From culture to fermentation in bulk

Introduction- Industrial fermentation takes into account many factors for economical and safe production of desired products such as substrates and its conversion, product selectivity, product titer, and productivity. But the most important factor in a Bioprocess industry is the procedure operator is opting while going in the process. As cells are living organisms, designing an upstream process is complex and many factors must be considered, i.e., oxygen dispersion, shear sensitivity of the cells, nutrient concentrations, etc. A minute miss in the process may delay batch productivity and it can only be diagnosed and corrected if the operator looks backwards and check all the points effecting batches. Let us discuss step by step procedure when we go from culture to fermentation:

Preparation of stock culture- Often neglected task in a laboratory is to maintain a collection of stock cultures (bacterial/fungal). Poorly labeled, dried-out cultures and mishandling of cultures are a few examples of how maintenance comes into account. It is recommended to prepare cell copies and store them for pre-poured ready-to-use culture media. Microbes are present as a mixed population in our environment and the culture that contains just one species are known as pure strain. In pure strain, cells have similar nature, identical morphology, and characteristics that show the same results in experiments. For the preparation of single strain stock culture, cells must be multiplied and stored. This process is a preserving method for single cultures which helps to make it feasible for people to study and experiment with their clinical aspects and physiology.

• Inoculation of single strain- For the effective batch it is important to achieve a good growth of individual colonies using the apt technique. But firstly, all cultures are checked visually before use for contamination. Also, the cells must have the capability to produce a batch, their viability is checked before going to the process. By simply picking out a single colony from a cell bank/ freshly isolated strain/ genetically modified strain, cells are cultured either by streak on solid media or in a liquid medium. Both work well but streaking onto agar plate are highly used by microbiologists. Streaks on the plate do not only distinguish contaminated strains from original strains but also mutant strains. The rationale behind this is to avoid the enrichment of contamination if already in the glycerol stock. To revive pure culture, first streak the culture on an agar plate then take a well-isolated single colony and inoculate in broth medium. Thus, the revived culture will be pure.

• Spreading of inoculum- Ensure that plates are sufficiently dry before use. It should also be noted that there should be no delay in spreading the inoculum once it has been applied to the plate as some cells will rapidly attach to the agar. The loop is immersed in the cell bank culture vial to subculture carefully without exposing it to cross-contamination chances, proper care should be taken not to contaminate the loop holder. The technique can also be replaced by pipetting 2-3 drops from vial over the plate containing cell growth media (basically LB agar media is used for general strains)

• Storage and labeling of culture media- method of storing cultures under conditions that suspend metabolism is so-called cryogenic storage, usually using liquid nitrogen. Suspensions of bacterial cells or fungal spores are prepared in a cryoprotectant medium, generally containing 10-15% glycerol to minimize damage during freezing. The suspension is then dispensed into suitable containers, such as small screw-capped vials, which are then immersed in, or suspended above, liquid nitrogen.

Inoculation of culture media- Using a sterile wire loop, obtain a sample of microbial culture (a small amount of bacterial colony). Hold the bottle or tube to be inoculated at an angle and rub the loop against the side of the container below the level of the fluid. Growth can be observed in the inoculated tube as turbidity or milky appearance after incubation at 37°C for 24 hours. The medium in which seed is inoculated is known as inoculation medium and subculture media is known as production medium, where both differ in volume present in the flask. Inoculation media can be used in the range of at least 25ml-50 ml whereas production media capacity can be above 200ml.

Subculture from inoculation media to production media- A few microliters from inoculation media are taken to be subculture and using aseptic technique transfer to an appropriate broth with a pipette into production media. Emulsify the organism using the inside surface of the container and gently agitate before incubation to distribute the organisms throughout the broth. Keeping the flask at 30-37°C incubator/ shaker incubator overnight or specific time, production media with viable cells are ready to use in the fermentation process.

Initiation and preparation-

1. The process is initiated by a seed stock whether wild type or genetically modified is added into the basic ingredients of a compatible media which include nutrients, stabilizers, antibiotics, antifoaming agents.

2. A UV visible spectrophotometer is used to monitor the optical density which is a measure of the concentration of cell the bioreactor. An offline PH meter should be available to help track the acid-based balance and adjust online measurements. Also, the microscope is used to examine the viability and physical properties of cells and their constituents.

3. The bioreactor is equipped with a water jacket around the vessel to regulate temperature and an integrated sensor to monitor key environmental factors including DO, pH, internal temperature, water jacket temperature, vessel pressure, an agitator dedicated ports for adding seed stock, media ingredients, separate ports for an acid-based supplement.

4. Air filters are applied for supply and exhaust valves for drawing samples and sample harvesting. Most fermentation and monitoring functions can be managed from the bioreactor dedicated process controller before the fermentation process can begin the area must be prepared.

5. Preparation includes removing equipment’s parts and material that won’t be used in the process, cleaning and sanitizing the area and equipment as required by the SOPs (Standard operating procedure). A less expensive, less labor-intensive, less chemical expose, and faster process to clean interior surfaces of a reactor like pipes, valves, and other utility parts without vessel disassembly is CLEAN IN PROCESS (CIP). Before giving steam sterilization, the reactor vessel is first washed with caustic soda, effective acid solution, and water so that all the debris is removed. After the CIP procedure, SIP i.e., STEAM IN PLACE is required to eliminate unwanted microorganisms which can grow naturally in the fermentation media and process equipment. All required material and documentation should be gathered, and prepared and process control software should be loaded and verified. The fermentation batch process will be guided and documented with the record known as BPR (Batch Process Record). This record leads the operator through the process step by step with each step requiring a sign-off and separate verification. This record also includes spaces for documenting key times activities and instrument readings.

The procedure-

1. The fermentation process begins with the expansion of bacterial seed stock. After removing specific strain from the freezer and thawing, inoculate a small number of cells in fresh media and keep the system in a shaker flask till the number of cells has reached the target amount.

2. The very next day, a few microliters from inoculation media are transferred to the production media comparatively in the larger flask (250-500ml flasks) again till they reach the log phase, the thriving cells are ready for fermentation.

3. Meanwhile, in the fermentation area, operators begin their screening with a complete check of all critical equipment valves, caps, and lines, hoses are tightened, probes are verified and calibrated and HPW (high Purity Water) in kg is added to the vessel. The bioreactor is brought up to normal process pressure and held there to check for leaks, the pressure is monitored over 30 mins period. If a leak is detected then first it is tested, corrected, and run once again till the reactor passes the test. After all tests and checks, the vessel is ready to mix the media in the vessel, the agitator is turned on and media ingredients are added.

4. Once all the initial ingredients in high purity water are added, all ports and valves of the vessels are closed immediately. All condensate valves are kept open so that the bioreactor begins a SIP cycle on its own. The target for sterilization is 121°C for 30 minutes and as soon as the temperature climbs to the targeted temperature the condensate valves are closed and the SIP cycle completes automatically. Both the vessel and media are sterile and ready to add other media components, if necessary, from the hose attached to the vessel, the connection is steamed to sterilize it, and separately sterilized antibiotic solution is pumped into the vessel.

5. A manual pH reading of the media is taken and the bioreactor is set up for its fermentation cycle. After the inoculation hoses connected to the reactor are steamed for 20 minutes, the expended seed production media is pumped into the reactor containing the media of fermentation.

6. Now, the operator takes zero hour reading and begins to regularly monitor batch temperature agitator RPMs oxygen level, pH vessel pressure, OD, air flow rate, glucose concentration (if added). OD values are taken every 2-4 hours, so they are graphed as well as documented when the targeted levels of OD are achieved, samples are drawn to check the percentage of cell solids.

7. The product is now referred to as broth which contains spent media, and cellular activity is completed as feed is exhausted and key nutrients are mostly consumed, the batch has recorded the desired concentration.

8. The batch is then cooled down, pumped into a broth tank, and labeled with the batch number, volume, time, and date. The fermentation process is now complete, the harvested broth will now move downstream for the recovery processes where the cells will be ruptured to free proteins and those proteins will be separated from the other broth components to get desired product.

Source of contamination- It is very hard to cope with the fact that due to contamination many batches are discarded at the cost of all efforts and input the operator has put in. To avoid such circumstances, it is essential to identify the root cause of the problem and thus eliminate the causes. There can be many sources for contamination if we start from the starting point till the fermentation list can be as follows:

1. Incomplete or poorly autoclaved flasks, Petri plates, and media during the sterilization procedure

2. Mishandling while inoculation

3. Already the presence of foreign germ in the seed

4. Misplacing of valves, seals, connection lines, and ill-constructed O-rings

5. No proper use of air filters and mixed gas supply

6. Unfiltered reagents or solutions added through port

7. Failure in obeying CIP and SIP SOPs of bioreactor

8. Mishandling while sampling

Finding contamination is the top priority. This sounds obvious, but a quick kill of the process will save cost and resources before any investigation has even started. Devise tests for each contamination possibility and use them consistently. Good record keeping will ensure the next problem is fixed at least as quickly as the last.

Reference source: #Sciencedirect #generalmicroscience