• Biomass Hydrolysis
    Bioprocess 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.

Hydrolysis of Lignocellulose

A major pathway by which many lignocellulosic feedstocks are processed is known as hydrolysis, where monomeric sugars are released from the lignocellulosic polysaccharides (i.e. cellulose and hemicellulose). Typically, these polysaccharides are hydrolysed by acid or, more commonly, by enzymes. The hydrolysis of cellulose will yield monomeric glucose (as cellulose is a hompolysaccharide, i.e only containing one type of sugar), whilst the hydrolysis of hemicellulose will yield a variety of different sugars covering the hexoses (6-carbon sugars) glucose, galactose, and mannose, and the pentoses (5-carbon sugars) xylose and arabinose, depending on the type of hemicellulose. Hydrolysis of hemicellulose can also yield uronic acids and acetyl groups.

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 (in some cases) hydrolyse hemicelluloses, hence separating the hydrolysate from cellulose which can then undergo more severe/targeted treatment.

Click below to read more about bioprocess development for biomass pretreatment.

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History of Hydrolysis of Lignocellulosic Biomass

Early Work (Acid Hydrolysis)

The potential of lignocellulosic biomass as a source of fermentable sugars was recognized as early as the late 19th century, with notable work by researchers such as Charles Tanret. However, the robust structure of lignocellulose—comprising cellulose, hemicellulose, and lignin—posed significant challenges. Acid hydrolysis emerged as an early method for biomass conversion, making use of sulphuric acid to cleave glycosidic bonds in cellulose and hemicellulose.

The acid hydrolysis of lignocellulose materials was commercialised in the late 19th century and several dilute-acid facilities existed in the USA, Germany, Japan, and Russia by World War 1 while concentrated acid hydrolysis facilities were being built between 1937 and the late 1960s. However, these were uneconomic where fossil fuels were available and very few facilities were operational at the end of the 20th century.

Early Enzymatic Hydrolysis

In the mid-20th century, a paradigm shift occurred with the introduction of enzymatic hydrolysis. Enzymes produced by microorganisms, such as Trichoderma reesei, were found to efficiently convert cellulose into glucose. This biological hydrolysis was more environmentally friendly and yielded higher sugar concentrations. Despite its promise, the cost of producing cellulase enzymes was prohibitive, which led to a research emphasis on reducing enzyme costs and improving their efficiency.

Research Advances

It was recognised that the recalcitrance of lignin remained a major challenge in developing efficient biological hydrolysis processes. As a result, pre-treatment processes were introduced to enhance the accessibility of cellulose and hemicellulose to enzymes. These processes include dilute acid pretreatment, steam explosion, and organosolv methods. Each of these methods disrupts the lignocellulosic structure in various ways, increasing the efficiency of subsequent enzymatic hydrolysis.

There were also significant advances, in the late 20th century and early 21st century, on the developement of enzymes that were more efficient in hydrolysing biomass and less sensitive to inhibition. Additionally, improvements were also made in the engineering of enzyme production systems and enzyme recycling technologies. These all helped to reduce the costs associated with the use of enzymes for hydrolysing biomass. As a result, there are a handful of commercial-scale biorefineries using enzymes for the hydrolysis of lignocellulosic feedstocks.

Ongoing Research

Research continues for developing improved pretreatments and more robust enzymes. Additionally, the early 21st century has seen a particular focus on the development of consolidated bioprocessing (CBP). This approach consolidates enzyme production, saccharification, and fermentation into a single step. CBP employs engineered microbes, such as Clostridium thermocellum, that can both produce cellulolytic enzymes and ferment sugars into ethanol. This process further simplifies the biomass-to-product conversion process and reduces costs.

Acid Hydrolysis of Lignocellulosic Biomass

Dilute-Acid Hydrolysis

This involves the use of acids, in relatively low concentrations and at elevated temperatures, to hydrolyse the biomass polysaccharides. The dynamics and outputs of the process are highly dependent on its severity, based on factors such as the type and concentration of acid and the temperatures used. Since cellulose requires more severe hydrolysis conditions than hemicellulose, the dilute-acid approach usually involves two stages. The fist stage focuses on the hydrolysis of hemicellulose and the second stage then involves more severe conditions that allow for cellulose to be hydrolysed.

Click below to read more about dilute-acid hydrolysis and bioprocess development to optimise this approach.

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Concentrated Acid Hydrolysis

Concentrated acid hydrolysis uses acids in higher concentrations, but at lower temperatures. As with dilute acid hydrolysis, the process usually involves two stages with the first focused on hemicellulose hydrolysis and the second, involving more severe process conditions, focused on cellulose hydrolysis. The use of concentrated acid can allow for higher cellulose-to-glucose yields and for less unwanted sugar degradation products than in dilute-acid hydrolysis. However, acid recovery and sugar separation are major challenges that must be addressed in order to develep a commercially-viable process.

Click below to read more about concentrated-acid hydrolysis and bioprocess development to optimise this approach.

Get more info...Concentrated Acid Hydrolysis

Enzymatic Hydrolysis of Lignocellulosic Biomass

Enzymatic hydrolysis is an alternative, biological, approach for obtaining monomeric sugars from lignocellulose. It involves the action of cellulases, for the hydrolysis of cellulose, and hemicellulases for the hydrolysis of hemicellulose. These enzymes can be produced from baceria and fungi, with a number of different types of enzymes required for effective hydrolysis of these polysaccharides. For example, cellulose hydrolysis involves the activities of endoglucanases, exoglucanases, and betaglucosidases.

Some of the different enzymatic hydrolysis technologies are listed and described below.

Separate Hydrolysis and Fermentation (SHF)

This process involves two distinct stages: hydrolysis, in which the cellulose and hemicellulose (if not already removed in the pretreatment) are broken down into simple sugars; and fermentation, where these sugars are transformed into biofuels.

Click below to read more about SHF and bioprocess development to optimise this approach.

Get more info...Separate Hydrolysis and Fermentation

Simultaneous Saccharification and Fermentation (SSF)

SSF involves the concurrent breakdown (hydrolysis) of cellulose (and hemicellulose, if present) into monomeric sugars (saccharification), and the conversion of these sugars into products via fermentation. Unlike in SHF, in SSF both stages take place in the same reactor.

Click below to read more about SSF and bioprocess development to optimise this approach.

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Simultaneous Saccharification and Co-Fermentation (SSCF)

SSCF is a modification of the Simultaneous Saccharification and Fermentation (SSF) method whereby the hemicellulose-derived pentose sugars are fermented alongside the hexose sugars.

Click below to read more about SSCF and bioprocess development to optimise this approach.

Get more info...Simultaneous Saccharification and Co-Fermentation

Consolidated Bioprocessing (CBP)

Consolidated Bioprocessing (CBP) is an even more integrated approach in which the enzyme production, hydrolysis, and fermentation steps all occur in one step and one reactor.

Click below to read more about CBP and bioprocess development to optimise this approach.

Get more info...Consolidated Bioprocessing

Bioprocess Development for Biomass Hydrolysis - How Celignis Works With Our Clients

At Celignis, we have the expertise and infrastructure to work on projects involving the hydrolysis of lignocellulosic biomass. Developing a fully-integrated process, starting with the original feedstock, involves a number of different steps, outlined below. We can work on projects where all these steps are undertaken or we can focus on the optimisation of a particular stage in the process. This approach is particularly relevant where our clients already have an existing technology and are looking for optimising the process at particular nodes. For example, we can work on the optimisation of the hydrolysis and fermentation stages using your already-pretreated biomass.

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.

2. Detailed Feedstock Analysis

In cases where you have already selected a feedstock for the bioprocess, we would then undertake a detailed compositional analysis (P10 or, ideally, P19) of representative samples of that feedstock.

In cases where the feedstock has not yet been selected we can review your list of candidate feedstocks, selecting top candidates based on our prior experience in their analysis and bioprocessing. If you do not have a list of candidate feedstocks then we can provide one, based on your location and the requirements outlined in Stage 1. We would then analyse in detail these priority feedstocks and come to a decision, based on the compositional data and other relevant factors (e.g. price, supply, consistency etc.) on a selected feedstock for the project.

At this point of the project, the Celignis Bioprocess team typically meet to discuss and prepare a project proposal for the development of a biomass hydrolysis bioprocess from this feedstock. After this proposal is reviewed by the client, and revised if needed, we are then ready to start work on the next Stages.

3. Pretreatment (Lab-Scale)

If our project involves the hydrolysis of a virgin biomass feedstock then it is likely that a pretreatment stage will be necessary in order to provide a suitable substrate for subsequent hydrolysis and downstream valorisation. Hence, this stage of the project will 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. Hydrolysis Optimisation

At this stage the focus is on optimising the hydrolysis of the pretreated biomass. The configuration of this stage will be highly dependent on what hydrolysis technology is being used. For example, in concentrated-acid hydrolysis there will be separate hydrolysis stages for the hemicellulose and cellulose.

If Simultaneous Saccharification and Fermentation (SSF), or Simultaneous Saccharification and Co-Fermentation (SSCF), are the hydrolysis technologies being optimised then this Stage will also involve the concurrent fermentation of the sugars liberated during the hydrolysis.

Whatever form this Stage takes, it will also involve a DoE being undertaken (like in Stage 3) where the process conditions are narrowed down. Again, these optimisation experiments are usually undertaken at the lab-scale in order to accelerate the outputs and reduce project costs.

This stage usually follows the pretreatment optimisation activities, however it is possible that there can be some overlap in order to reach the final project outputs more quickly.

5. Downstream Processing

Again, the work undertaken in this Stage will depend on the hydrolysis technology being optimised. For example, if Separate Hydrolysis and Fermentation (SHF) is the technology then this Stage will focus on the fermentation of the sugars hydrolysed in Stage 4. Similarly, fermentation will be the process to be optimised in dilute-acid hydrolysis and concentrated-acid hydrolysis technologies targeting biobased chemicals (such as ethanol) via biological conversion.

In contrast, in technologies (e.g. SSF and SSCF) where the main target product is already produced in Stage 3 then this Stage can focus on the processing of the side-streams obtained in Stage 3 (pretreament). For example, approaches to valorise the hydrolysed hemicellulose sugars in the liquid phase of an organosolv pretreatment.

It is possible for this Stage to run alongside Stage 4.

6. Product Recovery

Based on the outputs of the prior lab-scale Stages we can optimise the methods employed for separating and purifying the target products from the liquid streams. We can also potentially look at the recovery of other compounds from the hydorlysate/fermentation-broth.

It is possible for this Stage to run alongside Stage 4/5.

7. Valorisation of Remaining Biomass

Depending on the pretreatment, hydrolysis, and downstream processess employed in the project, we can look at valorising those biomass components that are not hydrolysed and converted to your target product.

For example, we can analyse the solid residue that remains after hydrolysis for its suitability for combustion.

8. Validation at Higher TRLs

Once we have concluded our optimisation of the biomass hydrolysis 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 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.

If we find that there are differences between the yield and compositions of the different 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.

9. 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 undertake sensitivity analyses to assess the effect of variable costs and revenues on the commercial viability of the process.

Our preferred approach is to include TEA studies at each stage of the development of the 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.

Biomass Hydrolysis Projects - Case Study

Bioethanol from Palm Residues

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

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 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. The next stage of the project then involved optimising the type and dosage of enzymes, as well as other factors such as the solid-loading, in order to maximise ethanol yields from the targeted biomass components.


Contact Celignis Bioprocess

With regards to the development and optimisation of biomass hydrolysis processes, 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.

Oscar Bedzo

Bioprocess Project Manager & Technoeconomic Analysis Lead


A dynamic, purpose-driven chemical engineer with expertise in bioprocess development, process design, simulation and techno-economic analysis over several years in the bioeconomy sector.

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.

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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The choice of pretreatment method varies with the type of biomass and the end-product requirements. At Celignis we can determine the most suitable pretreatment for your feedstock and determine the optimum conditions in lab-scale trials followed by higher TRL scale-ups.

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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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