The Importance of Downstream Processing in Bioprocess Development

Downstream Processing

Importance in Bioprocesses

Solid/Liquid Separation

• Filtration - This can be done using filter presses, filter-dryers, rotary vacuum filters, or membrane filters, among other equipment. The choice of filter depends on the characteristics of the solids and liquid, as well as the desired separation efficiency.
• Centrifugation - Centrifuges can effectively separate solids from liquids based on their differences in density.
• Sedimentation - In some cases, solids can be left to settle at the bottom of a vessel due to gravity, then removed from the liquid. This is often used in combination with other separation methods.
• Flotation - This involves the introduction of air bubbles into the liquid, which attach to the solid particles and cause them to float to the surface, where they can be removed.

Product Recovery

• Distillation - A widely-used technology for product recovery in bioprocesses, particularly for volatile products. For example, distillation is used to separate bioethanol from the fermentation broth due to its lower boiling point. For fuel-grade ethanol, azeotropic distillation may be needed.
• Gas Stripping and Liquid-Liquid Extraction - These techniques are often used to recover products that are toxic to the producing organisms or have higher boiling points. For example, in biobutanol production from lignocellulosic feedstocks, butanol recovery from the fermentation broth is challenging due to its toxicity to the microorganisms and its higher boiling point. Gas stripping and liquid-liquid extraction are among the techniques that have been explored for butanol recovery.
• Membrane-Based Separations - These techniques, which include ultrafiltration, electrodialysis, pervaporation, and Tangential Flow Filtration (TFF), are used when traditional methods are not efficient or generate large amounts of waste. One example of a product that can be recovered using this approach is lactic acid, derived from the fermentation of biomass-derived sugars. For this product membrane-based processes, such as ultrafiltration and electrodialysis, have been explored as alternatives to traditional methods like precipitation and ion exchange.
• Centrifugation and Chromatography - These techniques are commonly used for the recovery and purification of non-volatile products like enzymes. Using lignocellulosic feedstocks as a carbon source, enzymes such as cellulases and xylanases can be produced. The recovery process uses a combination of centrifugation to remove the microbial cells, ultrafiltration to concentrate the enzymes, and chromatography for further purification if needed.

Solvent Recovery

• Distillation - This involves the separation of substances based on their different boiling points. For instance, in the organosolv process, organic solvents such as ethanol or acetone are used to pretreat lignocellulosic biomass. Post-pretreatment, these solvents are typically recovered via distillation and then reused. Distillation is also used to recover ionic liquids in ionic liquid pretreatment, and in the separation of butanol and solvents in biobutanol production.
• Evaporation - This technique is used when the solvents are more volatile compared to the other components in the mixture. By supplying heat, the solvents evaporate, leaving the other components behind. The solvent vapours are then condensed and collected. An example of its application can be seen in certain processes that use organic solvents to extract lignin from lignocellulosic biomass.
• Liquid-Liquid Extraction - This method is used when the solvent forms two immiscible phases with water or other solvents. After creating two phases, they can be easily separated, which allows for the recovery of the solvent. A prominent example of this technique can be seen in biobutanol production. In the recovery phase, liquid-liquid extraction is often used, involving a solvent that can extract butanol from the fermentation broth. This solvent-butanol mixture is then subjected to distillation for the separation and recovery of both butanol and the solvent.

Product Purification

• Membrane Filtration - Membrane filtration technologies, such as microfiltration, ultrafiltration, nanofiltration, and Tangential Flow Filtration (TFF), can be used to separate the desired product based on size or molecular weight. For example, in the production of biofuels or biochemicals from lignocellulosic feedstocks, ultrafiltration can be used to concentrate the product and remove small impurities.
• Chromatography - Chromatography is a powerful technique for the separation and purification of products based on their different affinities for a stationary phase. For example, in the production of high-value chemicals or proteins from lignocellulosic feedstocks, chromatography can be used to purify the product to a high degree. Different types of chromatography, such as ion-exchange, affinity, or size-exclusion chromatography, can be used depending on the nature of the product and the impurities.
• Crystallization - Crystallization is often used for the purification of solid products. The product is first dissolved in a suitable solvent, and then conditions are set to allow the product to crystallize out, leaving impurities in the solvent. For example, in the production of organic acids from lignocellulosic feedstocks, crystallization can be used to purify the acid.
• Distillation - Distillation, as mentioned earlier, is not only used for product recovery but also for product purification. For example, bioethanol produced from lignocellulosic feedstocks often needs to be distilled multiple times to achieve the required purity for use as a fuel.
• Extraction - Liquid-liquid extraction can also be used for product purification, where the product is selectively dissolved in a solvent, separating it from impurities.

• Complexity - The product stream in a bioprocess often contains a complex mixture of the product, other metabolites, proteins, cells, and medium components. The product may also be present in very low concentrations. This complexity and the need for high purity often requires multiple downstream steps, each of which needs to be optimised and coordinated.
• Product Stability - Many bioproducts, such as proteins or enzymes, are sensitive to changes in temperature, pH, or shear stress, which can lead to product degradation during downstream processing. Maintaining the stability of the product throughout the downstream process can be challenging and may require the use of gentle separation techniques, stabilizing agents, or specific temperature and pH conditions.
• Scale-Up - While a downstream process might work well at a laboratory scale, scaling up to an industrial scale can pose several challenges. For example, the efficiency of a separation technique may decrease at a larger scale, or it may be more difficult to maintain consistent temperature or pH conditions. The cost of downstream processing can also increase significantly with scale.
• Cost - Downstream processing can account for a significant proportion of the total cost of a bioprocess, particularly for high-purity products. The use of expensive consumables, such as chromatography resins or membranes, and the energy cost of operations like distillation or centrifugation, can contribute to high downstream costs. Reducing these costs while maintaining product quality is a key challenge.
• Environmental Impact - Many downstream processes use solvents or generate waste streams that can have a significant environmental impact. Reducing this impact, through the use of green solvents, waste minimisation, or waste valorization strategies, is another important challenge.
• Regulatory Compliance - For bioproducts used in food, pharmaceutical, or medical applications, the downstream process needs to comply with strict regulatory standards. These may relate to the purity of the product, the removal of specific contaminants, or the validation of the downstream process. Meeting these standards can be technically challenging and add to the cost of the process.

Tangential Flow Filtration (TFF)

Filter Press

Filter Dryer

Supercritical CO₂ Extraction System

Distillation Equipment

Background

• Informed Process Design Decisions - Downstream processing often presents a significant challenge due to the complexity of the mixture obtained after conversion processes. This mixture includes the desired products and a variety of byproducts, unreacted feedstock, and impurities. TEA allows for comparisons to be made between different options for downstream processing, including product recovery, separation, and purification. By identifying the most cost-effective and technically feasible methods, TEA can inform critical design decisions that can influence the efficiency and economics of the overall process.
• Evaluation of Innovative Technologies - Technological innovation often plays a vital role in improving the efficiency and reducing the costs of downstream processing. However, these new technologies also come with risks, particularly related to scalability, performance stability, and regulatory acceptance. TEA can provide an objective assessment of these novel technologies by quantifying their potential economic benefits and associated risks, aiding in the decision-making process.
• Assessing Commercial Feasibility - Downstream processing can significantly impact the overall economics of a lignocellulosic bioprocess. It directly influences the cost of producing the final product, which in turn determines the product selling price. TEA allows the estimation of the product's market price based on the overall processing costs. The comparison of this price with existing market prices can provide insights into the commercial feasibility of the bioprocess.
• Guiding Future Research Directions - TEA provides a systematic way to identify the most expensive or technically challenging stages in downstream processing. By pointing out the areas that significantly impact the process economics or efficiency, it can guide the direction of future research and development efforts. This can lead to the generation of solutions that improve the overall process, enhance the product yield and quality, and reduce the associated costs.

Approach at Celignis

1. Initial TEA: At the beginning of the project, an initial TEA is conducted to evaluate the existing downstream processing options. This analysis includes an assessment of the technical challenges, performance, cost, and potential environmental impacts associated with each option. This stage provides an overview of the different strategies available and their relative merits and shortcomings, thereby setting the stage for informed decision-making.
2. Experimental Selection: Based on the outcomes of the initial TEA, a subset of promising downstream processing options is selected for further investigation. These options are then subjected to experimental testing in the laboratory to evaluate their performance under controlled conditions. This stage provides empirical data on each option’s efficacy and potential for optimisation.
3. Secondary TEA: - Following the experimental testing, a second TEA is conducted based on the results obtained. This analysis provides a refined understanding of the costs and performance of each option when applied in a real-world context. The findings from this secondary TEA inform the selection of the most appropriate downstream processing option for further development.
4. Process Optimisation: Once the most promising downstream processing option has been selected, further experimental work is carried out to optimise this process. These experiments aim to maximize efficiency, minimize costs, and ensure a high-quality end product. The optimisation phase is informed by the data and insights gathered from the previous experimental and TEA stages.
5. Validation at Higher TRLs: After optimising the selected process, it is then tested at higher Technology Readiness Levels (TRLs). These tests aim to validate the process under conditions that more closely resemble those of full-scale industrial operations. This stage can identify potential scale-up issues and provide data on how the process performs under realistic operating conditions.
6. Final TEA: In the final stage of the project, a detailed TEA is conducted based on the results obtained from the higher TRL testing. This final analysis evaluates the commercial-scale viability of the developed downstream process, taking into account all associated costs, potential revenues, and environmental impacts. This final TEA can help stakeholders make informed decisions about whether to invest in further development or commercial deployment of the process.