INTRODUCTION AND HISTORY

The heyday of continuous culture was in the 1960s, when its versatility and reproducibility were used to address fundamental problems in diverse microbiological fields such as biochemistry, ecology, genetics and physiology. The advent of molecular genetics in the 1970s and 1980s led to a decline in the popularity of continuous culture as a standard laboratory tool. The current trend of studying global proteomics, transcriptomics and metabolomics requires reproducible, reliable and biologically homogeneous datasets with which to approach a given problem.The kinetics of microbial growth are fundamental to every field of microbiology, be it physiology, genetics, ecology or biotechnology. The simultaneous development of the chemostat by Monod and Novick & Szilard allowed microbial physiologists to dissect bacterial growth independently of the physiochemical environment. The heyday of the chemostat was the 1960s, during which a multitude of studies investigated the physiology of various species under wide-ranging growth and environmental conditions. We have now entered the post-genomic era, where we have knowledge of microbial genomes, and cutting-edge technology is available to study global protein, mRNA and metabolite profiles. These so called ‘omic’ technologies provide the possibility to characterize cell physiology at a molecular level, providing temporal, spatial and even real-time information. One of the constraints upon this approach surrounds the cultivation of the organism concerned. In order to gather reproducible and meaningful information the cell population must be grown in a defined, ideally constant, controllable, set of physico-chemical conditions. Conducting such experiments in simple ‘batch culture’ systems results in dynamic physico-chemical conditions, producing complex data patterns that are often difficult or impossible to interpret. The ideal experimental system for such studies is therefore the chemostat, a continuous culture system that provides a constant environmental milieu.

CONTINUOUS CULTURE METHODS

Continuous culture systems have been widely used to culture microbes for industrial and research purposes. In recent years these culture techniques have found their way into the bioassay methods of ecotoxicology . The early development of a continuous culture system can be traced back to the work of Novik and Szilard who developed the first ”chemostat”, and by Monod (1950) who called his apparatus a “bactogen”. Batch and continuous culture systems differ in that in a continuous culture system, nutrients are supplied to the cell culture at a constant rate, and in order to maintain a constant volume, an equal volume of cell culture is removed. This allows the cell population to reach a “steady state” (ie. growth and cell division where the growth rate and the total number of cells per milliliter of culture remains constant).

CHEMOSTAT:A chemostat (from Chemical environment is static) is a continuous culture device used in microbiology for growing and harvesting microbes. It consists of two primary parts: a nutrient reservoir and a growth chamber. The most important feature of a chemostat is that all fermentation parameters; growth chamber volume, dissolved oxygen, nutrient concentrations, pH, cell density, etc, remain constant throughout the experiment.

Basics of chemostat culture:The work of Monod (1942) underpins chemostat theory, showing the importance of the relationship between specific growth rate (µ) of a microbial population and the substrate concentration (s). The familiar lag, exponential and stationary phases of microbial growth dissected by the seminal works of Monod are referred to as the growth cycle. However, this progression of events is not an inherent property of the organism but a result of its interaction with the physico-chemical environment in which it is growing . The use of continuous culture systems to uncouple growth from the transient conditions encountered in batch culture offers unlimited advantages to both the academic and industrial investigator. The system and term chemostat were invented by Novick & Szilard , although simultaneously and independently the ‘bactogène’, a virtually identical device, was developed by Monod . The basis of both is that the specific growth rate of an organism, relative to its theoretical maximum, is governed by the external substrate concentration of a limiting nutrient.

All continuous culture systems begin life as a batch culture, where growth proceeds via the familiar growth cycle. The addition of fresh nutrient-replete medium to such a culture during exponential growth at a suitable rate would allow growth to proceed at a given rate indefinitely. As a consequence of this, the culture volume and biomass would increase exponentially without the removal of culture at the rate of fresh medium input, as occurs in continuous culture. The advantage of this system is that the microbial population within the vessel then grows at a constant rate in a constant environment and assumes a ‘steady state’. Environmental factors such as pH, nutrient concentration (including oxygen and light) and metabolic products can be varied and controlled by the investigator. In experimental configurations the vessel in which the culture is growing is well mixed to ensure that the culture as a whole is homogeneous and that the various additions to the vessel do not result in chemical gradients. Mixing is, more often than not, facilitated by mechanically driven impellers providing mixing times of less than 5 s. Such configurations provide cultures in homogeneous steady states and this is the most common form of continuous culture.

WORKING OF CHEMOSTAT:An apparatus for the continuous cultivation of microorganisms or plant cells. The nutrients required for cell growth are supplied continuously to the culture vessel by a pump connected to a medium reservoir. The cells in the vessel grow continuously on these nutrients. Residual nutrients and cells are removed from the vessel (fermenter) at the same rate by an overflow, thus maintaining the culture in the fermenter at a constant volume.

Schematic representation of chemostat apparatus-

An important feature of chemostat cultivation is the dilution rate, defined as the volume of nutrient medium supplied per hour divided by the volume of the culture. During chemostat cultivation, an equilibrium is established (steady state) at which the growth rate of the cells equals the dilution rate. The higher the dilution rate, the faster the organisms are allowed to grow. Above a given dilution rate the cells will not be able to grow any faster, and the culture will be washed out of the fermenter. The chemostat thus offers the opportunity to study the properties of organisms at selected growth rates.

The nutrient medium which is fed to the fermenter contains an excess of all growth factors except one, the growth-limiting nutrient. The concentration of the cells (biomass) in the fermenter is dependent on the concentration of the growth-limiting nutrient in the medium feed. Upon entering the fermenter, the growth-limiting nutrient is consumed almost to completion, and only minute amounts of it may be found in the culture and the effluent. Initially, when few cells have been inoculated in the growth vessel, even the growth-limiting nutrient is in excess. Therefore, the microorganisms can grow at a rate exceeding their rate of removal. This growth of cells causes a fall in the level of the growth-limiting nutrient, gradually leading to a lower specific growth rate of the microorganisms. Once the specific rate of growth balances the removal of cells by dilution, a steady state is established in which both the cell density and the concentration of the growth-limiting nutrient remain constant. Thus the chemostat is a tool for the cultivation of microorganisms almost indefinitely in a constant physiological state.

To achieve a steady state, parameters other than the dilution rate and culture volume must be kept constant (for example, temperature and pH). The fermenter is stirred to provide a homogeneous suspension in which all individual cells in the culture come into contact with the growth-limiting nutrient immediately, and to achieve optimal distribution of air (oxygen) in the fermenter when aerobic cultures are in use.Laboratory chemostats usually contain 0.5 to 10.5 quarts (0.5 to 10 liters) of culture, but industrial chemostat cultivation can involve volumes up to 343,000 gal (1300 m³) for the continuous production of microbial biomass.

In many chemostat continuous culture systems, the nutrient medium is delivered to the culture at a constant rate by a peristaltic pump or solenoid gate system (Kubitschek 1970). The rate of media flow can be adjusted, and is often set at approximately 20% of culture volume per day. Air is pumped into the culture vessel through a sterile filter. This bubbling air has three effects: it supplies CO2 and O2 to the culture, aids in circulation and agitation of the cultures, and pressurizes the head space of the culture vessel so as to provide the force to “remove” an amount of media (and cells) equal to the volume of inflowing media. The culture may be aseptically sampled by opening the clamp on a sample port. The magnetic stirrer and aeration help to prevent the cells from collecting in the bottom of the culture vessel. A truly continuous culture will have the medium delivered at a constant volume per unit time. However it has been  experienced that delivery systems such as peristaltic pumps or solenoid gates are inherently unreliable. It is difficult to adjust such systems to deliver equal amounts of medium to several cultures simultaneously. This is what is needed if competition experiments are to be truly replicated. In order to deliver exactly the same amounts of medium to several cultures growing at once, a “semi-continuous” approach can be taken. In a semi-continuous system the fresh medium is delivered to the culture all at once, by simply opening a valve in the medium delivery line. Fresh medium flows into the culture vessel, and spent culture flows out into a collecting vessel. Once the required medium has entered the culture, the valve is closed, and the culture is allowed to grow for 24 hours, when the procedure is repeated. Our lab has used both methods and it has been shown that there are significant advantages in the consistent medium delivery of a semi-continuous system.

Diagramatic representation of a semi-continuous culture system

Control Factors: The growth and survival of bacteria depend on the close monitoring and control of many conditions within the chemostat such as the pH level, temperature, dissolved oxygen level, dilution rate, and agitation speed. As expected with CSTRs, the pumps delivering the fresh medium and removing the effluent are controlled such that the fluid volume in the vessel remains constant.

1.pH level -Different cells favor different pH environments. The operators need to determine an optimal pH and maintain the CSTR at it for efficient operation. Controlling the pH at a desired value during the process is extremely important because there is a tendency towards a lower pH associated with cell growth due to cell respiration (carbon dioxide is produced when cells respire and it forms carbonic acid which in turn causes a lower pH). Under extreme pH conditions, cells cannot grow properly, therefore appropriate action needs to be taken to restore the original pH (i.e. adding acid or base).

2.Temperature -Controlling the temperature is also crucial because cell growth can be significantly affected by environmental conditions. Choosing the appropriate temperature can maximize the cell growth rate as many of the enzymatic activates function the best at its optimal temperature due to the protein nature of enzymes.

3.Dilution rate -One of the important features of the chemostat is that it allows the operator to control the cell growth rate. Dilution rate is simply defined as the volumetric flow rate of nutrient supplied to the reactor divided by the volume of the culture (unit: time-1). While using a chemostat, it is useful to keep in mind that the specific growth rate of bacteria equals the dilution rate at steady state. At this steady state, the temperature, pH, flow rate, and feed substrate concentration will all remain stable. Similarly, the number of cells in the reactor, as well as the concentration of reactant and product in the effluent stream will remain constant.

In general, increasing the dilution rate will increase the growth of cells. However, the dilution rate still needs to be controlled relative to the specific growth rate to prevent wash-out. The dilution rate should be regulated so as to maximize the cell production rate. Figure 1 below shows how the dilution rate affects cell production rate(DC_C), cell concentration (C_C), and substrate concentration (C_S).

Figure 1: Cell concentration, cell production, and substrate concentration as a function of dilution rate

Initially, the rate of cell production increases as dilution rate increases. When D_maxprod is reached, the rate of cell production is at a maximum. This is the point where cells will not grow any faster. D = μ (dilution rate = specific growth rate) is also established at this point, where the steady-state equilibrium is reached. The concentration of cells (C_C) starts to decrease once the dilution rate exceeds the D_maxprod. The cell concentration will continue to decrease until it reaches a point where all cells are washed out. At this stage, there will be a steep increase in substrate concentration because fewer and fewer cells are present to consume the substrate.

4.Oxygen transfer rate -Since oxygen is an essential nutrient for all aerobic growth, maintaining an adequate supply of oxygen during aerobic processes is crucial. Therefore, in order to maximize the cell growth, optimization of oxygen transfer between the air bubbles and the cells becomes extremely important. The oxygen transfer rate (OTR) tells us how much oxygen is consumed per unit time when given concentrations of cells are cultured in the bioreactor. This relationship is expressed as below.

Oxygen Transfer Rate (OTR) = Q_O

Where C_C is simply the concentration of cell in the reactor and Q_O2 is the microbial respiration rate or specific oxygen uptake rate. The chemostat is a very convenient tool to study the growth of specific cells because it allows the operators to control the amount of oxygen supplied to the reactor. Therefore it is essential that the oxygen level be maintained at an appropriate level because the cell growth can be seriously limited if inadequate oxygen is supplied.

5.Agitation speed- A stirrer, usually automated and powered with a motor, mixes the contents of the chemostat to provide a homogeneous suspension. This enables individual cells in the culture to come into contact with the growth-limiting nutrient and to achieve optimal distribution of oxygen when aerobic cultures are present. Faster, more rigorous stirring expedites cell growth. Stirring may also be required to break agglutinations of bacterial cells that may form.

Some sources of concern are:

• Foaming results in overflow with the volume of liquid not exactly constant
• Some very fragile cells are ruptured when caught between the magnetic stirring bar and the glass of the vessel. Suspending the stirring bar usually corrects this fault.
• Changing pumping rate by turning the pump on and off over short time periods may not work because cells respond to sudden changes by altering their rates. Very short intervals of on/off are alright.
• Bacteria travel upstream quite easily. They will reach the reservoir of sterile medium quickly unless the liquid path is interrupted by an air break in which the medium falls in drops through air.

DEVICES OTHER THAN CHEMOSTAT

1. Turbidostat: A turbidostat is a continuous 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 it is similar. A chemostat technically has a fixed volume and flow rate – thus a fixed dilution rate. When the cells are uniform and at equilibrium, operation of a chemostat and turbidostat should be identical. It is only when classical chemostat assumptions are violated (for instance, out of equilibrium; or the cells are mutating) 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.While most turbidostats use a spectrophotometer/turbidometer to measure the optical density for control purposes, there exist other options, such as electrical conductivity.

2.Auxostat: An auxostat is a continuous culture device which, while in operation, uses feedback from a measurement taken on the growth chamber to control the media flow rate, maintaining the measurement at a constant. Auxo was the Greek goddess of spring growth, and as a prefix represents nutrients. However, the most typical auxostats are pH-auxostats , with feedback between the growth rate and a pH meter.Other auxostats may measure oxygen tension, ethanol concentrations, and sugar concentrations

^{APPLICATIONS  OF  CHEMOSTAT:}

^v how bacteria respond to different antibiotics. Bacteria are also used in the production of therapeutic Pharmaceuticals: Used to study a number of different bacteria, a specific example being analyzing proteins such as insulin for diabetics.

v  Manufacturing: Used to produce ethanol, the fermentation of sugar by bacteria takes place in a series of chemostats. Also, many different antibiotics are produced in chemostats.

v  Food Industry: Used in the production of fermented foods such as cheese.

v  Research: Used to collect data to be used in the creation of a mathematical model of growth for specific cells or organisms.

ADVANCEMENT IN CONTINUOUS CULTURE SYSTEMS

Improvement of a Continuous-Culture Apparatus for Long-Term Use

A glass and plastic apparatus was designed to meet requirements for continuous culture of cells. Some of the improvements incorporated into this apparatus include an all-glass growth vessel with a self-cleaning bottom, special compression fittings to connect tubing to glass tubing, a removable yield reservoir, and a nonwetting gas exhaust assembly. All portions of the system can be autoclaved for sterilization, and medium bottles and pump lines are replaced aseptically.The continuous production and harvest of uncontaminated cells for periods of several months are a major requirement in the design of a suitable culture system. Many continuous-culture units have been reported previously (1, 2), and some units are now commercially available. However, in these units, rubber, metals, and other sources of potentially toxic extractives are not prevented from direct contact with the culture. Factors that are toxic to some cells are known to leach from rubber stoppers, tubing, and some flexible polyethylenes (3). Even if these materials are nontoxic to the cells under culture, they are unknown factors  in investigations of the cellular products of antibiotic or other biological activity. Most of the systems previously described use cotton filters for the exhaust gas. Since this gas is wet, the filters eventually become moist and permit microorganisms to penetrate the growth system. Thecoupling of components is another disadvantage of most systems. When glass tubing with ground glass joints is used throughout, the system is rigid and inconvenient in many respects. Plastic tubing, when stretched over glass tubing, frequently becomes distorted during autoclaving and thereafter forms an unreliable seal. Moreover, removal and replacement of tubing units is difficult. These problems were resolved in the construction of the system described below, which was used to maintain cultures of Chlamydomonas and Chlorella (unicellular algae) without contamination for periods of up to 4 months.

APPLICATION OF CONTINUOUS CULTURE SYSTEMS

v  The combination of steady-state chemostat cultures and functional genomic techniques provides a sound experimental basis for the analysis of biological processes in micro-organisms . Studies such those of Hayes et al., Kolkman et al., Piper et al. and Wu et al.  highlight the enhanced reproducibility of transcriptome, proteome, metabolome and metabolic engineering data obtained from steady-state chemostat cultures. The carefully controlled and defined physiological conditions obtainable in chemostat cultures enable the acquisition of reliable biological samples for analysis by multiple functional genomic techniques, resulting in, to paraphrase Delneri et al., ‘a truly integrative biology of model organisms’

v  The use of continuous culture systems to study biofilm formation in Pseudomonas aeruginosa, using chemostat and continuous culture biofilm flow cells, has shown correlations between protein expression in planktonic cultures and during stages of biofilm formation (Sauer et al.),. More than 800 proteins were found to exhibit a sixfold or greater change in expression levels between planktonic and biofilm cultures. Many of the upregulated genes were involved in carbon catabolism and amino acid metabolism, and although the limiting nutrient was not specified, the prevalence of such proteins would suggest carbon limitation. This work serves to illustrate that apparently physically unlinked culture conditions can be studied using continuous culture strategies.

FUTURE PROSPECTIVES

The advantages of steady-state continuous culture systems as a means of biological investigation have been recognized since their invention in the 1950s. A resurgence in studies of evolutionary responses to environmental variation and of mutator genes facilitated by the chemostat is beginning to provide important biological insights into the functions and mechanisms of such genes and their obvious implications in processes such as acquired antimicrobial resistance. Recent advances in biological methods applied at the global level have led to a need for reproducible, biologically homogeneous and reliable data, and this has in turn led to a revival of the use of the chemostat. The advantages of continuous culture systems are proven in disciplines such as biochemistry and physiology on a gene-by-gene analysis basis; however it is only now that we are discovering the power of this technique at the whole-organism level. Of course, in the natural world, micro-organisms do not live in ideal, well-mixed, homogeneous conditions. At this point we would like to acknowledge a modification of the chemostat designated the ‘gradostat’ that allows cultures to be grown in the presence of solute gradients. We are not currently aware of any ‘omic’ studies using this system; however it can be only a matter of time before meaningful spatial and temporal experiments will be conducted under more ecologically relevant conditions.

REFFERENCES

• http://www.springerlink.com/content/r1215224pn487235/
• http://aem.asm.org/cgi/reprint/16/2/232.pdf
• http://controls.engin.umich.edu/wiki/index.php/Bacterial_Chemostat_Model
• http://www.midgard.liu.se/~b00perst/chemostat.pdf
• http://www.rpi.edu/dept/chem-eng/Biotech-Environ/Contin/chemosta.htm
• http://www.molecular-plant-biotechnology.info/single-cell-culture/types-of-open-cultures-chemostat-and-turbidostat.htm
• http://en.wikipedia.org/wiki/Turbidostat
• http://www.rpi.edu/dept/chem-eng/Biotech-Environ/Contin/chemosta.htm
• http://www.midgard.liu.se/~b00perst/chemostat.pdf