Continuous Culture

Continuous culture is a set of techniques used to reproducibly cultivate microorganisms at submaximal growth rates at different growth limitations in such a way that the culture conditions remain virtually constant (in 'steady state') over extended periods of time.

From: Encyclopedia of Microbiology (Third Edition) , 2009

Continuous Cultures (Chemostats)

J.G. Kuenen , O.J. Johnson , in Encyclopedia of Microbiology (Third Edition), 2009

Introduction

Continuous culture is a set of techniques used to reproducibly cultivate microorganisms at submaximal growth rates at different growth limitations in such a way that the culture conditions remain virtually constant (in 'steady state') over extended periods of time. In the steady state, the growth of organisms can be studied in great detail under precisely controlled physiochemical states. Such conditions are amenable to a great deal of mathematical modeling that enables powerful quantitative analysis of microbial activities. Continuous culture principles first appeared in the literature near the middle of the twentieth century, notably from work performed in the labs of Herbert, Monod, and Novick. Since that time, continuous culture techniques have become common tools in both research and industry. A large diversity of continuous culture applications exists, of which only a modest subset will be mentioned in the present work. Focus will be on a number of classic and a few up-to-date examples of the use of continuous cultivation in various applications. As will be described, the use of continuous culture has enabled studies into several ecological phenomena, including the relationship between growth rate and intracellular metabolic fluxes, the transcriptional responses of microorganisms to various nutrient limitations, the competitive strategies between microorganisms at low nutrient concentrations, as well as the selection and competition between spontaneous or designed mutants for biotechnological applications. As synergistic tools continue becoming more powerful and widely available, the number of uses and the value of the classic continuous culture techniques will likely continue growing at a comparable rate.

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Methodologies for bioactivity assay: cell study

Nan Shang , ... Jianping Wu , in Biologically Active Peptides, 2021

7.2.4.2 Continuous cultures

Continuous cultures are comprised of a single-cell type that can be serially propagated in culture either for a limited number of cell division or otherwise indefinitely. Continuous cell lines that can be propagated indefinitely generally have this ability because they have been transformed into tumor cells. Tumor cell lines are often derived from actual clinical tumors, but transformation may also be induced using viral oncogenes or by chemical treatment. Transformed cell lines present the advantage of almost limitless availability, but the disadvantage of having retained very little of the original in vivo characteristics.

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Methods in Methane Metabolism, Part A

John A. Leigh , in Methods in Enzymology, 2011

3.1 General considerations

Continuous culture systems have been described for M. maripaludis (Haydock et al., 2004) and M. thermautotrophicus (de Poorter et al., 2007). For M. maripaludis, we adopted an approach, detailed in Haydock et al., 2004, whereby a single known nutrient limits growth. We generally hold the dilution rate (and hence the growth rate) constant and limit the cell density by lowering the level of a given nutrient. Hydrogen becomes the limiting nutrient when its proportion in the gas mixture delivered to the chemostat vessel is lowered to the point where it limits the cell density at steady state. Table 6.1 shows an example of gas mixtures that are used for hydrogen limitation and hydrogen excess. A nutrient such as phosphate or ammonia is limiting when its concentration in the growth medium is lowered to the point where it similarly determines cell density. If the cell density is the same for each limiting nutrient, and the growth rate is also the same, differences between cultures result solely from which nutrient is limiting. In our studies, we regard a culture as experiencing hydrogen excess if its density is limited by some other nutrient such as phosphate or ammonia (Hendrickson et al., 2007; Xia et al., 2009). For the continuous culture of M. thermautotrophicus (de Poorter et al., 2007), the dilution rate and gassing rate were varied, resulting in cultures of different densities. Two kinds of culture were obtained: cultures whose density remained generally constant over a range of dilution rates and gassing rates were apparently limited by a nutrient in the growth medium, and cultures whose density decreased with increasing dilution rates, or increased with increasing gassing rates, were hydrogen-limited.

Table 6.1. Example of gas flow rates (ml/min) for growth of M. maripaludis in a chemostat under hydrogen limitation and hydrogen excess a

Hydrogen limitation b Hydrogen excess b
H2 20 110
N2 or Ar 125 35
CO2 40 40
H2S (1% in N2 or Ar) 15 15
a
These gas flow rates were determined for continuous culture of M. maripaludis at a specific growth rate of 0.083   h−   1 at a cell density (OD660) of 0.6 in a 1-l volume with an agitation rate of 1000   rpm. Appropriate values will vary with different organisms, different conditions, and different equipment.
b
Hydrogen limitation: This level of H2 holds the cell density (OD660) at about 0.6. Hydrogen excess: A limiting level of some nutrient in the medium (e.g., nitrogen or phosphate) holds the OD660 at about 0.6.

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Research on Nitrification and Related Processes, Part A

Annette Bollmann , ... Hendrikus J. Laanbroek , in Methods in Enzymology, 2011

2.3 General set-up of a continuous culture experiment

Continuous cultures have been used to enrich AOB that are adapted to low ammonium concentration ( Bollmann and Laanbroek, 2001), to conduct competition experiments between AOB and heterotrophic bacteria (Verhagen and Laanbroek, 1991), between different AOB (Bollmann et al., 2002), between AOB and NOB (Laanbroek and Gerards, 1993; Laanbroek et al., 1994), and to perform experiments that simulate environmental conditions (Bollmann and Laanbroek, 2002).

Continuous culture studies can be conducted in commercially available chemostats or self-built units (Fig. 3.1). Independent of which approach is used, the following conditions have to be adjusted and kept constant for controlled growth of AOB:

1.

Temperature: Most continuous culture vessels have a double-wall system so that the temperature can be controlled with an external thermostat. Another option is to place the vessel in a temperature-controlled water bath or room.

2.

Mixing of the culture: Commercially available chemostats have stirrer systems that mix the culture well. When using a self-built unit, a vessel with a flat bottom should be used. The vessel can be placed on a stirrer and the liquid is mixed by a stir bar. It is important to observe the chemostat over time to ensure that no wall growth is building up. If the wall growth builds up, the stirrer speed can be increased. However, often a biofilm that has been already developed on the wall cannot be removed by increasing the stirrer speed. Therefore, the experiment should be restarted with a higher stirrer speed to prevent wall growth from the beginning.

3.

O 2 partial pressure: Depending on the system, the O2 partial pressure is kept constant by bubbling the culture with air or an O2-containing gas mixture, or by changing the rate of mixing. Commercially available chemostats often have a unit that can be used to adjust the O2 partial pressure to a fixed value. Gases for bubbling must be sterilized by filtration through 0.2-μm air filters to ensure that the culture is not contaminated by air-borne bacteria. In a self-built unit, a sparger with an air pump can be used as an alternative; however, these lack the option to adjust the O2 partial pressure.

4.

pH value: The pH value is a very important factor while growing ammonia oxidizers. It can be adjusted and kept constant by controlling with a pH electrode and addition of alkaline. If no pH control unit is available, the pH of the culture can be kept constant by using buffer-containing medium. The pH of the culture should be checked regularly with an outside pH electrode and if necessary the pH in the vessel can be readjusted.

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Guide to Yeast Genetics: Functional Genomics, Proteomics, and Other Systems Analysis

Maitreya J. Dunham , in Methods in Enzymology, 2010

3.3 Turbidostats

Another continuous culture system, the turbidostat, first introduced by Bryson and Szybalski (1952), combines some properties of serial dilution and chemostats. Instead of adding new medium at a constant rate, in a turbidostat, cell density is held constant. This is achieved by a feedback loop allowing adjustment of the nutrient addition rate in response to changes in density, usually measured via light transmittance. Few commercial options appear to exist currently, but turbidostats can be built using modern microprocessor controlled peristaltic pumps. Designs using simple light-measurement devices can be found in textbooks (e.g., Norris and Ribbons, 1970), and variations using LED and photodiode components would be straightforward extensions.

The turbidostat provides selection on maximal growth rate while simultaneously maintaining other conditions constant. The media composition defines the selection pressure as in other systems. Very little has been published on yeast grown in turbidostats, although that is likely to change given the benefits that this system provides.

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Solvent Production

N. Qureshi , in Encyclopedia of Microbiology (Third Edition), 2009

Suspended cell continuous bioreactors

The continuous culture technique may be used to improve reactor productivity and study the physiology of the culture in steady state. For continuous fermentation of butanol, a number of studies have been carried out. Laboratory studies have demonstrated that due to production of fluctuating levels of solvents and complexity of butanol fermentation, the use of a single-stage continuous reactor does not seem to be practical at the industrial scale. In continuous culture, a serious problem exists in that solvent production may not be stable for long time periods and ultimately declines over time, with a concomitant increase in acid production. In a single-stage continuous system, high reactor productivity may be obtained; however, this occurs at the expense of low product concentration in the effluent when compared to that achieved in a batch process. In a single-stage continuous reactor, 15.9  g   l−1 total solvents was produced at a dilution rate of 0.1   h−1 resulting in a productivity of 1.5   g   l−1  h−1. In order to improve the productivity, the dilution rate was increased to 0.22   h−1, thus improving productivity to 2.55   g   l−1  h−1. However, the product concentration decreased to 12   g   l−1. In a more recent study, a continuous fermentation was run in order to study the effect of fermentation gases and dilution rate on solvent production. In this system, a productivity of 0.58   g   l−1  h−1 was obtained at a dilution rate of 0.07   h−1, thus producing 8.3   g   l−1 total solvents. In order to improve reactor productivity, a single-stage spin filter perfusion bioreactor has also been used. In the perfusion bioreactor, a maximum productivity of 1.14   g   l−1  h−1 was obtained; however, solvent concentration fluctuated over time.

Two or more multistage continuous fermentation systems have been investigated to reduce fluctuations and increase solvent concentration in the product stream. This is done by allowing growth, acid production, and solvent production to occur in separate bioreactors. In a two-stage continuous system, a solvent concentration of 18.2   g   l−1 was reported using C. acetobutylicum DSM 1731, which is comparable to the solvent concentration in a batch reactor. This type of multistage reactor system (7–11 fermentors in series) was successfully tested at the pilot plant and full scale plant level in Russia (then Soviet Union).

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Methods in Systems Biology

Catherine L. Winder , Karin Lanthaler , in Methods in Enzymology, 2011

2.4 Experimental time scale and replicates

In continuous cultures, adaptations of the microbial community to environment conditions increase with operation time and potentially selecting for mutations in the culture. If the mutants outcompete the parental strain, the genetic makeup of the population will alter potentially changing the phenotype of the culture. It is therefore essential to minimize the number of generations by which the culture is allowed to progress and monitor the growth rate to check that it does not change during the duration of the experiment.

It is important to include biological replicates in any experiment to assess both the biological and analytical (or technical) variability of the experiment. In microbial studies, biological replicates are those which originate from different cultures which have been grown in identical conditions and ideally from the same inoculum. Analytical (or technical) replicates result from either multiple samples from the same culture (at the same time point in time-dependent studies) or repeated measurements from the same biological sample. In our experiments, we perform a minimum of three biological replicates for measurements of the proteome and metabolome. In order to check the reproducibility of our cultures, we monitor a number of growth-associated parameters in the steady-state cultures before the collection of sample commences. These include (1) the growth rate, (2) the biomass yield (monitored in both the batch phase of growth and steady state), (3) the extracellular glucose concentration, (4) the purity of the culture, and (5) the off-gas analysis of CO2 and O2.

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Engineering Perspectives in Biotechnology

J.-J. Zhong , in Comprehensive Biotechnology (Third Edition), 2011

2.21.4.2 Continuous and Semicontinuous Culture

Theoretically, continuous culture is the most promising mode for obtaining high culture productivity. In a continuous culture, the nutrients consumed by the cells are continuously replenished by an inflow of fresh medium. A constant inflow of fresh medium is balanced by a constant efflux of spent medium plus cells. Consequently, a steady state will be developed at a dilution rate (equal to outflow rate/volume) less than the maximum specific growth rate of the culture. Under steady-state conditions, the average specific growth rate in the culture is identical to the dilution rate. The biomass concentration of the culture is then determined by the concentration of the growth-limiting nutrient in the influent. All culture parameters (biomass, biomass composition, and nutrients) remain constant for prolonged periods of time in a steady state. This balanced growth is a very attractive tool in studies on growth and production kinetics or cell physiology. 22

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Immobilized Cells

Richard U. Edgehill , in Progress in Biotechnology, 1996

Materials and Methods

Batch and continuous cultures of Arthrobacter strain ATCC 33790 were used for all of the studies. The mineral salts medium composition and culture preparation procedures have been previously described (7). The biofilm was grown on 3   mm glass beads packed in a tubular glass reactor of dimensions 2.5   x   13   cm. Feed was added dropwise through a glass medium break tube (New Brunswick Scientific, New Brunswick, New Jersey) with sterile air purge. The liquid was partially atomized resulting in a fairly uniform distribution of droplets over the packing. The feed pH was adjusted to 7.2-7.5.

Concentrations of PCP greater than 10   mg/l were analysed using UV spectrophotometry at 320   nm. The 4-aminoantipyrene method was used for lower concentrations (7). Nitrite and chloride were analysed using the colorimetric method described by Vogel and ion chromatography (Dionex 2010i), respectively (8).

To prepare the culture for examination under the Electron Microscope, a few glass beads were placed in a sealed container adjacent to a drop of 1% osmium tetroxide to allow osmium vapor fixation for 15   minutes. The beads were then sputter coated with gold and viewed in a JEOL 6400F Field Emission Scanning Electron Microscope.

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Immobilized Cells

Ralf Pörtner , Herbert Märkl , in Progress in Biotechnology, 1996

Fermentations

Stable conditions during continuous culture could be maintained over process times of several weeks with suspended and immobilized cells. Figure 3 compares continuous cultures in defined medium. In suspension the typical chemostat behaviour can be observed. With increasing dilution rate the concentration of suspended cells decreases until the dilution rate D reaches the maximal growth rate μmax. In case of immobilization (carrier concentration 7   g   l-   1) the cell concentration remained on a high level even at dilution rates above the maximal growth rate.

Figure 3. Relationship between cell concentration and dilution rate for suspended and immobilized Pyrococcus furiosus

Cell concentration related to the reactor volume; defined medium, carrier concentration 7   g   l-   1

In figure 4 the relationship between concentration of immobilized cells and carrier concentration is shown for defined medium and a dilution rate of D   =   0.5   h-   1. As expected the cell concentration increases with increasing carrier concentration. At appr. 25   g carrier l-   1, which corresponds to a volume fraction of appr. 20 %, a saturation seems to be reached.

Figure 4. Relationship between cell concentration (Pyrococcus furiosus, immobilized) and carrier concentration (AQUACEL), defined medium

The potential of the immobilization technology can be seen in figure 5, were the maximal cell number and amylase productivity for chemostat cultures with suspended cells and continuous cultures with immobilized cells in defined and complex medium are compared. In defined medium the cell concentration in the immobilized system is appr. 2 times higher than the maximum value in chemostat cultures of suspended cells, the amylase productivity appr. 3 times.

Figure 5. Volume specific amylase productivity and cell concentration for suspended and immobilized Pyrococcus furiosus

Suspension : defined medium; dilution rate D   =   0.1   h-   1

Immobilized : AQUACEL carrier, carrier concentration 23   g 1-   1, Dilution rate 0.5   h-   1, cell concentration related to the reactor volume, defined and complex medium

The highest cell concentration related to the total medium volume and amylase productivity was obtained for immobilized cultures in complex medium. The cell concentration was 4.6*109 cells ml-   1, 2.5 times higher compared to immobilized cultures in defined medium. This cell density corresponds to a number of 2.3* 1010 cells mlcarrier -   1 or 2*1011 cells gcarrier -   1The amylase productivity was 16 times higher compared to defined medium.

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