É esperado que a produção de combustíveis a partir de resíduos lignocelulósicos alcance representatividade na matriz energética mundial. Neste cenário, leveduras desempenharão importante papel como plataformas microbianas para os processos de conversão dos açúcares derivados da biomassa em etanol (de segunda geração) e em outros produtos de interesse. Apesar dos avanços consideráveis na área, a fermentação de hidrolisados lignocelulósicos ainda apresenta alguns desafios científicos e tecnológicos, como por exemplo, os problemas enfrentados na fermentação devido à presença de diversos inibidores oriundos dos processos de pré-tratamento e hidrólise da biomassa (tais como furaldeídos, compostos fenólicos e ácidos orgânicos). A geração destes inibidores reduz consideravelmente a eficiência da etapa fermentativa, e muitas vezes, inviabiliza o processo como um todo. Neste contexto, o presente trabalho estudou a identificação dos principais compostos lignocelulósicos inibidores na fermentação por leveduras presentes em cinco amostras de hidrolisados industriais. Ácido glicólico e ácido acético, dentre os ácidos orgânicos foram identificados em todas as amostras analisadas. Em relação aos furaldeídos, foram detectados furfural e 5- hidroximetil-furufural (HMF) em todas as amostras analisadas. Os principais e mais abundantes compostos fenólicos identificados pelo sistema foram ácido p-cumárico (em 4 de 5), ácido ferúlico (em 3 de 5) e ácido vanílico (em 3 de 5). Tendo os principais compostos quantificados e identificados, realizou-se um estudo de mínimas e máximas concentrações de alguns destes inibidores em quatro diferentes linhagens laboratoriais e industriais (CEN.PK113-7D, CEN.PK112, SA-1 e JAY270), a fim de verificar a toxicidade dos mesmos frente aos principais parâmetros cinéticos da fermentação (fase lag, µmáx, produção de biomassa e etanol). De forma geral, a linhagem industrial S. cerevisiae SA-1 se mostrou mais tolerante frente às demais linhagens, em praticamente todas as condições analisadas. Visto que a presença de algumas bactérias nas linhas de produção de etanol é sabida, procurou-se verificar a resistência destas na presença dos compostos inibitórios. Ao comparar a resistência frente aos inibidores entre bactérias lácticas e leveduras laboratoriais e industriais, notou-se uma potencial robustez das bactérias frente às leveduras, uma vez que as últimas foram incapazes de crescer no meio de cultivo acrescido de um coquetel de inibidores, enquanto que as bactérias apresentaram crescimento. Por fim, investigou-se a influência conjunta de três compostos inibitórios (ácido acético, HMF e ácido p-cumárico) sobre os principais parâmetros fisiológicos da linhagem SA-1, a mais tolerante das linhagens avaliadas. Assim, através de um delineamento composto central rotacional (DCCR), pode-se analisar estatisticamente os efeitos dos três inibidores, com o qual verificou-se que a maior toxicidade (redução da velocidade específica de crescimento) é exercida pelo HMF. De uma maneira geral, a linhagem SA-1 pode ser considerada uma plataforma microbiana com grande potencial de aplicação na produção de etanol de segunda geração, bem como no estudo das bases moleculares para tolerância aos inibidores oriundos desses processos. / It is expected that the production of fuels from lignocellulosic residues will be representative in the world energy matrix. In this scenario, yeasts will play an important role as microbial platforms for the conversion processes of sugars derived from biomass into ethanol and other products of interest. Despite the considerable advances in the area, the fermentation of lignocellulosic hydrolysates still presents some scientific and technological challenges, such as the problems faced in the fermentation due to the presence of several inhibitors from the processes of pretreatment and hydrolysis of biomass (such as furaldehydes, phenolic compounds and organic acids). The presence of these inhibitors considerably reduces the efficiency of the fermentation step, and often hampers the process itself. In this context, the present work aimed to identify major inhibitory lignocellulosic compounds for yeast fermentation present in five samples of industrial hydrolysates. Glycolic acid and acetic acids were identified in all samples analysed. In relation to furaldehydes, furfural and 5-hydroxymethyl-furfural (HMF) were detected in all samples as well. The major and most abundant phenolic compounds identified were p-coumaric acid (in 4 out of 5 samples), ferulic acid (in 3 out of 5) and vanillic acid (in 3 out of 5). After quantification of these compounds, minimum and maximum concentrations of the major ones were studied in four different laboratory and industrial strains (CEN.PK113-7D, CEN.PK112, SA-1 and JAY270), in order to verify their toxicity against physiological parameters such as elongation of lag phase and growth rate. In general, the S. cerevisiae SA-1 industrial strain was more tolerant in comparison to the other 3 strains for virtually all compounds investigated. Since bacteria is present during fuel ethanol production, , the resistance of these bacteria for the same inhibitory compounds was also evaluated. When comparing the resistance against inhibitors between lactic acid bacteria and yeast strains, a higher robustness of bacteria in relation to yeasts was observed, since yeasts were unable to grow in a medium supplemented with a cocktail of inhibitors, whereas bacteria did grow. Finally, the influence of three inhibitory compounds (acetic acid, HMF and p-coumaric acid) on the main physiological parameters of the strain SA-1, the most tolerant among the strains evaluated, was investigated. Thus, using a rotatable central composite design (RCCD), the effects of the three inhibitors were analysed, and it was found that the highest toxicity (reduction in specific growth rate) is caused by HMF. Overall, SA-1 is a promising platform yeast strain for second generation ethanol production and for understanding strain robustness toward lignocellulosic inhibitory compounds.

Les macrophages associés aux tumeurs (TAM) orchestrent l'inflammation nécessaire à la croissance tumorale (propriétés de type M1) et favorisent les métastases et l'angiogenèse (caractéristiques de type M2). Cependant, la nature des facteurs capables de conférer des propriétés M1 et M2 aux macrophages humains demeure inconnue. L'acide lactique (AL) est un métabolite produit par les cellules tumorales connu pour moduler les fonctions des cellules présentes dans le microenvironnement tumoral. Dans cette étude, nous avons analysé son impact sur la différenciation des monocytes humains. Les résultats montrent que l’AL induit la différentiation des monocytes (cultivées en présence de GM-CSF) en macrophages présentant un phénotype atypique (CD14high CD163high IL-10low IL-12low) et, de manière intéressante, des caractéristiques phénotypiques M1 (production de cytokines inflammatoires) et M2 (production de facteurs de croissance, expression de gènes prototypiques M2). Un profil similaire est obtenu lorsque les monocytes sont cultivés avec des cellules cancéreuses primaires glycolytiques. Ces effets de l'AL sur la polarisation des macrophages nécessitent l'entrée du lactate dans les cellules (via le transporteur MCT-1) et son oxydation en pyruvate et sont médiés par une stabilisation de HIF-1α et une consommation autocrine de M-CSF.Ces résultats (i) identifient l'AL comme un médiateur induisant la génération de macrophages humains présentant des caractéristiques M2 et des propriétés inflammatoires et (ii) renforcent l'intérêt de l’utilisation des inhibiteurs de la glycolyse aérobie pour moduler les fonctions des TAM. / In established tumors, tumor-associated macrophages (TAM) orchestrate unresolving cancer-related inflammation (M1-related properties) and favor tumor development, metastasis and angiogenesis (M2-like properties). However, to date, the nature of the polarization factor(s) able to confer M1 and M2 functional properties to human macrophages remains unknown.Lactic acid (LA), a metabolite produced at high levels in most established tumors, can impact the phenotype and functions of cells present in the tumor microenvironment. In this study, we analyzed the impact of LA on the human monocyte differentiation. Results showed that LA skews monocytes (differentiated in the presence of GM-CSF) into macrophages (GM+LA-Mφ) exhibiting an atypical CD14high CD163high IL-10low IL-12low phenotype. Interestingly they harbor M1 and M2 phenotypic features, as assessed the production of a wide variety of inflammatory and growth factors and the expression of prototypic M2-like genes. A similar profile is induced by culturing monocytes with glycolytic human primary cancer cells. These effects of LA on macrophage polarization require the entry of lactate into the cells (via the monocarboxylate transporter 1) and its oxidation into pyruvate and are mediated via HIF-1α stabilization and autocrine M-CSF consumption by differentiating cells. These results identify tumor-derived LA as a missing link reconciling the M2-like features of TAM with their inflammatory properties. They also reinforce the interest of aerobic glycolysis inhibitors to modulate the functions of TAM.

The objective of this study was to evaluate the survival of non-O157:H7 STEC, Salmonella spp., and S. aureus in dry fermented sausages. Chorizo and Landjager sausages were inoculated with individual bacterial cocktails and stuffed into natural casings. Temperature, relative humidity, pH, and water activity were monitored through fermentation, drying, and storage. Bacterial counts were determined by serial dilution and plated in triplicates on selective media. Plates were incubated at 37°C for 24 hours and colony forming units per gram (CFU/g) were observed. Results of the first study validate that contaminated raw materials contribute to pathogen survival and background bacteria outcompeted the starter culture. The pH critical limit of < 5.3 was met but there was no pathogen inhibition. Results from the second study confirm that pH and water activity are not enough to eliminate pathogens when post processing interventions are not used. Critical pH (< 5.3) and water activity (< 0.85) limits were met, but pathogens still survived. In chorizo, non-O157:H7 was recovered through enrichments until the end of the study. In landjager, non-O157:H7 STEC and Salmonella were recovered through enrichments until the end of the study.The studies suggest that sausages produced without post processing interventions are a health risk to consumers.

A deficiência de folato é um problema de saúde pública que vem sendo combatido através da fortificação obrigatória de ácido fólico. Entretanto, a ingestão excessiva pode causar efeitos colaterais indesejados. A forma natural da vitamina produzida por algumas cepas de bactérias láticas pode ser uma alternativa segura para aumentar a ingestão de folato pela população. Este trabalho visou selecionar as melhores cepas de BAL produtoras de folato, identificar as melhores condições para a produção da vitamina e avaliar a biodisponibilidade do folato produzido pelas cepas no leite fermentado, empregando-se testes em modelos animais. O estudo foi desenvolvido com cinco cepas de Streptococcus thermophilus (34v, 170v, 268v, 361v e 341 pc) e um cepa de Lactobacillus plantarum (16cv), todas boas produtoras de folato. As condições de produção e a combinação de cepas que resultaram na melhor produção folato foram utilizadas para produzir um leite biofermentado (BFM), avaliando-se a biodisponibilidade da vitamina produzida empregando-se um modelo animal de depleção/repleção, com camundongos Balb/c submetidos a 14 dias de depleção, seguido de 21 dias de repleção da vitamina. Após os 35 dias, os animais foram sacrificados, retirando-se sangue, rins, fígado, baço e intestino para quantificação de folato e avaliação histológica da mucosa intestinal. Todas as quantificações de folato foram feitas pelo método microbiológico. A melhor combinação de cepas (St. Thermophilus 34v + Lb. Plantarum 16cv) resultou na produção de 300 ng/mL de folato, sendo a temperatura de 42ºC melhor que 37ºC para a produção da vitamina. A produção de folato no leite na fermentação em pH controlado 6,0 foi 35% superior à observada no leite fermentado em pH livre. As concentrações de folato no baço e glóbulos vermelhos foram mais altas nos camundongos que ingeriram leite fermentado bioenriquecido (BFM) em relação aos demais grupos de animais. A relação vilosidade/cripta nos camundongos dos grupos BFM foi igual à observada nos animais que receberam leite suplementado com ácido fólico. Houve aumento da hemoglobina, hematócrito e hemácias nos camundongos que ingeriram BFM, evidenciando a bioeficácia do folato produzido. Esses resultados indicam que a produção de folato por bactérias láticas selecionadas em produtos fermentados pode ser uma boa alternativa para aumentar a ingestão de vitamina B9 pela população. / Folate deficiency is a public health problem that has been tackled through mandatory fortification of folic acid. However, excessive intake can cause unwanted side effects. The natural form of the vitamin produced by some strains of lactic acid bacteria (BAL) may be a safe alternative to increase folate intake by the population. The objective of this study was to select the best folate-producing BAL strains, to identify the best conditions for vitamin production and to evaluate the bioavailability of folate produced by the strains in fermented milk, using tests in animal models. The study was carried out with five strains of Streptococcus thermophilus (34v, 170v, 268v, 361v and 341 pc) and a strain of Lactobacillus plantarum (16 cv), all good folate producers. The conditions of production and the combination of strains that resulted in the best folate production were used to produce a biofermented milk (BFM), evaluating the bioavailability of the vitamin produced using a animal model of depletion/repletion with Balb/c mice submitted to 14 days of vitamin depletion, followed by 21 days of repletion. After 35 days, the animals were sacrificed, and blood, kidneys, liver, spleen and intestine were removed for quantification of folate and histological evaluation of the intestinal mucosa. All quantifications of folate were made by the microbiological method. The best combination of strains (St. thermophilus 34v + Lb. plantarum 16cv) resulted in the production of 300 ng / mL folate, the temperature being 42ºC better than 37ºC for vitamin production. The production of folate in milk in fermentation at controlled pH 6.0 was 35% higher than that observed in fermented milk without pH control. The villi/crypt ratio in the mice of the BFM group was the same as in animals receiving milk supplemented with folic acid. There was an increase in hemoglobin, hematocrit and red blood cells in the BFM group, evidencing the bioefficacy of the folate produced. These results indicate that folate production by selected lactic bacteria in fermented products may be a good alternative to increase vitamin B9 intake by the population.

Three effective solutions to dissolve the filter cake created by water-based drilling fluids weighted with Mn3O4 particles were developed. Hydrochloric acid at concentration lower than 5 wt% can dissolve most of Mn3O4-based filter cake. Dissolving the filter cake in two-stage treatment of enzyme and organic acid was effective and eliminated the associated drawbacks of using HCl. Finally, combining low and safe concentration of HCl with an organic acid in one-stage treatment was very effective. Hydrochloric acid (10-wt%) dissolved 78 wt% of Mn3O4-based filter cake at 250°F after 28 hours soaking time. However, Chlorine gas was detected during the reaction of 5 to 15-wt% HCl with Mn3O4 particles. At 190°F, 1- and 4-wt% HCl dissolved most Mn3O4 particles (up to 70-wt% solubility). Their reactions with Mn3O4 particles followed Eq. 8 at 190°F, which further confirmed the absence of chlorine gas production at HCl concentrations lower than 5-wt%. EDTA and DTPA at high pH (12) and acetic, propionic, butyric, and gluconic acids at low pH (3-5) showed very low solubilities of Mn3O4 particles. GLDA, citric, oxalic, and tartaric acids produced large amount of white precipitation upon the reactions with Mn3O4 particles. Similarly, DTPA will produce damaging material if used to dissolve Mn3O4-based filter cake in sandstone formation. At 4-wt% acid concentration, lactic, glycolic, and formic acids dissolved Mn3O4 particles up to 76 wt% solubility at 190°F. Malonic acid at lower concentration (2-wt%) dissolved 54 wt% of Mn3O4 particles at 190°F. Manganese tetraoxide particles were covered with polymeric material (starch), which significantly reduced the solubility of filter cake in organic acids. Therefore, there was a need to remove Mn3O4-based filter cake in two-stage treatment. Enzyme-A (10-wt%) and Precursor of lactic acid (12.5-wt%) dissolved 84 wt% of the filter cake. An innovative approach led to complete solubility of Mn3O4 particles when low and safe concentration of HCl (1-wt%) combined with 4-wt% lactic acid at 190°F. HCl (1-wt%) combined with lactic acid (4-wt%), dissolved 85 wt% of the Mn3O4-based filter cake after 18-22 hours soaking time at 250°F in one stage treatment.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(農学) / 甲第19046号 / 農博第2124号 / 新制||農||1032 / 31997 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 加納 健司, 教授 植田 充美 / 学位規則第4条第1項該当

D. Tech. Food Technology. / Discusses a range of lactobacilli and bifidobacteria were isolated from functional food products and pharmaceutical preparations and also obtained from culture collections. They were then subjected to phylogenetic analysis for accurate identification and classification and the probiotic properties of the organisms was evaluated. The isolates were then screened for inhibitory activity against a range of pathogenic bacteria and Candida albicans strains. Selected isolates that were found to have the necessary inhibitory and probiotic properties were recommended for inclusion in an envisaged synbiotic, maize-based beverage that would, in a subsequent study, be subjected to a nutritional intervention trial aimed at alleviating oral thrush in human patients. The specific objectives: to investigate and illustrate the superiority of rpoA and pheS gene sequencing compared to 16S rRNA gene sequencing in the identification and phylogenic assignment of Lactobacillus isolates ; to determine the precision of selected protein-coding gene sequencing in comparison with 16S rRNA gene sequencing for the discrimination and phylogenetic analysis of Bifidobacterium isolates ; to investigate the probiotic properties of selected bacterial strains in terms of antibacterial activity, anti-Candida activity, acid resistance, bile tolerance and antibiotic resistance ; to determine the potential of Lactobacillus isolates to inhibit the growth of each of seven Candida albicans strains in fermented maize gruel and to establish the factors contributing to Candida inhibition and to determine the antimicrobial and antioxidant activity of intracellular extracts and to elucidate compounds in methanol extracts from selected Lactobacillus strains.

Lactic acid bacteria produce pediocin-like bacteriocins designated as Class Ha. These antimicrobial peptides are antagonistic against Listeria monocytogenes and other closely related Gram-positive bacteria Self-protection of the producer organism is attributed to the immunity proteins, encoded by genes that are eo-transcribed with the structural gene that encode the bacteriocin. The lactic acid bacterium, Leuconostoc gelidum UAL 187-22 is immune to its own bacteriocin, leucocin A. This is accredited to its immunity protein and the possible absence of a receptor on its cytoplasmic membrane. Leucocin A was purified from the supernatant of 1. gelidum to 90% purity by ion-exhange chromatography and C18 reverse phase High Pressure Liquid Chromatography (RP-HPLC) eluted with an acetonitrile, 0.1% Triflouroacetic acid (TFA) gradient. The immunity gene was isolated from the same producer using the polymerase chain reaction from the recombinant plasmid pJF 5.5 using primers EAL-2 and EAL-3. The amplicon was truncated into versions A and B by removing the C- and N-terminals, with HaeIII and ClaI restriction enzymes, respectively. The amplicon and the truncated fragments A and B were cloned into pMALc2 to construct recombinant plasmids pKPl, pKPIA and pKPIB, correspondingly, which were transformed into Escherichia coli (E. coli) strain JMI03. Clones were confirmed by colony PCR and Southern blot hybridization. The recombinant clones were subsequently expressed as MBP-IP, MBP-IPA and MBP-IPB fusion proteins that were verified by Western blot using the anti-MBP antibody. Factor Xa protease was used to cleave MBP from the proteins of interest. The resulting pure immunity protein versions had an approximate molecular weight of slightly more that 10 kDa. The binding interactions of the purified immunity protein constructs and leucocin A were compared on the Biacore 2000 instrument with surface plasmon resonance. None of the immunity constructs interacted with leucocin A, however, the N-terminal region of the immunity protein interacted with the cytoplasmic extract. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

The blending of lignin as a component in a thermoplastic blend poses a challenge in the form of dispersion and compatibility. Polyesters such as poly(lactic acid) and poly(butylene adipate-co-terephthalate) offer the best opportunity of compatibility in melt blending with lignin due to their ability to form hydrogen bonds. The fractionation of lignin into more homogeneous fractions offers better dispersion and more consistent properties, retaining the toughness of the original polymer in addition to bridging stress transfer between PLA and PBAT. Functionalization of lignin was done by lactic acid grafting. The resulting blend of PLA/PBAT/modified fractionated lignin showed improved interaction between lignin and PLA, but reduced compatibility between lignin and PBAT. This thesis provides a deeper understanding on the effect of lignin heterogeneity, its fractions, and the functionalization of lignin on lignin and bioplastic blends to further the use of a largely produced industrial by-product in high value applications. / Natural Sciences and Engineering Research Council (NSERC) – Lignoworks Biomaterials and Chemicals Strategic Research Network, Canadian Foundation for Innovation (CFI), Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA)

To gain a better understanding of contaminating bacteria in bioethanol industry, we profiled the bacterial community structure in corn-based bioethanol fermentations and evaluated its correlation to environmental variables. Twenty-three batches of corn-mash sample were collected from six bioethanol facilities. The V4 region of the collective bacterial 16S rRNA genes was analyzed by Illumina Miseq sequencing to investigate the bacterial community structure. Non-metric multidimensional scaling (NMDS) ordination plots were constructed to visualize bacterial community structure groupings among different samples, as well as the effects of multiple environmental variables on community structure variation. Our results suggest that bacterial community structure is facility-specific, although there are two core bacterial phyla, Firmicutes and Proteobacteria. Feedstock, facility, and fermentation technology may explain the difference in community structure between different facilities. Lactic acid, the most important environmental variable that influences bacterial community structure grouping, could be utilized as an indicator of bacterial contamination. We also identified genes responsible for the multiple antibiotic-resistance phenotype of an Enterobacter cloacae strain isolated from a bioethanol fermentation facility. We performed PCR assays and revealed the presence of canonical genes encoding resistance to penicillin and erythromycin. However, a gene encoding resistance to virginiamycin was not detected.