Outer membrane porin M35 of Moraxella catarrhalis mediates susceptibility to aminopenicillins

BACKGROUND: The outer membrane protein M35 is a conserved porin of type 1 strains of the respiratory pathogen Moraxella catarrhalis. It was previously shown that M35 is involved in the uptake of essential nutrients required for bacterial growth and for nasal colonization in mice. The aim of this study was (i) to characterize the potential roles of M35 in the host-pathogen interactions considering the known multifunctionality of porins and (ii) to characterize the degree of conservation in the phylogenetic older subpopulation (type 2) of M. catarrhalis. RESULTS: Isogenic m35 mutants of the type 1 strains O35E, 300 and 415 were tested for their antimicrobial susceptibility against 15 different agents. Differences in the MIC (Minimum Inhibitory Concentration) between wild-type and mutant strains were found for eight antibiotics. For ampicillin and amoxicillin, we observed a statistically significant 2.5 to 2.9-fold MIC increase (p < 0.03) in the m35 mutants. Immunoblot analysis demonstrated that human saliva contains anti-M35 IgA. Wild-type strains and their respective m35 mutants were indistinguishable with respect to the phenotypes of autoagglutination, serum resistance, iron acquisition from human lactoferrin, adherence to and invasion of respiratory tract epithelial cells, and proinflammatory stimulation of human monocytes. DNA sequencing of m35 from the phylogenetic subpopulation type 2 strain 287 revealed 94.2% and 92.8% identity on the DNA and amino acid levels, respectively, in comparison with type 1 strains. CONCLUSION: The increase in MIC for ampicillin and amoxicillin, respectively, in the M35-deficient mutants indicates that this porin affects the outer membrane permeability for aminopenicillins in a clinically relevant manner. The presence of IgA antibodies in healthy human donors indicates that M35 is expressed in vivo and recognized as a mucosal antigen by the human host. However, immunoblot analysis of human saliva suggests the possibility of antigenic variation of immunoreactive epitopes, which warrants further analysis before M35 can be considered a potential vaccine candidate.

Decrease in pneumococcal co-colonization following vaccination with the seven-valent pneumococcal conjugate vaccine.

Understanding the epidemiology of pneumococcal co-colonization is important for monitoring vaccine effectiveness and the occurrence of horizontal gene transfer between pneumococcal strains. In this study we aimed to evaluate the impact of the seven-valent pneumococcal conjugate vaccine (PCV7) on pneumococcal co-colonization among Portuguese children. Nasopharyngeal samples from children up to 6 years old yielding a pneumococcal culture were clustered into three groups: pre-vaccine era (n = 173), unvaccinated children of the vaccine era (n = 169), and fully vaccinated children (4 doses; n = 150). Co-colonization, serotype identification, and relative serotype abundance were detected by analysis of DNA of the total bacterial growth of the primary culture plate using the plyNCR-RFLP method and a molecular serotyping microarray-based strategy. The plyNCR-RFLP method detected an overall co-colonization rate of 20.1%. Microarray analysis confirmed the plyNCR-RFLP results. Vaccination status was the only factor found to be significantly associated with co-colonization: co-colonization rates were significantly lower (p = 0.004; Fisher's exact test) among fully vaccinated children (8.0%) than among children from the pre-PCV7 era (17.3%) or unvaccinated children of the PCV7 era (18.3%). In the PCV7 era there were significantly less non-vaccine type (NVT) co-colonization events than would be expected based on the NVT distribution observed in the pre-PCV7 era (p = 0.024). In conclusion, vaccination with PCV7 resulted in a lower co-colonization rate due to an asymmetric distribution between NVTs found in single and co-colonized samples. We propose that some NVTs prevalent in the PCV7 era are more competitive than others, hampering their co-existence in the same niche. This result may have important implications since a decrease in co-colonization events is expected to translate in decreased opportunities for horizontal gene transfer, hindering pneumococcal evolution events such as acquisition of antibiotic resistance determinants or capsular switch. This might represent a novel potential benefit of conjugate vaccines.

Role of two UDP-Glycosyltransferases from the L group of arabidopsis in resistance against pseudomonas syringae

The role of the salicylic acid (SA) glycosides SA 2-O-β-D-glucose (SAG), SA glucose ester (SGE) and the glycosyl transferases UGT74F1 and UGT74F2 in the establishment of basal resistance of Arabidopsis against Pseudomonas syringae pv tomato DC3000 (Pst) was investigated. Both mutants altered in the corresponding glycosyl transferases (ugt74f1 and ugt74f2) were affected in their basal resistance against Pst. The mutant ugt74f1 showed enhanced susceptibility, while ugt74f2 showed enhanced resistance against the same pathogen. Both mutants have to some extent, altered levels of SAG and SGE compared to wild type plants, however, in response to the infection, ugt74f2 accumulated higher levels of free SA until 24 hpi compared to wild type plants while ugt74f1 accumulated lower SA levels. These SA levels correlated well with reduced expression in PR1 and EDS1 in ugt74f1. In contrast, ugt74f2 has enhanced expression of Enhanced Disease Susceptibility 1 (EDS1) but a strong reduction in the expression of several jasmonate (JA)-dependent genes. Bacterial infection interfered with the expression of Fatty Acid Desaturase (FAD), Lipoxygenase2 (LOX2), carboxyl methyltransferase1 (BSMT1) and 9-cis-epoxycarotenoid dioxygenase (NCED3) genes in ugt74f1, thus promoting an antagonistic effect with SA-signalling and leading to enhanced bacterial growth. UGT74F2 might be a target for bacterial effectors since bacterial mutants affected in effector synthesis were impaired in inducing UGT74F2 expression. These results suggest that UGT74F2 negatively influences the accumulation of free SA, hence leading to an increased susceptibility due to reduced SA levels and increased expression of the JA and ABA markers LOX-2, FAD and NCED-3.

Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization among dental professionals

Background. MRSA (methicillin-resistant Staphylococcus aureus) is a multi-drug resistant bacterium that is quite prevalent in social environments where close person-to-person contact and crowding are an issue. In dental settings, the likelihood of transmission of MRSA may be higher than among other healthcare practitioners because of the close proximity between a patient's nose (where MRSA colonizes) and the field of procedure (the mouth) to the dental professional. Objective. To estimate the prevalence of MRSA nasal colonization among dental professionals (dentists and dental hygienists) in the Greater Houston Metropolitan Area, Texas, and analyze its associations with demographic, professional and personal protective equipment-related variables. Methods. 800 dental professionals (400 dentists and 400 dental hygienists) were randomly selected in the Greater Houston Metropolitan Area. Multiple waves of nasal swab kits and a self-administered questionnaire were mailed to increase the response rate of the study population. The swabs were cultured on chromagenic agar growth medium and bacterial growth results were evaluated after 18 hours. Positively selected bacterial colonies were confirmed as MRSA by further culturing these isolated bacteria on blood agar plates. Associations between positive nasal swabs and self-reported professional practice patterns, personal protective equipment use and demographics were analyzed using multiple logistic regression. Main Results. Completed questionnaires and nasal swabs were received from 496 study participants (68%). Fourteen cultures were positive for MRSA (4.2% among dentists and 1.6% among dental hygienists, p=0.07). After adjusting for gender, dental hygienists had a significantly lower prevalence of nasal colonization of MRSA as compared to dentists (OR: 0.20, 95% CI: 0.05–0.75). No other significant associations or interactions were found. Conclusion. The prevalence of nasal colonization with MRSA among dentists is similar to that reported for health care workers in general, whereas prevalence among dental hygienists is only slightly above that of the general population (1%). Differences in practice patterns and use of personal protective equipment did not explain this difference in this study, and was possibly due either to residual confounding or unexplored risk factors. Increased prevalence of MRSA among dentists warrants further investigation as to the reason for the increased rate and to allow implementation of measures to avoid transmission and progression to disease. ^

Concentrations of total organic carbon on vertical profiles in waters of the Weddell Sea measured on water bottle sample during POLARSTERN cruise ANT-X/6

Concentrations of dissolved organic carbon (DOC) and nitrogen (DON) were measured during early austral Spring 1992 at a number of stations along the 6°W meridian between 47° and 60°S. This included the Polar Front in the north, the zone of melting sea-ice in the south, and waters of the Antarctic Circumpolar Current in between. Concentrations of DOC were low in deep water (34-38 ?M) with generally similar or slightly higher values in the surface mixed layer (38-55 ?M). DOC:DON ratios are wider in surface water than in deep water, i.e. surface accumulations contain relatively C-rich dissolved organic matter. The highly variable distribution of the surface DOC was not related to hydrographic or biotic features (fronts, plankton development) indicating the lability and transient occurrence of this material. Growth rates of bacteria were determined in subsamples from 51 0.8-?m-filtered batches of seawater incubated in the dark at in-situ temperature. Thymidine and leucine uptake and bacterial biomass change as well as changes in dissolved organic carbon in the batches, and oxygen consumption in parallel incubations correlated linearly over 2 weeks of incubation which allowed extrapolation to in-situ conditions. Bacterial growth in these experiments depended strongly on the amount of initial DOC. Growth in water from greater depth (1000 m) containing 38 ?M DOC was minimal, as were DOC-decrease and oxygen consumption. Higher rates were observed in surface water slightly enriched with DOC, and highest rates in surface water amended with DOC-rich melted sea ice. Bacterial growth efficiencies (biomass C-increase vs DOC consumed) were about 30%. The experiments showed that at least 40-60% of the DOC in excess of deep water concentrations was available to bacteria.

Seawater carbonate chemistry, biomass and metabolic rates (leucine incorporation, CO2 fixation and respiration) of Rhodobacteraceae (strain MED165) and Flavobacteriaceae (strain MED217) in a laboratory experiment

Experimental results related to the effects of ocean acidification on planktonic marine microbes are still rather inconsistent and occasionally contradictory. Moreover, laboratory or field experiments that address the effects of changes in CO2 concentrations on heterotrophic microbes are very scarce, despite the major role of these organisms in the marine carbon cycle. We tested the direct effect of an elevated CO2 concentration (1000 ppmv) on the biomass and metabolic rates (leucine incorporation, CO2 fixation and respiration) of 2 isolates belonging to 2 relevant marine bacterial families, Rhodobacteraceae (strain MED165) and Flavobacteriaceae (strain MED217). Our results demonstrate that, contrary to some expectations, high pCO2 did not negatively affect bacterial growth but increased growth efficiency in the case of MED217. The elevated partial pressure of CO2 (pCO2) caused, in both cases, higher rates of CO2 fixation in the dissolved fraction and, in the case of MED217, lower respiration rates. Both responses would tend to increase the pH of seawater acting as a negative feedback between elevated atmospheric CO2 concentrations and ocean acidification.

Differential in vitro and in vivo effect of barley cysteine and serine protease inhibitors on phytopathogenic microorganisms

Protease inhibitors from plants have been involved in defence mechanisms against pests and pathogens. Phytocystatins and trypsin/α-amylase inhibitors are two of the best characterized protease inhibitor families in plants. In barley, thirteen cystatins (HvCPI-1 to 13) and the BTI-CMe trypsin inhibitor have been previously studied. Their capacity to inhibit pest digestive proteases, and the negative in vivo effect caused by plants expressing these inhibitors on pests support the defence function of these proteins. Barley cystatins are also able to inhibit in vitro fungal growth. However, the antifungal effect of these inhibitors in vivo had not been previously tested. Moreover, their in vitro and in vivo effect on plant pathogenous bacteria is still unknown. In order to obtain new insights on this feature, in vitro assays were made against different bacterial and fungal pathogens of plants using the trypsin inhibitor BTI-CMe and the thirteen barley cystatins. Most barley cystatins and the BTI-CMe inhibitor were able to inhibit mycelial growth but no bacterial growth. Transgenic Arabidopsis plants independently expressing the BTI-CMe inhibitor and the cystatin HvCPI-6 were tested against the same bacterial and fungal pathogens. Neither the HvCPI-6 expressing transgenic plants nor the BTI-CMe ones were more resistant to plant pathogen fungi and bacteria than control Arabidopsis plants. The differences observed between the in vitro and in planta assays against phytopathogenic fungi are discussed

Programación y simulación de la comunicación intercelular mediante conjugación en el simulador multicelular GRO

Los conjuntos bacterianos son sistemas dinámicos difíciles de modelar debido a que las bacterias colaboran e intercambian información entre sí. Estos microorganismos procariotas pueden tomar decisiones por mayoría e intercambiar información genética importante que, por ejemplo, las haga resistentes a un antibiótico. El proceso de conjugación consiste en el intercambio de un plásmido de una bacteria con otra, permitiendo así que se transfieran propiedades. Estudios recientes han demostrado que estos plásmidos pueden ser reprogramados artificialmente para que la bacteria que lo contenga realice una función específica [1]. Entre la multitud de aplicaciones que supone esta idea, el proyecto europeo PLASWIRES está intentando demostrar que es posible usar organismos vivos como computadores distribuidos en paralelo y plásmidos como conexión entre ellos mediante conjugación. Por tanto, mediante una correcta programación de un plásmido, se puede conseguir, por ejemplo, hacer que una colonia de bacterias haga la función de un antibiótico o detecte otros plásmidos peligrosos en bacterias virulentas. El proceso experimental para demostrar esta idea puede llegar a ser algo lento y tedioso, por lo que es necesario el uso de simuladores que predigan su comportamiento. Debido a que el proyecto PLASWIRES se basa en la conjugación bacteriana, surge la necesidad de un simulador que reproduzca esta operación. El presente trabajo surge debido a la deficiencia del simulador GRO para reproducir la conjugación. En este documento se detallan las modificaciones necesarias para que GRO pueda representar este proceso, así como analizar los datos obtenidos e intentar ajustar el modelo a los datos obtenidos por el Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC). ---ABSTRACT---Bacterial colonies are dynamical systems difficult to model because bacteria collaborate and exchange information with each other. These prokaryotic organisms can make decisions by majority and exchange important genetic information, for example, make them resistant to an antibiotic. The conjugation process is the exchange of a plasmid from one bacterium to another, allowing both to have the same properties. Recent studies have shown that these plasmids can be artificially reprogrammed to make the bacteria that contain it to perform a specific function [1]. Among the multitude of applications involved in this idea, the European project PLASWIRES is attempting to prove that it is possible to use living organisms as parallel and distributed computers with plasmids acting as connectors between them through conjugation. Thus, by properly programming a plasmid, you can get a colony of bacteria that work as an antibiotic or detect hazardous plasmids in virulent bacteria. The experimental process to prove this idea can be slow and tedious, so the use of simulators to predict their behavior is required. Since PLASWIRES project is based on bacterial conjugation, a simulator that can reproduce this operation is required. This work arises due to the absence of the conjugation process in the simulator GRO. This document details the changes made to GRO to represent this process, analyze the data and try to adjust the model to the data obtained by the Institute of Biomedicine and Biotechnology of Cantabria ( IBBTEC ). This project has two main objectives, the first is to add the functionality of intercellular communication by conjugation to the simulator GRO, and the second is to use the experimental data obtained by the IBBTEC. To do this, the following points should be followed: • Study of conjugation biology as a mechanism of intercellular communication. • Design and implementation of the algorithm that simulates conjugation. • Experimental validation and model adjust to the experimental data on rates of conjugation and bacterial growth.

Diseño e implementación de un módulo de crecimiento bacteriano dependiente de nutrientes en el simulador GRO

Hace no más de una década que empezó a escucharse el término biología sintética. Este área de estudio emergente consiste en la ingeniería y programación de sistemas biológicos, tratando la biología como una tecnología programable a la que aplican los principios y metodologías de la ingeniería, con el fin de crear nuevas funcionalidades genéticas desde cero, procurando asÍ algún beneficio como por ejemplo, programar células bacterianas para producir biocombustibles. Sin embargo, para la creación de dichas funcionalidades es necesario conocer bien al organismo sobre el que se van a implantar. Por este motivo, los biólogos sintéticos emplean bacterias en sus estudios, ya que es la forma de vida más simple, está presente en prácticamente todos los nichos ecológicos, desempeña algunas de las funcionalidades vitales para los humanos y lo mas importante, se conoce prácticamente todo su material genético. Los experimentos son costosos en tiempo y dinero, siendo necesaria la ayuda de herramientas que faciliten esta labor, los simuladores. En PLASWIRES, proyecto europeo de biología sintética en el que se engloba este este trabajo, el simulador empleado es GRO. Sin embargo, en GRO el crecimiento de las bacterias ocurre de forma exponencial y sin restricciones, generando comportamientos poco realistas. Por ello, se ha considerado relevante en biología sintética, y en el simulador GRO en particular, disponer de un modelo de crecimiento bacteriano dependiente de los nutrientes. El desarrollo de este trabajo se centra en la implementación de un módulo de consumo de nutrientes en colonias de bacterias simuladas con GRO, introduciendo así la limitación de nutrientes y evitanto que las bacterias crezcan exponencialmente. Se han introducido nutrientes en el medio y la capacidad de consumirlos, con el objetivo de obtener un crecimiento ajustado al que ocurre en la naturaleza. Además, se ha desarrollado en GRO una nueva función de adquisición de volumen, que condiciona el volumen adquirido por cada bacteria en función de los nutrientes. La implentación de las dos aportaciones presentadas ha supuesto la adición de funcionalidad extra a GRO, convirtiéndolo en el único simulador de bacterias que tiene en cuenta el crecimiento bacteriano dependiente de nutrientes.---ABSTRACT---It has been in this last decade that the synthetic biology term began to be heard. This emergent area of study consists in the engineering and programming of biological systems, dealing with biology as a programable technology in which the engineering principles and methodologies are applied in order to create novel genetic functinalities from scratch, obtaining some advatages such as programmed bacteria in order to produce biofuels. However, to create this functionalities, it is necessary to know well the organisms in which they are going to be implemented. For this reason, synthetic biology researchers use bacteria, because it is the simplest life form, it can be found in almost all the ecological niche, it does some vital function to humans and, most important, almost all of its genetic information is known. Experiments are expensive in time and money, making it necessary to use tools to ease this task: the simulators. In PLASWIRES, the european synthetic biology project in which this work is included, the simulator used is GRO. However, the bacterial growth in GRO is exponential and it does not have restrictions, generating unrealistic behaviours. Therefore, it has been considered relevant in synthetic biology, and in a particular way in GRO, to provide a bacterial growth model dependent on nutrient. This work focuses on the implementation of a nutrient consumption module in bacteria colonies simulated with GRO, introducing a nuntrient limitation and avoiding the bacteria exponential growth. The module introduces nutrients and the capacity for bacteria to consume them, aiming to obtain realistic growth simulations that fit the observations made in nature. Moreover, an adquisition volumen function has been developed in GRO, determining the volumen depending on nutrients. This two contributions make GRO the only bacteria simulator that computes growth depending on nutrients

Ribosomal protein S7 from Escherichia coli uses the same determinants to bind 16S ribosomal RNA and its messenger RNA

Ribosomal protein S7 from Escherichia coli binds to the lower half of the 3′ major domain of 16S rRNA and initiates its folding. It also binds to its own mRNA, the str mRNA, and represses its translation. Using filter binding assays, we show in this study that the same mutations that interfere with S7 binding to 16S rRNA also weaken its affinity for its mRNA. This suggests that the same protein regions are responsible for mRNA and rRNA binding affinities, and that S7 recognizes identical sequence elements within the two RNA targets, although they have dissimilar secondary structures. Overexpression of S7 is known to inhibit bacterial growth. This phenotypic growth defect was relieved in cells overexpressing S7 mutants that bind poorly the str mRNA, confirming that growth impairment is controlled by the binding of S7 to its mRNA. Interestingly, a mutant with a short deletion at the C-terminus of S7 was more detrimental to cell growth than wild-type S7. This suggests that the C-terminal portion of S7 plays an important role in ribosome function, which is perturbed by the deletion.

Adenosine diphosphate sugar pyrophosphatase prevents glycogen biosynthesis in Escherichia coli

An adenosine diphosphate sugar pyrophosphatase (ASPPase, EC 3.6.1.21) has been characterized by using Escherichia coli. This enzyme, whose activities in the cell are inversely correlated with the intracellular glycogen content and the glucose concentration in the culture medium, hydrolyzes ADP-glucose, the precursor molecule of glycogen biosynthesis. ASPPase was purified to apparent homogeneity (over 3,000-fold), and sequence analyses revealed that it is a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated as “nudix” hydrolases. Insertional mutagenesis experiments leading to the inactivation of the ASPPase encoding gene, aspP, produced cells with marginally low enzymatic activities and higher glycogen content than wild-type bacteria. aspP was cloned into an expression vector and introduced into E. coli. Transformed cells were shown to contain a dramatically reduced amount of glycogen, as compared with the untransformed bacteria. No pleiotropic changes in the bacterial growth occurred in both the aspP-overexpressing and aspP-deficient strains. The overall results pinpoint the reaction catalyzed by ASPPase as a potential step of regulating glycogen biosynthesis in E. coli.

Desenvolvimento de hidroxiapatita contendo prata via precipitação e imersão: avaliação do efeito antimicrobiano

A utilização de substitutos ósseos para recuperação da função perdida é uma constante busca dentro da área médica. Por isso os biomateriais têm recebido uma atenção muito grande por parte da comunidade científica, dentre eles os materiais a base de fosfato de cálcio. A hidroxiapatita, Ca10 (PO4)6 (OH) 2, tem sido muito estudada, pois além de representar a constituição da massa dos ossos naturais e dentes em 30 a 70%, possui propriedades de bioatividade e osteocondutividade, favorecendo e auxiliando o crescimento do tecido ósseo. Em contrapartida, infecções bacterianas podem surgir após o implante ocasionando a perda da funcionalidade a curto e médio prazo. Várias alternativas estão sendo testadas, geralmente associadas ao uso de antibióticos convencionais incorporados aos biomateriais. Uma alternativa a tais antibióticos seria a utilização de metais que possuem propriedades antibacterianas. A prata (Ag) é conhecida como um metal bactericida e por isso ganhou lugar de destaque dentre os estudos como um aliado importante no controle das infecções pós-cirúrgicas. Este trabalho teve como objetivo sintetizar, caracterizar e avaliar o efeito antimicrobiano da adição de íons de prata em hidroxiapatita. Foram obtidos pós de hidroxiapatita contendo prata (HAAg), nas concentrações de 0,1M; 0,01M e 0,001M pelo método de precipitação em temperatura ambiente e por imersão do pó de hidroxiapatita em soluções aquosas. As fases cristalinas e os grupamentos iônicos foram analisados para cada condição por técnicas de difração de raios X (DRX) e espectroscopia no infravermelho (IV) respectivamente. As informações sobre a morfologia e identificação de elementos químicos foi realizado pela técnica de microscopia eletrônica de varredura com espectroscopia de energia dispersiva (MEV EDS). As avaliações antimicrobianas foram realizadas por ensaios qualitativos e quantitativos, o ensaio qualitativo utilizou o teste de halo de difusão em disco para Staphylococcus aureus e Escherichia coli e o ensaio quantitativo utilizou contagem de bactérias para as cepas de Staphylococcus aureus. Os resultados de DRX e IV indicaram que independentemente do método de obtenção da HAAg foi possível observar a presença de prata metálica caracterizada pelos picos em 2θ=38,1º e 44,3º nas amostras HAAg0,1Im, HAAg0,1Pr e HAAg0,01Pr. Observou-se também a presença de AgO, correspondente ao pico em 2θ=37,5º nas amostras de HAAg0,01Pr e HAAg0,001Pr. Nos espectros de IV estão presentes as bandas que caracterizam a fase HA, referentes aos grupamentos PO43-, OH- e CO32-. Analisados em conjunto os ensaios qualitativos e quantitativos, as amostras HAAg0,01Im e HAAg0,001Im sintetizadas por imersão indicaram os melhores resultados para o ensaio de disco difusão, por apresentarem formação de halo inibição do crescimento bacteriano para a bactéria S. aureus. Para os ensaios quantitativos as amostras obtidas por precipitação com concentrações 0,1M e 0,01M de prata apresentaram melhor resultado por inibirem o crescimento bacteriano para as cepas S. aureus.

Avaliação da contaminação conjunta de Dekkera bruxellensis e Lactobacillus fermentum sobre a fermentação alcoólica: efeito do substrato e formas de controle

As contaminações por leveduras selvagens e por bactérias no processo de produção de etanol combustível no Brasil causam prejuízos ao rendimento fermentativo e aumento de custos pelo uso de biocidas. No entanto, poucos estudos tem focado no efeito das contaminações conjuntas de leveduras selvagens e bactérias e as possíveis interações entre os micro-organismos, especialmente em função dos diferentes substratos de fermentação e das formas de controle. Este trabalho teve por objetivos verificar o efeito do substrato (caldo de cana e melaço) sobre o desenvolvimento das contaminações pela levedura da espécie Dekkera bruxellensis e pela bactéria Lactobacillus fermentum, em co-culturas com Saccharomyces cerevisiae (linhagem industrial PE-2) e possíveis formas de controle do crescimento dos contaminantes (pelo uso de metabissulfito de potássio e adição de etanol ao tratamento ácido) sem afetar a levedura do processo. Os testes foram realizados em condições de crescimento (substrato com 4 °Brix, culturas agitadas) e fermentação com reciclo celular (substrato com 16 °Brix, culturas estáticas). Houve interação entre as leveduras e a bactéria quando crescidas em caldo de cana 4 °Brix. A levedura industrial não foi afetada pela presença dos micro-organismos contaminantes, no entanto, para D. bruxellensis a presença de L. fermentum interferiu positivamente no crescimento, com aumento no número de UFC, e consequentemente inibição do crescimento da bactéria. Em melaço, houve um estímulo ao crescimento de L. fermentum quando em co-cultura com S. cerevisiae. Houve influência das contaminações sobre os parâmetros avaliados no experimento (pH, açúcar redutor total, etanol, glicerol e crescimento das células) e a contaminação conjunta de L. fermentum e D. bruxellensis potencializou o efeito das contaminações pelos micro-organismos isoladamente, tanto em caldo quanto em melaço. A adição de 13% de etanol à solução de ácido sulfúrico pH 2,0 no tratamento celular resultou em uma diminuição significativa no número de UFC de D. bruxellensis (entre 90-99%). A levedura PE-2 foi pouco afetada pelo tratamento proposto. A bactéria L. fermentum teve seu crescimento afetado em todas as combinações testadas. Como os experimentos foram feitos em co-culturas, verificouse que pode haver influência de um micro-organismo sobre a viabilidade do outro, dependendo da reação ao tratamento ácido-etanol. O metabissulfito de potássio (MBP), no intervalo entre 200-400 mg/L, foi eficaz para controlar o crescimento de D. bruxellensis dependendo do meio de cultura e linhagem. Quando adicionado (250 mg/L) à solução ácida (pH 2,0) no tratamento celular, um efeito significativo foi observado nas culturas mistas, pois ocorreu a inativação do SO2 pela S. cerevisiae e uma provável proteção das células de D. bruxellensis, não sendo essa levedura prejudicada pelo MBP. A resposta fisiológica de S. cerevisiae na presença de MBP pode explicar a diminuição significativa na produção de etanol. Quando o MBP foi adicionado ao meio de fermentação, resultou no controle da D. bruxellensis mas não em sua morte, com efeito menos intensivo sobre a eficiência fermentativa. Em cocultura com a adição de MBP, a eficiência fermentativa foi significativamente menor do que na ausência de MBP.

Kinetic model for selective cultivation of microfungi in a microscreen process for food processing wastewater treatment and biomass production

The research was aimed at developing a technology to combine the production of useful microfungi with the treatment of wastewater from food processing. A recycle bioreactor equipped with a micro-screen was developed as a wastewater treatment system on a laboratory scale to contain a Rhizopus culture and maintain its dominance under non-aseptic conditions. Competitive growth of bacteria was observed, but this was minimised by manipulation of the solids retention time and the hydraulic retention time. Removal of about 90% of the waste organic material (as BOD) from the wastewater was achieved simultaneously. Since essentially all fungi are retained behind the 100 mum aperture screen, the solids retention time could be controlled by the rate of harvesting. The hydraulic retention time was employed to control the bacterial growth as the bacteria were washed through the screen at a short HRT. A steady state model was developed to determine these two parameters. This model predicts the effluent quality. Experimental work is still needed to determine the growth characteristics of the selected fungal species under optimum conditions (pH and temperature).

Folate: Methods of analysis

Folates and its derivatives occur as polyglutamates in nature. The multiplicity of forms and the generally low levels in foods makes quantitative analysis of folate a difficult task. The assay of folates from foods generally involves three steps: liberation of folates from the cellular matrix; deconjugation from the polyglutamate to the mono and di-glutamate forms; and the detection of the biological activity or chemical concentration of the resulting folates. The detection methods used are the microbiological assay relying on the turbidimetric bacterial growth of Lactobacillus rhamnosus which is by far the most commonly used method; the HPLC and LC/MS techniques and bio-specific procedures. This review attempts to describe the methods along with the merits and demerits of using each of these methods.