‗n siftingsproses van 115 suikerriet rasse bestaan wat deur klassieke en presisie (geneties gemodifiseerde) teling gegenereer is. Die sifting was op agronomiese data gebaseer, asook op data van verdunde suur voorafbehandeling en ensimatiese hidrolise eksperimente wat op die bagasse fraksie van elke ras uitgevoer is. Die resultate het op groot variasie in die chemiese samestelling van die bagasse van verskillende rasse gedui. Die strukturele koolhidrate het tussen 66.6 en 77.6% droë massa (DM) gewissel, terwyl die lignien inhoud ‗n variasie van 14.4 en 23.1% DM getoon het. Verder het meeste van die presisie-teling variëteite ‗n hoër arabinoxilaan, maar ‗n laer lignien en as-inhoud as meeste van die klassieke teling rasse gehad. Die gekombineerde suikeropbrengs (GSO) van die bagasse na voorafbehandeling en ensimatiese hidrolise het ook beduidend tussen rasse gewissel, waar ‗n verskil van tot 27.9 g/100g (droë bagasse) waargeneem is. Daar was ‗n omgekeerde korrelasie tussen die gekombineerde suikeropbrengs en die lignien en as-inhoud gewees, maar die opbrengs het ‗n sterk positiewe korrelasie met die strukturele koolhidrate getoon. Die totale potensiële etanol opbrengs per hektaar wat vanaf die suikerriet se oplosbare en nie-oplosbare suikerinhoud bereken is, het ook beduidend tussen rasse verskil (8,602−18,244 L/ha), waar die potensiële etanol opbrengs van die bagasse gedeelte ongeveer een derde van die totale potensiële etanol opbrengs beslaan het. Interessante bevindinge het op sommige rasse met gekombineerde eienskappe van hoë potensiële opbrengs per hektaar asook ‗n hoë omskakelingsvermoë gedui. Derhalwe is ses variëteite (drie van elke telingstegnologie) as voorkeurvariëteite vir verdere studie gekies. Om die etanol opbrengs vanaf die bagasse te verbeter was voorafbehandeling van die voorkeurvariëteite geoptimeer, en waar industriële bagasse vir vergelykingsdoeleindes ingesluit was. Vir die optimering was dit ten doel gestel om die gekombineerde suikeropbrengs van die gekombineerde voorafbehandeling-hidrolise proses te maksimeer. ‗n Sentrale saamgestelde ontwerp (SSO) is gebruik om die effek van temperatuur, suurkonsentrasie en residensietyd op die responsveranderlikes vas te stel wat uiteindelik gebruik is om die maksimum gekombineerde suikeropbrengs te bepaal. Die optimering van die voorafbehandeling is op gram-skaal in ‗n 22.9 ml reaktor, asook op bank-skaal in ‗n 1000 ml reaktor uitgevoer. Beduidende verskille in die suikeropbrengs (xilose, glukose en gekombineerde suiker) is tussen die voorkeurrasse waargeneem. Tussen die rasse wat die beste gevaar het, asook die industriële bagasse, het die gekombineerde suikeropbrengs by optimale voorafbehandeling-hidrolise toestande onderskeidelik met tot 34.1% en 33% op gram-skaal en bank-skaal gevarieer. ‗n Hoë verhouding van koolhidrate tot lignien, asook ‗n lae as-inhoud het tot ‗n toename in die vrystelling van suiker uit die substraat gelei. By matige voorafbehandelingstoestande was die verskille in omskakelingseffektiwiteit tussen rasse groter as onder hewige toestande, wat daarop gedui het dat omskakelingseffektiwiteit grotendeels deur die eienskappe van die biomassa bepaal is. Verder is daar ook gedemonstreer dat die voorbehandelingsomstandighede met temperatuur tussen 184 en 200ºC en verandering van die residensietyd om 'n hewigheidsfaktor van tussen 3.51 en 3.96 te verskaf, 'n gemeenskaplike area gelewer het waar 95% van maksimum gekombineer suiker opbrengs (GSO) verkry kon word. Gelyktydige versuikering en fermentasie (GVF) is na voorafbehandeling op ongewaste, gepersde bagasse substraat by toestande vir die maksimum gekombineerde suikeropbrengs op bank-skaal uitgevoer. Bondel en voerbondel SSF voerstrategie by verskillende vaste ladings en ensiemdoserings is gebruik om 'n etanol konsentrasie van ten minste 40 g/L te bereik. Ná GVF was die algehele etanol opbrengs vir die voorkeurvariëteite (84.5–85.6%) beduidend beter relatief tot die industriële bagasse (74.8%). Die maksimum etanol opbrengs na SSF van die rasse met die beste prestasie was 48.6-51.3 g/L en 37.1-38.3 g/L vir rasse wat swak presteer het. Die etanol konsentrasie in die fermentasiesop was omgekeerd met lignien en die verhouding van xilose tot arabinose gekorreleer, maar was duidelik positief met die glukose opbrengs vanaf voorafbehandeling-hidrolise gekorreleer. ‗n Algemene assessering het op ‗n duidelike verbetering van die voorkeurvariëteite in terme van gekombineerde etanol opbrengs per hektaar gedui (71.1–90.7%), relatief tot die industriële suikerriet. Die prestasie in terme van etanol opbrengs van geselekteerde variëteite is oor 'n reeks oesjare ge-evalueer. Die resultate het aansienlike variasies in etanol opbrengs oor oesjare getoon. Die resultate het gewys dat die beste variëteite in terme van gekombineerde etanol opbrengs nie volhou is oor oeste nie. Die verskille in etanol opbrengste tussen variëteite was groter as die verskille oor oesjare. Verlengde ernstige droogte het die etanol opbrengs van alle variëteite met laer en intermediere lignien inhoud vir rietopbrengs aansienlik beinvloed, in vergelyking met dié wat die hoogste lignien inhoud gehad het. Die koolhidraatinhoud in die bagasse en suiker opbrengs/lewering tussen die oesjare het vir die meeste variëteite egter nie gewissel nie. Ter opsomming, die huidige studie verskaf bewyse van die impak van kultivarseleksie en voorbehandelings optimisering op die verhoging van die omskakelings-doeltreffendheid van bagasse. Die resultate wys dat variëteite met laer lignien- en asinhoud, en hoogs-gesubstitueerde xilaan hoër suiker- en etanol opbrengs gelewer het. Hierdie resultate stel voor dat verminderde voorbehandelingsvereistes bereik kan word sonder om die sap etanol en rietopbrengs per hektar te benadeel. Nieteenstaande, 'n poging om die lignien inhoud van die bagasse te verminder om die verwerkingsvereistes vir etanolproduksie te verminder, kan ook die verbetering van gewas-toleransie tov ernstige droogte-toestande teiken.

The fields of biodiesel and bioethanol research and development have largely developed independently of one another. Opportunities exist for greater integration of these processes that may result in decreased costs of production for both fuels.To that end, this work addresses the use of the starches and glycerol from processed algal biomass as substrates for fermentation by the yeasts Saccharomyces cerevisiae and Pachysolen tannophilus, respectively. Ethanol producers commonly employ the former yeast for ethanol production and include the latter yeast among candidate species for cellulosic ethanol production.A simple 95% ethanol extraction at 70°C followed by sulfuric acid hydrolysis at 121°C and 2 atm proved a sufficient pretreatment for S. cerevisiae fermentation of starch from Chlamydomonas reinhardtii mutant cw15. The maximum rate of ethanol production was observed as 14 mL/g-h and a maximum concentration of 0.9±0.01% (m/v) was observed by 28 hours. Some starch appeared invulnerable to hydrolysis.P. tannophilus fermentation of glycerol, both independently and among mixed substrates, was likewise demonstrated. It was found that glucose consumption preceded that of glycerol and xylose, but that the latter two substrates were consumed concurrently. Under aerobic, batch conditions, the maximum specific growth rate of the species on a 2% glycerol substrate was observed as 0.04/hr and the yield coefficient for conversion of glycerol to ethanol was 0.07 g/g. While the maximum observed concentration of ethanol in the glycerol-only fermentation was 0.1% m/v, that in mixed media containing 2% each glucose, xylose, and glycerol was 1.5%.Also investigated here was the flocculation of a mutant species of the algae C. reinhardtii by a combination of methanol and calcium. Algae harvest is typically an energy-intensive process, but the technique demonstrated here is not. Complete flocculation of cells was observed with only 5 minutes of mixing and less than 10 minutes of settling using 12 mM CaCl₂ and 4.6% methanol. Ethanol was observed to operate in the same capacity, intimating another area in which yeast bioethanol and algal biodiesel processes might enable one another. During growth, either an inhibitor of flocculation was produced or a facilitator was consumed.

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O rizoma de gengibre é amplamente comercializado em função de seu emprego na medicina popular, na alimentação, industrial, especialmente como matéria-prima para fabricação de bebidas, perfumes e produtos de confeitaria como pães, bolos, biscoitos e geléias. Frente ao conteúdo de amido presente neste rizoma, uma possibilidade de incremento da cadeia produtiva do gengibre seria a produção de bebida destilada, a qual poderia ser obtida a partir de rizomas de baixa qualidade para exportação e/ou a partir do resíduo da extração dos óleos essenciais por indústrias processadoras deste rizoma. Este trabalho teve por objetivo geral a avaliação dos parâmetros técnicos do processamento de gengibre para produção de aguardente e, como objetivos específicos: caracterizar a composição centesimal dos rizomas de gengibre; avaliar a influência de alguns parâmetros dos processos de hidrólise e fermentação no rendimento em açúcares e etanol; e caracterizar o destilado quanto aos parâmetros de qualidade para aguardente. O trabalho foi dividido em quatro experimentos. O primeiro experimento objetivou comparar o efeito de duas α-amilases na etapa de liquefação, bem como, a influência do tempo de ação da amiloglucosidase na etapa de sacarificação, sobre o perfil de açúcares e rendimento do processo. O segundo experimento objetivou estabelecer qual a melhor concentração enzimática para o processo de hidrólise e sacarificação. O terceiro experimento teve por objetivo verificar o efeito do tempo de fermentação, concentração de levedura e temperatura de fermentação sobre o perfil cromatográfico do vinho obtido. A partir das conclusões parciais dos experimentos anteriores foi realizado o quarto ensaio que teve por objetivo produzir e caracterizar a aguardente...
The rhizome of ginger is widely marketed on the basis of its use in folk medicine, food, industrial, especially as raw material for the manufacture of beverages, perfumes and sugar confectionery such as breads, cakes, cookies and jellies. Facing the content of starch in this rhizome, an opportunity to increase the productive chain of ginger would be the production of distilled drink, which could be obtained from rhizomes of low quality for export and / or from residue from the extraction of oil key industries for processing this rhizome. This study aimed at evaluating the general technical parameters of the processing of ginger for the production of spirits, and as specific objectives: characterization of the proximate composition of the rhizomes of ginger to evaluate the quality and quantity of raw materials needed for the process of hydrolysis -saccharification; evaluate the influence of some parameters of the process of hydrolysis, saccharification and fermentation on yield of sugar and ethanol; and characterize the ginger spirit. The work was experiments. The first experiment aimed to compare the effect of two α-amylases in the stage of liquefaction, as well as, the influence of time of amiloglucosidase action in the saccharification step, on the profile of sugars and yield of the process. The second experiment aimed to establish the best concentration enzymes for the process of hydrolysis and saccharification. The third experiment aimed to verify the effect of time of fermentation, the yeast concentration and temperature of fermentation on the chromatographic profile of the wine obtained. The fourth experiment was conducted based on partial findings of previous experiments and aimed to produce and characterize the spirit of ginger. The results showed that the enzyme... (Complete abstract click electronic access below)

The current research investigates the utilization of cotton gin waste as a feedstock to produce a value-added product - fuel ethanol. Cotton gin waste consists of pieces of burs, stems, motes (immature seeds) and cotton fiber, and is considered to be a lignocellulosic material. The three main chemical constituents are cellulose, hemicellulose, and lignin. Cellulose and hemicellulose are polysaccharides of primarily fermentable sugars, glucose and xylose respectively. Hemicellulose also includes small fractions of arabinose, galactose, and mannose, all of which are fermentable as well. The main issue in converting cotton gin waste to fuel ethanol is the accessibility of the polysaccharides for enzymatic breakdown into monosaccharides. This study focused on the use of steam explosion as the pretreatment method. Steam explosion treatment of biomass has been previously described to increase cellulose accessibility. The governing factors for the effectiveness of steam explosion are steam temperature and retention times. The two factors are combined into a single severity term, log(Ro). Following steam explosion pretreatment, cotton gin waste was subjected to enzyme hydrolysis using Primalco basic cellulase. The sugars released by enzyme hydrolysis were fermented by a genetically engineered Escherichia coli (Escherichia coli KO11). The effect of steam explosion pretreatment on ethanol production from cotton gin waste was studied using a statistically based experimental design. The results obtained from this study showed that steam exploded cotton gin waste is a heterogeneous material. Drying and milling of steam exploded cotton gin waste was necessary to reduce variability in compositional analysis. Raw cotton gin waste was found to have 52.3% fermentable sugars. The fiber loss during the steam explosion treatment was high, up to 24.1%. Xylan and glucan loss from the pretreatment was linear with respect to steam explosion severity. Steam explosion treatment on cotton gin waste increased the hydrolysis of cellulose by enzyme hydrolysis. Following 24 hours of enzyme hydrolysis, a maximum cellulose conversion of 66.9% was obtained at a severity of 4.68. Similarly, sugar to ethanol conversions were improved by steam explosion. Maximum sugar to ethanol conversion of 83.1% was observed at a severity of 3.56. The conclusions drawn from this study are the following: steam explosion was able to improve both glucose yields from enzyme hydrolysis and ethanol yields from fermentation. However, when analyzed on whole biomass, or starting material basis, it was found that the fiber loss incurred during steam explosion treatment negated the gain in ethanol yield.
Master of Science

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Na busca por fontes de energia renováveis e alternativas que reduzam a emissão de gases nocivos ao meio ambiente, diminuindo assim o uso de combustíveis fósseis em face da conscientização global, os biocombustíveis são uma fonte alternativa de energia, sendo principal destaque o etanol de 1ª e 2ª geração. Objetivou-se neste trabalho avaliar o potencial de diferentes cultivares de sorgo doce (Sorghum bicolor (L.) Moench.) para produção de etanol de 1ª geração a partir da fermentação do caldo dos colmos e etanol de 2ª geração a partir do substrato (bagaço). Para obtenção dos açúcares fermentescíveis a partir do bagaço, utilizou-se pré-tratamento ácido seguido de básico com auxílio de uma matriz de planejamento experimental 2³+3 pontos centrais para avaliar os efeitos das variáveis de entrada, tempo, temperatura e concentrações de ácido e base, verificando a solubilização da hemicelulose e lignina. Para avaliar a hidrólise enzimática verificou-se, por meio da aplicação de uma matriz de planejamento experimental 2²+3 pontos centrais, o efeito da carga enzimática e a razão massa seca de bagaço de sorgo e extrato enzimático. Foram usadas as enzimas comerciais Celluclast 1.5L da Novozyme e betaglicosidase da Prozyn na hidrólise enzimática para obtenção de açúcares fermentescíveis. A pesquisa foi desenvolvida no laboratório de engenharia bioquímica – LEB, na Universidade Federal de Campina Grande – UFCG, no estado da Paráiba e no Instituto Agronômico de Pernambuco – IPA, ambas localizadas na região do nordeste Brasileiro, a colheita das progens foi realizada na Estação Experimental do IPA no município de Vitória de Santo Antão, localizado na Zona da Mata de Pernambuco (lat: 35° 22' W, long: 08° 08' S, altitude: 146 m) no período de março/2013 a setembro/2017. Os resultados referentes aos dados agronômicos apresentaram diferenças significativas para um intervalo de confiança de 95% pelo teste Tukey. Dentre as sete cultivares pesquisadas as que obtiveram maiores teores de sólidos solúveis totais em campo foram entre BRS506, IPA2502, IPA EP17, IPA P222, IPA P134, IPA P228 e IPA SF15 com valores entre 12,3±0,08; 5,1±0,10; 16,8±0,40; 14,8±0,11; 7,1±0,10; 9,2±0,10 e 13,6±0,27 ºBrix, respectivamente. Os resultados referentes a fração sacarina (caldo do sorgo sacarino) foram analisados usando o teste de Tukey para um intervalo de confiança de 95%. Nos dados encontrados para concentração de etanol de 1ª geração, obtidos na fermentação dos caldos das cultivares que se destacaram foram IPA SF15, BRS506, IPA EP17, IPA P222 e IPA P134 com valores entre 55,72 ± 0,24; 45,46 ± 1,5; 44,6 ± 0,18; 33,78 ± 0,57 e 31,78 ± 0,28 g.L-1 respectivamente. As frações (bagaço) para avaliação na produção de etanol de 2ª geração, as cultivares IPA EP17 e BRS506, após pré-tratamento ácido seguido de básico, mostraram-se eficientes em concentrar a celulose, pela solubilização da hemicelulose e lignina, acarretando um aumento da celulose de 31,70 ± 0,49 para 60,42 ± 0,91% na variedade IPA-EP17 e para variedade BRS506 de 40,6 ± 1,1 para 64,52 ± 0,82% respectivamente. Os resultados encontrados no processo de hidrólise enzimática dos bagaços das variedades IPA EP17 para produção de glicose foi de 26,23 g L-1, nas condições de carga enzimática e razão (relação massa seca de bagaço) por volume de meio reacional (25 FPU/g e 2%) no tempo de 36 h de hidrólise e rotação constante de 150 RPM, e para BRS506 a produção de glicose foi de 25,17 g L-1, nas condições de carga enzimática e razão (relação massa seca de bagaço) por volume de meio reacional (25 FPU/g e 2%) no tempo de 36 h de hidrólise.
In the search for renewable energy sources and alternatives that reduce the emission of harmful gases to the environment, thus reducing the use of fossil fuels in the face of global awareness, biofuels present as an alternative, the main highlight being ethanol. The objective of this work was to evaluate the potential of different cultivars of sweet sorghum (Sorghum bicolor (L.) Moench.) For the production of ethanol of 1st generation from the fermentation of the broth the 2st generation ethanol from the substrate (bagasse). To obtain the fermentable sugars from the bagasse, acid pretreatment followed by base with the aid of experimental planning 2³ + 3 central points was applied to evaluate the effects of the variables of entry, time, temperature and acid and base concentrations, verifying the solubilization of hemicellulose and lignin. In order to evaluate the enzymatic hydrolysis, the effect of the enzymatic loading and the dry mass ratio of sorghum bagasse and enzyme extract wereverified using experimental planning design 2² + 3 central points. The commercial enzymes Celluclast 1.5L from Novozyme and beta-glucosidase from Prozyn wereused in the enzymatic hydrolysis to obtain fermentable sugars. The research was carried out at the biochemical-engineering laboratory - LEB, at the Federal University of Campina Grande - UFCG, in the state of Paráiba and at the Agronomic Institute of Pernambuco - IPA, both located in the northeast region of Brazil. The progenies were collected at the IPA Experimental Station in the city of Vitória de Santo Antão, located in the Zona da Mata of Pernambuco (lat: 35 ° 22 'W, long: 08 ° 08' S, altitude: 146 m) from March / 2013 to September / 2017. The results concerning agronomic data presented significant differences for a 95% confidence interval by the Tukey test. Among the seven cultivars studied, the highest soluble solids in the field were BRS506, IPA2502, IPA EP17, IPA P222, IPA P134, IPA P228 and IPA SF15 with values between 12,3 ± 0,08; 5,1 ± 0,10; 16,8 ± 0,40; 14,8 ± 0,11; 7,1 ± 0,10; 9,2 ± 0,10 and 13,6 ± 0,27 °Brix, respectively. Results for saccharin fraction (sorghum broth) were analyzed using the Tukey's test for a 95% confidence interval. In the data obtained for the concentration of ethanol of 1st generation, obtained in the fermentation of the broths of the cultivars that stood out were IPA SF15, BRS506, IPA EP17, IPA P222 and IPA P134 with values between 55,72 ± 0,24; 45,46 ± 1,5; 44,6 ± 0,18; 33,78 ± 0,57 and 31,78 ± 0,28 g.L-1 respectively. The fractions (bagasse) for evaluation in the production of second generation ethanol, IPA EP17 and BRS506, after acid pretreatment followed by basic, were efficient in concentrating the cellulose by the solubilization of hemicellulose and lignin, resulting in an increase of cellulose from 31,70 ± 0,49 to 60,42 ± 0,91% in the variety IPA-EP17 and for the BRS506 variety from 40,6 ± 1,1 to 64,52 ± 0,82% respectively. The results obtained in the enzymatic hydrolysis process for the IPA EP17 variety in the glucose production were 26,23 g L-1, for enzyme loading conditions and ratio (bagasse dry mass ratio) per reaction medium volume (25 FPU / and 2%) in the time of 36 hours of hydrolysis and constant rotation of 150 RPM, for BRS506 variety the glucose production was 25,17 g.L-1, for the conditions of enzymatic loading and ratio (bagasse dry mass ratio) by volume of reaction medium (25 FPU / g and 2%) in the time of 36 hours of hydrolysis.

Orientador: Magali Leonel
Banca: Manoel Lima Menezes
Banca: Waldemar Gastoni Venturini |Filho
Resumo: O rizoma de gengibre é amplamente comercializado em função de seu emprego na medicina popular, na alimentação, industrial, especialmente como matéria-prima para fabricação de bebidas, perfumes e produtos de confeitaria como pães, bolos, biscoitos e geléias. Frente ao conteúdo de amido presente neste rizoma, uma possibilidade de incremento da cadeia produtiva do gengibre seria a produção de bebida destilada, a qual poderia ser obtida a partir de rizomas de baixa qualidade para exportação e/ou a partir do resíduo da extração dos óleos essenciais por indústrias processadoras deste rizoma. Este trabalho teve por objetivo geral a avaliação dos parâmetros técnicos do processamento de gengibre para produção de aguardente e, como objetivos específicos: caracterizar a composição centesimal dos rizomas de gengibre; avaliar a influência de alguns parâmetros dos processos de hidrólise e fermentação no rendimento em açúcares e etanol; e caracterizar o destilado quanto aos parâmetros de qualidade para aguardente. O trabalho foi dividido em quatro experimentos. O primeiro experimento objetivou comparar o efeito de duas α-amilases na etapa de liquefação, bem como, a influência do tempo de ação da amiloglucosidase na etapa de sacarificação, sobre o perfil de açúcares e rendimento do processo. O segundo experimento objetivou estabelecer qual a melhor concentração enzimática para o processo de hidrólise e sacarificação. O terceiro experimento teve por objetivo verificar o efeito do tempo de fermentação, concentração de levedura e temperatura de fermentação sobre o perfil cromatográfico do vinho obtido. A partir das conclusões parciais dos experimentos anteriores foi realizado o quarto ensaio que teve por objetivo produzir e caracterizar a aguardente... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The rhizome of ginger is widely marketed on the basis of its use in folk medicine, food, industrial, especially as raw material for the manufacture of beverages, perfumes and sugar confectionery such as breads, cakes, cookies and jellies. Facing the content of starch in this rhizome, an opportunity to increase the productive chain of ginger would be the production of distilled drink, which could be obtained from rhizomes of low quality for export and / or from residue from the extraction of oil key industries for processing this rhizome. This study aimed at evaluating the general technical parameters of the processing of ginger for the production of spirits, and as specific objectives: characterization of the proximate composition of the rhizomes of ginger to evaluate the quality and quantity of raw materials needed for the process of hydrolysis -saccharification; evaluate the influence of some parameters of the process of hydrolysis, saccharification and fermentation on yield of sugar and ethanol; and characterize the ginger spirit. The work was experiments. The first experiment aimed to compare the effect of two α-amylases in the stage of liquefaction, as well as, the influence of time of amiloglucosidase action in the saccharification step, on the profile of sugars and yield of the process. The second experiment aimed to establish the best concentration enzymes for the process of hydrolysis and saccharification. The third experiment aimed to verify the effect of time of fermentation, the yeast concentration and temperature of fermentation on the chromatographic profile of the wine obtained. The fourth experiment was conducted based on partial findings of previous experiments and aimed to produce and characterize the spirit of ginger. The results showed that the enzyme... (Complete abstract click electronic access below)
Mestre

Doctor of Philosophy
Grain Science and Industry
Praveen V. Vadlani
Lignocellulosic biomass feedstocks are a sustainable resource required for rapid growth of bio-based industries. An integrated approach, including plant breeding, harvesting, handling, and conversion to fuels, chemicals and power, is required for the commercial viability of the lignocellulosic-based biorefineries. Optimization of conversion processes, including biomass pretreatment and hydrolysis, is a challenging task because of the distinct variations in composition and structure of biopolymers among biomass types. Efficient fermentation of biomass hydrolyzates comprising of different types of sugars is challenging. The purpose of this doctoral research was to evaluate and optimize the various processing steps in the entire the biomass value chain for efficient production of advanced biofuels and chemicals from diverse biomass feedstocks. Our results showed that densification of bulky biomass by pelleting to better streamline the handling and logistic issues improved pretreatment and hydrolysis efficiencies. Alkali pretreatment was significantly more effective than acid pretreatment at same processing conditions for grass and hardwood. The ethanol-isopropanol mixture, and glycerol with 0.4% (w/v) sodium hydroxide were the promising organic solvent systems for the pretreatment of corn stover (grass), and poplar (hardwood), respectively. None of the pretreatment methods used in this study worked well for Douglas fir (softwood), which indicates a need to further optimize appropriate processing conditions, better solvent and catalyst for effective pretreatment of this biomass. The brown midrib (bmr) mutations improved the biomass quality as a feedstock for biochemicals production in some sorghum cultivars and bmr types, while adverse effects were observed in others. These results indicated that each potential sorghum cultivar should be separately evaluated for each type of bmr mutation to develop the best sorghum line as an energy crop. Development of an appropriate biomass processing technology to generate separate cellulose and hemicellulose hydrolyzates is required for efficient 2,3-butanediol (BD) fermentation using a non-pathogenic bacterial strain, Bacillus licheniformis DSM 8785. This culture is significantly more efficient for BD fermentation in single sugar media than Klebsiella oxytoca ATCC 8724. Though K. oxytoca is a better culture reported so far for BD fermentation from diverse sugars media, but it is a biosafety level 2 organism, which limits its commercial potential.

Ogi is a fermented food made from maize, sorghum or millet which serves as complementary food for infants and breakfast for adults in Nigeria, West Africa. This study characterized the microbial diversity of maize and sorghum grains and ogi produced by their natural fermentation in an attempt to understand the roles of the key microbial species and the impact of the population dynamics and selected species on changes in nutritional composition and aroma notes of ogi during fermentation. A combined approach of culture dependent and culture independent methods of analysis was applied to investigate the microbial community of grains and ogi from two different sources. Microbial diversity and viable populations varied with the source of the grain. Bacterial and fungal genera identified with the partial 16S rRNA and 26S rRNA sequence analysis respectively in maize and sorghum were Bacillus, Enterobacter, Micrococcus, Kytococcus, Pantoea, Staphylococcus, Amycolatopsis, Methanoculleus, Aspergillus, Penicillium, Eupenicillium, Acremonium, Schizosaccharomyces, Meyerozyma, Hyphopichia, and Pichia in maize grains; Enterococcus, Enterobacter, Pantoea, Bifidobacterium, Aspergillus, Cladosporum, and Penicillium in maize ogi; Enterococcus, Enterobacter, Pantoea, Aeribacillus, Cyanobacterium, Acinetobacter, Fusarium and Trametes in sorghum grains; and Pediococcus, Lactobacillus, Enterococcus, Bacillus, Cladosporum and Penicillium in sorghum ogi. Similar species were observed in both sources of maize while those of sorghum differed slightly. Predominant microbes included species of Enterobacteriaceae and moulds. Acetic acid bacteria were not identified as part of the diverse community. Following the predominance of moulds during the natural fermentation, preliminary screening was performed by PCR using specific biosynthetic gene primers to test whether they are the mycotoxin producing species. None of the genes tested were detected by PCR thus they may not be the toxin producing species. Starch, non-starch polysaccharide (NSP), phytate and phenolic compounds were determined in the grains and respective ogi to ascertain the levels of these nutritionally important components in the naturally fermented ogi and the impact of the varying microbial populations on the fate of these compounds during fermentation. In the grains, the average starch and NSP contents in each case were 80.35 g/100g and 9.40g/100g in maize and 93.12 g/100g and 8.14 g/100g in sorghum. Out of the total in grain the average percentage recovery of starch and NSP respectively in the ogi showed 63% and 42% in maize and 58% and 27% in sorghum. Maize showed good starch and fibre (NSP) retention than sorghum after fermentation. To further understand the types and levels of polymers in NSP hydrolysis in ogi fermentation, HPLC analysis of the hydrolysed extract was performed. Glucose was entirely present in maize and sorghum ogi which represents the beta-D-glucans while arabinose and xylose (in maize only), mostly lost with the pomace, signify the arabinoxylans. Overall variations in the microbial populations of sorghum seemed causal to the difference in starch and NSP recoveries. Phytate was assessed based on release of total phosphorus in the samples by enzymatic and chemical methods. Recovery of phytate in the naturally fermented ogi ranged from 18-25% in maize and 40-48% in sorghum suggesting greater phytase activity and more nutrient bioavailability in maize ogi than in the sorghum. Greater activity in maize reflects the presence of phytate hydrolysing species such as Aspergillus in the grain. Total phenolic content (TPC) was assessed by Folin-Ciocalteu colorimetric method after direct extraction of samples by saponification. TPC in the original grains ranged from 410–437 mg GAE/100g in maize and 221–247 mg GAE/100g in sorghum. Due to the nutritional significance, the amount of phenolics that are either freely soluble or are covalently bound to the food matrix were assessed. Soluble phenolics in ogi ranged from 16-38% in maize and 32-49% in sorghum based on the total soluble fraction in the original grain. In all cases loss of soluble phenolics with the waste waters accounted for 12-25% and 31-39% with the pomace. Only the LAB population seemed to correlate with the release of phenolics in the natural fermentation. Given the higher value of soluble phenolics, naturally fermented sorghum ogi appeared to have higher antioxidant potential than the maize ogi. Furthermore an attempt was made to ascertain whether the use of selected microbes would improve the antioxidant properties and aroma of ogi while minimizing the incidence of pathogens due to chance inoculation. Thus the impact of selected LAB (Pediococcus pentosaceus) and fungi (T. hirsuta and A. zeae previously shown to have phytase activity) on changes in phytate, phenolics and aroma of ogi was assessed following a parallel experiment to the previous study but using autoclaved grains. Five fermentation treatments of the pure and co-cultures were investigated. Cell populations in all culture fermentations varied and reached the average maximum of log 6-9 cfu/ml. Changes in the distribution of bound and soluble phenolics were observed showing esterase activity. Leaching of phenolics was evident in all cases but was higher in the sorghum fermentations. Higher levels of soluble phenolics were recovered in pure culture fermented ogi using T. hirsuta or P. pentosaceus than in the natural fermentation having 76% and 45% of the original soluble fraction in maize and sorghum respectively. This suggests greater antioxidant potentials than the naturally fermented ogi. Pure culture fermentations using T. hirsuta and co-culture of P. pentosaceus with A. zeae reduced phytate by 97% and 96% in maize and sorghum ogi respectively showing greater phytase activity and more nutrient bioavailabilty in the ogi than in the natural fermentation. The aroma profile of ogi was analysed by solid-phase microextraction and gas chromatography-mass spectrophotometry (SPME GC-MS). Ethyl acetate, butyl acetate and ethyl hexanoate were observed as the key active aroma components in ogi. The ester, methyl thiobutanoate was found to be unique to the naturally fermented ogi suggesting that it may have been generated by species other than the selected starter organisms. Overall in both natural and starter culture fermentations, maize ogi showed high relative abundance of volatile components suggesting good substrate compatibility and utilization during fermentation. Thus compounds with high threshold values may be significant in the aroma notes of maize ogi. P. pentosaceus and T. hirsuta in pure and in co-culture fermentations produced ogi with aroma notes mostly related to the naturally fermented product. In conclusion the diversity and levels of the initial microflora and the structural composition of grain could be major factors contributing to the nutritional compositional changes in ogi fermentation.

Att rena vatten är kostsamt, från både en ekonomisk och miljömässig synpunkt, då behovet av fällningskemikalier, kolkällor och energi är stort. Det är därför önskvärt att titta på alternativa lösningar som möjliggör reningsverk att bli mer självförsörjande och kretsloppsanpassande. Hammarby Sjöstadverket är en pilotanläggning för avloppsvattenrening belägen i Stockholms sydöstra stadsdel i Henriksdal och ägs av IVL, Svenska Miljöinstitutet samt KTH. Sedan pilotanläggning byggdes år 2002, har flera olika reningstekniker utvärderats där fokus ligger på att sträva efter miljövänliga och kretsloppsanpassade system. Bland annat har en förfällningsteknik, så kallad trepunktsfällning utvärderats. Trepunktsfällningen innebär att ett metallsalt följt av två olika polymer tillsätts i flockningskammaren i inbördes ordning för att på så sätt kunna reducera en högre halt av det organiska materialet. Tidigare fällningsförsök på Hammarby Sjöstadsverket, har visat på att trepunktsfällningen kan avskilja det organiska materialet med upp till 90 %, vilket kan jämföras med en avskiljning på endast 75 % med vanlig förfällning. Detta examensarbete syftar till att validera redan framtagna resultat inom trepunktsfällning samt att genom biologisk hydrolys av primärslam, utvinna en kolkälla till efterdenitrifikationen innehållande så höga halter flyktiga fettsyror, Volatile Fatty Acids, (VFA) som möjligt samt undersöka kostnaderna för ett avloppsreningsverk med trepunktsfällning, biogasutvinning och uttag av intern kolkälla från primärslammet vid en uppskalning motsvarande 100 000 personekvivalenter. Fällningsförsöken utfördes i pilotskala med fällningskemikalien PAX XL-36 kombinerat med en lågmolekylär organisk polymer, Purfix-120, följt av en högmolekylär oorganisk polymer Superflock C-494. Syftet med trepunktsfällningen var att validera redan framtagna resultat inom trepunktsfällning och på så sätt avskilja så stora mänger organiskt material som möjligt i förfällningen så att ett primärslam innehållande en hög halt Chemical Oxygen Demand (COD) erhålls. Två olika doseringar med PAX XL-36 testades, 145 respektive 193,5 g/m3. Bäst reduktion av COD och fosfor erhölls då 193 g/m3 PAX XL-36 kombinerades med 60 g/m3 Purfix-120 samt 0,025 g/m3 Superflock C-494. Då erhölls en COD-reduktion på 75 % samt en totalfosforreduktion på 83 %. Genom hydrolys av primärslam i laboratorieskala undersöktes torrsubstanshalter (TS-halter) på 1, 2 respektive 3 % och uppehållstider på upptill åtta dagar. Syftet med hydrolysen var i detta fall att undersöka vilken torrsubstanshalt samt vilken uppehållstid som gav högst produktion av VFA. Försöken visade att en TS-halt på 3 % producerade högst andel VFA och att produktionen av VFA för samtliga TS-halter var som störst under dygn två. VFA- och COD-produktionen ökade linjärt för samtliga TS-halter fram till och med dygn fem. Efter dygn fem började produktionen av VFA och COD för TS-halterna 1 och 2 % avta något. TS 3 % visade dock inte samma avtagande trend för VFA.  Även den dagliga ammoniumhalten och pH undersöktes. Ammoniumhalten ökade i takt med att VFA-halten ökade. pH hade generellt sett en avtagande trend. Fyra olika denitrifikationsförsök genomfördes i laboratorieskala med det uttagna hydrolysatet där den tillsatta COD-halten antingen var 3,3 eller 4 gånger den initiala nitratkvävekoncentrationen. Syftet med denitrifikationsförsöken var att utvärdera det framtagna hydrolysatets funktion som en kolkälla. Denitrifikationsförsöken uppvisade denitrifikationshastigheter mellan 4,3 och 7 mg NO3-N/g volatile suspended solids*h med kol-kvävekvoter (C/N-kvoter) mellan 3,9 och 12,3 mg COD/mg NO3-N. Lägst C/N-kvot erhöll lägst denitrifikationshastighet. Dock kunde inte slutsatsen dras att högst C/N-kvot gav högst denitrifikatioshastighet. Även de ekonomiska aspekterna undersöktes i syfte att utreda kostnaderna för ett avloppsreningsverk med trepunktsfällning, biogasutvinning och uttag av intern kolkälla från primärslammet vid en uppskalning motsvarande 100 000 personekvivalenter. Uttaget av en intern kolkälla skulle trots förlust i biogasutvinning vara ekonomiskt gynnsamt. Vinsten, i form av att ej behöva inhandla en extern kolkälla, i detta fall etanol, motsvarar biogasförlustens belopp. Trepunktsfällningens fällningskemikaliekostnader var den största posten och uppgick till 8 060 000 kr. Denna kostnad kontra mindre energiförbrukning i biosteget undersöktes också. Här uppgick besparingarna i biosteget till ca 1/8 av fällningskemikaliekostnaderna. Då trepunktsfällningen endast kunde ge en 75-procentig COD-reduktion, vilket motsvarar en vanlig förfällning, anses trepunktsfällningen vara ekonomisk ogynnsam då den medför extra kostnader av polymerer.  Istället borde trepunktsfällningen ersättas med en förfällning. Sammanfattningsvis kan det konstateras att en intern kolkälla i form av hydrolyserat primärslam skulle kunna ersätta en extern kolkälla rent funktionsmässigt. Dock måste åtgärder göras för att minimera produktionen av ammonium under slamhydrolysen. Ur ett ekonomiskt perspektiv skulle utvinningen av en intern kolkälla endast vara ekonomisk gynnsam då trepunktsfällningen ersätts med förfällning.
Water treatment is costly, from both an economic and environmental point of view, since the need for precipitation chemicals, carbon sources and energy is high. It is therefore desirable to look for alternative solutions that enable plants to be more self-sustaining. Hammarby Sjöstadsverket is a pilot plant for wastewater treatment located in Henriksdal, a southeastern neighborhood of Stockholm. The plant is owned by IVL, Svenska Miljöinstitutet and Kungliga Tekniska Högskolan (KTH). Since the pilot plant was built in 2002, several different cleaning techniques have been evaluated with focus on striving for eco-friendly and eco-adapted systems. For instance, a pre-precipitation technique, so-called three step precipitation have been evaluated. The three step precipitation implicates that a metal salt followed by two different polymers are added in the flocculation chamber in the particular order to thereby enable to reduce a higher content of the organic material. In previous precipitation tests at Hammarby Sjöstadsverket, one managed to remove up to 90 % of the organic material using the three step precipitation (IVL, Hammarby Sjöstadsverket, 2011). This can be compared to a removal of only 75 % with ordinary pre-precipitation. This thesis aims to validate already obtained results within three step precipitation and with biological hydrolysis of primary sludge, extract a carbon source for post-denitrification containing as high concentrations of Volatile Fatty Acids (VFA) as possible and to investigate the cost of a wastewater treatment plant with three step precipitation, biogas generation and collection of internal carbon source from primary sludge at an upscaling corresponding to 100,000 person equivalents. Precipitation experiments were carried out in pilot scale with the precipitation chemicals, PAX-XL 36, combined with a low molecular weight organic polymer, Purfix-120, followed by a high molecular weight inorganic polymer, Super Flock C-494. The purpose of the three step precipitation was to validate the already produced results within three step precipitation and thus separate as large amounts of organic material as possible in the precipitation so that a primary sludge containing a high chemical oxygen demand (COD) can be obtained.Two different dosages of Purfix-120 were tested: 45 and 60 g/m3. The best reduction of COD and phosphorus were obtained when 193 g/m3 PAX XL-36 was combined with 60 g/m3 Purfix-120 and 0.025 g/m3 Super Flock C-494. Hereby a COD reduction of 75 % and a total phosphorus removal of 83 % were obtained. By hydrolysis of primary sludge in batch experiments Total Solid (TS) concentrations of 1, 2 and 3 % and residence times of up to eight days were examined. The purpose of the hydrolysis in this case was to investigate which TS concentration and residence time that gave the highest production of VFA. The experiments showed that a TS concentration of 3 % produced the highest amount of VFA and that the VFA production of the three different TS concentrations peaked at the second day. The VFA and COD production increased linearly for the three TS concentrations up until day five. After day five the COD and VFA production, for the TS concentrations of 1 and 2 %, started to decrease slightly. However TS 3 % did not show the same declining trend for VFA. Furthermore the daily ammonium and pH were investigated.  As the VFA and COD concentration increased the ammonium concentration increased as well. pH had overall a  decreasing trend. Four different denitrification tests were performed in batch experiments with the extracted hydrolyzate. The added COD content from the hydolyzate was either 3.3 or 4 times the initial nitrate concentration. The denitrification tests showed denitrification rates between 4.3 and 7 mg NO3-N / g volatile suspended solids * h with carbon-nitrogen ratios (C/N ratio) between 3.9 and 12.3 mg COD/mg NO3-N. The lowest C/N ratio received the lowest denitrification rate. However, it could not be concluded that the maximum C/N ratio had the highest denitrification rate. Futhermore the financial aspects were examined in order to investigate the cost of a wastewater treatment plant with three step precipitation, biogas generation and collection of internal carbon source from primary sludge at an upscaling corresponding to 100,000 person equivalents. The extraction of an internal carbon source would, despite loss of biogas production, be economically beneficial. Gain, in terms of not having to purchase an external carbon source, in this case ethanol, amounts to the amount of the biogas loss. The three step precipitation chemical costs were the largest item, amounting to 8,060,000 SEK. This cost versus less energy utilization in the biological step was also examined. In this case the savings in the biological step amounted to about 1/8 of precipitation chemical cost. Since the three step precipitation only managed to remove 75 % of the COD, a removal which corresponds to an ordinary pre-precipitation, the three step precipitation is considered to be economically unfavorable as it involves additional costs of polymers. Instead the three step precipitation should be replaced with a pre-precipitation. In summary it can be stated that an internal carbon source in the form of hydrolyzed primary sludge could replace an external carbon source in a functional way. However, steps must be taken to minimize the production of ammonium during sludge hydrolysis. From an economic perspective, the extraction of an internal carbon source would only be economical favorable if the three step precipitation is replaced with pre-precipitation.

No contexto energético o bioetanol tem recebido considerável atenção, por questões ambientais e também pela redução da dependência dos combustíveis de origem fóssil. Embora o Brasil seja o segundo maior produtor mundial de etanol, a produção está concentrada em determinadas regiões, enquanto o Rio Grande do Sul é basicamente um importador. Para mudar tal situação, a batata-doce é uma matéria-prima atrativa, por ser rica em amido e possuir características agronômicas vantajosas. Visando colaborar para a viabilização em maior escala da produção de etanol a partir de batata-doce, o presente trabalho reúne estudos sobre este tema. De trabalhos anteriores desenvolvidos no GIMSCOP, ficou o desafio da obtenção de teores de etanol no vinho entre 10 – 12% (v/v), o que foi alcançado neste trabalho com o aumento da concentração batata: água para 1,5 kg: 1 L. A enzima comercial Stargen 002, recomendada pelo fabricante para o amido granular, foi utilizada para a hidrólise. Em ensaios amido granular vs. batata pré-aquecida em banho-maria (a 76oC), foi obtido um teor de etanol de 9,25% (v/v) em 24 h com o aquecimento prévio, enquanto que com a batata-doce crua o teor foi de 6,13% (v/v) em 58 h. Portanto, o método com a batata pré-aquecida foi selecionado, onde após o pré-aquecimento três processos são conduzidos em única etapa: hidrólise, fermentação e redução da viscosidade. Como resultado de um planejamento composto central, a temperatura e o tempo do processo apresentaram efeitos significativos, com maior sensibilidade à temperatura do que ao tempo. Como a temperatura praticada já estava em torno do ótimo (34oC), o maior ganho foi na redução do tempo, de 24h para 19h. Objetivando aumentar a eficiência de conversão, foi avaliado o efeito do pré-aquecimento em micro-ondas e também do amadurecimento da batata pós-colheita (atuação das próprias amilases ao longo do tempo). O amadurecimento foi positivo, o pico na produção de etanol foi em 25 dias após a colheita. Não foi observado ganho no pré-aquecimento com micro-ondas. Na estimativa de custos do processo, a levedura e a matéria-prima implicaram nas maiores frações do custo total. Visando um futuro processo de destilação com injeção de CO2, é apresentado um estudo teórico-experimental sobre o equilíbrio da mistura água-etanol-CO2 incluindo o estudo experimental de injeção de CO2 em um vinho de batata-doce. Viu-se que são necessários aperfeiçoamentos no modelo e no aparato experimental. Contudo, os resultados experimentais mostraram que se conseguem maiores concentrações de etanol no condensado nas menores vazões de CO2. Foi identificada uma relação linear entre a umidade da batata-doce e o teor de açúcares redutores totais (ART), resultando em uma proposta de estimativa interessante pela praticidade, mas se aconselha que a curva seja construída com dados de um tipo de batata e de fontes controladas. Também, um bom ajuste de curva requer que o método analítico empregado na produção dos pontos seja confiável. Foi iniciado um estudo comparativo de métodos, que deve ser continuado a fim de maximizar a quantificação de glicose com o menor custo de análise.
In the energy context ethanol has received considerable attention due to environmental issues and also by reducing the fossil fuels dependence. Although Brazil is the second largest ethanol producer in the world, the production is concentrated in certain regions, whereas Rio Grande do Sul state is practically an exclusive importer. To change this situation, sweet potato is a very attractive feedstock, because it is rich in starch and has favorable agronomic characteristics. Aiming to contribute to the viability of ethanol production from sweet potato in larger scale, this work brings studies upon this topic. In GIMSCOP's previous works, there was the challenge of obtaining ethanol concentration in wine between 10 – 12% (v/v), which was achieved in this work, by increasing the concentration potato: water to 1.5 kg: 1 L. The commercial enzyme Stargen 002 was used for hydrolysis, the manufacturer indicates it to the granular starch. In experiments: granular starch vs. potato preheated in water bath (at 76oC), was obtained an ethanol content 9.25% (v/v) in 24 h when applying the preheating, whereas with raw sweet potato the ethanol content was 6.13% (v/v) in 58 h. Therefore, the preheating method was selected, where after preheating three processes are conducted in a single stage: hydrolysis, fermentation and reduction of viscosity. A central composite design was carried out, the temperature and process time showed significant effects, with higher sensitivity to temperature than time. However, the practiced temperature was already around the optimum (34oC), the most important gain was the time reducing, from 24 h to 19 h. In order to increase conversion efficiency, was evaluated the effect of pre-heating in microwave and also sweet potato ripening over post-harvest (action of own amylases over time). The ripening was positive, the ethanol production peak was at 25 days after harvest. There was no gain in the preheating with microwave. In the process's costs estimation, the yeast and feedstock resulted in the largest fraction of total cost. Aiming a future process of distillation with CO2 injection, this work presents a theoretical and experimental study of the mixture water-ethanol-CO2 equilibrium, also there is an experimental study of CO2 injection through a sweet potato wine. It was seen that improvement is needed in the model and the experimental apparatus. However, experimental results showed that higher ethanol concentrations can be obtained in the condensate with lower CO2 flows. A linear relationship was found between the sweet potato moisture and the total reducing sugars (TRS), resulting in an interesting proposal for estimating due to practicality, but it is advisable that the curve be constructed with data from a kind of sweet potato with controlled sources. Also, a good curve fitting requires a reliable analytical method when obtaining the experimental points. A comparative study between methods was initiated, and it must be continued, in order to maximize the glucose quantification with lower cost analysis.

El trabajo realizado en esta tesis se enmarca dentro de las principales líneas de investigación del grupo de compostaje (GICOM), por una parte, en la valorización de residuos sólidos por medio de la fermentación en estado sólido (FES) para la obtención de productos de valor añadido en consoncia con el nuevo paradigma de considerar los residuos como materias primas y, en segundo lugar, en la utilización de nanomateriales para potenciar el uso de los productos obtenidos mediante FES. La primera parte se centra en el aprovechamiento de residuos sólidos para la producción de enzimas de interés, concretamente proteasas. Las enzimas se produjeron a partir de la FES de la mezcla de residuo de pelo vacuno procedente de la industria de curtidos y lodos procedentes del proceso de digestión anaerobia utilizado como cosubstrato e inóculo. Por una parte se ha demostrado la capacidad de producir proteasas y sus posibles aplicaciones en la propia industria, ya que se han obtenido buenos resultados en el proceso del depilado del cuero. Además, se ha establecido un esquema para el tratamiento posterior a la fermentación, incluida la reutilización de los residuos generados a lo largo del proceso de modo que se aproxime a un sistema de residuo cero. La segunda parte del trabajo consistió en una exploración preliminar para determinar cuáles son los soportes más eficientes para inmovilizar las proteasas producidas por FES de cara a su uso continuado y su mejor preservación. Se utilizaron proteasas obtenidas a partir de la FES de residuos ricos en nitrógeno, pelo vacuno utilizado en la primera parte del trabajo y residuo de soja de una industrial alimentaria. Se evaluó la aplicabilidad, reutilización, reproducibilidad y coste de los soportes para su uso en la inmovilización de dichas proteasas. La evaluación se efectuó con 10 materiales considerando su bio-compatibilidad con las proteasas: nanopartículas de oro (NpAu), perlas de quitosano (húmedas y secas), perlas de quitosano con nanopartículas de oro (Quitosan/Au), zeolitas funcionalizadas, resina aniónica A520, resina aniónica con nanopartículas de oro (A520/Au), nanopartículas de óxido de hierro (Fe3O4) y nanopartículas de óxido de titanio (TiO2). La eficiencia de la inmovilización se determinó a partir del rendimiento de inmovilización y la carga enzimática de cada soporte (U/mg). Las nanopartículas de óxido de hierro resultaron ser un soporte prometedor gracias a su bajo coste y a su fácil separación de forma magnética, aumentado así sus posibilidades de reutilización. En último lugar, tanto las proteasas obtenidas a partir de los residuos de pelo como las de residuo de soja se inmovilizaron para ser evaluadas en la hidrólisis de proteínas, cuya eficiencia se comparó con la de las enzimas libres en el medio. Se utilizaron substratos de diferente origen (animales y vegetales): caseína, albúmina de huevo y proteína de salvado de avena, observado un efecto diferente según el origen de la proteasa utilizada. Estos resultados se corroboraron con los aminoácidos liberados tras la hidrólisis de las proteínas. En términos globales, este estudio representa una investigación multidisciplinaria que abarca, por una parte, la gestión y valorización de residuos orgánicos mediante el proceso de fermentación en estado sólido, con la producción y recuperación del producto de interés, incluyendo la etapa de postratamiento y así como la aplicabilidad del producto. Además, la immbolización de las proteasas sobre nanopartículas de bajo coste se ha demostrado efectiva en la hidrólisis de proteinas tipo, consistuyendo una alternativa de bajo coste (tanto su producción como el soporte) a las técnicas actuales.
In line with the main research lines of Composting research group (GICOM), this thesis focuses on the valorisation of solid organic residues and the utilisation of nanomaterials. Thus the thesis mutually linked the studies by transforming the solid waste using solid state fermentation (SSF) into value-added products and the use of nanoparticles to enhance the use of products obtained from SSF. The first major part covers the valorisation of solid waste and transforms it into valuable product namely protease. The protease was produced through the fermentation of hair waste and anaerobically digested sludge (ADS) acting as co-substrate and inoculum. For industrial application, the protease produced from this work has shown a good result on dehairing of cowhides. This chapter not only demonstrated from the production of protease until application on industrial activity but also exhibited the downstream processing in SSF and reutilisation of the remaining residues after SSF which was a crucial thing to establish a zero discharge. The second major part consisted of a preliminary screening work intending to determine the most suitable support for immobilisation of proteases. Proteases produced from two different protein-rich solid wastes: hair waste and soy fibre residue. The work aimed to find the feasible, reusable, reproducible, and cost-effective supports or carrier to be exploited for protease immobilisation. There were 10 materials were assessed for their biocompatibility with the protein: gold nanoparticles (AuNps), chitosan beads (wet and dry), chitosan beads coupled with gold nanoparticles (chitosan/Au), functionalised zeolite particles, the anionic resin (A520), anionic resin coupled with gold nanoparticles (A520/Au), magnetic iron oxide (Fe3O4) nanoparticles (MNPs), titanium oxide nanoparticles (TiO2_A and TiO2_B). The immobilisation efficiency was monitored based on immobilisation yield (% IY) and enzyme loading per carrier (U/mg). Iron oxide nanoparticles were shown as a promising support thanks to its low-cost and easy separation by a magnetic force, thus increasing their possibilities of reuse. In the last part of the thesis, both of the proteases from hair waste and soy fibre residue were immobilised on the support and were tested for application in protein hydrolysis. The efficiency of immobilised enzymes was compared with the free enzymes during the protein hydrolysis. Not to mention, the use of the different type of proteases (animal and plant origin) also was assessed during the protein hydrolysis of different type of protein (casein, egg white albumin, and oat bran protein isolate), since different protease produce different effect towards certain substrates. The amino acids released after hydrolysis reaction were well balanced with the degree of hydrolysis according to each protein substrates. Overall, the study represented a multidisciplinary research field spanning waste management using solid state fermentation including the downstream processing and its application. In addition, the immobilisation of proteases on low-cost nanoparticles has been shown to be effective in the hydrolysis of proteins, being a low-cost alternative (both production and support) to actual techniques.

C'est dans un contexte marqué par une demande croissante en énergie primaire, une diminution des ressources et dans un souci de protection de l'environnement que le biocarburant de 2ème génération est développé. Cependant, ce biocarburant est non viable économiquement. L’optimisation, le contrôle et la connaissance des cinétiques régissant les procédés de fabrication de ce bioéthanol sont donc des éléments capitaux. Dans cette étude, le potentiel de la calorimétrie isotherme pour surveiller les réactions d'hydrolyse et de fermentation est testé.Les résultats montrent que cette méthode est efficace. En effet, celle-ci a permis de mettre en évidence l'importance du ratio enzyme/substrat pour maximiser le rendement et de déterminer un meilleur cocktail composé de cellulases + cellobiose déshydrogénase (CDH) qui permet la production d'une certaine quantité d'acide gluconique, qui pourrait améliorer l'attractivité de ce biocarburant. Ces mêmes essais ont également permis de déterminer la chaleur de l'hydrolyse de la paille de blé, qui est 32,18 ± 3,18 J.g-1 (gramme de sucres produits).Les mesures obtenues ont été utilisées pour déterminer les constantes cinétiques des cellulases + CDH sur la paille de blé et les résultats montrent que ce cocktail enzymatique est plus rapide à 45 °C dans la gamme de températures testée (40-55°C) avec une vitesse de 7,36 ± 0,62 mmol/L.min.Par ailleurs, les essais avec un calorimètre à échelle laboratoire ont montré que même si celui-ci ne mesure pas avec précision les chaleurs engendrées par les réactions d'hydrolyse et de fermentation, celui-ci donne de bonnes indications sur le déroulement et l'avancement de ces réactions
Second generation biofuel is developed in a context marked by an increasing demand for primary energy, a decrease in resources and in environmental protection concernsHowever, this biofuel is not economically viable. Optimization, control and knowledge of the kinetics governing this bioethanol production processes are crucial elements.In this study the potential of isothermal calorimetry to monitor hydrolysis and fermentation reactions is tested.The results show that the isothermal calorimetry is an effective method. Indeed this method allowed determining that the substrate/enzyme ratio is an important parameter of the hydrolysis yield.Furthermore it has determined a better enzyme cocktail consisting of Cellulases + Cellobiose Dehydrogenase (CDH) which allows the production of a certain amount of gluconic acid, which could improve the attractiveness of these second-generation biofuels. These same tests also determined the hydrolysis heat of wheat straw which is 32.18 ± 3.18 J.g-1 (gram reducing sugars product).The measurements obtained were used to determine kinetic constants cellulases + CDH on wheat straw and the results show that this enzyme cocktail is faster at 45 ° C in the range of temperatures tested (40 - 55°C) with a speed of 7.36 ± 0.62 mmol/L.min.In addition, testing with a laboratory-scale calorimeter showed that even if this tool does not accurately measure the heat generated by the hydrolysis reaction and fermentation, it gives a good indication of the development and advancement of these reactions

Orientador: Hélia Harumi Sato
Texto em português e inglês
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: As proteases constituem um dos mais importantes grupos de enzimas produzidos comercialmente, apresentando diversas aplicações nas indústrias de alimentos e farmacêutica. A utilização de proteases na hidrólise enzimática de proteínas para obtenção de peptídeos com propriedades antioxidantes tem recebido grande notoriedade nas pesquisas científicas. Nesse contexto, o presente trabalho visou estudar a produção e caracterização bioquímica de protease de Aspergillus oryzae LBA 01 obtida por processo fermentativo em estado sólido e avaliar a aplicação desta protease e de preparações comerciais na hidrólise de proteínas para obtenção de hidrolisados com atividade antioxidante. A maior produção de protease por A. oryzae LBA 01 foi observada em meio de cultivo composto de farelo de trigo, peptona (2,0% p/p) e extrato de levedura (2,0% p/p) sob as seguintes condições: 50,0% de umidade inicial, inóculo de 107 esporos.g-1 e incubação a 23°C por 72h. A caracterização bioquímica, realizada por planejamento experimental, mostrou que a protease apresentou maior atividade na faixa de pH 5,0-5,5 e 55-60°C, e estabilidade no intervalo de pH 4,5-6,0 após 1h de tratamento na faixa de temperatura de 35-45°C. Proteína isolada de soja, soro de leite e clara de ovo apresentaram aumento expressivo nas suas propriedades antioxidantes quando hidrolisadas com diferentes proteases microbianas. A aplicação de protease comercial Flavourzyme® 500L, obtida de A. oryzae, para a hidrólise de proteína isolada de soja, resultou na obtenção de hidrolisados com maior atividade antioxidante quando comparados aos hidrolisados preparados com as proteases de A. oryzae LBA 01 e a protease comercial Alcalase® 2.4L de Bacillus licheniformis. As condições de hidrólise, definidas a partir de delineamento composto central rotacional (DCCR), foram: concentração de substrato de 90,0 mg.mL-1 e adição de 70,0 U de protease por mL de mistura reacional (U.mL-1), resultando em 775,17 e 11,83 Trolox EQ µmol.g-1, para os ensaios de ORAC e DPPH, respectivamente. Os hidrolisados de soro de leite com maior capacidade antioxidante foram obtidos com a protease de A. oryzae LBA 01. A adição de 70,0 U.mL-1 de protease a solução de soro de leite 80,0 mg.mL-1, resultou em 424,32 e 16,39 Trolox EQ µmol.g-1, para os ensaios de ORAC e DPPH, respectivamente. Na preparação de hidrolisados de proteínas de clara de ovo, a utilização de 30,0 mg.mL-1 de substrato e 20,0 U.mL-1 da protease comercial Flavourzyme® 500L de A. oryzae, resultou em 1.193,12 e 19,05 Trolox EQ µmol.g-1 para os ensaios de ORAC e DPPH, respectivamente. Os maiores valores de atividade antioxidante, para os três substratos, foram detectados entre 30 e 180 minutos de incubação, onde o grau de hidrólise variou de 40,0 a 66,0%. Os resultados obtidos mostraram que a preparação de protease de A. oryzae LBA 01 obtida por fermentação em estado sólido e posterior concentração por precipitação com sulfato de amônio, diálise e liofilização, apresentou atividade enzimática semelhante às preparações comerciais avaliadas, tendo, portanto, potencial para aplicação na hidrólise proteica. A hidrólise enzimática, nas condições de estudo avaliadas, aumentou de 2 a 23 vezes a capacidade antioxidante de proteína isolada de soja, soro de leite e clara de ovo, mostrando-se um processo eficaz para obtenção de peptídeos com atividade antioxidante
Abstract: Proteases are one of the most important groups of enzymes produced commercially, with several applications in the food and pharmaceutical industries. The use of proteases in the enzymatic hydrolysis of proteins to obtain peptides with antioxidant properties has gained great notoriety in scientific research. In this context, the main objectives of the present study were to optimize the production of the protease from Aspergillus oryzae LBA 01 by solid state fermentation, and to determine its biochemical characteristics. The application of this protease and of commercial preparations to protein hydrolysis, and the study of the antioxidant properties of the hydrolysates obtained, was evaluated. The optimum fermentation medium was composed of wheat bran, 2.0% (w/w) peptone and 2.0% (w/w) yeast extract, and the conditions for maximum protease production were an initial moisture content of 50.0%, an inoculum level of 107 spores.g-1 and incubation at 23°C for 72h. The biochemical characterization, evaluated using an experimental design, showed that the enzyme was most active in the pH range 5.0-5.5 and 55-60°C. The enzyme was stable from pH 4.5 to 6.0 after 1h incubation at 35-45°C. Soy protein isolate, bovine whey protein and egg white protein exhibited increases in antioxidant activity when hydrolyzed with the different microbial proteases. For the hydrolysis of soy protein isolate, application of the commercial protease Flavourzyme® 500L from A. oryzae resulted in hydrolysates with greater antioxidant activity as compared to hydrolysates prepared with the protease from A. oryzae LBA 01 and the commercial protease Alcalase® 2.4L from Bacillus licheniformis. The hydrolysis conditions, as defined by a central composite rotational design (CCRD), were: 90.0 mg.mL-1 substrate concentration plus 70.0 U of protease per mL of reaction mixture (U.mL-1), which resulted in 775.17 and 11.83 Trolox EQ µmol.g-1 as determined by the ORAC and DPPH assays, respectively. For the whey protein hydrolysates, the greatest antioxidant activity was obtained with the protease from A. oryzae LBA 01. According to the CCRD, the use of 80.0 mg.mL-1 of bovine whey protein and 70.0 U.mL-1 of protease resulted in 424.32 and 16.39 Trolox EQ µmol.g-1, respectively, as determined by the ORAC and DPPH assays. For the egg white protein, hydrolysis with 20.0 U.mL-1 of Flavourzyme® 500L from A. oryzae with 30.0 mg.mL-1 substrate concentration, resulted in 19.05 and 1,193.12 Trolox EQ µmol.g-1, respectively, as determined by the ORAC and DPPH assays. The maximum antioxidant activities were obtained in the range from 30 to 180 min of hydrolysis, with a degree of hydrolysis of about 40.0-66.0%. The results showed that the protease preparation from A. oryzae LBA 01 obtained by solid state fermentation produced enzymatic activity similar to that of the commercial preparations, and was an attractive enzyme to apply in protein hydrolysis. Under the conditions evaluated in this study, enzymatic hydrolysis resulted in a 2.0- to 23.0-fold increases in antioxidant activity for the soy protein isolate, bovine whey protein and egg white protein, being an effective process to obtain peptides with antioxidant activity
Mestrado
Ciência de Alimentos
Mestre em Ciência de Alimentos

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The present work was to isolate, select and cultivate microorganisms that produce lipases to apply the wastewater treatment industry of potato chips. For the isolation of microorganisms was used for industrial effluent and sewage treatment plant. The selection of the strain producing lipase was performed on solid medium containing tributyrin and in liquid medium, with p-nitrofenolpalmitato. Optimization of lipase production was performed as variables, the concentration of corn steep liquor, soybean oil and different mineral sources. The enzyme extract and the commercial enzyme selected strain were characterized on the optimal activity and stability at different temperatures and pH values and determined the molecular weight of the commercial enzyme. For the treatment of sewage, it has been characterized and has been the optimization of hydrolysis of triglycerides of the effluent having as variables, pH, enzyme concentration, emulsifier and the influence of fed batch and batch processes. Twenty-nine were isolated microorganisms were acquired and eleven research institutes. Of these, four microorganisms were pre-selected for their higher enzymatic activity. In the selection in submerged fermentation, enzymatic activity was the best strain of Burkholderia cepacia ATCC 25416. Optimization of lipase production was obtained with corn steep liquor (13.8 to 26.2 g.L-1) and sodium nitrate (1.5 to 3.9 g.L-1). In the characterization of the lipase enzyme extract and immobilized lipase from Burkholderia cepacia, the best results were found at 37ºC and pH 8.0. Were stable at 40°C, and thermosensitive at 50 and 60°C. The inactivation of lipases template denaturation followed first order. The molecular mass of commercial enzyme was 33 kDa. For the treatment of effluent, the maximum hydrolytic activity (0.310 U.mL-1) was observed when the volume of 7% gum arabic was set at 3 mL of pH between 7.47 to 7.92 and the concentration of enzyme 0.111 to 0.148 g.mL-1. The mathematical model obtained can be used for batch process and the predictive largest possible hydrolysis of the triglycerides present in the effluent.
O presente trabalho teve como objetivo isolar, selecionar e cultivar microrganismos produtores de lipases para aplicar no tratamento de efluente de indústria de batata frita. Para o isolamento de microrganismos foi utilizado efluente industrial e de Estação de Tratamento de Esgotos. A seleção da cepa produtora de lipase foi realizada em meio sólido, contendo tributirina e em meio líquido, com paranitrofenolpalmitato. A otimização da produção de lipase foi realizada tendo como variáveis, a concentração de água de maceração de milho, óleo de soja e diferentes fontes minerais. O extrato enzimático e a enzima comercial da cepa selecionada foram caracterizados acerca da atividade ótima e estabilidade em diferentes temperaturas e pHs e determinada a massa molecular da enzima comercial. Para o tratamento do efluente, o mesmo foi caracterizado e realizou-se otimização da hidrólise dos triacilglicerídeos do efluente, tendo como variáveis, o pH, concentração de enzima, agente emulsificante e a influência dos processos descontínuo e descontínuo alimentado. Foram isolados vinte e nove microrganismos e onze foram adquiridos de institutos de pesquisa. Destes, quatro microrganismos foram préselecionados por apresentarem maior atividade enzimática. Na seleção em fermentação submersa, a melhor atividade enzimática foi da cepa Burkholderia cepacia ATCC 25416. A otimização da produção de lipase foi obtida utilizando-se água de maceração de milho (13,8 a 26,2 g.L-1) e nitrato de sódio (1,5 a 3,9 g.L-1). Na caracterização das lipases do extrato enzimático e da lipase comercial de Burkholderia cepacia, os melhores resultados foram encontrados a 37°C e pH 8,0. Foram estáveis a 40ºC, e termossensíveis a 50 e 60ºC. A inativação das lipases seguiu modelo de desnaturação de primeira ordem. A massa molecular da enzima comercial foi de 33 kDa. Para o tratamento do efluente, a atividade hidrolítica máxima (0,310 U.mL-1) foi observada quando o volume de goma arábica a 7% foi fixado em 3 mL, entre o pH de 7,47 a 7,92 e concentração de enzima de 0,111 a 0,148 g.mL-1. O modelo matemático obtido pode ser utilizado para fins preditivos e o processo descontínuo possibilitou maior hidrólise dos triacilglicerídeos presentes no efluente.

Doctor of Philosophy
Department of Biological and Agricultural Engineering
Yong Cheng Shi
Donghai Wang
Cellulosic ethanol made from low cost lignocellulosic biomass has been considered as new generation transportation fuel with economic and environmental advantages. Photoperiod-sensitive (PS) sorghum, because of its high biomass yield (2.6 kg dry mass/m2), about 18% of soluble sugar in dry mass, and drought tolerance, is a promising biomass for ethanol production. The overall goals of this study are to develop an efficient approach to convert PS sorghum to ethanol and to understand the structural characteristics of biomass. For increasing the efficiency of biomass conversion, an integrated method, using diluted sulfuric acid pretreatment, has been developed to utilize both the structural polysaccharide (cellulose) and the soluble sugar (sucrose, glucose, and fructose) for fermentation. Response surface methodology was employed to optimize the pretreatment condition for maximizing the cellulose-glucose conversion. Simultaneous enzymatic hydrolysis and yeast fermentation was used for ethanol production. The effects of the buffer concentration, the inoculation dosage and time, and the fermentation temperature were investigated for maximizing ethanol yield. A total conversion efficiency of 77.2% and an ethanol concentration of 2.3% (v/v) were obtained after 72 h fermentation. About 210 kg (~266 Liters) ethanol could be produced from one ton dry mass of PS sorghum under the optimized condition. The structural features of the PS sorghum were studied using techniques including scanning electron microscopy and X-ray diffraction/scattering. Biomass at different botanic locations was investigated. Wide-angle X-ray diffraction (WAXD) study showed that the PS sorghum rind had oriented crystal peaks and the highest degree of crystallinity, whereas the crystalline structures of the inner pith and leaf were less ordered. The results from WAXD suggested that crystalline cellulose was melted at 120 °C before its significant degradation. Both the cellulose crystallinity and the crystal size at the dimension lateral to fiber direction increased as the temperature increased from 120 to 160 °C. The efficiency of enzymatic hydrolysis increased because the protective structure was damaged and most hemicellulose was removed, resulting in the increase in accessible area as suggested by small-angle X-ray scattering result of the increased length of microvoids. The results from WAXD also suggested a simultaneous hydrolysis and crystallization of cellulose by acid.

Orientador: Cláudio Cabello
Banca: Waldemar Gastoni Venturini Filho
Banca: Simone Damasceno
Resumo: Neste trabalho fez-se uma avaliação da utilização dos resíduos da agroindustrialização da mandioca como fonte de carbono para fermentação alcoólica, buscando um etanol, que possua qualidades adequadas para uso na indústria alimentícia. No sentido de promover uma alternativa viável, tanto tecnologicamente como economicamente, traçou-se um planejamento experimental que minimizasse os custos. Preocupou-se também, com a sustentabilidade ambiental do processo, pois a produção de álcool etílico pelo método aqui proposto, acena como uma alternativa para que o resíduo gerado, na indústria da mandioca, passe ao status de co-produto, gerando para a indústria não só receita como todos os benefícios mercadológicos de uma indústria limpa. O resíduo utilizado foi o amiláceo pré-hidrolizado que após o processo de fermentação foi destilado e teve seu produto separado e analisado. Desenvolveu-se uma coluna de destilação com recheio de vidro, para operação contínua a fim de estabelecer uma planta pré-piloto, onde ocorram os processos de hidrólise, fermentação e destilação. O substrato escolhido foi submetido a diversos ensaios e, o etanol obtido foi avaliado utilizando metodologias analíticas de derivatização. Para suprir as leveduras alcoólicas dos requisitos nutricionais necessários à sua manutenção durante o processo de fermentação, adicionou-se concentrações crescentes de mel residuário de sacarose aos substratos amiláceos previamente hidrolisados, nas proporções de 5, 10, 15 e 20% . Estas soluções foram padronizadas buscando manter a mesma concentração de carbono orgânico e na etapa subseqüente foram tratadas com enzimas amilolíticas para sacarificação completa. Após o inóculo com leveduras Saccharomyces cerevisiae, as fermentações foram...(Resumo completo, clicar acesso eletrônico abaixo)
Abstract: In this study an evaluation of the residue from the agricultural industrialization of cassava root is aimed. The main goal of this evaluation was towards the potential use of the root as a carbon source to the ethanol production via fermentation. The ethanol obtained must have characteristics good enough to be used in the food industry. Keeping in mind that, nowadays, industrial process must be sustainable and also economically feasible, an experimental scheme was designed. If the second goal may be proved, the first one appears immediatelly; due to promote an alternative use for a residue that otherwise may impact the environment. The experiment begins with the pre-hydrolysed amylaceous that is a by-product from the production of cassava flour. This starch was submited to fermentation under optimized conditions, the wine resultant went trough destilation, the final product was separated and analised. A destilation column, containing glass pearls, was designed in order to integrate a pilot project that may work in a process of continuos operation. This pilot plant was designed in order to achieve the process of hydrolysis, fermentation and destilation. The chosen substrate was then submited to various tests at diverse conditions and the ethanol obtained was evalueted trhough analytical methods with derivatization. In order to optimize the fermentation process by giving to the yeast better conditions of development, molasses, from the sucrose production, was added. The additions were of 5, 10, 15 and 20 %. These solutions were standardized in order to keep the same organic carbon content and after this were submited to a treatment with amylolitc enzymes. This treatment goal was to promote a complete saccharification...(Complete abstract, click electronic access below)
Mestre

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The objective of this work was to evaluate the hydrolysis and alcoholic fermentation stages of the sugar cane bagasse (SCB) for second generation ethanol production (2G). Hydrolysis experiments were carried out employing 8%, 10%, 15% and 20% (m/v) solid loads. Samples of in natura SCB and steam exploded SCB were submitted to different pretreatment sequences: water + 4.0% NaOH solution; 1.0% H2SO4 solution + 4.0% NaOH 4%; 1.0% H2SO4 solution + 7.0% NaOH solution; 7.0% NaOH solution; aqueous ammonia; water. Pretreated bagasse was characterized (chemical analysis and SEM) and the mass yield of each sequence was evaluated. This material was employed in hydrolysis experiments using different enzyme loads (33, 65 and 98 FPU/g-cellulose). Three different configurations were evaluated: SHF Separated Saccharification and Fermentation, SSF Simultaneous Hydrolysis and Fermentation, and FB Fed-Batch. In these experiments it was employed Accelerase 1500 enzymatic extract (Genencor). Concerning cellulose loss, SCB pretreated with water and 4.0% of NaOH solution presented the best result (23.9%). For this case, the hemicellulose and ligning removal was 73.7% and 79.1%, respectively. For the others pretreatment sequences the hemicellulose and lignin removal range from 72.3 to 92.2%. For lignin removal the values range from 60.0 and 88.9%. Experiments employing high solid load (20%) resulting in a more concentrated hydrolyzed (ca. 110 g/L). In this experiment it was achieved 64% of cellulose to glucose conversion in 34 hour. The fermentation experiments were carried out employing Saccharomyces cerevisiae yeast (lyophilized commercial). The yield ranges from 75 to 94%. Experiment conducted in the SSF configuration achieved 86.5% of cellulose to ethanol conversion in 14 hours. Experiment carried out in SHF configuration achieved 63.2% of cellulose to ethanol conversion in 37 hours. Experiments carried out in FB configuration reached larger amount of glucose with the same enzyme load employed in batch experiment and at the same time.
O objetivo deste trabalho foi avaliar as etapas de hidrolise enzimatica e fermentacao do caldo hidrolitico do bagaco de cana-de-acucar (BCA) para producao de etanol de segunda geracao (2G). Foram realizados experimentos de hidrolise enzimatica empregando cargas de solidos de 8%, 10%, 15% e 20% (m/v). Amostras de BCA innatura e explodido a vapor foram submetidas a diferentes sequencias de pretratamentos: agua + solucao de NaOH 4%; solucao de H2SO4 1% + solucao de NaOH 4%; solucao de H2SO4 1% + solucao de NaOH 7%; solucao NaOH 7%; amonia aquosa 15%; agua. As amostras de bagaco foram caracterizadas quimica e estruturalmente (MEV). Os rendimentos em cada sequencia de pre-tratamento foram calculados. O material pre-tratado foi submetido a experimento de hidrolise enzimatica com diferentes cargas de enzimas (33, 65 e 98 FPU/g de celulose) e configuracoes (SHF Separated Hydrolysis and Fermentation, SSF Simultaneous Saccharification and Fermentation, e BA Batelada Alimentada). Nestes experimentos foi utilizado extrato enzimatico Accelerase 1500 (Genencor). Na etapa de pre-tratamento a amostra de BCA in natura pre-tratada com agua e solucao de NaOH 4% obteve a menor perda de celulose (23,9%). A remocao de hemicelulose e lignina foi de 73,7% e 79,1%, respectivamente. As demais sequencias de pretratamento obtiveram valores de remocao de hemicelulose entre 72,3% a 92,2% e de remocao de lignina entre 60,0% a 88,9%. Hidrolises realizadas com alta carga de solido (20%) apresentaram um hidrolisado mais concentrado em glicose (ca. 110 g/l), obtendo-se 64% de conversao em 34 horas. Apos a etapa de hidrolise seguiuse a etapa fermentacao. Esses experimentos foram realizados com a levedura Saccharomyces cerevisiae (liofilizado comercial) obtendo-se rendimentos na faixa de 75 a 94%. Experimento realizado na configuracao SSF atingiu 86,5% de conversao de celulose a etanol em 14 horas de experimento. Experimento na configuracao SHF obteve 63,2% de conversao de celulose a etanol em 37 horas. Experimento em BA obteve maior massa de glicose com a mesma quantidade de enzima utilizada no experimento em batelada e no mesmo tempo.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Universidade Estadual Paulista (UNESP)
Neste trabalho fez-se uma avaliação da utilização dos resíduos da agroindustrialização da mandioca como fonte de carbono para fermentação alcoólica, buscando um etanol, que possua qualidades adequadas para uso na indústria alimentícia. No sentido de promover uma alternativa viável, tanto tecnologicamente como economicamente, traçou-se um planejamento experimental que minimizasse os custos. Preocupou-se também, com a sustentabilidade ambiental do processo, pois a produção de álcool etílico pelo método aqui proposto, acena como uma alternativa para que o resíduo gerado, na indústria da mandioca, passe ao status de co-produto, gerando para a indústria não só receita como todos os benefícios mercadológicos de uma indústria limpa. O resíduo utilizado foi o amiláceo pré-hidrolizado que após o processo de fermentação foi destilado e teve seu produto separado e analisado. Desenvolveu-se uma coluna de destilação com recheio de vidro, para operação contínua a fim de estabelecer uma planta pré-piloto, onde ocorram os processos de hidrólise, fermentação e destilação. O substrato escolhido foi submetido a diversos ensaios e, o etanol obtido foi avaliado utilizando metodologias analíticas de derivatização. Para suprir as leveduras alcoólicas dos requisitos nutricionais necessários à sua manutenção durante o processo de fermentação, adicionou-se concentrações crescentes de mel residuário de sacarose aos substratos amiláceos previamente hidrolisados, nas proporções de 5, 10, 15 e 20% . Estas soluções foram padronizadas buscando manter a mesma concentração de carbono orgânico e na etapa subseqüente foram tratadas com enzimas amilolíticas para sacarificação completa. Após o inóculo com leveduras Saccharomyces cerevisiae, as fermentações foram...
In this study an evaluation of the residue from the agricultural industrialization of cassava root is aimed. The main goal of this evaluation was towards the potential use of the root as a carbon source to the ethanol production via fermentation. The ethanol obtained must have characteristics good enough to be used in the food industry. Keeping in mind that, nowadays, industrial process must be sustainable and also economically feasible, an experimental scheme was designed. If the second goal may be proved, the first one appears immediatelly; due to promote an alternative use for a residue that otherwise may impact the environment. The experiment begins with the pre-hydrolysed amylaceous that is a by-product from the production of cassava flour. This starch was submited to fermentation under optimized conditions, the wine resultant went trough destilation, the final product was separated and analised. A destilation column, containing glass pearls, was designed in order to integrate a pilot project that may work in a process of continuos operation. This pilot plant was designed in order to achieve the process of hydrolysis, fermentation and destilation. The chosen substrate was then submited to various tests at diverse conditions and the ethanol obtained was evalueted trhough analytical methods with derivatization. In order to optimize the fermentation process by giving to the yeast better conditions of development, molasses, from the sucrose production, was added. The additions were of 5, 10, 15 and 20 %. These solutions were standardized in order to keep the same organic carbon content and after this were submited to a treatment with amylolitc enzymes. This treatment goal was to promote a complete saccharification...(Complete abstract, click electronic access below)

Fatores ambientais e econômicos impulsionam mundialmente a produção de biocombustíveis. No Brasil, o etanol, produzido da cana-de-açúcar, já é um biocombustível estabelecido e substitui ca. 40% da gasolina, representando 13% do total de energia necessária para transportes. Nesse cenário, o Rio Grande do Sul (RS) é um grande comprador de etanol, tendo produzido nos últimos anos apenas 2% do consumo estadual de etanol hidratado combustível. O estado também consome em média 600 milhões de litros de etanol anidro por ano, adicionados na proporção de 25% à gasolina comum, e, a partir de 2010, 460 milhões de litros de etanol por ano para a produção de polietileno verde. Essa conjuntura demonstra uma oportunidade para aumentar a produção local de etanol. O presente trabalho buscou primeiramente avaliar a viabilidade da produção de etanol no Rio Grande do Sul em um modelo de microusinas descentralizadas (ca. 1.000 L.dia-1). Para tanto, foram empregados os indicadores econômicos, como valor presente líquido, taxa interna de retorno de investimento e tempo de retorno de investimento. Foram comparados cenários que empregaram apenas cana-de-açúcar e combinações de cana-de-açúcar, sorgo sacarino, mandioca e batata-doce. A utilização de cana-de-açúcar sem o consórcio com outra cultura se mostrou inviável, exceto quando mais de 40% ou 80% da produção, para as produtividades de 80 e 50 t.ha-1 respectivamente, é destinada ao consumo próprio. Entre os cenários com combinação de culturas, aqueles que combinaram sorgo com cana-de-açúcar e sorgo com batata-doce foram os únicos que se mostraram viáveis quando toda a produção foi destina à venda para terceiros. Quando produtividades médias de cana-de-açúcar próximas a 80 t.ha-1 podem ser alcançadas, verificou-se que a combinação dessa cultura com sorgo sacarino apresentou o melhor potencial entre os cenários avaliados. Já para regiões onde esses valores não são atingidos, o consórcio de sorgo sacarino e batata-doce se mostrou a melhor opção. Posteriormente, foram realizados experimentos em shaker para estudar a influência da concentração de substrato e da proporção de enzima na hidrólise a frio da batata-doce, determinar o melhor pré-tratamento, verificar a necessidade de suplementação do meio e do controle de pH na condução das hidrólises e fermentações simultâneas e finalmente testar a melhor condição em biorreator. Para hidrolisar o amido, empregou a mistura de enzimas Stargen™ 002 e, para suplementar o meio, o fertilizante NITROFOS KL. Em todos os experimentos, usou-se a cultivar BRS Cuia, cuja caracterização indicou teor de carboidratos de 28,7%, possibilitando a produção de 185 L.t-1 de etanol e 7.400 L. ha-1. A metodologia de superfície de resposta indicou a condição 200 g.L-1 de batata-doce e 45 GAU.g de batata–doce-1 como a que apresentou o melhor compromisso entre alta taxa de formação de glicose na primeira hora (8,3 g.L-1.h-1) e baixo consumo de enzimas. O pré-tratamento de uma hora que levou a maior concentração de glicose (14,3 g.L-1) foi na temperatura de 52°C na presença da mistura de enzimas. O estudo da hidrólise e fermentação simultâneas mostrou que a suplementação do meio não apresenta influência significativa, enquanto o controle de pH aumentou em aproximadamente 40% a produção de etanol. Os testes em biorreator reproduziram os resultados anteriores mesmo sendo realizados em ambiente semiestéril, que se aproxima da condição industrial.
In Brazil, sugarcane ethanol is already a reality as a biofuel and replaces 40% of gasoline, meaning 13% of energy for transportation. In this scenario, Rio Grande do Sul has produced only 2% of the annual demand for hydrate ethanol in the last years; therefore it is a big importer of ethanol from other states. Additionally, it consumes every year 600 million liters of anhydrous ethanol mixed in the gasoline and 460 million liters for production of green plastic. These numbers highlight the opportunity of producing ethanol locally. In regard to these facts, the present work aimed to evaluate the feasibility of producing ethanol in small scale plants (ca. 1 000 L.dia-1) in Rio Grande do Sul. For this purpose, economic indicators, such as net present value, internal rate of return and payback period were employed. The compared scenarios involved combinations of sugar cane, sweet sorghum, cassava and sweet potato. When only sugarcane was used, the scenarios for 80 and 50 t.ha-1 were unfeasible if less than 40% or 80% of the production, respectively, was used by the own producer. Regarding the scenarios with mix of crops, the mix of sweet sorghum with sugar cane and sweet sorghum with sweet potato presented the best performances. For regions, where 80 t.ha-1 of sugar cane productivity can be achieved, it was verified that the first combination leads to the best result. For other regions, the combination of sweet sorghum and sweet potato presented itself as the more feasible scenario. As a consequence, in the second part of this work, the cold hydrolysis and fermentation of sweet potato was experimentally studied. For that, it was employed the sweet potato strain BRS Cuia, whose carbohydrate level reaches 28,7%. What it can be translated into a potential to produce 185 L.t-1 ethanol, or equivalently 7 400 L. ha-1. The enzymes blend adopted for the hydrolysis stage was Stargen™ 002, while the fertilizer NITROFOS KL was used for fermentation medium supplementation. The surface response method indicated 200 g.L-1 of sweet potato and 45 GAU.g of sweet potato-1 as the best balance between high glucose formation rate in the first hour (8,3 g.L-1.h-1) and low enzyme consume. The one hour pre-treatment that achieved the highest glucose concentration (14,3 g.L-1) was at 52°C in the presence of the enzymes blend. The study of the simultaneous hydrolysis and fermentation showed that the medium supplementation has no significant effect over the fermentation performance, while the pH control is beneficial, improving the ethanol production in 40%. Finally, the tests in bioreactor could reproduce the previous results, even though the experiments were carried out non-sterilely.

Atualmente o consumo de xarope de maltose vem crescendo devido ao seu uso em cervejarias e está substituindo progressivamente os adjuntos amiláceos. O xarope de maltose é, tradicionalmente, produzido por meio da hidrólise ácida e/ou enzimática de amido ou flocos de milho. Este trabalho teve como objetivo analisar a possibilidade de obtenção de maltose a partir de outras matérias-primas amiláceas como a mandioca e a puba (produto derivado da fermentação da mandioca) pela ação da a-amilase bacteriana e da a-amilase fúngica sem que fosse necessária a extração do amido. Amostras de mandioca e puba com 10, 20 e 30% de sólidos foram incubadas com a-amilase bacteriana termoestável durante 10, 20 e 30 minutos a 80 0 C, adicionando-se em seguida, µ-amilase fúngica e incubando-se as amostras durante 48 horas a 55 0 C. O grau de sacarificação, expresso em dextrose equivalente (DE), foi determinado pelo método DNS em vários intervalos de tempo. Glicose e maltose foram determinadas por HPLC após 48 horas de sacarificação. Os resultados mostraram que o tempo de ação da a-amilase bacteriana não causou diferenças significativas no grau de hidrólise entre as amostras, mas a concentração de sólidos influiu significativamente no grau da liquefação das amostras. O comportamento das curvas do grau de sacarificação foi semelhante para todos os tratamentos tanto da mandioca quanto da puba. O conteúdo de maltose nas amostras variou entre 30-60% e a glicose entre 0-10% caracterizando um xarope com alto teor de maltose. A eficiência de hidrólise ficou abaixo do esperado. Entretanto, esse fato pode ser explicado pela utilização da mandioca sem extração prévia do amido e pelas dificuldades na extração dos sólidos por centrifugação. Pode-se afirmar que tanto a mandioca quanto à puba podem ser utilizadas como matéria prima em substituição ao milho na obtenção de xarope de maltose através da hidrólise enzimática. A puba, porém, é de mais fácil manuseio sendo que o tratamento com 20% de sólidos, exposto durante 10 minutos a a-amilase bacteriana proporcionou maior rendimento, atingindo 4,2 kg de maltose e 0,3 kg de glicose por 100 kg de mandioca fresca além de proporcionar menor quantidade de resíduo sólidos de 13,7 kg.
Nowadays the consumption of maltose syrups is increasing due to its utilization in breweries where it replaces starch adjuncts. Traditionally maltose syrup has been produced from cornstarch or pellets using acid and/or enzymatic hydrolysis. The objective of this work was to evaluate the possibility of obtaining maltose from starch sources other than maize, such as cassava roots or puba, a fermented cassava product, without extraction of the starch, using bacterial a-amylase and fungal a-amylase for starch hydrolysis. Cassava and puba samples with 10, 20 and 30% dry matter were incubated with termostable bacterial a-amylase for 10, 20 and 30 minutes at 80 0 C, followed by the addition of fungal a-amylase. The samples were incubated for 48 hours at 55 0 C. The degree of saccharification, expressed as dextrose equivalent (DE), was determined by the DNS method. The glucose and maltose contents of the hydrolysate were determined after 48 hours by HPLC. The results showed that the time of action of a-amylase did not influence the degree of saccharification of the samples but the solids concentration significantly affected the hydrolysis degree. The saccharification degree curves were similar for both cassava and puba. The maltose content of the samples varied between 30-60% and the glucose content between 0-10%, which characterized them as "High maltose syrups". The hydrolysis efficiency was lower than expected. However, this fact could be explained by the use of cassava without extraction of the starch and by the difficulty of extracting the solids by centrifugation. It was concluded that for replacement of corn starch, both cassava roots and puba could be used as raw materials for maltose syrup production by enzymatic hydrolysis. However the puba was easier to handle than cassava. The puba treatment consisting of 20% of solids and 10 minutes exposure to a-amylase gave the highest yield reaching 4.2 kg of maltose and 0.3 kg of glucose per 100 kg of raw cassava, in addition to a lower solids residue of 13.7 kg.

Thermal processing has been used as a means of modifying the sensory aspects of wine. Cabernet Sauvignon wines were heated prior to dejuicing (3ºC per day from 25ºC to 42ºC) or after bottling (42ºC for 21 days) to determine the effects on total glycosides and glycosidic fractions. Total and phenol-free glycosidic concentrations in the wine and skins were quantified by analysis of glycosyl-glucose. Pre-dejuicing thermal vinification resulted in higher total glycosides (12%), phenol-free glycosides (18%), total hydroxycinnamates (16%), large polymeric pigments (LPP) (208%) small polymeric pigments (SPP) (41%), and lower monomeric pigments (42%) in wines. Skins had lower total glycosides (-16%), and no significant difference in phenol-free glycosides. Post-bottling heat treatment resulted in lower total (-15%) and phenol-free (-16%) glycosides, increased hue (25%), a 62% increase in LPP and a 29% decrease in monmeric pigments. A second study investigated the potential of enological spoilage microorganisms to affect wine aroma, flavor, and color. The activities of b-glucosidase were determined in model systems for fourteen strains of Brettanomyces bruxellensis yeast and nine strains of lactic acid bacteria (Oenococcus oeni). All Brettanomyces strains and seven Oenococcus strains exhibited enzymatic activity. B. bruxellensis b-glucosidase activity was primarily intracellular; O. oeni showed some extracellular activity. Yeasts and bacteria showing activity greater than 1000 nmole mL-1 g -1 for Brettanomyces, or 100 nmole mL-1 g -1 for Oenococcus, were evaluated for their effect on Viognier grape glycosides. Neither was active on native grape glycosides.
Master of Science

Atualmente, o aquecimento global, a deterioração ambiental, a eminente escassez das limitadas reservas de combustíveis fósseis e a forte demanda e dependência da matriz energética mundial face a esses combustíveis constituem sérias ameaças à sustentabilidade do desenvolvimento mundial, o que se têm revertido a favor de um crescente interesse por fontes alternativas de energia renováveis, dentre os quais a biomassa lignocelulósica se destaca como uma das mais promissoras. Neste contexto, a produção de etanol a partir do bagaço de cana-de-açúcar pela via enzimática e bioquímica se apresenta como uma das mais oportunas alternativas para a produção integrada e sustentável de biocombustível visando otimização de recursos, redução de resíduos e minimização de impactos ambientais nefastos. Todavia, o desenvolvimento de um pré-tratamento eficaz, a par da produção de enzimas hidrolíticas, constitui a chave para a viabilização da produção de etanol a partir de lignocelulósicos. Assim sendo, o propósito deste trabalho foi determinar os efeitos produzidos pelo pré-tratamento hidrotérmico catalisado com sulfito sobre a composição química do bagaço de cana e correlacionar esses valores com a eficiência da sacarificação enzimática e fermentabilidade do hidrolisado obtido desse bagaço. Após caracterização química inicial, o bagaço in natura foi pré-tratado com sulfito neutro e alcalino e verificou-se que a celulose foi recuperada quase que integralmente nas frações sólidas da reação. Por seu turno, as xilanas obtiveram solubilizações máximas de 72,0% e 35,4% enquanto a deslignificação máxima foi de 43,5% e 76,7%, para as reações catalisadas com sulfito neutro e sulfito alcalino respectivamente. Verificou-se maior efetividade do sulfito alcalino no enriquecimento de carboidratos no bagaço pré-tratado bem como uma maior deslignificação, pelo que, foi escolhido para as etapas seguintes do projeto. A partir de um planejamento fatorial 33, foram obtidos substratos pré-tratados que levaram aos máximos de sacarificação enzimática de 79,91% e 70,69% para celulose e xilanas, respectivamente, utilizando 10 UPF de celulases e 10 UCB de ?-glucosidase. Pela análise do rendimento global de glucanas foi definida a condição ótima para o prétratamento empregando 10% (m/m) de sulfito alcalino a 150ºC, por 30 min, o que foi corroborada pela análise estatística. Hidrolisados enzimáticos de bagaços pré-tratados em condições otimizadas foram fermentados a etanol com eficiência de, praticamente, 100% pela Saccharomyces cerevisiae PE-2 e 80,41% pela Scheffersomyces (Pichia) stipitis CBS 5773. Para as duas leveduras, os parâmetros fermentativos do hidrolisado foram em tudo semelhantes aos verificados para os meios semissintéticos contendo glicose e xilose puras. Conclui-se assim, que o pré-tratamento adotado neste trabalho apresentou grande potencialidade em gerar substratos com alta digestibilidade enzimática e hidrolisados tão fermentáveis quanto meios semissintéticos.
Currently, global warming, environmental degradation, imminent shortage of limited fossil fuel reserves and an intense demand and dependence of global energy on these fuels constitute serious threats to the sustainability of world development, which have led to a growing interest in alternative sources of renewable energy, among which lignocellulosic biomass stands out as one of the most promising. In this context, the production of ethanol from sugarcane bagasse by enzymatic and biochemical routes appears as the most promising alternative for integrated and sustainable production of biofuel aiming resource optimization, waste reduction and mitigation of adverse environmental impacts. However, the development of an effective pretreatment, as well as the production of hydrolytic enzymes, is the key to enabling the production of ethanol from lignocellulosics. Therefore, the purpose of this study was to determine the effects of hydrothermal pretreatment catalyzed by sulfite on the chemical composition of sugarcane bagasse and establish a correlation of these values with the efficiency of enzymatic saccharification and fermentability of these bagasse hydrolyzate. After the initial chemical characterization, the bagasse was pretreated with neutral and alkaline sulfite and was found that cellulose was almost fully recovered in the solid fraction of the reaction. In turn, xylan obtained its maximum solubilization of 72.0% and 35.4% while the maximum delignification was 43.5% and 76.7%, for the reactions catalyzed by neutral and alkaline sulfite, respectively. The alkaline sulfite was found more effective for carbohydrates enrichment in the pretreated bagasse and led to a greater delignification, therefore, it was chosen for the following stages of the project. From a 33 full factorial experiments were obtained pretreated substrates which led to maximum enzymatic saccharification of 79.91% and 70.69% for cellulose and xylans, respectively, using 10 FPU cellulases and 10 CBU ?- glucosidase. By analysis of the glucans overall yield the optimal condition for the pretreatment has been defined as 10% (w/w) alkaline sulfide at 150°C for 30 min, which was confirmed by statistical analysis. Enzymatic hydrolysates of bagasse pretreated in optimized conditions were fermented into ethanol with efficiency of virtually 100% by Saccharomyces cerevisiae PE-2 and 80.41% by Scheffersomyces (Pichia) stipitis CBS 5773. For both yeasts, the hydrolyzate fermentation parameters were at all similar to those seen for the semisynthetic media containing pure glucose and xylose. Therefore, we concluded that the pre-treatment used in this work showed great potential to generate substrates with high enzymatic digestibility and hydrolysates as fermentable as semisynthetic media.

A utilização de resíduos agroindustriais como fontes lignocelulósicas para a obtenção de diversos insumos químicos é uma alternativa para contribuir para a valorização destes subprodutos. Neste contexto, o etanol produzido a partir de materiais lignocelulósicos torna-se uma opção interessante para aumentar a produção deste combustível sem aumentar a área plantada das colheitas utilizadas para sua produção, já que a demanda de etanol vem aumentando cada vez mais nos últimos anos, com o objetivo de substituir o petróleo e seus derivados, contribuindo significativamente para a redução dos impactos negativos ao meio ambiente, tais como o aquecimento global provocado pela queima dos combustíveis fósseis. Para que a produção de etanol celulósico seja economicamente viável é necessário que estas fontes lignocelulósicas sejam fracionadas de forma a disponibilizar a maior quantidade de carboidratos possível para o processo de fermentação alcoólica. Neste trabalho propôs-se avaliar a sacarificação enzimática dos materiais, palha de cana-de-açúcar, bagaço de cana-deaçúcar e pseudocaule de bananeira, nas formas -in natura?, pré-tratada e deslignificada de forma a verificar o efeito do pré-tratamento e da deslignificação no aumento da conversão enzimática da celulose de cada biomassa vegetal. Todos os materiais lignocelulósicos foram caracterizados quimicamente nas formas -in natura?, pré-tratada e deslignificada. Análises de FTIR para cada biomassa comprovaram a alteração na estrutura química proporcionada pelo pré-tratamento e deslignificação destes materiais. O pré-tratamento com H2SO4 1,0% (m/v) a 120 °C por 10 min, seguido de deslignificação com NaOH 1,0% (m/v) a 100 °C por 1h, ambos em reator piloto agitado de 350 L promoveu uma solubilização de 88,8 % de hemicelulose e 77,9% de lignina para a palha de cana, e uma solubilização de 79,3% de hemicelulose e 62,3% de lignina para o pseudocaule de bananeira. Já o bagaço de cana foi pré-tratado hidrotermicamente em reator de 20 L, nas condições, 180 °C/10 min, 185 °C/10 min, 190 °C/10 min e 195 °C/10 min, sendo que esta última condição, seguida de deslignificação com NaOH a 1,0% (m/v) a 100 °C por 1h proporcionou uma solubilização de 95,8% de hemicelulose e 80,9% de lignina para este material. Os ensaios de conversão enzimática dos materiais lignocelulósicos mostraram que a conversão celulósica aumentou consideravelmente, para todos os materiais, após o pré-tratamento seguido de deslignificação, atingindo 85% de conversão para a palha de cana, 89,2% de conversão para o bagaço de cana e 61,0% de conversão para o pseudocaule de bananeira. Após as etapas de pré-tratamento e deslignificação alcalina, os materiais lignocelulósicos apresentaram uma estrutura morfológica modificada, com as células vegetais livres de células de parênquima, conforme verificado pelas análises de MEV, e com redução da cristalinidade da celulose remanescente, conforme mostrado pelas análises de Difratometria de Raios X. Ensaios de fermentabilidade dos hidrolisados celulósicos de bagaço de cana utilizando a levedura Candida guilliermondii mostraram uma boa resposta da levedura à produção de etanol alcançando uma concentração máxima de 20 g/L.
The use of agroindustrials residues about lignocellulosics sources to obtain many chemicals products it\'s an alternative to contribute for the valuation of these subproducts. In this context, the ethanol produced by lignocellulosic materials it´s an interesting option for increase the production of this fuel without increase the agriculture area for production of biofuels, since the ethanol demand has increasing even more in the last years, with the objective of substitute the oil and his derivates, contribute significatively for reduce of negative impacts for environment, about the greenhouse gas impacts produced by burning of fossil fuels. For that production of cellulosic ethanol will be economic favorable it´s necessary that lignocellulosics sources will be fractionates for to make it available many carbohydrates possible for alcohol fermentation processes. This work had objective to evaluate enzymatic saccharification of materials, sugar cane straw, sugar cane bagasse and pseudosteam of banana, in this raw, pretreated and delignificated forms, for analyze the effect of pretreatment and delignification on the increase of enzymatic saccharification of cellulose wich biomass. Every lignocellulosic materials went submitted a chemical characterization in this raw, pretreated and delignificated forms. FTIR analysis for each biomass confirmed the change of chemical structure challenge for pretreatment and delignification of this materials. The pretreatment with H2SO4 1.0% (m/v), 120 °C, 10 min, followed of delignification with NaOH 1.0% (m/v), 100 °C, 1h, both in 350 L agitated reactor, promoted a solubilization of 88.8% for hemicellulose and 77.9% of lignin for sugar cane straw, and a solubilization of 79.3% for hemicellulose and 62.3% of lignin for pseudosteam of banana. The sugar cane bagasse went carried hydrothermal processing pretreatment on reactor of 20 L, in this conditions, 180 °C/10 min, 185 °C/10 min, 190 °C/10 min e 195 °C/10 min, although in this last condition, followed of delignification with NaOH 1,0% (m/v), 100 °C for 1h to affored a solubilization of 95.8% for hemicellulose and 80.9% of lignin for this material. The experiments of enzymatic saccharification of lignocellulosic materials showed that cellulosic conversion increased considerably, for all materials, after pretreatment followed of delignification, attaining 85% of conversion for the sugar cane straw, 89.2% of conversion for the sugar cane bagasse and 61% of conversion for the pseudosteam of banana. After pretreatments steps and alkaline delignification, the lignocellulosic materials showed changed morphologic estrutural, with vegetable cells lived of parenchyme cells, according to the SEM analysis, and with reduce of cellulose cristallinity remaining, according to the X Ray diffraction analysis. Experiments of fermentation of cellulosics hydrolysates of sugar cane bagasse using Candida guilliermondii it has showed a good reply of yeast for the ethanol production aimed the máxime concentration of 20 g/L.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
Neste trabalho, a enzima β-galactosidase, que catalisa a hidrÃlise da lactose em glicose e galactose, foi produzida pelo cultivo do micro-organismo Kluyveromyces lactis NRRL Y1564 em soro de leite suplementado com extrato de levedura. A enzima à um metabÃlito intracelular, sendo a liberaÃÃo da enzima para o meio uma condiÃÃo essencial, por isso estudaram-se diferentes mÃtodos quÃmicos e mecÃnicos de extraÃÃo como, agitaÃÃo com pÃrolas de vidro, ruptura em ultrassom com pÃrolas de vidro, adiÃÃo de etanol e tolueno, observando tambÃm, seus efeitos na atividade enzimÃtica. Posteriormente, realizaram-se ensaios para estudar a influÃncia da temperatura (30, 34, 37 e 40 ÂC) na produÃÃo da enzima a partir do soro de queijo. Na extraÃÃo da enzima, o uso de esferas de vidro em vÃrtex foi o mÃtodo mais eficiente quando comparado com os outros avaliados. A enzima, nÃo sofreu inibiÃÃo ou desnaturaÃÃo quando incubadas com etanol, tolueno ou etanol-tolueno. A temperatura Ãtima de produÃÃo da enzima por K. lactis NRRL Y1564 foi 30 ÂC, com atividade enzimÃtica de 4418,37 U/g de cÃlulas em 12 h de fermentaÃÃo. A enzima produzida foi imobilizada em quitosana 2,0% m/v. Diferentes protocolos de ativaÃÃo usando glutaraldeÃdo, epicloridrina ou glicidol foram avaliados. Estudou-se tambÃm, o tempo de contato enzima-suporte (3, 5 e 10 horas) a fim de se obter um biocatalisador que apresentasse alto rendimento, atividade recuperada e tempo de meia-vida, visando a hidrÃlise da lactose em reator batelada e leito fixo. A influÃncia da temperatura e do pH na hidrÃlise da lactose foi avaliada, usando como substrato uma soluÃÃo sintÃtica (lactose 5,0% (m/v) e leite desnatado, contendo 4,3% m/v de lactose. O suporte que apresentou melhores resultados nos parÃmetros de imobilizaÃÃo foi a quitosana 2% reticulada com glutaraldeÃdo no tempo de imobilizaÃÃo de 5 horas. O biocatalisador produzido nesse estudo apresentou um fator de estabilidade de 17,37 vezes maior que a enzima solÃvel, com uma estabilidade de armazenamento de 100% quando armazenada a 4 ÂC por 90 dias. A temperatura Ãtima de hidrÃlise da lactose foi de 40 ÂC e o pH Ãtimo foi 7,0 . A conversÃo da lactose a 40 ÂC para este derivado (3,0 U/g) foi em mÃdia 53% em 10 ciclos (bateladas consecutivas). Em reator batelada, a conversÃo em glicose a partir da hidrÃlise da lactose, usando soluÃÃo sintÃtica, foi aproximadamente 86 % para a enzima solÃvel (3,8 U/mL) e 83 % para a enzima imobilizada (3,8 U/g). A conversÃo obtida na hidrÃlise do leite desnatado foi de 17 % para a enzima solÃvel e 20 % para a enzima imobilizada.
In this work, the enzyme β-galactosidase which catalyzes the hydrolysis of lactose to glucose and galactose, was produced by cultivating the micro-organism Kluyveromyces lactis NRRL Y1564 in whey supplemented with yeast extract. The enzyme is an intracellular metabolite, the release enzyme is very important, therefore were studied various chemical and mechanical methods of extraction and stirring with glass beads, sonication, addition of ethanol and toluene, noting also their effects on enzymatic activity. Subsequently, trials were carried out to study the influence of temperature (30, 34, 37 and 40 Â C) in the production of the enzyme from the cheese whey. In the enzyme extraction, using glass beads by a vÃrtex was more efficient method compared to others evaluated. The enzyme not presented inhibited or denatured when incubated with ethanol, toluene or ethanol-toluene. The optimum temperature for enzyme production by K. lactis NRRL Y1564 was 30 ÂC with enzymatic activity of 4418.37 U/g of cells at 12 h of fermentation. The enzyme produced was immobilized on chitosan 2.0% w/v. Different activation protocols using glutaraldehyde, epichlorohydrin or glycidol were evaluated. Was also studied, the contact time the enzyme-carrier (3, 5, and 10 hours) to obtain a biocatalyst to produce high yield, recovered activity and half-life in order to hydrolysis of lactose in batch reactor and fixed bed. The influence of temperature and pH on the hydrolysis of lactose was evaluated using as substrate a synthetic solution (lactose 5.0% (w/v) in potassium phosphate buffer 100 mM with 0.1 mM MnCl2) and skimmed milk containing 4.3% w/v lactose. The support shows better results in the parameters of immobilization was chitosan 2% actived with glutaraldehyde and contact time of 5 hours. The biocatalyst produced in this study showed a stability factor of 17.37 and a storage stability of 100% when stored at 4 ÂC for 90 days. Temperature optimum hydrolysis of lactose was 40 ÂC and the optimal pH 7.0. The conversion of lactose to 40 ÂC for this derivative (3.0 U/g) was on average 53% in 10 cycles (consecutive batches). In batch reactor, the conversion to glucose by the hydrolysis of lactose using synthetic solution was approximately 86% for the soluble enzyme (3.8 U/mL) and 83% of the immobilized enzyme (3.8 U/g). The conversion obtained in the hydrolysis of skim milk was 17% for the soluble enzyme and 20% for the immobilized enzyme.

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Atualmente, a matriz energética brasileira centraliza toda a sua produção de bioetanol a partir da cana-de-açúcar, no entanto, tendo em vista a vasta biodiversidade encontrada em seu território, o Brasil dispõe de uma grande variedade de resíduos agrícolas e agroindustriais cujo bioprocessamento para a produção de bioetanol desperta um grande interesse econômico e social. Nesse contexto, a presente pesquisa teve como objetivo estudar o aproveitamento de sementes de jaca (resíduo) como fonte de amido para a produção de bioetanol. Visando à máxima remoção do amido presente nas sementes de jaca, as mesmas foram submetidas a etapas de extração e purificação. Parâmetros físico-químicos foram determinados tanto para as sementes quanto para o amido extraído das sementes de jaca. O material amiláceo obtido foi, então, submetido a tratamentos hidrolíticos, empregando os ácidos sulfúrico e fosfórico como catalisadores, seguindo um planejamento fatorial completo 23 com três pontos centrais. Foram definidas como variáveis independentes, a temperatura (80, 100 e 120 °C), o tempo de reação (30, 60 e 90 min.) e a concentração do ácido (1, 3 e 5%), e como variáveis dependentes, as concentrações de glicose e hidroximetilfurfural (g.L1 ) presentes nos licores hidrolisados. A fim de observar e avaliar o efeito dos tratamentos ácidos, realizados nas condições otimizadas de operação, sobre a superfície dos grânulos do amido extraído de sementes de jaca, a técnica de microscopia eletrônica de varredura (MEV) foi empregada. Os licores hidrolisados pelos ácidos sulfúrico e fosfórico, obtidos nas condições otimizadas de operação, foram submetidos a ensaios fermentativos em uma câmara incubadora com agitação orbital a 160 rpm, sob temperatura controlada de 32 ºC, empregando como microrganismo, a levedura industrial Saccharomyces cerevisiae Y904, e como nutrientes, o composto nutricional industrial Nitrofós. O material amiláceo extraído das sementes de jaca apresentou elevado teor de amido (85,5%). Maiores concentrações de glicose, 32,98 e 31,23 g.L-1 para os tratamentos hidrolíticos com os ácidos sulfúrico e fosfórico, respectivamente, concomitantemente, com concentrações de hidroximetilfurfural (HMF) que não inibissem o processo fermentativo, 0,53 e 0,08 g.L-1 para os tratamentos com os ácidos sulfúrico e fosfórico, respectivamente, foram encontradas no ensaio executado nas condições mais severas estabelecidas para a matriz de planejamento, sendo elas: 120 °C, concentração do ácido a 5% e 90 min. de reação. O tratamento hidrolítico do amido conduzido com o ácido sulfúrico apresentou maior eficiência de hidrólise (70,13%) quando comparada a eficiência alcançada pelo tratamento empregando-se o ácido fosfórico (66,41%). As micrografias revelaram os aspectos morfológicos característicos dos grânulos de amido de sementes de jaca, constatando-se formatos, predominantemente, elipsoides e hemisféricos, e tamanhos variando de 4 a 9 μm, bem como suas modificações estruturais causadas pela ação dos ácidos durante os tratamentos hidrolíticos. Os ensaios fermentativos realizados com os licores hidrolisados pelos ácidos sulfúrico e fosfórico apresentaram eficiências de fermentação de 97,85 e 95,89%, respectivamente, indicando, portanto, que a levedura foi capaz de metabolizar, praticamente, todo o substrato limitante (glicose) presente nos licores em etanol. Considerando os resultados obtidos em todas as etapas realizadas durante a presente pesquisa, conclui-se que o amido extraído de sementes de jaca apresentou-se como uma matéria-prima promissora para a produção de bioetanol.
Currently, the Brazilian energy matrix centralizes all their production of bioethanol from sugar cane, however, in view of the vast biodiversity found in its territory, Brazil has a wide variety of agricultural and agro-industrial waste whose bioprocessing for bioethanol production arouses a great economic and social interest. In this context, the present research was to study the use of jackfruit seeds (residue) as a source of starch for the production of bioethanol. For ultimate removal of this starch in jackfruit seeds, they were subjected to extraction and purification steps. Physical and chemical parameters were determined for both the seeds and the starch extracted from jackfruit seeds. The starch material obtained was then subjected to hydrolytic treatments using sulfuric and phosphoric acids as catalysts, following a full 23 factorial design with three central points. We defined as independent variables the temperature (80, 100, 120 °C), the reaction time (30, 60 and 90 min.) and the concentration of the acid (1, 3 and 5%), the dependent variables were the glucose concentrations and hydroxymethylfurfural (g.L-1) present in the hydrolyzed liquor. In order to observe and evaluate the effect of acid treatments, based on the optimized operating conditions on the surface of starch granules extracted from jackfruit seeds, scanning electron microscopy technique (SEM) was used. The liquors hydrolyzed by sulfuric and phosphoric acids, obtained in the optimized operating conditions, underwent fermentation trials in a dry incubator with orbital shaking at 160 rpm, under controlled temperature of 32 °C, using industrial yeast Saccharomyces cerevisiae Y904 as microorganism, and the industrial nutritional compound Nitrophós as nutrients. The starch material extracted from jackfruit seeds showed high starch content (85.5%). We found higher concentrations of glucose, 32.98 and 31.23 g.L-1 to hydrolytic treatment with sulfuric and phosphoric acids respectively, concurrently, with concentrations of hydroxymethylfurfural (HMF) which would not inhibit the fermentative process, 0.53 and 0, 08 g.L-1 for treatment with sulfuric and phosphoric acids, respectively. These were found in the test run under the most severe conditions attached to the planning matrix, namely, 120 °C, 5% acid concentration and 90 min. reaction. The hydrolytic treatment of starch conducted with sulfuric acid hydrolysis showed higher efficiency (70.13%) compared to treatment efficiency achieved by employing phosphoric acid (66.41%). The micrographs revealed the characteristic morphology of jackfruit seed starch granules, confirming hemisphere and ellipsoid formats, and sizes ranging from 4 to 9 μm, as well as its structural modifications caused by the action of acid during the hydrolytic treatment . The fermentation trials with liquors hydrolyzed by sulfuric and phosphoric acids showed fermentation efficiencies of 97.85 and 95.89%, respectively, thus indicating that the yeast was capable of metabolizing substantially all the limiting substrate (glucose) present in liquors from ethanol. Considering the results obtained in all steps undertaken during this research, it is concluded that the extracted starch jackfruit seeds appeared as a promising raw material for the production of bioethanol.

Thesis (MBA (Business Management))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: The objective of the study was to establish whether a small-scale biomass renewable energy project that uses waste fruit and pomace as the predominant feedstock could be a feasible clean development mechanism (CDM) project. The study was based on the solid waste streams of a pome fruit processor. Renewable energy technologies that convert biomass into a methane rich gas were evaluated. These included the various anaerobic digestion technologies that yield biogas and gasification technologies that yield syngas. Gasification was not found to be a feasible technology due to the moisture content requirement of less than 20% against that of the biomass of 70-80% and due to the low bulk density of the biomass of 250 kg/m3 versus the required minimum of 500kg/m3. The biogas could either be fired in a combined heat and power (CHP) unit or in a dual fired burner with heavy furnace oil (HFO). Feeding the thermal energy and electricity from the CHP unit back to the processor or supplying the electricity to the national electricity grid was considered. The plant would supply electricity to the national electricity grid as an Independent Power Producer (IPP). Eskom would be the renewable energy purchasing agent (REPA) that purchases the electricity at an anticipated R0.96/kWh under a power purchase agreement (PPA) in terms of the renewable energy feed-in tariff phase II (REFIT) guidelines. The anticipated revenue for electricity was six fold the coal offset cost of R0.16/kWh, while there was no demand for the heat energy after the processing season, thus firing biogas in a dual fired burner was not feasible. The most feasible technology was the anaerobic digestion of the biomass using a continuous stirred tank reactor (CSTR) process followed by a CHP unit that feeds the heat to the processing facility and the electricity into the national electricity grid. Waste fruit and wet pomace would be codigested with abattoir waste during the operating season. Dried pomace, abattoir waste, waste fruit and any other non-woody material from the surrounding farms and packhouses would be digested after the season to optimize the utilisation of the plant capacity. A capacity of 11 dry tons per day for the case study at a cost of R13 138 889 yielded an IRR of 15.2% and a net present value (NPV) of - R1 498 616 based on a discount rate of 18%, when excluding revenue from the sale of carbon emission reductions (CERs). Therefore, without the sale of CERs, this project would be rejected as a non-feasible investment. The project is eligible for CDM registration as a small-scale renewable energy project activity. No barriers were identified that would prevent the registration of this project activity as a CDM project in South Africa. The sale of CERs increased the NPV of the optimum solution to R156 483 and the IRR to 18.3%. Registering the project as a CDM project activity improved the feasibility of the project to the extent that it was marginally feasible. The fermentation and hydrolysis of the biomass into bioethanol was investigated as an alternative to the generation of heat and electricity. Although the technology is proven, no facilities were identified that use spoilt apples or apple pomace as the substrate. This increased the risk of the project and a discount rate of 24% was set when calculating the project NPV. This project activity yielded an NPV of - R1 296 057 when excluding CDM revenue. The feasibility improved to an NPV of - R263 507 and an IRR of 23.4% when taking the additional revenue from the sale of CERs into account. This project alternative was only marginally out of the money. With the development of newer technology, this alternative could prove to be more attractive in the future.

Bioethanol is an environmentally friendly fuel, obtained from different renewable sources, which can be sweetened raw materials, starch or cellulose. One of the factors that influence cell proliferation and transformation efficiency of sugar into ethanol is the need of nutritional yeast during ethanol fermentation processes. The use of cassava for the production of ethanol as an alternative presents itself, generating mainly the improvement in income distribution in regions of low population density. However, further study is needed to process improvement, contributing to its viability. This study evaluates the fermentation kinetics of enzymatic and acid hydrolysates with and without nutritional supplementation, obtained mixing two types of cassava grown in the municipality of Junqueiro-AL. Fermentations were conducted in simple batch in oven at 30°C, with commercial yeast and nutritional supplement made with nutrient used in distilleries in the State. The kinetics of the fermentation process was monitored by determining the cell concentration (X), total soluble solids (Brix), reducing sugars (AR), total reducing sugars (ART) and ethanol (E). At the end of fermentation parameters were determined conversion factor substrate in cells (YX/S), conversion factor substrate in ethanol (YE/S) conversion factor cell in ethanol (YE/X), ethanol productivity (PE), cells productivity (PX), fermentation efficiency (nf (%)) and process efficiency (np (%)). The results showed that the use of acid and enzymatic hydrolysis treatment of flour generated sufficient sugar for the fermentations, showing satisfactory yields, being 80,15% ± 0,78 for the acid hydrolysis, and 98,02 ± 0,26 for enzymatic hydrolysis. The results obtained at the end of fermentation for the medium based of acid hydrolyzate without nutritional supplementation (HASN), the medium based of acid hydrolyzate with nutritional supplementation (HACN), the medium based of enzymatic hydrolyzate without nutritional supplementation (HESN) and the medium based of enzymatic hydrolyzate with nutritional supplement (HECN), in that order, were: 48,09, 48,11, 49,59 and 49,45 g/L for ethanol concentration; 1,57, 1,57, 1,63 and 1,60 g/L.h for productivity in ethanol (PE); 80,23, 82,14, 84,15 and 80,23% for the fermentation efficiency (nf (%)); and 76,45, 76,37, 82,40 and 79,86% for process efficiency (np (%)).
O etanol é um biocombustível ambientalmente correto, obtido de diversas fontes renováveis, que podem ser matérias-primas açucaradas, amiláceas ou celulósicas. Um dos fatores que influenciam na multiplicação celular e na eficiência de transformação de açúcar em etanol é a necessidade nutricional das leveduras durante o processo de fermentação etanólica. A utilização de mandioca para a fabricação de etanol apresenta-se como alternativa, gerando, principalmente, a melhoria na distribuição de renda nas regiões de baixa densidade populacional. Todavia, ainda é necessário estudos para aprimoramento do processo, contribuindo para sua viabilidade. Este trabalho avalia a cinética da fermentação dos hidrolisados ácido e enzimático, com e sem complementação nutricional, obtidos da mistura de dois tipos de farinha de mandioca cultivada no município de Junqueiro-AL. As fermentações foram conduzidas em batelada simples, em estufa à 30°C, com levedura comercial (fermento Fermix), vendida na forma de fermento em pó seco, e complementação nutricional feita com nutriente utilizado nas destilarias do estado. A cinética do processo fermentativo foi acompanhada através das determinações de concentração celular (X), sólidos solúveis totais (Brix), açúcares redutores (AR), açúcares redutores totais (ART) e etanol (E). Ao final das fermentações foram determinados os parâmetros fator de conversão substrato em células (YX/S), fator de conversão substrato em etanol (YE/S), fator de conversão células em etanol (YE/X), produtividade em etanol (PE), produtividade em células (PX), eficiência de fermentação (nf (%)) e eficiência de processo (np (%)). Os resultados mostraram que o emprego dos tratamentos ácido e enzimático na hidrólise da farinha gerou açúcar suficiente para as fermentações, apresentando rendimentos satisfatórios, sendo 80,15% ± 0,78 para a hidrólise ácida e 98,02 ± 0,26 para a hidrólise enzimática. Os resultados obtidos no final das fermentações para o meio à base de hidrolisado ácido sem complementação nutricional (HASN), o meio à base de hidrolisado ácido com complementação nutricional (HACN), o meio à base de hidrolisado enzimático sem complementação nutricional (HESN) e o meio à base de hidrolisado enzimático com complementação nutricional (HECN), nessa ordem, foram: 48,09, 48,11, 49,59 e 49,45 g/L para a concentração de etanol; 1,57, 1,57, 1,63 e 1,60 g/L.h para a produtividade em etanol (PE); 80,23, 82,19, 84,15 e 80,23% para a eficiência de fermentação (nf (%)); e 76,46, 76,37, 82,40 e 79,86% para a eficiência de processo (np (%)).

In the diploma thesis are discussed the process of enzymatic hydrolysis of waste paper as a source for the production of bioethanol by bacteria Zymomonas mobilis. In the theoretical part summarize basic information about particular methods of hydrolysis, about paper used as a raw material for enzymatic hydrolysis, about possibilities of the fermentative production of bioethanol focusing on the method of simultaneous saccharification and fermentation comparison with enzymatic hydrolysis and fermentation. Suitable microorganisms for ethanolic fermentation and simultaneous saccharification and fermentation and their advantages and disadvantages, are further discussed in this part as well. The theoretical part ends with the suggestion of the technological process for production of bioetanol. It covers all necessary steps from the input of raw material to the separation of produced ethanol. In the experimental part various parameters of hydrolysis, fermentation and simultaneous saccharification and fermentation were optimized using enzymes from Novozymes® company and the Zymomonas mobilis CCM2770 and Zymomonas mobilis LMG457 bacterium. The conversion rate of paper cellulose to gluckose and production of ethanol were observed by HPLC/RI method. Type of buffer, quantity of cells, enzyme and substrate were optimized in order to maximize the efficiency of the process. All experiments were performed on paper containing high amount of cellulose and for comparison on standard medium which contains gluckose. The highest yields was achieved with the use of Novozymes® Cellulosic ethanol enzyme Kit. The strain Zymomonas mobilis LMG457 has demonstrated as a better producer.

L’objectif de ce travail est d’étudier la valorisation des produits connexes de l’industrie gabonaise du bois d’okoumé dans un contexte de bioraffinerie. La production de bioéthanol cellulosique et de lignine en se basant sur un procédé de prétraitement transposable à l’échelle industrielle a été étudiée. Nos travaux, réalisés à l’échelle du laboratoire, portent sur (1) l’optimisation du prétraitement par explosion à la vapeur (EV), (2) l’extraction de la lignine de bois explosé par percolation avec de l’éthanol comme solvant, (3) la caractérisation des biopolymères (cellulose et lignine) et l’étude de l’impact des paramètres du prétraitement sur leur structure chimique et (4) la conversion du bois prétraité en éthanol par hydrolyse enzymatique et fermentation. Dans un premier temps nous nous sommes intéressés à optimiser les différents paramètres du procédé EV (granulométrie, pré-imprégnation, temps de séjour et température) concernant les teneurs en glucose, xylose et en lignine du bois explosé. L’impact du procédé sur la production de produits de dégradation des sucres (furfural et le HMF ) potentiellement inhibiteurs sur les enzymes et microorganismes a également été étudié. Il a été constaté que la taille des particules est un paramètre important à considérer dans l’amélioration du procédé, les particules fines conduisant à une dégradation importante de la cellulose durant le prétraitement. L’influence de la concentration en acide, utilisé pour imprégner la biomasse, ainsi que des paramètres du procédé (temps de séjour et température) sur l’hydrolyse des biopolymères (lignine et hémicelluloses) ont ensuite été étudiés. Des conditions permettant d’optimiser des teneurs en cellulose (> 59%) dans la matière prétraitée ont été retenues. Afin de mieux comprendre l’impact du prétraitement sur la lignine extraite à partir du bois explosé et anticiper une valorisation future, des analyses spectroscopiques (RMN HSQC et 31P) et chromatographiques (SEC) ont été réalisées. Une diminution de la teneur en β-O-4 est observée avec l’augmentation de la sévérité du traitement d’EV avec une dégradation quasi-totale des unités β-O-4 dans des conditions de plus grande sévérité (T = 210°C, t = 2,5 min et H2SO4 = 0,5%). Des phénomènes de recondensation par voie radicalaire de la lignine ont également été observés. La cellulose des différentes fractions solides récupérées après traitement EV a également été caractérisée par SEC, DRX pour étudier l’effet du traitement et identifier les paramètres clés dans l’amélioration de l’efficacition de la conversion de la cellulose en glucose. Une augmentation de la cristallinité avec l’augmentation de sévérité a été observée avec une chute importante du DP de la cellulose pour les EV réalisées avec imprégnation acide. La digestibilité enzymatique des résidus cellulosiques après EV par T. Reesei et l’extraction des lignines ont été étudiées. Les principaux paramètres affectant positivement la réactivité aux enzymes sont l’élimination d’une partie des polymères non cellulosiques et la chute de DP de la cellulose. A forte sévérité d’EV, une baisse de réactivité a été attribuée à la formation de pseudolignines. Les conditions optimales d’EV en vu d’une hydrolyse enzymatique sont les suivantes : T = 210°C, t = 5 min et H2SO4 = 0,25%. Une bonne fermentabilité des hydrolysats par Saccharomyces Cerevisiae a été obtenue. A partir de 100 Kg d’aubier d’okoumé, il est possible dans les conditions de notre étude de produire 11,9 Kg d’éthanol
The objective of this work is to study the valorization of co-products from the Gabonese okoumé wood industry in a biorefinery context. The production of cellulosic bioethanol and lignin based on a pre-treatment process that can be transposed to an industrial scale was studied. Our work, carried out on a laboratory scale, focused on (1) optimization of pre-treatment by steam explosion (SE), (2) extraction of lignin from exploded wood by percolation with ethanol as solvent, (3) characterization of biopolymers (cellulose and lignin) and study of the impact of pre-treatment parameters on their chemical structure, and (3) conversion of pre-treated wood to ethanol by enzymatic hydrolysis and fermentation. We were first interested in optimizing the different parameters of the SE process (particle size, preimpregnation, SE residence time and temperature) concerning the glucose, xylose and lignin contents of the exploded wood. The impact of the process on the production of sugar degradation products (furfural and HMF) potentially inhibiting enzymes and microorganisms was also studied. It was found that particle size is an important parameter to consider in process improvement, as fine particles lead to significant degradation of cellulose during pre-treatment. The influence of the concentration of acid, used to impregnate the biomass prior to SE, as well as process parameters (residence time and temperature) on the hydrolysis of biopolymers (lignin and hemicelluloses) was then studied. Conditions for optimizing cellulose contents (> 59%) in the pre-treated material were selected. In order to better understand the impact of pre-treatment on the lignin extracted from exploded wood and to anticipate future SE lignin utilizations, spectroscopic (HSQC and 31P NMR) and chromatographic (SEC) analyses were carried out. A decrease in β-O-4 content is observed with the increase in the severity of the treatment of SE with an almost total degradation of the β-O-4 units under conditions of highest severity (T = 210°C, t = 2.5 min and H2SO4 = 0.5%). Recondensation of lignin through radical coupling processes was also observed. The celluloses of the different solid fractions recovered after EV treatment were also characterized by SEC, DRX to study the impact of the treatment and to identify the key parameters in the improvement of the efficiency of cellulose to glucose conversion. An increase in crystallinity with increasing severity was observed with a significant drop in cellulose DP for acid impregnated SE. The enzymatic digestibility of cellulosic residues after SE and lignin extraction by T. Reesei was studied. The main parameters positively affecting the enzymic reactivity are the removal of part of the non-cellulosic polymers and the drop of cellulose DP. At high SE severity, a decrease in reactivity was attributed to the formation of pseudolignins. The optimal conditions for SE before enzymatic hydrolysis are as follows: T = 210°C, t = 5 min and H2SO4 = 0.25%. Good fermentability of the hydrolysates by Saccharomyces Cerevisiae was obtained. From 100 Kg of okoumé sapwood, it is possible to produce 11.9 Kg of ethanol

Petroleum and its derivatives make up the largest portion of Brazil’s current energy matrix. It is well known that these are non-renewable and pollution-causing sources of energy; as such, the last decades have been marked by research and development of new, cleaner technologies. One of them being the hydrolysis of lignocellulosic materials to produce bioethanol. Brazil has an ample variety and large volumes of lignocellulosic biomass like corn husk and corncob. However, these biomass sources have complex structures which give rise to difficulties during the posterior stages of hydrolysis. Thus, it becomes necessary to conduct a pretreatment in order to alter these complex structures and make them more accessible to attack by microorganisms and enzymes. This work investigated two pretreatment methods for biomass coming from corn husk and corncob: pretreatment with dilute sulfuric acid and hydrothermal pretreatment; aiming to determine which method was more suitable for the given experimental conditions. An experimental design method was used to determine the best operating conditions for the acid pretreatment. Chemical characterization was used to evaluate the efficiency of the hydrothermal pretreatment. Both biomass sources were analyzed via Superior Calorific Power (SCP) resulting in17.276,5 J/g for corn husk and 17.872,0 J/g for corncob. Regarding the hydrothermal pretreatment, results showed reduction of the three components of biomass: cellulose, hemicellulose and lignin. Ideally, cellulose should not be degraded during the pretreatment. Therefore, the conditions leading to the lowest cellulose removal were determined: corn husk pretreatment at 170°C for 15 minutes and corncob pretreatment at 195°C for 10 minutes. The factors evaluated during the sulfuric acid pretreatment were: concentration, heating time and temperature. The two-variable factorial experiment with three replicates suggested that the highest reduced sugar percentages are obtained with low acid concentrations and high temperatures. Moreover, time was not a significant factor. Test 3 and 12 yielded the best results for both sources of biomass. The conditions for test 3 were: 120°C, a 15 minutes heating time and 0,5% sulfuric acid which resulted in 50,9% reduced sugars for corn husk and 42,6% for corncob. The conditions for test 12 were: 110°C, a 7 minutes heating time, and 2% sulfuric acid which resulted in 59,4% reduced sugars for corn husk and 61,5% for corncob.
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No panorama energético atual, nota-se que a utilização do petróleo e seus derivados ainda têm representado a maior fonte de energia em nosso país. Visto que essas fontes são não renováveis e poluentes, a necessidade de utilização de outras rotas, que sejam mais limpas, tem crescido ao longo dos anos. Quando se trata de materiais lignocelulósicos para a produção do bioetanol, encontra-se uma ampla variedade de biomassas, como é o caso da palha e do sabugo do milho. Porém essas biomassas possuem uma estrutura complexa, que dificulta as etapas posteriores de hidrólise. Logo, torna-se imprescindível a realização de um pré-tratamento que irá alterar essa estrutura, tornando-a mais acessível ao ataque de microrganismos e enzimas. Nesse trabalho, dois tipos de pré-tratamentos foram estudados, o pré-tratamento com ácido sulfúrico diluído e o pré-tratamento hidrotérmico, com o intuito de avaliar o mais viável, levando em conta todos os fatores envolvidos. O planejamento experimental foi uma ferramenta utilizada para a escolha das melhores condições operacionais do pré-tratamento com ácido diluído. Uma caracterização química foi realizada para avaliar a eficiência do pré-tratamento hidrotérmico. Análises de poder calorífico superior (PCS) também foram realizadas com as duas matérias-primas e os resultados do PCS foram de 17.276,5 J/g para a palha de milho e de 17.872,0 J/g para o sabugo. Em relação ao pré-tratamento hidrotérmico realizado, os resultados mostraram redução nos três componentes: celulose, hemicelulose e lignina. O¬¬¬¬ ideal é que a celulose sofra desorganização, contudo que não seja degradada, porém aqui se avaliou a melhor condição pela menor remoção de celulose, que foi dada com o pré-tratamento da palha de milho sob as condições de 170°C por 15 minutos e com o pré- tratamento do sabugo sob as condições de 195°C por 10 minutos. Já com relação ao pré-tratamento ácido, que ocorreu com utilização dos fatores: concentração de ácido sulfúrico, temperatura e tempo de aquecimento, um planejamento experimental em estrela com triplicata no ponto central mostrou que maiores percentuais de ART são dados quando se trabalha com baixas concentrações de ácido e elevadas temperaturas, na faixa investigada. O fator tempo não se mostrou significativo. As melhores condições foram obtidas nos ensaios 3 e 12 para ambas as biomassas. Com a temperatura de 120°C, tempo de 15 minutos e 0,5% de concentração de ácido sulfúrico, condição 3, obteve-se 50,9% de ART para a palha e 42,6% para o sabugo. Já com a temperatura de 110°C, tempo de 7 minutos e 2% de ácido, que foi a condição 12, obteve-se 59,4% de ART para a palha e 61,5% para o sabugo.

This diploma thesis deals with the optimization of the production of ethanol from waste paper using yeast. There were used 4 kinds of paper as a substrate - office paper, non-recycled workbook, recycled workbook and newspaper. All papers were pretreated with the following procedures: grinding, microwaves + NaOH, microwave + H2SO4 and microwave + H2SO4 + NaOH. The glucose concentration was determined in enzymatic hydrolysis by HPLC. Saccharomyces cerevisiae were chosen for ethanol production. The production of ethanol was carried out with all the pretreated papers in simultaneous saccharification and fermentation. During hydrolysis, the pretreated papers have reached the highest results in the combination with microwave + H2SO4 + NaOH. Non-recycled workbook was the only exception, where the highest concentration of glucose has been obtained by the pretreatment of microwaves + H2SO4. Following results have been acquired: office paper 24,69 gdm-3, non-recycled workbook 22,47 gdm-3, recycled workbook 16,94 gdm-3 and newspapers 15,36 gdm-3. SSF was carried out again with all the papers and their pretreatments. The highest concentration of ethanol has been achieved in microwave pretreatment + H2SO4 + NaOH. The highest overall concentration has been gained from the office paper, amounted to 16,98 gdm-3. The maximum concentration of ethanol for non-recycled workbook has been 15,25 gdm-3, for recycled workbook 12,2 gdm-3 and for newspapers 12,59 gdm-3.

Diploma thesis deals with use of Zymomonas mobilis for the production of bioethanol from waste paper. There were used three kinds of substrate (cardboard, drawing and office paper) to optimize of bioethanol production. Individual papers were subjected to the same pre treatment, namely a milling, a combination of microwave irradiation and NaOH, a combination of microwave irradiation and H2SO4 and combination microwave irradiation, H2SO4 and NaOH. The substrates were decomposed by enzymatic hydrolysis after pre treatment to evaluate the best pre-treatment. Simultaneous saccharification and fermentation was carried out for each substrate (with two of the best pre-treatment). The samples were taken during the hydrolysis and the simultaneous saccharification and fermentation, and were determined by HPLC. Growth curves of Zymomonas mobilis were constructed, as the most appropriate for SSF was chosen temperature of 40 ° C in which the exponential phase took place at the time of 6 15 hours. During hydrolysis was monitored glucose concentration in the solution. The maximum concentration of glucose was in the cardboard (microwaves + H2SO4 + NaOH) 16.46 gdm-3, a drawing (microwaves + H2SO4 + NaOH) 31.78 gdm-3, and office paper (microwaves + H2SO4) 25.04 gdm-3. The concentration of ethanol for SSF was highest in the same cases as in the hydrolysis. The cardboard was the maximum concentration of bio ethanol 9.5 gdm-3, for the drawing 16.1 gdm-3 and for the office paper 12.13 gdm-3.

Este trabalho avaliou a extração de xilose da palha de cana-de-açúcar sob diferentes condições de hidrólise por ácido sulfúrico diluído visando à obtenção de um hidrolisado hemicelulósico rico em xilose e ainda a fermentabilidade deste para a bioconversão a etanol por Pichia stipitis Y-7124. Inicialmente caracterizou-se a palha de cana e a fração hemicelulósica representou 30,79% da massa seca da palha, enquanto que a celulose representou 40,84% e a lignina 25,80%. Verificou-se ainda que 26,57% da palha corresponde à xilose, representando 86,27% de sua fração hemicelulósica. As hidrólises ácidas foram realizadas de acordo com um planejamento experimental fatorial fracionado 24-1 visando-se à resposta concentração de xilose (g/L) no hidrolisado hemicelulósico. Levou-se em consideração os fatores: tempo, temperatura, concentração da solução ácida e a relação sólido:líquido. A melhor resposta para a concentração de xilose foi encontrada nas condições do ponto central, com a média da concentração igual a 31,70 g/L. A análise de variância foi realizada para um screening do planejamento e observou-se que apenas os fatores concentração da solução ácida e temperatura foram significativos ao nível de 95% de confiança. Em seguida realizou-se ensaios para otimização da hidrólise ácida considerando os fatores temperatura e concentração da solução ácida. As condições de ótimo encontradas foram 130 ºC e a solução de ácido sulfúrico em 2,9% m/v, empregadas com uma relação sólido:líquido igual a 1:4 (g/mL) por 30 minutos de reação. Estas condições de ótimo permitiram a obtenção de um hidrolisado hemicelulósico com concentração igual a 56,5 g/L de xilose, correspondendo à extração de 85,1% da xilose da palha. Posteriormente, ensaios de fermentação foram conduzidos com o hidrolisado hemicelulósico obtido, visando à produção de etanol por P. stipitis em frascos Erlenmeyer de 250 mL com 100 mL de meio hidrolisado para avaliar a fermentabilidade do hidrolisado obtido bem como a necessidade de suplementação nutricional e utilizou-se um planejamento experimental fatorial 23 de face centrada para verificar a influência dos fatores: concentração de ureia, concentração de MgSO4*7H2O e concentração de extrato de levedura. Os ensaios foram conduzidos a 200 rpm e 30 ºC. Verificou-se que o extrato de levedura (5 g/L) exerceu maior influência sobre o processo fermentativo e que o MgSO4*7H2O (0,5 g/L) não apresentou significância estatística. A melhor condição na fermentação para produção de etanol por P. stipitis encontrada foi empregando-se extrato de levedura (5 g/L), ureia (5 g/L) e MgSO4*7H2O (1,0 g/L), em que se obteve um fator de rendimento em etanol YP/S igual a 0,38 g/g e produtividade volumétrica igual a 0,41 g/L.h, em 60 horas de fermentação.
This work evaluated the sugar cane straw xylose extration under different diluted sulfuric acid hydrolysis conditions aiming at the attainment of hemicellulosic hydrolysate rich in xylose and their fermentability for ethanol bioconversion by Pichia stipitis Y 7124. Previous assays for chemical characterization of the sugar cane straw has been carried. The hemicellulosic fraction represented 30.79% of the dry straw mass, whereas the cellulose represented 40.84% and the lignin represented 25,80%. It was verified despite 26,57% of the straw corresponded to xylose, representing 86,27% of its hemicellulosic fraction. The acid hydrolysis was carried out using an experimental design 24-1 aiming at it the xylose concentration (g/L) response on the hemicellulosic hydrolysate regarding the factors: time, temperature, acid solution concentration and the ratio solid:liquid. The best result for the xylose concentration has been found on the center point conditions, with concentration of 31,70 g/L. The analysis of variance was carried out to one screening of the design and it was observed that only the parameters temperature and acid solution concentration has been significant at 95% reliable level. After that acid hydrolysis assays has been carried regarding the factors temperature and acid solution concentration aiming the acid hydrolysis optimization. The optimum point conditions were 130 ºC and the sulfuric acid solution of 2,9% m/v, carried with the ratio solid:liquid of 1:4 (g/mL) at 30 minutes of reaction. Later, fermentation assays was carried out with the hemicellulosic hydrolysate obtained in 250 mL Erlenmeyer flasks containing 100 mL of hydrolysate to evaluate the hydrolysate fermentability obtained as well tha needless of nutritional supplementation and for it used an experimental design 23 of face centered to verify the influence of the factors: urea concentration, MgSO4*7H2O concentration and yeast extract concentration. Assays has been carried at 30 ºC and 200 rpm. It was verified that the yeast extract (5 g/L) exerts greater influence on the fermentation and the MgSO4*7H2O (0,5 g/L) did not present statistic significance. The best conditions were achieved using yeast extract (5 g/L), urea (5 g/L) and MgSO4*7H2O (0,5 g/L). with ethanol yield and volumetric productivity of 0,38 g/g and 0,41 g/L.h, respectively, after 60 hours of fermentation.

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A matriz energética mundial é baseada em fontes fósseis e renováveis. No Brasil, o bioetanol é gerado principalmente a partir da cana-de-açúcar. Resíduos agroindustriais (fontes celulósicas ou amiláceas) despontam como biomassas alternativas à cana-de-açúcar, para aumentar a competitividade deste combustível renovável frente aos de origem fóssil e também favorecer a sustentabilidade e a segurança alimentar e energética, pois são ricos em polissacarídeos não diretamente fermentescíveis, abundantes (problema ambiental) e apresentam baixo valor comercial. O farelo de mandioca é um exemplo de resíduo sólido gerado na produção de fécula (amido) e farinha de mandioca que ainda contém, em média, 75% de amido. Consequentemente, deve ser previamente hidrolisado e posteriormente fermentado por leveduras do gênero Saccharomyces para gerar etanol. O objetivo deste estudo foi produzir bioetanol a partir de hidrolisados enzimáticos de farelo de mandioca, usando levedura álcool resistente (AR). Primeiramente, a concentração de açúcares obtida a partir da hidrólise enzimática foi verificada através de um planejamento fatorial completo (24), com triplicata no ponto central, a fim de investigar a influência dos seguintes fatores na hidrólise: concentração de α-amilase (Termamyl 2X), tempo de liquefação, concentração de glucoamilase (AMG 300L) e o tempo sacarificação. A condição de hidrólise mais favorável foi a do ensaio com 0,517 mL de AMG/g amido, 0,270 mL de Termamyl/g amido, 1h de tempo de liquefação e 2h de tempo de sacarificação. O caldo resultante da condição escolhida alcançou altas concentrações de glicose (160 g/L). Os ensaios de fermentação alcoólica foram realizados em duplicata em biorreator de 3L, em regime de batelada, a 30C, 100 rpm e pH 5,5. Cerca de 3 g/L (massa seca) de uma linhagem de levedura álcool tolerante, Saccharomyces cerevisiae Hansen BY4741, crescida por 12h em meio YEDP (2% de glicose) foram usados como inóculo. O mosto consistiu de um litro de hidrolisado (160 g/L de glicose) fortificado com extrato de levedura (1%) e peptona de carne (1%), além da adição de um antiespumante (Tween 80) na concentração de 0,05% (m/v). Em 30 horas de fermentação, a média da concentração de etanol obtida foi de 65 g/L. A eficiência foi de 87,6% e o rendimento e a produtividade foram 0,448 e 2,16 g/L.h, respectivamente. Os resultados indicaram a aplicabilidade do farelo de mandioca como matéria-prima para a produção de bioetanol
The world energy matrix is based on fossil and renewable sources. In Brazil, bioethanol is generated mainly from sugarcane. Agro-industrial wastes (cellulosic or starchy sources) emerge as an alternative to sugarcane biomass, in order to increase this renewable fuel competitiveness against fossil ones, and also promote sustainability, food security and energy security, because they are rich in polysaccharides (not directly fermentable), abundant (environmental problem), and have low commercial value. The cassava bagasse is an example of a solid waste originated from starch and cassava flour industries, which still contains on average 75% of starch. Consequently, it should be hydrolyzed and then fermented by Saccharomyces yeasts in order to generate ethanol. This study aimed to produce bioethanol from enzymatic hydrolysate of cassava bagasse, using alcohol resistant (AR) yeast. At first, the concentration of sugars obtained from enzymatic hydrolysis was verified using a full factorial design (24) with triplicate at the center point to investigate the influence of α-amylase concentration (Termamyl 2X), liquefaction time, glucoamylase concentration (AMG 300L), and saccharification time. The best condition of hydrolysis was 0,270 mL of Termamyl/g starch, 1h of liquefaction, 0,517 mL of AMG/g starch, and 2h of saccharification. The resultant syrup of the chosen condition achieves high levels of glucose (160 g/L). Alcoholic fermentation assays were performed in duplicate in a 3L bioreactor under batch regime at 30C, 100 rpm and pH 5.5. About 3 g/L (dry weight) of an alcohol tolerant yeast strain, Saccharomyces cerevisiae Hansen BY4741, grown for 12 h in YEDP medium (2% glucose), were used as inoculum. The fermentation broth consisted of one liter of hydrolysate (160 g/L of glucose) supplemented with yeast extract (1%) and meat peptone (1%), plus the addition of an antifoam (Tween 80) in a concentration of 0.05% (w/v). At 30 hours of fermentation, the average ethanol concentration obtained was 65 g/L. The efficiency was 87.6% and the yield and the productivity were, respectively, 0.448 and 2.16 g/L.h. The results indicated the applicability of cassava bagasse as raw material for bioethanol production

In diploma thesis the process of enzymatic hydrolysis of waste paper as a source for the production of liquid biofuels is discused. It follows directly the homonymous diploma thesis from Ing. Brummer, and it is based on the findings, which were solved and decided in previous work. In the theoretical part there is a summarization of basic information on the enzymatic hydrolysis of waste paper and the associated influences of various factors of the rate and degree of hydrolysis. Higher attention is paid to a waste cardboard and its pretreatment methods due to the maximalization of the yield of hydrolysis. The next part summarizes options of the fermentative production of biofuels, focusing on the method of simultaneous saccharification and fermentation, where the further appropriate organism for ethanol fermentation is discussed. The last part is about the technological process from the raw material input to the separation of ethanol. In the experimental section the pre-treatment of waste paper in order to maximize the efficiency of hydrolysis was examined. The best results were achieved using a vibratory mill. In addition, various parameters for simultaneous saccharification and fermentation were optimized using enzymes from Novozymes® company and the yeast Saccharomyces cerevisiae. The conversion rate of waste paper cellulose to reducing sugars was observed by spectrophotometric method by Somogyi - Nelson and the amount of produced ethanol was quantified using HPLC / RI. As a part of this thesis some conditions (amount of enzyme, substrate, nutrients, yeasts, temperature, pH, type of buffer) were optimized to maximize the effectiveness of the overall process. All experiments were carried out on corrugated cardboard, which was chosen as the most promising material for hydrolysis that was among the waste paper pulp in diploma thesis by Ing. Brummer.