Dissertations / Theses on the topic 'Gas-fermentation'

Ethanol produced from lignocellulosic agricultural products and waste is an environmentally-friendly alternative to petroleum-derived fuel. Lignocellulosic biomass is gasified producing synthesis gas, which is composed of CO, CO2, and H2. Synthesis gas is fermented via anaerobic biocatalyst. The bacterium was grown in a fructose-rich medium then concentrated in ethanol production medium for synthesis gas fermentation. While the known ethanol-producing bacterium Clostridium ljungdahlii was used to provide baseline values for synthesis gas utilization and ethanol production, synthesis gas fermentation were conduced with a culture discovered at Mississippi State University. Additionally, efforts were made to isolate other anaerobic cultures capable of fermenting synthesis gas to ethanol.

Access to renewable energy and reduction of carbon emissions represent two major issues facing humankind in the twenty first century and beyond. The underlying driving forces behind both are multi-faceted and often intrinsically connected, ranging from environmental concerns over climate change to improving economic security through self-sustaining energy production. Possible solutions to reliance on non-renewable, carbon-emitting fossil fuels have been explored over recent decades, with significant interest placed on biofuels. Due to ease of integration into liquid-based petrochemical fuel infrastructure, these renewable alternatives have been a consistent topic of both industrial and academic interest. Despite offering renewable energy, conventional crop-based biofuel production has faced criticism due to consumption of land, water and other resources associated with agriculture. Acetogens provide a solution to conventional biofuel production due to their utilisation of carbon monoxide and carbon dioxide gas as carbon and energy sources, rather than plant matter. This allows generation of a range of chemical products from a broad range of sources, including industrial waste gases and gasified solid waste. Acetogens offer the double benefit of both renewable energy production, and carbon emission sequestation. This study outlines the development of genetic tools to provide a foundation for using synthetic biology approaches to improve performance of acetogens as industrial chassis. Specifically, development of tools and techniques for the acetogen Clostridium autoethanogenum are described, with further applications of such technology to other Clostridia.

Determining the redox potential of a system can be a useful tool in evaluating the thermodynamic changes that occur over a period of time. Currently, fermentation industries use pH measurements to assess the progress of fermentation and growth of lactic acid bacteria (LAB). Redox potential (Eh) measures the transfer of electrons within a system, rather than solely the concentration of hydrogen ions, as measured by pH. Continuous monitoring of fermentation by Eh may allow for more descriptive analysis of the metabolic process and could offer a method for the earlier prediction of spoilage by yeasts. The trends in redox potential of fermenting cucumbers were observed to evaluate the possible application of this parameter in monitoring the development of fermentation. Additionally, an evaluation of the effect of gas purging on microbial growth during the fermentation was conducted using redox potential trends as a monitoring tool. Cucumbers were packed and brined using sodium chloride, calcium chloride, and acetic acid and the redox potential monitored during fermentation. Examination of both pasteurized-inoculated jars and non-pasteurized jars was conducted in this study. In addition, changes in redox potential were measured in fermentations that were purged with nitrogen, oxygen, hydrogen, or left untreated. Brine samples were analyzed for microbial counts, pH and substrates and products of fermentation. Under conditions representative of a standard cucumber fermentation, a dramatic decrease in the redox potential was observed during the first day, concomitantly with the initiation of the log phase of bacterial growth. However, in the presence of spoilage yeast, redox potential remained low over this initial period. The progress of a fermentation sparged with nitrogen, oxygen, or no treatment produced similar Eh trends. The Eh trend was more reduced under hydrogen sparging. A heightened growth rate for LAB, more efficient production of lactic acid, and lower production of ethanol occurred with nitrogen sparging. The evidence suggests that the growth of yeasts in a cucumber fermentation could be detected based upon differences in the redox potential. Distinct variations in Eh were still recorded after pH values decreased and remained constant, suggesting the valuable possible application of Eh to monitor industrial cucumber fermentations. Additionally, changes in redox potential were affected by gases introduced during cucumber fermentations and sparging with nitrogen could have substantial benefits to the industry.

The objectives of this work were to investigate the effects of unconventional oils rich in phenolic compounds and rumen adaption on methane (CH4) gas production and rumen fermentation characteristics under in vitro rumen conditions. For this purpose, two sets of trials were conducted. In the first trial, the effects of blackberry, blueberry, raspberry, pomegranate, black seed and hemp oils on CH4 production and fermentation were examined in three 24 h batch culture experiments. Treatments in each experiment consisted of control (no oil supplement), control plus corn oil, or control plus two of the unconventional oils. Oils were added to rumen cultures at 500 mg/L (equivalent to 3.3 g oil/kg of diet dry matter (DM)). After 24 h of incubation, CH4 production was not different between the control and the corn oil treatments. Of the six unconventional oils tested, only hemp and blueberry oils reduced (P<0.05) CH4 production by 9-16% relative to the control and corn oil treatments. No significant differences were observed between treatments in dry matter digestibility (DMD) or total volatile fatty acids (tVFA). Except for a reduction (P<0.05) in acetate concentration with the raspberry oil, and an increase (P<0.05) in valerate concentration with the pomegranate oil, all other treatments had similar VFA concentrations. In the second trial, the effects of adding oregano essential oil (OEO) to adapted and unadapted rumen cultures on CH4 production and rumen fermentation were evaluated under in vitro condition. Rumen cultures were obtained from continues culture fermenters fed a control diet or control diet plus OEO at 250 mg/day for 10 days. The addition of OEO decreased (P<0.05) ii CH4 production only in adapted cultures. Total VFA and acetate concentrations were greater (P<0.05) in the unadapted than adapted cultures and their concentrations decreased (P<0.05) with the addition of OEO particularly when added to the adapted cultures. Propionate concentrations were also greater (P<0.05) in the unadapted than the adapted cultures and concentrations decreased (P<0.05) with the addition of OEO. Dry matter degradability and total gas production decreased (P<0.03) with the addition of OEO in both cultures and total gas production tended (P<0.13) to be lower when added to the adapted cultures. In conclusion, our results showed that hemp and blueberry oils were moderately effective in reducing rumen CH4 formation without compromising rumen fermentation and digestibility. Oregano Essential oil addition negatively affected rumen fermentation in both adapted and unadapted cultures and the effect was greater in the adapted cultures. The greater effects of OEO on CH4 production in the adapted cultures most likely due to the lower fermentation efficiency in these cultures.

Ce travail décrit les équilibres gaz-liquide lors des fermentations alcooliques en conditions œnologiques, en se focalisant sur les composés d'arôme les plus abondants : isobutanol, alcool isoamylique, acétate d'éthyle, acétate d'isoamyle et hexanoate d'éthyle. Les coefficients de partage à l'équilibre (ki) de ces molécules sont quantifiés, grâce à la méthode PRV (Phase Ratio Variation), en précisant l'effet de la composition du milieu et de la température. Grâce à la mise en œuvre de fermentations spécifiques pendant lesquelles la vitesse de dégagement de CO2 est contrôlée (grâce à une perfusion d'azote ammoniacal), il a été montré que le dégagement de CO2 n'avait pas d'effet sur le rapport de concentrations entre phase gaz et liquide et pouvait être assimilé au ki. Une démarche de modélisation est ensuite effectuée, pour estimer la valeur du rapport de concentrations entre phase liquide et gaz pour ces composés (à l'exception de l'acétate d'éthyle et de l'alcool isoamylique), à tout moment de la fermentation et quelle que soit la température, y compris en conditions de non isothermie. Le modèle, basé sur l'équilibre des phases gaz et liquide, conduit à une erreur moyenne d'estimation inférieure à 10%. Grâce à ce modèle, il devient possible de réaliser des bilans de production des différents composés tout au long de la fermentation, à partir de leur seule mesure dans la phase gaz. Ces bilans permettent de différencier (i) la quantité globale produite, représentative des potentialités de la levure (intérêt microbiologique), (ii) la quantité restant dans la phase liquide (intérêt organoleptique) et (ii) la quantité perdue dans le gaz effluent (intérêt technologique). Il est ainsi mis en évidence que les pertes d'esters sont très importantes. Par exemple, à 20°C, elles atteignent respectivement 44 % et 25 % pour l'hexanoate d'éthyle et l'acétate d'isoamyle. Grâce à un dispositif de suivi en ligne permettant des mesures très fréquentes (une par heure), les vitesses - et les vitesses spécifiques - de production et de pertes sont calculées. Elles constituent de nouvelles informations utiles à la fois pour l'étude du métabolisme (suivi de la dynamique de flux métaboliques) et pour mieux raisonner les conduites de fermentation, notamment le régime de température. L'impact de ce régime de température est étudié plus en détail en réalisant des bilans de production lors de fermentations conduites avec la même souche de levure et le même moût. Les résultats obtenus montrent que si l'on ne prend en compte que les concentrations dans la phase liquide - généralement les seules informations disponibles - on surestime fortement l'impact de la température sur le métabolisme de composés fortement volatils, tels que les esters
The gas-liquid partitioning during winemaking fermentations was studied, with a focus on the main aroma compounds: isobutanol, isoamyl alcohol, ethyl acetate, isoamyl acetate, ethyl hexanoate. The partition coefficients (ki) of these molecules were quantified, using the PRV (Phase Ratio Variation) method. The influence of the must composition and temperature was assessed. The absence of any effect of CO2 production on gaz-liquid ratio compared to ki value was established by running specific fermentations in which the rate of CO2 production was kept constant by perfusion with assimilable nitrogen. A model was then elaborated to calculate gaz-liquid ratio of these molecules (excepted ethyl acetate and isoamyl alcohol) at any fermentation progress and at any temperature, including anisothermal fermentations. The model based on the equilibrium between the gas and liquid phases predicted ki with less than 10% error. Using this model, balances were c alculated, with a differentiation between (i) the total production, representative of the yeast potential (microbiological interest), (ii) the amount remaining in the fermenting must (organoleptic interest) and (iii) the amount lost in the exhaust CO2 (technological interest). High losses of esters were observed. For example, at 20°C, they represented 44% and 25 % for ethyl hexanoate and isoamyl acetate, respectively. Using an on line monitoring with a high frequency of measurements (one per hour), rates and specific rates of production were calculated. These new data are useful both for studies on metabolism (dynamics of metabolic fluxes) and for improving fermentation control, in particular temperature profile. The impact of temperature was assessed in more detail by comparing balances of production during fermentations run with the same yeast strain and must. These experiments demonstrated that the effect of temperature on the yeast metabolism was highly overestimated whe n only considering the concentrations in the liquid, i.e. usually the only available information

Two types of in situ product recovery processes are investigated: gas stripping for solvent removal in continuous butanol fermentation and reverse osmosis for acid separation and purification in a continuous butyric acid fermentation. Gas stripping and reverse osmosis are easy to operate and design processes that can be integrated to fermentation processes.

A method to improve production of Fab' fragments using gas blending and pH-stat feeding strategy in a fermentation process with Escherichia coli has been developed. Regime analysis together with design of experiments (DoE) has been used to evaluate the effect of gas blending and feeding strategy on a Fab' fermentation process. The economic implications of the fermentation strategy have also been considered. Batch-feeding fermentations carried out at 20 L and 450 L scale indicated that cascade control was not sufficient to maintain a constant DOT level throughout the fermentation. DOT levels dropped to zero during induction phase at both scales. Regime analysis was performed based on experimental KLa determination. KLa values of -400 h"1 were observed at 20 L and 450 L scale. Comparison of time of oxygen consumption (tQc) and time of oxygen transfer (t0i) suggested that 02 limitation is present in this fermentation process and that this worsens as the scale increases. A gas blending system at 20 L scale was proposed to address this problem. A factorial 22 experimental design was executed to evaluate independently the effects and interactions of two main engineering factors (related to oxygen transfer in the broth) on Fab' titre: DOT level and agitation rate. Gas blending was successful in maintaining constant levels of DOT at 20 L scale. Fab' production was increased by 77 % at of agitation rate of 500 rpm independent of the DOT level, compared to operation at the same scale but without gas blending. High levels of product localisation in the periplasm of 84 - 93% were also obtained. Furthermore, based on t0c and t0T, it could be suggested that no oxygen limitation is likely to occur in the fermentations performed with gas blending, regardless of the agitation rate. Batch-fed fermentations either with or without gas blending carried out at 20 L scale indicated the presence of glycerol oscillations. A pH-stat feeding strategy in a gas blending system was implemented to address this problem. Results showed that a 2-fold increase in the production of Fab' at 20 L scale, compared to a fermentation operated in a pulsed fed- batch mode could be achieved. A consistently high level (>90%) of product localisation in the periplasm was also achieved and no negative impact on product recovery was observed. Finally, a preliminary economic analysis was performed to estimate the production costs in £/mg of product. The effects of gas blending and feeding strategy at 20 L scale on the final product cost were evaluated by comparing: batch-fed non gas-blending, batch-fed gas blending, and pH-stat gas blending fermentations. A fermentation production cost of £1.45/mg of Fab' was estimated for a pH-stat gas blending system. This resulted in cost savings of up to 75% when compared to the production cost of £5.80/mg of Fab' in the batch-fed system without gas blending. The results obtained in this work provide an impetus for further studies to evaluate the potential of gas blending and pH-stat feeding strategy for the industrial production of Fab' antibody fragments. The use of statistical design of experiments together with regime analysis was found to be a very useful tool to gain a better understanding of this system.

A microbubble dispersion (MBD) was used to supply oxygen for an aerobic fermentation of Baker's yeast. The 1-liter microbubble dispersion generator supplied bubbles for 20-liter and 50-liter working volume fermentations in a 72-liter pilot scale fermenter. The microbubbles were stabilized by the surfactants naturally present in the culturing broth medium. The growth patterns of yeast Saccharomyces cerevisiae, cultured at agitation speeds of 150 rpm and 500 rpm, were compared for oxygen supplied by ordinary air sparging and by MBD sparging. Both air sparged and MBD systems were supplied air at equivalent volumetric flow rates. The volumetric oxygen transfer coefficients (KLa) were estimated by the yield coefficient method. The KLa values increased from 142.5 to 458.3 h-1 and from 136.1 to 473.3 h-1 for 20- and 50- liter runs, respectively, as the agitation speed was increased from 150 to 500 rpm in the ordinary air sparged fermentations. The oxygen transfer coefficients in the MBD sparged fermentations were found to be independent of the fermenter agitation speed at approximately 480 h-1 for 20-liter runs and 340 h-1 for 50-liter runs. The growth rates for MBD at 150 rpm were essentially equivalent with air sparged fermentations at 500 rpm. The total power consumption per unit volume of broth for the 150 rpm, MBD fermentation was 68% lower than the 500 rpm, air sparged run for the 20-liter fermentations and was 55% lower for the 50-liter fermentations.
Master of Science

Due to environmental challenges, depleting oil resources, rising cost of oil and instability in oil-producing countries, biofuel production has attracted a lot of attention in recent decades. Biobutanol is one of the biofuels showing the most potential as an alternative for partly replacing petroleum-based fuels. Both researchers and industrialists are currently working at developing an energy-effective process to produce biobutanol at a large scale. Acetone-butanol-ethanol (ABE) fermentation is the biological process of biobutanol production and Clostridia are the most common bacteria used to produce biobutanol. However, there are several challenges in the butanol bioproduction process that should be addressed to make this process economically viable. The main challenge in the biobutanol production process is the low concentration of butanol in the ABE fermentation broth. It is therefore important to develop an efficient separation method. Several separation methods such as distillation, liquid-liquid extraction (LLE), pervaporation, gas stripping and adsorption have been considered to recover butanol from dilute solutions and ABE fermentation broths. Adsorption is considered as one of the most promising methods due to its high performance and energy efficiency for butanol separation. In this study, the focus was on developing an efficient separation method for butanol recovery from dilute model solution and fermentation broth using adsorption. A comprehensive adsorbent screening was first carried out to identify the best commercially available adsorbent among a series of potentially promising adsorbents. Activated carbon (AC) F-400 was selected for further experimentation since it showed high adsorption capacity and adsorption rate in addition to high selectivity toward butanol. AC F-400 was then tested extensively in packed adsorption column experiments for binary and ABE model solutions and fermentation broths to investigate the competitive adsorption between butanol and other components present in ABE broths. The results showed that the butanol adsorption capacity was not affected by the presence of ethanol, glucose and xylose while the presence of acetone led to a slight decrease in adsorption capacity at low butanol concentrations. On the other hand, the presence of acids (acetic acid and butyric acid) in the ABE broth showed a significant effect on the butanol adsorption capacity over a wide ii range of butanol concentration and this effect was more pronounced for butyric acid. At the end, different competitive adsorption isotherm models were also studied to appropriately represent the behaviour of the competitive adsorption. Desorption of butanol was subsequently investigated to evaluate both the desorption capacity of butanol and the capability of the adsorbent particles to be used for multiple adsorption-desorption cycles. The results of this set of experiments showed that AC F-400 can retain its initial adsorption capacity after 6 adsorption/desorption cycles. The recovery of butanol from butanol-water (1.5 wt%) binary and ABE model solutions was 84 and 80% with butanol adsorption capacity of 302 and 171 mg/g, respectively. The combination of adsorption and gas stripping techniques was also studied to investigate the performance of CO2 gas stripping of solvents from the model solutions and fermentation broths followed by adsorption. The results showed that the butanol adsorption capacity of the overall system for binary solutions (260 mg/g for a binary butanol-water solution of 15 g/L with vapour phase concentration of 5.8 mg/L), ABE model solutions (192 mg/g for a corresponding vapour concentration of 5.2 mg/L) and ABE fermentation broths (247 mg/g for a corresponding vapour phase concentration of 2.5 mg/L) was higher than what has been published in the literature. Finally, a model was developed and adequately validated the experimental data to predict the behaviour of the ABE compounds in a packed bed adsorption column for butanol separation from dilute solutions.

Purple threeawn (Aristida purpurea Nutt.) is a native bunch grass that is avoided by grazers. It is capable of dominating old cropland and overgrazed pastures, limiting livestock carrying capacity, and degrading wildlife habitat. Traditional management tools have had little impact on threeawn dominance in semiarid regions of the west. Our objectives were to: 1) assess fire and nitrogen treatment effects on threeawn forage quality at various phenological stages to test their potential as pretreatments in a grazing strategy and 2) examine a threeawn-dominated plant community?s response to prescribed fire, nitrogen addition, and clipping. Fire improved threeawn forage quality with greater improvements in early phenological stages. Nitrogen had little effect on forage quality. Fire and nitrogen reduced threeawn while increasing cool season grasses. Light and moderate clipping following fire did not improve the efficacy of fire. Fire appears to an effective preliminary treatment to improve the chance of herbivory.

Thesis (MSc)--Stellenbosch University, 2011.
ENGLISH ABSTRACT: This study focused on the potential of comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOF-MS) for the improved analysis of volatile wine constituents. Solid phase microextraction (SPME) in combination with GC×GC-TOF-MS was successfully used for the detailed investigation of the impact of three commercial Oenococcus oeni lactic acid bacteria (LAB) strains on the volatile composition of Pinotage wines subjected to malolactic fermentation (MLF). Due to increased separation power and enhanced sensitivity obtained by using two orthogonal separations coupled with the structural information provided by deconvoluted TOF-MS spectra, GC×GC-TOF-MS allowed for the identification and semi-quantitative analysis of much larger numbers of compounds compared to previous studies applying one-dimensional gas chromatography. The combination of univariate and multivariate statistical assessment was used as a powerful tool for data interpretation. The obtained results contribute significantly to the understanding of the impact of MLF on the volatile composition of Pinotage wine Some compounds have been linked to MLF for the first time. Moreover, the impact of these commercial starter cultures on the composition of volatile sulfur and nitrogen compounds in the same wines was studied by one-dimensional gas chromatographic methods with headspace injection and solid supported liquid-liquid extraction together with sulfur selective detection and tandem mass spectrometry. This study demonstrated also for the time, the impact of MLF on the composition of volatile sulfur and nitrogen compounds in Pinotage wine. GC×GC-TOF-MS was further used for the evaluation of the suitability of a new phase for stir bar sorptive extraction (SBSE) analysis of wine volatiles. Despite instrumental complications, beneficial extraction properties of the new stir bar phase for especially more polar compounds could be demonstrated. In addition, the extraction ability of this novel phase was evaluated for the analysis of selected thiazoles in wine using heart-cutting two dimensional gas chromatography in combination with nitrogen selective detection. Advantageous extraction performance of the new stir bar phase compared to a conventional polydimethylsiloxane (PDMS) phase for the determined thiazoles was demonstrated.
AFRIKAANSE OPSOMMING: Hierdie studie het gefokus daarop om die potensiaal van omvattende tweedimensionele gaschromatografie gekombineer met vlugtyd massaspektrometrie (GC×GC-TOF-MS) vir die verbeterde analise van vlugtige wynkomponente te ondersoek. Soliede fase mikro-ekstraksie (SPME) in kombinasie met GC×GC TOF MS is met sukses aangewend vir ‘n ondersoek na die impak van drie kommersiële Oenococcus oeni melksuur bakteria (LAB) rasse op die samestelling van die vlugtige fraksie van Pinotage wyne wat appelmelksuurgisting (AMG) ondergaan het. As gevolg van die verbeterde skeidingsvermoë en die verhoogte sensitiwiteit wat verkry word deur twee ortogonale skeidings te kombineer, tesame met die inligting aangaande die molekulêre struktuur wat die die gedekonvoleerde TOF massaspektra verskaf, maak GC×GC-TOF-MS die identifikasie en semi-kwantitatiewe analise van aansienlik meer komponente, in vergelyking met die gebruik van een-dimensionele gaschromatografie, moontlik. Die kombinasie van monoveranderlike asook multiveranderlike statistiese evaluering is gebruik as ‘n kragtige tegniek vir data interpretasie. Die resultate wat verkry is dra tot ‘n groot mate by tot die ontrafeling en begrip aangaande die impak wat AMG op die samestelling van vlugtige komponente in Pinotage wyn het. Daar word ook vir die eerste keer aangetoon dat somminge komponente verband te hou met AMG. Aanvullend hiertoe is die impak wat hierdie kommersiële kulture (wat gebruik word om fermentasie te inisieer) op die voorkoms van swawel en stikstof bevattende vlugtige komponente het bestudeer deur gebruik te maak van een-dimensionele gaschromatografiese metodes met ‘headspace’ inspuiting en vloeistof-voeistof ekstraksie tesame met swawel en stikstof selektiewe deteksie en tandem massaspektrometrie. Hierdie ondersoek werp lig, ook vir die eerste keer, op die samestelling van vlugtige swawel en stikstof bevattende komponente in Pinotage wyn. GC×GC-TOF-MS is ook gebruik vir die evalueering van die toepaslikheid van ‘n nuwe stasionêre fase vir gebruik met roerstaaf sorptiewe ekstraksie (SBSE) vir die analisering van vlugtige komponente in wyn. Ten spyte van instrumentele komplikasies, is die voordele wat hierdie nuwe fase vir die ekstraksie van vernaamlik meer polêre komponete aangetoon. Vervolgens is die ekstraksievermoë van hierdie nuwe fase vir die analise van sekere tiasole in wyn met ‘heart-cutting’ twedimensionaly gaschromatografie in kombinasie met stikstof-selektiewe deteksie gedemonstreer. Verbeterde ekstraksie van die nuwe roerstaaf fase vir die analise van tiasole, in vergelyking met ‘n tradisionele polydimethylsiloxane (PDMS) fase is voorts aangetoon.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第20434号
農博第2219号
新制||農||1049(附属図書館)
学位論文||H29||N5055(農学部図書室)
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 廣岡 博之, 教授 松井 徹, 教授 久米 新一
学位規則第4条第1項該当

Due to the warming of the climate system, public opinion has increased its interest in greenhouse gas, with particular predilection for methane (CH4 ) and their relationship with livestock sector. For this reason, several researches have proposed different nutritional strategies to reduce methane production in ruminants. The two main aims of this thesis were: i) to study methodological factors affecting in vitro CH4 production and to develop a new approach to measure in vitro CH4 production; ii) to evaluate effectiveness of some nutritional strategies on in vitro CH4 reduction. The objectives have been achieved through the research activities and reported in five experimental contributions. The first contribution aimed to analyse specific factors affecting in vitro gas production (GP), CH4 production and their ratio by a meta-analysis approach using 274 control treatments from 39 scientific papers published over 12 years. The common factors included in the analysis were: the pressure in the GP equipment (constant or increasing), the incubation time (24 or ≥48 h), the timing of rumen fluid collection (before or after feeding of donor animals), the presence of N in the buffer solution (presence or absence), and the ratio between amount of buffered rumen fluid and feed sample (<130 or 130-140 or >140 ml/g DM). The NDF content of feed sample incubated (392 ± 175.3 g/kg) was considered as a continuous variable. Results showed that GP measures were influenced mainly by the incubation time; values of CH4 production were affected mainly by the ratio between amount of buffered rumen fluid and feed sample; values of CH4 proportion (CH4 /GP) were mainly affected by timing of rumen fluid collection. Results suggest that factors considered in the meta-analysis explained a significant part (about 65%) of variability of GP and CH4 measures obtained in vitro. Thus, a higher standardization of specific methodological protocols would be desirable in order to compare results of different trials. The second contribution compared two in vitro techniques (closed or vented bottles) of sampling and measuring CH4 production from ruminant feeds. Five feeds (meadow hay, ryegrass hay, corn grain, dry sugar beet pulp, and expeller flaxseed) were tested. The experimental design was: 2 incubations×5 feeds×3 replications×2 gas sampling techniques, plus 4 blanks (bottles without feed sample), for a total of 64 bottles incubated. Half of the bottles were not vented during the incubation, whereas the remaining were vented at a fixed pressure and gas was collected into a tight bag connected to each bottle. At the end of each incubation, gas samples were sampled from the headspace of closed bottles or from headspace and bags of vented bottles and analyzed for CH4 concentration. GP values were adjusted or not for the amount of CO2 solubilized in the fermentation fluid. Values of CH4 concentration (ml CH4 /100 ml gas) and production (ml CH4 /g DM) were computed using corrected or uncorrected GP values. Closed bottles showed lower uncorrected GP (−18%) compared with vented bottles, whereas the correction for dissolved gas reduced but did not remove differences between techniques. Closed bottles showed unadjusted CH4 concentrations 23% greater than that of vented bottles, but the adjustment of measurements for solubilized CO2 reduced but did not remove this difference. Adjusted CH4 production was not influenced by technique. Closed bottles provide good measurements of CH4 production but not of GP. Venting of bottles at low pressure permits a reliable evaluation of total GP and CH4 production. The third contribution evaluated the influence that changes in main dietary constituents (starch:ADF ratio, protein and lipid contents) could have on in vitro GP, CH4 concentration and production. All changes have been made within ranges of diets used in intensive farms of the North Italy. A reference diet was used (273, 361, 158, and 33 g/kg DM of starch, aNDF, CP, and lipids, respectively). Other 6 diets had a smaller or a greater starch:ADF ratio (0.40 or 1.77, respectively), CP content (115 or 194 g/kg DM, respectively), or lipid content (26 or 61 g/kg DM, respectively), compared to the reference diet. The experimental design was: 4 incubations × 7 diets × 5 replications, plus 20 blanks (5 blanks/run), for a total of 160 bottles. Bottles were vented at fixed pressure and at the end of each incubation, gas samples were collected from the bottle headspace and analyzed for CH4 . The proportion of CH4 lost with venting was estimated. An increasing starch:ADF ratio reduced GP per g DM and per g TDMd, increased the amount of CH4 produced per g DM, whereas CH4 produced per g TDMd was unchanged. The increase of CP content decreased GP, whereas CH4 production was reduced in low protein diet only when expressed as proportion of GP. The increase of lipid content reduced GP per g DM, but no influences were observed on CH4 values. Results of this study showed that the magnitude of effects exerted by the dietary changes on in vitro fermentation was small. The fourth contribution aimed to explore effects of 4 pure plant extracts (allyl-sulfyde; cinnamaldehyde; eugenol; limonene) and 1 synthetic compound (monensin), all with antimicrobial properties, added to a commercial diet for dairy cows, on GP, CH4 concentration and production. Two dosages were tested: 3 or 30 mg/g of diet for pure plant extracts, 0.015 or 0.030 mg/g of diet for monensin. Incubation procedures were the same as the previous experiment. The experimental design was: 4 incubations×5 additives×2 dosages×3 replications, plus 12 bottles as control (without additive; 3/run) and 12 bottles as blanks (3/run), for a total of 144 bottles. Results showed that low dosages of all compounds did not exert any effect on in vitro GP and CH4 production. Compared to the control, high dosage of allyl-sulfyde, cinnamaldehyde, eugenol, limonene, and monensin significantly reduced in vitro GP (ml/g DM; -16%, -12%, -9%, -38%, -12%, respectively). In vitro CH 4 production was significantly reduced only by high dosage of allyl-sulfyde, cinnamaldehyde, limonene, and monensin (-32%, -12%, -43%, -18%, respectively, compared to the control). Only high dosage of allyl-sulfyde and limonene significantly reduce CH4 proportion (-18%, -12% respectively, compared to the control). The most promising results were observed for cinnamaldehyde, as the depression of CH4 production was not accompanied by a reduction of in vitro degradability. The fifth contribution investigated the possibility to reduce in vitro gas and CH4 production by combining a direct inhibitor of methanogenisis (chloroform) with two indirect inhibitors: i) nitrate, acting as a H2 sink, and ii) saponins, acting as antiprotozoal agent. The experiment was conducted using a semi-continuous flow system (RUSITEC ®). A diet, based on grass hay, was incubated alone (diet 1) or supplemented with 31.5 g/kg of nitrate (diet 2) or with 50 g/kg of saponins extracted from ivy extract (diet 3). Three diets were incubated without or with chloroform (2 µL/L), obtaining a total of 6 dietary treatments. Each of the 6 dietary treatments was incubated in four vessels, for a total of 24 vessels. Each vessel was inoculated with 800 ml of buffered rumen fluid. Whole incubation lasted 21 d. Total GP (on average 2.56 L/d) was not affected by diet or chloroform addition. When incubated alone, chloroform, nitrate and saponins reduced daily CH4 production by 96, 66 and 22%, respectively. However, when chloroform was combined with indirect inhibitors, neither nitrate or saponins had any additional effect in chloroform treated vessels on in vitro CH4 production. Results suggest that no synergistic effects emerged between the direct inhibitor and the two indirect inhibitors.
Il recente scenario del riscaldamento climatico globale, ha portato ad un maggiore interesse dell’opinione pubblica riguardo ai gas che causano l’effetto serra, ponendo particolare attenzione al gas metano (CH4 ) e alla sua relazione con il settore zootecnico. Per questa ragione, numerosi gruppi di ricerca hanno proposto delle strategie nutrizionali per ridurre le emissioni di CH4 da parte dei ruminanti. Nella presente tesi sono stati sviluppati due obiettivi principali. Il primo riguardava lo studio dei fattori metodologici che influenzano la produzione in vitro del CH4 e lo sviluppo di un nuovo approccio per la misurazione dello stesso gas prodotto durante delle fermentazioni in vitro. In secondo luogo, si è inteso valutare l’efficacia di alcune strategie nutrizionali sulla riduzione della produzione di CH4 . Tali obiettivi sono stati raggiunti attraverso cinque diverse attività di ricerca. Nel primo lavoro è stato valutato l’effetto che specifici fattori metodologici possono avere sulla produzione in vitro di gas e di CH4 (espresso sia in termini di produzione che di proporzione). Questa analisi è stata effettuata attraverso un approccio di meta-analisi utilizzando 274 osservazioni, che rappresentavano i trattamenti di controllo di 39 articoli scientifici pubblicati negli ultimi 12 anni. I fattori considerati erano: la pressione che si forma nello spazio di testa dello strumento utilizzato per le fermentazioni in vitro (costante o incrementale), il tempo di incubazione (24 o ≥48 ore), il momento di raccolta del liquido ruminale (prima o dopo la somministrazione del pasto agli animali donatori), la presenza di azoto nella composizione della saliva artificiale (presenza o assenza), e il rapporto tra la miscela di liquido ruminale e saliva artificiale sul campione alimentare incubato (130 or 130-140 or >140 ml/g DM). Questi cinque fattori sono stati considerati come variabili discrete, invece il contenuto di NDF del campione alimentare incubato (392 ± 175.3 g/kg) è stato considerato come variabile continua. I risultati hanno mostrato che la produzione totale di gas è principalmente influenzata dal tempo di incubazione, mentre la produzione di CH4 è influenzata soprattutto dal rapporto tra la miscela di liquido ruminale e saliva artificiale sul campione alimentare incubato. Quando invece il CH4 viene espresso in termini di proporzione sul gas totale prodotto, i valori sono principalmente influenzati dal momento di raccolta del liquido ruminale. Inoltre, questa meta-analisi dimostra che i fattori considerati spiegavano una parte considerevole (circa il 65 %) della variabilità dei dati di gas e CH4 prodotti durante le fermentazioni in vitro. Quindi, sarebbe desiderabile una maggiore standardizzazione dei protocolli metodologici internazionali, in modo da facilitare il confronto di dati ottenuti in diversi sperimentazioni. Il secondo contributo sperimentale ha inteso mettere a punto una procedura di raccolta e di misurazione del CH4 prodotto, utilizzando due diverse tipologie di fermentazione in vitro: i) un “sistema aperto”, con sfiato regolare del gas all’interno di un sacchetto connesso alle bottiglie di fermentazione, dal quale viene prelevato il campione di gas per l’analisi del CH4 ; ii) un “sistema chiuso”, con accumulo progressivo dei gas di fermentazione nello spazio di testa delle bottiglie, dal quale viene prelevato il campione per l’analisi del CH4 . Per le fermentazioni sono stati usati cinque alimenti singoli utilizzati nell’alimentazione dei ruminanti (fieno polifita, loietto, farina di mais, panello di lino e polpe di bietola). Il disegno sperimentale prevedeva: 2 incubazioni × 5 alimenti × 3 replicazioni per alimento × 2 tecniche di campionamento del gas, più 4 bianchi (bottiglie incubate senza campione alimentare), per un totale di 64 bottiglie incubate. Metà delle bottiglie non venivano sfiatate, mentre le rimanenti venivano sfiatate a pressione fissa e il gas era raccolto in un sacchetto a tenuta connesso ad ogni bottiglia. Alla fine di ogni incubazione, il gas veniva campionato dallo spazio di testa delle bottiglie utilizzate per il sistema chiuso o dallo spazio di testa e dal sacchetto delle bottiglie utilizzate per il sistema aperto. Tutti i campioni sono stati poi analizzati per quantificare la concentrazione di CH4 . I valori di gas prodotto venivano o meno corretti per la quantità di CO2 che si era disciolta nel liquido di fermentazione. I valori di produzione (ml CH4 /g DM) e di proporzione (ml CH4 /100 ml gas) di CH4 sono stati calcolati utilizzando i valori corretti o non corretti di produzione di gas totale. Il sistema chiuso ha mostrato una produzione di gas totale non corretto inferiore (-18%) rispetto al sistema aperto, mentre la correzione del gas ha ridotto ma non rimosso le differenze tra le due tecniche. Le bottiglie chiuse hanno mostrato una proporzione di CH4 non corretto superiore (+23%) rispetto al sistema aperto, mentre la correzione ha ridotto ma non rimosso le differenze tra le due tecniche. La produzione di CH4 corretto non è stata influenzata dalla tecnica utilizzata. Concludendo, il sistema chiuso non offre buone misurazioni della produzione del gas, mentre il sistema aperto consente una valutazione attendibile sia del gas che del CH4 prodotto. Nel terzo contributo sperimentale è stato valutato l’effetto che le variazioni quantitative dei principali componenti chimici (rapporto amido:ADF, contenuto proteico e contenuto lipidico) di diete per vacche da latte, posso avere sulla produzione di gas e CH4 . Le variazioni sono state fatte tenendo conto degli intervalli di fibra, amido, proteina e lipidi effettivamente utilizzate negli allevamenti intensivi del Nord Italia. La dieta di riferimento utilizzata aveva la seguente composizione chimica: 273, 361, 158, e 33 g/kg SS di amido, NDF, CP, e lipidi, rispettivamente. Le altre 6 diete avevano un minore o maggiore rapporto di amido:ADF (0.40 or 1.77, rispettivamente), o di contenuto proteico (115 or 194 g/kg DM, rispettivamente), o di contenuto lipidico (26 or 61 g/kg DM, rispettivamente), rispetto alla dieta di riferimento. Il disegno sperimentale prevedeva: 4 incubazioni × 7 diete × 5 replicazioni per dieta, più 20 bianchi (bottiglie incubate senza campione alimentare), per un totale di 160 bottiglie incubate. Il gas prodotto veniva regolarmente sfiatato a pressione fissa e alla fine di ogni incubazione un campione di gas veniva raccolto dallo spazio di testa delle bottiglie e veniva analizzato per misurare la concentrazione di CH4 . La quantità di CH4 perso durante lo sfiato del gas di fermentazione è stata stimata. I risultati mostrano che all’aumentare del rapporto amido:ADF si riduce la produzione di gas (per g di SS e per g di degradabilità "vera" della SS), aumenta la produzione di CH4 (per g di SS), mentre la produzione di CH4 espressa come g di degradabilità "vera" della SS non varia. All’aumentare del contenuto proteico si riduce la produzione di gas, mentre la proporzione di CH4 è stata ridotta solo nella dieta ipoproteica. L’aumento del contenuto lipidico ha ridotto la produzione di gas (per g di SS), ma non ha influenzato i valori di CH4 . Si può quindi concludere che l'entità degli effetti, esercitati dalle variazioni quantitative dei principali componenti chimici delle diete sulla fermentazione in vitro, è stata quasi inesistente. Il quarto lavoro ha inteso valutare l’effetto di quattro estratti puri di piante (allil-sulfide, cinnamaldeide, eugenolo e limonene) e di un composto sintetico (monensin), tutti con proprietà antimicrobiche, utilizzati come additivi di una dieta per vacche da latte, sulla produzione di gas e CH4 . Sono stati utilizzati due diversi dosaggi: 3 or 30 mg/g di per gli estratti puri di piante, 0.015 or 0.030 mg/g di dieta per il monensin. Le procedure di incubazioni utilizzate erano le stesse dell’esperimento precedente. Il disegno sperimentale prevedeva: 4 incubazioni × 5 additivi × 2 dosaggi × 3 replicazioni, più 12 bianchi (bottiglie incubate senza campione alimentare, 3 per incubazione), e 12 controlli (dieta incubata senza additivi, 3 per incubazione) per un totale di 144 bottiglie incubate. I risultati hanno mostrato che tutti i composti testati con il basso dosaggio non hanno mai influenzato la produzione in vitro di gas e CH4 . Rispetto al controllo, gli alti dosaggi di allil- sulfide, cinnamaldeide, eugenolo, limonene, e monensin hanno ridotto significativamente la produzione in vitro di gas (ml/g DM; -16%, -12%, -9%, -38%, -12%, rispettivamente). La produzione in vitro di CH4 è stata significativamente ridotta solo dell’alto dosaggio di allil-sulfide, cinnamaldeide, limonene, e monensin (-32%, -12%, -43%, -18%, rispetto al controllo). Solo gli alti dosaggi di allil- sulfide e limonene hanno ridotto significativamente anche la proporzione di CH4 (-18% e -12% rispetto al controllo). I risultati più promettenti sono stati osservati per la cinnamaldeide, che ha depresso la produzione CH4 senza influenzare negativamente gli altri parametri fermentativi. Il quinto e ultimo contributo sperimentale ha valutato la possibilità di ridurre la produzione in vitro di gas e CH4 attraverso la combinazione di un diretto inibitore del CH4 (cloroformio) con due inibitori indiretti: i) i nitrati, che agiscono come accettori di idrogeno e ii) le saponine, dei noti agenti antiprotozoari. L’esperimento è stato condotto utilizzando un sistema di simulazione ruminale a flusso semi-continuo (RUSITEC®). Una dieta base è stata incubata singolarmente (dieta 1) o addizionata con 31.5 g/ kg di nitrati (dieta 2) o con 50 g/kg saponine ottenute dall’estratto d’edera (dieta 3). Queste tre diete sono state incubate senza o con l’aggiunta di cloroformio (2 µL/L), ottenendo un totale di 6 trattamenti testati. Ogni trattamento è stato incubato in quattro bottiglioni, per un totale di 24 bottiglioni. L’intera incubazione è durata 21 giorni. I risultati mostrano che la produzione totale di gas (in media 2.56 l/d) non è stata influenzata dalla dieta o dalla presenza del cloroformio. Quando utilizzati singolarmente, cloroformio, nitrati e saponine riducono la produzione giornaliera di CH4 del 96, 66 e 22%, rispettivamente. Nonostante ciò, quando il cloroformio era combinato con inibitori indiretti, non si è evidenziato nessun effetto addizionale sulla riduzione di CH4 prodotto. Concludendo, si può quindi affermare che nessun effetto sinergico è emerso tra l’inibitore diretto di CH4 e i due inibitori indiretti.

Thesis (MScEng (Process Engineering))--Stellenbosch University, 2010.
ENGLISH ABSTRACT: Recently, improved technologies have been developed for the biobutanol fermentation process: higher butanol concentrations and productivities are achieved during fermentation, and separation and purification techniques are less energy intensive. This may result in an economically viable process when compared to the petrochemical pathway for butanol production. The objective of this study is to develop process models to compare different possible process designs for biobutanol production from sugarcane molasses. Some of the best improved strains, which include Clostridium acetobutylicum PCSIR-10 and Clostridium beijerinckii BA101, produce total solvent concentrations of up to 24 g/L. Among the novel technologies for fermentation and downstream processing, fedbatch fermentation with in situ product recovery by gas-stripping, followed by either liquid-liquid extraction or adsorption, appears to be the most promising techniques for current industrial application. Incorporating these technologies into a biorefinery concept will contribute toward the development of an economically viable process. In this study three process routes are developed. The first two process routes incorporate well established industrial technologies: Process Route 1 consist of batch fermentation and steam stripping distillation, while in Process Route 2, some of the distillation columns is replaced with a liquid-liquid extraction column. The third process route incorporates fed-batch fermentation and gas-stripping, an unproven technology on industrial scale. Process modelling in ASPEN PLUS® and economic analyses in ASPEN Icarus® are performed to determine the economic feasibility of these biobutanol production process designs. Process Route 3 proved to be the only profitable design in current economic conditions. For the latter process, the first order estimate of the total project capital cost is $187 345 000.00 (IRR: 35.96%). Improved fermentation strains currently available are not sufficient to attain a profitable process design without implementation of advanced processing techniques. Gas stripping is shown to be the single most effective process step (of those evaluated in this study) which can be employed on an industrial scale to improve process economics of biobutanol production.
AFRIKAANSE OPSOMMING: Onlangse verbeteringe in die tegnologie vir die vervaardiging van butanol via die fermentasie roete het tot gevolg dat: hoër butanol konsentrasies en produktiwiteit verkry kan word tydens die fermentasie proses, en energie verbruik tydens skeiding-en suiweringsprosesse laer is. Hierdie verbeteringe kan daartoe lei dat biobutanol op ʼn ekonomiese vlak kan kompeteer met die petrochemiese vervaardigings proses vir butanol. Die doelwit van die studie is om proses modelle te ontwikkel waarmee verskillende proses ontwerpe vir die vervaardiging van biobutanol vanaf suikerriet melasse vergelyk kan word. Verbeterde fermentasie organismes, wat insluit Clostridium acetobutylicum PCSIR-10 en Clostridium beijerinckii BA101, het die vermoë om ABE konsentrasies so hoog as 24 g/L te produseer. Wat nuwe tegnologie vir fermentasie en skeidingprosesse behels, wil dit voorkom of wisselvoer fermentasie met gelyktydige verwydering van produkte deur gasstroping, gevolg deur of vloeistof-vloeistof ekstraksie of adsorpsie, van die mees belowende tegnieke is om tans in die nywerheid te implementeer. Deur hierdie tegnologie in ʼn bioraffinadery konsep te inkorporeer sal bydra tot die ontwikkeling van ʼn ekonomies lewensvatbare proses. Drie prosesserings roetes word in die studie ontwikkel. Die eerste twee maak gebruik van goed gevestigde industriële tegnologie: Proses Roete 1 implementeer enkellading fermentasie en stoom stroping distillasie, terwyl in Proses Roete 2 van die distilasiekolomme vervang word met ʼn vloeistof-vloeistof ekstraksiekolom. Die derde proses roete maak gebruik van wisselvoer fermentasie met gelyktydige verwydering van produkte deur gas stroping. Die tegnologie is nog nie in die nywerheid bewys of gevestig nie. Om die ekonomiese uitvoerbaarheid van die proses ontwerpe te bepaal word proses modellering uitgevoer in ASPEN PLUS® en ekonomiese analises in ASPEN Icarus® gedoen. Proses Roete 3 is die enigste ontwerp wat winsgewend is in huidige ekonomiese toestande. Die eerste orde koste beraming van die laasgenoemde projek se totale kapitale koste is $187 345 000.00 (opbrengskoers: 35.96%). Die verbeterde fermentasie organismes wat tans beskikbaar is, is nie voldoende om ʼn proses winsgewend te maak nie; gevorderde proses tegnologie moet geïmplementeer word. Gasstroping is bewys as die mees effektiewe proses stap (getoets in die studie) wat op industriële skaal geïmplementeer kan word om die winsgewendheid van die biobutanol proses te verbeter.
Centre for Renewable and Sustainable Energy Studies

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Funda??o de Amparo ? Pesquisa do Estado de S?o Paulo - FAPESP
Cacha?a corresponds to a beverage with characteristic flavor and aroma, constituted mainly of alcohol and water plus some other components formed in small amounts during the process of fermentation, distillation and aging, and known as the secondary products of alcoholic fermentation. The yeasts and fermentation conditions are considered to be the factors that influence the flavor of alcoholic beverages, since the majority of the secondary compounds responsible for the chemical and sensory quality of the beverage are formed during fermentation. The use of selected yeasts for the production of cane spirit has been studied, with a view to increasing productivity, gaining technological advantages and improving the sensory characteristics of the beverage. This study aimed to produce cacha?a in pilot and alembic scales and evaluate the chemical composition of the beverages produced by selected strains of S. cerevisiae; verify the presence of the selected yeast at the end of the fermentation process on a pilot scale and carry out the evaluation of fermentation parameters of the selected strain in relation to commercial and wild inoculums. The following 16 strains of Saccharomyces cerevisiae were tested: SC52, SC60, SC82, SC91, SC102, SC114, SC129, SC138, SC174, SC177, SC179, SC184, SC219, SC220, SC225 and SC229. Among these, only the SC82 strain could pursue multiplication and fermentation scheme steps to produce cacha?a in pilot and alembic scales. By RFLP mtDNA technique was possible to verify the permanence and dominance of selected yeast (SC82) at the end of fermentation process done in pilot scale. In the evaluation of fermentation parameters to S. cerevisiae strain SC82 had a shorter fermentation time (14h average) and a higher yield (48%), greater efficiency (93.94%) and a higher productivity (2.35 g/Lh) at the end of the third fermentation carried out in relation to commercial and wild inoculums. Regarding the results of the chemical analysis of cacha?a produced in pilot and still scale, only the higher alcohol levels were above that allowed by Brazilian legislation in both produced beverages.
A cacha?a corresponde a uma bebida dotada de sabor e aroma caracter?stico, sendo constitu?da principalmente de ?lcool e ?gua e de outros componentes, formados em pequenas quantidades durante o processo de fermenta??o, destila??o e envelhecimento, os quais recebem a denomina??o de produtos secund?rios da fermenta??o alco?lica. As leveduras e as condi??es de fermenta??o s?o apontadas como fatores que influenciam no sabor das bebidas alco?licas, pois a maioria dos compostos secund?rios respons?veis pela qualidade qu?mica e sensorial da bebida ? formada durante a fermenta??o. A utiliza??o de leveduras selecionadas para produ??o de cacha?a apresenta vantagens tecnol?gicas, como permite minimizar contamina??es indesej?veis, reduz o tempo de fermenta??o, aumenta a produtividade e melhora as caracter?sticas qu?micas e sensoriais da bebida. O presente trabalho teve como objetivos produzir cacha?a em escala piloto e de alambique e avaliar a composi??o qu?mica das bebidas produzidas por linhagens selecionadas de S. cerevisiae; verificar a presen?a da levedura selecionada no final do processo fermentativo em escala piloto e realizar a avalia??o de par?metros fermentativos da cepa selecionada em rela??o a in?culos comercial e selvagem. Foram testadas 16 cepas de Saccharomyces cerevisiae: SC52, SC60, SC82, SC91, SC102, SC114, SC129, SC138, SC174, SC177, SC179, SC184, SC219, SC220, SC225 e SC229. Destas, apenas a cepa SC82 conseguiu prosseguir as etapas do esquema de multiplica??o em escala piloto e em escala de alambique, bem como nas etapas do processo de fermenta??o em escala piloto e de alambique e produzir cacha?a. Atrav?s da t?cnica de RFLPmtDNA foi poss?vel verificar a perman?ncia e domin?ncia da levedura selecionada (SC82) no final do processo fermentativo feito em escala piloto. Na avalia??o dos par?metros fermentativos a S. cerevisiae cepa SC82 apresentou um menor tempo de fermenta??o (m?dia de 14 h) e um maior rendimento (48 %), uma maior efici?ncia (93,94 %) e uma produtividade (2,35 g/Lh) superior ao final da terceira fermenta??o realizada em rela??o ao in?culo comercial e ao in?culo selvagem. Em rela??o aos resultados das an?lises qu?micas da cacha?a produzida em escala piloto e da produzida em escala de alambique, apenas os teores de alco?is superiores estavam acima do permitido pela legisla??o brasileira nas duas bebidas produzidas.

The whey generated during cheese curd manufacture is a byproduct of great importance for cheese industry. Since a large volume of whey is produced and it is rich in several nutrients, the utilization of this product gains importance. The objective of this work was to develop a biotechnological process for obtaining compounds of higher aggregated values by promoting fermentation of whey from cheese curd manufacture. Several fermentations utilizing whey as a substrate were undertaken by using the yeast kluyveromices lactis in the initial concentrations of 9.0 g/L and 19.0g/L, and with the non-sterilized whey in its natural form as obtained in the process of manufacture of cheese curd. In relation to physico-chemical characteristics, increase in protein and lactose consumption was observed in all the fermentations undertaken and these values were relatively more when yeast concentration used for fermentation was of 9.0 g/L. The microbial growth curves were constructed for all the fermentations and fermented samples withdrawn characterizing each phase (14, 30 and 62 hours of fermentation) were analyzed in a gas chromatographic system coupled with several detectors for the presence of volatile compounds. The results demonstrated that between 200 to 252 volatile constituents were captured by dynamic headspace method, between 141 to 235 by liquid-liquid extraction and between 149 to 301 by simultaneous distillation and extraction method. In the volatiles extracted from different fermented products, the presence of compounds such as 2-hexadecenal, octadecanoico acid, hexadecanamide, 3-hexenol was revealed and these are relatively costly compounds as their market price varies from 70.06 to 194.50 US$/100 mL. It could be concluded from the results obtained in this study that the whey fermentation by usage of kluyveromices lactis is a promising biotechnological process for obtaining volatile compounds of higher commercial values.
O soro de queijo é um subproduto de importante relevância na indústria queijeira. Tendo em vista o elevado volume produzido e sua composição nutricional, o aproveitamento deste produto é de suma importância. O objetivo deste trabalho foi desenvolver um processo biotecnológico para obtenção de compostos de alto valor agregado através da fermentação do soro de queijo coalho. Foram realizadas fermentações com o soro utilizando a levedura kluyveromices lactis nas concentrações iniciais de 9,0 g/L e 19,0g/L e com o soro in natura (não esterilizado). Em relação às características físico-químicas observou-se que houve acentuado consumo de proteínas e lactose e aumento no teor de acidez total em todas as fermentações realizadas, sendo obtido valores maiores durante a fermentação do soro com a levedura na concentração inicial de 9,0 g/L. Determinou-se a curva de crescimento microbiano para todas as fermentações, onde coletou-se uma amostra de fermentado de cada fase (14, 30 e 62 horas de fermentação) para serem analisadas por cromatografia gasosa acoplada com diversos detectores para identificação de compostos voláteis. Os resultados demonstraram que de 200 a 252 constituintes voláteis foram capturados pelo método de extração headspace dinâmico, entre 141 a 235 pelo método líquido-líquido e entre 149 a 301 pelo método de destilação e extração simultânea. Nos voláteis extraídos dos produtos fermentados identificou-se a presença de compostos tais como 2-hexadecenal, ácido octadecanóico, hexadecanamida, 3-hexenol e outros, os quais apresentam custo extremamente elevado no mercado mundial variando de 70,06 a 194,50 US$/100 mL. Contudo, verificou-se através deste trabalho que a fermentação do soro de queijo coalho usando a kluyveromices lactis consiste em um processo biotecnológico promissor para a obtenção de compostos voláteis de grande valor comercial.

A produção de metano entérico está entre as principais fontes de emissão de gases de efeito estufa dentre as atividades agropecuárias, além de gerar perda energética ao animal de até 12% da energia bruta consumida. Assim, o objetivo deste trabalho foi avaliar o uso de nitrato de cálcio encapsulado na alimentação de ruminantes como estratégia nutricional a mitigação de metano entérico. O experimento consistiu de duas fases. Fase I: Foram testadas dietas suplementadas com produto comercial de nitrato de cálcio encapsulado utilizando a técnica semiautomática de produção de gases in vitro. Meio grama de substrato com 50 mL de meio de incubação e 25 mL de inóculo ruminal foram incubados em frascos de vidro (160 mL) à 39 ºC por 24 horas para determinação da melhor dieta a ser testada in vivo. O primeiro ensaio testou a associação entre a monensina (dietas com e sem adição de monensina) e doses de nitrato encapsulado (0; 1,5 e 3% da matéria seca (MS)) para mitigação de metano in vitro. Não foi observada interação entre monensina e nitrato para as variáveis testadas. O segundo ensaio in vitro testou a interação do tipo de dieta com duas relações concentrado:volumoso, 20:80 e 80:20, e a inclusão de doses de nitrato encapsulado (0; 1,5; 3 e 4,5% MS). Embora não foi observado efeito associativo entre dieta e nitrato para redução de metano, foi observada mudança nos produtos da fermentação ruminal, com redução de propionato, em decorrência da concorrência de nitrato e propianogênicas por hidrogênio mais escasso em dietas com menor fermentação. Fase II: Conforme os resultados obtidos na Fase I, na segunda fase foi avaliado o efeito associativo da relação de concentrado:volumoso da dieta e a dose de nitrato sobre a emissão de metano, constituintes ruminais e toxicidade do nitrato in vivo. Utilizou-se seis borregos canulados no rúmen, distribuídos em delineamento experimental quadrado latino 6 x 6, em fatorial 2 x 3. Os fatores foram tipo de dieta (relação concentrado:volumoso 20:80 e 80:20) e inclusão de doses de nitrato encapsulado na dieta (0; 1,5 e 3% MS) em substituição gradual ao farelo de soja, totalizando seis tratamentos. Os teores de substituição do farelo de soja pelo nitrato foram em equivalente proteico de maneira a deixar as dietas isonitrogenadas. Os animais foram adaptados gradualmente a oferta de nitrato dietético para evitar problemas com toxidez. A análise de toxicidade foi avaliada pela taxa de metahemoglobina no sangue dos ovinos 3 horas após a alimentação. Nitrato reduziu a produção de metano em ambas as dietas. Os níveis de metahemoglobina no sangue dos animais não foram alterados pela adição de nitrato. Foi observado efeito associativo entre o tipo de dieta e nitrato para os produtos da fermentação ruminal, como acetato, que aumentou linearmente nas dietas com 80% de concentrado quando nitrato foi adicionado. Concluí-se que nitrato, utilizado de forma segura, é uma promissora estratégia para redução de metano entérico independentemente do tipo de dieta com que está sendo suplementado
Production of enteric methane is among the leading sources of greenhouse gas emissions from agricultural activities and generate energy loss to the animal up to 12% of gross energy consumption. The objective of this study was to evaluate the use of calcium nitrate encapsulated in ruminant feed as a nutritional strategy for mitigation of enteric methane. The experiment consisted of two phases. Phase I: tested diets were supplemented with encapsulated calcium nitrate using a semi-automatic in vitro gas production technique Half gram of substrate with 50 mL of incubation medium, and 25 mL of rumen fluid were incubated in glass bottles (160 ml) at 39 °C for 24 hours to determine the best diets to be tested in vivo. The first trial tested the association between monensin (diets with and without monensin) and encapsulated nitrate levels (0, 1.5 and 3% of dry matter (DM)) for in vitro methane mitigation. There was no interaction between monensin and nitrate for the tested variables. The second in vitro assay tested the interaction of diet type with two concentrate:forage ratios, 20:80 and 80:20, and the inclusion of encapsulated nitrate levels (0, 1.5, 3 and 4.5% DM). Although it was not observed associative effect between diet and nitrate for reduction of methane, it was observed change in the ruminal fermentation products. Phase II: According to the results obtained in Phase I, in the second phase we evaluated the associative effect of concentrate: forage ratio of diet and the dose of nitrate on the methane emission, ruminal constituents and nitrate toxicity in vivo. We used six lambs cannulated in the rumen, distributed in Latin square design 6 x 6 in factorial 2 x 3. The factors were type of diet (concentrate:forage ratios 20:80 and 80:20) and inclusion of encapsulated nitrate doses in the diet (0, 1.5 and 3% DM) in the gradual replacement of soybean meal, a total of six treatments. The replacement levels of soybean meal by nitrate were in protein equivalent so as to leave the diets isonitrogenous. The animals were gradually adapted to dietary nitrate supply to avoid problems with toxicity. The analysis of toxicity were evaluated by the rate of methemoglobin in the blood of sheep 3 hours after feeding. Methemoglobin levels in blood of animals were not changed by the addition of nitrate. Associative effect was observed between the type of diet and nitrate for ruminal fermentation products such as acetate, which decreased linearly in the diets with 80% forage when nitrate was added. It can be concluded that nitrate used securely is a promising strategy for reducing enteric methane independently of the type of diet being supplemented

The goal of this work was to establish the suitable and limiting concentrations of Zn, Cu and Mn compounds during syngas fermentation. The results showed that cells encased in polyvinylidene difluoride (PVDF) membranes had a faster accumulation of methane in reactors containing fermentation medium dosed with 5 mg/L of each heavy metal compared to free cells. It was also revealed that total inhibition of biohydrogen production occurred in medium containing 5 mg/L Cu, 30 mg/L Zn and 140 mg/L Mn while the most suitable metal concentration level was 0.1 mg/L Cu, 0.6 mg/L and 2.8 mg/L Mn. In addition, a comparison test showed that for the most suitable metal concentration in the medium, rate of performance at pH 6 and 7 was higher than at pH 5.

A new method of in situ extraction of alcohols from fermentation broth was investigated. The extraction method exploited the latent advantages of the non-equilibrium phase interaction of the fluid system in the flash tank to effectively recover the alcohol. Carbon dioxide gas ranging from 4.2L/min to 12.6L/min was used to continuously strip 2 and 12% (v/v) ethanol solution in a fermentor with a recycle. Ethanol and water in the stripped gas was recovered by compressing and then flashing into a flash tank that was maintained at 5 to 70bar and 5 to 55oC where two immiscible phases comprising CO2-rich phase (top layer) and H2O-rich phase (bottom layer) were formed. The H2O-rich bottom layer was collected as the Bottoms. The CO2-rich phase was continuously throttled producing a condensate (Tops) as a result of the Joule-Thompson cooling effect. The total ethanol recovered from the extraction scheme was 46.0 to 80% for the fermentor containing 2% (v/v) ethanol and 57 to 89% for the fermentor containing 12% (v/v) ethanol. The concentration of ethanol in the Bottoms ranged from 8.0 to 14.9 %(v/v) for the extraction from the 2 %(v/v) ethanol solution and 40.0 to 53.8 %(v/v) for the 12% (v/v) fermentor ethanol extraction. The Bottoms concentration showed a fourfold increase compared to the feed. The ethanol concentration of the Tops were much higher with the highest at approx. 90% (v/v) ethanol, however the yields were extremely low. Compression work required ranged from 6.4 to 20.1 MJ/kg ethanol recovered from the gas stream in the case of 12% (v/v) ethanol in fermentor. The energy requirement for the 2% (v/v) extraction was 84MJ/kg recovered ethanol. The measured Joule-Thompson cooling effect for the extraction scheme was in the range of 10 to 20% the work of compressing the gas. The lowest measured throttle valve temperature was -47oC at the flash tank conditions of 70bar and 25oC. Optimization of the extraction scheme showed that increasing the temperature of the flash tank reduced the amount of ethanol recovered. Increasing the pressure of the flash tank increased the total ethanol recovered but beyond 45bar it appeared to reduce the yield. The 12.6L/min carbon dioxide flow rate favored the high pressure(70bar) extraction whiles 4.2L/min appeared to favor the low pressure(40bar) extraction. The studies showed that the extraction method could potentially be used to recover ethanol and other fermentation products.
Master of Science

La micro-oxygénation des vins, par la dispersion de bulles d’oxygène, est une pratique de plus en plus utilisée dans le domaine de l’œnologie. Cette technique n’est pas toujours convenablement maitrisée par manque de connaissances scientifiques sur les paramètres régissant le transfert de l’oxygène. La recherche s'est focalisée sur l'étude des coefficients de transfert en fonction des composés du vin (CO2, éthanol, sucrose, consommateurs d’oxygène) et des conditions opératoires (type de diffuseur, température, rapport entre hauteur et diamètre du contenant de liquide). Les résultats montrent que lors de la micro-oxygénation, le dioxyde de carbone dissous et le sucrose ont une incidence négative sur le transfert alors que la présence d’éthanol améliore le transfert. En ce qui concerne les conditions opératoires, l’augmentation de débit de gaz et l’augmentation de rapport entre la hauteur et le diamètre de la cuve de micro-oxygénation joue positivement sur le transfert d’oxygène La surface spécifique des bulles et le coefficient de transfert de matière ont pu être dissociés pour les vins. La nature tensio-active des composés du vin semble être un élément important sur le transfert de matière. Les connaissances acquises ont été appliquées à la micro-oxygénation au cours de deux étapes de l'élaboration des vins : la fermentation alcoolique avec la maitrise de l’apport d’oxygène et la simulation de la technique d’élevage en barrique par micro-oxygénation couplée à l’ajout de copeaux de bois. Une nouvelle approche concerne l'étude d'un contacteur membranaire qui permet le transfert d’oxygène par diffusion
Micro-oxygenation of the wines, by the dispersion of oxygen bubbles, is a practice increasingly used in oenology. This technique is not always suitably controlled for lack of scientific knowledge on the parameters governing the transfer of oxygen. Research was focused on the study of transfer coefficients in function of wine components (CO2, ethanol, sucrose, consuming oxygen) and of operating conditions (type of diffuser, temperature, relationship between height and diameter of the container of liquid). The results show that during micro-oxygenation, the dissolved carbon dioxide and the sucrose have a negative incidence on the transfer whereas the presence of ethanol improves the transfer. As operating conditions are concerned, the increase in gas output and the increase in micro-oxygenation tank height/diameter ratio positively influence oxygen transfer. For wines, the specific surface of the bubbles and the mass transfer coefficient could be dissociated. The surfactant nature of wine components seems to be the most important factor in mass transfer. The knowledge so acquired was applied to micro-oxygenation during two stages of wine making: alcoholic fermentation with the oxygen yield control and the simulation of ageing technique in barrels coupled with the wood chips addition. A new approach relates to the study of a membrane contactor application allowing the oxygen transfer by diffusion

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In the ethanol fermentation processes, the product inhibition phenomenon limits the ethanol concentration in the fermented wine around 10% v.v-1, which results in a large volume of vinasse (around 12Lvinasse/Lethanol) and high level of energy consumption by distillation (around 2,5 Kgvapor/Lethanol). Alternative technologies designed to remove ethanol from the fermentation broth have been studied in the literature. One example is the operation of ethanol entrainment by a carrier gas, known as stripping, which in addition to the removal of the ethanol, a reduction of the temperature of the medium is expected due to the vaporization. In this context, the stripping operation could be used to control the ethanol concentration and cooling the fermentation process, as it could be used after the fermentation to promote the ethanol separation, being an alternative to distillation, reducing the energy consumption. In this study it was analyzed the stripping operation using CO2 (generated during the fermentation) in pilot plant. A factorial design was applied to evaluate the influence of the initial temperature of the solution (T0) and specific flow rate of the carrier gas (CO2) on the following stripping performance parameters: entrainment factor (FA), concentration factor (FC), and temperature reduction factor (FT). Furthermore, a mathematical model was proposed to describe the ethanol mass (mET) and the temperature (T) changes during the stripping operation. It has evaluated the performance of the ethanol recovery system and an extractive fermentation. The results showed that FA and FT were positively affected by T0, because the temperature increasing leads to greater vaporization, promoting greater entrainment of the ethanol and the high extraction of energy from the medium. The FA was also positively influenced by CO2, because it promotes greater mass transfer rate and contact area between the bubbles and liquid phase. The CO2 influenced the FT positively, but only for smaller CO2. Suppose that, for larger CO2, the equilibrium was not reached due the lower residence time of the bubbles in the wine, by removing smaller amount of energy from the liquid phase. The FC was negatively influenced by T0, because of the entrainment of a larger amount of water together with the ethanol has occurred. From the simulated results it was observed that, for smaller CO2, the expected results for the proposed model were close to the experimental, with maximum errors of 10%, concluding that the model is valid, i.e., the equilibrium was reached and the effect of mechanical entrainment was negligible. However, for larger CO2, the errors were higher (20%), concluding that the system has not reached the equilibrium and the effect of mechanical entrainment was significant. The ethanol recovery system of the CO2 stream presented 66.88 and 40.93% efficiency for the assays carried out at 33 and 65ºC, respectively, therefore with a low performance. In the extractive fermentation it was observed that the removal of the ethanol from the medium reduced the product inhibition and increased the substrate consumption rate. The CO2 flowthrough the fermentation medium (CO2 of 1.0 vvm) contributed to the reduction in the temperature of the medium in 41%. Furthermore, there was a decrease in the values of the volumetric flow rates of the cooling water, around 34%, in extractive fermentation when compared to conventional fermentation, indicating a potential savings of the 34% on inputs in the cooling towers and, also, saving energy in pumping this water.
Na fermentação alcoólica, o fenômeno da inibição pelo produto limita a concentração de etanol no vinho ao redor de 10 ºGL, o que resulta em grande volume de vinhaça (ca. 12 Lvinhaça/Letanol) e alto consumo de vapor na etapa de destilação (ca. 2,5 kgvapor/Letanol). Tecnologias para remover etanol do vinho vêm sendo estudadas. Exemplo é a operação de arraste de etanol por um gás, conhecida como stripping, que além da remoção de etanol, provoca também a redução da temperatura do meio reacional devido à sua vaporização. Neste contexto, a operação de stripping poderia ser utilizada tanto no controle da concentração de etanol e na redução da temperatura do meio fermentativo, como poderia ser utilizada após a fermentação para a extração de etanol do vinho, sendo uma alternativa à destilação, reduzindo o consumo de vapor da planta. Neste trabalho avaliou-se a operação de stripping, em escala piloto, utilizando CO2 (gerado no próprio processo industrial). Um planejamento fatorial foi utilizado para avaliar a influência das variáveis independentes, temperatura inicial do vinho (T0) e vazão específica de CO2 (CO2), nos parâmetros de desempenho: Fator de Arraste (FA), Fator de Concentração (FC) e Fator de Redução de Temperatura (FT). Além disso, desenvolveu-se um modelo para prever as variações da massa de etanol (mET) e da temperatura (T) do sistema durante a operação de stripping. Avaliou-se, também, o desempenho de um sistema de recuperação de etanol e de uma fermentação extrativa. Os resultados mostraram que o FA e o FT foram positivamente influenciados por T0, pois o aumento da temperatura gera maior vaporização, o que contribui para maior arraste de etanol e maior retirada de energia do meio. O FA foi também influenciado positivamente pela CO2, pois promove maior transferência de massa e área de contato entre as bolhas e o vinho. A CO2 influenciou positivamente o FT, porém, apenas para as menores CO2. Supõe-se que, para as maiores CO2, as bolhas não atingiram o equilíbrio devido ao menor tempo de residência das bolhas no vinho, retirando menor quantidade de energia do meio. O FC foi influenciado negativamente por T0, pois ocorre o arraste de uma maior quantidade de água juntamente com o etanol. A partir dos resultados da simulação observou-se que, para as menores CO2, os resultados previstos pelo modelo proposto ficaram próximos aos experimentais, com desvios máximos de 10%, concluindo-se que o modelo foi válido, ou seja, atingiu o equilíbrio e o efeito da sublação foi desprezível. Porém, para as maiores CO2, os desvios foram maiores (20%), concluindo-se que o sistema não atingiu o equilíbrio e o efeito da sublação foi significativo. O sistema de recuperação de etanol da corrente de CO2 mostrou eficiência de 66,88 e 40,93%, para os ensaios a 33º e 65 ºC, respectivamente, apresentando, portanto, um baixo desempenho. Notou-se que na fermentação extrativa houve a remoção de etanol do meio, reduzindo a inibição pelo produto e aumentando a velocidade de consumo de substrato. A passagem do CO2 pelo meio fermentativo (CO2 de 1 vvm) contribuiu para redução da temperatura do meio em 41%. Além disso, houve o decréscimo dos valores das vazões volumétricas da água de resfriamento, em torno de 34%, nas fermentações extrativas quando comparadas com as convencionais, indicando uma potencial economia de 34% em insumos nas torres de resfriamento e, também, economia de energia no bombeamento desta água.

The thesis discusses the use of biomass for electricity island operation. The first chapter explains the concept of biomass and its distribution. It also shows the potential and availability of biomass. The second chapter describes the transformation of biomass into a suitable fuel form. The third to fifth chapter describes the conversion of biomass as a fuel into electrical energy through combustion, Stirling and steam engine. In these chapters there are brought the following parameters and prices of these motors and single-phase and three-phase alternators that can be connected to these engines. Other chapters describe the production of electricity in line with consumption, where the cost per hour to run engines for the various types of fuel and price of the electricity produced in kilowatt hours is calculated. In the last chapter of the thesis there is described the possibility of accumulation of electrical energy.

This study aimed to evaluate the effect of different dose levels of aguamiel (Agave atrovirens) on in vitro cecal gas, methane (CH4) and carbon dioxide (CO2) productions of five forage species (Avena sativa (hay)), Moringa oleifera, Caesalpinia coriacea, Salix babylonica and Eichhornia crassipes) using inocula from horse. The forage samples were incubated with three doses of aguamiel: 0, 34 and 68 μg of aguamiel/g dry matter (DM) of substrate. Cecal inocula were collected from 4 adult female Criolla horses (3 to 4 years of age and weighing 300 ± 15.0 kg) grazed on native grasses for about eight hours without supplementation. Forage type affected (P < .001) cecal asymptotic, rate and lag time of gas, CH4 and CO2 productions (mL/g DM) and pH and DM degradability. Aguamiel dose had linear and quadratic effects (P < .05) on the asymptotic and rate of CH4 productions and rate and lag time of CO2 productions (mL/g DM). Forage type x aguamiel dose interactions were significant (P < .05) for asymptotic, rate and lag time of gas, CH4 and CO2 productions (mL/g DM). Forage species effects were pronounced (P < .05) on CH4 and CO2 productions (mL/g incubated and degraded DM) and proportional CH4 production at all hours of incubation, except for CO2 production (mL/g incubated DM). Aguamiel dose affected (P < .05) CO2 production (mL/g incubated DM) and proportional CO2 production at the incubated hours. Forage type x aguamiel dose interactions were observed (P < .05) for CO2 production (mL/g incubated DM) and proportional CO2 production at the incubated hours but had no impact of CH4 production. It is concluded that addition of aguamiel to five forage species affected fermentation kinetics of GP resulting in different in vitro cecal gas, methane and carbon dioxide productions from these substrates.

This study aimed to evaluate the effect of different dose levels of aguamiel (Agave atrovirens) on in vitro cecal gas, methane (CH4) and carbon dioxide (CO2) productions of five forage species (Avena sativa (hay)), Moringa oleifera, Caesalpinia coriacea, Salix babylonica and Eichhornia crassipes) using inocula from horse. The forage samples were incubated with three doses of aguamiel: 0, 34 and 68 μg of aguamiel/g dry matter (DM) of substrate. Cecal inocula were collected from 4 adult female Criolla horses (3 to 4 years of age and weighing 300 ± 15.0 kg) grazed on native grasses for about eight hours without supplementation. Forage type affected (P < .001) cecal asymptotic, rate and lag time of gas, CH4 and CO2 productions (mL/g DM) and pH and DM degradability. Aguamiel dose had linear and quadratic effects (P < .05) on the asymptotic and rate of CH4 productions and rate and lag time of CO2 productions (mL/g DM). Forage type x aguamiel dose interactions were significant (P < .05) for asymptotic, rate and lag time of gas, CH4 and CO2 productions (mL/g DM). Forage species effects were pronounced (P < .05) on CH4 and CO2 productions (mL/g incubated and degraded DM) and proportional CH4 production at all hours of incubation, except for CO2 production (mL/g incubated DM). Aguamiel dose affected (P < .05) CO2 production (mL/g incubated DM) and proportional CO2 production at the incubated hours. Forage type x aguamiel dose interactions were observed (P < .05) for CO2 production (mL/g incubated DM) and proportional CO2 production at the incubated hours but had no impact of CH4 production. It is concluded that addition of aguamiel to five forage species affected fermentation kinetics of GP resulting in different in vitro cecal gas, methane and carbon dioxide productions from these substrates.

碩士
國立成功大學
化學工程學系碩博士班
97
We investigated the impact of gas-stripping on the in situ removal of acetone, butanol, (AB) from batch reactor fermentation broth. The strain, Clostridium beijerinckii, was not affected adversely by gas stripping. A sizable improvement in the productivity and yield was observed in this research in comparison with the non-integrated process. In an integrated process of AB fermentation-recovery using Clostridium beijerinckii, AB productivity and yield were improved up to 50 and 10.8%, respectively, as compared to the controled batch fermentation data. In a batch reactor Clostridium beijerinckii utilized 30.9 g glucose L-1 and produced 11.3 g total AB L-1, while in the integrated process it utilized 38.8 g glucose L-1 and produced total AB of 15.3 g L-1. In the integrated process, acids were totally converted to solvents when compared to the non-integrated process (batch fermentation) which retained residual acids at the end of fermentation. In situ removal of AB by gas stripping has been informed to be one of the most important techniques of solvent removal. During these researches we were capable to maintain the AB concentration in the fermentation broth below toxic levels. Another study was carried out to study the effect of trace metal addition for butanol production The results show that this bacterial need a selected nutrient with selected concentration. Trace metal addition to the new medium showed that magnesium and calcium enhanced the butanol yield. The results showed that 1.2 g L-1 MgSO4 enhanced the butanol yield from 0.073 g g-1 glucose utilized to 0.115 g g-1 glucose utilized with biomass concentration of 5.35 g L-1. Magnesium and calcium addition were better for growth and for butanol production. Biomass concentration increases as trace metal increases in the fermentation medium. The highest biomass concentration was 5.35 g L-1 at 1.2 g L-1 MgSO4, 5.98 g L-1 at 6 g L-1 MgSO4 and 4.86 g L-1 at 1.1 g L-1 CaCl2, suggested that magnesium and calcium trace metal are necessary for biomass production. The effect of orange peel concentration on ethanol production by the ethanologenic bacterium Saccharomyces carlsbergensis was investigated. Orange peel was added in various amounts to determine its effects on ethanol production. Fermentation of sugar solutions was conducted at 30 oC. The ethanol produced by Saccharomyces carlsbergensis was determined after 0, 13, 21, 27, 35, 41, and 48 h. At 1, 1.5, 2, 2.5 g per 150 mL orange peel concentration, 0.13, 0.10, 0.09, and 0.08% (v/v) ethanol was achieved respectively, while 0.07% (v/v) was realized at 3 g per 150 ml orange peel concentration. The effect of initial orange peel concentration over the range of 1 to 3 g of dissolved solids per 150 ml greatly decreased the ethanol production from 0.13% (v/v) until 0.07% (v/v).

碩士
國立中興大學
化學工程學系所
102
A novel integrated in situ extraction-gas stripping process (designated as process 3 in this study) is proposed for running batch Acetone-Butanol-Ethanol (ABE) fermentation. Process 3 consists of two butanol separation processes where butanol is firstly extracted by oleyl alcohol during ABE fermentation. Simultaneously, gas stripping is performed directly on the oleyl alcohol phase where extracted butanol is separated from the fermentor. The butanol productivity and yield of 0.28±0.01 g·L-1·h-1 and 0.226±0.001 g-butanol/g-glucose were achieved with process 3. The ABE productivity and yield were 0.46±0.01 g·L-1·h-1 and 0.374±0.002 g-ABE/g-glucose. The glucose utilization was 97% where the initial glucose consumption was 121±2 g/L. The butanol and ABE concentrations of 93-113 and 166-204 g/L in the condensate can be achieved. This study demonstrated that an enhanced ABE fermentation can be achieved with process 3 while a high purity of solvents can be simultaneously obtained.For fed-batch fermentation,148.1 g/L ABE (82.2 g/L butanol, 49.5 g/L acetone, 16.4 g/L ethanol) were produced from 309.7 g/L glucose in four feeding cycles over 216 h. The overall productivity and yield were 0.69 g/L h and 0.48 g/g for ABE and 0.38 g/L h and 0.27 g/g for butanol, respectively. The higher productivity was attributed to increased viable cell density in the fed-batch fermentation. For fed-batch fermentation system,when the cooling temperature decreased from 2 ◦C to –196 ◦C, the butanol concentration in the condensate increased from 25-35 g/L to 150-220 g/L.The results suggested that a lower cooling temperature would be better for condensing and recovering butanol vapor in a more concentrated state because of higher selectivity over water.

碩士
朝陽科技大學
應用化學系碩士班
99
A real time quantitative analysis of flavors by FD-ESI-MS was developed in our lab to detect the acidic gas during fermentation which associated with the pH value in solution. However, the bare ESI source result in unstable data and the detection limit is not good enough. Our purpose is to improve this method to get a stable and lower limit detection results. The first improvement is putting the source inside a glass cover to reduce the interference of ambient environment, then (a) apply home-made nanoelectrospray ion source to couple with the sample gas, (b) compare the sensitivities by introducing sample gas from one-side or both-side of the glass cover to fuse with electrospray droplets. There are four parameters needed to be properly adjusted. They include: （1）ESI Voltage（2）ESI flow rate（3）carrier gas flow rate（4）the length of sample tube tip cutting. All measurements are under negative ion mode. The experimental results showed that（a）fusing of nano-ESI droplet with gaseous molecules can not reduce the detection limit, and （b）the detection limit（1×10-7 M）of both-sides introduced gas sample is better than that of one-side introduced gas sample which is 1×10-6 M. The optimization conditions for both-sides introduced gas sample are 3 kV of ESI voltage, 5 μL/ min of ESI flow rate, and 400 c.c./min of carrier gas flow rate. The best position for ES capillary tip is 2~5 mm to MS inlet, and there is 1~5 mm from the ES capillary tip to the tip of gas sample introducing tube. The angle between these two tips is about 85 degree. After optimization, the calibration curve was obtained by the ratio of peak height of different concentrations of sample acids to peak high of internal standard acid（TFA）. This method was applied to obtain quantitative information of flavor during the fermentation process of milk. Quantitative analysis is focusing on lactic acid and acetic acid. Pyruvic acid and pentanoic acid are also detected at 42 hours and 48 hours fermentation. Experimental results show the change of measured flavor amount is agree with the decrease of pH value. The slopes of calibration curve for lactic acid and acetic acid are also similar. Comparing the results of this improvement FD-ESI/MS method with the results of previous FD-ESI/MS method, the LOD for lactic acid and acetic acid in later one is 1×10-7 M each, This is 10 times better than previous one which is 1×10-6 M each. Meanwhile, the measured develop trends for lactic acid and acetic acid are much coincident and smooth than that of previous one. This could be resulted from higher stability after improvement.

碩士
國立臺灣師範大學
化學系
103
This study was conducted to a general understanding of the use of a milli-whistle as a gas chromatography (GC) detector in gas analysis.The milli-whistle is connected to the outlet of a GC capillary, and when the eluted gases and the GC carrier gas pass through it, a sound with a fundamental frequency is produced. The sound wave can be picked up by a microphone, and after a fast Fourier transform, the online data obtained for frequency-change vs. retention time constitute a new method for detecting gases with LabVIEW(Laboratory Virtual Engineer Workbench).In order to improve the efficiency of fermentation, in this study, yeast strain, Saccharomyces cerevisiae, using electrostatic spinning technology coated inside to the PLLA (poly-L-lactide; PLLA) microtubule arrays film. Yeast activated in film will not be affected. The film can be recycled and continue to use, reduce the cost of making ethanol. In this study, the fermentation broth and yeast that has been coated inside to fibers put into the container together to ferment. Using of program control self-assembled solenoid valve, the fermentation process produces ethanol and carbon dioxide vapors, etc., at 5-minute intervals, automatically injected into the capillary column to separate and record. Experimental results show that the use of our developed device, we can immediately detect and track fermentation. The experiment apparatus can detect at least 4 days with fully automated. According to the amount of change in frequency, it can instantly detect the concentration of ethanol that is in fermentation broth。

Bioprocesses carried out for the production of fuels and other value-added co-products require effective process control strategies. The objective of this research is to apply Titrimetric Off-Gas Analysis (TOGA) for the on-line estimation of fermentation end products using Clostridium thermocellum. The hydrogen ion production, gaseous H2 and CO2, soluble H2 and CO2, as well as ethanol in the liquid phase and vapour phase, were monitored. All parameters, except the dissolved gases, showed good correlation with concurrent off-line analysis. The resulting mass and electron balances were close to theoretical values, and not significantly different from those determined using off-line analysis. Liquid-to-gas mass transfer limitations caused supersaturation of H2(aq) for a wide-range of operating conditions, and on average, ranged between 8-14 times the expected value at thermodynamic equilibrium. The supersaturation of CO2(aq) was conditional, and could be alleviated by increased sparging at agitation such that no significant mass transfer limitation was present. Simultaneous data on ethanol, CO2, and H2 could be obtained with the MIMS probe placed adjacent to the liquid surface in the reactor headspace. From this data, a metabolic model was proposed for the on-line estimation of formate and acetate using a mass balance and an electron balance. The model estimated formate concentrations with reasonable accuracy. Acetate predictions agreed with the qualitative trends, but the concentrations were inaccurate in comparison with off-line analysis. It was demonstrated that the sensor could provide on-line information on all major end-products synthesized by C. thermocellum. In conclusion, TOGA is a valuable instrument for the on-line monitoring and study of fermentation processes for cellulosic biofuels production

碩士
國立中山大學
環境工程研究所
94
Kitchen waste compositing plants emit odorous gas streams with sulfur-, nitrogen-, and oxygen-containing compounds and other hydrocarbons. A pilot-scale biotrickling filter with a space of 0.3 m square and 1.0 m height packed with fern chips was used for removing the odorous components from the kitchen waste compositing gas. An average weight ratio of “kitchen waste: bulking material: seeding compost” of 90:4.5:5.5 was used to prepare the compositing material for producing the odorous gas for test. The kitchen waste was composed of residual material from food preparation and meal wastes. The bulking material was either wood trimmings or dried leaves and the seeding material was a blend of manure and bird feather compost. Experiments indicate that the composting material could develop to 32-55 oC during a composting period of 6 weeks and the vented gas contained ammonia, amines, mercaptans, and hydrogen sulfide to maximum values of 700, 1,000, 53, and 1.0 ppm, respectively. A maximum odor concentration of 23,000 was obtained and the odor intensity was closely related to mercaptans in the vented gas. Results indicate that by using the bio-treated effluent of the school-owned domestic wastewater treatment plant as a supplemental water and nutrition sources for the biotrickling filter, 0.5-5 and 1-15 ppm of ammonia and amines, respectively, in the introduced odorous gas could completely be removed at conditions of empty-bed-retention-times (EBRT) of 15 s and liquid/gas flow ratio (L/G) of 0.003 m3/m3. Particularly, with an EBRT of 7 s at a fixed L/G of 0.002, 99.7% of odor intensity (dilution to the threshold ratio, DT) in the influent gas with a DT of 5,500 could be removed. Instead of effluent wastewater, by supplementing tap water with 25 mg/L of milk powder to the biotrickling filter, results indicate that with an EBRT of 7 s at a fixed L/G of 0.002, 99.7% of odor intensity in the influent gas with a DT of 13,000 could be removed. Milk powder supplementation gave better performance than the effluent wastewater one.

Tese de doutoramento em Chemical and Biological Engineering
One of the major drawbacks of syngas fermentation is the limited gas-liquid mass transfer that generally limits productivities. Most research has been focusing in increasing the kLa (volumetric gas-liquid mass transfer coefficient), investigating different reactor typologies and gas dispersion devices. However, the driving force for mass transfer can also be increased, for instance, by operating at increased pressure. This thesis aims to explore the effect of increased pressure in syngas fermentation to improve the gas solubility, evaluating the effect on the biocatalysts and on the process itself. The starting point of this work was to use an adapted culture (from a syngas converting reactor) as inoculum. This culture was able to mainly produce methane and acetate from syngas. Methane was not produced directly from CO, but via the conversion of acetate and H2 by the bacteria present in the mixed culture (Acetobacterium and Sporomusa species). Later on, this culture was subsequentially transferred with syngas, originating an enriched culture mainly composed by Acetobacterium and Methanospirillum species that, besides acetate and methane, was able to produce propionate from syngas. From that highly enriched culture a new strain of Acetobacterium wieringae, strain JM, was isolated and characterized. This highly enriched culture was tested in an axial agitation reactor (AAR) with different initial syngas (CO, H2 and CO2 (60:30:10 %, v/v)) pressures, from 100 kPa to 600 kPa. No substrate inhibition was observed, even at the highest pressure and an increase of 45 % in titres (of acetate and propionate) were obtained. Moreover, the increase of pressure resulted in a shift in the metabolic pathways from acetate towards propionate, which is an uncommon product of syngas fermentation. The effect of pressure on the conversion of syngas by anaerobic mixed sludge was then studied using two different reactor typologies, AAR and GLR (gas-lift reactor). Initial syngas pressures of 100 kPa, 300 kPa and 500 kPa were tested. Overall, the GLR showed better performance in terms of CO consumption rates and product titres. The main product obtained was methane and the results showed, for the first time, that methanogenic activity was not inhibited with initial syngas pressures up to 500 kPa, achieving methane yields of 75 % for the GLR, and 92 % for the AAR. At 300 kPa and 500 kPa, volatile fatty acids were also produced, namely acetate, proprionate and n-butyrate. Propionate was the most abundant acid produced, reaching 4.4 mM at 300 kPa and 4.8 mM at 500 kPa in the AAR. Overall, the use of moderate pressures in syngas fermentation was shown beneficial, as it resulted in better productivities and titres without having significant detrimental effects on cell growth. The increase of pressure also did not inhibit methanogenesis. Moreover, higher pressures seem to induce the production of different chemicals, broadening the product spectrum of syngas fermentation. In this way, these findings could be the basis of new developments in the industrialization of syngas fermentation to produce platform chemicals and/or for biomethanation processes.
Um dos principais desafios da fermentação do gás de síntese é a limitação na transferência de massa gás-líquido que geralmente limita a produtividade. A maioria das pesquisas têm-se concentrado no aumento do kLa (coeficiente de transferência de massa gás-líquido volumétrico), investigando diferentes tipologias de reatores e dispositivos de dispersão de gás. No entanto, a força motriz para a transferência de massa também pode ser aumentada, por exemplo, operando sistemas pressurizados. Esta tese tem como objetivo explorar o efeito do aumento da pressão na fermentação de gás de síntese para melhorar a solubilidade do gás, avaliando o efeito nos biocatalisadores e no próprio processo. O ponto de partida deste trabalho foi a utilização de uma cultura adaptada (de um reator de conversão de gás de síntese) como inóculo. Essa cultura foi capaz de produzir principalmente metano e acetato a partir do gás de síntese. O metano não foi produzido diretamente do CO, mas através da conversão de acetato e H2 pelas bactérias presentes na cultura mista (espécies dos géneros Acetobacterium e Sporomusa). Essa cultura foi consecutivamente transferida usando gás de síntese como substrato, originando uma cultura enriquecida composta principalmente por espécies de Acetobacterium e de Methanospirillum que, além de acetato e metano, foi capaz de produzir propionato a partir de gás de síntese. A partir dessa cultura altamente enriquecida, uma nova strain de Acetobacterium wieringae, strain JM, foi isolada e caracterizada. Esta cultura altamente enriquecida foi testada num reator de agitação axial (AAR) com diferentes pressões de gás de síntese (CO, H2 e CO2 (60:30:10%, v/v)), de 100 kPa a 600 kPa. Não foi observada qualquer inibição pelo substrato, mesmo na pressão mais elevada e obteve-se um aumento de 45 % na produção final de acetato e propionato. Além disso, o aumento da pressão originou um desvio metabólico de acetato para propionato, que é um produto incomum da fermentação do gás de síntese. O efeito da pressão na conversão de gás de síntese utilizando biomassa anaeróbia foi também estudado usando duas tipologias de reatores diferentes, AAR e GLR (reator gas-lift). Foram testadas as seguintes pressões iniciais de gás de síntese: 100 kPa, 300 kPa e 500 kPa. O GLR apresentou melhor desempenho em termos de taxas de consumo de CO e de concentração final de produtos. O principal produto obtido foi o metano e os resultados mostraram, pela primeira vez, que a atividade metalogénica não foi inibida com pressões iniciais de syngas de até 500 kPa, atingindo rendimentos de metano de 75 % para o GLR e 92 % para o AAR. A 300 kPa e 500 kPa, também foram produzidos ácidos gordos voláteis, nomeadamente, acetato, propionato e n-butirato. O propionato foi o ácido mais abundante produzido, atingindo 4,4 mM a 300 kPa e 4,8 mM a 500 kPa no AAR. No geral, o uso de pressões moderadas na fermentação do gás de síntese mostrou-se benéfico, pois resultou em melhores produtividades e concentrações finais de produto sem ter efeitos prejudiciais significativos no crescimento celular. O aumento da pressão também não inibiu a metanogénese. Além disso, pressões mais altas parecem induzir a produção de diferentes produtos químicos, ampliando o espectro de produtos da fermentação do gás de síntese. Desta forma, estas descobertas podem ser a base de novos desenvolvimentos na industrialização da fermentação do gás de síntese para a produção de produtos químicos e/ou para processos de biometanação.

碩士
大同大學
生物工程研究所
89
Control of nutrient feeding using exit gas data and comparison of pH-stat and glucose-stat for fed-batch fermentation of recombinant microorganism was investigated. E. coli XL1 Blue was cultured by carbon dioxide evolution rate (CER) fed-batch cultivation, the cell density reached 26.5 gDCW/L with feeding proportionality parameter set at 6.6 in 34 h; when feeding proportionality parameter set at 5.2, the productivity of E. coli XL1 Blue was 1.515 gDCW/L/h. In contrast with CER strategy, the productivity of using glucose-stat and pH-stat as feeding strategy were 0.704 gDCW/L/h and 0.649 gDCW/L/h, respectively. To comparing CER strategy applying on other recombinant microorganism, fed-batch of E. coli BL21 and S. cerevisiae KC1/L8a were investigated. It was found that this feeding strategy could be applied successfully; E. coli BL21 and S. cerevisiae KC1/L8a reached 21.5 gDCW/L in 16 h and 18.7 gDCW/L in 39 h, respectively. In this report, we also used CER strategy fed-batch culture of E. coli XL1 Blue on pilot-plant fermenter (35 L). In this case, it should be considered that the capacity of oxygen supply of pilot-plant fermenter to adjust feeding proportionality parameter dynamically. The result of fed-batch of E. coli XL1 Blue reached 23.5 gDCW/L in 41 h.

Кваліфікаційна робота присвячена характеристиці властивостей молочнокислих бактерій, які використовуються в заквасках для ферментованих молочних продуктів. Відібрано протеїназо-позитивні штами лактококів та встановлено їхню протеолітичну активність, проаналізовано кислотоутворювальну здатність, вплив видового складу закваски на тривалість згортання казеїну Роботу присвячено розробці та обґрунтуванні технології приготування рідинних систем (опари), виготовленої із частковим зменшенням води за рахунок використання соку червоного буряка. Проведено аналітичний огляд інформаційних джерел вітчизняних та іноземних авторів стосовно досвіду й перспектив удосконалення технології хліба, проаналізовано принципи структуроутворення опари, розглянуто перспективи використання червоного буряка у технології хлібопекарської промисловості. Відповідно аналітичних досліджень встановлено, що рідка опара при структуроутворенні залежить від багатьох параметрів процесу та сировини, що зумовлює вибір цих характеристик для проведення експериментів.
The work is devoted to the development and substantiation of the technology of preparation of liquid systems (mash), made with partial reduction of water due to the use of red beet juice. An analytical review of information sources of domestic and foreign authors on the experience and prospects of improving the technology of bread, analyzed the principles of structure of the dough, considered the prospects for the use of red beets in the technology of the baking industry. According to analytical studies, it is established that the liquid vapor in the structure depends on many process parameters and raw materials, which determines the choice of these characteristics for experiments.
ВСТУП 1 Технологічна частина 1.1 Обґрунтування вибору технологічної схеми 1.2 Технологічні розрахунки 1.3 Розрахунок продуктивності печей 1.4 Розрахунок пофазних рецептур 1.5 Розрахунок виходу виробів 1.6 Розрахунок виробничих рецептур і вибір технологічних параметрів 1.7 Розрахунок витрат сировини і площ для її зберігання 1.8 Розрахунок і вибір технологічного обладнання 2 Науково-дослідна частина 2.1 Аспекти технології та деяке обладнання змішування водно-борошняних сумішей 2.2 Характеристика та склад борошна, як основної сировини 2.3 Узагальнена характеристика способів замішування тіста 2.3.1Приготування тіста опарним способом 2.4 Фізичні взаємодії води та борошна при їх змішуванні 2.5 Основи процесу змішування 2.5.1Стан та принцип дії обладнання для змішування опари Висновки та завдання досліджень 3 Мета, об’єкт, предмет та метод дослідження 3.1 Задачі дослідження 3.2 Організація проведення досліджень 3.3 Предмети і матеріали дослідження 3.3.1 Опис експериментальної установки 4.1 Дослідження впливу соку червоного буряка на біохімічні процеси в опарі 4.2 Визначення коефіцієнта неоднорідності 4.3 Статистична обробка даних 4.4 Реологічні властивості системи 5 Оцінка економічної ефективності реалізації нової технології 6 Охорона праці та безпека в надзвичайних ситуаціях 6.1 6.1. Аналіз виробничого травматизму 6.2 Заходи щодо техніки безпеки і промислової санітарії 6.3 Аналіз основних шкідливих і небезпечних факторів 6.4 Освітлення 6.5 Пропозиції по покращенню умов праці. Висновки 6.6 Безпека в надзвичайних ситуаціях Висновки Список використаних літературних джерел Додатки