Monitoreo descriptivo de parásitos de origen fecal hallados en hortalizas de hojas

p.271-276El objetivo de este trabajo fue contabilizar la presencia de protozoos, huevos y larvas de helmintos en muestras de hortalizas de hoja para consumo fresco, que se comercializan en el Mercado Central de Buenos Aires. Las especies analizadas fueron: lechuga, radicheta, berro y perejil. Las muestras se procesaron por los métodos de Baerman y Flotación. Los resultados obtenidos indican que: 1- Existe contaminación parasitaria: sobre un total de 98 muestras analizadas durante 12 meses, el 27,5por ciento estaban contaminadas. 2- La especie más contaminada fue berro (66,7por ciento), luego radicheta (31,8por ciento), lechuga (21,4por ciento) y, finalmente, perejil (13,6por ciento). 3- El parásito más frecuente fue Entamoeba coli (44,4por ciento) y en segundo término E.hystolitica (22,4por ciento). 4- El sur y sudoeste del conurbano bonaerense fue la zona de mayor contaminación (66,6por ciento). La presencia de los contaminantes en las hortalizas estudiadas demuestra el desconocimiento de los riesgos potenciales que ciertas prácticas agrícolas pueden acarrear a los consumidores

Citrate as Cost-Efficient NADPH Regenerating Agent

The economically efficient utilization of NAD(P)H-dependent enzymes requires the regeneration of consumed reduction equivalents. Classically, this is done by substrate supplementation, and if necessary by addition of one or more enzymes. The simplest method thereof is whole cell NADPH regeneration. In this context we now present an easy-to-apply whole cell cofactor regeneration approach, which can especially be used in screening applications. Simply by applying citrate to a buffer or directly using citrate/-phosphate buffer NADPH can be regenerated by native enzymes of the TCA cycle, practically present in all aerobic living organisms. Apart from viable-culturable cells, this regeneration approach can also be applied with lyophilized cells and even crude cell extracts. This is exemplarily shown for the synthesis of 1-phenylethanol from acetophenone with several oxidoreductases. The mechanism of NADPH regeneration by TCA cycle enzymes was further investigated by a transient isotopic labeling experiment feeding [1,5-13C]citrate. This revealed that the regeneration mechanism can further be optimized by genetic modification of two competing internal citrate metabolism pathways, the glyoxylate shunt, and the glutamate dehydrogenase

Extensive exometabolome analysis reveals extended overflow metabolism in various microorganisms

Overflow metabolism is well known for yeast, bacteria and mammalian cells. It typically occurs under glucose excess conditions and is characterized by excretions of by-products such as ethanol, acetate or lactate. This phenomenon, also denoted the short-term Crabtree effect, has been extensively studied over the past few decades, however, its basic regulatory mechanism and functional role in metabolism is still unknown. Here we present a comprehensive quantitative and time-dependent analysis of the exometabolome of Escherichia coli, Corynebacterium glutamicum, Bacillus licheniformis, and Saccharomyces cerevisiae during well-controlled bioreactor cultivations. Most surprisingly, in all cases a great diversity of central metabolic intermediates and amino acids is found in the culture medium with extracellular concentrations varying in the micromolar range. Different hypotheses for these observations are formulated and experimentally tested. As a result, the intermediates in the culture medium during batch growth must originate from passive or active transportation due to a new phenomenon termed “extended” overflow metabolism. Moreover, we provide broad evidence that this could be a common feature of all microorganism species when cultivated under conditions of carbon excess and non-inhibited carbon uptake. In turn, this finding has consequences for metabolite balancing and, particularly, for intracellular metabolite quantification and (13)C-metabolic flux analysis

Rapid and comprehensive evaluation of microalgal fatty acids via untargeted gas chromatography and time-of-flight mass spectrometry

Due to their high triacylglyceride content, microalgae are intensively investigated for bio‐economy and food applications. However, lipid analysis is a laborious task incorporating extraction, transesterification and typically gas chromatographic measurement. Co‐elution induces a significant risk of fatty acid misidentification and thus, additional purification steps like thin layer chromatography are needed. Contrary to database matching approaches, solely targeted analysis is facilitated as compound identification is driven by matching retention times or indices with standard substances. In this context, a rapid workflow for the analysis of algal fatty acids is presented. In‐situ transesterification was used to simplify sample preparation and conditions were optimized towards fast processing. If results are needed at the very day of sampling, direct processing without a preceding drying step is feasible to obtain a rough estimate about the occurrence of the major compounds. Coupling gas chromatography and time‐of‐flight mass spectrometry enables untargeted analysis. Unknown compounds may be identified by structural reconstruction of their respective fragmentation patterns and by database matching to routinely avoid mismatches by co‐elution of disturbing agents. The developed workflow was successfully applied to derive the exact stereochemistry of all fatty acids from Chlorella vulgaris and a systematic shift depending on physiological state of the cells was confirmed

The linkage between nutrient supply, intracellular enzyme abundances and bacterial growth: New evidences from the central carbon metabolism of Corynebacterium glutamicum

Corynebacterium glutamicum serves as important production host for small molecular compounds that are derived from precursor molecules of the central carbon metabolism. It is therefore a well-studied model organism of industrial biotechnology. However, a deeper understanding of the regulatory principles underlying the synthesis of central metabolic enzymes under different environmental conditions as well as its impact on cell growth is still missing. We studied enzyme abundances in C. glutamicum in response to growth on: (i) one limiting carbon source by sampling chemostat and fed-batch cultivations and (ii) changing carbon sources provided in excess by sampling batch cultivations. The targeted quantification of 20 central metabolic enzymes by isotope dilution mass spectrometry revealed that cells maintain stable enzyme concentrations when grown on d-glucose as single carbon and energy source and, most importantly, independent of its availability. By contrast, switching from d-glucose to d-fructose, d-mannose, d-arabitol, acetate, l-lactate or l-glutamate results in highly specific enzyme regulation patterns that can partly be explained by the activity of known transcriptional regulators. Based on these experimental results we propose a simple framework for modeling cell population growth as a nested function of nutrient supply and intracellular enzyme abundances. In summary, our study extends the basis for the formulation of predictive mechanistic models of bacterial growth, applicable in industrial bioprocess development

Elucidating cellular mechanisms of Saccharomyces cerevisiae tolerant to combined lignocellulosic-derived inhibitors using high-throughput phenotyping and multiomics analyses

A robust cell factory that can tolerate combined inhibitory lignocellulosic compounds is essential for the cost-effective lignocellulose-based production of second-generation bioethanol and other bulk chemicals. Following high-throughput phenotyping of a yeast genomic overexpression library, we identified a Saccharomyces cerevisiae mutant (denoted AFb.01) with improved growth and fermentation performance under combined toxicity of acetic acid and furfural. AFb.01 carries overexpression of TRX1, which encodes for thioredoxin, a cellular redox machinery. Through comparative proteomics and metabolomics, the resulting cell-wide changes in the mutant were elucidated and these primarily target on the maintenance of energy and redox homeostasis and the minimization of stress-induced cell damages. In particular, the upregulation of the stress-response proteins Hsp26p and Fmp16p conferred tolerance of AFb.01 against protein denaturation and DNA damage. Moreover, increased levels of protectant metabolites such as trehalose, fatty acids, GABA and putrescine provided additional defense mechanisms for the mutant against oxidative and redox stresses. Future studies will concentrate on targeted genetic engineering to validate these mechanisms as well as to support the creation of more robust yeast strains, applicable for industrial, cost-competitive biorefinery production