Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Industrial Protein Synthesis Using Recombinant Microorganisms
Enhancing Fermentation Efficiency

Regardless of the bioreactor type, parameters such as dissolved oxygen concentration, pH, Temperature, and agitation intensity must be strictly controlled during Fermentation. Too great a variation in any of these can significantly reduce Cell Growth Rate and the Stability of the protein product.

Optimal growth of E. coli and many other microorganisms used as expression systems for recombinant Proteins typically requires a well-aerated culture medium. The maximum oxygen utilization rate during fermentation, Q_max, depends on The Cell mass X, the maximum specific growth rate μ_mах, and the growth yield based on oxygen consumption Y_O2. This relationship is expressed by the following formula:

Class="center">Q_max = Xμ_max/Y_О2.

Since oxygen is poorly soluble in Water (0.0084 g/L at 25 °C), it must be supplied to the medium continuously. Typically, sterilized air is sparged through the fermenter for aeration. However, this generates bubbles in the medium, and if they are too large, The rate of oxygen transfer to the Cells will be insufficient to support their growth. Therefore, during fermentation, it is necessary to monitor the dissolved oxygen content in the medium using a special sensor, ensure its uniform distribution throughout the volume, and maintain thorough agitation of the culture to ensure efficient bubble dispersion.

Most microorganisms grow best at a pH between 5.5 and 8.5. It should be borne in mind, however, that cellular metabolites released into the culture medium can alter its pH. Therefore, the pH must be carefully monitored during fermentation, and acid or alkali must be added to the fermenter as needed. These additions must be well mixed with the medium and uniformly distributed throughout the entire volume.

Another parameter critical to the success of fermentation is temperature. If it is below the optimum, Microbial growth slows down and metabolic activity decreases. Conversely, if the temperature is too high, it can lead to premature induction of Protein Synthesis (if controlled by a temperature-sensitive repressor) or the induction of heat Shock proteins, which activates cellular proteinases and reduces the yield of the protein product.

Thorough mixing of the culture is necessary, first, to ensure uniform delivery of nutrients to the cells and, second, to prevent the accumulation of toxic metabolic byproducts in any localized pocket of the bioreactor. Efficient mixing is relatively easy to achieve in small-scale cultivation, but in large-scale cultivation, maintaining the homogeneity of the culture medium becomes one of the major challenges.

Agitation of the culture medium also affects other parameters: the rate of oxygen transfer from gas bubbles to the liquid medium and then from the medium to the cells; Heat transfer efficiency; the accuracy of measuring metabolite concentrations in the culture broth; and the dispersion efficiency of added Reagents (acids, bases, nutrients, etc.). Based on all this, one might assume that the more vigorously the culture is agitated, the better it grows. However, excessive agitation can generate shear forces that are damaging to bacterial and mammalian cells, or cause a temperature rise that also affects their viability. Therefore, as always, a balance must be struck between The Need for thorough mixing and the preservation of cell integrity.

There is another aspect of large-scale fermentation that is unrelated to the technical side of the process but concerns whether recombinant microorganisms are used. In most countries, large-scale cultivation of recombinant microorganisms is subject to specific rules and regulations. Although most recombinant microorganisms pose no danger, it is crucial to prevent their accidental release into the environment. To achieve this, reliable containment systems are used to prevent the escape of live recombinant organisms or to limit their spread if a leak does occur. Furthermore, prior to final disposal from the facility, all recombinant microorganisms must be inactivated in accordance with specific guidelines. Spent culture medium must also be tested for the presence of viable microorganisms to prevent their release into the environment. High-Density Cultures Generally speaking, when producing foreign proteins using recombinant E. coli, the guiding principle is that maximizing the final cell density also maximizes the product yield. In fed-batch fermenters, the concentration of recombinant E. coli cells can reach 50 grams of dry cell weight per liter of medium (and in some cases >100 g/L). (The dry cell weight of E. coli is approximately 20–25% of the wet cell weight.)

One way to increase cell density is to optimize the culture medium. It should be noted that certain nutrients, including carbon and nitrogen sources, inhibit cell growth at excessively high concentrations. Glucose inhibits growth at concentrations >50 g/L, ammonia at >3 g/L, iron →1.15 g/L, magnesium → 8.7 g/L, phosphorus →10 g/L, and zinc → 0.038 g/L. Therefore, simply increasing the nutrient content in the culture medium during batch fermentation will not yield the desired results. Furthermore, because the composition of complex media such as peptone or Yeast extract can vary slightly from batch to batch, fermentation in them is not always reproducible.

Acetate, which can inhibit cell growth, is produced by E. coli when grown under oxygen-limited but glucose-excess conditions. Structure/149.html">The problem of its formation can be resolved by using glycerol instead of glucose as a carbon source, lowering the temperature, or using a recombinant E. coli strain capable of converting acetate into less toxic substances (see Chapter 6).

Oxygen limitation can also occur in high-density cultures. To avoid this, the air supply rate (sparging) or the agitation speed is increased, or both. Additionally, pure oxygen can be supplied to the culture instead of air (which contains only 20% oxygen), or cells can be grown under pressure to increase oxygen solubility. Alternatively, it has been proposed to express the Vitreoscilla Hemoglobin Gene in host E. coli cells, which would significantly increase oxygen uptake by the growing cells.

High cell density is most commonly achieved using a fed-batch cultivation mode. The nutrient feeding strategy can vary: continuous, stepwise, or exponential. In continuous feeding, constant amounts of nutrients are added to the medium throughout the fermentation. However, under these conditions, the specific growth rate continuously declines. In stepwise feeding, nutrients are added in increasing amounts as the cell concentration rises, largely compensating for the decline in specific growth rate. In exponential feeding, nutrients are added at a rate that maintains a constant cell growth rate. Fed-batch nutrient delivery can be automated based on real-time measurements of the limiting Substrate Concentration (e.g., glucose) in the medium during fermentation.

Bioreactors

A brief review of the biotechnology literature might suggest that the number of bioreactor types is limitless. In reality, however, all bioreactors can be classified into three main groups:

✵ stirred-tank reactors (Fig. 16.4, A)

✵ bubble Column reactors, in which air or another gas is sparged to mix the contents (Fig. 16.4, B)

✵ airlift bioreactors with internal (Fig. 16.4, C) or external (Fig. 16.4, D) loop recirculation; mixing and Circulation of the culture medium in these systems are driven by a gas flow (usually air), which creates a density gradient between the upper and lower Regions of the culture medium.

Bioreactors of the first type are the most widely used. They offer the following advantages:

✵ allow for easy adjustment of process conditions

✵ are readily available commercially

✵ provide efficient gas delivery to growing cells (in engineering terms, they have a high volumetric mass transfer coefficient, k_La)

✵ have a long history of use for cultivating various microorganisms.

Fig. 16.4. Different types of bioreactors (simplified diagram). A. Stirred-tank bioreactor. B. Bubble column. C. Airlift bioreactor with internal loop. D. Airlift bioreactor with external loop. Arrows indicate the direction of the culture medium flow.

In stirred-tank bioreactors, gas (typically air) is supplied to the culture medium under pressure through a sparger—either a ring with multiple small holes or a single-orifice tube. The former produces small air bubbles and ensures their more even distribution; however, tube spargers are used more frequently because they clog less often. To distribute the gas evenly throughout the bioreactor volume, one or more impellers are used. They break up large air bubbles, disperse them throughout the Reactor, and increase their residence time in the culture medium. Under vigorous agitation, the average bubble size in large bioreactors is virtually independent of the sparger orifice size. Gas distribution efficiency depends primarily on the impeller type, rotational speed, and the PHYSICOCHEMICAL PROPERTIES OF the medium. If the bioreactor is too large and the gas entering from the sparger is unevenly distributed throughout the volume, even vigorous agitation will fail to homogenize the medium.

Many culture media are highly corrosive, and to prevent corrosive or mechanical damage to the bioreactor walls, they are typically made of stainless steel or Glass. Glass components are generally used only in laboratory-scale bioreactors with a capacity of less than 50 L.

The size of a bioreactor is limited by its ability to efficiently dissipate heat generated by microorganisms during METABOLISM and released As a result of agitation. If heat dissipation is insufficient, the temperature of the medium may exceed critical levels, reducing product yield. To remove heat, a cooling jacket or internal cooling coils are used. Internal cooling is more efficient, but the coils often become coated with a layer of growing cells, which hinders cooling and sometimes interferes with the vigorous agitation of the culture medium.

Contamination of the fermenter by Fungi or Bacteria poses a major threat. Therefore, bioreactors are designed to be sterilizable, typically using pressurized steam. There must be no "dead zones" inside the reactor that are inaccessible to steam during sterilization. All Valves, sensors, inlets, and outlets must be treated. When designing, engineers often face a dilemma: whether to use the maximum number of sensors for complete control over the fermentation process or to limit them to a minimum set to make maintaining sterility easier.

Vigorous agitation of the culture medium during fermentation often leads to foaming. This can wet the exhaust filter through which air exits the bioreactor, reducing air flow, and can also allow foreign microorganisms to enter the reactor. To control foaming, chemical antifoaming agents or mechanical foam breakers are used. However, the presence of chemical agents can impair oxygen transfer and sometimes inhibit cellular Enzymes, reducing the growth rate of microorganisms. Furthermore, if antifoams are not removed, they can contaminate the final product. The foaming problem can be solved by leaving a sufficiently large headspace at the top of the bioreactor for air bubbles to burst. However, this reduces the working volume of the reactor by about 25%.

All these considerations also apply to "pneumatic" reactors such as bubble columns and airlift bioreactors. Thus, ensuring sterility, constant pH, and temperature are key requirements for any cultivation method, regardless of the bioreactor design.

The Design Features of bubble columns and airlift bioreactors offer several advantages over stirred-tank reactors. "Pneumatic" reactors are more cost-effective because mixing is achieved by an upward flow of air (or another gas in the case of anaerobic microorganisms) rather than an energy-intensive mechanical impeller. Furthermore, the absence of a mechanical stirrer eliminates one of the pathways for foreign microorganisms to enter the bioreactor. In pneumatic bioreactors, the culture medium does not experience such intense hydrodynamic shear stress (the sliding of liquid layers relative to each other), and in airlift bioreactors, mixing is more uniform throughout the volume. Reducing shear effects is highly important for the following reasons:

✵ recombinant microbial cells are more fragile than untransformed cells because a portion of their energy resources is spent on the synthesis of foreign proteins, resulting in a less robust Cell wall

✵ the most common cellular response to external stress is a decrease in The amount of all synthesized proteins, including recombinant ones

✵ shear effects can alter the physical and Chemical properties of cells, making subsequent Processing more difficult. For example, the amount of cell-surface Polysaccharides may increase, leading to poorer conditions for Cell Harvesting and lysis, as well as complicating the purification of the recombinant protein.

In bubble columns, air is supplied under high pressure to the bottom of the bioreactor; as they rise, small air bubbles coalesce, leading to uneven gas distribution. In addition, high-pressure air supply can cause excessive foaming. All of this limits the versatility of these designs, narrows the range of viable operating conditions, and restricts the maximum feasible size of bubble columns.

Airlift bioreactors can be used both in pilot-scale setups and for industrial fermentation. Gas is supplied to the bottom of a vertical channel. As it rises, it entrains the liquid toward the top of the channel—the gas-liquid separator—where it is partially vented. The denser, deaerated liquid then descends through another vertical channel to the bottom of the reactor, and the process repeats. Thus, the culture medium, along with the cells, continuously circulates within the bioreactor.

Airlift bioreactors are of two main types. In the first type, the reactor consists of a single vessel with an internal draft tube that facilitates liquid circulation (internal-loop reactors) (Fig. 16.4, C). In the second type, the culture medium flows through separate, independent channels (external-loop reactor) (Fig. 16.4, D). The design of internal-loop airlift reactors is simpler, but once built, their volume and circulation rate remain fixed. In contrast, an external-loop bioreactor can be modified to accommodate different fermentation conditions.

Airlift bioreactors are generally more efficient than bubble columns, especially for high-density or high-viscosity microbial Suspensions. Mixing is more effective, and the problem of bubble coalescence is less pronounced. In particularly large airlift fermenters, such as the 1,500,000 L fermenter by ICI (England) designed for Single-Cell Protein production, the time required for cells to complete a full cycle in the reactor is quite significant. To supply them with substrates throughout their movement with the liquid flow, substrates were introduced at multiple points along the entire length of the reactor.