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Table of Contents

• Introduction
• 1. Sterile environment
• 2. Biomass concentration
• 3. Temperature
• 4. pH
• 5. Oxygen concentration (pO2)
• 6. Agitation
• Sources

Introduction

Bioreactors provide a controlled environment for the chosen production organism to achieve optimal growth and product formation. The bioreactor can be thought of as the heart of the bioprocess, where a biological product is being produced.

In order to obtain the product of continuous desired quality, the bioreactor has to be continuously operating in optimal conditions. The performance of the bioreactor and consequently the bioprocess depends on some key parameters, such as sterile environment, biomass concentration, temperature, pH, oxygen concentration, and agitation. These parameters must be tightly monitored and controlled.

In this blog the key parameters that influence the functioning of the bioreactor will be discussed.

1. Sterile environment

The first thing that comes to mind, when setting up a bioprocess is preventing contamination of the culture. Maintaining a pure culture and keeping the bioreactor running under aseptic conditions for days or months is a key step in bioreactor design.

Slow-growing cultures are especially prone to contamination, thus keeping the bioreactor free of unwanted organisms acts as the first line of defence. Especially, since contamination of the culture is the cause of 3% - 5% of industrial fermentations being lost.

The frequency and causes of the possible contaminations vary, depending on the organism used and the product produced. A higher risk of contamination is present in the processes where complex and nutrient-rich media is used. When this is combined with slow-growing cells such as mammalian, the contamination rate can be as high as 17%. 

Big industrial bioreactors are sterilized through in situ steam sterilization under pressure, where all air in the vessel and pipes is replaced by steam.

After sterilization is finished, all air and medium entering the bioreactor vessel must be sterile. In order to reduce the contamination risk, filters can be fitted to gas lines and positive air pressure is maintained. All gaps in the vessel must be sealed, as they pose a possible entry point for the contaminants.

2. Biomass concentration

Biomass concentration in the bioreactor must be high enough through the bioprocess run to result in a high yield.

In the case of yeast or bacterial culture, an industrial reactor can in theory be inoculated with a single cell.

Contrary to microbial cultures, in the case of animal cells minimum density needs to be achieved for the cell culture to start growing. The density usually lies between 5*104 and 3*105 cells per cm3, however, it depends on many factors, such as type of cell, media composition, reactor conditions,… In order to reach the minimum inoculation density, a pre-culture is needed.

When the cell density is lower than this minimum density the cells will not grow. Below a certain cell density growth factors are not produced in sufficient amounts to sustain cell growth.

3. Temperature

Every organism used in a bioprocess has an optimal growth temperature, that should be sustained throughout the bioprocess run. Most bioprocess temperatures are within the range of 30 °C to 37 °C. The temperature must be tightly controlled to within about 1 °C. Higher temperature deviations can have a dramatic effect on cell viability, while lower temperatures slow down cell metabolism.

During the bioprocess, cell density increases, and consequently the metabolic activity increases, which generates a substantial amount of heat. The generated heat must be removed before the temperature in the vessel increases.

Thus, efficient heat transfer to operate and maintain the system at the optimal growth temperature for the working organism is needed.

The temperature in a bioreactor can be managed by removing excess heat using an external jacket or coil, through which cooling water is circulated. Or in the case of adding heat, steam is used for circulation.

Alternatively, the equipment can be located internally, such as helical or baffle coils.

An additional option for maintaining a set temperature is to pump liquid from the reactor through a separate heat exchange unit.

In the table below advantages and disadvantages of the internal and external heat-transfer equipment are listed.

Due to the predominant risk of failure and contamination with internal coils, external jackets are a preferred choice for cell cultures, with low cooling requirements.

4. pH

Maintaining the right pH in the bioreactor is important for cell metabolism and to mimic cells’ natural environment. The pH is maintained in the range naturally with correct buffers, usually containing bicarbonate buffer. The pH is measured using a calibrated pH electrode.

Due to cell metabolism producing CO2 and lactate culture medium can become more and more acidic over time. The pH may be regulated by the addition of acid or base or by regulating the amount of CO2 blown over the surface. This is usually managed automatically by a controller, compares the measured pH to the defined setpoint.

The pH range for most common culture cell types is given in the table below.

5. Oxygen concentration (pO2)

Oxygen is one of the most critical nutrients for cultivating aerobic organisms. It is needed in order for cell metabolism to operate efficiently and allow the cells to grow. However, oxygen has low solubility in the liquid phase. This leads to one of the main problems in cell culture, which is the transfer of sufficient oxygen into the medium.

Initially, the demand for oxygen in the cell culture is low, due to the fact that the cell density is low. Over time cell mass increases and consequently the demand for oxygen supply increases. At a certain point, O2 solubility becomes limiting for cell growth, as the oxygen transfer rate into the bulk liquid is not rapid enough for the cell metabolism. In cell culturing the latter wants to be avoided, as it leads to cell death and consequently lower yield and lower quality product.

There is a whole blog post dedicated to oxygen in cell culturing. To satisfy your curiosity read.

6. Agitation

The natural environment of the cells requires fluids in order for all the biological and chemical processes to take place. Similarly, it is key to recreate a fluid environment in the bioreactor so the bioprocess can run efficiently. Fluids in a bioreactor are always in motion due to applying agitation.

Effective agitation is important to achieve efficient mixing. It is needed to get a homogenous distribution of the culture, temperature, pH, nutrients, and oxygen in the bioreactor and prevent settling. The agitation system consists of an impeller and a motor. The size and type of the impeller and where it is positioned play a key role in achieving efficient mixing.

If agitation is too high, it can cause high shear rates (as we've already covered in a previous blog post all about shear forces). When shear forces exerted on the cells are too large the consequence is cell death. This is undesirable as it leads to a decrease in viable cells and the release of cell contents - overall leading to lower product quality and yield.

Sources

• Stem Cells Culture Bioreactor Fluid Flow, Shear Stress and Microcarriers Dispersion Analysis Using Computational Fluid Dynamics: http://www.biotechrep.ir/article_69217_d41d8f11ebd299013d747fa36e9f3321.pdf
• Mass transfer and shear in an airlift bioreactor: Using a mathematical model to improve reactor design and performance (https://www.sciencedirect.com/science/article/pii/S0009250911000583)
• Introduction to CFD (https://www.sciencedirect.com/science/article/pii/B9780128015674000015)
• Bioprocess Engineering Principles (Second Edition) - Mass Transfer (https://www.sciencedirect.com/science/article/pii/B9780122208515000101)
• Macintyre F. 1972. Flow patterns in breaking bubbles. J Geophys Res 77: 5211–5228.
• Papoutsakis ET. 1991a. Fluid-mechanical damage of animal cells in bioreactors. Trends Biotechnol 9:427–437. Papoutsakis ET. 1991.
• Media additives for protecting freely suspended animal cells against agitation and aeration damage. Trends Biotechnology 9:316–324.
• J.D. Michaels, J.E. Nowak, A.K. Mallik, K. Koczo, D.T. Wasan, E.T. Papoutsakis, Interfacial properties of cell culture media with cell-protecting additives, Biotechnol. Bioeng. 47 (1995) 420_430.
• Mechanisms of animal cell damage associated with gas bubbles and cell protection by medium additives (https://www.sciencedirect.com/science/article/abs/pii/0168165695001337)