Dissertations / Theses on the topic 'Bacterial volatiles'

The aims of this project were to study the immobilisation of microorganisms and the use of immobilised cell preparations in biochemical reactors. One particular process, the biodegradation of volatile fatty acids (VFAs), was chosen as a model system. Volatile fatty acids are compounds which are commonly found in odorous wastes and so can present a pollution problem. A bacterium was isolated, which was capable of VFA degradation in a minimal medium. The organism was identified as a strain of Alcaligenes denitrificans. The strain was able to grow on, and degrade, individual straight chain VFAs and mixtures, at concentrations much higher than those used in the isolation conditions. The strain was found to grow at a wide range of pH values, and a moderately wide range of growth temperatures. The strain was also tested for the degradation of VFAs in piggery slurry, but was found to be less effective than the natural population of organisms present in the waste. This bacterium was used to assess various immobilisation techniques, and their suitability for use in bioreactors. Four gel entrapment systems were tested. Conventional polyacrylamide and aluminium alginate gels both resulted in loss of cell viability. Calcium alginate was found to be too fragile for use in bioreactors, and only polyacrylamide hydrazide gel was found to be suitable. Beads of polyacrylamide hydrazide were used for longer term operation in a bubble column reactor, in a series of experiments to study the effects of changes in operating conditions, on bioreactor efficiency. Mathematical correlations were developed to explain the effects. Other parameters such as the mass transfer coefficients were calculated, to assist in the prediction of scale up. The second immobilisation system tested was adsorption to inorganic matrices. Four different types of particle were tested for their ability to adsorb non-growing cells from solution. The capacity to adsorb cells was related to the surface properties of the particles. Celite diatomaceous earth particles were found to have the greatest capacity to adsorb cells. Celite beads could be seeded in this manner, and then operated in a bubble column bioreactor. A biofilm was formed on the beads, which was capable of steady state biodegradation when the reactor was operated at dilution rates above the theoretical maximum for free cell growth. Bubble columns were the most suitable reactor of those tested for use with immobilised cell preparations. Mixing in these reactors was sufficient to provide good mass transfer, but not so violent as to disrupt the immobilised cell particles. Cell immobilisation by adsorption onto Celite was found to have several advantages over the other systems tested. The matrix could adsorb large quantities of cells, resulting in rapid biofilm formation and was also relatively cheap. Therefore, this appears to be an excellent new technique, and its potential applications in industrial processes are discussed.

Vinasse is the liquid waste removed from the base of sugarcane ethanol distillation columns at a ratio of 12-15 liters per liter of alcohol, resulting in an estimated production of approx. 350 billion liters in 2012/2013 in Brazil. Vinasse has a low pH and high chemical oxygen demand, which can cause land desertification when indiscriminately used as fertilizer. Also, underground water contamination is being observed in some regions. We evaluated the potential of vinasse as nutrient source for biohydrogen and volatile fatty acids production by means of anaerobic consortia. Two different vinasse-based media were proposed, using sugarcane juice or molasses as carbon source, and were compared to fermentation in a sucrosesupplemented medium. Pure cultures (4) and consortia (7) were cultured in the propose media and evaluated for volatile fatty acids (VFAs) and biohydrogen production. The consortium LPBAH1, originated from faeces of fruit bat, was selected for fermentation of vinasse supplemented with sugarcane juice and resulted in a higher H2 yield of 7.14 molH2/molsucrose and hydrogen content in biogas of approx. 31% after process optimization. Similarly, the optimized process using the consortium LPBAH2, originated from a lake of a dairy farm, resulted in 3.66 molH2/molsucrose and 32.7% hydrogen content in biogas. The proposed process is of great importance for giving a more rational destination to vinasse and expanding Brazilian energy matrix, reducing the dependence of fossil fuels.

La production de microalgues en hétérotrophie présente plusieurs avantages pour la production de biocarburants par rapport à la production autotrophe, comme une productivité plus importante en termes de biomasse et de lipides. Cependant, le développement industriel de ce procédé est limité par les coûts de productions associés au substrat organique (i.e. glucose) et à ceux liés à la stérilisation des fermenteurs. Les effluents de fermentation sombre, composés principalement d’acétate et de butyrate, pourraient être utilisés comme milieux de culture peu onéreux pour la culture hétérotrophe ou mixotrophe de microalgues. Les objectifs de cette thèse étaient i) de mieux appréhender la croissance algale sur des mélanges variés d’acétate et de butyrate en fonction de la présence ou l’absence de lumière et de la température de croissance et ii) d’évaluer la faisabilité d’utiliser des effluents de fermentation non stérilisés pour soutenir la croissance de microalgues oléagineuses. Tout d’abord, un modèle basé sur des bilans de masse a été construit afin de caractériser la croissance hétérotrophe de Chlorella sorokiniana et Auxenochlorella protothecoides (taux de croissance et rendements) sur des mélanges d’acétate et de butyrate. Les résultats ont montré que le rapport acétate:butyrate et la concentration en butyrate étaient deux paramètres clés pour soutenir la croissance hétérotrophe. Puis, il a été démontré que la présence de lumière et l’utilisation d’une température suboptimale (30 °C) pour la croissance algale permettaient de réduire l’inhibition du butyrate en permettant une production de biomasse autotrophe ou en améliorant la croissance sur acétate. Enfin, il a été montré que les microalgues peuvent être compétitives sur l’acétate lors de la croissance sur des effluents bruts de fermentation sombre en présence de bactéries fermentaires, grâce à la croissance rapide des microalgues sur acétate (1.75 j-1) et à un changement drastique des conditions de culture peu favorables à la croissance des bactéries d’origine fermentaire
Growing microalgae in heterotrophic mode present several advantages over autotrophic mode such as a higher productivity in terms of biomass and lipids for biofuels production. Nevertheless, this process is limited by the production cost associated with the organic substrate (i.e. glucose) and fermenters sterilization costs. Dark fermentation effluents, mainly composed of acetate and butyrate, could be used as a low-cost medium to grow microalgae heterotrophically or mixotrophically. The aims of this PhD were i) to optimize microalgae growth on various mixtures of fermentations metabolites using the presence or absence light and different cultivation temperatures and ii) to assess the feasibility of using unsterilized fermentation effluents. First, a model based on mass balance was built to characterize heterotrophic growth rates and yields when Chlorella sorokiniana and Auxenochlorella protothecoides were supplemented with different mixtures of acetate and butyrate. Results showed that the acetate:butyrate ratio and the butyrate concentration per se were two key parameters for promoting heterotrophic growth. Then, further studies showed that the presence of light and the use of suboptimal temperature (30 °C) could reduce the butyrate inhibition on growth by either triggering autotrophic production of biomass or enhancing growth on acetate. Finally, it was shown that microalgae could outcompete fermentation bacteria for acetate when growing on raw dark fermentation effluents, thanks to a fast algal growth on acetate (1.75 d-1) and a drastic change of culture conditions to the detrimental of bacterial growth

Soybean milk is a nutritional food consisting high protein content, unsaturated fatty acids and no cholesterol. However, soymilk and soy products are commonly characterised by a beany off-flavour leading to low-consumer acceptability in Western cultures. The aims of the cUJ:rent study are to reduce the main off-flavour, such as hexanal, by preparing soymilk freshly with control processing steps and investigating lactic acid bacteria to explor~the reduction of hexanal and enhance desirable volatile compounds in soymilk. Soybeans soaked in NaHC03 had an appearance and colour of soymilk better than the unsoaked soybeans. In addition, soymilk from soybeans soaked in purified water, dehulled soybeans, and boiled in NaHC03for 10 min, produced good qualities in total solids, and whiter than soymilk boiled for 30 min. The chosen procedure for the production of the soymilk, a blended (for 2 min) soybean milk was mixed by mixer emulsifier at 8000 rev min-I for 5 min, homogenized at 200 kg sq cm-I and finally separated the milk with centrifugation at 169 x g for 5 min. This soymilk was rich in total solids content (77.8 g kg-I). Soy yogl:mrt has a weak viscoelastic behaviour. The optimised ratio of soybeans: water (1 : 5) was used to prepare soy yoghurt. The aroma profiles ofsoymilk were identified by GC-MS analysis. The main volatile compound in soymilk was hexanal. Other volatiles present in high amounts were 1-octen-3-01, 1-hexanol, pentanal, 2-pentyl furan and 1-pentanol. Selected Strep. thermophilus and Lact. casei mixture produced acetaldehyde, diacetyl andacetoin and also reduced hexanal. The survival in soy yoghurt ofLact. casei was better than that ofLact. delbrueckii ssp. bulgaricus during 48h of incubation. Strep. thermophilus preferred to utIlIze sucrose to glucose whIle Lact. casei utilized glucose. Strep. thermophilus and Lact. casei did not reduce stachyose and raffinose. Strep. thermophilus and Lact. casei in single or mixture culture reduced hexanal to hexanol, and the hexanol was further reduced during the 24 h of the fermentation. The reduction ofhexanal to hexanol in soy yoghurt is desirable because hexanol has a weaker flavour than hexanal. Soy yoghurt fermented by mixed cultures ofStrep. thermophilus and Lact. casei had characteristics of yoghurt with a good curd and a pH of4.5-5.6. Strep. f' thermophilus grew rapidly during the first 6 h, and then grew slowly during the next 18 h of incubation. Lact. casei grew continually during 24h of incubation. Soy yoghurt fennented by mixed cultures contained desirable aroma compounds, including acetaldehyde, diacetyl, 2,3-pentanedione and acetoin. The soy yoghurts from soymilks with added glucose or sucrose had increased levels ofpyruvate, 2,3- pentanedione and diacetyl, suggesting that sugars contribute to the production of these products. The amino acids, methionine and threonine, did not contribute to the production of acetaldehyde in yoghurt but promoted ethanol fonnation. The microstructure of soy yoghurt was studied by scanning electron microscope. Soy yoghurt contained a gel network with branched chains and Strep. thermophilus and Lact. casei were found in the spaces or embedded in the netWork.

Un des enjeux majeurs dans la gestion de la plaie de pied diabétique est l’obtention d’informations permettant d’anticiper l’évolution de ces infections. Actuellement, il n’existe pas d’outils suffisamment efficaces qui permettent de distinguer une plaie colonisée d’une plaie infectée. L’approche proposée est basée sur discrimination de plusieurs bactéries fréquemment retrouvées dans les plaies chroniques de pied diabétique à partir de leur profil métabolique, et plus particulièrement des métabolites volatils qu’elles produisent. En effet, le dynamisme du métabolisme bactérien serait à même de mettre en évidence les changements qui s’opèrent dans la plaie. Dans un premier temps, une nouvelle méthodologie de concentration des métabolites volatils par Stir Bar Sorptive Extraction (SBSE) a été développée. Elle est basée sur l’utilisation de barreaux qui sont placés à la fois dans le milieu de culture et en espace de tête, suivie d’une analyse par GC-MS. La méthode a ensuite été comparée avec une autre méthode de concentration utilisant des fibres (la SPME) et a montré une meilleure capacité de concentration, permettant ainsi une détection plus sensible. Cette méthodologie a ensuite été utilisée pour suivre la production métabolique de six souches bactériennes cultivées dans des conditions mimant la plaie chronique. Grâce à leur profil métabolique, il a été possible de distinguer des espèces bactériennes. De plus, de manière plus surprenante, il a été possible de distinguer deux souches de Staphylococcus aureus présentant des profils de virulence différents. Enfin, une étude en co-culture a mis en évidence que 83% des métabolites produit en culture simple étaient retrouvés, prouvant l’intérêt de la méthodologie pour distinguer des souches bactériennes d’une même espèce au sein d’une plaie
One of the major challenges in the management of diabetic foot wounds is to obtain information to anticipate the evolution of these infections. Currently, there are no sufficiently effective tools to distinguish a colonized wound to an infected wound. The proposed approach is based on the discrimination of several bacteria frequently found in chronic diabetic foot wounds from their metabolic profile, and more specifically the volatile metabolites they produce. Indeed, the dynamism of bacterial metabolism would be able to highlight the changes that are occurring in the wound. First, a new methodology for the concentration of volatile metabolites by Stir Bar Sorptive Extraction (SBSE) was developed. It is based on the use of stir bars that are placed both in the culture medium and in headspace, followed by GC-MS analysis. The method was then compared with another concentration method using the fibres (SPME) and we highlighted a better concentration capacity with a more sensitive detection. This methodology was then used to monitor the metabolic production of six bacterial strains grown under conditions mimicking the chronic wound. Their metabolic profile allowed us to distinguish bacterial species. Moreover, more surprisingly, it was possible to distinguish two strains of Staphylococcus aureus with different virulence profiles. Finally, a co-culture was performed and we showed that 83% of the metabolites produced in simple culture were found, proving the interest of the methodology to distinguish bacterial strains of the same species within a wound

Cheese is a fermented product in which bacteria contribute to different flavours and textures. In order to understand the microbial processes in cheese, it is necessary to not only look at the genomic information in bacteria. The metabolome consists of a complete collection of metabolites in a biological sample. These metabolites are small molecules with a Mr >1.5 kDa, including flavour compounds. During the ripening process of cheese, many microbiological and biochemical changes occur that give cheese a diversity of textures and flavours. Proteins that go through proteolysis and amino acid catabolism are of great importance in the development of flavour in cheese, regardless of variety. Even though techniques for measurements of metabolites have existed for a long time, there are some unique challenges by analysing of several metabolites in parallel in a biological sample that promotes different metabolic pathways. Metabolic fingerprinting is the most common approach used in metabolomics, which is based on statistical analysis that through algorithms presents differences between samples. The electronic nose is able to identify the sum of volatile metabolites in a food, which is unlike the gas chromatograph that identifies individual metabolites. The aim of this review is to evaluate the use of metabolomics of selected Enterobacteriaceae together with electronic nose technology in order to analyse possible patterns of volatile metabolites produced in soft cheese. By this we hope to evaluate potential application of this approach in food quality control and microbial contamination screening. The pilot study was done together with the center for AASS, Örebro University where bacteria were analysed using the electronic nose NST3320. The study showed that it is possible to discriminate between Enterobacteriaceae, Staphylococcus aureus and cheese-associated bacteria, but also between the Enterobacteriaceae species Escherichia coli, Hafnia alvei and Klebsiella neumoniae. It is important to consider the gas sensors gradually lose their ability to detect substances after continual use, in which they need to be replaced with new gas sensors. Further, data processing requires special knowledge and can be hard to handle if the expertise is lacking. We believe that there is evidence that metabolomics together with the electronic nose have future prospects in terms of quality control and microbial contamination screening.

Les bactéries lactiques (LAB) ont de bonnes capacités de croissance sur différentes matrices alimentaires et produisent des activités enzymatiques très diverses qui sont notamment capables de modifier positivement les propriétés organoleptiques des aliments fermentés. Par conséquent, la sélection des ferments (starters) LAB possédant de bonnes capacités métaboliques et des activités enzymatiques intéressantes envers la matrice végétale pourrait améliorer les profils aromatiques des aliments fermentés. Le principal objectif de cette étude était d'améliorer les profils aromatiques des tomates fermentées en utilisant la voie biotechnologique. Pour y parvenir, premièrement, 200 LAB isolés à partir d'aliments fermentés cambodgiens et vietnamiens ont été criblées pour leur activité β-glucosidase et les isolats en double identifiés par analyse RAPD-PCR ont été rejetés. Ainsi, 40 souches étaient positives pour la β-glucosidase en utilisant le p-nitrophényl-β-D-glucopyranoside comme substrat. Parmi eux, 14 présentaient une activité supérieure à 10 nmol/min/mg de poids sec. Treize ont été identifiés comme Lactobacillus (Lactiplantibacillus) plantarum et un comme Lactobacillus (Lactiplantibacillus) pentosus. Quatre souches de phénotypes différents pour l'activité β-glucosidase ont été testées pour l'activité ADH. La capacité de réduction la plus élevée pour l'hexanal et le (E)-2-hexénal a été obtenue pour Lactobacillus (Limosilactobacillus) fermentum V013-1A pour laquelle aucune activité β-glucosidase n'était détectable. Les trois autres souches (L. plantarum C022-2B, C022-3B et V0023-4B2) présentaient une capacité de réduction plus faible et uniquement pour l'hexanal. Deuxièmement, les purées de tomates ont été fermentées avec ces quatre souches individuellement pour évaluer leur capacité à libérer des composés volatils à partir des précurseurs d’arômes de tomate. Cinquante-huit composés volatils ont été identifiés et quantifiés par HS-SPME/GC-MS. Les tomates non traitées étaient riches en aldéhydes. Les tomates fermentées avec les souches de L. plantarum étaient riches en cétones tandis que celles avec L. fermentum étaient riches en alcools. Cependant, pour la génération de terpénoïdes apportant des notes fruitées et florales, notre criblage de l'activité β-glucosidase n'a pas pu expliquer les différences entre les souches. Pour l'activité ADH, L. fermentum a montré une activité élevée dans la fermentation car la plupart des aldéhydes et cétones cibles ont disparu et ont été remplacés par leurs alcools correspondants. Les souches de L. plantarum ont montré une activité plus faible mais avec une importante diversité de sélectivité du substrat. Une meilleure connaissance de la fonctionnalité de chaque souche LAB dans la matrice alimentaire permettra de prédire et de façonner les profils aromatiques des aliments fermentés
Lactic acid bacteria (LAB) have good growth capacities on various food matrices and produce very diverse enzymatic activities which are notably capable of positively modifying the organoleptic properties of fermented foods. Therefore, the selection of the LAB starters possessing good metabolic abilities and interesting enzymatic activities towards the plant matrix could improve the aroma profiles of fermented foods. The main objective of this study was to enhance the aroma profiles of fermented tomatoes by using the biotechnological pathway. To achieve this, firstly, 200 LAB isolated from Cambodian and Vietnamese fermented foods were screened for their β-glucosidase activity and duplicate isolates identified through RAPD-PCR analysis were discarded. Thereby, 40 strains were found positive for β-glucosidase using p-nitrophenyl-β-D-glucopyranoside as substrate. Among them, 14 displayed an activity greater than 10 nmol/min/mg dry cell. Thirteen were identified as Lactobacillus (Lactiplantibacillus) plantarum and one as Lactobacillus (Lactiplantibacillus) pentosus. Four strains of different phenotypes for β-glucosidase activity were tested for ADH activity. The highest reduction ability for hexanal and (E)-2-hexenal was obtained for Lactobacillus (Limosilactobacillus) fermentum V013-1A for which no β-glucosidase activity was detectable. The three other strains (L. plantarum C022-2B, C022-3B and V0023-4B2) exhibited a lower reduction ability and only for hexanal. Secondly, mashed tomatoes were fermented with these four strains individually to evaluate their ability to release volatile compounds from the tomato aroma precursors. Fifty-eight volatile compounds were identified and quantified by HS-SPME/GC-MS. Untreated tomatoes were rich in aldehydes. The tomatoes fermented with L. plantarum strains were rich in ketones whereas those with L. fermentum were rich in alcohols. However, for the generation of terpenoids that provide fruity and floral notes, our screening of β-glucosidase activity was not able to explain the differences among the strains. For ADH activity, L. fermentum exhibited high activity in fermentation as most of the target aldehydes and ketones disappeared and were replaced by their corresponding alcohols. The L. plantarum strains exhibited a lower activity, but with an important substrate-selectivity diversity. A better knowledge of the functionality of each LAB strain in the food matrix will permit to predict and shape the aroma profiles of fermented food

The isolation of a number of strains of bacteria able to grow on dimethyl disulphide (DMDS) and dimethyl sulphide (DNS) as sole sources of energy is described. The isolates came from diverse habitats including soil, peat, marine mud and a freshwater pond, and were morphologically and physiologically best described as thlobacilli capable of growth as Calvin cycle autotrophs on inorganic sulphur compounds, methylated sulphides or thiocyanate. One Isolate (E6) was examined in detail and analysis of its DNA showed a mean mol% G - C content of 60.5 ± 1.0 which is In the normal range of f. thioparua. Substrate oxidation kinetics indicated that methanethiol (NT), sulphide, formaldehyde and formate could be implicated as intermediates in DNDS metabolism. Growth yields in chemostat culture on DMDS Indicated that energy conservation was probably coupled to the oxidation of formaldehyde and sulphide (derived from DMDS via NT) to CO and sulphide. Further evidence for the proposed oxidation pathway of DMDS was provided by demonstration of activities of a previously uncharacterised NADH-dependent DMDS 'reductase', NT oxidase, catalase, MAD4-dependent formaldehyde and formate dehydrogenases, rlbulose 1,5 - blsphosphate carboxylase and a sulphide oxidising system. This is the first demonstration of the Isolation of organisms into pure culture that are capable of growth on DMDS as sole energy substrate. T. thioparua strain Tk-m was found to be capable of growth on carbon disulphide (CS2) and carbonyl sulphide (COS). During growth on CS2, GC/MS analysis of the chloroform-extractable volatiles from the culture medium showed the formation of COS as a transient intermediate In CS2 metabolism. Anaerobic Incubation of call suspensions with CS2 also showed the production of hydrogen sulphilde (H2S) into the headspace. The proposed pathway of CS2 metabolism by T. thioparua strain TK-m most likely involved its reductive cleavage to COS and »¿S, COS then undergoin3 similar hydrolysis to CO- and HjS. This serves as the first detailed study of microbial CS2 metabolism.

Mestrado em Biotecnologia - Biotecnologia Industrial e Ambiental
Polyhydroxybutyrate is a type of biodegradable plastic, fully synthesized by bacteria, with similar properties to the ones of conventional plastics. This biopolymer can be produced by mixed cultures (activated sludge form waste water treatment plants) using the volatile fatty acids present in waste streams. Although the huge potential of this process, its application for the industrial production of PHB still lacks development. Throughout this work, three different strategies to obtain PHB-producing bacteria by using waste streams were tested. In the first one PHB-producing bacteria were first selected by aerobic dynamic feeding conditions, while simultaneously providing hydrogen gas, followed by an accumulation stage. In the second strategy the conventional aerobic dynamic feeding conditions were imposed, followed by an accumulation stage. And a third one, where a mixed culture was straightly submitted to the accumulation stage, without previous selection. Aerobic dynamic feeding was operated in cycles og 8 hours (3 cycles per day). In the first two strategies, feast phase was intended to last 2h30 and the famine 5h30 for a feast/famine ratio of 0.45. While the accumulation stage lasted 22 hours. High biomass concentration were achieved using strategy 1, in a stable reactor, and it was possible to accumulate PHB up to 59 % of the VSS with a PHB production yield of 0.30 g SLB/g COD fed. The second strategy resulted in less stable rectors, and a PHB content of 40 % of the VSS was achieved, but with PHB production yields as low as 0.09 g PHB/ g COD. Furthermore, it was no always possible to produce PHB as carbon source seemed to be directed to other metabolic pathways. A PHB production yields of 0.31 g PHB/g COD consumed was achieved with the third strategy, although only with a PHB content of 21 % of VSS. The production of PHB was verified firstly by a thermogravimetric method developed at Avecom previously to this work. This method was replaced by other that comprises the extraction of PHB using 1,2 propylene carbonate as solvent. The development of this method is also addressed in this project.
Polihidroxibutirato é um tipo de plástico biodegradável, completamente sintetizado por bactérias, com propriedades semelhantes aos plásticos convencionais. Este biopolímero pode ser produzito por culturas mistas (lamas ativadas de estações de tratamento de águas) usando os ácidos orgânicos voláteis presentes no efluente. Embora este processo apresente um enorme potencial, ainda é necessário o seu desenvolvimento para a sua aplicação na produção de PHB a escala industrial. Durante este trabalho, foram testadas três estratégias diferentes para a seleção de bactérias produtoras de PHB forma testadas. A primeira, em que as bactérias produtoras de PHB foram primeiro selecionadas por condições de alimentação dinâmica aeróbia, com a alimentação simultânea de hidrogénio, seguida de uma fase de acumulação. Uma segunda estratégia, onde a alimentação dinâmica aeróbia foi utilizada, seguida de uma etapa de acumulação. E uma terceira, em que uma cultura mista foi imediatamente submetida a uma fase de acumulação, sem seleção prévia. Nas duas primeiras estratégias a alimentação dinâmica aeróbia consistiu em ciclos de 8 horas (3 ciclos por dia), em que se pretendeu-se que a fase de fartura durasse 2h30 A fase de fome por seu lado durou 5h30 para um rácio fome/fartura de 0.45. A fase de acumulação durou 22 horas. Foram atingidas altas concentrações de biomassa usando a estratégia 1, num reactor estável, em que foi possível atingir um conteúdo em PHB de 59 % dos SSV, com um rendimento de produção de PHB de 0.30 g SLB/g CQO alimentado. A segunda estratégia resultou num reactor menos estável. Um conteúdo em PHB de 40 % dos SSV foi obtido, embora o rendimento de produção de PHB tenho sido só 0.09 g PHB/g CQO. Para além isso, nem sempre foi possível produzir PHB, visto que a fonte de carbono parecia ser direcionada para outras vias metabólicas. Foi atingido um rendimento de produção de PHB de 0.31 PHB/g CQO na terceira estratégia, no entanto o conteúdo em PHB foi só 21 % dos SSV. A produção de PHB foi inicialmente verificada por um método termogavimétrico desenvolvido na Avecom previamente a este trabalho. Este método foi posteriormente substituído por outro que envolve a extração de PHB usando carbonato de propileno como solvente. O desenvolvimento deste método é abordado no presente trabalho.

La présence de microorganismes peut être révélée par des métabolites volatils caractéristiques. Cette approche est particulièrement intéressante pour la détection non-invasive de pathogènes dans des échantillons complexes comme les matrices alimentaires, les échantillons sanguins, ou encore les plaies chroniques. Des capteurs nanoporeux à grande surface spécifique ont été préparés par voie sol-gel (xérogels) ; leur rôle est à la fois de capturer, concentrer et permettre une détection optique des Composés Organiques Volatils (COV) microbiens. Des capteurs dopés avec une molécule sonde, l'acide 5,5′ dithiobis 2 nitrobenzoïque, ont été développés pour mettre en évidence le sulfure d'hydrogène (H2S) produit par Salmonella, un pathogène d'intérêt dans le domaine de l'agroalimentaire. La capture d'H2S provoque un changement de couleur du capteur dès 5 ppm. Une partie du travail de recherche porte également sur la détection de métabolites dits « exogènes », libérés suite à l'hydrolyse d'un substrat enzymatique. C'est alors l'activité enzymatique qui est spécifique du micro-organisme ciblé. Deux COV exogènes sont envisagés : la β naphthylamine (β NA) et le 2 nitrophénol (2 NP). La première est issue d'activités enzymatiques peptidases, le second est issu d'activités glycosidases ou estérases. Pour ce dernier, une détection directe est possible dès 14 ppb grâce à son absorbance intrinsèque dans le visible. Après un travail sur la composition chimique des xérogels, une mise en forme originale par moulage des gels en forme de coin de cube permet une lecture de l'absorbance des xérogels en réflexion. Enfin, les capteurs obtenus ont été testés vis-à-vis de COV générés par 3 pathogènes: Salmonella, Escherichia coli et Staphylococcus aureus dans des matrices complexes (sang et échantillons alimentaires)
The presence of micro-organisms can be revealed by specific volatile metabolites. This approach is interesting for the non-invasive detection of pathogenic species in complex samples, such as food, blood or exudate. Nanoporous materials developing a high surface area have been prepared by sol-gel process (xerogels). They trap, concentrate and reveal the presence of microbial Volatile Organic Compounds (VOC) by means of an optical detection. Sensors have been doped with a probe molecule (5,5′ dithiobis 2 nitrobenzoic acid) in order to detect hydrogen sulfide emitted by foodborne pathogen Salmonella. The colour of sensor changes in the presence of 5 ppm of H2S. Another detection method is the use of enzymatic substrates which release exogenous VOCs. In this approach, the enzymatic activity is specific to the targeted pathogenic bacteria. Sensors have been developed for two exogenous VOCs: β naphthylamine (β NA) and 2 nitrophenol (2-NP). β NA is issued from peptidase activity, whereas 2 NP is produced by glycosidase or esterase activity. The latter can be detected above 14 ppb through absorbance in the visible region. The work focused both on the chemical composition of the xerogels and on their shape. After molding the xerogels into a trihedral prism (“corner reflector”), the absorbance can be easily monitored using the reflected light. VOCs produced by 3 pathogenic bacteria, Salmonella, Escherichia coli and Staphylococcus aureus, in complex media (blood and food samples) have been monitored with the obtained sensors

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior
FundaÃÃo de Amparo à Pesquisa do Estado do CearÃ
O ambiente marinho possui grande biodiversidade de algas marinhas, sendo uma rica fonte natural de muitos compostos biologicamente ativos, como, compostos orgÃnicos volÃteis, carotenoides, clorofilas, ficobilinas, terpenos, esteroides, compostos fenÃlicos, alcaloides, polissacarÃdeos, vitaminas e Ãcidos graxos saturados e poli-insaturados, tornando-as cada vez mais procuradas para fins comerciais. O objetivo deste trabalho foi realizar bioprospecÃÃo e avaliar atividades biolÃgicas de produtos naturais das algas marinhas vermelhas Pterocladiella capillcea e Osmundaria obtusiloba. Os extratos etanÃlicos a 70% (EtOH 70%) das algas apresentaram os maiores valores do conteÃdo fenÃlico total (CFT) comparados aos extratos hexÃnicos (Hex). Os resultados do DPPH dos extratos Hex e EtOH 70% da O. obtusiloba foram maiores (43,46% e 99,47%) do que aqueles da P. capillacea (33,04% e 40,81%), na concentraÃÃo de 1.000 μg/mL. Quanto à habilidade de quelaÃÃo de Ãons ferrosos (FIC), observou-se um comportamento inverso, os extratos da P. capillacea apresentaram atividade superior aos da O. obtusiloba. Todos os extratos apresentaram um baixo poder de reduÃÃo de Ãons fÃrricos (FRAP), com variaÃÃo da densidade Ãptica entre 0,054 e 0,180. As atividades antioxidantes de todos os extratos algÃceos, avaliadas pela degradaÃÃo do β-caroteno (BCB), foram superiores a 40%. NÃo foi observada atividade antibacteriana contra as estirpes bacterianas testadas. Entretanto, os extratos das duas espÃcies foram capazes de aglutinar cÃlulas bacterianas Gram positivas de Staphylococcus aureus e Gram negativas de Escherichia coli, Salmonella sorovar Infantis multirresistente e Vibrio harveyi. Os compostos orgÃnicos volÃteis (COVs) e os Ãcidos graxos das algas marinhas vermelhas P. capillacea e O. obtusiloba foram analisados qualitativamente por cromatografia gasosa acoplada à espectrometria de massas (CG-EM) e quantitativamente por cromatografia gasosa (CG) equipada com detector de ionizaÃÃo de chama (DIC). Quanto aos COVs, ao todo foram identificados 31 constituintes diferentes nas espÃcies de algas, sendo alguns deles comuns Ãs duas. Em P. capillacea, dos 21 constituintes identificados, os majoritÃrios foram hexanal (50,4%), 2-pentilfurano (9,2%) e heneicosano (8,8%). Em O. obtusiloba dos dos 21 constituintes identificados, os majoritÃrios foram heneicosano (57,3%), hexanal (20,5%) e 1-pentadeceno (2,6%). Nove Ãcidos graxos foram identificados por CG-EM nas duas espÃcies. Em P. capillacea, os Ãcidos graxos majoritÃrios foram Ãcido palmÃtico (88,8%), Ãcido oleico (3,1%), Ãcido araquidÃnico (2,0%) e Ãcido eicosapentaenoico (1,9%). Em O. obtusiloba, os Ãcidos palmÃtico (55,6%), eicosapentaenoico (9,1%), oleico (8,9%) e araquidÃnico (8,5%) foram os majoritÃrios. A capacidade de sequestro do radical DPPH dos Ãcidos graxos apresentou uma atividade moderada variando de 25,90% a 29,97%. Os Ãcidos graxos de P. capillacea (31,18%) apresentaram uma moderada atividade FIC e os de O. obtusiloba (17,17%), fraca. O FRAP dos Ãcidos graxos de P. capillacea e O. obtusiloba apresentou baixa atividade. Com relaÃÃo ao BCB, a alga P. capillacea apresentou uma atividade de 61,24% na concentraÃÃo de 12,5 μg/mL e O. obtusiloba apresentou uma atividade de 49,13% na concentraÃÃo de 50 μg/mL. Este à o primeiro relato sobre identificaÃÃo e quantificaÃÃo de COVs, de compostos lipofÃlicos em extratos brutos das rodÃfitas P. capillacea e O. obtusiloba na costa Nordeste do Brasil.
There is a great diversity of seaweeds in marine environment that are natural rich sources of a variety of biologically active compounds, such as, volatile organic compounds, carotenoids, chlorophyls, phycobillins, terpenes, steroids, phenolic compounds, alkaloids, polysaccharides, vitamins, saturated and unsaturated fatty acids, one reason for becoming desirable for commercial uses. The objective of this work was to carry on a bioprospection and the evaluation of the biological activity of the natural products from the red marine algae Pterocladiella capillacea and Osmundaria obtusiloba. The 70% ethanolic extracts (70% EtOH) from both species showed higher values of total phenolic compounds (TPC) than hexanic (Hex) ones. The results of DPPH of both extracts, Hex and 70% EtOH, from O. obtusiloba were higher (43.46% and 99.47%) than those from P. capillacea (33.04% and 40.81%), at 1,000 μg/mL concentration. In contrast, the ferrous ion chelation (FIC) activity from P. capillacea extracts was superior to those of O. obtusiloba. All extracts showed low ferric ion reduction (FRAP) activity, with optical density varying from 0.054 to 0.180. The antioxidant activities of all agal extracts, measured by β-carotene bleaching (BCB), were above 40%. No antibacterial activity was observed against the tested strains. However, the extracts of both algae were capable of agglutinating bacterial cells: Gram positive Staphylococcus aureus and Gram negative Escherichia coli, multi-resistant Salmonella sorovar Infantis and Vibrio harveyi. The volatile organic compounds (VOCs) and the fatty acids of the red marine algae P. capillacea and O. obtusiloba were analyzed qualitatively by gas chromatography-mass spectrometry (GC-MS) and quantitatively by gas chromatography with a flame ionization detector (GC-FID). Thirty-one different VOCs were identified in these alga species, some of which are common to both. In P. capillacea, out of twenty-one identified compounds, the major were hexanal (50.4%), 2-pentylfuran (9.2%), and heneicosane (8.8%). On the other hand, in O. obtusiloba, out of twenty-one identified compounds, the major were heneicosane (57.3%), hexanal (20.5%) and 1-pentadecane (2.6%). Nine fatty acids were identified by GC-MS in the two species. In P. capillacea, the principal fatty acids were palmitic acid (88.8%), oleic acid (3.1%), arachidodic acid (2.0%), and eicosapentaenoic acid (1.9%). In O. obtusiloba, palmitic acid (55.6%), eicosapentaenoic (9.1%), oleic (8.9%), and arachidonic (8.5%) were the most important fatty acids found. The scavenging of DPPH free radical of the fatty acids from both species showed a moderate activity, which varied from 25.90% to 29.97%. Fatty acids obtained from P. capillacea (31.18%) exhibited moderate FIC activity, whereas those from O. obtusiloba (17.17%), just weak. The FRAP measured from the fatty acids from both P. capillacea and O. obtusiloba showed low activity. Considering the BCB, the activity of P. capillacea was 61.24% in the 12.5 μg/mL concentration, and for O. obtusiloba, BCB activity was 49.13% in the 50 μg/mL concentration. This is the first report on the identification and quantification of VOC, from crude extracts of the red marine algae P. capillacea and O. obtusiloba, collected in the Northeast coast of Brazil.

Submitted by Ariane Souza (arianedocarmosouza@gmail.com) on 2018-08-14T02:02:56Z No. of bitstreams: 2 Ariane do Carmo Souza dissertação mestrado.pdf: 1195184 bytes, checksum: e6271bca7a0f0772ac6bb6269483b8fd (MD5) Carta para repositorio Ariane do Carmo Souza.pdf: 105567 bytes, checksum: 2517d898996d34fae9bd02b616df2176 (MD5)
Approved for entry into archive by Alini Demarchi (ri.bar@ufscar.br) on 2018-08-17T12:57:39Z (GMT) No. of bitstreams: 2 Ariane do Carmo Souza dissertação mestrado.pdf: 1195184 bytes, checksum: e6271bca7a0f0772ac6bb6269483b8fd (MD5) Carta para repositorio Ariane do Carmo Souza.pdf: 105567 bytes, checksum: 2517d898996d34fae9bd02b616df2176 (MD5)
Approved for entry into archive by Alini Demarchi (ri.bar@ufscar.br) on 2018-08-17T12:57:52Z (GMT) No. of bitstreams: 2 Ariane do Carmo Souza dissertação mestrado.pdf: 1195184 bytes, checksum: e6271bca7a0f0772ac6bb6269483b8fd (MD5) Carta para repositorio Ariane do Carmo Souza.pdf: 105567 bytes, checksum: 2517d898996d34fae9bd02b616df2176 (MD5)
Made available in DSpace on 2018-09-17T19:25:10Z (GMT). No. of bitstreams: 2 Ariane do Carmo Souza dissertação mestrado.pdf: 1195184 bytes, checksum: e6271bca7a0f0772ac6bb6269483b8fd (MD5) Carta para repositorio Ariane do Carmo Souza.pdf: 105567 bytes, checksum: 2517d898996d34fae9bd02b616df2176 (MD5) Previous issue date: 2018-06-14
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
The alternaria brown spot, caused by Alternaria alternata f sp. citri, causes large economic damages in tangor Murcott (Citrus sinensis Osbeck x Citrus reticulata [L.] Blanco). Its control is carried out through the spraying of agrochemicals, implying up to 15 pulverizations per year, which causes an increase in the production costs of the cultures and damages to the environment. As an alternative, the use of microorganisms, in particular Bacillus spp., has been used to diseases’ control. Therefore, the aim of this work was to evaluate the viability of Bacillus spp in in vitro and in vivo conditions. The methodologies were based on the interactions between biological control agents (Bacillus spp.) and the phytopathogen A. alternata, evaluated by the paired culture technique, by the production of volatile, thermostable and cell-free metabolites by different Bacillus spp. isolates. The molecular identification of the isolates tested and the efficacy of bacterial isolates were evaluated in leaves and plants under greenhouse conditions. The results showed that most of the isolates affected the development of phytopathogen and produced some types of metabolite, being antibiosis one of the probable mechanisms of action of the bacterium. The isolates ACB-01, ACB-07, ACB-08, ACB-18 and ACB-57 presented potential for disease control of A. alternata.
A mancha marrom de alternaria, causada por Alternaria alternata f sp. citri, causa grandes danos econômicos em tangor Murcott (Citrus sinensis L. Osbeck x Citrus reticulata [L.] Blanco). Seu controle é realizado através de pulverizações com agroquímicos, implicando em até 15 pulverizações por ano, o que acarreta em aumento no custo de produção da cultura e prejuízos ao meio ambiente. Como alternativa, o uso de microrganismos, em particular, as bactérias do gênero Bacillus spp., têm sido empregadas para o controle de doenças. Portanto, esse trabalho teve por objetivo avaliar em condições in vitro e in vivo a viabilidade de 47 isolados de Bacillus spp. para o controle da doença. As metodologias foram embasadas nas interações entre agentes de controle biológico (Bacillus spp.) e o fitopatógeno A. alternata avaliadas pela técnica de cultivo pareado, pela produção de metabólitos voláteis, termoestáveis e livre de células por diferentes isolados de Bacillus spp.. Realizou-se, ainda, a identificação molecular dos isolados testados e a eficácia dos isolados da bactéria em folhas destacadas e em plantas, sob condições de casa de vegetação. Os resultados obtidos mostraram que a maioria dos isolados afetou o desenvolvimento do fitopatógeno e produziram algum tipo de metabólito, sendo, a antibiose um dos prováveis mecanismos de ação da bactéria. Os isolados ACB-01, ACB-07, ACB-08, ACB-18 e ACB-57 apresentaram potencial para o biocontrole de A. alternata.

Si la sueur fraîchement émise par le corps humain est inodore, la dégradation de celle-ci par la flore bactérienne cutanée produit des composés volatils malodorants, responsables des odeurs de transpiration. Les odeurs de transpiration apparaissent également sur les vêtements au cours de leur utilisation, particulièrement sur les textiles réalisés en fibres synthétiques. Ce travail a pour but d’améliorer la compréhension du phénomène d’émanation d’odeurs en étudiant l’effet du sujet testé, l’effet de la flore bactérienne et l’effet du textile sur les émissions de composés volatils malodorants.L’intérêt de ce travail réside dans l’approche globale de la problématique des odeurs de transpiration et dans la diversité des méthodes de mesure mises en place, tant dans l’étude de la flore microbiologique que dans les méthodes de mesures des composés odorants émis.Dans un premier temps, le dénombrement simultané de la flore bactérienne sur la peau et sur le vêtement a été réalisé sur un échantillon de 15 sujets à l’issue d’un exercice physique. Cette expérimentation a permis d’évaluer le taux de transfert bactérien moyen lors d’une activité sportive et d’étudier son rôle dans l’émission d’odeurs. Ensuite, afin d’affiner ces résultats, une méthode basée sur la biologie moléculaire a été mise en place pour réaliser le suivi qualitatif de la stabilité de la flore commensale axillaire d’un sujet pendant 3 mois. Le transfert bactérien spécifique entre la peau du testeur et le vêtement a été étudié pour 4 matières textiles sélectionnées (dont le coton et le PET). Ceci a permis de déterminer le rôle du transfert bactérien spécifique dans l’émission des odeurs à partir de textile.Enfin, le dernier chapitre est consacré à l’étude de l’émission de composés volatils et odorants à l’aide de mesures olfactives et d’un nez électronique au cours du temps par 8 composants textiles sélectionnés. Après traitement statistique par analyse en composante principale et étude détaillée des mesures, 9 composés chimiques ont été identifiés comme indicateurs d’un comportement textile malodorant. Ces derniers pourraient être utilisés dans la mise en place d’une méthode ciblée de mesure physico-chimique des mauvaises odeurs.Ce travail a permis de déterminer l’impact de chacun des facteurs sujet, flore bactérienne et textile dans l’émission d’odeurs. En outre, ce travail ouvre des perspectives sur l’étude des contaminations bactériennes par contact, mais également dans l’étude des odeurs, sur les phénomènes de désorption de molécules volatiles à partir de différentes matrices textiles et sur les solutions pouvant être envisagées pour limiter les émissions odorantes à partir de textiles
Fresh human sweat is odorless. Odoriferous volatile compounds are produced by the metabolism of bacteria living on the skin, generating strong malodor. Sweaty body odors do also appear on clothes during use, and especially on synthetic fabrics. The aim of this document is to improve understanding of odor emission by investigating subject effect, microbiota effect and fabric effect on the emission of odoriferous volatile compounds.Odors of perspiration are hereby globally approached with a wide use of methods and experimental devices, for microbial flora study as well as for odoriferous volatile compounds emission study.First, microflora enumeration has been simultaneously processed on the skin and on the fabric after exercise for 15 subjects. This experiment allowed an evaluation of the average bacterial transfer yield during physical activity and the beginning of the investigation of its effect on odor emission.A molecular biology methodology has then been developed in order to refine these results. Monitoring of qualitative composition of the microbiota has been performed to study the stability of the armpit’s ecosystem on a subject during 3 months. Specific microbial transfer from subject’s skin to clothe has been performed for 4 textile fabrics (including cotton and PET). This leaded to characterize the effect of specific bacterial transfer on odor emission from fabric.The last chapter is dedicated to the study of the emission of odoriferous volatile compounds over time using olfactory measurements and electronic nose for 8 selected fabrics. Principal component analysis targeted 9 chemical compounds that have been selected as malodorous behavior indicators for a given fabric. Those 9 compounds could be used for setting up a fitted physicochemical method of malodor.To conclude, this study helped to understand the effect of 3 factors in odor perception from a fabric after sport : subject, microbial flora and fabric. Perspectives have been charted on contact microbial contamination, but also on odor, and especially on desorption of odoriferous volatile molecules from a textile or knitted matrix. The solutions that could be used to limit malodorous emission from fabrics have also been discussed

La détection et l'identification de bactéries pathogènes revêt une grande importance dans de nombreux domaines tels que la santé et l'industrie agroalimentaire. Dans ce contexte, les travaux de thèse s'intéressent à détection non invasive de Salmonella via la fraction volatile de son métabolome dont les métabolites volatils caractéristiques sont le sulfure d'hydrogène et la cadavérine. Ils illustrent également le concept de substrats osmogènes libérant des mCOV exogènes sous l'action d'enzyme spécifique d'Escherichia coli. Un premier capteur colorimétrique capable de distinguer le sulfure d'hydrogène du méthanethiol a été préparé. Il s'agit d'une matrice de silicate nanoporeuse dopée avec les réactifs N,N-diméthyl-p-phénylènediamine et ions Fe3+. Une bonne stabilité de l'intermédiaire réactionnel issu de ces réactifs, la quinonediimine (QD), est obtenue pour une forte concentration d'acide chlorhydrique. La réaction entre QD et 1000 ppm de sulfure d'hydrogène et de méthanethiol entraîne l'apparition respective d'une coloration verte et rouge-marron du capteur. Le capteur fluorimétrique de cadavérine, basé sur la formation d'un complexe fluorescent entre le Naphthol AS-BI déméthylé (ArOH) et la cadavérine, permet de détecter 250 ppb de cadavérine. La preuve de concept de substrats osmogènes a été illustrée avec la détection de p-nitrophénol (pNP) et de β-naphthylamine (β-NA) libérés en présence d'enzymes de E. coli, β-D-glucuronidase et L-alanine- β-naphthylamidase. Les capteurs nanoporeux produits, de taille de pores contrôlée, peuvent détecter 100 ppm de pNP, composé coloré (jaune) et 100 ppm de β-NA, composé fluorescent, ou encore 100 ppm de β-NA par dérivation chimique de ce dernier avec le diméthyl-p-aminocinnamaldéhyde (formation d'un produit rouge). En milieu biologique, l'eau est un interférent majeur.

Thesis (Ph. D.)--Georgia State University, 2006.
Title from title screen. George E. Pierce,committee chair; Eric S. Gilbert, Sidney A. Crow, committee members. Electronic text (135 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Aug. 20, 2007. Includes bibliographical references (p. 120-124).

Anopheles gambiae sensu lato mosquitoes are among the dominant malaria vectors in sub-Saharan Africa. However, not much is known about the oviposition behaviour of these species necessary for the development of malaria vector control strategies. With the aim of investigating cues associated with selected oviposition sites, artificial oviposition sites- ponds (soil mixed with water) were set-up in an open field at Mbita, Western Kenya in 2012 and 2013. Ponds were allowed to be colonized by wild An. gambiae s.l.. The numbers of Anopheles early instar larvae were counted and used as a proxy for oviposition preference. Water samples were then analysed for physicochemical, bacterial and chemical profiles. The bacterial profiles were analysed using denaturing gradient gel electrophoresis (DGGE) and the chemical profiles with gas chromatography-mass spectrometry (GC-MS). The detection of possible oviposition cues from oviposition substrates requires sensitive analytical methods. Volatiles detection was improved seven times. The detection of bacteria deoxyribonucleic acid (DNA) bands with DGGE was also improved to a minimum DNA concentration of 50 ng/µl. Results showed that ponds were colonized differently. Fresh ponds were preferred over slightly older ponds. Bacterial analysis revealed a low number of bacteria colony forming units (CFU) in preferred ponds. Some volatiles, including: 6,10-dimethyl-5,9-undecadien-2-one (geranylacetone) and 4-ethylbenzaldehyde, were associated with the oviposition preferred pond. In addition, low pH and high turbidity were associated with the ponds selected for oviposition. Finally, fungi isolated from the rhizomes of nut grass yielded a promising array of volatiles of which one is known to attract oviposition site seeking malaria mosquitoes. This finding opens the door for a cost effective and environmental friendly method of using fungi in an “attract and kill” strategy targeting malaria vectors.
Myggor i Anopheles gambiae sensu lato komplexet tillhör de myggor som är bäst på att sprida malaria parasiter i afrika söder om Sahara. Kunskapen om de här myggornas äggläggningsbeteende är begränsad. Den här kunskapen behövs för att kunna utveckla nya och förbättra tillgängliga malaria vektor kontroll metoder. Nya metoder som kan komplettera de som används idag (insecticides treated nets (ITNs) och indoor residual spraying (IRS)) behövs eftersom de metoderna har problem med resistensutveckling. Två studier utfördes på icipe fältstation i Mbita västra Kenya under 2012 och 2013 med målet att identifiera faktorer som påverkar myggornas äggläggningsbeteende. Baljor fyllda med en blandning av jord och vatten (äggläggningssubstrat) användes för att tillverka artificiella äggläggningsplatser som liknar de vattenpölar som de här myggarterna gärna lägger ägg i. Baljorna koloniserades av vilda myggor och antalet mygglarver som detekterades i baljorna jämfördes och användes som en proxy för äggläggningspreferens. Fysikaliska och kemiska parametrar mättes på jordvattenblandningarna i baljorna och prover togs för att analysera bakteriepopulationer med hjälp av denaturing gradient gel electrophoresis (DGGE) och flyktiga ämnen med hjälp av gas-kromatografi kopplat till mass-spektrometri (GC-MS). För att kunna detektera de låga halter av flyktiga ämnen och bakterier som fanns i de här proverna krävdes det känsliga metoder. Antalet flyktiga ämnen som kunde detekteras ökades sju gånger genom att tillsätta NaCl till vattenproverna innan doften insamlades och termisk desorption användes istället för lösningsmedels desorption. För att förbättra detektionsgränsen för bakterier amplifierades bakterie-DNA i två PCR reaktioner som sedan mixades och koncentrerades. Resultaten från fältstudierna med baljorna visade att de koloniserades olika av Anopheles myggorna. Baljor med nyblandat substrat innehöll dubbelt så många mygglarver som baljor med jord-vattensubstrat som åldrats under en längre tid. Lägre mängd bakterier, lägre pH och högre grumlighet var gemensamt för de baljor som myggorna föredrog. De flyktiga ämnen som detekterades i de olika baljor varierade mellan olika försök och inget ämne fanns med i alla upprepningar av ett försök. Trots det detekterades några ämnen oftare i de baljor som myggorna föredrog att lägga ägg jämfört med de med en mindre mängd mygglarver. De inkluderar geranylacetone och 4-ethylbenzaldehyde. Svampar isolerades från rotstockar av gräs som fanns i den jord som användes för att göra äggläggningssubstraten i fältstudierna. De flyktiga ämnen som avgavs från svampkulturerna analyserades. Bland annat så identifierades ett ämne som fungerar som en äggläggningsattrahent för An. gambiae s.l. myggor.Resultaten från den här avhandlingen kommer att kunna användas för att utveckla miljövänliga ”attract and kill” metoder för att kontrollera malaria myggor.

QC 20160211

L’accroissement de la population humaine, l’agriculture intensive et le développement industriel créent une pollution de l’air qui aujourd’hui devient préoccupante pour notre santé et notre environnement. Si la qualité de l’air extérieur fait l’objet depuis plusieurs décennies de règlementations qui permettent aujourd’hui de constater une diminution globale de la pollution dans les grandes agglomérations européennes, la pollution de l’air intérieur a quant à elle été longtemps sous-estimée. En effet, avec le développement de matériaux composites pour la construction et l’ameublement, la gamme de polluants de l’air intérieur s’est très largement agrandie et les concentrations ont globalement augmenté. Plusieurs études ont ainsi montré que de nombreux composés organiques volatils étaient détectés dans l’air intérieur à des concentrations bien plus élevées qu’à l’extérieur. D’autre part, la modification des modes de vie sédentaires et citadines ont pour conséquence une augmentation du temps passé dans des espaces confinés comme les logements, les lieux de travail et les transports en commun. Le simple renouvellement de l’air intérieur par de l’air extérieur devenant de moins en moins satisfaisant dans les grandes agglomérations, de nouvelles méthodes de traitement sont actuellement développées pour diminuer les concentrations de ces polluants tout en limitant la consommation d’énergie. La photocatalyse, en tant que procédé d’oxydation avancé fait partie des technologies intéressantes pour minéraliser des composés organiques volatils (COV). Après un rapide rappel du contexte sociétal de la pollution atmosphérique, les conditions de mesures et les méthodes possibles pour le traitement de cette pollution sont présentées. Le chapitre suivant regroupe les résultats sur le développement de matériaux photocatalytiques innovants et la mesure de leur efficacité. La première partie de ce chapitre fait le bilan des réacteurs photocatalytiques adaptés à l’étude de réactions à l’interface solide-gaz et résume les nombreuses difficultés liées à l’évaluation des performances de divers matériaux dans des conditions le plus souvent difficilement comparables. Dans la seconde partie, un premier matériau composite constitué de film polymère et de dioxyde de titane a été caractérisé par sa capacité à oxyder un composé volatil, le diméthyle disulfure, utilisé en agriculture pour la fumigation. Le développement d’un second matériau photocatalytique original, constitué de fibres de TiO2 pur a, quant à lui, été caractérisé par sa capacité à minéraliser des COV représentatifs de la pollution de l’air intérieur (acétone, heptane, toluène). Les deux dernières parties de ce chapitre se situent à l’interface entre la photochimie et la biologie. Dans un premier temps, la capacité d’inactivation bactérienne d’un textile « intelligent » sur lequel sont fixées des particules de dioxyde de titane couplées à un photosensibilisateur a été étudiée et l’efficacité sous rayonnement visible de ce tissu original a été analysée. L’impact de la pollution de l’air intérieur sur des cellules de la peau fait l’objet de la dernière partie de ce chapitre. Pour cela un montage permettant d’exposer des cellules de kératinocytes en culture, mais également des biopsies de peau humaine, à des concentrations contrôlées en COV a été mis au point. Nous avons ainsi pu mettre en évidence une réponse cellulaire à ce stress environnemental et préciser l’origine de ce stress. Enfin ce travail se termine par une ouverture sur des projets de recherche actuellement en cours ayant pour objet la mesure des espèces réactives de l’oxygène impliquées dans les réactions photochimiques et le développement de nouveau matériaux hybrides polymère/photosensibilisateurs. Des idées de projets à l’interface de la photochimie et de la biologie ouvrent de nouvelles perspectives à la suite de ces premiers résultats
The increase of human population, the modern agriculture and industrial development generate air pollution, which is nowadays worrying for health and environment. Since several decades, outdoor air pollution has been regulated giving rise a global decrease of pollution in the most important European cities. However indoor air pollution was neglected for a long time. Indeed with development of composite materials for building and furnishing, the number of air pollutants strongly increased together with their concentrations. Several studies have thus demonstrated that numerous volatile organic compounds (VOC) were detected indoor at much higher concentration than outdoor. Moreover, due to the modification of sedentary and urban lifestyles, the time spent in confined spaces like housing, working places and public transportation increases. It is less and less satisfactory to simply renew indoor air with outdoor air in most of urban agglomerations. Accordingly, new processes for air treatment are developed in order to decrease indoor air pollutant concentrations while limiting energetic consumption. Photocatalysis is an advanced oxidation process potentially interesting for VOC removal. After a short reminder on the societal context of atmospheric pollution, measurement and treatment methods are presented in chapters I and II. The following chapter gathers the results obtained on the development of new photocatalytic materials and on the measure of their efficiency. The first part of this chapter is devoted to an overview of photocatalytic reactors for gas solid reactions and summarizes the numerous problems arising from the comparison of different materials under various conditions, which are not always similar. In the second part, a composite material made of titanium dioxide encapsulated in a polymer film is characterized and used for the oxidation of a volatile compound used for agricultural fumigation, dimethyl disulfide. The spectroscopic analysis led to the optimization of the material as a function of its thickness and its titanium dioxide loading. A second innovative photocatalytic material made of pure TiO2 fibers is characterized by its mineralization ability of representative indoor air VOC (acetone, heptane, and toluene). The performance of this material is compared to that of a commercial one, Quartzel ® made of TiO2 deposited on quartz fibers, under strictly identical conditions. The two last parts of this chapter are at the interface between photochemistry and biology. In a first strep, bacterial inactivation by a smart textile where titanium dioxide particles coupled with a photosensitizer is studded under visible light. In the last part, the impact of indoor air pollution on skin cells is presented. A dedicated device allowing keratinocytes culture cells and skin biopsies exposures to controlled VOC concentrations is developed. It is thus possible to evidence and to determine the origin of the cellular response to this environmental stress. At last, new research projects for a near future are then presented. They concern the determination of reactive oxygen species involved in photochemical reactions and the development of new hybrid polymers encapsulating photosensitizing molecules. Prospective ideas at the interface of photochemistry and biology conclude this memory

L'objectif principal de ce travail est d'étudier les mécanismes d'oxydation lors de la photocatalyse (TiO2 irradié sous UV-A) appliquée à la dégradation des microorganismes et leurs effets sur les composants cellulaires. L'étude de l'efficacité antimicrobienne de TiO2 sur un panel de microorganismes (bactéries, spores, champignons) réalisée dans différents milieux (TiO2 en milieu riche, 'sec', en phase liquide) montre l'influence des méthodes d'évaluation, de test et de comptage sur les efficacités d'inactivation. Des études menées en présence de molécules scavengers d'anions superoxydes (O2°-) mettent en évidence l'implication des O2°- dans l'effet antibactérien et dans la peroxydation lipidique. Au niveau protéomique, diverses cibles d'action potentielles du TiO2 sont aussi proposées. Enfin, une partie applicative détermine l'efficacité antimicrobienne de dispositifs photocatalytiques équipés de mousses alvéolaires de β-SiC et de diodes électroluminescentes, et met en avant les propriétés auto-désinfectantes sous lumière solaire de textiles photocatalytiques fonctionnalisés par la technique layer-by-layer.

Plants harbor bacteria which can provide them with benefits such as growth promotion, enhancement of nutrient uptake, defense against pathogens and predators, and improvement of tolerance to abiotic factors such as drought. The development of biotechnological applications using these bacteria has been the focus of research and interest from the scientific community and agricultural sector. While crops have received significant attention, plant species growing in natural environments were neglected and should also be explored. This is particularly important in the paradigm of climate change and its threat to plant productivity, especially due to drought and desertification. This thesis aimed to study the diversity of bacteria living in legume root nodules from wild areas in continental Portugal., to understand if the site of origin and climate conditions influence bacterial osmotolerance, and to evaluate the plant growth promotion abilities of bacterial strains towards the non-legume plant model Arabidopsis thaliana. The bacterial strains isolated from different wild legume species were characterized by BOX-PCR and partial 16S rRNA gene sequencing. These strains, which belonged mainly to the genera Flavobacterium and Pseudomonas, were used to evaluate their plant growth promoting abilities (production of siderophores, indol acetic acid, emission of volatiles) and their tolerance to osmotic stress. A representative set of strains from the different genera was used to study the mechanisms behind the response to osmotic stress (osmolytes and antioxidant mechanisms). It was possible to suggest intracellular alginate as a new mechanism of bacterial osmotolerance. In this thesis, a particular focus has been given to the mechanism of plant growth promotion by bacterial volatiles. The volatile metabolites released by two bacterial strains (Flavobacterium sp. D9 and Rhizobium sp. E20-8) were captured using solid-phase microextraction and analyzed by gas chromatography – mass spectrometry and two-dimensional gas chromatography – mass spectrometry. The effects of bacterial volatiles emission on several physiological and biochemical endpoints of A. thaliana were also evaluated, in control and osmotic stress (drought). Globally, the results of this thesis evidenced the potential of wild legume plant species as sources of bacteria promoting the growth and tolerance to drought of plant species, including legumes and non-legumes, which should be the used as an agricultural practice to increase crop production, particularly in drought conditions.
As plantas são habitadas por bactérias que lhes podem trazer benefícios, como por exemplo a promoção do seu crescimento, a melhoria da aquisição de nutrientes, defesa contra agentes patogénicos e predadores, e melhoria da tolerância a fatores abióticos como por exemplo a seca. O desenvolvimento de aplicações biotecnológicas utilizando estas bactérias desde há muito que interessa a comunidade científica e o setor da agricultura. Enquanto que as espécies de plantas cultivadas têm recebido bastante atenção, as espécies de plantas a crescer em ambientes naturais têm sido negligenciadas e também devem ser exploradas nesse sentido. Este interesse é redobrado num contexto de ameaça à produtividade agrícola por parte das alterações climáticas, e em particular a seca e associada desertificação. Esta tese teve como objetivos explorar a diversidade de bactérias existentes nos nódulos das raízes de plantas leguminosas a crescer em ambiente selvagem em Portugal continental, perceber se as condições edafoclimáticas do local de origem afetam a osmotolerância das bactérias, e avaliar as capacidades de promoção de crescimento de uma planta modelo, não leguminosa, a Arabidopsis thaliana. As estirpes bacterianas isoladas a partir de várias espécies de plantas leguminosas a crescer em Portugal continental foram caracterizadas por BOX-PCR fingerprinting e amplificação parcial e sequenciação do gene que codifica para o 16S rRNA. Estas estirpes, predominantemente dos géneros Flavobacterium e Pseudomonas, foram utilizadas para avaliar a sua capacidade de evidenciar capacidades promotoras do crescimento das plantas (produção de sideróforos, ácido indol acético, emissão de compostos voláteis) e a sua tolerância ao stress osmótico. Um conjunto representativo de estirpes de diferentes géneros foi utilizado para estudar os mecanismos de resposta ao stress osmótico (osmólitos e mecanismos antioxidantes), tendo sido possível apontar o alginato intracelular como um possível novo mecanismo de osmotolerância bacteriana. Nesta tese, um enfoque particular foi dado ao mecanismo de promoção de crescimento de plantas através da emissão de metabolitos voláteis pelas bactérias. Os metabolitos voláteis emitidos por duas estipes bacterianas (Flavobacterium sp. D9 e Rhizobium sp. E20-8), promotoras do crescimento de A. thaliana, foram captados por microextração em fase sólida e analisados por cromatográfica gasosa e cromatografia gasosa bidimensional e espectrometria de massa. Foram também avaliados os efeitos da emissão de voláteis em diversos parâmetros fisiológicos e bioquímicos das plantas, em condições controlo e condições de stress osmótico (seca). Globalmente, os resultados desta tese evidenciam o potencial das espécies de leguminosas selvagens como fontes de bactérias que promovem o crescimento e tolerância à seca de espécies de plantas, incluindo leguminosas e não leguminosas, e que devem ser usadas como prática agrícola para aumentar a produtividade, particularmente em condições de seca.
Programa Doutoral em Biologia e Ecologia das Alterações Globais

Volatile sulfur compounds are the primary cause of bad breath. They are a byproduct of bacterial metabolism and can be difficult to eliminate because they generally originate on the dorsum of the tongue, an area often missed during oral hygiene practices. Chronic bad breath, or halitosis, can be a cause of extreme anxiety. Indeed, halitosis has been proven to affect people across the globe. However, doctors and dentists are generally unaware of the causes of this disease. While poor oral hygiene is the most obvious cause of halitosis, many sufferers in fact have scrupulous oral hygiene practices. Little is known about the development of the disease; researchers have instead focused on which mouth rinses are the most effective and which bacteria are the most likely culprits.

Bacterial volatile organic compounds (VOCs) are signal molecules that may have beneficial roles in the soil-plant-microbiome ecosystem. In this Ph.D. thesis, I aimed to assess and characterize the role of bacterial VOCs in plant tolerance to drought and in the biocontrol of fungal pathogens. I started by studying two root endophytic bacteria isolated from pepper plants cultivated under desert farming conditions. They showed an enhancement of pepper tolerance to drought stress and an amelioration of its physiological status. Moreover, they induced the expression of a vacuolar pyrophosphatase proton pump (V-PPase), implicated in the regulation of the vacuolar osmotic pressure, facilitating water uptake. Besides, the exposure of Arabidopsis thaliana plants, grown under salinity stress, to the volatile 2,3-butanediol, described for its plant growth promotion (PGP) potential, enhanced the plants tolerance to salinity, proving the potential involvement of this volatile in the osmotic stress resistance mechanism. Then, I studied VOCs released by three bacteria associated to healthy rice plants. Their released VOCs mixtures modified the color pattern of Magnaporthe oryzae, the agent of the rice blast disease, and protected rice from the pathogen infection. A significant reduction of melanin production, sporulation and appressoria formation was measured in presence of the bacterial VOCs, without major effects on mycelial proliferation. 1-butanol-3-methyl, one of the nine VOCs co-produced by the studied bacteria, proved its potential of reducing M. oryzae melanin in vitro. In vivo tests confirmed the infection inhibition effects mediated by the rice-bacterial VOCs, with a reduction of 94% of the disease incidence. Lastly, I compared the genomes of the five bacteria considered in the previous experimental studies. The PGP traits and the VOCs pathways identified from the genome analyses confirmed the effects observed with the in vitro and in vivo assays, revealing a complex mode of promotion and protection offered by the studied plant-associated bacteria. In conclusion, plant-associated bacterial VOCs can play potentially important roles in modulating plant drought tolerance and reducing fungal virulence. Such biological resources represent novel tools to counteract the deleterious effects of abiotic and biotic stresses and have the potential to be exploited for sustainable approaches in agriculture.

碩士
國立屏東科技大學
熱帶農業暨國際合作研究所
94
This study was investigated the volatile chemical components and their antimicrobial activities of perilla (Perilla frutescens Britt;) in five species, Steam distillation was used to extract the volatile chemical components, which were then separated and examined with GC and GC/MS. An ensuing analysis of their antimicrobial activities was conducted. From the analysis, thirty-six chemical components were found contained in the leaves of perilla. The analysis further showed that perillaldehyde, d-limonene, linalool, α-pinene, β-pinene, and eugenol were the main volatile chemical components. The main element ‘perillaldehyde’ was then extracted and analyzed for its antimicrobial activities. Paper Disk Diffusion Technique was used to test five species of bacteria separately. The results showed that perillaldehyde was able to inhibit Staphylococcus aureus. To obtain the minimum inhibitory concentration (MIC), the standard samples of perillaldehyde were diluted to different concentrations so as to test the changes in their inhibitory activity. The findings showed that perillaldehyde at 30% concentration was inhibitory effects on Staphylococcus aureus. Keywords：volatile chemical components, antimicrobial activities, perilla, perillaldehyde

腐乳是中國傳統豆類發酵製品，具有綿軟的口感和特殊的風味。其是豆腐通過真菌固態發酵，并加入鹽，米酒和香料等進行後期熟化而成的產品。本文的研究分為兩部份，第一部份對腐乳發酵過程中的毛胚，鹽胚，熟化第一天，熟化一個月以及熟化六個月的腐乳樣本進行採樣，并採用傳統微生物培養法和克隆文庫法對每個階段真菌和細菌的生態結構和動態變化進行研究。第二部份重點比較了四株腐乳產品中分離的微生物和購自台灣生物資源保存及研究中心的四株細菌的產揮發性含硫化合物能力，并挑選了最高產的一株微生物進行紫外誘變，最後獲得理想的突變株。本研究的結論如下：
1. 真菌和細菌的總數均是在毛胚階段為最高，在進入熟化階段后開始下降。在傳統微生物培養方法下分別分離出了三株真菌和九株細菌，通過18S rDNA和16 rDNA測序，發現絲孢酵母屬（Trichosporon spp.）是真菌中的優勢菌種，蠟狀芽孢桿菌（Bacillus cereus）和解澱粉芽孢桿菌（Bacillus amyloliquefaciens）為細菌中的優勢菌種；
2. 本研究建立了五個真菌18S rDNA克隆文庫和五個細菌16 rDNA克隆文庫用于研究真菌和細菌的生態結構和動態變化。通過聚合酶鏈式反應-限制性片段長度多態性（PCR-RFLP）的研究，分別在真菌和細菌克隆文庫中發現23和38種圖譜類型，并計算其相應比例。在進行真菌細菌測序之後，對優勢菌群進行了定性和定量分析；
3. 在對比傳統微生物培養方法和克隆文庫技術的結果后發現，二者的結果存有差異，有些在克隆文庫中鑒定到的微生物在傳統培養方法中未能分離鑒定，而有些微生物則只能在傳統培養方法中被分離鑒定。因此，本研究中將這兩種方法結合有助於我們更為全面、客觀地研究腐乳發酵過程中真菌和細菌的生態結構和多樣性。
4. 對四株腐乳中分離純化的微生物和四株外來購入細菌的產揮發性含硫化合物能力進行比較，結果發現，從腐乳產品中分離純化的B-1菌株擁有最高的產揮發性含硫化合物能力，通過紫外誘變后，突變株#3在產揮發性含硫化合物以及L-蛋氨酸代謝酶活力都比初始菌株有了顯著的提升。B-1菌經測序比對后鑒定為絲孢酵母（Trichosporon sp.）。
本研究結果對于傳統腐乳發酵的有效控制和現代腐乳生產工藝的建立有一定指導意義，並且對於腐乳產品中的風味物質，特別是揮發性含硫化合物的產生和優化提供信息。
Sufu (fermented soybean curd) is a soft creamy cheese-type product with a pronounced flavor and is made by fungal solid state fermentation of tofu (soybean curd) followed by aging in brine containing salt and alcohol. In first part of this research, the eco-structure and the dynamic changes of microbes during sufu production process (Pehtze, Salted pehtze, 0 Month sufu, 1 Month sufu and 6 Month sufu sample) were studied by combined microbiology techniques, including plate culture, 16S rDNA and 18S rDNA clone library and restriction fragment length polymorphism (RFLP) analysis. The second part of this research focus on the comparison of volatile sulfur-containing compounds (VSCs) production ability within isolated strains in sufu product and bacteria purchased commercially, the strain that possessed highest ability was selected and followed by a UV mutation experiment, finally obtained the desired mutant. The results of this research are as followed:
1. The population of both fungi and bacteria were all at highest number in Pehtze stage and started to decrease in ripening stages. A combined total of three and nine living strains of fungi and bacteria were obtained from the plate culture, respectively. Through 18S rDNA and 16S rDNA sequencing, Trichosporon spp. was the dominant fungi and Bacillus cereus and Bacillus amyloliquefaciens were the dominant bacteria;
2. Five 18S rDNA clone libraries and five 16S rDNA clone libraries from different stages of sufu production were constructed to analyze the structure and dynamic changes of fungi and bacteria. A total of 23 and 38 RFLP patterns were found, and the ratio of each pattern were calculated. After sequencing, qualitative and quantitative analysis on the dynamic changes of dominant strains was performed;
3. After comparing the results of plate culture and clone library, it was found that there were some differences between the two. Some strains were only found in clone library while some only found in plate culture approach. Therefore, the combination of the two microbiology methods will help us to objectively and completely analyze the structure and dynamic changes of microbes in the sufu production process;
4. The ability to produce VSCs within four strains (B-1, B-2, B-3 & B-4) isolated from a commercial sufu manufacturing process and four commercial strains (B. acetylicum, L. Lactics, S. thermophilus and L. Paracasei) were compared. Results showed that B-1 possessed both the highest VSCs production ability and L-methionine metabolism enzymatic activities among the eight strains. After UV light mutagenesis of B-1 strain, its mutant #3 significantly increased in DMDS and DMTS production and all four L-methionine-related enzymatic activities in reference to that of the starting strain (B-1). B-1 was identified as Trichosporon sp. by sequencing.
These results would make a profound significance on the control of traditional sufu production and the development of new technology for modern sufu manufacturing. They will also help to provide some important information of optimal production of VSCs in sufu ripening and the overall flavor in sufu product.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Huang, Ruolan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 106-117).
Abstracts also in Chinese.
Abstract --- p.i
摘要 --- p.iii
Acknowledgement --- p.v
Table of contents --- p.vi
List of Figures --- p.x
List of Tables --- p.xiii
Chapter Chapter 1 --- : Introduction --- p.1
Chapter 1.1 --- Sufu --- p.1
Chapter 1.1.1 --- Classification --- p.1
Chapter 1.1.1.1 --- Classified by processing technology --- p.1
Chapter 1.1.1.2 --- Classified by color and flavor --- p.1
Chapter 1.1.1.3 --- Other classifications --- p.2
Chapter 1.1.2 --- Typical commercial manufacturing process --- p.2
Chapter 1.1.2.1 --- Production process of naturally fermented sufu --- p.2
Chapter 1.2.2.2 --- Production process of traditional mold-based sufu --- p.5
Chapter 1.2.2.3 --- Production process of traditional bacteria-based sufu --- p.5
Chapter 1.2.2.4 --- Acceleration of sufu ripening process --- p.6
Chapter 1.1.3 --- Important ingredients in sufu production --- p.6
Chapter 1.1.4 --- Flavor components in sufu --- p.7
Chapter 1.1.4.1 --- Volatile flavor components --- p.7
Chapter 1.1.4.2 --- Essential odor in sufu product --- p.8
Chapter 1.1.4.3 --- Volatile sulfur compounds in sufu --- p.9
Chapter 1.1.4.4 --- Using Head Space-Solid phase Microextraction (HS-SPME) to analyze the volatile sulfur components --- p.9
Chapter 1.1.5 --- Relationship between microbes and sufu --- p.12
Chapter 1.1.5.1 --- Microbes involved in fermentation process --- p.13
Chapter 1.1.5.2 --- Microbial changes during the production of sufu --- p.14
Chapter 1.1.6 --- Study on microbial ecology in food product --- p.15
Chapter 1.1.6.1 --- PCR-based molecular techniques --- p.16
Chapter 1.1.6.2 --- Non-PCR based molecular techniques --- p.16
Chapter 1.1.6.3 --- The common techniques used in microbial ecology research --- p.17
Chapter 1.1.6.4 --- Microbial ecology study by molecular biological techniques --- p.18
Chapter 1.2 --- Objectives --- p.19
Chapter Chapter 2 --- : Analysis of fungi diversity during sufu fermentation process --- p.21
Chapter 2.1 --- Introduction --- p.21
Chapter 2.2 --- Materials and methods --- p.21
Chapter 2.2.1 --- Sample collection and preparation --- p.22
Chapter 2.2.2 --- Plate count of fungi during sufu fermentation process --- p.22
Chapter 2.2.3 --- Change of pH values and moisture content --- p.22
Chapter 2.2.4 --- Total DNA extraction from fungi --- p.23
Chapter 2.2.5 --- Preparation of competent cell --- p.23
Chapter 2.2.6 --- 18S rDNA PCR amplification and construction of 18S rDNA clone library --- p.24
Chapter 2.2.7 --- RFLP analysis of 18S rDNA clone library --- p.25
Chapter 2.2.8 --- DNA sequencing for fungi identification --- p.26
Chapter 2.2.9 --- Analysis of the diversity of 18S clone library --- p.26
Chapter 2.2.10 --- Frequency percentage analysis --- p.27
Chapter 2.2.11 --- Enzyme Solutions --- p.27
Chapter 2.2.12 --- Determination of protease activity --- p.28
Chapter 2.2.13 --- Determination of lipase activity --- p.29
Chapter 2.2.11 --- Microtox test --- p.30
Chapter 2.2.12 --- Statistical analysis --- p.30
Chapter 2.3 --- Results and discussion --- p.30
Chapter 2.3.1 --- Fungi growth on plate counting result --- p.30
Chapter 2.3.2 --- Changes in pH and moisture content of sufu during production --- p.33
Chapter 2.3.3 --- Construction and selection of 18S rDNA clone library --- p.35
Chapter 2.3.4 --- Fungal diversity based on 18S rDNA clone library analysis --- p.38
Chapter 2.3.5 --- Protease and lipase activities in Actinomucor elegans and Trichosporon japonicum --- p.45
Chapter 2.3.5.1 --- Protease activity --- p.46
Chapter 2.3.5.2 --- Lipase activity --- p.47
Chapter 2.3.6 --- Toxicity of Actinomucor elegans and Trichosporon japonicum --- p.49
Chapter 2.3.7 --- Analysis of fungi eco-structure and function during sufu fermentation process --- p.50
Chapter 2.3.8 --- The influence of PCR bias and artifact --- p.53
Chapter 2.2 --- Summary --- p.55
Chapter Chapter 3 --- : Analysis of bacteria diversity during sufu fermentation process --- p.57
Chapter 3.1 --- Introduction --- p.57
Chapter 3.2 --- Materials and methods --- p.57
Chapter 3.2.1 --- Sample collection and preparation --- p.57
Chapter 3.2.2 --- Plate count of bacteria during sufu fermentation process --- p.57
Chapter 3.2.3 --- Total DNA extraction from bacteria --- p.58
Chapter 3.2.4 --- Preparation of competent cell --- p.58
Chapter 3.2.5 --- 16S rDNA PCR amplification and construction of 16S rDNA clone library --- p.58
Chapter 3.2.6 --- RFLP analysis of 16S rDNA clone library --- p.59
Chapter 3.2.7 --- DNA sequencing for bacteria identification --- p.60
Chapter 3.2.8 --- Analysis of the diversity of 16S rDNA clone library --- p.60
Chapter 3.3 --- Results and discussion --- p.60
Chapter 3.3.1 --- Bacteria growth on plate counting result --- p.60
Chapter 3.3.2 --- Construction and selection of 16S rDNA clone library --- p.63
Chapter 3.3.3 --- 16S rDNA clone library analysis of bacteria diversity --- p.65
Chapter 3.3.4 --- Analysis of bacteria eco-structure and function during sufu fermentation process --- p.74
Chapter 3.4 --- Summary --- p.77
Chapter Chapter 4 --- : Screening the mutant possess higher capacity of forming volatile sulfur compounds (VSCs) from non-starter microbes of sufu product --- p.80
Chapter 4.1 --- Introduction --- p.80
Chapter 4.2 --- Materials and methods --- p.82
Chapter 4.2.1 --- Strains and culture conditions --- p.82
Chapter 4.2.2 --- Head space-solid phase microextraction (HS-SPME) analysis --- p.83
Chapter 4.2.3 --- Gas Chromatography-Mass Spectrometry (GC-MS) analysis --- p.84
Chapter 4.2.4 --- UV mutation --- p.85
Chapter 4.2.5 --- Ellman’s method --- p.86
Chapter 4.2.6 --- Preparation of cell-free extracts (CFE) for enzymatic assays --- p.86
Chapter 4.2.7 --- Enzymatic assay --- p.86
Chapter 4.2.7.1 --- L-methionine aminotransferase activity assay --- p.86
Chapter 4.2.7.2 --- L-methionine demethiolase activity assay --- p.87
Chapter 4.2.7.3 --- α-keto acid decarboxylase activity assay --- p.87
Chapter 4.2.7.4 --- C-S lyase activity --- p.88
Chapter 4.2.8 --- Statistical analysis --- p.88
Chapter 4.3 --- Results and discussion --- p.89
Chapter 4.3.1 --- Optimization of SPME extraction condition --- p.89
Chapter 4.3.2 --- Selecting the start strain --- p.90
Chapter 4.3.4.1 --- Comparison of VSCs production ability --- p.90
Chapter 4.3.4.2 --- Comparison of enzymatic activity in L-methionine metabolism --- p.92
Chapter 4.3.3 --- Optimization of UV exposure time --- p.95
Chapter 4.3.4 --- Screening the mutants --- p.96
Chapter 4.3.4.1 --- Comparison of VSCs production ability among the mutants --- p.96
Chapter 4.3.4.2 --- Comparison of the L-methionine related enzymatic activities among the mutants --- p.99
Chapter 4.3.4.3 --- Identified of strian B-1 --- p.101
Chapter 4.4 --- Summary --- p.102
Chapter Chapter 5 --- : General conclusions and future work --- p.103
References --- p.106

碩士
國立成功大學
生命科學系
104
Microbes affect plant growth through several mechanisms. One of the mechanisms affected by microbial volatile organic compounds (mVOCs) are gaseous molecules released by microbes. There are lots of reports that focus on the influence by mixing mVOCs. Just a few researches have analyzed the compositions of mVOCs. Here I analyzed two volatile organic compounds, which are released by Enterobacter aerogenes, a plant growth-inhibiting bacteria. I found the major inhibiting compound, C2, the second large compound in E. aerogenes mVOCs. Using next generation sequencing technique, I found that the key genes of autophagy, exocyst and asparagines synthesis were up regulated when tobacco was exposed to mVOCs of E. aerogenes or C2 compound. To further confirm how vesicle trafficking results in tobacco defense mechanism, virus-induced gene silence (VIGS) was performed to silence Exo70, an important gene participates in autophagy process. NbExo70 silenced tobacco was sensitive to mVOCs of E. aerogenes and C2 compound. H2O2 was not accumulated in leaves when NbExo70 silenced tobacco exposed to mVOCs of E. aerogenes and C2 compound. According to the results I can conclude that Exo70 induces the accumulation of H2O2. The accumulation of H2O2 would induce hypersensitive response programmed cell death(HR-PCD). When the level of H2O2 was abnormal accumulation, it would disturbe the HR-PCD of plant. Thus, Exo70 would help the autophagosomal degradation of H2O2 signaling related protein. NbExo70 silenced tobacco has less deposition in callose, but when it is exposed to mVOCs of E. aerogenes and C2 compound the deposition in callose will be higher than wild-type. It seems that Exo70 is involved in callose deposition, but when under the stress tobacco will turn on another mechanism.

The research presented in this thesis explores the interactions between certain phenolic compounds that are naturally present in wines and the microorganisms that are generally present in this medium. The interactions studied include the effects on microbial growth and metabolism, the intra-specific diversity of Oenococcus oeni and the evolution of volatile and non-volatile compounds during malolactic fermentations (MLF) and subsequent storage. The ability of several strains of lactic acid bacteria (LAB) to release hydroxycinnamic acids (HCA) from their tartrate derivative forms ((hydroxy)cinnamoyl-tartaric acids) was also studied. Metabarcoding analysis of 16 different wines analyzed at the post-malolactic stage was performed using sequence data from 16S (for bacteria) and ITS2 (for fungi) amplicons. Similar patterns were observed in all wines at this level of discrimination, with Saccharomyces cerevisiae yeasts and bacteria from the genera Acetobacter, Gluconobacter and Swaminathania being the most abundant taxa. Concerning the profiles of phenolics and volatiles, red and white wines were grouped separately, and French and Spanish wines tended to cluster together. The effects of kaempferol, trans-caffeic and trans-caftaric acids (added at 10 mg/L) on the microbial growth and metabolism were explored in non-inoculated and inoculated wine (O. oeni) and wine mixed with MRS (de Man, Rogosa & Sharpe). The strongest impact on microbial growth, malolactic and general metabolic activity in LAB, was noted for kaempferol, with trans-caftaric acid showing the weakest. The effects of each phenolic compound varied according to the medium used, the type of MLF (inoculated or not) and the O. oeni strain inoculated (OenosTM or CH35TM). Using concentrations normally encountered in wines as a reference, flavan-3-ols ((+)-catechin and (-)-epicatechin), flavonols (kaempferol and quercetin), HCA (trans-p-coumaric and trans-ferulic acids) and trans-resveratrol were studied in experiments with non-inoculated wines and wines inoculated with OenosTM. Depending on its concentration, (+)-catechin positively impacted the yeast population, activated or delayed malic acid degradation or inhibited the growth of bacteria. All phenolics tested, at all concentrations tested, delayed citrate consumption in inoculated samples. The effect of flavonols and trans-resveratrol on the O. oeni diversity was dependent on the strains under study. An increase in trans-p-coumaric and trans-ferulic acid levels appeared to induce the release of trans-caffeic acid from one of its precursors, possibly via an increase in cinnamoyl esterase activity of some strains. Flavan-3-ols and flavonols inhibited the growth of these bacteria during storage. The phenolics tested affected specific enzymatic systems responsible for the production and degradation of important metabolites involved in the organoleptic quality of the wines. Cinnamoyl esterase (CE) activity of wine microbes can be relevant as it confers the capacity to modulate the phenolic acid composition of a wine by liberating these from otherwise seemingly biologically unavailable, tartrate derivatives. Five commercial strains of O. oeni were studied in this respect, three exhibiting CE activity (OenosTM, CiNeTM and CH35TM) and two not (CH16TM and CH11TM). CE activity was detected in cell-free extracts of the three CE positive (CE+) strains and one of the CE negative (CE-) strains. From comparative genome analysis, no gene exclusive to the 3 CE+ was detected and the inference that membrane transport differences might be behind differences in CE activity was also not conclusively supported. This hypothesis needs to be further explored as does the possibility of the involvement of wine molecules and of more than one enzyme in the CE activity.
A investigação apresentada nesta tese explora as interações entre certos compostos fenólicos naturalmente presentes nos vinhos e os microrganismos geralmente presentes neste mesmo meio. As interações estudadas incluem os efeitos no crescimento e metabolismo microbiano, a diversidade intra-específica de Oenococcus oeni e a evolução de compostos voláteis e não voláteis durante a fermentação malolática (FML) e subsequente armazenamento dos vinhos. Foi também estudada a capacidade de várias estirpes de bactérias de ácido láctico (BAL) para liberar ácidos hidroxicinâmicos (AHCs) a partir dos correspondens ácidos hidroxicinamoil-tartáricos . A análise de metabolismo de 16 vinhos diferentes analisados na fase pós-malolática foi realizada usando dados de sequência de amplicons 16S (para bactérias) e ITS2 (para fungos). Foram observados padrões semelhantes em todos os vinhos neste nível de discriminação, tendo sido os géneros Saccharomyces cerevisiae e Acetobacter, Gluconobacter e Swaminathania, os mais abundantes. No que diz respeito aos perfis de compostos fenólicos e voláteis, os vinhos tintos e brancos foram agrupados separadamente e os vinhos franceses e espanhóis tenderam a agruparse em conjunto. Os efeitos do kaempferol e dos ácidos trans-caféico e trans-caftárico (adicionados a 10 mg/L) no crescimento e metabolismo microbiano foram explorados em vinho puro e em vinho misturado com o meio de cultura MRS (de Man, Rogosa & Sharpe) em experiencias inoculadas e não-inoculadas com O.oeni. O impacto mais forte no crescimento microbiano, atividade malolática e atividade metabólica geral no BAL, foi observado para o kaempferol, tendo o ácido trans-cafárico mostrando o efeito mais fraco. Os efeitos do cada composto fenólico variaram de acordo com o meio utilizado, o tipo de FML (com ou sem inoculação) e a estirpe de O. oeni inoculada (OenosTM ou CH35TM). Utilizando concentrações encontradas no vinho como referência, foram estudados vários compostos fenólicos, nomeadamente: flavan-3-óis ((+)-catequina e (-)-epicatequina), flavonóis (kaempferol e quercetina), HCA (ácidos trans-p-cumárico e trans-ferúlico) e trans-resveratrol em experiências com vinhos não inoculados e inoculados com OenosTM. Dependendo na sua concentração, a presença de (+)-catequina causou um impacto positivo na população de leveduras, ativou ou retardou a degradação do L-ácido málico ou inibiu o crescimento de bactérias. Todos os fenólicos testados, em todas as concentrações, atrasaram o consumo de ácido cítrico por BAL nas amostras inoculadas. O efeito de flavonóis e de trans-resveratrol na diversidade de O. oeni foi estudado, tendo-se observado ser dependente das estirpes em estudo. O aumento nos níveis de ácido transp-cumárico e trans-ferúlico pareceu induzir a liberação de ácido trans-caféico a partir de um dos seus precursores, possivelmente através de um aumento na atividade da cinamoil-esterase de algumas estirpes. Os flavan-3-óis e flavonóis testados inibiram o crescimento destas bactérias durante o armazenamento. Os compostos fenólicos testados afetaram sistemas enzimáticos responsáveis pela produção e degradação de importantes metabolitos envolvidos na qualidade organolética dos vinhos. A presença da enzima cinamoil-esterase (CE) nos microrganismos do vinho é relevante, uma vez que confere a capacidade de modular a composição de ácidos fenólicos de um vinho (que, de outro modo, não biologicamente disponíveis) através da quebra da ligação éster dos ácidos iii hidroxicinamoil-tartáricos. Cinco estirpes comerciais de O. oeni foram estudadas a este respeito, três exibindo atividade de CE (OenosTM, CiNeTM e CH35TM) e duas não (CH16TM e CH11TM). A atividade de CE foi detetada em extratos livres de células das três estirpes CE positiva (CE+) e uma das estirpes CE negativa (CE-). A partir da análise comparativa do genoma, não foi detetado nenhum gene exclusivo para as três estirpes CE+ e a inferência de que as diferenças na capacidade de transporte da membrana pudessem estar por trás das diferenças na atividade do CE, também não foi apoiada conclusivamente. Esta faceta merece ser mais explorada, assim como a possibilidade do envolvimento de moléculas de vinho e de mais enzimas na atividade da CE.