Cell culture fermentation basics of investing

In non-coalescing fluids, in which bubbles tend to stay separate, the mass transfer coefficient can be up to two times higher. For homogeneous flow, a similar expression for KLa applies; however, the gas hold-up is higher, because of the longer residence time of the bubbles in the liquid: 1.

Increase the transfer coefficient, KL; however, in practice this is a fairly constant parameter, not much influenced by the bubble diameter or medium properties. Increase the oxygen solubility via higher absolute and partial oxygen pressure. Ensure that, in stirred vessels, the operation is in the desired loading flow regime. Prevent coalescent broth properties by proper medium design and, especially, limited antifoam dosing.

When the cooling capacity is insufficient, the broth temperature will rise, which will negatively affect the process performance. In the overall heat balance, the microbial heat produced in the process reaction is the largest contributor. For aerobic fermentations, there is a direct link with oxygen consumption: for each mole of oxygen consumed, kJ heat are produced, 13 which is equivalent to — kJ per mole product.

For anaerobic processes, this is usually a factor of ten lower but, because anaerobic fermentations are often carried out at a larger scale, with less area per volume available for cooling, cooling strategies are equally important.

In STRs, the second most important source of heat production is the impeller. The advantage of the BC and the ALR is then clear because there is no impeller and pneumatic power input of gas at fermentation temperature has no heat effect. There can be other sources of heat as well, such as hot, sterilized feed streams or hot, compressed gases that are introduced in the broth.

A possible sink of heat is evaporation; water has a high enthalpy of vaporization, and therefore, water evaporation can have a considerable cooling effect. Water evaporation as a heat sink is more prominent in BC than in STR, as a BC is typically operated at larger airflow as well as lower headspace pressure, both of which promote the water evaporation rate.

The cumulative heat generated then needs to be transferred away from the fermenter. This can be done in various ways, for example via coils either mounted as a long spiral inside the reactor or welded at the outside wall as half-pipes , the vessel wall, baffles or via an external brothloop with a heat exchanger in which the heat is transferred to cooling water.

A series of steps is required to transport heat from the broth inside the fermenter via the coils to the cooling water. First, there is convective flow of heat from the bulk of the liquid to the coil. Second, there is transfer through liquid films outside and inside the coil and conduction through the coil material, and third there is convective heat flow away from the system via the cooling water.

In addition, there can be fouling layers at the broth and coolant sides that add additional resistance and further reduce the overall heat transport rate. There are two central terms that are used to quantify heat transfer. The heat capacity is thus related to the convection of the cooling fluid.

These terms, together with the temperature driving forces, determine the heat transfer and heat removal rates per volume: 1. The Stanton number, St, is a very important quantity in the design of a cooling system. It is a dimensionless number that indicates the ratio of the transfer and convective capacities of a system: 1. If the Stanton number is much lower than one, then there is an overcapacity of the cooling water flow and the temperature of the cooling water at inflow is close to the temperature at outflow.

A St far away from one indicates a wasteful investment. As an alternative to using coils for cooling, an external cooling loop can be applied. This presents some challenges, but also brings opportunities. The main advantage of external cooling is that there is greater freedom in design, meaning that the external loop can be designed in such a way that a lot more heat is transferred. Using a cooling coil may result in a slightly lower temperature in the thin liquid film on the broth side.

In comparison, the external loop can easily generate five degrees of cooling for all broth elements every 10 or 20 minutes. This is very effective, but the relatively high shear rate can be challenging for the microorganisms, and they also have to be capable of handling such cyclic temperature changes cold shocks. Another disadvantage of external cooling is that an extra pump is needed, which requires a capital investment as well as operational costs for electricity and maintenance and risks of contamination.

In terms of PI, heat management can be improved by a combination of: Improving the product yield on feedstock, which lowers the heat production stoichiometrically coupled to oxygen consumption and heat formation.

Maximizing the overall heat transfer coefficient via better flow around the cooling area, proper cleaning of both the broth and coolant side and a sufficiently thin wall design. Maximizing the cooling area in the fermenter, or applying an external cooling loop with a cooling machine. At this point, it is stated that, in large-scale bioreactors in which substrates glucose, oxygen are gradually supplied, there will always be concentration gradients, but the magnitude depends on the specific uptake kinetics and the mixing conditions.

This is consistent with the liquid circulation flow rates that are determined by the impeller action and gas supply. In the impeller flooding regime, tm is roughly twice as low, i. Comparing these results, it is clear that general mixing in a BC is significantly faster than in a multi-impeller STR see Figure 1. For stirred tank reactors STRs , the higher the aspect ratio, the more impellers are needed. A lower mixing number means better mixing. At this point, still, it is reiterated that a long mixing time per se is not enough to confirm a mixing issue.

The key indicator is the consumption time of the limiting substrate, which is strongly dependent on the residual broth concentration: only when this is shorter than the circulation time, will there then be significant concentration gradients and the cells may suffer.

Black box models must then be replaced by metabolically structured models. There, local depletion of substrate or accumulation of product will also slow down the reaction rate. However, microbial cells have a level of complexity that sets them apart from chemical catalysts: the genetic control system will react to gradients, as a result of which, even when the cells are back in a region with optimal conditions, the enzyme levels may have become suboptimal.

In addition, the different histories of the individual cells will result in population heterogeneity, which can further deteriorate the overall performance. These aspects are essential in metabolically structured models. The question is now if and how the PI tools could be applied to this field. If we take another look at the fundamentals of PI, as outlined in Figure 1. The ultimate objective is to go anaerobic, where the qo hits the zero limit Figure 1.

Maximize homogeneity: minimize the impact of gradients in substrate concentration, temperature and shear rate via reducing the average broth circulation time, minimizing zones with extremely long circulation time, elevating the residual substrate concentrations via adjusting the uptake kinetics, creating organisms with lower viscosity at equal biomass concentration and creating organisms that are more robust against the gradients and inhibitory levels of reaction products including CO2 , i.

Note that higher rates see previous point generally conflict with the aim of maximizing homogeneity. Arrange smart integration: combine multiple operations. The most impactful option, and fundamental to fermentation in general, is that organisms harbor efficient metabolic networks that are ideally programmed to maximize the flux to a desired end-product and minimize unwanted by-products. The overall conversion of a low cost, renewable feedstock into a valuable product can easily comprise 10 or 20 biocatalytic steps that are highly specific and of which part is irreversible, allowing near complete conversions.

The latter also minimizes the need for a plug flow type of operation, as recommended for chemical processes. Fermentations can be an alternative for processes with multiple chemical reaction steps, usually separated in different operations. Other integration possibilities are moving from batch to continuous processing see example 1. These four points make clear that, for FI, there are complementary biological and technological solutions. For example, a better transfer of CO2 away from the cells could very well relieve inhibition, but at the same time, a more CO2-tolerant host could present an alternative solution.

A further note is made regarding the preceding analysis of fluid flow patterns in different reactors. These are dependent on geometry e. Regime changes switches between flow patterns have consequences for the rates of mixing, cooling and mass transfer, which therefore also depend on geometry, scale and power input.

When designing and scaling up , one should be aware to be, and remain, at the intended, optimal flow regime, and at the same time, remain in the right kinetic regime. An optimal balance between transport and kinetic rates will allow proper scaling, both scale-up and scale-down, and a minimum cost level at large scale.

The four FI principles will be illustrated with four examples. The two main production countries are Brazil, which produces ethanol from sugar cane juice in continuous or fed-batch mode, and the USA, which uses corn syrup and fed-batch operation. The main cost factor in ethanol production is the cost of sugar.

With a maximum theoretical yield of 2 mol ethanol per mol glucose 0. It is therefore no surprise that all commercial processes operate with an actual yield of more than 0. Further improvement of the yield is now pursued by diverging co-products, e. The benefits are a short process and a high ethanol titer, i. Because of this, alternative configurations have been proposed and implemented, which are: the fed-batch mode, including extension variants such as repeated fed-batch, where a small part e.

There are many publications that advocate active immobilization or membrane techniques; however, these are still rarely seen in industrial practice. An exception is yeast flocculation, which can be applied to keep cells in the bioreactor and is widely applied in brewing. Typically, products are made in agitated bioreactors smaller than 10 m3, with relatively low power input such that mixing and mass transfer seriously limit the reaction rate and cell concentrations are not higher than about — cells per mL i.

The low power input is due to the perceived shear sensitivity of the applied cell lines, although it has been convincingly demonstrated that transport issues are more likely causing problems, e. The perfusion allows the inhibitory lactate and ammonia to be kept to low levels, due to fast removal, while keeping the cells in the reactor. Thus, the cell density has increased 10—fold to values of cells per mL, which is equivalent to about 50 kg cell dry matter per m3, i.

Because of the FI steps made, cultivation can be done in 10— times smaller vessels. This brings several synergistic advantages: Mixing problems will be less prominent because of shorter distances. Cooling will be facilitated because of a higher vessel wall area per volume albeit possibly counteracted by a higher rate of heat formation. Cell retention systems are far easier to implement. Disposable reactors can be introduced, minimizing cleaning efforts and risks for cross contamination.

Modular solutions become possible. For example, after intensification by a factor 20, a certain amount of product can be made in four disposable, well-characterized vessels of liter instead of one large steel bioreactor of 20 m3. Manufacturing flexibility is increased, which helps to support gradual market introduction and varying market demands for the product.

Related to these advances in upstream processing, it is no surprise that the main cost for manufacturing of biopharmaceuticals has shifted to the downstream section, and further efforts should be focused on this discipline. The fermentation is usually performed in aerobic BC fermenters, where the feeding rate of molasses and production of the cells is limited by oxygen transfer, under conditions that the local dissolved oxygen concentration is close to zero. In the following example, based on ref.

Groen et al. With a headspace pressure of 1 bar absolute and using a solubility for air at this pressure of 0. Because eqn 1. The injection nozzle of the pure oxygen created a supersonic shock wave and a bimodal bubble size distribution with peaks at a mean bubble diameter of 0.

The latter was similar to the 4—6 mm air bubbles that were injected using a standard sparger. Finally, in large-scale fermentation this additional mass transfer capacity was utilized by feeding more molasses, and indeed, the bakers' yeast productivity was enhanced 3.

One might wonder how these results compare to the simpler application of oxygen enriched air to increase the oxygen partial pressure and solubility. It can be calculated how much an additional supply of pure oxygen, with an oxygen : air ratio of 1 : 6 through the same sparger, would increase the oxygen mass transfer rate. The oxygen fraction in the inlet gas is then As a result, the average oxygen solubility is increased from 0.

Improvement of the biokinetics via strain development has long been recognized as a key success factor for fermentation. The area is very diverse, with examples in all product segments. For example, the performance of many antibiotics fermentations has been improved several orders of magnitude over periods of decades via introduction of classically improved strains — that is via random mutagenesis.

Or, in more recent years, it has become possible to rationally design and introduce metabolic pathways capable of synthesizing non-natural chemicals, such as BDO, caprolactam and other monomers for plastics, 8,10 or tailored antibiotics such as adipoylADCA, 6 that have replaced processes with multiple chemical reaction steps by one single, efficient fermentation step. It is underlined see Section 1.

Of course, the research on one-pot synthesis concepts is also intensive in chemical processes and could provide synergistic advantages as well e. In practice, there are restrictions to which level this is possible. In general, the maximum production rate in fermenters is restricted by six main factors: 16 Mass transfer.

Moreover, for larger fermenters, the impeller motor can never be bigger than about 1 MW. Superficial gas velocities, corrected for local pressure, can be increased to about 0. In order to avoid operation in the inefficient impeller flooding and complete gas recirculation regimes, the ratio between power input by impeller and gas needs to be between one and five, and preferably between one and two for energy efficiency reasons.

The headspace pressure can be elevated to 2—3 bar, but not higher because of process safety risks and costs associated with construction of large pressure vessels, CO2 inhibition and inefficient compressor performance.

To get an impression, applying eqn 1. Smaller vessels will allow a relatively higher impeller power input and then they will outcompete the BC. In Table 1. In addition, a higher aspect ratio from three to five increases the mixing time a factor of two to three.

In absolute terms, in the BC examples, a mixing time of about 60 s would mean an average circulation loop time of only 15 s, which is much lower than usually considered in currently published scale-down studies. Further, STR's with a high aspect ratio perform relatively poorly in terms of mixing.

Heat transfer. Because of the direct link between oxygen consumption and heat production, the examples in Table 1. In the m3 vessel, an additional area could be installed of m2 to solve the issue, e. In general, cooling will not present a serious issue, although a good design is important.

Increasingly, we may start to think of plant-based, fermentation-derived, and cultivated products as an intersecting venn diagram rather than as three distinct categories. For plant-based meat, eggs, and dairy, traditional fermentation can help optimize the digestibility, taste, texture, and nutrients of existing plant-based ingredients. Ingredients made with biomass fermentation or precision fermentation can also be combined with plant-based ingredients to make better plant-based meat.

On the cultivated meat side, precision fermentation can help efficiently produce nutrients and growth factors for cell culture media. Furthermore, proteins such as collagen or fibronectin produced through fermentation may serve as key animal-free components of scaffolding for more complex cultivated meat products.

Why is fermentation important? Alternative protein production is generally much more efficient than conventional meat production. The different types of fermentation offer several unique advantages that can further increase the efficiency of the alternative protein sector as a whole. Traditional fermentation can help us make better use of existing ingredients. Further, we can minimize food waste and transform low-value agricultural sidestreams into nutritious and delicious food using fermentation.

Biomass fermentation is one of the most efficient ways to produce lots of protein. The microorganisms used in fermentation reproduce and grow very quickly. The doubling time of these microorganisms is hours, compared to months or years for animals. Bioreactors are also very space-efficient. When facilities are scaled up, fermentation can produce many tons of biomass every hour. Many of these organisms are incredibly high in protein content.

For species used for biomass fermentation, protein content is more than 50 percent by dry weight. Compare that to about a quarter for conventional beef. Mycelium, microalgae, microbes, and fermented plant proteins can provide the sensory experiences and positive nutritional aspects of animal products but without undesirable substances, such as cholesterol, antibiotics, and hormones.

Efficient protein production is good not just for human health but for planetary health. This efficiency reduces pollutants and GHG emissions and saves water and land, allowing our food system to be much more sustainable. Fermentation can also utilize feedstocks that are low-cost industrial or agricultural side streams or waste streams. This lowers both variable and external costs associated with production, such as transportation of inputs. Feedstock diversification will enable local production that leverages readily available feedstocks without long-distance shipping.

What needs to be done to advance fermentation? Opportunities for advancing fermentation can be segmented into five key areas spanning the value chain: target selection and design, strain development, feedstock optimization, bioprocess design, and end-product formulation and manufacturing.

Although a number of organisms are already used by the fermentation sector, most possibilities have not been fully explored. Existing lines can be further modified for greater efficiency or target specificity.

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Lag phase occurs when cells are acclimatizing to culture conditions and are not dividing. Log phase occurs when cells are actively dividing. This is the best phase for cell experimentation and data collection. Cells should be sub-cultured when they reach late log phase. This occurs just before overcrowding. When cells approach overcrowding, cell growth slows. This is known as stationary phase or plateau phase.

Cells in this phase are at risk of cellular stress. When the natural process of cell death predominates, a cell population is considered to be in death phase, also referred to as decline phase. When the log of the cell count over time is graphed, it generates a sigmoidal curve as depicted in Figure 2. It is important to note that the amount of time spent in each phase differs between individual cell lines and cultures. An important metric to describe monolayer cell culture is confluence.

It is the percentage of the culture vessel surface area that appears covered by a layer of cells when observed by microscopy. The schematic in Figure 3 shows examples of different cell confluences. Cell density is used to describe cells that are grown in suspension. This image was created with BioRender. Choosing a cell line There are thousands of established cells lines used in laboratories around the world that can be purchased from commercial or non-profit suppliers cell banks.

It is critical to obtain cell lines from reputable suppliers as cells are verified and contamination-free. Obtaining cell lines from other laboratories has a high risk of contamination and lack of cell line validation and is therefore advised against. The criteria in Table 4 should be taken into consideration when selecting the appropriate cell line for an experiment.

The cell line chosen will largely depend on the nature and requirements of the experiment to be performed. Table 4: Criteria to consider when selecting a cell line. If not, non-human and non-primate cell lines usually require reduced biosafety restrictions which may be favorable.

Functional characteristics Use an appropriate cell line for your experiment. For example, liver- and kidney-derived cell lines may be more suitable for toxicity testing. They withstand rigorous treatment, require large amounts of air, and need shearing impellers to break apart the clumps.

Mammalian cells have no cell walls and are fragile and sensitive. Vessels for these cells come equipped with impellers that gently mix the media and minimize shearing. Mammalian cell culture vessels are available in water jacketed and non-jacketed configurations depending on the desired method of temperature control.

Determine what size vessel your research requires. Vessels are available in three sizes: 3-, 7-, and liters. A 3-liter system is ideal when you are conducting very basic or introductory research. Once optimal cell growth is achieved scale up to a larger vessel. For sizes larger than 15 liters, call for more information. Controllers and Accessories Temperature Control 1A.

A fermentation process at an incorrect temperature will result in poor growth, low production, or cell death. There are three ways to control temperature in fermentation: 1 a water-jacketed vessel 1B , 2 a heat exchanger, or 3 a heating blanket. Water-jacketed vessels and heat exchangers require a circulating water bath 1C. Heating blankets wrap around nonjacketed vessels and supply uniform heat. However, they also require a temperature controller. Use a pH controller 2B in conjunction with a peristaltic pump 2C to add acids or bases to your culture.

DO Probe: DO and proper aeration are essential in the fermentation process. Dissolved oxygen controllers 3B drive air pumps 9 that add oxygen, air, or nitrogen to the vessel according to the needs of your process. CO2 is introduced into the system during aeration.

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Mammalian cell culture vessels are available in water jacketed and non-jacketed configurations depending on the desired method of temperature control. Determine what size vessel your research requires. Vessels are available in three sizes: 3-, 7-, and liters. A 3-liter system is ideal when you are conducting very basic or introductory research. Once optimal cell growth is achieved scale up to a larger vessel. For sizes larger than 15 liters, call for more information. Controllers and Accessories Temperature Control 1A.

A fermentation process at an incorrect temperature will result in poor growth, low production, or cell death. There are three ways to control temperature in fermentation: 1 a water-jacketed vessel 1B , 2 a heat exchanger, or 3 a heating blanket. Water-jacketed vessels and heat exchangers require a circulating water bath 1C. Heating blankets wrap around nonjacketed vessels and supply uniform heat.

However, they also require a temperature controller. Use a pH controller 2B in conjunction with a peristaltic pump 2C to add acids or bases to your culture. DO Probe: DO and proper aeration are essential in the fermentation process. Dissolved oxygen controllers 3B drive air pumps 9 that add oxygen, air, or nitrogen to the vessel according to the needs of your process.

CO2 is introduced into the system during aeration. Too much CO2 can cause the culture to become acidic; too little can cause it to become basic. Antifoam Control not pictured : Microbial fermentation requires a high amount of agitation and aeration which often results in excessive foaming.

There are two reasons to control foam: 1 If foam levels get too high, foam will be forced out of the ports in the headplate, opening the system to contamination; and 2 Excessive foam inhibits oxygen transfer. This occurs just before overcrowding. When cells approach overcrowding, cell growth slows.

This is known as stationary phase or plateau phase. Cells in this phase are at risk of cellular stress. When the natural process of cell death predominates, a cell population is considered to be in death phase, also referred to as decline phase. When the log of the cell count over time is graphed, it generates a sigmoidal curve as depicted in Figure 2. It is important to note that the amount of time spent in each phase differs between individual cell lines and cultures. An important metric to describe monolayer cell culture is confluence.

It is the percentage of the culture vessel surface area that appears covered by a layer of cells when observed by microscopy. The schematic in Figure 3 shows examples of different cell confluences. Cell density is used to describe cells that are grown in suspension. This image was created with BioRender. Choosing a cell line There are thousands of established cells lines used in laboratories around the world that can be purchased from commercial or non-profit suppliers cell banks.

It is critical to obtain cell lines from reputable suppliers as cells are verified and contamination-free. Obtaining cell lines from other laboratories has a high risk of contamination and lack of cell line validation and is therefore advised against. The criteria in Table 4 should be taken into consideration when selecting the appropriate cell line for an experiment.

The cell line chosen will largely depend on the nature and requirements of the experiment to be performed. Table 4: Criteria to consider when selecting a cell line. If not, non-human and non-primate cell lines usually require reduced biosafety restrictions which may be favorable. Functional characteristics Use an appropriate cell line for your experiment. For example, liver- and kidney-derived cell lines may be more suitable for toxicity testing.

Finite or immortalized Finite cell lines are more functionally relevant as they have not undergone immortalization, however immortalized cell lines are often easier to maintain and clone. Normal or transformed Transformed cell lines have an increased growth rate and higher plating efficiency which is favorable, but the cells have undergone a permanent genetic change. Does this potentially impact your experiment?

Growth conditions and characteristics Is growth rate, cloning efficiency, or saturation density important for your experiment?