• Pretreatment of Biomass
    Process Development
    At Celignis Biomass Lab


Lignocellulosic Biomass

Lignocellulosic biomass is defined as a plant, or plant-derived, material that is mostly composed of cellulose, hemicellulose, and lignin. Lignocellulosic feedstocks are highly abundant, covering many biomass types including grasses, wood, energy crops (e.g. Miscanthus and coppices), agricultural residues (e.g. straws and corn stover), and municipal wastes.

Lignocellulosic feedstocks are highly abundant and can often be sourced sustainably, at low cost, without leading to land-use conflicts. As a result, there is currently great interest in obtaining chemicals, fuels, and biomaterials from such biomass.

Biomass Hydrolysis

A major pathway by which many lignocellulosic feedstocks are processed is known as hydrolysis, where sugars are released from the lignocellulosic polysaccharides (i.e. cellulose and hemicellulose).

However, the hydrolysis of lignocellulosic polysaccharides is not easy and is influenced by the complex inter-associations between hemicellulose and cellulose and between these polysaccharides and lignin in the lignocellulosic matrix. In particular, the crystalline nature of much cellulose and the existence of a physical barrier of lignin surrounding the cellulose fibres are said to be major contributors to the recalcitrance of cellulose.

The mechanism of hydrolysis is further complicated by the fact that different process intensities are required for the hydrolysis of cellulose versus hemicellulose. The more intense conditions required for cellulose hydrolysis may degrade the sugars hydrolysed from hemicellulose (to products such as furfural and formic acid).

For this reason, most hydrolysis technologies employ pre-treatment processes that aim to break apart the matrix (and in particular the associations between lignin and cellulose), reduce cellulose crystallinty, and hydrolyse hemicelluloses, hence separating the hydrolysate from cellulose which can then undergo more severe/targeted treatment.

Get more info...Biomass Hydrolysis

Need for Pretreatment

For this reason, most hydrolysis technologies employ pre-treatment processes that aim to break apart the matrix (and in particular the associations between lignin and cellulose), reduce cellulose crystallinty, and hydrolyse hemicelluloses, hence separating the hydrolysate from cellulose which can then undergo more severe/targeted treatment.

How to Develop a Sustainable Biomass Pretreatment Process

There are several important considerations when developing a biomass pretreatment technology, including:

Feedstock Chemistry

The chemical composition of lignocellulosic feedstocks varies greatly. This variability if seen not only in the relative proportions of the different polymers of lignocellulose (cellulose, hemicellulose, lignin) but also in the compositions of each of these polymers.

The particular chemistry of the feedstock is a crucial factor when deciding on an optimal pretreatment, since some types of biomass (due to their chemistry) are unsuitable for certain pretreatment technologies. For instance, steam explosion is much less effective as a pretreatment on softwoods compared with hardwoods due to the fact that softwood hemicelluloses do not contain acetyl groups (which aids the mechanisms of steam explosion pretreatment). Similarly, a feedstock with a very high lignin content may be more suited to organosolv pretreatments that can separate the lignin from the cellulose, allowing the feedstock to be more accessible to cellulase enzymes.

The content and composition of extractives is also an important consideration when designing a pre-treatment process since these can complicate downstream valorisation stages.

Lignocellulose Fractionation

Some pretreatment processes (e.g. steam explosion) focus primarily on a disruption of the lignocellulose matrix to allow for it to be more readily hydrolysed in downstream processes. However, other technologies (e.g. when using acids, alkalis, solvents, or hydrothermal pretreatment) involve the production of two (or more) process streams with the primary lignocellulose polymers (cellulose, hemicellulose, lignin) displaying a preference for one of these streams.

For example, an organosolv approach employing acetone as the solvent (as was the case in our CBE project UNRAVEL) would result in the hemicellulose and lignin in the liquid phase with the solid phase mostly composed of cellulose.

Hence, if your target for biomass valorisation is different downstream processes for each polymer then certain types of pretreatments that allows for such fractionation of biomass chemistry should be preferred.

Downstream Technologies

There are different ways in which the output streams (solid and liquid) of the pretreatment process can be valorised. For example, the hemicellulose sugars that become separated into the liquid phase in a number of pretreatment processes (e.g. acid pretreatments and hydrothermal pretreatments) can be biologically-processed (e.g. fermented to ethanol) or chemically/catalytically processed (e.g. catalytic production of xylitol from xylose).

Each type of downstream valorisation process will have its own preferred conditions and requirements for the composition of the stream being processed. For example, many organisms used for fermentations may have their activities inhibited by the presence of degradation products of the sugars (e.g. furfural is the degradation product of xylose and arabinose). This would lead to a preference for a pretreatment that minimises the formation of inhibitors (for example hydrothermal pretreatment may be favoured over an acid pretreatment in this case). Conversely, if you are using a downstream technology that is not affected by these inhibitors then other aspects of the pretreatment become more important (for example, the process with the lower CAPEX/OPEX or the process which provides the highest yields of monomeric sugars).

Commercial & Environmental Viability

For a bioprocess to be commercially-viable it is necessary for the revenues associated with it to exceed the operating costs and that these operating margins cover the CAPEX in a reasonable timeframe.

Some pretreatments can have significantly higher CAPEX and/or OPEX than others. However this does not mean that they are not commercially viable since they may provide more valuable products. An example would be organosolv technologies which typically have higher OPEX costs (due to chemicals usage and the energy costs of product/solvent recovery) and CAPEX costs. However, organosolvs can produce a high-quality lignin with signficant revenue-generation potential. For this potential to be realised pretreatment optimisation should focus on the use of a feedstock, and the development of a process, that will achieve the product requirements for entering these higher-value markets.

Similarly, bioprocesses targeting products to replace fossil-derived products should be demonstrably more sustainable. Hence, the pretreatment and downstream steps should be optimised so that the greenhouse gas emmissions, and environmental impacts of the whole process, represent a signficant improvement on the status-quo.

At Celignis we are experts in the technoeconomic analysis (TEA) of bioprocesses and consider this, as well as the environmental impacts, at all stages of process development.

Types of Biomass Pretreament

Mechanical Pretreatment

Mechanical pretreatments of biomass usually focus on a reduction in the particle size of the feedstock, allowing for more surface-area availability in downstream processes.

Get more info...Mechanical Pretreatment

Steam Pretreatment

Steam pretreatment uses high-pressure steam at elevated temperatures. In steam-explosion pretreatment this is followed by rapid decompression which physically disrupts the biomass structure.

Get more info...Steam Pretreatment

Hydrothermal Pretreatment

Hydrothermal pretreatment, also know as Liquid Hot Water (LHW) pretreatment, does not involve the use of any chemicals but operates at elevated temperatures and pressures

Get more info...Hydrothermal Pretreatment

Acid Pretreatment

This pretreatment involves treating the biomass with a dilute solution of a strong acid at elevated temperatures. A primary target is the hydrolysis of hemicellulose into monomeric sugars.

Get more info...Acid Pretreatment

Alkali Pretreatment

Alkali pretreatment uses chemicals (e.g. sodium hydroxide, potassium hydroxide, calcium hydroxide) to disrupt the complex structure of lignocellulosic biomass with lignin removal a primary target.

Get more info...Acid Pretreatment

Organosolv Pretreatment

Organosolv pretreatment fractionates biomass into its three major components (cellulose, hemicellulose, and lignin), with obtaining a lignin of a high-purity and quality being a particular target.

Get more info...Organosolv Pretreatment

Other Pretreatments

This page covers some other pretreatments: ionic liquids pretreatment, hydrogen-peroxide pretreatment, ultrasonic pretreatment, microwave pretreatment, fungal pretreatment, and bacterial pretreatment.

Get more info...Other Pretreatments


How Celignis Develops Biomass Pretreatments for Our Clients

1. Understanding Your Requirements

Prior to undertaking bioprocess projects we learn from our clients what their targets are from the process as well as whether there are any restrictions or requirements that may need to form the boundaries of the work that we undertake. These help to guide us to then prepare a potential bioprocess development project.

For example, in the context of pretreatment, the primary target of one client may be to achieve the greatest possible glucose yields from cellulose, with the valorisation of the other main components of biomass (e.g. lignin and hemicellulose) being of much lesser importance. In contrast, another client may be particularly focused on the production of oligomers from hemicellulose, with the efficient hydrolysis of cellulose being lower down in their list of priorities. Such differing targets are likely to lead to either different pretreatment technologies being used in each case and/or in the selection of different process parameter ranges for the experiments to be undertaken.

Similarly, a requirement from a client for no chemicals to be used would narrow-down the range of options that we could consider for the pretreatment process.

2. Detailed Feedstock Analysis

A thorough understanding of the chemistry of a feedstock is a crucial component in designing an effective pretreatment process. Detailed compositional analysis is also necessary in order to accurately determine process yields and efficiencies.

We recommend that each feedstock that is to be investigated for pretreatment be analysed with one of our in-depth lignocellulose analysis packages (P10 or, ideally, P19). We advise that these analyses are undertaken prior to the start of the bioprocess project as the resulting data will allow us to consider the appropriate pretreatment technologies and process conditions for any particular feedstock.

Hence, based on the analytical results and the client's requirements, the Celignis Bioprocess team will then meet to discuss and prepare a project proposal for biomass pretreatment development and optimisation. After this proposal is reviewed by the client, and revised if needed, we are then ready to start work on developing the pretreatment process.

3. Lab-Scale Pretreatments

Our projects usually involve undertaking a number of pretreatment experiments, covering a variety of process conditions. We follow a scientifically-based Design of Experiments (DoE) protocol where the criteria and boundaries for this DoE are formulated in close collaboration with our clients, considering the chemistry of the feedstock(s) and our understandings of the mechanisms of biomass pretreatment.

We usually recommend that these initial optimisation experiments are undertaken at the lab-scale (around TRL3) in order to reduce costs and the length of the project. For each experiment we analyse the solid and liquid outputs of the pretreatment process, leading to a detailed data-set where effects of process conditions on the yield and composition of the various streams can be explored and mapped.

We can also undertake a second iteration of lab-scale experiments in order to fine-tune the conditions based on the knowledge gained in the initial experiments.

4. Downstream Valorisation Experiments

The analytical results from our lab-scale experiments will provide us with valuable data on the composition of the various output streams (e.g. solid and liquid) from the pretreatment process. However, to fully evaluate these streams for their suitability for the planned applications (for example, ethanol from the cellulosic fraction) we advise that their downstream processing is tested.

We can undertake a wide variety of tests here, based on our client's requirements and the targeted end products. The results from these experiments allow for a more thorough evaluation of the pretreatment process and for the conditions to be optimised according to the final product requirements, rather than just according to the chemical composition of the pretreatment's output streams.

We advise that these initial downstream valorisation experiments are also undertaken at the lab-scale, using the outputs of Stage 3. This will enable us to gather more data, at lower costs, allowing for the pretreatment process to be optimised most effectively.

5. Validation at Higher TRLs

Once we have concluded our optimisation of the pretreatment process conditions at the lab-scale we can then test those conditions at higher technology readiness levels (TRLs). The scales at which we can operate are dependent on the type of pretreatment technology employed, but can reach up to 100 litres.

We have all of the necessary downstream equipment to efficiently handle the solid and liquid streams arising from these scaled-up activities and we can also undertake scaled-up processing of these different fractions from the preatment, for example high-TRL enzymatic hydrolysis runs for the solids and fermentation runs for the liquid stream.

If we find that there are differences between the yield and compositions of the different post-pretreatment streams, compared with our lab-scale experiments, then we can explore the potential reasons for these and work on final tweaks to optimise the bioprocess for higher TRLs.

6. Technoeconomic Analysis (TEA)

The Celignis team, including Oscar our chief TEA expert, can undertake a detailed technoeconomic analysis of the developed process. We apply accurate and realistic costing models to determine the CAPEX and OPEX of simulated and pilot scale processes which are then used to determine key economic indicators such as IRR, NPV and payback periods.

Within these TEAs we can consider the pretreatment as a discrete independent module or we can evaluate the biomass valorisation process as a whole, evaluating the impact of the pretreatment on the whole biorefinery concept. We also undertake sensitivity analyses to assess the effect of variable costs and revenues on the commercial viability of the pre-treatment process.

Our preferred approach is to include TEA studies at each stage of the development of the pretreatment bioprocess, so that the process can be optimised in a commercially-relevant way, followed by a more detailed TEA after the process has been optimised and tested at higher TRL levels.

Click here to read more about the technoeconomic analysis (TEA) services offered by Celignis.

Bioprocess Pretreatment Projects - Case Studies

Pretreatment of Palm Residues

Celignis undertook a bioprocess development project for a client, based in the Middle East, that was targeting the production of ethanol from the residues of local palm trees. This was a lab-scale vertically-integrated project covering pretreatment, hydrolysis, and fermentation.

The client initially had a certain type of pretreatment technology in mind and requested that we undertake a series of experiments to assess it. However, based on our initial compositional analysis of the feedstocks, we had reservations that the chosen pretreatment would be suitable for such biomass. We discussed this with the client and it was agreed that three different types of pretreatments were tested, with each pretreatment type being undertaken a number of times in order to allow for an initial evaluation on the effects of varying the process parameters on the yield and compositions of the output streams.

The results from these initial pretreatment experiments confirmed Celignis's reservations regarding the originally-chosen pretreatment and resulted in the pretreatment technology that we recommended, based on the feedstock compositional data, being selected for further study.

There then followed a more extensive series of lab-scale experiments focused on optimising the pretreatment conditions so that the yields and commercial viability of the process as a whole could be improved.

Pre-Pretreatment Extraction

Extractives, if present in signficant quantities in the feedstock, can present a challenge to certain pretreatment technologies. For example, in organosolv pretreatments the extractives typically end-up in the liquid output stream of the process, along with lignin and hemicellulose sugars. At that point it can be hard to separate these extractives from the lignin meaning that the final lignin product may have a lower purity and, potentially, a lower market value.

In the CBE project UNRAVEL, completed in 2022 and focused on the development of biomass pretreatment, one of Celignis's main roles was to optimise an extraction process focused on removing the extractives prior to the pretreatment stage. Complete removal of all extractives can be a costly process in terms of chemical and energy requirements. Hence, the primary objective of the work was to develop an optimised pre-extraction process that produced a substrate suitable for the pretreatment process while minimising the cost and complexity of the process.

Celignis undertook a detailed and extensive DoE, considering a wide variety of parameters including feedstock, particle size, temperature, residence-time, solvent(s) used, and extraction cycles. The final output of the work was a protocol, optimised for each feedstock, for extractives removal prior to the main pretreatment process.

Prodution of Oligomers from Hemicellulose

Celignis undertook a bioprocess development project for a client where the focus was on the optimisation of process conditions to allow for increased yields of oligomeric sugars from the hemicellulose of an agricultural residue feedstock.

The approach was challenging as the target was efficient hydrolysis of hemicellulose to oligomers without those oligomers then being further hydrolysed to monomeric sugars.

The Celignis Bioprocess team spent some time working with our client in order to understand their targets and their restrictions in terms of chemical and energy usage. We then formulated a DoE to explore the effects of different types of pretreatments and process conditions on the yield of oligomers and on the oligomer/monomer ratios.

The results of our experiments allowed for a much more focused narrowing-down of the process conditions that would allow oligomers to be produced in high yields and at enhanced purities.

Pretreatment of Paper Industry Residues

In this project we worked with a client on evaluating processes for producing sugars in high yields from recycled paper streams. Along with other aspects, the project involved evaluating different process conditions for the selected pretreatment technology and then assessing the effect of these pretreatments on the subsequent enzymatic hydrolysis of the solids.

How our Bioprocess Development Services Work

Our Bioprocess Development Services can work on the evaluation and optimisation of a particular node in the biomass processing technology or can involve the development of a bespoke vertically-integrated technology for your chosen feedstock and/or product. Click here to see how our Bioprocess Development Services work and how we devise and undertake a project.

With regards to the pretreatment of biomass, the Celignis Bioprocess team members with the most experience in undertaking such projects are listed below. Feel free to contact them to discuss potential projects.

Lalitha Gottumukkala

Founder of Celignis Bioprocess, CIO of Celignis


Has a deep understanding of all biological and chemical aspects of bioproceses. Has developed Celignis into a renowned provider of bioprocess development services to a global network of clients.

Dan Hayes

Celignis CEO And Founder

PhD (Analytical Chemistry)

Dreamer and achiever. Took Celignis from a concept in a research project to being the bioeconomy's premier provider of analytical and bioprocessing expertise.

Sajna KV

Bioanalysis Developer


Our Biomass Detective! Designs, tests, optimizes and validates robust analytical methods for our bioprocess development projects. Such bespoke analysis is key to developing an optimised bioprocess.

Other Celignis Services for Bioprocess Development

Global Recognition as Bioprocess Experts

Celignis provides valued services to over 1000 clients. We understand how the focus of bioprocess projects can differ between countries and have advised a global network of clients. We also have customs-exemptions for samples sent to us allowing us to quickly get to work no matter where our clients are based.

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Biomass can be rich in bioactive compounds of high value for food, feed, cosmetic, and pharmaceutical applications. We develop bespoke extraction methods suitable for your needs with high selectivity, efficiency and low environmental impact.

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For the hydrolysis of lignocellulosic biomass to monomeric sugars either chemical or biological approaches can be used. At Celignis Bioprocess we can use both methods at scales ranging from flask-level to 100-litres. We have particular expertise in the optimisation of conditions for enzymatic hydrolysis.

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Enzymes are biological catalysts that have a wide variety of applicaitons in the bioeconomy, ranging from the liberation of sugars from lignocellulosic biomass to the functionalisation of biomass-derived chemicals and materials for higher-value applications. We are experts in the design and use of enzymatic approaches.

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Development of fermentation processes requires knowledge of an array of important factors including: biomass, the microbes used, nutrient media, and fermentation conditions. We're experienced in many fermentations and can help you determine and optimise yields of an array of different fermentation products.

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Downstream Processing

How the various outputs (solid and liquid) of a bioprocess are dealt with is often overlooked until later in bioprocess development, leading to excessive costs and complications. We consider and tackle these issues, and others such as product recovery, early-on as being integral to the bioprocess.

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Lab-Scale Optimisations

We consider that optimising a bioprocess at the lab-scale is the most cost-effective approach to explore a range of different scenarios in search of optimal process conditions. Based on the outputs of these experiments we can then test the chosen set of conditions at higher TRL levels.

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TRL Scale-Up

At our dedicated Celignis Bioprocess laboratories we have all the necessary upstream and downstream apparatus to undertake bioprocess projects up to a tehcnology readiness level (TRL) of 6, with reactor and processing capacities of up to 100 litres.

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Technoeconomic Analyses

Our technoeconomic experts can evaluate your bioprocess, considering various scale, technology, and feedstock options. We apply accurate costing models to determine CAPEX/OPEX of simulated and pilot-scale processes which are then used to determine key economic indicators (e.g. IRR, NPV).

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Biobased Chemicals

A large array of chemicals and materials are possible from biomass and wastes. These can involve chemical or biological approaches, or a combination of the two. Based on your desired end-product we can design and test the most appropriate bioprocess.

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From Process Refinements to an Entire New Process

We work closely with you to understand your objectives and timelines. We then propose a project, usually covering a series of deliverables and stage-gates. Often our projects involve optimising conditions at the lab-scale before replicating the conditions at higher TRL levels.

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Research Collaborations

Celignis is active in several bioprocess research projects. These include projects funded by the EU's CBE-JU, with Celignis being a Full Industry Member of the BIC. We're open to participating in future collaborative research projects where our extensive infrastructure and expertise in bioprocesses can be leveraged.

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