Applications of microbial fermentations for production of gluten-free products and perspectives

A gluten-free (GF) diet is recognised as being the only accepted treatment for celiac disease—a permanent autoimmune enteropathy triggered by the ingestion of gluten-containing cereals. The bakery products available in today’s gluten-free market are characterised by lower palatability than their conventional counterparts and may lead to nutritional deficiencies of vitamins, minerals and fibre. Thus, the production of high-quality gluten-free products has become a very important socioeconomical issue. Microbial fermentation by means of lactic acid bacteria and yeast is one of the most ecological/economical methods of producing and preserving food. In this review, the role of a fermentation process for improving the quality of GF products and for developing a new concept of GF products with nutraceutical and health-promoting characteristics will be examined.     

Using flow cytometry to quantify microbial heterogeneity

Flow cytometry is a powerful technique for the study of single cells, and thus it is of particular utility in the study of heterogeneity in microbial populations. This review seeks to highlight the role of flow cytometric analyses in studies of microbial heterogeneity, drawing wherever possible on recently published research articles. Whilst microbial heterogeneity is well documented in both natural and laboratory environments, the underlying causes are less well understood. Possible sources for the heterogeneity that is observed in microbial systems are discussed, together with the flow cytometric tools that aid its study. The role of flow cytometry in molecular biology is discussed with reference to gene reporter systems, which enable heterogeneity of gene expression to be monitored. With the recent sequencing of a variety of microbial genomes, it is anticipated that flow cytometry will have an increasing role to play in studying the effects of gene expression and mutation on heterogeneity, and in resolving the interactions of genetics and physiology.     

Using multi-parameter flow cytometry as a novel approach for physiological characterization of bacteria in microbial fuel cells

This work demonstrates, for the first time, the potential of using multi-parameter flow cytometry to monitor changes in the microbial cytoplasmic membrane integrity and polarization during microbial fuel cells (MFC) operation. Such information is crucial to follow the dynamics of bacteria colonization of the electrodes and their viability maintenance during electrical current production. Interestingly, the results show that during voltage production, the electrostatic gradients of the bacteria cytoplasmic membrane are disturbed, leading to depolarization of a subpopulation (where less than 40% of the cells were polarized). Once the voltage dropped, due to substrate limitation, several cells in the anode supernatant restored their polarized state. This process was reversible and observed over more than 4 cycles of fresh substrate addition. Similar power outputs induced similar membrane polarization results, regardless of the substrate used. The percentage of non-viable cells was maintained constant during current production. This study opens new opportunities to monitor cell behavior, and thus increase the knowledge of dynamic mechanisms responsible for current production at the individual cell level. This technique could be of great interest for the development of new MFC configurations and optimization of MFC operation conditions toward increased performance.     

Selection and optimization of microbial hosts for biofuels production

Currently, the predominant microbially produced biofuel is starch- or sugar-derived ethanol. However, ethanol is not an ideal fuel molecule, and lignocellulosic feedstocks are considerably more abundant than both starch and sugar. Thus, many improvements in both the feedstock and the fuel have been proposed. In this paper, we examine the prospects for bioproduction of four second-generation biofuels (n-butanol, 2-butanol, terpenoids, or higher lipids) from four feedstocks (sugars and starches, lignocellulosics, syngas, and atmospheric carbon dioxide). The principal obstacle to commercial production of these fuels is that microbial catalysts of robust yields, productivities, and titers have yet to be developed. Suitable microbial hosts for biofuel production must tolerate process stresses such as end-product toxicity and tolerance to fermentation inhibitors in order to achieve high yields and titers. We tested seven fast-growing host organisms for tolerance to production stresses, and discuss several metabolic engineering strategies for the improvement of biofuels production.     

Versatile microbial surface-display for environmental remediation and biofuels production

Surface display is a powerful technique that uses natural microbial functional components to express proteins or peptides on the cell exterior. Since the reporting of the first surface-display system in the mid-1980s, a variety of new systems have been reported for yeast, Gram-positive and Gram-negative bacteria. Non-conventional display methods are emerging, eliminating the generation of genetically modified microorganisms. Cells with surface display are used as biocatalysts, biosorbents and biostimulants. Microbial cell-surface display has proven to be extremely important for numerous applications, ranging from combinatorial library screening and protein engineering to bioremediation and biofuels production.     

New method to characterize microbial diversity using flow cytometry

The majority of microorganisms have yet to be cultivated and represent a vast uncharacterized and untapped resource. Here, we report the utilization of a combination of flow cytometry, cultivation, and molecular genetics to develop new methodologies to access and characterize biodiversity in microbial samples. We demonstrate that fluorescent dyes and combinations of dyes can selectively stain portions of bacterial populations that can be isolated as sub-populations using fluorescence-activated cell sorting (FACS). Microbial sub-populations obtained by FACS differ substantially from the original microbial population, as demonstrated by denaturing gradient gel electrophoresis and determination of 16S rRNA gene sequences. These sub-populations can subsequently be used to inoculate microbial growth media, allowing the isolation of different microbial species from those that can be readily cultivated from the original sample using the same microbial growth media. When this technique was applied to the analysis of activated-sludge and Yellowstone Lake hydrothermal vent samples, comparative analysis of 16S rDNA sequences revealed that FACS allowed the detection of numerous bacterial species, including previously unknown species, not readily detectable in the original sample due to low relative abundance. This approach may result in a convenient methodology to more thoroughly characterize microbial biodiversity.     

Metabolic engineering of microbial pathways for advanced biofuels production

Production of biofuels from renewable resources such as cellulosic biomass provides a source of liquid transportation fuel to replace petroleum-based fuels. This endeavor requires the conversion of cellulosic biomass into simple sugars, and the conversion of simple sugars into biofuels. Recently, microorganisms have been engineered to convert simple sugars into several types of biofuels, such as alcohols, fatty acid alkyl esters, alkanes, and terpenes, with high titers and yields. Here, we review recently engineered biosynthetic pathways from the well-characterized microorganisms Escherichia coli and Saccharomyces cerevisiae for the production of several advanced biofuels.     

Applications of flow cytometry to hematopoietic stem cell transplantation

Applications of flow cytometry to clinical and experimental hematopoietic stem cell transplantation (HSCT) are discussed in this review covering the following topics: diagnosis and classification of lymphohematologic disorders, quantitation of hematopoietic progenitors in the graft, lymphohematopoietic reconstitution following HSCT and animal models of human HSCT. At the end, the utilization of flow cytometry in clinical HSCT by Brazilian transplant centers is briefly reviewed.     

Applications of flow cytometry to ecotoxicity testing using microalgae

Flow cytometry is a rapid method for the quantitative measurement of light scattering and fluorescent properties of cells. Although this technique has been widely applied to biomedical and environmental studies, its potential as a tool in ecotoxicological studies has not yet been fully exploited. This article describes the application of flow cytometry to the development of bioassays with marine and freshwater algae for assessing the bioavailability of contaminants in waters and sediments.     

Monitoring Rhodotorula glutinis CCMI 145 physiological response and oil production growing on xylose and glucose using multi-parameter flow cytometry

Flow cytometry was used to monitor the lipid content, viability and intrinsic light scatter properties of Rhodotorula glutinis CCMI 145 cells growing on batch cultures using xylose and glucose as carbon sources. The highest lipid content was observed for cells grown on glucose, at the end of the exponential phase (17.8% w/w). The proportion of cells stained with PI attaining 77% at the end of the glucose growth. Cells growing on xylose produced a maximum lipid content of 10.6% (w/w), at the stationary phase. An increase in the proportion of cells stained with PI was observed, reaching 29% at the end of xylose growth. Changes in the side and forward light scatter detected during the yeast batch cultures supported that R. glutinis cells grown on glucose experienced harsher conditions, resulting in a high level of cytoplasmic membrane damage, which did not occur when R. glutinis cells grew on xylose.     

Applications of flow cytometry in cellular pharmacology

Cellular pharmacology is defined as the study of drug effects on various cell functions. Flow cytometry enriches cellular pharmacology by the following possibilities for efficient analysis. Firstly, the determination of toxic concentrations can be approached by the assessment of cell viability. However, due to the existence of many fluorescent DNA probes, most studies are devoted to the investigation of products acting on cell division, particularly in the area of antineoplastic drugs. The effects of drugs on respiration can be approached by analysis of mitochondrial activities. On the other hand, the studies of drug actions on cell differentiation functions have been started using antisera or monoclonal antibodies to cell-specific proteins such as collagen and keratin. Flow cytometry appears to be more and more important in the progress of cellular toxicology and pharmacology.     

Differential response of human basophil activation markers: a multi-parameter flow cytometry approach

Background

Basophils are circulating cells involved in hypersensitivity reactions and allergy but many aspects of their activation, including the sensitivity to external triggering factors and the molecular aspects of cell responses, are still to be focused. In this context, polychromatic flow cytometry (PFC) is a proper tool to investigate basophil function, as it allows to distinguish the expression of several membrane markers upon activation in multiple experimental conditions.

Methods

Cell suspensions were prepared from leukocyte buffy coat of K2-EDTA anticoagulated blood specimens; about 1500-2500 cellular events for each tested sample, gated in the lymphocyte CD45dim area and then electronically purified as HLADRnon expressing/CD123bright, were identified as basophilic cells. Basophil activation with fMLP, anti-IgE and calcium ionophore A23187 was evaluated by studying up-regulation of the indicated membrane markers with a two-laser six-color PFC protocol.

Results

Following stimulation, CD63, CD13, CD45 and the ectoenzyme CD203c up-regulated their membrane expression, while CD69 did not; CD63 expression occurred immediately (within 60 sec) but only in a minority of basophils, even at optimal agonist doses (in 33% and 14% of basophils, following fMLP and anti-IgE stimulation respectively). CD203c up-regulation occurred in the whole basophil population, even in CD63non expressing cells. Dose-dependence curves revealed CD203c as a more sensitive marker than CD63, in response to fMLP but not in response to anti-IgE and to calcium ionophore.

Conclusion

Use of polychromatic flow cytometry allowed efficient basophil electronic purification and identification of different behaviors of the major activation markers. The simultaneous use of two markers of activation and careful choice of activator are essential steps for reliable assessment of human basophil functions.     

Fatty acid alkyl esters: perspectives for production of alternative biofuels

The global economy heads for a severe energy crisis: whereas the energy demand is going to rise, easily accessible sources of crude oil are expected to be depleted in only 10–20 years. Since a serious decline of oil supply and an associated collapse of the economy might be reality very soon, alternative energies and also biofuels that replace fossil fuels must be established. In addition, these alternatives should not further impair the environment and climate. About 90% of the biofuel market is currently captured by bioethanol and biodiesel. Biodiesel is composed of fatty acid alkyl esters (FAAE) and can be synthesized by chemical, enzymatic, or in vivo catalysis mainly from renewable resources. Biodiesel is already established as it is compatible with the existing fuel infrastructure, non-toxic, and has superior combustion characteristics than fossil diesel; and in 2008, the global production was 12.2 million tons. The biotechnological production of FAAE from low cost and abundant feedstocks like biomass will enable an appreciable substitution of petroleum diesel. To overcome high costs for immobilized enzymes, the in vivo synthesis of FAAE using bacteria represents a promising approach. This article points to the potential of different FAAE as alternative biofuels, e.g., by comparing their fuel properties. In addition to conventional production processes, this review presents natural and genetically engineered biological systems capable of in vivo FAAE synthesis.     

Application of multi-parameter flow cytometry using fluorescent probes to study substrate toxicity in the indene bioconversion

The bioconversion of indene to cis-(1S,2R) indandiol, a potential key intermediate in the synthesis of Merck's HIV protease inhibitor, CRIXIVAN trade mark, can be achieved using a Rhodococcus strain. This study using Rhodococcus I24 reports on the application of multiparameter flow cytometry for the measurement of cell physiological properties based on cytoplasmic membrane (CM) integrity and membrane depolarization as indicators of toxic effects of the substrate, indene. Quantification of intact polarized CM, intact depolarized CM and permeabilized CM of a large population of bacterial cells has been conducted using specific intracellular and membrane-binding fluorescent stains. Measurements of oxygen uptake rate (OUR) and optical density (OD) as indicators of metabolic activity and biomass growth, respectively, were also made. Indene concentrations of up to 0.25 g/L (0.037 g indene/g dry cell weight) did not significantly (<5% compared to control) affect cell light-scattering properties, intact CM, membrane polarization, respiratory activity, or biomass growth. Between this value and 1.5 g/L (0.221 g indene/g dry cell weight), the changes in intact CM, respiratory activity and biomass growth were relatively insignificant (<5% compared to control), although dissipation of the membrane potential of a significant proportion of the cell population occurred at 0.50 g/L (0.074 g indene/g dry cell weight). At 2.5 g/L (0.368 g indene/g dry cell weight) there was a significant increase in the dead cell population, accompanied by changes in the extracellular cationic concentrations and substantial decrease in respiratory activity. The primary effect of indene toxicity was the disruption of the proton motive force across the cytoplasmic membrane which drives the formation of ATP. The disruption of the proton motive force may have been due to the measured changes in proton permeability across the membrane. In addition, indene may have directly inhibited the membrane-bound enzymes related to respiratory activity. The overall consequence of this was reduced respiratory activity and biomass growth. The cell physiological properties measured via flow cytometry are important for understanding the effects of toxicity at the cellular level which neither measurements of biomass growth or indandiol formation rates can provide since both are cell averaged measurements. The technique described here can also be used as a generic tool for measuring cell membrane properties in response to toxicity of other indene-resistant strains that may be possible to use as recombinant hosts to perform the biotransformation of indene. This study has demonstrated that flow cytometry is a powerful tool for the measurement of cell physiological properties to assess solvent toxicity on whole cell biocatalysts.     

Induction studies with Escherichia coli expressing recombinant interleukin-13 using multi-parameter flow cytometry

The expression of interleukin-13 (IL13) following induction with IPTG in Escherichia coli results in metabolic changes as indicated by multi-parameter flow cytometry and traditional methods of fermentation profiling (O₂ uptake rate, CO₂ evolution rate and optical density measurements). Induction early in the rapid growth phase was optimal although this led to lower overall biomass concentrations and lower maximum specific growth rates. In contrast, induction in the mid-rapid growth phase was the most detrimental to cell quality as measured by cytoplamsic membrane depolarisation.     

流式细胞技术在微生物检测领域的应用研究

研究应用流式细胞技术(flow cytometry method,FCM)进行快速微生物检测的方法。与传统微生物检测方法相比,FCM法更快速和准确。经Pearson相关系数分析表明,在一定浓度范围内,FCM检测法与标准平板检测法(SPC)具有极强线性相关性。经Q检验法分析,FCM检测法具有良好的重复性。由此可见,FCM法可成为一种替代传统微生物检测法的自动化仪器检测新技术。     

Carbon-isotopic analysis of microbial cells sorted by flow cytometry

One of the outstanding current problems in both geobiology and environmental microbiology is the quantitative analysis of in situ microbial metabolic activities. Techniques capable of such analysis would have wide application, from quantifying natural rates of biogeochemical cycling to identifying the metabolic activity of uncultured organisms. We describe here a method that represents one step towards that goal, namely the high‐precision measurement of ¹³C in specific populations of microbial cells that are purified by fluorescence‐activated cell sorting (FACS). Sorted cells are concentrated on a Teflon membrane filter, and their ¹³C content is measured by coupling an isotope ratio mass spectrometer (IRMS) with a home‐built spooling wire microcombustion (SWiM) apparatus. The combined instrumentation provides measurements of δ¹³C in whole cells with precision better than 0.2‰ for samples containing as little as 25 ng of carbon. When losses associated with sample handling are taken into account, isotopic analyses require sorting roughly 10⁴ eukaryotic or 10⁷ bacterial cells per sample. Coupled with ¹³C‐labelled substrate additions, this approach has the potential to directly quantify uptake of metabolites in specific populations of sorted cells. The high precision afforded by SWiM‐IRMS also permits useful studies of natural abundance variations in ¹³C. The approach is equally applicable to specific populations of cells sorted from multicellular organisms.     

An evaluation of the anti-bacterial action of ceramic powder slurries using multi-parameter flow cytometry

Multi-parameter flow cytometric techniques have been used to study the effects of three ceramic powders CaO, MgO and ZnO on the physiology of individual, exponentially growing E. coli cells. Whilst all three powders inhibited reproductive growth, depending on their concentration, the mechanism of action of CaO and MgO was different to that of ZnO as shown by fluorescent staining techniques developed in our laboratory.