Gas chromatographic acquisition of representative 'Total' cellular fatty acid profiles from bacteria or bacteria-containing samples (e.g., environmental or clinical materials) tends to be dependent on the method used to released the fatty acids and convert them to derivatives suitable for analysis. Alkaline saponification or interesterification methods, while preserving acid-sensitive components such as cyclopropane fatty acids, are often insufficient to release amide-linked components, such as hydroxylated fatty acids. Acid-catalyzed hydrolyses or interesterifications, on the other hand, while more efficiently releasing the predominantly amide-linked hydroxylated components, have been shown to cause severe and unpredictable degradation of cyclopropane fatty acids. We report studies of a single-tube method involving sequential alkaline/acid release of fatty acids in which fatty acids released by the alkaline step are partitioned into an organic epiphase during the aqueous acid hydrolysis step. After hydrolysis, the epiphase and the released fatty acids are extracted into an hypophasic solvend and esterified at moderate temperature under relatively low acid concentrations. Under these conditions, cyclopropane as well as hydroxylated fatty acids are recovered in high yield.

Previous studies have shown that pullulan films are able to stabilize enzymes and other labile molecules from thermal and oxidative degradation. Solutions made with commercially available pullulan are extremely viscous and difficult to process limiting the ability to use low-cost printing systems, such as inkjet printers, to format pullulan-containing. In this work, we show that pullulan can be made printable by decreasing its chain length by acid hydrolysis. The acid hydrolysis reaction was modelled using statistical software; the molecular weight of pullulan decreased with increasing reaction time, temperature and acid concentration. Interactions between time and temperature, and temperature and acid concentration were determined to be significant to the reaction as well. The mechanical properties and oxygen permeability of films made from pullulan with different molecular weights were also measured. The films were found to have similar tensile properties and oxygen permeabilities to each other and to those obtained using native pullulan. Using a thermally unstable enzyme (acetylcholinesterase) and an easily oxidizable small molecule (indoxyl acetate) as test materials, it was found that these films have the same ability to stabilize the enzyme and to serve as an oxygen barrier, as the films made with native pullulan. It was also found that pullulan is inkjet printable as long as the molecular weight is 56 kDa. Poor jetting and clogging of the printhead was observed when pullulan with a molecular weight higher than this threshold was used. Microarray printing was also demonstrated by a printing acetylcholinesterase/pullulan in nano-sized volumes using a Dimatix inkjet printer and showing activity of the enzyme after printing and storage at ambient conditions. Proof of concept of microarray printing opens up the potential for future applications of pullulan in other high throughput applications. / Thesis / Master of Applied Science (MASc)

<p> The c^13 isotope effect in acid and alkaline hydrolysis of both ethyl and tertiary butyl benzoate-carboxyl-c^14 have been measured. Significant isotope effects were found in all the hydrolyses except acid hydrolysis of the tertiary butyl oster. These results are additional evidence for the suggestion of Choen and Schneider that alkyl oxygen rather than normal acyl oxygen bond cleavage occurs when tertiary butyl benzoate is hydrolyzed in acid solution. No appreciable c^13 isotope effect was found to occur in thermal, aqueous, or acid-catalysed decarboxylation of anthranilic acid. From this observation and other experimental evidence, it is concluded that anthranilic acid decarboxylates by a bimolecular process in which the attack of the hydrogen ion is rate controlling. It is further suggested that the hydrogen ion attack is on the zwitterion at the c carbon position. Both the c^13 and the c^14 isotope effect in the decarboxylation of mesitoic acid were measured using a mass spectrometer. The c^14 isotope effect was found to be two and one half times the magnitude of the c^13 isotope effect instead of twice as present theory predicts. </p> / Thesis / Doctor of Philosophy (PhD)

Plastics are highly useful economically because of their resistance to diverse types of environmental and chemical agents and their ability to be molded into many types of products. Globally, plastic production is greater than 20 million metric tons per year. However, their widespread use and often their disposable nature results in significant plastic accumulation in the environment. Plastics are made of hydrocarbons, materials that are biodegradable depending on their molecular structure and size. It is hypothesized that pre-treatment of plastic materials could enhance their bioavailability, facilitating their microbial biodegradation. In this dissertation, a process was developed to treat nylon 6,6 polymers by acid hydrolysis to produce a microbial growth medium. The chemical composition of the medium was determined by low pressure liquid chromatography-spectrophotometry and electrospray ionization mass spectrometry and found that the medium was a mixture of molecules with molecular weight > 800 m/z and with similar chemical characteristics to polyamines. There was steady growth of Pseudomonas putida KT2440 in the medium with concomitant substrate biodegradation. Notably, the yeast Yarowia lipolytica grew well in the medium when supplemented with yeast extract. A similar medium derived from nylon 6,6 containing nylon-derived particles supported the growth of Beijerinckia sp. and Streptomyces sp. BAS1. Confocal laser scanning microscopy and flame ionization gas chromatography were used to identify and quantify the production of polyhydroxybutyrate, a type of “bioplastic”. The aforementioned microorganisms were cultivated in a bench-scale bioreactor that was developed as part of this dissertation. The bioreactor had a novel impeller design resulting in enhanced mixing and rotation and also a modular format allowing for diverse configurations. The bioreactor was notable for its durability and low cost. A detailed description of its design is included in the appendices. In summary, plastic materials can potentially be processed into growth media for microorganisms and can be used for production of value-added products. The media described herein can be used in bioconversion processes using a bioreactor.

O Brasil é um país com forte produção agrícola, produzindo anualmente uma grande quantidade de biomassa vegetal, proveniente de resíduos agroflorestais, como o bagaço de cana-de-açúcar, a casca de café, entre outros. As biomassas de origem vegetal são constituídas basicamente por frações de celulose, hemicelulose e lignina que encontram-se intimamente associadas dando origem a uma estrutura recalcitrante do vegetal. O presente estudo teve como objetivo contribuir para o emprego de uma nova matéria-prima, a casca de café, para obtenção de produtos com valor agregado. Foi analisado a composição química da casca de café para determinar os valores de compostos extrativos, celulose, hemicelulose, lignina e cinzas. Foi analisado também diferentes condições de pré-tratamento ácido e pré-tratamento alcalino, seguido de sacarificação, da casca de café. Os hidrolisados obtidos foram submetidos à fermentação pelas leveduras Scheffersomyces shehatae UFMG-HM 52.2 e Candida guilliermondii FTI 20037 para produção de etanol e xilitol, respectivamente e Saccharomyces cerevisiae 174 para produção de etanol pelos métodos SHF (Separate Hydrolysis and Fermentation) e SSF (Simultaneous Saccharification and Fermentation). A caracterização química da casca de café apresentou concentrações de 38,05% de compostos extrativos, 24% de celulose, 19% de hemicelulose, 13,68% de lignina e cerca de 0,36% em cinzas. As melhores condições de pré-tratamento ácido forneceram um hidrolisado com 31,35 g/L de xilose, 12,42 g/L de glicose, 1,25 g/L de ácido acético e pH de 0,8. A fermentação do hidrolisado ácido produziu 6,1 g/L de etanol, com um Yp/s de 0,27 g/g. A fermentação do hidrolisado hemicelulósico de casca de café para produção de xilitol apresentou valores de 2,82 g/L do produto, com um Yp/s de 0,16 g/g. A produção de etanol pelo método SHF a partir do hidrolisado enzimático da casca de café foi de 4,89 g/L nas primeiras 12 horas de fermentação, com Yp/s de 0,20 g/g. A fermentação pelo método SSF produziu 4,66 g/L de etanol, com um Yp/s de 0,17 g/g de etanol no período de 18 horas de fermentação. Frente a isto é possível concluir que a casca de café é uma biomassa com potencial para uso em processos biotecnológicos na produção de compostos com valor agregado como etanol e xilitol. / Brazil is a country with strong agriculture, producing a large amount of plant biomass from agroindustrial waste, such as sugarcane bagasse, coffee husk, among others. Biomasses from plants are basically constituted of cellulose, hemicellulose and lignin, which are deeply associated, resulting in a recalcitrant structure in the plant. The present study aimed at contributing for the application of a new feedstock, coffee husk, for obtaining value-added products. The chemical composition of the coffee husk was analyzed in order to determine values of extractive compounds, cellulose, hemicellulose, lignin and ashes. It was also analyzed different conditions of acid pretreatment and alkaline pretreatment, followed by saccharification, of coffee husks in order to improve the release of sugars. The hydrolysates were fermented by the yeasts Scheffersomyces shehatae UFMG-HM 52.2 and Candida guilliermondii FTI 20037 for the production of ethanol and xylitol, respectively, and by the yeast Saccharomyces cerevisiae 174 for the production of ethanol through SHF (Separate Hydrolysis and Fermentation) and SSF (Simultaneous Saccharification and Fermentation) methods. Chemical characterization of the coffee husk presented 38.05% of extractive compounds, 24% of cellulose, 19% of hemicellulose, 13.68% of lignin and around 0.36% of ashes. The best conditions for acid pretreatment yielded 31.35 g/L in xylose, 12.42 g/L glucose and 1.25 g/L acetic acid in 0.8 pH. Acid hydrolysate fermentation of coffee husk produced 6.1 g/L of ethanol, with an YP/S of 0.16 g/g. Ethanol production through SHF methods from enzymatic hydrolysate of coffee husk yielded 4.89 g/L in the first 12 hours of the process, with an YP/S of 0.20 g/g. SSF process yielded 4.66 g/L of ethanol with YP/S of 0.17 g/g after 18 hours of fermentation. It is possible to conclude, thus, that coffee husk is a biomass with potential for biotechnological applications in the production of value-added compounds, such as ethanol and xylitol.

Nos últimos anos, pesquisas sobre a produção de nanocristais de amido (NCA) receberam interesse crescente devido a suas diversas aplicações, principalmente como material de reforço de matrizes poliméricas. Nesse contexto, o amido de quinoa (AQ) apresenta características desejáveis na produção de NCA tais como tamanho de grânulo pequeno e conteúdo de amilose relativamente baixo. Assim, o objetivo desta pesquisa foi produzir NCA de quinoa (NCAQ) por hidrólise ácida em diferentes temperaturas (30, 35 e 40) °C. Além disso, foi estudado o efeito da adição dos NCAQ nas propriedades estruturais e físicas de filmes de amido de mandioca. O AQ apresentou diferentes percentagens de hidrólise, no quinto dia, 63%, 73% e 91% para (30, 35 e 40) °C, respectivamente. O AQ (k = 0,59 dias-1) foi hidrolisado mais rápido que o amido de milho ceroso (k = 0,39 dias-1) a 40 °C. O rendimento dos NCAQ diminuiu com o incremento da temperatura de 30 a 40 °C; enquanto que a cristalinidade relativa dos NCAQ não foi alterada (~35%). A morfologia dos NCAQ produzidos a 30 °C foi irregular com tamanho micrométrico, enquanto que os produzidos a 35 e 40 °C apresentaram forma de paralelepípedo com tamanhos entre (50 e 100) nm e (400 e 900) nm (agregados). O diâmetro hidrodinâmico e as propriedades térmicas dos NCAQ diminuíram com o aumento da temperatura da hidrólise; enquanto que a intensidade das bandas FTIR e o potencial zeta aumentaram. As propriedades indicaram que NCAQ foram produzidos somente a (35 e 40) °C com rendimentos de 22,7% e 6,8%, respectivamente. Dessa forma, considerando o rendimento e a temperatura de transição, os NCAQ produzidos a 35 °C foram selecionados para aplicação em filmes de amido de mandioca. Os filmes foram preparados pela técnica do casting, com 4 g de amido de mandioca/100 g de dispersão filmogênica; 25 g glicerol/ 100 g de amido; e 0; 2,5; 5,0 e 7,5 g de NCAQ/ 100 g de amido. Os difratogramas de raios X confirmaram a presença dos NCAQ nos filmes. A adição de NCAQ nos filmes aumentou a rugosidade e o ângulo de contato em concentrações de 5% e 7,5%, a resistência à tração e o módulo elástico, os parâmetros de cor L* e a* em concentrações 7,5%, e a opacidade; enquanto que diminuiu a deformação na ruptura, a permeabilidade ao vapor de água na concentração de 5%, e o brilho. Outras propriedades dos filmes como espessura, umidade, solubilidade, propriedades térmicas não foram alteradas pela adição de NCAQ. Os resultados indicaram que os NCAQ produzidos a 35 °C podem ser usados como reforço em filmes nanocompósitos para melhorar suas propriedades mecânicas. / Recently researches on starch nanocrystals (SNC) production have become of interest due to their many applications, especially as reinforcement in polymeric matrices. Quinoa starch (QS) has desirable characteristics for SNC production such as small granule size and relatively low amylose content. Thus, the objective of this research was to produce quinoa SNC (QSNC) by acid hydrolysis at different temperatures (30, 35 and 40) °C. Furthermore, the effect of QSNC addition on the structural and physical properties of cassava starch films was studied. QS presented different percentages of hydrolysis on the fifth day, 63%, 73% and 91% for (30, 35 and 40) °C, respectively. QS (0.59 days-1) was hydrolyzed more rapidly than waxy maize starch (0.39 days-1) at 40 °C. QSNC yields decreased with temperature increase from (30 to 40) °C, while the relative crystallinity was not altered (~35%). The morphology of QSNC produced at 30 °C was irregular with micrometric size while those produced at 35 °C and 40 °C presented parallelepiped shapes with sizes between 50 nm and 100 nm and 400 nm to 900 nm (aggregates). The hydrodynamic diameter and the thermal properties of QSNC decreased with temperature increase, while the FTIR band intensities and the zeta potential increased. The properties indicated that quinoa QSNC were only obtained at (35 and 40) °C with yields of 22.8% and 6.8%, respectively. QSNC produced at 40 °C presented lower yield and crystallinity than waxy maize SNC, but a lower hydrodynamic diameter. Thus, based on the yield and transition temperature, QSNC produced at 35 °C was selected for application in cassava starch films. The films were prepared by casting technique, with 4 g of cassava starch / 100 g of film forming dispersion; 25 g glycerol / 100 g starch; and 0; 2.5; 5.0 and 7.5 g of QSNC / 100 g of starch. X-ray diffractograms confirmed the presence of QSNC in the films. Addition of QSNC to films increased the roughness and the contact angle at 5.0% and 7.5% concentrations, the tensile strength and elastic modulus, the color parameters L* and a* at 7.5% concentration, and the opacity; while decreasing deformation at break, water vapor permeability at 5.0% concentration, and gloss. Other film properties such as thickness, moisture content, solubility, thermal properties were not affected by QSNC addition. The results indicated that the QSNC produced at 35 ° C can be used as reinforcement in nanocomposite films to improve their mechanical properties.

For the citrus processing industry the disposal of fresh peels has become a major concern for manyfactories. Orange peels are the major solid by-product. Dried orange peels have a high content ofpectin, cellulose and hemicellulose, which make it suitable as fermentation substrate when hydrolyzed.The present work aims at utilizing orange peels for the production of ethanol by using the fungusMucor indicus. Hence, producing a valuable product from the orange peel waste. The biomass growthwas also examined, since the biomass of the fungus can be processed into chitosan, which also is avaluable material.The work was first focused on examining the fungus ability to assimilate galacturonic acid and severalother sugars present in orange peel hydrolyzate (fructose, glucose, galactose, arabionose, and xylose).Fructose and glucose are the sugars which are consumed the fastest whereas arabinose, xylose andgalacturonic acid are assimilated much slower.One problem when using orange peels as raw material is its content of peel oils (mainly D-limonene),which has an immense antimicrobial effect on many microorganism even at low concentrations. Inorder to study M. indicus sensitivity to peel oil the fungus was grown in medium containing differentconcentrations of D-limonene.At very low limonene concentrations the fungal growth was delayed only modestly, hence a couple ofhours when starting from spores and almost nothing when starting with biomass. Increasing theconcentration to 0.25% (v/v) and above halted the growth to a large extent. However, the fungus wasable to grow even at a limonene concentration of 1.0%, although, at very reduced rate. Cultivationsstarted from spore-solution were more sensitive than those started with biomass.Orange peels were hydrolyzed by two different methods to fermentable sugars, namely by dilute acidhydrolysis (0.5% (v/v) H2SO4) at 150 °C and by enzymatic hydrolysis by cellulase, pectinase and β-glucosidase. The fungus was able to produce ethanol with a maximum yield of about 0.36 g/g after 24h when grown on acid hydrolyzed orange peels both by aerobic and anaerobic cultivation. Apreliminary aerobic cultivation on enzymatic hydrolyzed orange peels gave a maximum ethanol yieldof 0.33 g/g after 26 h.The major metabolite produced during the cultivations was ethanol. Apart from ethanol, glycerol wasthe only component produced in significant amounts. In cultivations performed aerobically on acidandenzymatic hydrolyzed orange peels the glycerol yields were 0.048 g/g after 24 h.Two different techniques were also examined in order to evaluate if the methods could be use asbiomass determining methods when solid particles are present in the culture medium. The problemwith solid particles is that they will be buried inside the fungal biomass matrix. Hence makingseparation impossible prior to dry weight determination in the ordinary way. However, none of themethods involving chitin extraction or chitosan extraction did show any good results.The results from the present work are rather clear, M. indicus was able to grow and produce bothethanol and biomass even when limonene was present in the culture medium. The maximum ethanolyield was achieved after about 24 h in cultivations performed on both acid hydrolyzed and enzymatichydrolyzed orange peels. However, in order to say if the method can be applicable at industrial scaleand made economically feasible the subject has to be investigated further.

Barley spent grain, the main residue of the brewing industry, is a lignocellulosic material, which could be considered a potential raw material for ethanol production. In this work, spent grains generated in a microbrewery were fractionated by acid hydrolysis and delignification. The investigated sort of barley spent grains had high carbohydrate content, accounting for 60% of the dry matter, while its lignin content was lower than that reported for other sorts of spent grains. Since the used spent grains contained residual starch different treatment approaches were used for separating their main components without affecting the sugars generated by starch hydrolysis. Two kinds of acid hydrolysis processes, namely single-step and two-step hydrolysis, were used for solubilising the carbohydrate fraction. Single-step hydrolysis was performed either at 100oC or at 121oC. In the two-step approach, a second hydrolysis, at 121oC, was performed after the first hydrolysis step. The dilute-acid hydrolysis at 100oC removed all the starch, whereas the hydrolysis at 121oC removed also a part of the hemicelluloses in addition to starch. During the second hydrolysis step, the content of easily hydrolysable polysaccharides decreased from 32.5 to 7.6% in the material pre-hydrolyzed at 100oC and from 20.3 to 10.6% in the material pre-hydrolyzed at 121oC. The amount of easily hydrolysable polysaccharides removed in the second step corresponded to 83% and 81.5% of the total removed matter in the materials pre-hydrolyzed at 100 and 121oC, respectively. In the next step, acetosolv and alkaline delignification, either alone or combined with acid hydrolysis, were used for dissolving the lignin fraction. A higher solubilisation occurred after alkaline delignification, where 83% of the initial material was removed. Only 34% of the initial lignin was removed by direct acetosolv, while the combined acid hydrolysis/acetosolv approach resulted in lignin removal between 70 and 75%. However, the resulted pulp still contained important amount of lignin. The acid prehydrolysis was also beneficial for alkaline delignification, but the effect was less noticeable than for acetosolv. Lignin removal increased from 95% in direct alkaline delignification to nearly 100% in the acid hydrolysis-assisted alkaline treatment. Two different methods were carried out for lignin precipitation. In the liquid fraction obtained by acetosolv, lignin was precipitated by water addition after concentration of the liquors to 75% of the initial volume. Although the visual inspection of the liquors after water addition revealed a relatively good lignin precipitation, the separation by filtration of the precipitated material was difficult, apparently due to the small particle size of precipitated lignin molecules. Some improvement was observed for the combined treatments, especially for those including two-step acid hydrolysis. The best recovery, 54% of the precipitated lignin, occurred for the process including consecutive acid hydrolyses at 100 and then at 121oC before acetosolv. In the alkaline liquors, lignin was precipitated by pH adjustment to 2.0 by HCl. Around 40.5% of the solubilised lignin was precipitated, and it increased to 85-100% when combined treatments were applied. The best results were achieved upon the treatment including acid prehydrolysis at 121oC before alkaline process.

Blood group glycolipids are important tools in the study of microbial receptor interactions and other biological phenomena. Presently blood group glycolipids of interest are isolated from biological samples. However, all glycolipids are not readily available due to the low frequency of some phenotypes in the general population. The ability to acquire the rare glycolipids from the degradation of common glycolipids would be a useful alternative to trying to obtain the molecules from biological sources.This research set out to establish the ability of blood group glycolipids to be degraded into useful glycolipids in a controlled manner by acid hydrolysis and possibly metal catalysis. The initial experiments investigated the degradation/hydrolysis of the more readily available glycolipid globoside with a range of salts and acids to establish degradation concepts such as; temperature, type of acid, acid concentration, and the role of metal ions in glycolipid degradation. These concepts then led to a series of degradation experiments with the blood group glycolipids Leb and ALeb. These glycolipids were incubated with a range of acid concentrations and varying temperatures. Thin layer chromatography separation and chemical and immunochemical staining were the main methods used to identify the products of degradation.It was established that metal ions were not directly involved in the catalysis of glycolipids in the short-term, however some metal ions were indirectly implicated in their degradation due to their ability to form acid solutions. Acid hydrolysis was established as the principle mechanism for glycan chain degradation. In general it was found that the glycan chain primarily lost its fucose groups (in no particular order) and was then followed by sequential degradation of the remaining glycan chain. The glycan chain also appeared to have a protective function on the ceramide moiety. Degradation of globoside established a simple sequential pathway of glycan chain reduction from the non-reducing end. Blood group glycolipids ALeb and Leb first lost their fucose side groups followed by sequential reduction of the glycan chain. Although not fully controllable, degradation of Leb was able to produce Lea, Led and Lec. In contrast degradation of ALeb did not produce any Lea or Led. Instead A-type 1 and two novel A-like structures, 'linear A' and 'GalNAc-Lea' were generated. Lec was only produced from ALeb in extremely acidic conditions. This research established the ability to generate, by acid hydrolysis, a range of rare and "unnatural" novel glycolipids from more commonly available structures. It is of interest that the so-called unnatural glycolipids obtained from the acid hydrolysis of ALeb may, in theory, occur naturally in the acid environment of the stomach, and as such could have the potential to be implicated in disease. It is probable that by applying the principles learned here, a range of novel and natural structures suitable for use in the study of biological interactions can be obtained.

Blood group glycolipids are important tools in the study of microbial receptor interactions and other biological phenomena. Presently blood group glycolipids of interest are isolated from biological samples. However, all glycolipids are not readily available due to the low frequency of some phenotypes in the general population. The ability to acquire the rare glycolipids from the degradation of common glycolipids would be a useful alternative to trying to obtain the molecules from biological sources.This research set out to establish the ability of blood group glycolipids to be degraded into useful glycolipids in a controlled manner by acid hydrolysis and possibly metal catalysis. The initial experiments investigated the degradation/hydrolysis of the more readily available glycolipid globoside with a range of salts and acids to establish degradation concepts such as; temperature, type of acid, acid concentration, and the role of metal ions in glycolipid degradation. These concepts then led to a series of degradation experiments with the blood group glycolipids Leb and ALeb. These glycolipids were incubated with a range of acid concentrations and varying temperatures. Thin layer chromatography separation and chemical and immunochemical staining were the main methods used to identify the products of degradation.It was established that metal ions were not directly involved in the catalysis of glycolipids in the short-term, however some metal ions were indirectly implicated in their degradation due to their ability to form acid solutions. Acid hydrolysis was established as the principle mechanism for glycan chain degradation. In general it was found that the glycan chain primarily lost its fucose groups (in no particular order) and was then followed by sequential degradation of the remaining glycan chain. The glycan chain also appeared to have a protective function on the ceramide moiety. Degradation of globoside established a simple sequential pathway of glycan chain reduction from the non-reducing end. Blood group glycolipids ALeb and Leb first lost their fucose side groups followed by sequential reduction of the glycan chain. Although not fully controllable, degradation of Leb was able to produce Lea, Led and Lec. In contrast degradation of ALeb did not produce any Lea or Led. Instead A-type 1 and two novel A-like structures, 'linear A' and 'GalNAc-Lea' were generated. Lec was only produced from ALeb in extremely acidic conditions. This research established the ability to generate, by acid hydrolysis, a range of rare and "unnatural" novel glycolipids from more commonly available structures. It is of interest that the so-called unnatural glycolipids obtained from the acid hydrolysis of ALeb may, in theory, occur naturally in the acid environment of the stomach, and as such could have the potential to be implicated in disease. It is probable that by applying the principles learned here, a range of novel and natural structures suitable for use in the study of biological interactions can be obtained.