An Introduction to Bioreactors and Fermentation 🔬

The world’s population growth, heading towards 8.5 billion (UN News), is posing serious challenges to our food and healthcare systems. Traditional farming methods are struggling with rising costs and the advancing threat of global warming. Additionally, the increasing pharmaceutical demand necessitates biological mass production for the population and emphasizes the need for efficient alternatives on a large scale.

This article explores bioreactors and fermentation as innovative solutions to these pressing issues.

Istiklal Avenue, Istanbul. Istanbul is the most populated city in Europe, with an official population of 15.6 million (Wiki) (Image: IstTourStud).

Meet precision fermentation — a technology with roots dating back thousands of years, combined with today’s advanced laboratory techniques, poised to revolutionize how we produce essential goods.

In the following, I will handle bioreactors, mention fermentation and proteins, and comprehensively discuss the challenges bioreactors face.

Bioreactors: The Powerhouses of Biotechnology

Think of a bioreactor as a five-star hotel for microorganisms.

A bioreactor is not just a “living flask” where we let cells grow, reproduce, and synthesize substances we are interested in.

It is a luxurious and highly sterile habitat where we supply the cells and microorganisms with their needs and regularly check the input and output variables. This ensures they comfortably produce proteins (enzymes and antibodies), antibiotics, chemicals, pigments, lipids, and almost anything imaginable.

This technology can even provide food and oxygen for astronauts in space stations.

by Dall-E.

Bioreactors come in various types, each suited for different applications and organisms. Stirred tank bioreactors are powerful mixers, ideal for processes requiring intensive agitation.

Airlift bioreactors use air to circulate cells and nutrients, making them suitable for delicate cells needing gentle mixing.

Membrane bioreactors provide a stable environment, perfect for applications requiring high levels of purity and separation.

Two stirred tank bioreactors are described below, typically used to prepare “the carbon-rich meal” for microorganisms.

They don’t look like a luxurious restaurant though!

Left: Two feed fermenters and stirred tanks are in the picture at the LIVZYM Enzyme Facility in Kocaeli, Türkiye. Feed fermenters produce carbon sources for fed-batch reactors, where microorganisms are fed with carbon sources to initiate the production. Right: A closer look from the top.

Bioreactors play a crucial role in biotechnology, expanding production to an industrial scale.

0,1 m³ fed-batch reactor at LIVZYM Enzymes Facility. A bioreactor in this size can be called as pilot scale.

A 20 m³ fed-batch fermenter is shown in the picture. Measuring nozzles, a temperature sensor (blue), and the mechanical seal of the bioreactor are visible on top. The mechanical seal (dark blue) is one of the most complex parts of a bioreactor to produce, as it is designed to maintain sterile conditions while containing a sealing fluid (such as 80%-glycerin or sterilized water) as part of the “double-acting mechanical seal.”

These bioreactors often require strict sterile installation procedures and design features to ensure cleanliness and prevent contamination.

This allows for the effective cleaning of bioreactor parts and the removal of any microorganisms left from previous batches.

By maintaining such sterile conditions, the risk of contamination in subsequent production cycles is significantly reduced.

When I say a sterile design, I mean it very seriously!

Sampling valve from bottom side of a bioreactor is given. Mind the vapor pipe merged into the valve coming from the upper side, which depicts one fundamental way of sterilization of reactor parts by vapor pressure. Such design features play a crucial role in maintaining sterilization throughout the whole process.

Fermentation Unveiled: The Science Behind the Process

Fermentation, classically defined, is a biological process in which microorganisms such as bacteria, yeast, and fungi convert carbohydrates into energy and various byproducts such as alcohol, acids (e.g., lactic acid), and gases (e.g., carbon dioxide) under anaerobic conditions, meaning without the involvement of an oxidizing agent. This process is what gives products like yogurt and beer their unique flavors.

Fermentation: because who needs oxygen anyway?

Louis Pasteur demonstrated that fermentation is caused by microorganisms, specifically yeast. He described fermentation as “la vie sans l’air,” or “life without air.”

In one notable instance, Pasteur saved a brewery manager by identifying the cause of acetic acid production instead of alcohol, which was due to bacterial contamination.

This discovery not only saved the brewery but also led to the development of methods to prevent such spoilage in the future. (ExpYea).​

Thank you Pasteur for saving the taste of Weihenstephaner!

Louis Pasteur (1822–1895) was a French biologist and chemist renowned for his discoveries in microbiology, including the principles of vaccination, microbial fermentation, and pasteurization. Image source: Louis Pasteur, Wikipedia (public domain).

However, fermentation is a diverse process. Different microorganisms utilize various mechanisms to convert glucose into energy, showcasing the incredible variety within fermentation processes.

This diversity allows for the production of a wide range of products, depending on the specific microorganisms and conditions involved(RockEdu).

Precision fermentation uses organisms or cells to produce specific, high-value compounds such as enzymes, chemical or biological drugs, bioconjugates, and other valuable substances.

Unlike classic fermentation, industrial fermentation can be conducted under both aerobic and anaerobic conditions. This process is utilized in food production, biofuel generation, and industrial applications for making enzymes, pharmaceuticals, proteins, and bio-based chemicals.

By genetically modifying microorganisms to produce targeted molecules, precision fermentation offers a promising alternative to traditional agricultural practices, efficiently and sustainably producing and filtering essential components for traditional food, such as proteins, sugars, and lipids. This technology enables the production of synthetic meat, ice cream, and more!

Moreover, techniques like CRISPR, gene knockout/knock-in, recombinant DNA technology, and metabolic engineering furthermore enhance these microorganisms’ production capabilities.

This technology offers furthermore significant environmental benefits by reducing greenhouse gas emissions and land use, and it holds substantial economic potential by providing scalable and cost-effective alternatives to animal-based products.

With precision fermentation, life’s about to get much easier for vegans — delicious animal-free goodies are here, and cows can finally take that well-deserved vacation!

A 2019 study found that precision fermentation requires almost 1,700 times less land to produce the same amount of protein as soy farming in the US.

This highlights precision fermentation’s potential to produce food more sustainably and reduce environmental pressures from conventional agriculture​ (Animal Agriculture and Climate Change)​.

Ancient Roots of Fermentation

Fermentation is actually not a new term, it is an ancient technique used by various cultures around the world.

The Chinese have a long history of producing fermented alcoholic beverages and leavened bread, dating back thousands of years.

These early fermentation practices were crucial in developing food preservation and flavor enhancement techniques​ (Fermentology)​.

Early Neolithic jars with high flaring necks and rims from Jiahu (Henan province, China), dating to around 7050–6600 B.C.E., were analyzed via C-14 testing of carbonized fruit seeds and rice grains by Zhang Chi and Hsiao-chun Hung (Jiahu). Researchers discovered that these jars contained a mixed fermented beverage made from rice, honey, and fruit. (PennMuseum)

A portion of an ancient Egyptian artifact depicting grape cultivation and winemaking. Source: Ägyptischer Maler um 1500 v. Chr. (Wikimedia)

In Turkish and Greek cultures, yoghurt is also a well-known fermented product. Yoghurt has been a staple in these regions for centuries, made by fermenting milk with lactic acid bacteria.

This process not only extends the shelf life of milk but also adds unique flavors and textures, making yoghurt a versatile and nutritious food​ (Oxford Academic). Delicious!..

An Example of a Bioreactor Product: Proteins

A cell is the smallest unit of life, capable of independent function and reproduction. A human cell is estimated to contain approximately 20,000 to 25,000 different types of “proteins”, while a single cell can contain roughly 2 billion to 10 billion protein molecules (BioNumbers)​​.

The word “protein” is derived from the Greek word “proteios,” meaning “of the first rank” or “primary.” The term was coined in 1838 by the Swedish scientist Jöns Berzelius to reflect the importance of this group of molecules. Proteins are fundamental components of all living cells and play critical roles in various biological processes, including catalyzing metabolic reactions, DNA replication, and transporting molecules within cells (Britannica).

Given that the average size of a proteins are measured in nanometers, while a human cell is usually micrometers across, it is clear that the interior of a cell is a bustling environment, crowded with numerous proteins and other molecules performing biological functions, such as enzyme activity and signal transduction.

Proteins gain their functionality through their 3D structures. This image shows one of the earliest depictions of myoglobin, published by John Kendrew and his colleagues in 1958. Balls in different tones represent heavy metals attached to the polypeptide chain. The marks on the scale are 1 Å apart (nature).

A 3D model of a protein structure showing its secondary and tertiary features. The red regions represent alpha helices, while the yellow regions represent beta sheets, with lines indicating the polypeptide backbone. This visualization helps understand the protein’s folding and function (Umass).

The scale difference between proteins and the cell itself suggests a complex and dynamic environment where countless biochemical reactions and interactions occur simultaneously.

Below is a snippet from a protein simulation, illustrating how densely packed the inside of a cell is.

Similar to a metro ride in Tokyo!

This illustration presents a dynamic molecular representation of the bacterial cytoplasm, offering an impressive view of its densely populated environment. It incorporates 50 of the most prevalent macromolecule types found in Escherichia coli, achieving a total concentration of 275 grams per liter (CellimLi).

In precision fermentation, microorganisms are genetically to enable the targeted production of specific proteins, vitamins, flavors. We knock out unnecessary genes.

For instance, Aspergillus oryzae, a fungus, can be genetically optimized to increase amylase secretion for industrial needs.

However, these genetic modifications can make the organism more vulnerable to external risks, as disrupting the expression of certain genes can lead to unintended consequences like the overproduction or underproduction of specific metabolites.

Such outcomes can disrupt the cell’s equilibrium and capacity to adapt to environmental shifts.

Moreover, organisms engineered for industrial processes are often designed for highly controlled conditions, including temperature, pH, and nutrient levels.

While such optimization improves efficiency under ideal conditions, it renders these organisms less resilient to deviations from these set parameters.

As a result, genetically modified organisms become more sensitive and require stringent protection against environmental changes and contamination from competitive species.

Otherwise, we may end up with a production batch failure.

Remember, bioreactors are like toddlers: they need constant attention and the right conditions to thrive.

From Lab to Industry: The Complexities of Scaling Up

Raising a child may seem demanding, but at least they are not a bioreactor.

We have already mentioned that a bioreactor is a device or system that provides a controlled environment for the growth of cells or microorganisms under specific conditions to produce biological products.

However, one major challenge in the biotech industry is expanding production

Scaling it up!

Rigid-walled stirred-tank bioreactors for cultivating human and animal cells desgined by Eppendorf, ranging from 100 ml to 40 L.

Bioreactors often considered as one of the most difficult pieces of equipment to scale (Fredrik Lundström, 2018).

Let me tell you why Fredrik was right.

Let’s assume we have successfully achieved optimal conditions at both laboratory scale (5L) and pilot scale (100L).

While aiming to let organisms grow under these optimal conditions, we regulate parameters such as pH and heat exchange rates to keep them “happy.”

However, scaling up to industrial levels often presents unexpected challenges because many parameters do not directly correlate with size.

In scaling up, maintaining geometric similarity, impeller tip speed, power per unit volume (P/V), oxygen transfer rate, and other factors across different scales is crucial.

These parameters often interact in unpredictable ways, leading to difficulties in maintaining optimal conditions for microorganism growth and production.

Thus, the scale-up demands substantial effort, relying on rules of thumb and trial-and-error methods, is often limited to mechanistic model approximations.

High initial investment required for bioreactors pose on the other hand other significant challenges. A 100m³ bioreactor can cost up to 10 million dollars.

There is no universally accepted methodology for determining the suitable scale-up criteria and their operational ranges, as these are process-specific, requiring a range of experiments for each new production process and scaling operation.

Establishing a stable and effective production line requires significant time, financial investment, and extensive experience.

The complexity of the scaling process means that even slight parameter deviations can lead to significant issues.

However, the future brings promising solutions, thanks to the digital revolution:

1. Digital Twins: Creating digital replicas of bioreactors to simulate and optimize the scaling process without physical trials.
2. Machine Learning and AI: Utilizing data-driven approaches to predict and adjust parameters for optimal performance.
3. Advanced Sensors and Automation: Implementing real-time monitoring and automated adjustments to maintain precise control over production conditions.
4. Computational Fluid Dynamics (CFD): Using simulations to understand and optimize fluid flow and mixing within bioreactors at different scales.
5. Hybrid Models: Combining mechanistic and data-driven models to enhance the predictability and efficiency of bioprocesses.

Left: Construction of a tube bundle (Rohrbündel) of a bioreactor for numerical computer modeling. Right: CFD (Computational Fluid Dynamics) analysis of it is shown, with colors representing the degree of damage or wear due to vortices occurring inside the bioreactor as a result of the stirring process. “Niedrig” (low) and “Hoch” (high) indicate the damage levels. Influencing factors include the density and viscosity of the fluid, stirring speed, and stirrer type (e.g., axial or radial flow). etc. (Source: Bioprozesstechnik, 4th Edition)

This image presents color maps showing the volumetric mass transfer coefficient (kLa) in three different bioreactors: a 2,000-liter single-use bioreactor, a 5,000-liter, and a 10,000-liter stainless steel bioreactor, from left to right. The CFD analysis on the left illustrates fluid dynamics in these cylindrical and cuboid bioreactors of various volumes. The color scale represents kLa values, indicating the rate of oxygen transfer within the bioreactors, highlighting how geometry and size influence oxygen transfer efficiency (Source: BioTech&BioEng) .

These methods aim to establish reliable know-how and make bioreactors reproducible, shaping the future of humanity in the process. Will they succeed?

I can’t see the future!

I will discuss these opportunities, use-cases and many more in the future. If you enjoyed this topic, I highly encourage you to do some further research.

Thanks for reading!