For a sustainable future, there is a call to increase the market share of bio-based technologies and materials. Microbial-based technologies have the potential and the ability to contribute substantively on many levels to global efforts to achieve sustainability. Development and utilization of microbial technologies is, however, an extensive process involving numerous steps, including the discovery of novel technologies and the development of industrially viable production systems. In the presented thesis, individual steps of microbial biotechnology development were addressed. In the first part of the study, a variety of methodological approaches were employed in order to study the effect of the anthropogenic activity (i.e., decades lasting production of penicillin G) on the structure of soil microbial communities. Moreover, both cultivable and non-cultivable fractions of populations were subjected to functional screening in order to unravel the biotechnological potential of the microorganisms in terms of production of enzymes involved in biotransformation of beta-lactam antibiotics: penicillin G acylase (PGA) and alpha amino acid ester hydrolase (AEH). Our results indicated that the impacted communities harbour a microbial community with increased diversity and richness. However, on the...

Orientador: Lucia Regina Durrant / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-11T09:19:17Z (GMT). No. of bitstreams: 1 Silva_IsisSerrano_D.pdf: 2225247 bytes, checksum: 9106b26119b1fee141fe2a577f21f1e9 (MD5) Previous issue date: 2007 / Resumo: A biodegradação de poluentes de petróleo em um solo contaminado foi acompanhada neste estudo, avaliando o comportamento da microbiota do solo durante a utilização de hidrocarbonetos policíclicos aromáticos (HPAs) como fontes de carbono. Através da biomassa microbiana, da taxa de respiração no solo, bem como do quociente metabólico (eficiência em degradar os compostos recalcitrantes em questão), foi possível uma avaliação do impacto na microbiota nativa do solo contaminado e nos diferentes microcosmos bioaumentados com os consórcios de bactérias, fungos, e uma mistura destes consórcios por 12 semanas. Tanto a microbiota nativa quanto os solos bioaumentados demonstraram uma rápida resposta à adaptação neste ambiente contaminado pelo aumento da biomassa e das taxas metabólicas. Durante o período de biodegradação dos HPAs, valores de evolução de CO2 foram diminuindo e a biomassa se manteve em crescimento estável, indicando menos gasto de energia para os microrganismos sobreviverem neste solo impactado, como resposta à boa competitividade e eficiência da microbiota nativa e bioaumentada. A biodegradação dos HPAs e a presença de metabólitos intermediários foram também avaliados, apresentando uma rápida redução das concentrações dos HPAs de baixo peso molecular (menores que 4 anéis aromáticos) em comparação com os de alto peso molecular, devido à sua biodisponibilidade e alta atividade microbiana de degradação. Todavia, a bioaumentação não demonstrou ser melhor que a microbiota nativa na degradação dos HPAs. É provável que o mecanismo de cooperação metabólica por co-metabolismo tenha sido realizado pela comunidade do solo, uma vez que vários HPAs complexos foram degradados. Diversos estudos apontam a presença de HPAs de menor peso molecular e seus respectivos produtos como indutores da degradação dos HPAs de maior peso molecular. Os metabólitos intermediários produzidos foram assimilados pelos microrganismos em processo cooperativo no solo, sendo responsáveis pela indução de enzimas e suas respectivas vias degradativas. Os distúrbios causados no solo pela poluição com HPAs normalmente estimulam o crescimento de microrganismos capazes de sobreviver nestes compostos, causando mudanças na estrutura microbiana do solo devido às suas adaptações nos processos co-metabólicos para a manutenção da comunidade. Neste trabalho, um solo impactado com HPAs, estocado por vários anos sob refrigeração foi analisado quanto à habilidade da microbiota nativa em crescer em alguns HPAs, individualmente, ou ainda em mistura complexa. Perfis moleculares de microbiota foram observados pela técnica de PCR-DGGE utilizando fragmentos do gene RNA ribossômico 16S durante 4 semanas. O número de bandas observadas foi interpretado como os membros dominantes na comunidade, e os diferentes perfis mostraram diferentes dinâmicas de degradação dos HPAs em meio contendo ou não fatores essenciais de crescimento (micronutrientes e vitaminas). Espécies ativas metabolicamente mostraram-se predominantes na interação com a comunidade e na cooperação catabólica. Após enriquecimento do solo original por 6 meses, apenas duas bandas foram visulizadas em gel, correspondendo à duas colônias morfologicamente diferentes isoladas em meio ágar, sendo identificadas como sendo do gênero Pseudomona, mais provavelmente Pseudomonas stutzeri (98% de similaridade). Esta espécie possui alta capacidade de transformação natural no solo, gerando mutantes. Diferenças genéticas entre as colônias de P. stutzeri foram confirmadas por PFGE, as quais apresentaram bandas para os genes catabólicos nahA-dioxigenase, catecol-1,2 e 2,3-dioxigenases, responsáveis por codificar as respectivas enzimas atuantes nas principais vias metabólicas de degradação dos HPAs / Abstract: The biodegradation of petroleum derivatives in a contaminated soil was evaluated in this study monitoring the behavior of the soil microbiota when using polycyclic aromatic hydrocarbons (PAHs) as carbon sources. Analyzing C-biomass, respiration rates (CO2 evolution) and metabolic quotient (which means the efficiency in degrading PAHs), it was possible to evaluate the impact of the contaminated soil in the native microbiota and soil bioaugmented with bacterial and fungal consortia or a mixture of these consortia for 12 weeks in microcosms. Even the native microbiota or the bioaugmented soils performed a rapid response regarding to their adaptation into contaminated environment due to the increase of biomass and respiration rates. During the PAH biodegradation period, CO2 evolution values remained steady, indicating less loss of energy to the survival microorganism into the impacted soil, a good competitiveness, and also an efficiency of both native and bioaugmented populations. The biodegradation of PAHs and the production of metabolic intermediates were assessed, and low-molecular-weight (LMW) PAHs (less than 4 rings) had fast reduction in their concentrations comparing with the high-molecular-weight (HMW) PAHs, probably due to their bioavailability and the high microbial activity. Bioaugmentations were not better than native microbiota in the PAHs degradation performances, and it is believed that the cooperative mechanism under co-metabolism could be responsible for the degradation of HMW-PAHs. Metabolic intermediates were assimilated by microorganisms in a cooperative process, inducing some key-catabolic pathways enzymes. The pollution of soil by PAHs could stimulate the growth of organisms capable of surviving in the presence of these compounds, changing the soil microbial structure due a catabolic adaptation process. In this study, another PAH-impacted soil, collected from a manufacturing gas plant and stored for several years was analyzed regarding to the remained ability of the native microbiota to grow on individual PAHs or on its mixtures. Molecular profiles of the microbial community were observed using PCR-DGGE of the 16S rDNA fragment for 4 weeks. The number of bands was interpretated as dominant members into the community, and differences in profiles showed different PAH degradation dynamics in mineral medium with or without micronutrients and vitamins. Catabolically activated species were predominant in the community, and after several enrichment steps for 6 months, only two bands were observed in DGGE, corresponding to two colonies showing morphological differences, identified as Pseudomonas genus, very close to Pseudomonas stutzeri (98% of similarity). This specie has high capacity of natural transformation in soil, generating some mutants. Genetical differences were found between colonies using PFGE, and the presence of catabolic genes as nahA-dioxygenase, cathecol-1,2- and 2,3- dioxygenases were confirmed by PCR products in agarose gel electrophoreses / Doutorado / Doutor em Ciência de Alimentos

Thesis (PhD (Chemical Engineering))--Cape Peninsula University of Technology, 2019 / The agro-processing industry is currently facing losses due to microbial spoilage of agricultural produce and associated value-added products such as beverages. Decay and undesired fermentation of fruit and beverages by fungal, yeast and bacterial spoilage organisms are among the major contributors of product losses in the food industry. When looking at the different level of food spoilage, it is common to find different spoilage organisms occurring in the same food item; which usually requires food producers to utilise a mixture of synthetic preservatives for spoilage organism control. Some of the synthetic chemical compounds with growth inhibition properties that have been used in food preservation are sulphur dioxide, benzoic, lactic, sorbic and acetic acid. These compounds act against a variety of spoilage microorganisms. In post-harvest control of fungi, triazoles, hydroanilide fenhexamid, dicarboximides and succinate dehydrogenase are also being used. Some spoilage organisms have been found to be resistant to the use of synthetic chemical preservatives which usually favour the use of higher dosage of preservatives in food. The use of synthetic chemicals as preservative and as postharvest control agents has been found to present serious health risks such as cardiovascular diseases, muscles and stomach pains, eyesight and skin damages and impairment of brain functions. The problem posed by the current use of synthetic chemicals in food put pressure on food producers and exporters to seek alternatives that will allow for the eradication of the use of synthetic chemicals as preservative in beverages and as postharvest control agents on fruits. Yeasts have been found to have the ability to grow at a faster rate on cheap media and to colonise dried surfaces rapidly. It has also been found that yeasts produce extracellular compounds of proteinaceous and volatile organic nature with growth inhibition properties against spoilage organisms. The current findings lack some engineering concept that could assist in the design of a production system for high scale production of biopreservation compounds from yeasts. The availability of a cost effective production media, the growth and production kinetics data using a cheaply available nutrient sources as well as the biological thermodynamic data are some of the gaps in biopreservation bioprospecting. Although several yeasts have already been studied to have great inhibition properties against fruit fungal pathogens, it was still unclear what was the minimum inoculum dose to be able to have a fungistatic and fungicidal effect on the growth of fruit spoilage organisms. The concept of combination of biopreservatives and the interaction effect of their biopreservation activity against consortia of spoilage organisms has also been lacking. As an attempt to seek alternatives to the use of synthetic chemicals as preservatives or postharvest control agents, Candida pyralidae Y1117, Pichia kluyveri Y1125 and Pichia kluyveri Y1164 strains were assessed for antimicrobial activity against spoilage yeasts (Dekkera bruxellensis, Dekkera anomala, Zygosaccharomyces bailii) and spoilage fungi (Botrytis cinerea, Colletotrichum acutatum and Rhizopus stolonifer). As alternative to refined media, a cost effective approach was explored whereby the use of agro-waste, i.e. grape pomace extracts (GPE), as production medium for biopreservation compounds, was studied. Production kinetics using modified existing models, subsequent to optimization using response surface methodology (RSM) for biopreservation compounds production was studied for the three biocontrol yeasts using GPE broth as the fermentation medium. The evaluation of the interaction study between mixtures of crude biopreservatives against consortia of common spoilage organisms present in beverages was also conducted by producing the crude biopreservation compounds separately from yeasts and then formulating growth inhibition combinations (GICs); GIC 1 (Candida pyralidae Y1117 and Pichia kluyveri Y1125); GIC 2 (C. pyralidae Y1117 and P. kluyveri Y1164), GIC 3 (P. kluyveri Y1125 and Pichia kluyveri Y1164); GIC 4 (C. pyralidae, P. kluyveri Y1125 and P. kluyveri Y1164). The spoilage organism consortia combinations, i.e. SC1, D. anomala and D. bruxellensis; SC2 (D. anomala and Z. bailii); SC3 (D. bruxellensis and Z. bailii) and SC4 (D. anomala, D. bruxellensis and Z. bailii) were also prepared. This study also investigated the effect of varying inoculum dose (ID) of Candida pyralidae strain Y1117, Pichia kluyveri Y1125 and Pichia kluyveri Y1164 on the biocontrol of Botrytis cinerea by contaminating the headspace of the growth medium with a fungal plug subsequent to biotreatment with different initial inoculum dose of the respective biocontrol yeasts. Finally, grape pomace extracts was used as fermentation medium to study the biological thermodynamics of biopreservation compound production from the three biocontrol yeasts. The results obtained demonstrated some interesting results. The antagonistic properties of C. pyralidae and P. kluyveri were observed on cheap solidified medium (grape pomace extracts) as well as on fruits (grapes and apples). These yeasts produced extracellular volatile organic compounds (VOCs) that could be responsible for yeast and fungal growth inhibition. Twenty-five VOCs in the category of alcohols, organic acids and esters were identified by GC-MS. The results of the kinetic study showed that the highest volumetric zone of inhibition (VZI) was 1.24 L contaminated solidified media (CSM) per mL biopreservation compounds used (BCU) when Candida pyralidae Y1117 was inoculated in a pH 3-diluted GPE broth (150 g L−1) incubated at 25 °C for 24 h. Similar conditions were applied for Pichia kluyveri Y1125 and P. kluyveri Y1164, albeit under slightly elongated fermentation periods (up to 28 h), prior to the attainment of a maximum VZI of only 0.72 and 0.76 L CSM mL−1 ACU, respectively. The potential biopreservation compounds produced were identified to be isoamyl acetate, isoamyl alcohol, 2-phenyl ethylacetate and 2-phenyl ethanol. The growth inhibition interaction study showed a variation in growth inhibition proficiency depending on the spoilage organisms or the consortia of spoilage organisms being deactivated. It was then suggested that, a food environment contaminated with a consortium of spoilage organisms can be controlled by employing either the crude biopreservation compounds from individual yeast or those of the following yeast combinations, GIC1-4, which showed a better growth inhibition proficiency against SC1-3. The fungistatic and fungicidal effects on the fungal pathogen were dose dependent. The fungistatic characteristics against Botrytis cinerea were displayed after 7 days when 102-105 cells mL-1 of Candida pyralidae Y1117, Pichia kluyveri Y1125 and Pichia kluyveri Y1164 were independently used in-vitro and in-vivo. However, 106-108 cells mL-1 inoculum doses displayed fungicidal characteristics. Additionally, the fungicidal property of yeasts studied was also confirmed on table grape (in vivo studies) using closed jar method. The biological thermodynamic study showed that, dried biomass molecular weight of 28.9 g/C-mol, 29.163 g/C-mol, and 27.176 g/C-mol were obtained for Candida pyralidae strain Y1117, Pichia kluyveri Y1125 and Pichia kluyveri Y1164 respectively. The results obtained successfully established useful biological thermodynamic data applicable to the design of adequate biopreservatives production system from yeasts using cheaply available nutrients source.

This thesis examined the use of two-phase partitioning bioreactors (TPPBs) for the biodegradation of poorly water-soluble compounds. TPPBs are stirred tank bioreactors composed of a biocatalyst-containing aqueous phase and an immiscible second phase containing large amounts of poorly water-soluble or toxic substrates. Degradation of the bioavailable substrate in the aqueous phase will result in equilibrium-driven partitioning of additional substrate from the immiscible phase into the aqueous phase, theoretically allowing for complete substrate degradation. Fundamental work was undertaken with the PCB-degrading organisms Burkholderia xenovorans LB400 in liquid-liquid and solid-liquid TPPBs. Initially biphenyl was used as the sole carbon source due to its hydrophobic nature and structural similarity to the environmentally relevant PCBs. The critical LogKO/W (octanol/water partitioning coefficient) of the organism was determined to be 5.5 and its growth kinetics on biphenyl were determined in a liquid-liquid TPPB. A polymer selection strategy for solid-liquid TPPBs was developed in the next chapter, and it was shown in the following chapter that biphenyl degradation in solid-liquid TPPBs was mass transfer limited, as described mathematically utilising the previously estimated microbial kinetics. The fundamental knowledge gained in the early chapters was then applied to the degradation of PCBs by the same organism. It was shown that the aqueous phase availability of PCBs is the rate-limiting step in biphasic bioreactors, and not the mass transfer rate. The low specific microbial degradation rates, resulting from substrate-limited growth were addressed with increased biomass concentrations; however, it was also found that an additional carbon source was required to maintain microbial activity over an extended period of time. Pyruvic acid was selected as a carbon source which, once added to actively PCB-degrading cells, maintained the cells’ activity towards PCBs and up to 85 % of 100 mg l-1 was degraded in 15 h. It was shown as the final contribution in this thesis that TPPBs can be combined with a PCB soil extraction step as a potential remediation scheme for PCB contaminated soil. PCBs were extracted from soil with polymer beads (up to 75 % removal), followed by biodegradation of the PCBs in a solid-liquid TPPB in which PCBs were delivered to the degrading organism from the same polymer. / Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2007-08-07 16:11:00.494