Today, fermentation technology is at the heart of manufacturing industries spanning from pharmaceuticals and agriculture to biofuels and environmental management. The growing need for microbial products is pushing the boundaries of current fermentation technologies to deliver effective, stable, and safe products at scale. In order to achieve the desired outputs, microbial producers usually employ one of the two basic fermentation types – solid-state fermentation (SSF), or liquid-state fermentation (LSF; also known as submerged fermentation (SmF)). Both are highly valuable tools able to produce a wide range of microbial ingredients, but both come with their own set of limitations and challenges. This short guide explores the essentials of the two fermentation types, technical approaches and challenges that come with them, as well as a new emerging approach – pseudo-solid-state fermentation.

Solid-State Fermentation (SSF)

SSF involves the production of microbes on a solid, non-soluble substrate without free-flowing water. Some of the most common substrates for SSF include grains like rice, barley, wheat, as well as corn, sugarcane, and soybean. The purpose of the substrate is to provide physical support and a source of food to the microbes.

Microbes

SSF can produce a wide range of bacteria, fungi, and yeast. It provides the greatest advantages in filamentous fungi fermentation since these fungi naturally grow on solid surfaces and require physical support to develop their mycelia.

Bioreactor Concepts

According to their mode of operation, there are four basic types of bioreactors used in SSF – tray bioreactors, packed-bed reactors, air pressure pulsation bioreactors, and intermittent or continuously mixed bioreactors. All four have the goal of ensuring an even distribution of nutrients, oxygen, and microbial biomass, but they achieve it in different ways.

• Tray bioreactors are the most common ones and include stacked flat trays on which the substrate is spread in thin layers.
• Packed bed bioreactors, also known as cylindrical bioreactors, are composed of cylindrical shells with convex heads; the shells are positioned vertically and use gravity to ensure the flow of air and nutrients through the packed substrate.
• Air pressure pulsation bioreactors, as their name indicates, use air pressure pulsation to ensure even microbial growth.
• Intermittent or continuously mixed bioreactors use mechanical stirring mechanisms to mix the substrate periodically or continuously.

Industrial applications of SSF

Food ingredients (flavors, organic acids, xanthan gum, etc.)

Enzymes used in detergents and biofuel production (cellulases, amylases, and proteases)

Pharmaceuticals

Fugal biocontrol agents.

Liquid-State Fermentation (LSF)

Microbes cultivated in LSF grow in a liquid substrate that contains nutrient and oxygen levels optimized according to their needs. This method is commonly used for bacteria fermentation, but it is also widely employed in fungi fermentation. Since it is significantly easier to ensure uniform conditions in a liquid substrate, LSF enables a high level of precision in regulation of the microbial activity and product yield. This is the greatest advantage LSF offers over SSF.

Microbes

LSF can produce a wider range of microbes compared to SSF, as it offers a greater level of bioprocess control and approaches suitable for microbes that require specific conditions or are more sensitive.

Bioreactor Concepts

LSF is typically carried out in stirred-tank bioreactors, airlift bioreactors, or bubble column bioreactors.

• Stirred-tank bioreactors use mechanical agitators that continuously mix the substrate to ensure uniform distribution of nutrients and maintain optimal growth conditions.
• Bubble column bioreactors rely solely on air bubbles for mixing and oxygenation, making them energy-efficient and easy to scale up.
• Airlift bioreactors use air bubbles that provide oxygen and mix the substrate without mechanical agitation, which is more favorable for sensitive microbes like filamentous fungi.

Industrial applications of LSF

Pharmaceuticals (antibiotics, vaccines, and other microbially-based pharmaceutical compounds)  

  Biofuels (ethanol and biogas from yeasts or bacteria)

Food ingredients (alcoholic beverages, yogurt)

Challenges Related to Solid-State and Liquid-State Fermentation

All technologies come with a set of benefits, but also limitations and challenges. These challenges become especially evident in the fermentation and scale up of sensitive organisms, like Gram-negative bacteria and filamentous fungi.

Limitations of SSF

• Heat Accumulation: SSF generates significant metabolic heat, which can accumulate in the substrate and inhibit microbial growth or enzyme activity if not effectively managed.
• Moisture Control: Maintaining optimal and uniform moisture levels in a solid substrate is rather difficult, especially at greater volumes. Too much moisture in the substrate can create a lack of oxygen and invite contaminants, while too little can inhibit microbial growth.
• Scale-Up Complexity: Any type of SSF process scale-up comes with very challenging limitations, as the possibility of ensuring consistent aeration, temperature control, and nutrient distribution decreases significantly as the volumes go up.
• Process Control: Compared to LSF, SSF is more limited in controlling production parameters like pH and oxygen levels, which can lead to inconsistent product quality between the batches.
• Contamination Risks: The solid substrate may invite unwanted organisms if not sterilized properly and if the moisture content isn’t adequately controlled during the fermentation process.
• Product Quality: Biomass produced via SSF is bound to have some substrate residue in the end product, which lowers product purity and quality.

Limitations of LSF

• Substrate Costs: Specialized substrates for LSF can be very costly at larger production scales.
• Oxygen Transfer: Microbes produced via LSF can suffer from inadequate oxygen transfer, especially in high-density cultures. Lack of oxygen limits microbial growth, affecting yield and product efficacy.
• Shear Sensitivity: Many fungal species are sensitive to mechanical stress, and mixing can damage their hyphal structures and reduce productivity.
• High Water and Energy Demand: LSF processes require significant amounts of water and energy, making them costly and less sustainable, especially at larger scales (e.g. industrial scale).
• Contamination Risks: The nutrient-rich liquid substrate is highly susceptible to contamination, creating a need for stringent aseptic techniques that increase operational costs.

	Solid-State Fermentation	Liquid-State Fermentation
Substrate	Insoluble, solid substrates	Soluble substrates
Water consumption	Limited	High
Aeration	Easy	Limited
pH control	Limited	Easy
Temperature control	Limited	Easy
Energy consumption	Low	High
Waste production	Low	High
Hardware	Low-cost	High-cost
Scale-up	Highly limited	Easy
Product quality	Limited	High

Adapted from: Augur, Christopher & Viniegra-González, Gustavo. (1998). Molecular techniques applied to fungal strain upgradation capability related to SSF cultures.

The Best of Both Worlds – Pseudo-Solid-State Fermentation (PSSF)

How to achieve efficiency, scalability, versatility, and a high level of process control of LSF, while cutting down costs and lowering contamination risks? The answer to this question lies in a novel fermentation approach – Pseudo-Solid-State Fermentation (PSSF). This approach uses natural biopolymers as a substrate, which solves the problems of contamination and uneven nutrient and oxygen distribution, at lower costs.

PSSF makes the term “unculturable” obsolete, as it enables fine-tuning of production parameters at each step of the fermentation process and circumvents approaches that interfere with microbial growth (e.g. mechanical mixing). The biopolymer substrate is easily removed in downstream processing via drying, leaving little to no residue in the end product. These advantages of PSSF are ensuring high product quality throughout the whole process, yielding superior results, and enabling a wide range of applications.

Continuous improvements in bioreactor design and process engineering are unlocking novel fermentation technologies and making fermentation increasingly competitive as a manufacturing technology. These improvements are necessary to enable practical applications of the accumulated knowledge in microbiology, genetics, and strain engineering, and bring the much-anticipated biological revolution to traditional manufacturing industries.

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