Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2018-01-19T11:21:58Z No. of bitstreams: 1 texto completo.pdf: 2459203 bytes, checksum: bc78901467b027107f9d6f9397026427 (MD5) / Made available in DSpace on 2018-01-19T11:21:58Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2459203 bytes, checksum: bc78901467b027107f9d6f9397026427 (MD5) Previous issue date: 2017-07-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A antibioticoterapia é a forma de tratamento mais utilizada para o combate de doenças causadas por bactérias, tanto na medicina humana quanto na veterinária. Entretanto, o uso indiscriminado desses compostos favorece a seleção de bactérias resistentes. Uma alternativa no combate a estes patógenos é a utilização de óleos essenciais. Estes são compostos sintetizados pelas plantas a partir do metabolismo secundário e desempenham funções de atração de polinizadores e alelopatia. Neste trabalho, foi analisado o efeito antimicrobiano e antibiofilme de óleos essenciais contra isolados da bactéria Actinobacillus pleuropneumoniae, agente causal da pleuropneumonia suína. Essa doença é de grande importância na suinocultura devido às significativas perdas econômicas. Recentemente, foi observado que isolados desta bactéria apresentam genes de resistência a diversos antibióticos comerciais, tornando a prospecção e desenvolvimento de novos fármacos uma medida imediata para melhor controle da doença. Para isso, dezoito óleos essenciais foram utilizados para triagem de atividade antimicrobiana e antibiofilme em quatro isolados de A. pleuropneumoniae sorotipo 8 (MV518, MV780, MV1022 e MIDG2331) e em um isolado do sorotipo 1 (S1). Oito óleos essenciais apresentaram atividade antibacteriana, sendo, então, selecionados: canela, coentro, hortelã pimenta, hortelã do campo, tomilho, manjerona, eucalipto e louro. Estes óleos foram analisados por cromatografia gasosa para avaliar seus componentes. Os testes de concentração inibitória mínima (CIM) e de concentração bactericida mínima (CBM) foram realizados com os oito óleos ativos. Os valores de CIM e CBM foram idênticos para todos os óleos em todos os isolados, com valores variando de 5 mg/mL a 0,3125 mg/mL. O óleo de coentro (0,3125 mg/mL) e o de canela (0,625 mg/mL) apresentaram valores de CIM e CBM mais baixas para a maioria dos isolados testados, sendo considerados os mais efetivos. Adicionalmente, foram realizadas as análises de rompimento do biofilme pré-formado e da inibição do biofilme em formação. Seis óleos, na maior concentração (4xCIM), foram capazes de romper em mais de 30 % o biofilme pré-formado das bactérias analisadas, sendo que os óleos de canela e eucalipto em sua menor concentração analisada (1/8xCIM), foram capazes de romper o biofilme do isolado MV1022 em 21,29 % e 30,68 %, respectivamente. Na avaliação do biofilme em formação, cinco óleos foram capazes de inibi-lo em mais de 30 %. Entretanto, apesar do efeito no rompimento e formação do biofilme, os óleos de tomilho e coentro, dependendo da concentração utilizada, induziram o aumento na formação do biofilme. Esses resultados evidenciam a possível utilização destes óleos essenciais como sanitizantes e no combate a patógenos bacterianos. Assim, esses óleos se tornam uma possível alternativa no tratamento a doenças causadas por bactérias resistentes, a exemplo da A. pleuropneumoniae. / Antibiotic therapy is the most used form of treatment against diseases caused by bacteria, both in human and veterinary medicine. However, the indiscriminate use of these substances causes the selection of resistant bacteria. An alternative strategy of treatment against these pathogens is the utilization of essential oils. These are synthesized by aromatic plants from the secondary metabolism and perform functions of the attraction of pollinators and allelopathy. In this work, we analyzed the antimicrobial and antibiofilm effect of essential oils in isolates of Actinobacillus pleuropneumoniae, causative agent of swine pleuropneumonia. This disease is of great importance in swine farming due to significant economic losses. Recently, it was observed that isolates of this bacterium present resistance genes to several commercial antibiotics, which makes the need for prospecting and development of new drugs an immediate measure for better control of the disease. For this, eighteen essential oils were initially used for screening antimicrobial and antibiofilm activity in four isolates of A. pleuropneumoniae serotype 8 (MV518, MV780, MV1022 and MIDG2331) and a serotype 1 (S1) isolate. Eight essential oils showed antibacterial activity and were selected for future analyses: cinnamon, coriander, peppermint, mint, thyme, marjoram, eucalyptus, and laurel. These oils were analyzed by gas chromatography and presented antimicrobial components in their composition. The minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) tests were performed with the eight active oils. MIC and MBC values were identical for all oils in all isolates, with values ranging from 5 mg/mL to 0.3125 mg/mL. Coriander oil (0.3125 mg / mL) and cinnamon oil (0.625 mg / mL) showed lower MIC and MBC for most of the tested isolates, being considered the most effective oil. In addition, the analyses of rupture of the preformed biofilm and the inhibition of the biofilm in formation were performed. Six oils, in the highest concentration (4xMIC), were able to disrupt in more than 30% the preformed biofilm of the analyzed bacteria, being the oils of cinnamon and eucalyptus in its lowest concentration analyzed (1/8xCIM), were able to break the biofilm of isolate MV1022 in 21.29 % and 30.68 %, respectively. In the evaluation of the biofilm in formation, five oils were able to inhibit this biofilm in more than 30 %. However, despite the effect on biofilm breakdown and formation, the thyme and coriander oils, depending on the concentration used, induced increased biofilm formation. These results show the possible use of essential oils as sanitizers and in the treatment against bacterial pathogens. Thus, these oils become a possible alternative in the treatment of diseases caused by resistant bacteria, such as A. pleuropneumoniae.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-09-05T18:15:16Z No. of bitstreams: 1 texto completo.pdf: 693325 bytes, checksum: 2afbc494cbadf4704a75769d027d64d1 (MD5) / Made available in DSpace on 2016-09-05T18:15:16Z (GMT). No. of bitstreams: 1 texto completo.pdf: 693325 bytes, checksum: 2afbc494cbadf4704a75769d027d64d1 (MD5) Previous issue date: 2015-02-24 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Actinobacillus pleuropneumoniae é o agente etiológico da pleuropneumonia suína, uma severa enfermidade que acomete suínos de todas as idades, gerando perdas econômicas significativas para a suinocultura mundial. Embora os 15 sorotipos conhecidos dessa bactéria possam causar a doença, existem diferenças marcantes de virulência entre eles. A virulência de A. pleuropneumoniae é multifatorial e está relacionada à composição e estrutura de polissacarídeos da cápsula, LPS, e toxinas da família RTX, além desses fatores, a aderência em forma de biofilme e a resistência a agentes antimicrobianos podem ser determinantes para virulência. Este trabalho estabeleceu um modelo de infecção alternativo para o estudo de A. pleuropneumoniae, utilizando larvas de Galleria mellonella e, posteriormente esse modelo foi usado para investigar a virulência de isolados clínicos de A. pleuropneumoniae sorotipo 8. Os mesmos isolados foram avaliados quanto ao potencial de formação de biofilme e resistência a antimicrobianos comumente empregados em campo. A partir dessas informações, isolados clínicos com diferenças significativas na virulência, no potencial de formação de biofilme e no perfil de resistência foram selecionados para o sequenciamento genômico. Os resultados mostraram que o modelo de infecção A. pleuropneumoniae – G. mellonella é capaz de diferenciar níveis de virulência de isolados clínicos de mesmo sorotipo, além de permitir a avaliação da eficiência de agentes antimicrobianos contra este patógeno. O modelo também mostrou eficiência para diferenciar virulência entre linhagens selvagem e mutante da mesma bactéria. Uma análise de correlação entre os dados de virulência, formação de biofilme e resistência a antimicrobianos permitiu que seis isolados fossem selecionados para o sequenciamento. Com a montagem e anotação foi possível verificar que os genomas de A. pleuropneumoniae sorotipo 8 apresentam tamanho de 2,2 ± 0,004 Mpb, com o conteúdo GC de 40,33% ± 0,263 e regiões codificadoras com uma média de tamanho de 817,3 ± 6,8 pb. As regiões codificadoras correspondem a 89,05% ± 0,13 do genoma, das quais a maior parte foi anotada como genes funcionais, o que permitirá a realização de estudos comparativos. Estes genomas apresentam em média 79,5 ± 24,05 genes exclusivos, revelando a alta variabilidade genética dessa espécie, que pode estar relacionada com a variação da virulência entre os isolados estudados. / Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a severe disease that affects pigs of all ages, causing significant economic losses to the swine industry worldwide. Although the 15 serotypes of this bacterium are known to cause the disease, there are marked differences in virulence between them. A. pleuropneumoniae virulence is multifactorial and involves capsular polysaccharides, LPS, and toxins of the RTX family. In addition to these factors, the adhesion in biofilm form and resistance to antimicrobial agents may be determinant for virulence. This work has established an alternative infection model for the study of A. pleuropneumoniae, using larvae of Galleria mellonella and this model was subsequently used to investigate the virulence of clinical isolates of A. pleuropneumoniae serotype 8. The same isolates were evaluated for biofilm formation potential and resistance to antimicrobials commonly used in the field. From this information, clinical isolates with significant differences in virulence, biofilm formation potential and resistance profile were selected for genomic sequencing. Results show that the A. pleuropneumoniae - G. mellonella infection model is capable of differentiating levels of virulence of clinical isolates of the same serotype. Furthermore, it can be used to evaluate the effectiveness of antimicrobial agents against this pathogen. The model also showed efficiency to differentiate the virulence between wild and mutant strains of the same bacteria. A correlation analysis between the virulence data, biofilm formation and antibiotic resistance allowed six isolates to be selected for genome sequencing. With the assembly and annotation, we found that the genomes of A. pleuropneumoniae serotype 8 present size of 2.2 ± 0.004 Mpb, with GC content of 40.33% ± 0.263 and coding regions with an average size of 817.3 ± 6.8 bp. The coding regions correspond to 89.05 ± 0.13% of the genome, most of which was recorded as functional genes, enabling the comparative studies with these genomes. These genomes have 79.5 ± 24.05 exclusive proteins, revealing the high genetic variability of the species, which may be related to the variation in virulence between these isolates.

Aggregatibacter actinomycetemcomitans is a gram-negative, facultative anaerobic coccobacillus bacterium that colonizes the human oral cavity. Regarding the bacterial consortium found into biofilms, previous studies have suggested that the occurrence of particular serotypes of A. actinomycetemcomitans may be related to other oral microorganisms. Interestingly, the relations among these bacterial species seem to be under influence of geographic and ethnical aspects. Objective: This study aimed to evaluate the frequency of A. actinomycetemcomitans serotype-specific antigens and its association with Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and Campilobacter rectus in Brazilian subjects. Method: A wide group of subjects (n=1320) had their periodontal status determined and were screened for the subgingival presence of A. actinomycetemcomitans serotypes. Then, a total of 263 individuals A. actinomycetemcomitans positive for serotypes A, B, C and E were considered in this survey. Subsequently, in this same group of positive subjects, the presence of P. gingivalis, P. intermedia, T. forsythia and C. rectus from periodontal pockets were also evaluated by PCR. Result: Out of 263 subjects investigated, A. actinomycetemcomitans serotype A was detected in 28.5% subjects; serotype B in 15.97%; serotype C in 51.71% and serotype E in 3.80% subjects. The frequency of serotype A of A. actinomycetemcomitans was significantly higher in C. rectus positive subjects than in P. gingivalis, P. intermedia or T. forsythia positive subjects. Additionally, the frequency of serotypes B and C were significantly higher in both C. rectus and T. forsythia positive subjects in comparison to P. gingivalis and P. intermedia positive subjects. Meanwhile, the frequency of serotype E was not associated with the presence of the other periodontal pathogens investigated. Conclusion: We observed a positive association between A. actinomycetemcomitans serotypes A with C. rectus and between B and C serotypes with C. rectus and T. forsythia. / Aggregatibacter actinomycetemcomitans é uma bactéria do tipo cocobacilo anaeróbia facultativa gram-negativa que coloniza a cavidade oral humana. Levando em consideração as associações bacterianas encontradas no biofilme dentário, estudos prévios têm sugerido que a ocorrência de sorotipos específicos de A. actinomycetemcomitans pode estar relacionada à ocorrência de outros micro-organismos orais. Curiosamente, as relações entre essas espécies de bactérias parecem sofrer influência de aspectos geográficos e étnicos. Objetivo: Este estudo tem como objetivo avaliar a frequência de sorotipo-específicos de A. Actinomycetemcomitans e suas associações com Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia e Campilobacter rectus em indivíduos brasileiros. Materiais e Métodos: Um extenso grupo de indivíduos (n=1320) teve sua condição periodontal determinada e foram selecionados pela presença subgengival de algum sorotipo de A. actinomycetemcomitans. Sendo assim, um total de 263 indivíduos positivos para os sorotipos A, B, C e E foram considerados nessa pesquisa. Posteriormente, nesse mesmo grupo de indivíduos positivos, foram avaliadas, por PCR, a presença de P. gingivalis, P. intermedia, T. forsythia e C. rectus nas bolsas periodontais. Resultados: Dos 263 indivíduos investigados, o sorotipo A de A. actinomycetemcomitans foi detectado em 28,5% dos casos, o sorotipo B em 15,97%, o sorotipo C em 51,71% e o sorotipo E em 3,80% dos indivíduos. A frequência do sorotipo A de A. actinomycetencomitans foi significativamente maior em indivíduos positivos para C. rectus do que em indivíduos positivos para P. gingivalis, P. intermedia ou T. forsythia. Além disso, a frequência de sorotipos B e C foi significativamente maior tanto em indivíduos positivos para C. rectus quanto em T. forsythia quando comparados a indivíduos positivos para P. gingivalis e P. intermedia. Entretanto, a frequência do sorotipo E não foi associada com a presença de nenhum dos periodontopatógenos investigados. Conclusão: Observamos uma associação positiva entre o sorotipo A de A. actinomycetemcomitans com C. rectus e entre o sorotipo B e C com C. rectus e T. forsythia.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Aggregatibacter actinomycetemcomitans à um patÃgeno intensamente relacionado com a etiologia da periodontite agressiva. O objetivo deste estudo foi avaliar, via reaÃÃo em cadeia da polimerase, a presenÃa de clones de A. actinomycetemcomitans que exibem alta ou mÃnima leucotoxicidade em indivÃduos portadores de periodontite agressiva (PAG) e em seus membros familiares (FAM). Trinta e cinco indivÃduos com PAG (33,9  7,1 anos), 33 FAM (22,8  11,4 anos) e 41 portadores de periodontite crÃnica (PC) (44,1  9,4 anos) foram analisados clinicamente pelo Ãndice de placa (IP), Ãndice gengival (IG), profundidade de sondagem (PS) e nÃvel de inserÃÃo clÃnico (NIC). Amostras de biofilme subgengival foram coletadas de todos os indivÃduos do sÃtio interproximal com maior PS e maior NIC e analisadas microbiologicamente por meio de PCR, quanto à presenÃa de A. actinomycetemcomitans e de seus clones leucotÃxicos. MÃdias de PS e NIC desses sÃtios foram: PAG â 9,8 mm e 10,7 mm, FAM â 5,3 mm e 4,6 mm e PC â 8,2 mm e 9,4 mm, tendo sido observadas diferenÃas estatisticamente significantes entre os grupos. A presenÃa de A. actinomycetemcomitans foi observada em 23 (51,1%) portadores de periodontite agressiva e em 16 (30,1%) portadores de periodontite crÃnica. Dentre eles, 37 (94,8%) exibiram clones de mÃnima leucotoxicidade e 2 (5,1%) clones de alta leucotoxicidade, um do grupo PAG e outro do grupo FAM, tambÃm com doenÃa agressiva. Quanto à condiÃÃo periodontal do grupo FAM, 30,3% apresentaram periodontite agressiva, 36,3% periodontite crÃnica e 33,3% estavam periodontalmente saudÃveis ou mostravam gengivite. Dentre eles, 12,2% estavam positivos para a presenÃa de A. actinomycetemcomitans. Maiores valores de PS e NIC foram observados nos pacientes com periodontite agressiva positivos para A. actinomycetemcomitans quando comparados Ãqueles pacientes negativos, com diferenÃa estatisticamente significante. A presenÃa de A. actinomycetemcomitans foi associada à condiÃÃo clinica de periodontite agressiva (odds ratio=3,1; intervalo de confianÃa: 1,4-7,0; p=0,009). A maioria destes indivÃduos exibiu uma forma generalizada de doenÃa e estavam positivos para clones de mÃnima leucotoxicidade de A. actinomycetemcomitans. Os familiares externaram em sua maioria algum tipo de doenÃa periodontal crÃnica ou agressiva. / Aggregatibacter actinomycetemcomitans is a pathogen strongly associated with the etiology of aggressive periodontitis. The purpose of this study was to evaluate by polymerase chain reaction (PCR) the presence of highly and minimally leukotoxic clones of A. actinomycetemcomitans in patients with aggressive periodontitis (AgP) and their family members (FM). Thirty five patients with AgP (33,9  7,1 years), 33 FM (22,8  11,4 years) and 41 patients with chronic periodontitis (CP) (44,1  9,4 years) were clinical analyzed through plaque index (PI), gingival index (GI), probing depth (PD) and clinical attachment level (CAL). Subgingival biofilm samples were collected from the interproximal periodontal sites (> PD and > CAL) of each patient and their microbiological analysis were taken by PCR, concerning A. actinomycetemcomitans was observed and its leukotoxic clones. The PD and CAL average of this sites were: AgP â 9,8 mm e 10,7 mm, FM â 5,3 mm e 4,6 mm e CP â 8,2 mm e 9,4 mm. Statistically significant difference was observed among the groups. The presence of A. actinomycetemcomitans was observed in 23 (51,1%) patients with aggressive periodontitis and in others ones, 16 (30,1%) with chronic periodontitis. Among them 37 (94,8%) showed minimally leukotoxic clones and 2 (5,1%) highly leukotoxic clone, one was AgP group, the other was FM group, also with aggressive disease. Concerning to periodontal status of the FM group, 30,3% had aggressive periodontitis, 36,3% chronic periodontitis and 33,3% were periodontally normal or had gingivitis. Among them 12,2% were A. actinomycetemcomitans positive. The higher values of PD and CAL were observed in aggressive periodontitis patients that were A. actinomycetemcomitans positive when compared with A. actinomycetemcomitans negative patients (p<0.05). The presence of A. actinomycetemcomitans was correlated with aggressive periodontitis clinical status (Odds ratio=3,1; CI: 1,4-7,0; p=0,009). The majority of the patients with aggressive periodontitis exhibited a generalized form of disease and were positives to minimally leukotoxic A. actinomycetemcomitans clones. The family members had any type of chronic or aggressive periodontal disease.

Please read the abstract in the section front of this document. / Dissertation (MEng)--University of Pretoria, 2012. / Chemical Engineering / unrestricted

Actinobacillus succinogenes cells were grown on Poraver® support particles in a packed-bed reactor. Dilution rates (D) of 0.054–0.72 h-1 were investigated. Glucose was used as substrate. CO2 (g) was bubbled into a complex medium to satisfy the fixation requirements and maintain anaerobic conditions. At D ≥ 0.31 h-1, an initial glucose concentration of 35 g.L-1 was used; at lower dilution rates, this was increased to 60 g.L-1 in order to avoid substrate limitations. By-product formation included acetic and formic acids. A maximum productivity of 10.7 g.L-1 was obtained at D = 0.7 h-1. It was found that the system provided repeatable results at a given D. The longest steady state period was maintained for about 97 h at D = 0.31 h-1. Steady state stability was maintained for > 72 h at D < 0.31 h-1. For periods longer than 75 h, however, inhibitory acid titres resulted in a gradual decline in productivity. At higher dilution rates, long-term stability could not be maintained. The low acid titres produced significant biofilm sloughing following aggressive biofilm growth, resulting in oscillatory system behaviour. For fermentation times < 115 h, the dilution rate was secondary to the attachment area in determining the total biomass at steady state. Total biomass values were then used to determine specific rates. A clear trend was observed, with the specific glucose consumption rate, and specific acid production rates, increasing with increasing D. This was explained by assuming a maintenance-driven system at all Ds studied. A product analysis indicated that at ΔS < 15 g.L-1, pyruvate formate lyase was the preferred oxidative route. A shift to the pyruvate dehydrogenase pathway occurred at higher ΔS values, so that the highest YSS values obtained exceeded 0.85 g.g-1. A decrease in C3 by-product formation resulted in high YSS values being maintained, indicating an additional, unknown source of nicotinamide adenine dinucleotide (NADH). It is recommended that any process utilising immobilised A. succinogenes cells should operate at an intermediate D, in order to maintain long-term reactor stability, high productivities and good yields. / Dissertation (MEng)--University of Pretoria, 2013. / gm2014 / Chemical Engineering / unrestricted

Continuous fermentations were performed in a novel external-recycle, biofilm reactor using D-glucose and CO2 as carbon substrates. Corn steep liquor (CSL) and yeast extract (YE) served as nitrogen sources. In anaerobic fermentations using medium containing CSL and YE, succinic acid (SA) yields were found to be an increasing function of glucose consumption. The ratio of SA to the major by-product, acetic acid (YAASA), increased from 2.4 g g-1 at a glucose consumption of 15 g L-1, to 5.7 g g-1 at a glucose consumption of 46 g L-1. For medium containing no CSL, YAASA remained near 1.97 g g-1, exceeding this for cases where biofilm grown on CSL-containing medium was present. The ratio of formic acid to acetic acid (YAAFA), for CSL-containing medium, decreased from an equimolar value (0.77 g g-1) at a glucose consumption of 10 g L-1 to zero at 46 g L-1 glucose consumed. In contrast, YAAFA for YE-only medium remained at 0.77 g g-1. Therefore, pyruvate was metabolised solely by pyruvate-formate lyase when no CSL was present. The highest SA yield obtained on glucose, SA titre and SA productivity were 0.91 g g-1, 48.5 g L-1 and 9.4 g L-1 h-1, respectively, all for medium containing CSL. Medium that included CSL significantly outperformed medium that excluded CSL, achieving 64%, 21% and 203% greater SA titres, yields on glucose and productivities respectively. Metabolic flux analyses based on the established C3 and C4 metabolic pathways of Actinobacillus succinogenes revealed that the increase in YAASA, for CSL-containing fermentations, could not be attributed to the decrease in formate and biomass formation, and that an additional source of reducing power was present. The fraction of reducing power (NADH) unaccounted for increased with glucose consumption, suggesting that the maintenance or non-growth metabolism encountered at higher SA titres differs from the growth metabolism. It is postulated that the additional reducing power originates from an active pentose phosphate pathway in non-growing cells or from an undetected component(s) in the fermentation medium. No major metabolic flux variations were found in fermentations that excluded CSL. / Dissertation (MEng)--University of Pretoria, 2013. / gm2014 / Chemical Engineering / unrestricted

Biofilms of Actinobacillus succinogenes, grown in a biofilm reactor system, were investigated for structure and cell viability, through microscopic visualisation with a confocal scanning laser microscope (CSLM) and a scanning electron microscope (SEM). Biofilms were sampled and visualised at steady state conditions with the broth containing succinic acid titres between 15 and 21 g/L. All sampled biofilm was 6 days old. Six-day-old biofilms of A. succinogenes showed a heterogeneous biofilm architecture composed of cell micro-colony pillars which varied considerably in thickness, area and shape. Microcolony pillars consisted of a densely packed entanglement of sessile cells. Quantitative analysis revealed that the pillars were mostly large, with a mean pillar diameter of 170 m and a mean thickness of 92 m, although pillar diameter and thickness were variable as they ranged from 25 – 500 m and 30 – 300 m, respectively. In the regions close to the substratum surface, pillars were characterised by having defined borders with a network of channels ranging from 40 – 200 m in width separating them. However, towards the middle of the biofilm depth some of the pillars coalesced. For this reason low cross sectional area coverage of biofilm consistently occurred at the bottom portion of the biofilm whilst the highest coverage was in the middle portion of the biofilm. Regarding cell morphology, very large differences were observed. Planktonic cells were rod-shaped, whereas sessile cells expressed an elongated rod morphology and thus were much longer and thinner compared with planktonic cells. Planktonic cells were 1 – 2 m thick and 4 – 5 m long, while sessile cells were 0.5 – 1 m thick and 5 – 100 m long. Long sessile cells resulted in extensive tangling in microcolony pillars, which may have contributed to the structural stability of the pillars. Fibre-like connections of constant diameter were observed between cells, and between the cells and surface. The diameter of these connections was approximately 20 – 30 nm. Viability stains showed that in the bottom portion (from 0 - 20 m above the substratum surface) of the biofilm, most of the cells were dead. However, the portion of covered area attributed to living cells increased past the middle of the biofilm towards the top part of the biofilm. A high percentage of living cells was thus found towards the top part of the biofilm. Overall, 65% (with 2% standard deviation) of the entire biofilm was composed of dead cells. In this way, the results show that operation at high acid conditions comes at a cost of low overall biomass productivity due to decreased active biomass. / Dissertation (MEng)--University of Pretoria, 2017. / Chemical Engineering / MEng / Unrestricted

Biofilms of the bovine rumen bacterium Actinobacillus succinogenes have demonstrated their exceptional capabilities as biocatalysts for high productivity, titre and yield production of succinic acid (SA). Succinic acid is set to become a significant building block chemical in the biobased economy. Although substantial progress has been made towards understanding the productive aspect of this microorganism with regard to its metabolic limits and performance on unrefined biorefinery stream substrates, more research is still required to address other challenges. One aspect is to understand how the biofilm biocatalyst is affected by bioreactor conditions, which would help in developing stable and highly active biofilms. For this reason the aim of this thesis was (i) to characterise how the accumulation of acid metabolites in continuous operation impacts A. succinogenes biofilms with respect to biofilm development, biofilm structure and cell activity within the biofilm, (ii) to show how shear conditions in the fermenter can be used to manipulate the biofilm structure and viable cell content of biofilms, leading to improved cell-based succinic acid productivities, and lastly (iii) to investigate the internal mass transfer effects on biofilm performance, further showing the role played by differences in shear and acid accumulation conditions in this respect. The first part of the study addressed the interaction between the biofilm and the accumulation of metabolites produced. The results showed that biofilms of A. succinogenes develop rapidly and with high activity when cultivated under low product accumulation (LPA) conditions (< 10 g L-1 SA). High product accumulation (HPA) conditions considerably slowed down biofilm development, and increased cell mortality. Under HPA conditions some cells exhibited severe elongation while maintaining a cross-sectional diameter like the rod/cocci-shaped cells predominantly found in LPA conditions. The elongated cells formed in HPA conditions were found to be more viable and thus more resistant than the clusters of rod-shaped or cocci-shaped cells. The global microscopic structure of the HPA biofilms also differed significantly from that of the LPA biofilms. Although both exhibited shedding after 4 days of growth, the LPA biofilms were more homogenous (less patchy), thicker and had high viability throughout the biofilm depth. In the second part of the study, two custom-designed bioreactors were used to evaluate the effect of shear on the biofilms. The first bioreactor allowed for in situ removal of small biofilm samples used for microscopic imaging. The second bioreactor allowed for complete removal of all biofilm and was used to analyse biofilm composition and productivity. Results clearly indicated that high shear biofilm cultivation in LPA conditions has beneficial morphological, viability and cell-based productivity characteristics. The smooth, low-porosity biofilms obtained under high shear and LPA conditions had an average cell viability of 79% (over a 3-day cultivation period) compared with the low shear value of 57%, also developed under LPA conditions. The EPS content of the high shear biofilm was 58% compared with 7% of the low shear equivalent. The cell-based (EPS excluded) succinic acid productivity for the high shear biofilm was 2.4 g g-1DCW h-1 compared with the 0.8 g g-1DCW h-1 for the low shear biofilm. This threefold increase in productivity obtained from the second bioreactor corresponded to the cell viability differences obtained from the first bioreactor. Clear evidence was provided for shear-induced shaping of the biofilm which resulted in improved volumetric glucose turnover attributes within the biofilm matrix. The last section of the study investigated internal mass transfer effects in biofilm fermentations of Actinobacillus succinogenes by performing batch fermentations using attached and resuspended biofilms as biocatalysts. In the latter, the biofilms were resuspended after initial development to simulate mass transfer-free fermentations. Intrinsic kinetics for succinic acid production obtained from resuspended fermentations predicted faster production rates than for the attached biofilm runs (biofilm thicknesses in the range of 120–200 µm), indicating internal mass transfer limitations. A developed biofilm reaction diffusion model gave good prediction of attached biofilm batch operation results by accounting for internal mass transfer in the biofilm. Biofilm effectiveness factors ranged from 75% to 97% for all batches at the inception of batch conditions, but increased with the progression of batch operation due to the increased succinic acid titres which inhibited the production rates. Analysis of pseudo-steady-state continuous fermentation data from the literature, as well as from the second part of the study, using the model developed, showed that active biofilm thickness and effectiveness factors were dependent on the shear conditions and succinic acid titres in the biofilm reactors. A simplified algorithm was developed to estimate the pseudo-steady-state glucose penetration and biofilm effectiveness of A. succinogenes biofilms without the requirement to solve the overall mass transfer model. The results clearly showed that internal mass transfer needs to be considered in biofilm fermentations involving A. succinogenes as high biomass concentrations may not always equate to increased productivities if mass transfer effects dominate. / Thesis (PhD)--University of Pretoria, 2020. / NRF / Chemical Engineering / PhD / Unrestricted

Dans ce projet, la bactérie du rumen des bovins Actinobacillus succinogenes souche 130Z est utilisée pour fermenter le lactose et le lactosérum pour en produire de l'acide succinique qui a des larges applications industrielles. Le but de ce travail est de déterminer les conditions optimales de fermentation afin de produire l'acide succinique à des grandes concentrations et avec des bonnes valeurs de rendement et de productivité. Les paramètres à optimiser en mode de fermentation batch sont les concentrations initiales du lactose (25, 50 et 75 g/1) et d'extrait de levure (7,5, 10 g/1), et le taux de C02 (0.16, 0.32 et 0.80 vvm) injecté dans le milieu de fermentation. Par la suite, produire l'acide succinique dans ces conditions optimales en mode fed batch à partir du lactose et du lactosérum. En mode de fermentation batch, les résultats obtenus montrent que les concentrations optimales d'extrait de levure et du lactose sont respectivement 7.5 et 25 g/1 et le taux de COT le plus adéquat pour la production de l'acide succinique est 0.32 wm. Tout d'abord, il n'y a pas de différences pour les deux concentrations de levure étudiées. En ce qui concerne la concentration initiale du lactose, et pour les différents taux de CO2 étudiés, les valeurs de rendement et de productivité obtenues pour une concentration initiale du lactose égale à 25 g/1 sont supérieures à celles de 50 et 75 g/1. En effet, des concentrations initiales de lactose supérieures à 25 g/1 exercent une inhibition sur la croissance bactérienne et par conséquent la consommation du lactose et la production de l'acide succinique. La concentration maximale d'acide succinique obtenue à partir de 25 g/1 du lactose, 7.5 g/1 d'extrait de levure et 0.32 vvm de CO2 est égale à 18.7 g/1, les valeurs de rendement et de productivité sont respectivement 76% et 1.52 g/l/h. Dans les mêmes conditions que la fermentation du lactose, la fermentation batch du lactosérum (concentration initiale 33.5 g/1) a donné 13.86 g/1 d'acide succinique, un rendement de 61% et une productivité de 0,97 g/l/h. Les valeurs des rapports massiques AS/AA et AS/AF (2.43 et 3.96 respectivement) obtenus au cours de la fermentation batch du lactosérum sont inférieurs à ceux obtenus dans le cas du lactose (8.47 et 14.6 respectivement). Ceci montre que la fermentation du lactosérum favorise le développement de la biomasse, et la production des sous-produits acides de la fermentation (acides acétique et formique) par rapport à la fermentation du lactose. Les résultats de la fermentation fed batch du lactose et du lactosérum montrent que l'acide succinique a pu être produit à des grandes concentrations (environ 55 et 50.5 g/1 respectivement) comparativement à la fermentation batch. Dans le cas du lactosérum, la fermentation fed batch a permis une augmentation de rendement et de la productivité (67% et 1.28 g/l/h à 24h). Mais dans le cas du lactose, les valeurs de rendement et de la productivité sont similaires à celle de la fermentation batch (70% et 1.52g/l/h à 24h). Comme dans le cas de la fermentation batch, plus de biomasse et de sous-produits de fermentation sont produits en fermentation fed batch du lactosérum par rapport à ceux du lactose. Les différents acides organiques (acide succinique, acétique et formique) testés seuls ou en combinaison exercent une inhibition de la croissance bactérienne contre les deux souches E. coli et L. ivanovii. À faible concentration C4 (1.1, 0.8 et 0.35 mg/ml d'acide succinique, acétique et formique respectivement), le mélange (AS+AA+AF) a permis d'inhiber efficacement la croissance bactérienne par rapport aux associations deux à deux (AS+AA, AS+AF et AA+AF). Le mélange d'acides a permis 73 et 92 % d'inhibition à 12h pour E. coli et L. ivanovii respectivement. Ceci permet de conclure qu'il ya un effet synergique entre les trois acides organiques testés.