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

Molecular Biotechnology of Microbial Systems
Industrial Protein Synthesis Using Recombinant Microorganisms
Microbial Growth

Microorganisms can be grown in a batch fermenter, a fed-batch fermenter, or in a continuous culture (Fig. 16.2). In the first case, microorganisms are grown under sterile conditions without adding fresh culture medium during Fermentation. In the second case, increasing amounts of nutrients are periodically added to the culture during fermentation, while the culture medium is not removed until the process is complete. In continuous fermentation, fresh culture medium is continuously fed into the fermenter, and an equal volume of Cell suspension is simultaneously withdrawn. In all cases, oxygen (usually as sterile air) is bubbled through the medium if necessary, and an antifoaming agent and (if needed) an acid or base are added.

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Fig. 16.2. Time course of cell and substrate concentrations in batch culture (A), fed-batch culture (B), and continuous culture (C).

Batch culture

During batch fermentation, the COMPOSITION OF THE culture medium, the concentration of microorganisms (biomass concentration), The chemical composition of the Cells, and The amount of protein product or metabolite depend on the growth phase, cellular METABOLISM, and nutrient availability. Six main growth phases are distinguished: the lag phase, the acceleration phase, the logarithmic (log) or exponential phase, the deceleration phase, the stationary phase, and the death phase (Fig. 16.3).

Typically, following the inoculation of a sterile culture medium, there is no immediate increase in cell number. During a certain period, known as the lag phase, the cells adapt to their new environment, such as a different pH or nutrient concentration. During this adaptation, new, previously inactive metabolic pathways may be induced. A lag phase is observed whenever the inoculum is obtained from a culture whose growth has ceased due to substrate depletion or product inhibition (i.e., a stationary phase culture). The duration of the lag phase depends on how long the inoculum cells remained in the stationary phase and how much the medium in which the culture grew differed from the new, fresh culture medium. However, if the inoculum is a culture in the exponential phase, a distinct lag phase may be absent, and growth will begin immediately after inoculation. Between the lag and exponential phases, There is a brief period called the acceleration phase, during which the Cell Growth Rate increases until it reaches a constant value.

Fig. 16.3. Growth curve of a bacterial culture during batch fermentation. 1 — lag phase, 2 — acceleration phase, 3 — exponential phase, 4 — deceleration phase, 5 — stationary phase, 6 — death phase.

During the exponential phase, cells undergo several divisions, and the specific growth rate remains constant. When substrate (nutrients) is in excess and in the absence of growth inhibition by any compound present in the culture medium, the specific growth rate is independent of the Substrate Concentration. Under these conditions, the growth curve can be described mathematically, allowing biotechnologists to model the process and subsequently scale it up. The increase in cell mass over time, dX/dt, is equal to the product of the specific growth rate μ and the biomass X:

Similarly, the increase in cell number dN/dt is equal to the product of the specific rate μ and The Cell number N:

The specific rate μ depends on the concentration of the limiting substrate (carbon or nitrogen source) S, the maximum specific growth rate μ_mах, and the substrate-specific constant K_s.

Both S and K_s have the dimension of concentration (g/L or M).

Sometimes, instead of the specific growth rate, the doubling time or generation time td = ln2/μ is used. This is the time required for the cell number or biomass to double under specific conditions. For unicellular microorganisms, the value of μ_mах is typically in the range of 2.1 to 0.086 h^-1, which corresponds to a doubling time of approximately 20 min to 8 h.

When the substrate is in excess (i.e., S >> K_s), μ = μ_mах, and the maximum growth rate of the culture in the exponential phase is achieved. Typically, the value of K_s is so small that the substrate concentration rarely becomes comparable to K_s during the exponential phase. For example, in the case of Escherichia coli, K_s for glucose is approximately 1 mg/L, whereas the initial glucose concentration in the medium is usually around 10,000 mg/L. However, at the end of the exponential phase, little substrate remains, and S may fall below K_s. When S < K_s, the deceleration phase rapidly ensues. It can be very brief or even virtually unnoticeable because, due to the large number of cells at the end of the exponential phase, the substrate can be consumed very quickly.

As a result of the depletion of the limiting substrate (e.g., carbon source) or the accumulation of growth-inhibiting metabolic products, the increase in cell number gradually ceases, and the culture enters the stationary phase. During this time, the biomass remains constant, but metabolism often undergoes drastic changes. It is during this period that compounds of commercial interest (secondary metabolites), such as Antibiotics, are frequently synthesized. The duration of the stationary phase depends on the specific Organism and growth conditions.

In the death phase, the energy reserves of the cells are exhausted, and metabolism ceases. In most Industrial processes, fermentation is stopped and cells are harvested before the onset of the death phase.

Fed-batch culture

In this case, substrate is periodically added to the fermenter, and the final product is harvested only upon completion of the process. Substrate addition leads to the extension of the exponential and stationary phases, and to an increase in biomass and the amount of metabolites synthesized during the stationary phase (e.g., antibiotics). However, during the stationary phase, microorganisms often synthesize Proteolytic Enzymes (proteinases) that degrade any Proteins they produce. Therefore, if the goal of fermentation is to obtain protein products, the process must be stopped before transitioning into this phase. Direct measurement of substrate concentration during fermentation is often difficult, so to determine when to add the next portion of substrate, other indicators correlating with its consumption must be used, such as the amount of organic acids synthesized, the pH value, or the amount of CO₂ produced. Generally speaking, fed-batch fermenters require continuous and more rigorous control than simple batch fermenters and are therefore used less frequently. However, they offer several advantages for developing protein production systems using recombinant microorganisms, and are thus becoming increasingly popular.

Periodic addition of substrate to a growing culture of recombinant microorganisms extends the exponential phase and delays the onset of the stationary phase, during which cellular stress responses are initiated, proteinase synthesis occurs, and other metabolic changes take place that reduce the yield of recombinant protein. To maintain host cell metabolism, the amount of added substrate must be constantly increased. To ensure continuous synthesis and Stability of the recombinant protein, the process must be carefully monitored, and substrate (a carbon and nitrogen source along with Trace Elements) must be added as soon as the need arises. Depending on the genotype of the microorganism and The Nature of the recombinant protein, the product yield in fed-batch fermentation can increase by 25–1000% compared to simple batch fermentation.

Fed-batch fermentation can be used to cultivate not only microorganisms but also mammalian and insect cells. This is highly important because: 1) such cultures are increasingly used to obtain protein products of medical significance; 2) without periodic substrate addition, animal cells do not synthesize foreign proteins very efficiently.

Continuous culture

In continuous fermentation, steady-state conditions—i.e., conditions under which dX/dt = 0—are maintained because, at a constant bioreactor volume, the loss of cells (product removal) is precisely balanced by their increase through division. More formally, for a continuous process in a steady state, the dilution rate D, defined as the medium flow rate F divided by the constant volume of medium V in the bioreactor,

D - F/V,

is equal to the specific growth rate μ:

D = (dX/dt)(l/X) = μ.

To obtain a continuous culture with constant hydrodynamic characteristics, it is necessary to establish conditions under which the specific growth rate is below the maximum value μ_max. To achieve this, the pump controlling the feed rate F must be adjusted to maintain a constant culture volume V in the bioreactor.

The primary goal of industrial fermentation is to maximize product yield while minimizing costs. This can be achieved by designing a custom, highly efficient fermenter configuration for each specific process. Generally speaking, continuous fermentation is not very widely used in industry, primarily because researchers have accumulated the most experience working with batch cultures. However, the cost of producing a given amount of biomass in a continuous fermenter is much lower than in a batch fermenter. This cost reduction is due to the following factors.

✵ To obtain a given amount of product, continuous fermentation requires smaller bioreactors than batch fermentation.

✵ In batch fermentation, large-scale equipment is required for Cell Harvesting, Cell Disruption, and subsequent purification of the protein product or metabolite synthesized by the microorganism. In contrast, synthesis in a continuous fermenter occurs gradually, allowing for less bulky equipment.

✵ A continuous fermenter does not experience the downtime of a batch fermenter, which must be periodically discharged and prepared for the next run. Bioreactor downtime due to maintenance, cleaning, or sterilization is the primary cause of reduced process efficiency. With continuous fermentation, this downtime is significantly reduced.

✵ The physiological state of most cells in a continuous fermentation is uniform, resulting in more consistent synthesis. In batch fermentation, however, slight variations in cell harvesting times—ranging from mid-exponential to late-exponential phase—can lead to significant inconsistency.

Continuous fermentation has already been used for the industrial PRODUCTION OF SINGLE-cell proteins, antibiotics, and organic Solvents.

However, this method also has its drawbacks.

✵ Continuous fermentation times can sometimes reach 500–1000 hours, during which some cells may lose their recombinant Plasmids. Plasmid-free cells typically expend less energy and divide faster than plasmid-bearing cells; consequently, product yield may decline over time due to a decrease in the number of producing cells. This problem could be resolved by integrating the cloned Gene into the host genome.

✵ It is extremely difficult to maintain sterile conditions in industrial-scale facilities over long periods. Additionally, continuous processes require sterile backup equipment, which significantly increases capital costs.

✵ The quality standards for culture medium components used in large-scale fermentation are not as stringent as those for laboratory-scale media; they can vary from batch to batch, which may alter cell physiology and reduce productivity.

The reputation of batch fermentation as a highly reliable system hinders the transition to any other type of fermentation, even though continuous operation is more efficient. Nevertheless, several laboratory-scale (up to 10 L) and pilot-scale (up to 1000 L) systems have recently been developed for continuous and fed-batch fermentation to produce proteins using recombinant microorganisms. This suggests that the wider industrial adoption of continuous and fed-batch fermenters is only a matter of time.