Orientador: Marcelo Cristianini / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-03T20:56:17Z (GMT). No. of bitstreams: 1 Senatore_MarcelaAndreaDuranHaun_M.pdf: 684290 bytes, checksum: 522970fe79261c7d2890c0a19a39b587 (MD5) Previous issue date: 2004 / Resumo: Um dos problemas que enfrenta a indústria de laticínios é a recontaminação do leite decorrente da formação de biofilmes em tanques de armazenamento e trocadores de calor. A tecnologia de esterilização de materiais por gás plasma tem sido utilizada com sucesso na esterilização de instrumentos cirúrgicos, oftálmicos e dentários. Os objetivos deste trabalho foram avaliar a eficiência de um sistema de esterilização a gás plasma sob células de Pseudomonas fluorescens aderidas em placas de aço inoxidável utilizando o leite como substrato. Foram avaliadas as variáveis tempo de pré plasma, tempo de exposição ao plasma e potência do mesmo na remoção do biofilme. As placas de aço inoxidável com a bactéria aderida foram submetidas a um tratamento com gás plasma, que foi formado a partir de um produto comercial composto por ácido peracético, peróxido de hidrogênio e ácido acético. Dois sistemas modelo foram utilizados para simular a formação de biofilmes de Pseudomonas fluorescens, um dinâmico e outro estático, simulando trocadores de calor e tanques de armazenamento, respectivamente. Através do modelo estático foi possível obter contagens acima de 105 UFC por placa de aço inoxidável, mesmo após três ciclos de lavagem das placas em água estéril sob agitação. Foi observado um efeito positivo na inativação de P. fluorescens em função do tempo de pré-plasma do sanificante. Exposições acima de 7 minutos foram capazes de produzir reduções superiores a dois ciclos logarítmicos na inativação do microrganismo. Através de um planejamento experimental fatorial 23 foi demosntrado que as variáveis tempo de pré-plasma, tempo de exposição ao plasma e potência do plasma apresentaram efeitos positivos na inativação de Pseudomonas fluorescens. O tempo de exposição (min.) apresentou o maior efeito na destruição da bactéria; mas sendo um pouco superior à potência do plasma (w) / Abstract: One of the problems that food industry is facing is the milk recontamination through biofims formation on storage tanks and heat exchanger. The technology of sterilization for materials through gas plasma has been used with succesfull for cirurgycal, ophathalmic and dentistry equipment. The goals of this work was to evaluate the efficiency of a gas plasma sterilization system on Pseudomonas fluorescens cells adhered on stainless steel plates using milk as substrate. Time of pre plasma, plasma exposition and potency (Watts) were evaluated as independent variables on cell destruction. The stainless steel plates were submitted to gas plasma treatment originated from a commercial product composed of peracetic acid, hydrogen peroxide and acetic acid. Two model systems were used to simulate a biofilm formation of Pseudomonas fluorescens, one dynamic and one static, in order to simulate heat exchangers and storage tanks, respectively. Through static model it was possible to get counts over 105 UFC/plate after washing three times using sterile water under stirring conditions. It was observed a positive effect on P. fluorescens inactivation by pre-plasma in a time dependent way. Expositions over seven minutes were capable to produce reductions higher than two logarithmic cycles on microorganism inactivation. A 23 factorial design indicated that the pre-plasma time, time exposition and potency showed positive effects on Pseudomonas fluorescens inactivation. Time exposition (min) was the most effective variable on bacteria destruction, being a little higher than plasma potency (w) / Mestrado / Mestre em Tecnologia de Alimentos

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Persistence of Listeria monocytogenes in food processing plants is a huge health and economic burden. Biofilms are considered to be one of the major mechanisms by which this pathogen persists within these environments. Studies so far have mostly used optimal growth conditions in their investigations which may not provide a realistic understanding of the biofilm forming abilities of L. monocytogenes in food processing plants. Therefore the aim of this study was to 1) establish a model (12 ºC, Beef Broth) that closely relates to the food processing environment 2) screen 66 isolates of L. monocytogenes from food and clinical sources and determine their biofilm forming phenotypes (non-, weak, moderate and strong formers) and 3) analyze the correlation between biofilm formation phenotypes and biofilm associated genes detected using polymerase chain reaction (PCR) and Basic Local Alignment Search Tool (BLAST) for whole genome sequences. Biofilm formation established at 12 ºC in Beef Broth was the most consistent and quantifiable at day 9 of incubation. Subsequently, 66 isolates were screened using this model, resulting in 60 isolates being identified as strong biofilm formers, 5 isolates as moderate biofilm formers and 1 isolate as a weak biofilm former. Twenty biofilm associated genes were analyzed using PCR in 27 representative isolates. Out of the 20 genes, at least 17 of them were detected in all the tested isolates. Out of the 106 biofilm associated genes analyzed using BLAST, all the isolates were found to show the presence of at least 92 genes. In conclusion, there was no obvious correlation between the presence/absence of the genes selected for analysis and the ability to form biofilms using approaches performed in this study. However, the model established in the study will be useful in further analysis (transcription and translation studies) of genetic markers responsible for biofilm formation of L. monocytogenes under food processing conditions.

In nature, urea is hydrolyzed to ammonia and bicarbonate primarily by enzymes called ureases. As urine waste contains multiple important plant nutrients, there is interest in the waste treatment field to use urine waste products as plant fertilizers. Since urease enzymes are usually found in biofilms, one can prevent nitrogen loss in urine waste by preventing biofilm formation in the surrounding environment. In recent years, many new strategies to prevent microbial growth have been developed, especially within the field of nanoscience. The aim of this master's thesis was to develop a method for growing and analyzing urease-active biofilms and to investigate whether super-acidic metal- oxide surfaces could prevent biofilm growth. In this project, the methods are divided into two sections: methods for producing super-acidic metal-oxide surfaces and methods for growing and analyzing biofilms. The method for growing biofilms was developed through successive experiments, with the results of one experiment being used to design the next. Three batches of antimicrobial plates were manufactured, and seven biofilm experiments were conducted. In these experiments, biofilms were able to grow on antimicrobial plates, but the results were somewhat inconclusive. The biofilms were analyzed by microscopy, since no quantitative analysis method was successful in this study.

There is an urgent need to develop ammonia removal treatment systems for municipal and industrial wastewater treatment due to the increasingly stringent ammonia effluent discharge regulations implemented by Canada, the United States, and the European Union. The objective of this dissertation is to develop new understanding and advance the current design and operation of total ammonia nitrogen (TAN) removal via the moving bed biofilm reactor technology (MBBR) for municipal and industrial wastewaters. The first specific objective is to develop a passive, low operationally intensive, efficient and robust design strategy for municipal wastewater treatment to achieve partial nitritation (PN) as a pre-treatment to anammox treatment without using control strategies such as operating at low dissolved oxygen, or the use of inhibitors. This first objective includes developing new knowledge of the biofilm, biomass and microbiome of attached growth PN systems. The second specific objective is to investigate the impact of defining a maximum biofilm thickness, via bio-carrier design, to enhance the effects of free nitrous acid inhibition for PN of municipal wastewaters. The third objective is to investigate the effect of influent copper concentration on nitrifying MBBR systems over long-term operations, to demonstrate the feasibility of the nitrifying MBBR as a solution for TAN removal from gold mining wastewaters. The results pertaining to the first objective, achieved via a study investigating the operation of a nitrifying moving bed biofilm reactor at elevated TAN surface area loading rates (SALRs) of 3, 4, 5, and 6.5 g TAN/m²∙d with the aim of achieving passive PN, demonstrates that operating at a TAN SALR value of 6.5 g TAN/m²∙d can achieve PN without restricting dissolved oxygen or using inhibitors. Operating at a TAN SALR value of 6.5 g TAN/m²∙d achieves a TAN surface area removal rate (SARR) of 3.5 g TAN/m²∙d, and a nitrite accumulation of 99.8% of the oxidized TAN, demonstrating the suppression of nitrite oxidizing bacteria (NOB) activity, while achieving elevated TAN SARR values. At the molecular-scale, there is a statistically significant change in the ammonia oxidizing bacteria (AOB) to NOB ratio from 1:2.6 to 8.7:1 as the TAN SALR increases from 3 to 6.5 g TAN/m²∙d; however, even at a TAN SALR value of 6.5 g TAN/m²∙d there is an NOB abundance of approximately 2%; thus demonstrating that NOB remain present in the biofilm, while their activity is suppressed by operation at elevated TAN SALR values. Furthermore, this system was shown to achieve stable PN consistently for over a period of 10 months of operation, demonstrating a robust, passive, low operational strategy for attached growth PN. The second objective of this dissertation is addressed through a study that compared the carrier design of defined maximal biofilm thickness (z-prototype carrier) to undefined maximal biofilm thickness (chip-prototype carrier) for PN via free nitrous acid inhibition of tertiary, low carbon, municipal wastewaters. The study demonstrates that defined maximal biofilm thickness is a preferred design choice to achieve attached growth PN. The chip-prototype carrier shows biofilm thicknesses and biofilm mass values that are ten-fold higher than the z-prototype carrier, which is shown to contribute to the impact of free nitrous acid on AOB and NOB activities. The z-prototype carrier shows PN is achieved after 3 hours of exposure to free nitrous acid while the chip-prototype carrier does not achieve PN within this same time of exposure. Therefore, the defined maximal biofilm thickness carrier is identified in this research as the preferred design option to achieve attached growth PN for municipal, low carbon, tertiary wastewater treatment. The results of the third objective, achieved via a study investigating the effects of influent copper concentrations on nitrifying MBBR during long term operations to gold mining wastewaters, demonstrates that there is no AOB inhibition in attached growth systems exposed to 0.1, 0.3, 0.45, and 0.6 mg Cu/L for long exposure times. A trend of increasing nitrite accumulation with increasing influent copper concentrations is shown, indicating that NOB inhibition occurs at influent copper concentrations of 0.3 mg Cu/L and greater, with the greatest NOB inhibition observed with an influent copper concentration of 0.6 mg/L. There is no statistically significant difference in biofilm characteristics at the copper concentrations tested; however, there is a trend of increasing biofilm thickness and biofilm roughness with increasing copper concentrations. This study demonstrates the resilience of the nitrifying biofilm to copper inhibition and demonstrates that the nitrifying MBBR is a promising system for removing TAN in mining wastewater in the presence of copper.

Center for Disease Control and Food and Drug Administration reports indicate that cantaloupe is one of the five most likely fruits and vegetables to cause a foodborne disease outbreak. Cantaloupe is a potential hazardous food based on the FDA food code since it is capable of supporting pathogen growth due to its low acidity and high moisture content. The objectives of this study were to determine the effect of strain and temperature on growth and biofilm formation of L. monocytogenes and Salmonella spp. in cantaloupe flesh and peel extracts on different food-contact processing surfaces. Growth of L. monocytogenes and Salmonella strains was greater in high cantaloupe flesh and peel extract concentration at 22°C and 10°C. In 50 mg/ml of cantaloupe flesh or peel extract, the cell numbers of L. monocytogenes and Salmonella increased by 5.0-5.5 log CFU/ml in 40 h at 22°C and 1-3.5 log CFU/ml in 72 h at 10°C. In 2 mg/ml of cantaloupe flesh or peel extract, the cell numbers of L. monocytogenes and Salmonella increased by 4.0-4.5 log CFU/ml in 72 h at 22°C but no change in log CFU/ml in 72 h at 10°C. There were no differences (P ˃ 0.05) among L. monocytogenes orSalmonella strains for biofilm formation in cantaloupe extracts, but biofilm formation was greater (P < 0.05) at high temperature and high cantaloupe flesh or peel extract concentration. In 50 mg/ml cantaloupe flesh or peel extract, L. monocytogenes and Salmonella produced biofilms of 7 log CFU/coupon in 4 days at 22°C and 4-5 log CFU/coupon in 7 days at 10°C. In 2 mg/ml cantaloupe flesh or peel extract, L. monocytogenes and Salmonella produced biofilms of 5-6 log CFU/coupon in 4 days at 22°C and 3-4 log CFU/coupon in 7 days at 10°C. L. monocytogenes and Salmonella spp. formed less biofilms (P < 0.05) on buna-n rubber when compared to stainless steel, polyethylene and polyurethane surfaces. These findings indicate that a very low concentration of nutrients that are leaked from cantaloupe flesh or peel can induce growth and biofilm formation in L. monocytogenes and Salmonella spp. on different food-contact surfaces.

The effect of exposure to sub-lethal chlorine stress on Salmonella enterica serotypes Typhimurium and Heidelberg was examined in this study. Both serotypes demonstrated an acquired tolerance to chlorine with the adapted cells growing in concentrations above the MIC. Chlorine induced a morphological change to the rugose variant in Salmonella. The biofilm formation of the adapted and control cells was tested on food-contact surfaces at room temperature and 37°C. The chlorineapted rugose formed stronger biofilms (P < 0.05) when compared to smooth (adapted and control) on both surfaces tested and at both temperatures. The possibility of crossaptation to antibiotics and low pH was evaluated. Adapted rugose showed reduced susceptibility against some of the antibiotics tested. Chlorine does not aid in the survival of Salmonella enterica at low pH. Chlorine stress can select for tolerant Salmonella cells that attach strongly to food-contact surfaces and after some time may become less susceptible to antimicrobials.

Biofilm formation within burn wounds pose numerous health-related problems as they prolong recovery, inhibit antimicrobial treatments, and serve as a reservoir to spread new infections. In the United States alone there are half a million burn wounds each year. These burn wounds result in tens of thousands of patients to be admitted to hospitals and thousands of deaths. Burn wound infections alone account for over half of these deaths. Currently, standard models of burn wound biofilms, both in-vivo and in-vitro, have their benefits and limitations. These models include skin explants, animal models, and complex growth media. For the examination of microbial biofilms and rapid screening of potential antimicrobial topical treatments, a physiologically relevant media that more closely mimics what would be found in the host’s tissue would be advantageous. This pilot study was conducted to examine different formulations of a synthetic tissue-like media, the biofilm growth of common burn wound infectious microbes, and served as a high-throughput means of testing current and potentially new antimicrobials. Our laboratory has begun characterizing a new antimicrobial gel and its ability to eradicate microorganisms that commonly infect burn wounds, specifically focusing on the common wound microbe Staphylococcus aureus. Utilizing a constitutively expressed green fluorescent protein, both the growth on the textured media, as well as, biofilm inhibition by the antimicrobial gel showed significant reduction in S. aureus. On a molecular level, we examined biofilm gene expression, via reverse transcription polymerase chain reaction, of adhesion, quorum sensing, and drug resistance markers in our new model in conjunction with our antimicrobial gel. Our new synthetic wound-like media supported the growth of S. aureus and was successful in its ability to quickly screen different formulations of topical antimicrobial treatments. The antimicrobial gel produced significant reduction of S. aureus burden. The results of this study indicate that our formulated synthetic burn wound media model supports microbial growth, is efficient in the ability to rapidly screen antimicrobials, and could lead to a better understanding of the etiology of burn wound infections.

The James River Treatment Plant (JRTP) operated a 2 MGD Integrated Fixed Film Activated Sludge (IFAS) demonstration process from November 2007 to April 2009 to explore IFAS performance and investigate IFAS technology as an option for a full scale plant upgrade in response to stricter nutrient discharge limits in the James River Basin. During the study, nitrification kinetics for both ammonia and nitrite oxidizing bacteria and plastic biofilm carrier biomass content were monitored on a near-weekly basis comparing the IFAS media, the IFAS process mixed liquor, and mixed liquor from the full-scale activated sludge process. Carrier biomass content is variable with respect to temperature and process SRT and relates to the localization of nitrification activity in the IFAS basin. Similar to trends observed for carrier biomass content (Regmi, 2008), ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) activity also shifted from the fixed film to the suspended phase as water temperatures increased and vice versa as the temperature decreased. The data suggest that AOB activity occurs on the surface of the biofilm carriers, while NOB activity remains deeper in the biofilm. During the highest temperatures observed in the IFAS tank, AOB activity on the media contributed as little as 30% of the total nitrification activity in the basin, and after temperatures dropped below 20 °C, AOB activity in the fixed film phase made up 75% of the total activity in the IFAS basin. During the warmest period of the summer, the media still retained more than 60% of the total NOB activity, and more than 90% of the total NOB activity during the period of coldest water temperature. This trend also points out that some AOB and NOB activity remained in the mixed liquor, even during the coldest periods. The retention of nitrification activity in the mixed liquor indicates that the constant sloughing of biomass off of the carriers allowed for autotrophic activity, even during washout conditions. Carrier biomass content and nitrification rates on the IFAS media remained constant along the length of the basin, indicating that the IFAS tank is will mixed with respect to biomass growth, although there was a concentration gradient for soluble species (NH₄-N, NO₂-N, NO₃-N). In addition to the weekly nitrification rate measurements, experiments were also conducted to determine how operational inputs such as dissolved oxygen (DO) and mixing affect the nitrification rates. Mixing intensity had a clear impact on nitrification rates by increasing the velocity gradient in the bulk liquid and decreasing the mass transfer boundary layer mass transfer resistance. At higher mixing intensities, advection through the mass transfer boundary layer increased making substrate more available to the biofilm. The affect of mixing was much more profound at low DO, whereas increased mixing had less effect on nitrification rates at higher bulk liquid DO. DO also affected nitrification rates, such that as DO increased it penetrated deeper into the biofilm increasing the nitrification rate in a linear fashion until the biofilm became saturated. Another aspect of the research was modeling effective half saturation effects for AOB and NOB activity in the fixed film phase. The modeling work demonstrated that KS for AOB activity on the media was similar to accepted suspended growth KS values, while KS for NOB activity on the media was considerably higher than suspended growth KS. This trend indicates that nitrite was not as bioavailable in the biofilm and resists diffusion into the deeper part of the biofilm where NOB activity takes place. KO for both AOB and NOB activity in the biofilm was higher than typical suspended growth values because of boundary layer and biofilm diffusion resistances. In addition, the presence of readily degradable organics did not significantly affect nitrification rates on the media, but did reduce nitrification rates in the mixed liquor. That, combined with low chemical oxygen demand (COD) uptake rates indicates that little heterotrophic activity is occurring on the media. / Master of Science

Wetland systems have been widely studied and found to have enhanced capacity to transform meaningful amounts of nitrate (NO3-N) from shallow subsurface water before the improved-quality water is delivered to lake, river, and groundwater systems. Wetland are characterized by the abundant presence of electron donors and acceptors (i.e., organic carbon & NO3-N, respectively) as well as anoxic and reducing conditions which are crucial for supporting denitrification processes and the reduction of excessive nitrate levels in the environment. When favorable conditions within the wetland systems are not present, denitrification is often limited to the biofilm-protected bacteria hosted on the sediment surfaces. However, there is still a need to determine if floodplain wetlands are being utilized to their maximum potential in excess nitrate removal.