• Other Biomass
    Bioprocess Development


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.

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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.

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In other pages we cover a number of the most commonly-used pretreatment methods for lignocellulosic biomass. These include: Steam Pretreatment (including steam-explosion pretreatment), Hydrothermal Pretreatment, Dilute-Acid Pretreatment, Alkali Pretreatment, Organosolv Pretreatment, and Mechanical Pretreatment.

However, there are also other means by which biomass can be pretreated, presented below.

Other Biomass Pretreatments

Ionic-Liquid Biomass Pretreatment

Ionic liquids are salts in which the ions are poorly coordinated, resulting in these solvents being liquid below 100oC, or even at room temperature. They have gained a lot of interest in recent years for the pretreatment of lignocellulosic biomass due to their unique properties. For example, ionic liquids can possess excellent thermal stability, negligible vapor pressure, and high solvation power, making them ideal candidates for biomass pretreatment.

Ionic liquids can dissolve or swell lignocellulosic materials, disrupting the crystalline structure of cellulose and making the biomass more amenable to enzymatic hydrolysis. This approach improves the subsequent enzymatic and microbial digestibility of the biomass, ultimately enhancing the yield of biomass-derived sugars.

The most commonly used ionic liquid for biomass pretreatment is 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), but a wide range of other ionic liquids have also been explored. These solvents are effective in disrupting the strong hydrogen bonds that hold together the cellulose and hemicellulose polymers in the plant cell wall.

However, despite their advantages, there are also significant challenges associated with the use of ionic liquids for biomass pretreatment. These include high cost, potential inhibition of subsequent biological processes, and the need for recovery and recycling to make the process economically and environmentally feasible. Also, some ionic liquids do not remove lignin from the biomass, which can inhibit subsequent enzymatic hydrolysis. As a result, many research efforts are focused on finding synergistic combinations of ionic liquid pretreatment with other pretreatment methods or developing new types of ionic liquids that can also remove or modify lignin.

Hydrogen-Peroxide Pretreatment of Biomass

Hydrogen peroxide (H2O2) pretreatment primarily targets the lignin in the biomass. This works by oxidizing the phenolic structures of lignin, breaking it down into smaller fragments and increasing the accessibility of cellulose and hemicellulose to enzymatic hydrolysis.

The effectiveness of hydrogen peroxide pretreatment can be influenced by several factors, including:
  • H2O2 Concentration - Higher concentrations typically lead to more significant lignin removal, but this must be balanced against the potential for cellulose degradation and the costs of the pretreatment.
  • Temperature - Similarly, higher temperatures can increase the rate of lignin removal but can also increase the risk of cellulose degradation.
  • pH - The pH can significantly influence the effectiveness of hydrogen peroxide pretreatment. Often, an alkaline pH is used, as this facilitates the cleavage of ether bonds in lignin.
  • Reaction Time - Longer reaction times can lead to more extensive lignin removal. However, extended reaction times may also result in greater cellulose degradation.
One of the main advantages of using hydrogen peroxide for biomass pretreatment is that it is relatively environmentally benign. Hydrogen peroxide decomposes into water and oxygen, avoiding the environmental issues associated with the disposal of other chemical pretreatment residues. However, the energy requirements for producing hydrogen peroxide and the need to carefully control the pretreatment conditions to prevent excessive cellulose degradation are challenges that need to be addressed to make this method more economically viable.

The Milox Process is a type of hydrogen peroxide pretreatment where formic acid is mixed with H2O2, leading to the generation of peroxyformic acid. This allows for a more extensive oxidation of lignin and for the hydrolysis of the hemicellulose to monomers or their degradation products (e.g. furfural).

Ultrasonic Biomass Pretreatment

Ultrasonic pretreatment is a physical method used to improve the conversion of lignocellulosic biomass into fermentable sugars. Ultrasonication involves the use of high-frequency sound waves to disrupt the structure of the biomass, making the cellulose more accessible to enzymatic hydrolysis.

When high-frequency sound waves pass through a liquid medium, they cause rapid pressure changes that lead to the formation, growth, and eventual collapse of tiny bubbles in a process known as cavitation. The collapse of these bubbles generates intense local heat and pressure, which can disrupt the structure of lignocellulosic biomass.

Ultrasonic pretreatment can be used alone or in combination with other pretreatments. When used alone, howeverm it is typically less effective at breaking down the lignocellulosic structure than when combined with other methods.

Microwave-Assisted Biomass Pretreatment

Microwave-assisted pretreatment is a physical method used to enhance the conversion of lignocellulosic biomass. It uses the electromagnetic energy generated by microwaves to heat the biomass, often in combination with chemical reagents, such as acids, alkalis, or ionic liquids.

When microwaves pass through the biomass, they generate heat by causing polar molecules, particularly water, to vibrate. This rapid heating can help to break down the complex structure of the biomass, increasing the accessibility of the cellulose and hemicellulose to enzymatic hydrolysis.

Microwave-assisted pretreatment has several potential advantages over conventional heating methods. One of these is the speed and uniformity by which heating can be achieved. As a result of this direct and uniform heating, the process can be more energy-efficient than conventional heating methods. However, the cost of microwave equipment can be high and the efficiency of microwave heating can depend on the moisture content and other characteristics of the biomass.

Fungal Biomass Pretreatment

In fungal pretreatment the primary goal is to break down lignin. The two main types of fungi used in fungal pretreatment are white-rot and brown-rot fungi, descrbed below.

  1. White-rot Fungi: These are the most efficient lignin-degrading organisms. They secrete a range of enzymes, including lignin peroxidases, manganese peroxidases, and laccases, which work together to degrade lignin. The white-rot fungus Phanerochaete chrysosporium is the most extensively studied for lignocellulosic pretreatment. Other species such as Trametes versicolor and Pleurotus ostreatus are also used.
  2. Brown-rot Fungi: These fungi preferentially degrade cellulose and hemicellulose, leaving a brownish residue rich in lignin. They produce a range of cellulases and hemicellulases, as well as non-enzymatic Fenton chemistry systems that generate highly reactive hydroxyl radicals capable of attacking lignin.
Fungal pretreatment has some significant advantages. It operates under mild conditions, typically at ambient temperatures and pressures, and does not require harsh chemicals.

However, there are also some challenges associated with fungal pretreatment. For example, the degradation process is relatively slow, often requiring several weeks to achieve substantial lignin degradation. This can significantly limit the applicability of this approach for large-scale industrial processes. Additionally, controlling the growth and metabolic activity of the fungi can be complex, requiring careful monitoring and control of factors such as pH, temperature, and moisture content.

The use of genetically-modified fungi that produce improved or additional enzymes, or that grow more rapidly or under more varied conditions, is an area of active research. Another approach is to use the enzymes produced by these fungi, rather than the whole organism, to break down the biomass.

Bacterial Biomass Pretreatment

Bacterial pretreatment of lignocellulosic biomass involves the use of certain bacteria that produce cellulases, hemicellulases, and ligninases, enzymes that can degrade cellulose, hemicellulose, and lignin respectively. This approach aims to increase the accessibility and digestibility of biomass for subsequent bioconversion processes. Different bacterial species have been studied for this purpose:
  • Clostridium Species - These are anaerobic bacteria that produce cellulolytic and hemicellulolytic enzymes. Certain species like Clostridium thermocellum are thermophiles, which makes them suitable for high-temperature pretreatment processes.
  • Bacillus Species - These bacteria can produce cellulases and hemicellulases. Some strains, such as Bacillus subtilis, have been genetically modified to overproduce these enzymes, enhancing their biomass degrading capabilities.
  • Actinobacteria - This is a large group of bacteria that includes many species capable of degrading lignocellulose. For instance, some species of Thermobifida are known to degrade cellulose and hemicellulose, and certain strains have been shown to degrade lignin.
  • Lignin-Degrading Bacteria - Some bacteria, such as Rhodococcus jostii and Nocardia iowensis, have been found to degrade lignin, although this process is generally less efficient in bacteria than in fungi.
The advantages of bacterial pretreatment include: its operation under mild conditions; the absence of harsh chemicals; and the potential for high selectivity towards particular biomass components. Some bacteria can also tolerate or even metabolize inhibitors produced during biomass pretreatment, which can be an advantage in subsequent bioconversion steps.

However, similar to fungal pretreatment, bacterial pretreatment also has its challenges. The process can be relatively slow and requires precise control over the growth conditions. Some bacteria can be pathogenic, raising safety and containment issues. Furthermore, lignin degradation by bacteria is generally less efficient compared to white-rot fungi.

Ongoing research is focused on improving the efficiency of bacterial pretreatment through genetic engineering of bacteria to enhance their enzyme production and tolerance to inhibitors. Another area of interest is the co-culturing of different bacterial species or bacteria with fungi to achieve more efficient biomass degradation.

Pretreatment Bioprocess Development at Celignis

Whether you have an exisiting pretreatment process that you think can be improved, or if you want to evaluate a pretreatment method for your feedstock(s), we are here to help.

Our initial step would involve learning from you what your targets are for the pretreatment and what the subsequent stages of the process, and final products, should be.

Then we would undergo a detailed compositional analysis of the feedstock in order to gain an understanding of its suitability for pretreatment and to consider appropriate process conditions.

We would then work on a design of experiments (DoE) for pre-treatments to explore the effects of different process conditions.

There would be detailed analysis of the liquid and solid outputs of these pretreatments, comparing these results with the targeted aims of the pretreatment and downstream valorisation.

If subsequent enzymatic hydrolysis of the solid residue is the planned next step in the process then we can undertaken lab-scale enzymatic hydrolysis experiments on the residues from this DoE. We can also use the cellulose-derived sugars as a substrate for fermentation, combining the approaches if required (i.e. simultaneous saccharification and fermentation).

If there are also plans for downstream valorisation of the liquid fraction we can also assess the suitability of various hydrolysates from the DoE for such processes.

Following a review of the results from the DoE we can then work on sequential scaling-up of the process. The outputs of these pretreatments can also be tested for their downstream valorisations. Any deviations from the outputs at lower TRLs can be assessed and remedying approaches undertaken.

The commercial viability of pretreatment is particularly important and should be considered throughout the project. We usually recommend to our clients that we undertake interative techno-economic analyses (TEA) at various stages of the bioprocess development, so that the experimental and scale-up work are framed in a commercially-relevant context.

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Other 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.

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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.

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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

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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.

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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.

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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.

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Pretreatment Homepage

Click here to read more about the important factors to consider when designing a biomass pretreatment process and to read about how we undertake our pretreatment development projects with our clients.

Get more info...Pretreatment Homepage


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.

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.