Size reduction is also known as comminution. Decreasing particle size of biomass improves accessibility to plant cell wall carbohydrates for chemical and biochemical depolymerization. It can also increase the bulk density for storage and transportation. There is a cost of energy when using mechanical size reduction. For example, 20-40 kWh/metric tons are needed to reduce the size of hardwood chips to coarse particles of 0.6-2.0 mm in size, and kWhs typically have a cost of anywhere from $0.04-0.10 per kWh. To reduce the size of particles to a fine size (0.15-0.30 mm), 100-200 kWh/ton is required.

There are multiple methods used to reduce the size of particles, and the method used will depend on whether the sample is dry or wet. There are hammer mills (a repetitive hammering of the sample), knife mills (a rotating knife slices the sample), and ball mills (the sample is put into a container with metal balls and rolled). Sometimes the sample has to be shredded and dried before using some of these techniques.

Samples can also be “densified.” Samples can be mixed with some sort of binder (to keep the materials together, like glue) and pushed into shape, or pelletized. This increases the bulk density (i.e., from 80-150 kg/m³ for straw or 200 kg/m³ for sawdust to 600-700 kg/m³ after densification). This can lower transportation costs, reduce the storage volume, and make handling easier. After densification, the materials usually have lower moisture content.

Pelletized biomass.

Credit: Marcus Kauffman via Flickr

The mechanism for low pH treatments is the hydrolysis of hemicellulose. Hydrolysis is a reaction with water, where acid is added to the water to accelerate the reaction time. Several acids can be used, including dilute sulfuric acid (H₂SO₄), gaseous sulfur dioxide (SO₂), hydrochloric acid (HCl), phosphoric acid (H₃PO₄), and oxalic acid (C₂H₂O₄). Because it is a reaction, the key parameters affecting it include temperature, time, acid concentration, and moisture content of the biomass. The following reactions can take place: hemicellulose can be solubilized, lignin can be separated, acetyl groups are removed, and the surface of the biomass becomes more accessible. As an example, the α-1,4 bond is broken by the water and acid to yield two glucose units. An enzyme, amylase, can also promote the reaction. The addition of acid and elevated temperature increases the rate of reaction.

Not only is acid used to facilitate hydrolysis, but acid-catalyzed dehydration of sugars can form furans, which can break down into organic acids such as formic acid and levulinic acid. These compounds can be toxic to the enzymes that are used in sugar fermentation. So after the reaction, the residual acid must be neutralized, and inhibitors formed or released during pretreatment must be reduced. Two methods are to use calcium oxide (also known as overliming) or ammonium hydroxide.

Calcium oxide is cheap, forms gypsum during the process, and has a loss of sugar of ~10%, with the necessity of by-product removal and disposal. The reaction is shown below in Reaction 1.

$${\text{Reaction 1: CaO + H} }_2{\text{SO} }_4\to {\text{H}}_2{\text{O + CaSO} }_4$$

The advantage of using ammonium hydroxide is that less sugar is lost and less waste is generated, but the cost is higher. The reaction is shown below in Reaction 2.

$${\text{Reaction 2: 2NH} }_4{\text{OH + H} }_2{\text{SO} }_4\to 2{\text{H}}_2{\text{O + (NH} }_4{\text{)}}_2{\text{SO} }_4$$

The figures below show process diagrams of typical configurations and reaction conditions for sulfuric acid and SO₂.

Pretreatment can also take place in neutral pH water. There are two pathways that can occur. One is when acidic compounds are released from acetylated hemicellulose, mainly acetic acid. This is also called autohydrolysis. Water can also dissociate as the temperature and pressure increase to near the supercritical point (approximately 374°C, 3200 psi), into H+ and OH−, and as this happens, the water behaves like an acid/base system. It is done in water without added chemicals, either in liquid hot water, steam explosion, or water near the supercritical point. The key parameters are time, temperature, and moisture content, and the effects are similar to low pH methods. A schematic for liquid hot water processing is shown in the figure below.

One process, developed by Inbicon, is a counter-current multi-stage hot water pretreatment process. There is a pilot-scale unit at Skærbæk, Denmark. It is a three-stage process using hot water (hydrothermal) at 80°C, 160-200 °C, and 190-230°C. After the first stage, a liquid composed of C5 molasses (sugar) is taken out of the process, which is used for animal feed. After the third stage, the fiber fraction contains cellulose and lignin. Bioethanol and solid fuel for heat and power are produced when using enzymes, yeast, and fermentation. The figure below shows the before and after pretreatment of wheat straw (the raw wheat straw and the cellulosic-lignin portion).

The next pretreatment processes to discuss are at high pH. The high pH removes the lignin portion of biomass through the breaking of ether linkages (R-O-R’) that hold aromatic phenolic compounds together; ring opening can also take place. It is a depolymerization process. There are several processes and bases used, including lime, calcium carbonate, potassium hydroxide, sodium hydroxide, and aqueous ammonia. Key parameters include temperature, reaction time, concentration of base, moisture of the feed material, as well as oxidizing agents. The effects include the removal of most of the lignin, some removal of hemicellulose, and the removal of acetyl links between lignin and hemicellulose.

Lignin is most prominent in grasses and woody biomass. It composes 6-35% of lignocellulosic biomass, depending on the type of grass or wood. Lignin is comprised of crosslinked, branched, monoaromatic units with methoxy and propyl alcohol functional groups. These are shown in the first figure below. The second figure below shows a model of a lignin molecule and how the aromatic monomers are linked together.

There are two possible outcomes for the chemistry behind the high pH treatment: 1) one is essentially a degradation reaction that liberates lignin fragments and leads to lignin dissolution, and 2) the other is condensation reactions that increase the molecular size of lignin fragments and result in lignin precipitation. As you can see, lignin is a complicated molecule, with a variety of linkages, so reactions are complicated due to lignin complexity. The addition of oxidizing agents greatly improves delignification.

There are multiple processes that have been developed for this type of treatment. The first figure below shows the lime pretreatment process flow diagram. The pretreatment can be done under various conditions, such as oxidative and non-oxidative conditions, short-term high temperature (100-200°C, 1-6 h), and long-term low temperature (25-65°C, 1-8 weeks). The second figure below shows the soaking in aqueous ammonia (SAA) process flow diagram.

One of the more developed high-pH processes is the ammonia fiber expansion (AFEX) process. Lignocellulosic biomass is soaked in liquid ammonia (causing swelling) followed by the rapid release of pressure (causing expansion). Anhydrous liquid ammonia is used, and key parameters include temperature, residence time, ammonia concentration, and moisture content of the biomass. During this process, there is virtually no compositional change, but lignin is relocated, cellulose is decrystallized, and hemicellulose is depolymerized. This method increases the size and number of micropores in the cell wall to allow for greater accessibility of chemicals for the following stages of processing. A process schematic is shown below.

The next process type is using an organic solvent, such as the Organosolv (OS) process or the Cellulose solvent- and Organic Solvent-based LIgnocellulose Fractionation (COSLIF) process. For OS pretreatment, the main mechanism involves the dissolution of lignin by organic solvent and then re-precipitation by adding an antisolvent, such as acidified water. This method was first introduced as a pulping method for papermaking. The organic solvents commonly used are acetone, ethanol, methanol, etc., in an aqueous solution of 20-60% water. Key parameters include temperature, residence time, chemical addition, and water concentration. The effect is to: separate lignin from lignocellulosic biomass; solubilize hemicellulose; and increase pore size and surface area in the cell wall. Below is a schematic of a process diagram for OS pretreatment.

Another organic solvent-based process is cellulose-solvent and organic-solvent lignocellulose fractionation (COSLIF). For this process, an organic solvent is introduced to dissolve cellulose prior to Organosolv processing. Below is a schematic of COSLIF processing.

One of the more usual methods of pretreatment of biomass uses ionic liquids. Ionic liquids (ILs) are organic salts that usually melt below 100°C and are strong solvation agents. A common salt that we are all familiar with is table salt, sodium chloride, NaCl. If dissolved in water, it separates into the ions of Na+ and Cl-, but it is not an organic salt like ILs. It has interesting properties, including the fact that, depending on the IL, it can solubilize whole cellulosic biomass or selectively dissolve components, e.g., lignin and cellulose. It is relatively easy to separate the dissolvent component from the organic salt by using an anti-solvent such as water, methanol, or ethanol. When cellulose has been dissolved in organic liquid and then re-precipitated by an anti-solvent, cellulose is less crystalline and easier to break down. Unfortunately, this is still a costly method of pretreatment, as there is difficulty in recycling ILs, and the ILs can be toxic to the enzymes and microbes used in processing cellulose to ethanol. One such IL is known as EmimAc (1-ethyl-3-methylimidazolium acetate), and is able to completely solubilize both cellulose and lignin in switchgrass. The first figure below shows the chemical structure of EmimAc and the change in cellulose after reprecipitation using an antisolvent (T = 120°C). The second figure below shows a schematic of the process diagram.

The last technology we will look at is biological pretreatment. Lignin is removed from lignocellulosic biomass through lignin-degrading microorganisms. Key parameters are temperature, cultivation time, nutrient addition, and selectivity on lignin. Some of the lignin-degrading enzymes include lignin peroxidase, manganese peroxidase, laccase, and xylanase. Advantages to using a system such as this include: no chemicals, mild conditions (ambient temperature and pressure), low energy and low capital outlay, and less enzyme use later on. However, pretreatments take days to weeks, loss of cellulose and hemicellulose, contaminants, and additional pretreatment for higher sugar yield.

Of the methods we’ve discussed, there are pretreatment options that lead the others (some under commercialization). The current leading pretreatment options include dilute acid, AFEX, liquid hot water, lime, and aqueous ammonia, with dilute acid and water, AFEX, and lime under commercialization. The figure below shows switchgrass before pretreatment and after several pretreatment options, i.e., AFEX, dilute acid, liquid hot water, lime, and soaking in aqueous ammonia (SAA).

The Resulting Switchgrass Solids after Different Pretreatment Technologies.

Click Here for a text description of the switchgrass solids.

The image displays six samples of switchgrass, each subjected to a different pretreatment method, arranged side by side for visual comparison. The samples are labeled from A to F, with each label corresponding to a specific treatment:

• A. Control – Untreated switchgrass, serving as the baseline for comparison.
• B. AFEX – Treated with Ammonia Fiber Expansion, a method used to enhance biomass digestibility.
• C. Dil. Acid – Treated with dilute acid, commonly used to break down hemicellulose and improve enzymatic access.
• D. LHW – Treated with Liquid Hot Water, a hydrothermal method that disrupts plant cell wall structure.
• E. Lime – Treated with calcium hydroxide (lime), which helps in delignification and cellulose exposure.
• F. SAA – Treated with Sulfite-Assisted Alkaline pretreatment, aimed at removing lignin and enhancing cellulose accessibility.

A scale bar in the bottom right corner indicates 5 mm, providing a reference for the size and physical changes in the switchgrass samples due to each treatment. The image highlights the visual differences in texture, color, and structure among the treated and untreated samples, which are important for evaluating the effectiveness of each pretreatment method in biofuel or biochemical production processes.

Credit: Donohoe et al., 2011. Bioresour. Technol.

To summarize the methods of pretreatment, the table below shows some of these pretreatment methods and the major and minor effects on lignocellulosic biomass. All methods (AFEX, dilute acid, lime, liquid hot water, soaking aqueous ammonia, and treatment with SO2) affect increasing surface area, removing hemicellulose, and altering lignin structure. Only AFEX, lime, and SAA pretreatments remove lignin, and AFEX and SAA decrystallize cellulose.

This table shows the conditions for ideal pretreatment of lignocellulosic biomass for dilute acid, steam explosion, AFEX, and liquid hot water.

^a Modified from (86); AFEX ratings from Bruce Dale (personal communication).

^b For grasses, data for wood not available.

Credit: Lynd, 1996. Annual Rev. Energy Environ., 21: 403-465