Types of Bioreactors for Bacteriophages Production

Regarding the different types of bioreactor systems, the most relevant are batch, single-stage continuous, multi-stage continuous and two-stage cycling. In order to obtain a production process with greater bacteriophage titers, it is essential that the type of bioreactor chosen allow the establishing of optimal operating and infection conditions. ⁴¹

The most common type of bacteriophage production is based on a batch operation (Figure 2.8). According to this approach, the host bacterial population grows up to a certain optical density, in which the phage infection is initiated for obtaining a specific initial MOI. Then, both bacterial and bacteriophage populations grow until the bacteriophages takes over and the lysis of bacterial cells occurs, in a process highly dependent on the population dynamics and the transient conditions. ⁴¹

In this way, the initial conditions of infection such as the initial MOI, the infection load and the infection volume significantly influence the final titer obtained in the bacteriophage production for a given set of processing parameters. ⁴¹

When the objective is to obtain greater final phage titers and lower levels of cell debris, the recommend methodology is to initiate the bacteriophage infection at a low initial infection load and a low initial MOI, to allow the host bacterial population to grow to a high cell number before population-wide lysis occurs. On the other hand, if the main goal is to perform shorter batches, the most adequate approach can be the use of a high initial MOI with a large infection load. This is possible because a large portion of the host bacterial population is rapidly infected, and a rapid population-wide lysis is obtained with few bacteriophage replication cycles. ⁴¹

The main advantages and disadvantages/limitations of a batch operation for the bacteriophage production in bioreactor are summarized in Table 2.1.

Table 2.1 – Advantages and limitations of a batch operation for the bacteriophage production in bioreactor.

Information adapted from Sauvageau et al. and Agboluaje et al. ^8,41.

Advantages Limitations

Robustness Low throughput

High phage titers Large equipment volumes

Ease of operation and control Downtime required for preparation Sterilization and cleaning of the bioreactor

relative to the production time Potential batch-to-batch variations

Large footprints

Regarding the single-stage continuous modes of operation for bacteriophage production (Figure 2.9), the most commonly known are the chemostat and the turbidostat, which are associated with Figure 2.8 – Simplified schematic representation of a batch bioreactor used for bacteriophage production.

Adapted from Agboluaje et al. ⁴¹.

bacteriophage progeny at high volumetric throughput. However, these two approaches are not recommended for a bacteriophage-based therapeutic production in large scale due to the dynamic nature of the interactions between the bacterial host and the bacteriophages, with the rise of mutations and host-bacteriophage coevolution. Therefore, the underlying selective pressures present in chemostats can lead to the assortment of significant mutations in both bacteria and bacteriophages genomes, which makes it very difficult to operate the process at steady state, a crucial prerequisite for reliable continuous production in terms of the product quality and regulatory issues. ⁴¹

Moreover, the residence time distributions in continuous stirred-

tank reactors is another important factor since the several host cells will spend different amounts of time in the bioreactor, being possible that some of them leave the reaction vessel without being infected by the bacteriophage. Then, a very controlled balance between the rates of nutrients addition (named dilution rate), of host cell proliferation, of host cell infection and of cell lysis is required, because any variation in these parameters can lead to a different steady state, in terms of host cells concentration and of final phage titer, or to the washout and loss of bacteriophage production. Another relevant factor is the host cell population density for which the infection is justified, that is, the threshold population density. Above this value, the phage infection will totally destroy the host population, resulting in a bacteriophage washout. Below this threshold value, the infection transmission rate will be very low, with the consequent end of the infection process. ⁴¹

The study of Nabergoj et al. is an example of the application of a continuous culture operating system, specifically a chemostat, with the objective of understanding the effect of bacterial growth rate on bacteriophage population growth rate, adsorption constant, latent period and burst size. The researchers used a model system composed by the bacterium E. coli K-12 and the well-studied bacteriophage T4, having obtained results that effectively proved the important influence of bacterial growth rate on the bacteriophage population growth rate and on the corresponding phage growth parameters previously mentioned. ¹⁰

Additionally, there is another type of continuous bioreactor that enable a more efficient process for the bacteriophage production, named two-stage or multistage continuous operation (Figure 2.10). The general methodology applied in this operation approach consists in the separation of the host bacterial growth from the phage infection process. In the first stage the bacterial cells are grown in the absence of bacteriophages inside the bioreactor while in the second stage it is performed the

phage infection using the host cells from the first stage. It is possible the operation of the two stages with different sets of experimental conditions, each optimal for their specific purpose (optimal growth or infection). ⁴¹

Figure 2.9 – Simplified schematic representation of a single-stage continuous bioreactor used for bacteriophage production. Adapted from Agboluaje et al. ⁴¹.

Figure 2.10 – Simplified schematic representation of a two-stage continuous bioreactor used for bacteriophage production.

Adapted from Agboluaje et al. ⁴¹.

The two-stage continuous operation mode is a very attractive alternative to the single-stage continuous process due to the improvement of many aspects of the latter. Better threshold population densities, greater phage titers, the robustness of the process, and the low probability of coevolution are some of the interesting characteristics of this continuous operation mode. It is important to note that the threshold population density needs to be maintained in order to ensure that the infection process occurs in the second stage. For maintaining a steady state infection, the rate of host cells fed to the second bioreactor should be proportional to the infection rate, otherwise a higher quantity of phages will leave the fermenter without infecting a host cell, resulting in the bacteriophage washout. Furthermore, since the majority of the host bacterial cell growth occurs in the first stage, without the presence of bacteriophages, it is significantly reduced the possibility of emergence a bacterial mutation that leads to the bacteriophage resistance. Regarding the limitations of this type of bioreactor, one of the most relevant is the existence in both stages of a residence time distribution. This, on the one hand, can lead to the appearance of a resistant bacterial host because some host cells have the possibility to replicate in the second stage. On the other hand, it means that various uninfected host bacterial cells are present in the outlet stream, which is not the ideal situation. ⁴¹

Finally, it is also possible the production of bacteriophages in bioreactors using a two-stage or multi-stage semi-continuous operation (Figure 2.11). The methodology is similar to that previously described for the two-stage continuous operating mode. Initially, in the first stage, the uninfected bacterial host cells are grown in a process performed as a sequential batch bioreactor or as a self-cycling fermentation.

Then, in the second stage, it occurs the bacteriophage infection, with a small portion of the bacteriophages produced in a previous infection cycle being used to infect the host bacterial fed from the first stage. ⁴¹

The two-stage semi-continuous operation mode combines advantages from a batch process, namely the robustness and the high phage titers obtained, and also from a continuous approach, specifically the high volumetric throughput, the reduced downtime per production time and the smaller equipment footprints. Furthermore, the issues related to the residence time distribution are not present, and also the possibility of coevolution is significantly reduced, since all host bacterial cells are destroyed and removed between infection cycles. However, this type of bioreactor has some limitations in the monitoring and control of the process, whereby it is required additional research for the development of more sophisticated monitoring and control strategies. ^8,41

An example of the development of a semi-continuous or self-cycling process for the production of bacteriophages, with two independently-controlled stages, is described in the study of Sauvageau et al.. The researchers used a model system composed by the bacterium E. coli and the bacteriophage T4 in order to verify the feasibility of a new process on the production of a virulent bacteriophage.

The first stage, having only the uninfected bacterial host cells growing, was operated under the principles of self-cycling fermentation (SCF). This is an automated and a non-steady state method that Figure 2.11 – Simplified schematic representation of a two-stage semi- continuous bioreactor used for bacteriophage production. Adapted from Agboluaje et al. ⁴¹.

uses a control parameter associated with cell growth, in this case the carbon dioxide evolution rate (CER), in order to keep the cell population in the exponential growth phase and simultaneously to synchronize the host bacteria.The second stage, in which the bacterial cells from the SCF stage were infected by the bacteriophages, was operated using an automated cycling mode named self-cycling infection (SCI), taking in account the CER and other associated data as the control parameters. After the ending of each infection cycle, the bacteriophages were harvested, and it was initiated a new infection process through the addition of host bacterial cells from the SCF stage. With the several results obtained, this semi-continuous mode of operation allowed the occurrence of stable infection cycles, with all the phage titers obtained being reproducible between different cycles and very similar to those achieved in batch mode with the same operation conditions. Some crucial characteristics of this process were the production of phages in different cycles with no significant variations in terms of infectivity as well as the improvement of the specific phage productivity in the infection stage. Therefore, the authors demonstrated a significant potential of this SCF/SCI method for upstream and downstream steps optimization, due to the decrease in the process time and in the associated costs. ⁸