Chemostat | |
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Industry | Biological engineering |
Application | Research and Industry |
A chemostat (from chemical environment is static) is a bioreactor to which fresh medium is continuously added, while culture liquid containing left over nutrients, metabolic end products and microorganisms is continuously removed at the same rate to keep the culture volume constant. [2] [3] By changing the rate with which medium is added to the bioreactor the specific growth rate of the microorganism can be easily controlled within limits.
One of the most important features of chemostats is that microorganisms can be grown in a physiological steady state under constant environmental conditions. In this steady state, growth occurs at a constant specific growth rate and all culture parameters remain constant (culture volume, dissolved oxygen concentration, nutrient and product concentrations, pH, cell density, etc.). In addition, environmental conditions can be controlled by the experimenter. [4] Microorganisms growing in chemostats usually reach a steady state because of a negative feedback between growth rate and nutrient consumption: if a low number of cells are present in the bioreactor, the cells can grow at growth rates higher than the dilution rate as they consume little nutrient so growth is less limited by the addition of limiting nutrient with the inflowing fresh medium. The limiting nutrient is a nutrient essential for growth, present in the medium at a limiting concentration (all other nutrients are usually supplied in surplus). However, the higher the number of cells becomes, the more nutrient is consumed, lowering the concentration of the limiting nutrient. In turn, this will reduce the specific growth rate of the cells, which will lead to a decline in the number of cells as they keep being removed from the system with the outflow. This results in a steady state. Due to self-regulation, the steady state is stable. This enables the experimenter to control the specific growth rate of the microorganisms by changing the speed of the pump feeding fresh medium into the vessel.
Another important feature of chemostats and other continuous culture systems is that they are well-mixed so that environmental conditions are homogenous or uniform and microorganisms are randomly dispersed and encounter each other randomly. Therefore, competition and other interactions in the chemostat are global, in contrast to biofilms.
The rate of nutrient exchange is expressed as the dilution rate D. At steady state, the specific growth rate μ of the micro-organism is equal to the dilution rate D. The dilution rate is defined as the flow of medium per unit of time, F, over the volume V of culture in the bioreactor
Specific growth rate μ is inversely related to the time it takes the biomass to double, called doubling time td, by:
Therefore, the doubling time td becomes a function of dilution rate D in steady state:
Each microorganism growing on a particular substrate has a maximal specific growth rate μmax (the rate of growth observed if growth is limited by internal constraints rather than external nutrients). If a dilution rate is chosen that is higher than μmax, the cells cannot grow at a rate as fast as the rate with which they are being removed so the culture will not be able to sustain itself in the bioreactor, and will wash out.
However, since the concentration of the limiting nutrient in the chemostat cannot exceed the concentration in the feed, the specific growth rate that the cells can reach in the chemostat is usually slightly lower than the maximal specific growth rate because specific growth rate usually increases with nutrient concentration as described by the kinetics of the Monod equation. [ citation needed ] The highest specific growth' rates (μmax) cells can attain is equal to the critical dilution rate (D'c):
where S is the substrate or nutrient concentration in the chemostat and KS is the half-saturation constant (this equation assumes Monod kinetics).
Chemostats in research are used for investigations in cell biology, as a source for large volumes of uniform cells or protein. The chemostat is often used to gather steady state data about an organism in order to generate a mathematical model relating to its metabolic processes. Chemostats are also used as microcosms in ecology [5] [6] and evolutionary biology. [7] [8] [9] [10] In the one case, mutation/selection is a nuisance, in the other case, it is the desired process under study. Chemostats can also be used to enrich for specific types of bacterial mutants in culture such as auxotrophs or those that are resistant to antibiotics or bacteriophages for further scientific study. [11] Variations in the dilution rate permit the study of the metabolic strategies pursued by the organisms at different growth rates. [12] [13]
Competition for single and multiple resources, the evolution of resource acquisition and utilization pathways, cross-feeding/symbiosis, [14] [15] antagonism, predation, and competition among predators have all been studied in ecology and evolutionary biology using chemostats. [16] [17] [18]
Chemostats are frequently used in the industrial manufacturing of ethanol. In this case, several chemostats are used in series, each maintained at decreasing sugar concentrations.[ citation needed ] The chemostat also serves as an experimental model of continuous cell cultures in the biotechnological industry. [13]
Continuous efforts to remedy each defect lead to variations on the basic chemostat quite regularly. Examples in the literature are numerous.
This section relies largely or entirely on a single source .(December 2019) |
Fermentation setups closely related to the chemostats are the turbidostat, the auxostat and the retentostat. In retentostats, culture liquid is also removed from the bioreactor, but a filter retains the biomass. In this case, the biomass concentration increases until the nutrient requirement for biomass maintenance has become equal to the amount of limiting nutrient that can be consumed.
Bacterial growth is proliferation of bacterium into two daughter cells, in a process called binary fission. Providing no mutation event occurs, the resulting daughter cells are genetically identical to the original cell. Hence, bacterial growth occurs. Both daughter cells from the division do not necessarily survive. However, if the surviving number exceeds unity on average, the bacterial population undergoes exponential growth. The measurement of an exponential bacterial growth curve in batch culture was traditionally a part of the training of all microbiologists; the basic means requires bacterial enumeration by direct and individual, direct and bulk (biomass), indirect and individual, or indirect and bulk methods. Models reconcile theory with the measurements.
An agar plate is a Petri dish that contains a growth medium solidified with agar, used to culture microorganisms. Sometimes selective compounds are added to influence growth, such as antibiotics.
The lactose operon is an operon required for the transport and metabolism of lactose in E. coli and many other enteric bacteria. Although glucose is the preferred carbon source for most enteric bacteria, the lac operon allows for the effective digestion of lactose when glucose is not available through the activity of β-galactosidase. Gene regulation of the lac operon was the first genetic regulatory mechanism to be understood clearly, so it has become a foremost example of prokaryotic gene regulation. It is often discussed in introductory molecular and cellular biology classes for this reason. This lactose metabolism system was used by François Jacob and Jacques Monod to determine how a biological cell knows which enzyme to synthesize. Their work on the lac operon won them the Nobel Prize in Physiology in 1965.
A bioreactor is any manufactured device or system that supports a biologically active environment. In one case, a bioreactor is a vessel in which a chemical process is carried out which involves organisms or biochemically active substances derived from such organisms. This process can either be aerobic or anaerobic. These bioreactors are commonly cylindrical, ranging in size from litres to cubic metres, and are often made of stainless steel. It may also refer to a device or system designed to grow cells or tissues in the context of cell culture. These devices are being developed for use in tissue engineering or biochemical/bioprocess engineering.
A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation or small plants like the moss Physcomitrella patens. Different types of media are used for growing different types of cells.
The Luria–Delbrück experiment (1943) demonstrated that in bacteria, genetic mutations arise in the absence of selective pressure rather than being a response to it. Thus, it concluded Darwin's theory of natural selection acting on random mutations applies to bacteria as well as to more complex organisms. Max Delbrück and Salvador Luria won the 1969 Nobel Prize in Physiology or Medicine in part for this work.
Industrial fermentation is the intentional use of fermentation in manufacturing processes. In addition to the mass production of fermented foods and drinks, industrial fermentation has widespread applications in chemical industry. Commodity chemicals, such as acetic acid, citric acid, and ethanol are made by fermentation. Moreover, nearly all commercially produced industrial enzymes, such as lipase, invertase and rennet, are made by fermentation with genetically modified microbes. In some cases, production of biomass itself is the objective, as is the case for single-cell proteins, baker's yeast, and starter cultures for lactic acid bacteria used in cheesemaking.
In microbiology, a colony-forming unit is a unit which estimates the number of microbial cells in a sample that are viable, able to multiply via binary fission under the controlled conditions. Counting with colony-forming units requires culturing the microbes and counts only viable cells, in contrast with microscopic examination which counts all cells, living or dead. The visual appearance of a colony in a cell culture requires significant growth, and when counting colonies, it is uncertain if the colony arose from a single cell or a group of cells. Expressing results as colony-forming units reflects this uncertainty.
A photobioreactor (PBR) refers to any cultivation system designed for growing photoautotrophic organisms using artificial light sources or solar light to facilitate photosynthesis. Photobioreactors are typically used to cultivate microalgae, cyanobacteria, and some mosses. Photobioreactors can be open systems, such as raceway ponds, which rely upon natural sources of light and carbon dioxide. Closed photobioreactors are flexible systems that can be controlled to the physiological requirements of the cultured organism, resulting in optimal growth rates and purity levels. Photobioreactors are typically used for the cultivation of bioactive compounds for biofuels, pharmaceuticals, and other industrial uses.
Adaptive mutation, also called directed mutation or directed mutagenesis is a controversial evolutionary theory. It posits that mutations, or genetic changes, are much less random and more purposeful than traditional evolution, implying that organisms can respond to environmental stresses by directing mutations to certain genes or areas of the genome. There have been a wide variety of experiments trying to support the idea of adaptive mutation, at least in microorganisms.
In microbiology, streaking is a technique used to isolate a pure strain from a single species of microorganism, often bacteria. Samples can then be taken from the resulting colonies and a microbiological culture can be grown on a new plate so that the organism can be identified, studied, or tested.
Fed-batch culture is, in the broadest sense, defined as an operational technique in biotechnological processes where one or more nutrients (substrates) are fed (supplied) to the bioreactor during cultivation and in which the product(s) remain in the bioreactor until the end of the run. An alternative description of the method is that of a culture in which "a base medium supports initial cell culture and a feed medium is added to prevent nutrient depletion". It is also a type of semi-batch culture. In some cases, all the nutrients are fed into the bioreactor. The advantage of the fed-batch culture is that one can control concentration of fed-substrate in the culture liquid at arbitrarily desired levels.
A turbidostat is a continuous microbiological culture device, similar to a chemostat or an auxostat, which has feedback between the turbidity of the culture vessel and the dilution rate. The theoretical relationship between growth in a chemostat and growth in a turbidostat is somewhat complex, in part because they are similar. A chemostat has a fixed volume and flow rate, and thus a fixed dilution rate. A turbidostat dynamically adjusts the flow rate to make the turbidity constant. At steady state, operation of both the chemostat and turbidostat are identical. It is only when classical chemostat assumptions are violated that a turbidostat is functionally different. One case may be while cells are growing at their maximum growth rate, in which case it is difficult to set a chemostat to the appropriate constant dilution rate.
The E. coli long-term evolution experiment (LTEE) is an ongoing study in experimental evolution begun by Richard Lenski at the University of California, Irvine, carried on by Lenski and colleagues at Michigan State University, and currently overseen by Jeffrey E. Barrick at the University of Texas at Austin. It has been tracking genetic changes in 12 initially identical populations of asexual Escherichia coli bacteria since 24 February 1988. Lenski performed the 10,000th transfer of the experiment on March 13, 2017. The populations reached over 73,000 generations in early 2020, shortly before being frozen because of the COVID-19 pandemic. In September 2020, the LTEE experiment was resumed using the frozen stocks.
A moss bioreactor is a photobioreactor used for the cultivation and propagation of mosses. It is usually used in molecular farming for the production of recombinant protein using transgenic moss. In environmental science moss bioreactors are used to multiply peat mosses e.g. by the Mossclone consortium to monitor air pollution.
The Monod equation is a mathematical model for the growth of microorganisms. It is named for Jacques Monod, who proposed using an equation of this form to relate microbial growth rates in an aqueous environment to the concentration of a limiting nutrient. The Monod equation has the same form as the Michaelis–Menten equation, but differs in that it is empirical while the latter is based on theoretical considerations.
Changestat is a continuous cultivation method that is used for acquiring quantitative data of a microorganism's metabolism at various environmental conditions within a single experiment. Every changestat always starts as a continuous cultivation experiment, but after reaching steady state, smooth and slow change of an environmental parameter is applied. Two most common changestat techniques are accelerostat (A-stat) and dilution rate stat (D-stat).
A Hollow fiber bioreactor is a 3 dimensional cell culturing system based on hollow fibers, which are small, semi-permeable capillary membranes arranged in parallel array with a typical molecular weight cut-off (MWCO) range of 10-30 kDa. These hollow fiber membranes are often bundled and housed within tubular polycarbonate shells to create hollow fiber bioreactor cartridges. Within the cartridges, which are also fitted with inlet and outlet ports, are two compartments: the intracapillary (IC) space within the hollow fibers, and the extracapillary (EC) space surrounding the hollow fibers.
Synechococcus elongatus is a unicellular cyanobacterium that has a rapid autotrophic growth comparable to yeast. Its ability to grow rapidly using sunlight has implications for biotechnological applications, especially when incorporating genetic modification.
Substrate inhibition in bioreactors occurs when the concentration of substrate exceeds the optimal parameters and reduces the growth rate of the cells within the bioreactor. This is often confused with substrate limitation, which describes environments in which cell growth is limited due to of low substrate. Limited conditions can be modeled with the Monod equation; however, the Monod equation is no longer suitable in substrate inhibiting conditions. A Monod deviation, such as the Haldane (Andrew) equation, is more suitable for substrate inhibiting conditions. These cell growth models are analogous to equations that describe enzyme kinetics, although, unlike enzyme kinetics parameters, cell growth parameters are generally empirically estimated.