The influence of cultivation methods on Shewanella oneidensis physiology and proteome expression

High-throughput analyses that are central to microbial systems biology and ecophysiology research benefit from highly homogeneous and physiologically well-defined cell cultures. While attention has focused on the technical variation associated with high-throughput technologies, biological variation introduced as a function of cell cultivation methods has been largely overlooked. This study evaluated the impact of cultivation methods, controlled batch or continuous culture in bioreactors versus shake flasks, on the reproducibility of global proteome measurements in Shewanella oneidensis MR-1. Variability in dissolved oxygen concentration and consumption rate, metabolite profiles, and proteome was greater in shake flask than controlled batch or chemostat cultures. Proteins indicative of suboxic and anaerobic growth (e.g., fumarate reductase and decaheme c-type cytochromes) were more abundant in cells from shake flasks compared to bioreactor cultures, a finding consistent with data demonstrating that “aerobic” flask cultures were O₂ deficient due to poor mass transfer kinetics. The work described herein establishes the necessity of controlled cultivation for ensuring highly reproducible and homogenous microbial cultures. By decreasing cell to cell variability, higher quality samples will allow for the interpretive accuracy necessary for drawing conclusions relevant to microbial systems biology research.     

Plant cell and tissue culture: alternatives for metabolite production

Plant cell culture systems represent a potential renewable source of valuable medicinals, flavours, essences and colourants that cannot be produced by microbial cells or chemical syntheses. However, only a few cultures produce these compounds in commercially useful amounts. The low productivities are associated with our poor understanding of the biochemistry of these systems. Recent advances in molecular biology, enzymology, physiology and fermentation technology of plant cell cultures suggest that these systems will become a viable source of important natural products. This review examines the sate of the art of production of medicinal plant secondary metabolites by plant cell cultures.     

Tissue culture on a chip: Developmental biology applications of self‐organized capillary networks in microfluidic devices

Organ culture systems are used to elucidate the mechanisms of pattern formation in developmental biology. Various organ culture techniques have been used, but the lack of microcirculation in such cultures impedes the long‐term maintenance of larger tissues. Recent advances in microfluidic devices now enable us to utilize self‐organized perfusable capillary networks in organ cultures. In this review, we will overview past approaches to organ culture and current technical advances in microfluidic devices, and discuss possible applications of microfluidics towards the study of developmental biology.     

An in vitro approach to ruminant mammary gland biology

This review discusses both fundamental and applied in vitro studies on ruminant mammary gland biology and summarizes progress made over the last decade in development of in vitro techniques to study growth, function and pathology of the mammary gland. The advantages and limitations of different in vitro systems are considered including explant cultures, primary cell cultures and immortalized lines of mammary-derived cells from cow, sheep and goat. The cell growth, differentiation and response to lactogenic hormones and growth factors are discussed as well as the relevance of the cell behavior in different culture conditions.     

Continuous cultures limited by a gaseous substrate: Development of a simple, unstructured mathematical model and experimental verification with Methanobacterium thermoautotrophicum

This article presents a simple, unstructured mathematical model describing microbial growth in continuous culture limited by a gaseous substrate. The model predicts constant gas conversion rates and a decreasing biomass concentration with increasing dilution rate. It has been found that the parameters influencing growth are primarily the gas transfer rate and the dilution rate. Furthermore, it is shown that, for correct simulation of growth, the influence of gaseous substrate consumption on the effective gas flow through the system has to be taken into account.Continuous cultures of Methanobacterium thermoautotrophicum were performed at three different gassing rates. In addition to the measurement of the rates of biomass production, product formation, and substrate consumption, microbial heat dissipation was assessed using a reaction calorimeter. For the on-line measurement of the concentration of the growth-limiting substrate, H(2), a specially developed probe has been used. Experimental data from continuous cultures were in good agreement with the model simulations. An increase in gassing rate enhanced gaseous substrate consumption and methane production rates. However, the biomass yield as well as the specific conversion rates remained constant, irrespective of the gassing rate. It was found that growth performance in continuous culture limited by a gaseous substrate is substantially different from "classic" continuous culture in which the limiting substrate is provided by the liquid feed. In this report, the differences between both continuous culture systems are discussed.     

合成生物学伦理、法律与社会问题探讨

合成生物学是以基因组学、系统生物学知识和分子生物学技术为基础,综合了科学与工程的一门新兴交叉学科。它使生命科学和生物技术研发进入了以人工设计、合成自然界中原本不曾出现的人造生命体系,以及对这些人工体系进行体内、体外优化,或利用这些人造生命体系研究自然生命规律为目标的新时代。然而,合成生物学研究在迅速发展、表现出巨大潜力和应用前景的同时,也引发了社会各界对相关社会、伦理、安全,以及知识产权等问题的重视与讨论。就世界各国针对合成生命对传统意义上生命概念的挑战、合成生物学产品存在的潜在风险危害、合成生物学研究的风险评估与监管等问题进行回顾综述和相关探讨。     

合成生物学技术在环境保护中的应用

合成生物学是一个基于生物学和工程学原理的科学领域,其目的是重新设计和重组微生物,以优化或创建具有增强功能的新生物系统。该领域利用分子工具、系统生物学和遗传框架的重编程,从而构建合成途径以获得具有替代功能的微生物。传统上,合成生物学方法通常旨在开发具有成本效益的微生物细胞工厂进而从可再生资源中生产化学物质。然而,近年来合成生物学技术开始在环境保护中发挥着更直接的作用。本综述介绍了基因工程中的合成生物学工具,讨论了基于基因工程的微生物修复策略,强调了合成生物学技术可以通过响应特定污染物进行生物修复来保护环境。其中,规律间隔成簇短回文重复序列(Clustered Regularly Interspersed Short Palindromic Repeats, CRISPR)技术在基因工程细菌和古细菌的生物修复中得到了广泛应用,生物修复领域也出现了很多新的先进技术,包括生物膜工程、人工微生物群落的构建、基因驱动、酶和蛋白质工程等。有了这些新的技术和工具,生物修复将成为当今最好和最有效的污染物去除方式之一。     

污染土壤微生物群落结构分子鉴定技术研究进展

土壤微生物群落结构多样性检测是土壤修复、监测、评估时的一个重要参数。由于绝大多数微生物在实验室条件下是不可培养,因而早期依赖于微生物培养的检测结果代表性不强。20世纪90年代以来,不依赖于微生物培养的分子生物学方法是研究微生物群落结构的重要手段。该文对近年来土壤污染微生物群落结构研究所采用的主要分子生物学方法按照其原理进行了比较、分析、总结。根据不同技术的灵敏度、优缺点分析了其适用范围。指出了目前技术中存在的一些共性问题和缺陷并展望了土壤修复领域分子生物学技术的发展趋势。     

Studies on the Growth in Culture of Plant Cells: XII. A VERSATILE SYSTEM FOR THE LARGE SCALE BATCH OR CONTINUOUS CULTURE OF PLANT CELL SUSPENSIONS

A versatile large-scale batch culture unit has been developedfor the growth of plant cell suspension cultures. This unithas been modified to permit of intermittent or continuous renewalof culture medium and, in a modified form, incorporated intoopen continuous culture systems of the chemostat and turbidostattype. A fully automatic culture sampler has been incorporatedinto the basic culture unit. The culture systems described havebeen successfully operated using a cell suspension derived fromAcer pseudoplatanus and results are presented demonstratingsynchronous growth in batch culture, prolonged logarithmic increassein cell number under conditions of high aeration and culturemedium renewal, and steady states of growth resulting from automaticregulation of the optical density of the cell suspension andfrom fixed rates of displacement of cell suspension by new medium.The potentialities of the culture systems are discussed in thelight of the experimental results presented.     

Dynamics of microbial propagation: Models considering inhibitors and variable cell composition

Mathematical models for microbial growth in batch and continuous cultures are formulated. The models have been referred to as distributed models since the microbial population in a culture is looked upon as protoplasmic mass distributed uniformly throughout the culture. Growth is regarded as the increase in this mass by conversion of medium components into biological mass and metabolic products. Two sets of models have been presented. The first arise from introducing additional considerations into the model proposed by Monod to account for the stationary phase and the phase of decline in a batch culture. These have been referred to as unstructured, distributed models since they do not recognize any form of structure in the protoplasmic mass. The models in the second set are referred to as structured, distributed models. Structure is introduced by considering the protoplasmic mass to be composed of two groups of substances which interact with each other and with substances in the environment to produce growth. The structured models account for the dependence of growth on the past, history of the cells; thus they predict all growth phases observed in batch cultures, whereas the unstructured models do not predict a lag phase. The full implications of the models for continuous propagation, as determined by the method of stability analysis and transient calculations, are discussed. The models prediet a number of new results and should be confronted with experiments.     

Dynamic and steady state studies of phenol biodegradation in pure and mixed cultures.

The microbial degradation of phenol by pure and mixed cultures of Pseudomonas putida was studied in batch, phenol-stat, and continuous culture systems. In the continuous culture runs, both steady state and transient experiments were performed. From these experiments, a model for the kinetic behavior of the organisms was evolved and an analysis performed on the stability and dynamic behavior of pure and mixed cultures. The results indicate that it should be possible to achieve phenol removal from wastewaters down to levels of 1-2 ppm in a single state system. However, because of the effect of substrate inhibition on kinetic behavior of the microorganisms, long lasting transients can occur. The transient behavior of such systems cannot be solely determined from mumax or Ks parameters, but must include a consideration of the transient size and response characteristic of the organism.     

The energetics of microbes at slow growth rates: Maintenance energies and dormant organisms

In continuous cultures at slow growth rates (less than about 10% maximum) bacterial growth yields from the carbon and energy source are higher than those expected. To account for this deviation it is proposed that dormant or non-viable cells with zero maintenance energy are generated at slow growth rates. From the growth yield variation, it is shown, the dormant fraction of the culture can be calculated. The few quantitative data available on the viability of bacteria in chemostat steady states at very slow growth rates are in agreement with the hypothesis. It is suggested that enzymic, chemical and morphological characters also may be used to distinguish between the growing and dormant fractions of a culture.The model provides a unifying theory for studies of microbial function at slow growth rates, which is a field of great practical importance.     

Maximization of steady-state bacterial production in a chemostat with pH and substrate control.

This analytical study deals with the steady-state behavior and control of microbial growth in continuous cultures. A second order Haldane-Monod model of continuous cultures is used as a basis for study of the effects of the adjustment of pH by the addition of acidic (or basic) materials. The treatment of a hydrogen ion concentration, in addition to substrate and microbial concentrations as state variables, results in a third order system of equations describing the process. The analysis of the system in equilibrium yields several admissible steady states, that is, steady states which satisfy all constraints. An optimal control problem is formulated and subsequently solved to maximize steady-state microbial production.     

Advances in molecular cloning

“Molecular cloning” meaning creation of recombinant DNA molecules has impelled advancement throughout life sciences. DNA manipulation has become easy due to powerful tools showing exponential growth in applications and sophistication of recombinant DNA technology. Cloning genes has become simple what led to an explosion in the understanding of gene function by seamlessly stitching together multiple DNA fragments or by the use of swappable gene cassettes, maximizing swiftness and litheness. A novel archetype might materialize in the near future with synthetic biology techniques that will facilitate quicker assembly and iteration of DNA clones, accelerating the progress of gene therapy vectors, recombinant protein production processes and new vaccines by in vitro chemical synthesis of any in silico-specified DNA construct. The advent of innovative cloning techniques has opened the door to more refined applications such as identification and mapping of epigenetic modifications and high-throughput assembly of combinatorial libraries. In this review, we will examine the major breakthroughs in cloning techniques and their applications in various areas of biological research that have evolved mainly due to easy construction of novel expression systems.     

Using single‐cell genomics to understand developmental processes and cell fate decisions

High‐throughput ‐omics techniques have revolutionised biology, allowing for thorough and unbiased characterisation of the molecular states of biological systems. However, cellular decision‐making is inherently a unicellular process to which “bulk” ‐omics techniques are poorly suited, as they capture ensemble averages of cell states. Recently developed single‐cell methods bridge this gap, allowing high‐throughput molecular surveys of individual cells. In this review, we cover core concepts of analysis of single‐cell gene expression data and highlight areas of developmental biology where single‐cell techniques have made important contributions. These include understanding of cell‐to‐cell heterogeneity, the tracing of differentiation pathways, quantification of gene expression from specific alleles, and the future directions of cell lineage tracing and spatial gene expression analysis.     

Muscle cell culture as a tool in animal growth research

Muscle cell culture techniques have been used for several years in research on muscle growth and development. Several types of culture systems have been devised, including primary cultures from embryonic or postnatal muscle and myogenic cell lines. In addition, serum-free and serum-containing media have been developed to address specific muscle development questions. Many of these questions center around muscle cell differentiation and muscle cell physiology; and, more recently, muscle cell cultures have been used as bioassay tools for examining growth physiology in domestic animals. In our laboratory, skeletal muscle satellite cells have been studied in vitro to evaluate the effect of several protein hormones and growth factors on satellite cell proliferation and differentiation. Of the hormones examined, only the insulin-like growth factors/somatomedins and fibroblast growth factor have been shown to have a stimulatory effect on proliferation that could be physiologically significant. None of the major anterior pituitary hormones interacted directly with satellite cells to stimulate proliferation. With advances in serum-free medium formulations and cell separation techniques, more information can be obtained from experiments with muscle cell cultures. With appropriate design and interpretation, our knowledge of muscle growth in domestic animals will be expanded.     

Growth Kinetics of Suspended Microbial Cells: From Single-Substrate-Controlled Growth to Mixed-Substrate Kinetics

Growth kinetics, i.e., the relationship between specific growth rate and the concentration of a substrate, is one of the basic tools in microbiology. However, despite more than half a century of research, many fundamental questions about the validity and application of growth kinetics as observed in the laboratory to environmental growth conditions are still unanswered. For pure cultures growing with single substrates, enormous inconsistencies exist in the growth kinetic data reported. The low quality of experimental data has so far hampered the comparison and validation of the different growth models proposed, and only recently have data collected from nutrient-controlled chemostat cultures allowed us to compare different kinetic models on a statistical basis. The problems are mainly due to (i) the analytical difficulty in measuring substrates at growth-controlling concentrations and (ii) the fact that during a kinetic experiment, particularly in batch systems, microorganisms alter their kinetic properties because of adaptation to the changing environment. For example, for Escherichia coli growing with glucose, a physiological long-term adaptation results in a change in K_S for glucose from some 5 mg liter⁻¹ to ca. 30 μg liter⁻¹. The data suggest that a dilemma exists, namely, that either “intrinsic” K_S (under substrate-controlled conditions in chemostat culture) or μ_max (under substrate-excess conditions in batch culture) can be measured but both cannot be determined at the same time. The above-described conventional growth kinetics derived from single-substrate-controlled laboratory experiments have invariably been used for describing both growth and substrate utilization in ecosystems. However, in nature, microbial cells are exposed to a wide spectrum of potential substrates, many of which they utilize simultaneously (in particular carbon sources). The kinetic data available to date for growth of pure cultures in carbon-controlled continuous culture with defined mixtures of two or more carbon sources (including pollutants) clearly demonstrate that simultaneous utilization results in lowered residual steady-state concentrations of all substrates. This should result in a competitive advantage of a cell capable of mixed-substrate growth because it can grow much faster at low substrate concentrations than one would expect from single-substrate kinetics. Additionally, the relevance of the kinetic principles obtained from defined culture systems with single, mixed, or multicomponent substrates to the kinetics of pollutant degradation as it occurs in the presence of alternative carbon sources in complex environmental systems is discussed. The presented overview indicates that many of the environmentally relevant apects in growth kinetics are still waiting to be discovered, established, and exploited.