Strategy for selection of methods for separation of bioparticles from particle mixtures

The desired product of bioprocesses is often produced in particulate form, either as an inclusion body (IB) or as a crystal. Particle harvesting is then a crucial and attractive form of product recovery. Because the liquid phase often contains other bioparticles, such as cell debris, whole cells, particulate biocatalysts or particulate by-products, the recovery of product particles is a complex process. In most cases, the particulate product is purified using selective solubilization or extraction. However, if selective particle recovery is possible, the already high purity of the particles makes this downstream process more favorable. This work gives an overview of typical bioparticle mixtures that are encountered in industrial biotechnology and the various driving forces that may be used for particle-particle separation, such as the centrifugal force, the magnetic force, the electric force, and forces related to interfaces. By coupling these driving forces to the resisting forces, the limitations of using these driving forces with respect to particle size are calculated. It shows that centrifugation is not a general solution for particle-particle separation in biotechnology because the particle sizes of product and contaminating particles are often very small, thus, causing their settling velocities to be too low for efficient separation by centrifugation. Examples of such separation problems are the recovery of IBs or virus-like particles (VLPs) from (microbial) cell debris. In these cases, separation processes that use electrical forces or fluid-fluid interfaces show to have a large potential for particle-particle separation. These methods are not yet commonly applied for large-scale particle-particle separation in biotechnology and more research is required on the separation techniques and on particle characterization to facilitate successful application of these methods in industry.     

A Predictive Model of Bacterial Foraging by Means of Freely Released Extracellular Enzymes

Abstract Extracellular enzymes are important agents for microbial foraging and material cycling in diverse natural and man-made systems. Their abundance and effects are analyzed empirically on scales much larger than the forager. Here, we use a modelling approach to analyze the potential costs and benefits, to an individual immobile microbe, of freely releasing extracellular enzymes into a fluid-bathed, stable matrix of both inert and food-containing particles. The target environments are marine aggregates and sediments, but the results extend to biofilms, bioreactors, soils, stored foods, teeth, gut contents, and even soft tissues attacked by disease organisms. Model predictions, consistent with macroscopic observations of enzyme activity in laboratory and environmental samples, include: support of significant bacterial growth by cell-free enzymes; preponderance of particle-attached, as opposed to dissolved, cell-free enzymes; solubilization of particulate substrates in excess of resident microbe growth requirements; and constitutive, abundant enzyme release in some environments. Feeding with cell-free enzymes appears to be limited to substrates within a well-defined distance of the enzyme source. Fluxes of dissolved organic material out of pelagic oceanic aggregates and marine sediments, and difficulty detecting dissolved enzymes in such environments, may reflect characteristics of cell-free enzyme foraging and properties of the enzymes. Our calculations further suggest that cell-free enzymes may often be used by microorganisms as the fastest means to search for food. Received: 3 June 1997; Accepted: 25 November 1997     

The role of cytochrome P450 monooxygenases in microbial fatty acid metabolism

Cytochrome P450 monooxygenases (P450s) are a diverse collection of enzymes acting on various endogenous and xenobiotic molecules. Most of them catalyse hydroxylation reactions and one group of possible substrates are fatty acids and their related structures. In this minireview, the significance of P450s in microbial fatty acid conversion is described. Bacteria and yeasts possess various P450 systems involved in alkane and fatty acid degradation, and often several enzymes with different activities and specificities are retrieved in one organism. Furthermore, P450s take part in the formation of fatty acid-based secondary metabolites. Finally, there are a substantial number of microbial P450s displaying activity towards fatty acids, but to which no biological role could be assigned despite the often quite intense research.     

Aquatic microfauna alter larval food resources and affect development and biomass of West Nile and Saint Louis encephalitis vector Culex nigripalpus (Diptera: Culicidae)

Ciliate protists and rotifers are ubiquitous in aquatic habitats and can comprise a significant portion of the microbial food resources available to larval mosquitoes, often showing substantial declines in abundance in the presence of mosquito larvae. This top‐down regulation of protists is reported to be strong for mosquitoes inhabiting small aquatic containers such as pitcher plants or tree holes, but the nature of these interactions with larval mosquitoes developing in other aquatic habitats is poorly understood. We examined the effects of these two microbial groups on lower trophic level microbial food resources, such as bacteria, small flagellates, and organic particles, in the water column, and on Culex larval development and adult production. In three independent laboratory experiments using two microeukaryote species (one ciliate protist and one rotifer) acquired from field larval mosquito habitats and cultured in the laboratory, we determined the effects of Culex nigripalpus larval grazing on water column microbial dynamics, while simultaneously monitoring larval growth and development. The results revealed previously unknown interactions that were different from the top‐down regulation of microbial groups by mosquito larvae in other systems. Both ciliates and rotifers, singly or in combination, altered other microbial populations and inhibited mosquito growth. It is likely that these microeukaryotes, instead of serving as food resources, competed with early instar mosquito larvae for microbes such as small flagellates and bacteria in a density‐dependent manner. These findings help our understanding of the basic larval biology of Culex mosquitoes, variation in mosquito production among various larval habitats, and may have implications for existing vector control strategies and for developing novel microbial‐based control methods.     

The cellulose paradox: pollutant par excellence and/or a reclaimable natural resource?

The various aspects of cellulose as a pollutant are considered in view of its lack of toxicity on the one hand and its recalcitrant durable nature on the other. The microbial degradation of cellulosics is discussed, and the contrast between its success in handling natural cellulosic wastes versus its failure to cope with man-made refuse is described. Research carried out in the past decade has demonstrated that cellulolytic organisms are provided with cell surface multifunctional multienzyme conglomerates, called cellulosomes, which are capable of solubilizing solid cellulosic substrates. The intriguing properties of such complexes include their cohesive nature, their many enzymatic components, and a characteristic glycosylated cellulose-binding, scaffolding component. The latter appears to serve as a substrate-targeting carrier, which delivers the other (hydrolytic) components to the cellulose. Progress in establishing efficient model systems for in vitro solubilization of purified cellulose or natural cellulosic substrates has been achieved using purified cellulosome preparations, fortified with -glucosidase and pectinase. The latter enzymes were required in order to alleviate the phenomenon of product inhibition which reduces the efficiency of the free cellulosome. Such combined enzyme systems are proposed as examples of future tailor-made cellulolytic systems for the degradation of natural cellulosics.     

Microbial attachment to particles in marine and freshwater ecosystems

Scanning electron microscopy observations ofin situ suspended marine and freshwater particles show diverse but similar modes of bacterial and fungal attachment. A survey of Sierra Nevada mountain lakes and pelagic and near-shore waters in the Pacific Ocean indicates that attachment is most noticeable in the near-surface waters where fresh dissolved and particulate input of carbon from phytoplankton and elevated temperatures favor microbial growth. The most common modes of attachment are: adhesive stalk formation, growth on adhesive webs, attachment by the use of pili-like appendages and slimy capsular secretions, and molecular or chemical sorption without the use of visualized structural appendages. Attached microbial growth is accelerated when particulate substrates are supplied, even when they are not rich in organic nutrients. This is the case in the Lake Tahoe basin, where microflora attached to eroded silts can significantly modify the organic carbon and nutrient content of such minerogenous particles.     

Oxidative stress-induced DNA damage by particulate air pollution

Exposure to ambient air particulate matter (PM) is associated with pulmonary and cardiovascular diseases and cancer. The mechanisms of PM-induced health effects are believed to involve inflammation and oxidative stress. The oxidative stress mediated by PM may arise from direct generation of reactive oxygen species from the surface of particles, soluble compounds such as transition metals or organic compounds, altered function of mitochondria or NADPH-oxidase, and activation of inflammatory cells capable of generating ROS and reactive nitrogen species. Resulting oxidative DNA damage may be implicated in cancer risk and may serve as marker for oxidative stress relevant for other ailments caused by particulate air pollution. There is overwhelming evidence from animal experimental models, cell culture experiments, and cell free systems that exposure to diesel exhaust and diesel exhaust particles causes oxidative DNA damage. Similarly, various preparations of ambient air PM induce oxidative DNA damage in in vitro systems, whereas in vivo studies are scarce. Studies with various model/surrogate particle preparations, such as carbon black, suggest that the surface area is the most important determinant of effect for ultrafine particles (diameter less than 100 nm), whereas chemical composition may be more important for larger particles. The knowledge concerning mechanisms of action of PM has prompted the use of markers of oxidative stress and DNA damage for human biomonitoring in relation to ambient air. By means of personal monitoring and biomarkers a few studies have attempted to characterize individual exposure, explore mechanisms and identify significant sources to size fractions of ambient air PM with respect to relevant biological effects. In these studies guanine oxidation in DNA has been correlated with exposure to PM(2.5) and ultrafine particles outdoor and indoor. Oxidative stress-induced DNA damage appears to an important mechanism of action of urban particulate air pollution. Related biomarkers and personal monitoring may be useful tools for risk characterization.     

Extracellular enzymes in terrestrial, freshwater, and marine environments: perspectives on system variability and common research needs

Extracellular enzymes produced by heterotrophic microbial communities are major drivers of carbon and nutrient cycling in terrestrial, freshwater, and marine environments. Although carbon and nutrient cycles are coupled on global scales, studies of extracellular enzymes associated with terrestrial, freshwater, and marine microbial communities are not often compared across ecosystems. In part, this disconnect arises because the environmental parameters that control enzyme activities in terrestrial and freshwater systems, such as temperature, pH, and moisture content, have little explanatory power for patterns of enzyme activities in marine systems. Instead, factors such as the functional diversity of microbial communities may explain varying patterns of enzyme activities observed in the ocean to date. In any case, many studies across systems focus on similar issues that highlight the commonalities of microbial community organization. Examples include the effective lifetime of enzymes released into the environment; the extent to which microbial communities coordinate enzyme expression to decompose complex organic substrates; and the influence of microbial community composition on enzyme activities and kinetics. Here we review the often-disparate research foci in terrestrial, freshwater, and marine environments. We consider the extent to which environmental factors may regulate extracellular enzyme activities within each ecosystem, and highlight commonalities and current methodological challenges to identify research questions that may aid in integrating cross-system perspectives in the future.     

Mathematical Modelling of Anaerobic Reactors Treating Domestic Wastewater: Rational Criteria for Model Use

Anaerobic digestion modelling is an established method for assessing anaerobic wastewater treatment for design, systems analysis, operational analysis, and control. Anaerobic treatment of domestic wastewater is a relatively new, but rapidly maturing technology, especially in developing countries, where the combination of low cost, and moderate-good performance are particularly attractive. The key emerging technology is high-rate anaerobic treatment, particularly UASB reactors. Systems modelling can potentially offer a number of advantages to this field, and the key motivations for modelling have been identified as operational analysis, technology development, and model-based design. Design is particularly important, as it determines capital cost, a key motivation for implementers. Published modelling studies for anaerobic domestic sewage treatment are limited in number, but well directed at specific issues. Most have a low structural complexity, with first order kinetics, as compared to the more commonly used Monod kinetics. This review addresses the use of anaerobic models in general, application of models to domestic sewage systems, and evaluates future requirements for models that need to address the key motivations of operational analysis, technology development, and model-based design. For operational analysis and technology development, a complex model such as the ADM1 is recommended, with further extensions as required to address factors such as sulphate reduction. For design, the critical issSues are hydraulics and particles (i.e., biomass and solid substrate) modelling. Therefore, the kinetic structure should be relatively simple (at least two-step), but the hydraulic and particulate model should be relatively complex.     

Microbial synthetic biology for human therapeutics

The emerging field of synthetic biology holds tremendous potential for developing novel drugs to treat various human conditions. The current study discusses the scope of synthetic biology for human therapeutics via microbial approach. In this context, synthetic biology aims at designing, engineering and building new microbial synthetic cells that do not pre-exist in nature as well as re-engineer existing microbes for synthesis of therapeutic products. It is expected that the construction of novel microbial genetic circuitry for human therapeutics will greatly benefit from the data generated by ??omics?? approaches and multidisciplinary nature of synthetic biology. Development of novel antimicrobial drugs and vaccines by engineering microbial systems are a promising area of research in the field of synthetic biology for human theragnostics. Expression of plant based medicinal compounds in the microbial system using synthetic biology tools is another avenue dealt in the present study. Additionally, the study suggest that the traditional medicinal knowledge can do value addition for developing novel drugs in the microbial systems using synthetic biology tools. The presented work envisions the success of synthetic biology for human therapeutics via microbial approach in a holistic manner. Keeping this in view, various legal and socio-ethical concerns emerging from the use of synthetic biology via microbial approach such as patenting, biosafety and biosecurity issues have been touched upon in the later sections.     

Production of natural value-added compounds: an insight into the eugenol biotransformation pathway

During the past few years, the production of natural value-added compounds from microbial sources has gained tremendous importance. Due to an increase in consumer demand for natural products, various food and pharmaceutical industries are continuously in search of novel metabolites obtained from microbial biotransformation. The exploitation of microbial biosynthetic pathways is both feasible and cost effective in the production of natural compounds. The environmentally compatible nature of these products is one major reason for their increasing demand. Novel approaches for natural product biogeneration will take advantage of the current studies on biotechnology, biochemical pathways and microbiology. The interest of the scientific community has shifted toward the use of microbial bioconversion for the production of valuable compounds from natural substrates. The present review focuses on eugenol biotransformation by microorganisms resulting in the formation of various value-added products such as ferulic acid, coniferyl alcohol, vanillin and vanillic acid.     

稳定性同位素探测技术在微生物生态学研究中的应用

稳定性同位素标记技术同分子生物学技术相结合而发展起来的稳定性同位素探测技术（stableisotope probing,SIP）,在对各种环境中微生物群落组成进行遗传分类学鉴定的同时,可确定其在环境过程中的功能,提供复杂群落中微生物相互作用及其代谢功能的大量信息,具有广阔的应用前景.其基本原理是：将原位或微宇宙（microcosm）的环境样品暴露于稳定性同位素富集的基质中,这些样品中存在的某些微生物能够以基质中的稳定（性同位素为碳源或氮源进行物质代谢并满足其自身生长需要,基质中的稳定性同位素被吸收同化进入微生物体内,参与各类物质如核酸（DNA和RNA）及磷脂脂肪酸（PLFA）等的生物合成,通过提取、分离、纯化、分析这些微生物体内稳定性同位素标记的生物标志物,从而将微生物的组成与其功能联系起来.在介绍稳定性同位素培养基质的选择及标记方法、合适的生物标志物的选择及提取分离方法的基础上,举例阐述了此项技术在甲基营养菌、有机污染物降解菌、根际微生物生态、互营微生物、宏基因组学等方面的应用.     

Web of ecological interactions in an experimental gut microbiota

The dynamics of all ecosystems are dictated by intrinsic, density‐dependent mechanisms and by density‐independent environmental forcing. In spite of the importance of the gastrointestinal microbiota in health and disease, the ecology of this system remains largely unknown. Here, we take an ecological approach to gut microbial community analysis, with statistical modelling of time series data from chemostats. This approach removes effects of host forcing, allowing us to describe a network of intrinsic interactions determining the dynamic structure of an experimental gut microbiota. Surprisingly, the main colonization pattern in this simplified model system resembled that of the human infant gut, suggesting a potentially important role of density‐dependent interactions in the early gut microbiota. Knowledge of ecological structures in microbial systems may provide us with a means of controlling such systems by modifying the strength and nature of interactions among microbes and between the microbes and their environment.     

Mass loss and qualitative change in stream-incubated fine particulate organic matter derived from leaves differing in rate of processing

The initial quantitative breakdown of fine particulate organic matter (FPOM) was investigated by measuring the loss (over 73 days) of substrate mass of particles of known size ranges (53–125 µm, 125–250 µm, 250–500 µm, 500 µm-1 mm) and derived from known organic sources (Alnus rubra, Acer macrophyllum, Polystichum munitum). Qualitative examinations (organic content, C : N ratio) also were made. Particles ranging from 500 µm to 1 mm in diameter differed greatly from particles in other size ranges, and results of studies with these particles closely resembled results of coarse particulate (CPOM) leaf pack studies. Despite variation, alder particles generally exhibited the greatest mass loss, those of sword-fern, the least, and mass loss of bigleaf maple particles was intermediate. Organic contents of all particle substrates decreased over time. In general, the C : N ratios of alder particles increased, those of bigleaf maple decreased, and those of sword-fern exhibited little change. All particle substrates were incubated in the field in vials, which allowed for influx of natural detritus of unknown source and period of residence. Given the overall abundance and prevalence of the FPOM resource in lotic systems, standardization of a procedure such as that used in this investigation would be useful in extending understanding of stream system processes, including detrital processing and decomposition.     

Polysaccharases for microbial exopolysaccharides

Microbial exopolysaccharides (EPS) are the substrates for a wide range of enzymes most of which are highly specific. The enzymes are either endoglycanases or polysaccharide lyases and their specificity is determined by carbohydrate structure with uronic acids often playing a major role. The presence of various acyl substituents frequently has little effect on the action of many of the polysaccharases but markedly inhibits some of the polysaccharide lyases including alginate and gellan lyases. The commonest sources of such enzymes can be either microorganisms or bacteriophages. These specific polysaccharide-degrading enzymes can yield oligosaccharide fragments, which are amenable to NMR and other analytical techniques. They have thus proved to be extremely useful in providing information about microbial polysaccharide structures and were routinely used in many such studies. Complex systems containing various mixtures of enzymes may also be effective in the absence of single enzymes but may be difficult to obtain with reproducible activities. Such preparations may also cause extensive degradation of the polysaccharide structure and thus prove less useful in providing information. Commercially available enzyme preparations have seldom proved capable of degrading microbial heteropolysaccharides, although some are active against bacterial alginates and homopolysaccharides including bacterial cellulose and curdlan.     

Lipases in lipophilization reactions

Lipases are used in various sectors, as pharmaceutical, food or detergency industry. Their advantage versus classical chemical catalysts is that they exhibit a better selectivity and operate in milder reaction conditions. Theses enzymes can also be used in lipophilization reactions corresponding to the grafting of a lipophilic moiety to a hydrophilic one such as sugar, amino acids and proteins, or phenolic compounds. The major difficulty to overcome in such enzyme-catalyzed reaction resides in the fact that the two involved substrates greatly differ in term of polarity and solvent affinity. Therefore, several key parameters are to be considered in order to achieve the reaction in satisfactory kinetics and yields. The present review discusses the nature of such parameters (eg solvent nature, water activity, chemical modification of substrates) and illustrates their effect with examples of lipase-catalyzed lipophilization reactions of various sugar, amino acids or phenolic derivatives.     

Effects of elevated atmospheric CO2 concentrations on soil microorganisms

Effects of elevated CO(2) on soil microorganisms are known to be mediated by various interactions with plants, for which such effects are relatively poorly documented. In this review, we summarize and synthesize results from studies assessing impacts of elevated CO(2) on soil ecosystems, focusing primarily on plants and a variety the of microbial processes. The processes considered include changes in microbial biomass of C and N, microbial number, respiration rates, organic matter decomposition, soil enzyme activities, microbial community composition, and functional groups of bacteria mediating trace gas emission such as methane and nitrous oxide. Elevated CO(2) in atmosphere may enhance certain microbial processes such as CH(4) emission from wetlands due to enhanced carbon supply from plants. However, responses of extracellular enzyme activities and microbial community structure are still controversy, because interferences with other factors such as the types of plants, nutrient availabilitial in soil, soil types, analysis methods, and types of CO(2) fumigation systems are not fully understood.     

Lipid based particulate formulations for the delivery of antigen

Particulate adjuvant systems are largely classified according to their functional characteristics, such as the nature of the typical immune response they induce, or their perceived mode of action. From a formulation science perspective, it is practical to classify antigen delivery systems according to the physical nature of the formulations. This article discusses lipid based particulate systems, grouped according to the nature of their predominant lipid constituent.     

Limited versus unlimited membership in microbial communities: evaluation and experimental tests of some paradigms

Recent developments in community ecology have allowed for the synthesis of community models based on principles of limited and unlimited membership. In this discussion, these developments are used as a framework for evaluating the validity of three paradigms that have constrained research on aquatic microbial communities. Because microbes are considered to possess global distributions, species availability is not generally considered to be an important factor determining microbial community composition in most habitats. Requirements for the global distribution of a species are not the same as those for unlimited availability. Rates of propagule transport to isolated and newly formed aquatic systems ( 4 years old) are low enough to have a strong effect on microbial community composition. Natural aquatic systems may require several years to accumulate a full complement of species adapted to environmental conditions at a particular time. Except under extreme circumstances, environmental conditions are not considered to constrain membership in aquatic microbial communities. Most evidence for this contention is based on an inability to detect simple relationships between species distributions and levels of individual environmental parameters. Environmental measurements are often made at a spatial scale much greater than that of the local environment of microbes. Biotic interactions, such as competition, are generally considered to be the predominant force structuring aquatic microbial communities. Although there is an extensive laboratory database to suggest the importance of different types of species interactions, there have been few field studies to confirm this. A general research protocol is described to test predictions derived from current theory of microbial community organization. A mesocosm approach is advocated in order to incorporate crucial aspects of environmental realism into experimental designs while maintaining some of the control found in the laboratory.