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Hydrolysis is a chemical process, where a molecule is cleaved into two parts by adding a molecule of water. One fragment of the molecule gains a hydrogen ion (H + ). The other group collects the remaining hydroxyl group (OH ).

After pre-treatment of lignocellulosic material, the cellulose is prepared for hydrolysis, which means, that a molecule is cleaved by the addition of a water molecule:

Because of the presence of nonglucan components (e.g. lignin and hemicellulose), hydrolysis of lignocellulosic biomass is more complicated and difficult than that of pure cellulose.

This hydrolytic reaction is catalysed by either dilute acid, concentrated acid or enzymes (so-called cellulases). Catalysis with enzymes (cellulases) has many advantages, because of the mild conditions (pH 4.8 and temperature 318-323 K), therefore obtaining high yields, as well as lowering maintenance costs (compared to alkaline and acid hydrolysis).

Mostly, hydrolysis process for converting cellulose into glucose is accomplished with cellulolytic enzymes or sulphuric acid of varying concentrations.

Acid hydrolysis

Acid hydrolysis of lignocellulosic biomass produce xylose from xylan, but the cellulosic and lignin fractions remain unchanged. Due to its amorphous structure, xylan is more susceptible to hydrolysis by mild acid treatment. Cellulose needs severe treatment conditions because of its crystalline nature. Xylose is degraded to furfural and other inhibitory-acting condensation by-products (e.g. 5-hydroxymethyl furfural (HMF), acetate, hydroxybenzaldehyde (HBA), siringaldedyde (SGA) and vanillin), which have an inhibitory effect on microorganisms (e.g. on yeast cells).

Acid-catalyzed hydrolysis is a complex heterogeneous reaction, involving physical factors, as well as the hydrolytic chemical reaction. In figure 1, the molecular mechanism of acid-catalyzed hydrolysis of cellulose (cleavage of β-1-4-glycosidic bond) is described. Monosaccharide products can be further reduced into undesirable chemicals. Depending on the permeate composition, a number of possible side reactions can occur.

Figure 1: Schematic mechanism of acid catalyzed hydrolysis of β-1-4 glucan (source: Xiang et al. 2003)

with   I: anhydro glucose unit plus H* radical

        II: anhydro glucose intermediate including O* radical (with high energy)

       II´: anhydro glucose intermediate including O* radical (without high energy)

       III: fragment from anhydro glucose unit includes C* radical

      III´: anhydro glucose intermediate includes C* radical

Generally, two main types of acid hydrolysis can be used:

  1. Dilute acid
  2. Concentrated acid

Dilute acid hydrolysis

This technique is the oldest hydrolysis technology, used to produce bioethanol from cellulosic biomass. Conversion of hemicellulose fraction is done at lower temperatures (compared to cellulosic fraction), where dilute sulphuric acid (conc. 1 %) is added to the biomass at high temperatures (about 488 K). Because of less efficient sugar recovery (about 50 %), a major challenge in the development of a more efficient process, obtaining glucose yields higher than 70 % and minimizing glucose decomposition has been started.

Dilute acid hydrolysis is done in two stages: (Figure 2)

1. stage: is performed at low temperatures, to maximize the yield from hemicellulose, as well as to recover the C5-sugars.

2. stage: is done at higher temperatures, to optimize the cellulose portion of the feedstock, as well as to recover the C6-sugars.

Figure 2: Schematic demonstration of a dilute acid hydrolysis (2-stage process) and separate fermentation of pentose and hexose sugars (source: adapted from Chandel et al. 2007).

Dilute acid hydrolysis has a big advantage, concerning the fast rate of reaction, facilitating continuous processing. The huge disadvantage is the low obtained sugar yield. Another disadvantage is the size-reduction of used feedstocks (maximum particle dimension: a few millimeteres), in order to allow adequate acid penetration.

Concentrated acid hydrolysis

With concentrated acid hydrolysis, an almost complete and fast conversion of cellulose to glucose and hemicellulose to C5-sugars is provided. For being economical, a cost effectively recovery of the used acid has to be provided. This process needs relatively mild temperatures and pressures (only needed, when pumping materials from vessel to vessel). Reaction times with concentrated acid (e.g. 70 % sulphuric acid) are with 2 – 4 hours (at 313-323 K) longer than with diluted acids. Due to lower temperatures and pressures, sugar degradation is minimized.

The hydrolyzed material is then washed to recover the sugars. In the next step, the cellulosic fraction has to be depolymerized. The solid residue from first stage is de-watered and for further cellulose hydrolysis, it is immersed in 30 - 40% sulfuric acid for 50 min at 373 K.

The high sugar recovery efficiency, as well as the potential for cost reduction are the most significant advantages of concentrated acid hydrolysis process.

Disadvantageous is, that concentrated sulphuric or hydrochloric acids are difficult to work with. Additionally, a recovery process and a reconcentration step are advisable in order to work economically.

Enzymatic hydrolysis

Enzymatic hydrolysis is another basic method. Enzymes are naturally found plant proteins, capable of causing certain chemical reactions to occur. Enzymatic hydrolysis can be divided into two major developments:

1. Enzymatic conversion method

2. Direct microbial conversion method.

Enzymatic hydrolysis of lignocellulosic materials is naturally a very slow process, because of structural parameters of the substrate, e.g. lignin and hemicellulose, surface area and cellulose crystallinity. Since enzymatic hydrolysis of lignocellulose usually cause solubilization of V20% of the originally present glucan, some form of pre-treatment for increase of amenability to enzymatic hydrolysis is included in most process concepts for biological conversion of lignocellulose.

Economically, enzymatic hydrolysis is cheap compared to acid or alkaline hydrolysis. One reason for this is the usually mild conditions, used for enzymatic hydrolysis (pH 4.8 and temperature 318--323 K) as well as the absence of corrosion problems. Furthermore, with this technique, high yields of glucose can be achieved.

A big disadvantage is, that during enzymatic hydrolysis of cellulosic biomass, numerous factors (both substrate- and enzyme-related factors) can inhibit the catalytic activity of the cellulase (enzyme) mixture. Also disadvantageous is the difficult recycling/recovery process of cellulases (enzymes), because of a possible adsorption onto lignocellulosic substrates.

Cellulose degradation through enzymatic hydrolysis is a complicate process, which takes place at a solid-liquid phase boundary. There are 3 processes, which take place simultaneously when cellulases (enzymes) act in vitro on insoluble cellulosic substrates:

1. Residual (not yet solubilized) solid-phase cellulose changes chemically and physically

2. Release of soluble intermediates from the surface of reacting cellulose molecules (primary hydrolysis)

3. Hydrolysis of soluble intermediates to lower molecular weight intermediates and finally to glucose (secondary hydrolysis)

Generally, degradation of cellulose by enzymatic hydrolysis is characterized by a rapid initial phase, then a slow secondary phase (which lasts until all substrate is consumed) follows.


Cellulases (enzymes), used for hydrolysis of lignocellulosic materials are produced from both bacteria and fungi, which can be aerobic, anaerobic, mesophilic and thermophilic.

For examples, bacteria belonging to beneath listed genera can produce cellulases:

  • Clostridium
  • Cellulomonas
  • Bacillus
  • Thermomonospora
  • Ruminococcus
  • Bacteriodes
  • Erwinia
  • Acetovibrio
  • Microbispora
  • Streptomyces

Filamentous fungi are the major source of cellulases and hemicellulases. Wild type and mutant strains of Trichoderma sp. (T. viride, T. reesei, T. longibrachiatum) have long been considered to be the most productive and powerful destroyers of crystalline cellulose.

There are 3 main groups of cellulose-decompositioning enzymes, also named cellulases:

  1. Endoglucanases or endo-1,4-β-glucanases (EG); (EC
  2. Exoglucanases or ellobiohydrolases (CBH); (EC, EC
  3. ß-Glucosidases (BGL); (EC

EG have an important role in the cellulose hydrolysis. They cleave cellulose chains randomly and thus encourage strong degradation. Accessible intramolecular β-1,4-glucosidic bonds of cellulose chains are hydrolyzed randomly by EG, in order to produce new chain ends. Exoglucanases cut cellulose chains at the ends to release soluble cellobiose or glucose. Cellobiose is hydrolyzed to glucose by BGLs, in order to eliminate cellobiose inhibition. Additionally, BGL complete the hydrolysis process through catalysis of the hydrolysis of cellobiose to glucose.

In the above classification the enzymes are divided after their effectiveness.

Figure 3: Schematic demonstration of cellulase-effectiveness

A lot of cellulase-producing microorganism have been determined, particularly aerobe fungi.

One of the cellulase producers most commonly used in industry is the microorganism filamentous fungi Trichoderma reesei. Although this organism has already been studied (for over than 50 years), its mechanisms of cellulase induction is still not fully understood and cellulase cultivation processes only rely on the results of previous experiments. A big advantage is, that T. reesei produces cellulases of all 3 main groups (named above), as well as that it possesses a huge exo-endoglucanase relation.

Trichoderma reesei Cellulases

T. reesei produces 2 different cellobiohydrolases (= Exoglucanases; CBH I and CBH II), several endoglucanaes (EG I – EG V) and 2 ß-glucosidases. Therefore, all categories of possible cellulases are covered.

The characterisation of cellolytical proteins is obvious in the following table 1.

Table 1: List of characterisation patterns of cellolytical proteins.

CBH I (Cel7A)

One suppose, that Cellobiohydralse I (CBH I) is the key enzyme for the biodegradation of crystalline cellulose, because with 60 – 70 % it forms the main percentage of the cellulose complex. CBH I decomposes cellulose from the not-reducing end, it is a low specific, heavily binding enzyme, which leaves the bounded substrate not before complete biodegradation.

CBH I is inhibited by the end product cellobiose.


Cellobiohydrolase II (CBH II) is similar structured to CBH I, but it decomposes cellulose from the not-reducing end. CBH II is a member of the exoglucanases.

Endoglucanases I and II

Endoglucanase I (EG I) has high activity on soluble cellulose, Endoglucanase II (EG II) on not-soluble cellulose. EG I is not inhibited by cellobiose (in contrast to CBH I).


ß-glucosidases (BGL) is produced only in small amounts. The main task is the biodegradation of cellobiose to glucose.

Separate hydrolysis and fermentation (SHF)

The SHF-process consists of a separately performed enzymatic hydrolysis and a separately done fermentation step. During SHF-process, the joint liquid flow from both hydrolysis reactors first enters the glucose fermentation reactor, afterwards, the mixture is distilled (remove of bioethanol), unconverted xylose (C5-sugar) is left behind. The remaining xylose is fermented to bioethanol, as well as distilled in a second reactor.

In figure 4, the SHF with separate pentose (C5) and hexose (C6) sugars and combined sugar fermentation are shown. Compared to above explained SHF-process, there are a few advantages;

  • higher final bioethanol yield
  • less energy is required
  • production costs are minimized

Generally, the major advantage of SHF is, that hydrolysis and fermentation occur at optimum conditions.

Big disadvantage of SHF is, that cellulolytic enzymes are "end-product"-inhibited. That means, that the rate of hydrolysis is progressively reduced, when glucose and cellobiose accumulate.

Figure 4: SHF-process with separately performed hydrolysis and fermentation.

Simultaneous saccharification and fermentation (SSF)

In this process, the enzymatic hydrolysis and fermentation are performed in a combined step - the so-called simultaneous SSF. This process is often done in combination with dilute-acid or high-temperature hot-water pre-treatment. During SSF, the enzymes (cellulases and xylanases) convert the carbohydrate polymers to fermentable sugars. Unfortunately, the enzymes are susceptible to feedback inhibition by the products (glucose, xylose, cellobiose, and other oligosaccharides.

In figure 5, a SSF-process with combined sugars (C5 and C6) fermentation is demonstrated.

Figure 5: SSF-process with combined sugars fermentation.

Advantages of SSF:

  • Hydrolysis rate is increased by conversion of sugars, that inhibit the cellulase activity
  • Higher bioethanol yields
  • Lower amounts of enzyme (end-product inhibition is relieved by yeast fermentation)
  • Increased efficiency of product formation with enhanced bioethanol concentration (up to 5 % w/w)
  • Lower requirements for sterile conditions (glucose is removed immediately and bioethanol is produced)
  • Shorter process time
  • Less reactor volume

Disadvantages of SSF:

  • Use of different temperature optima for saccharification and fermentation
  • Low pH (less than pH 5,0)
  • High temperature ( >313 K are necessary for enzymatic hydrolysis; may affect adversely fungal cell growth)

SSF is a batch process, which uses natural heterogeneous materials (e.g. complex polymers, such as lignin, pectin and lignocelluloses).

Simultaneous saccharification and co-fermentation (SSCF)

This technology has developed more recently, because of being advantageous for the simultaneous fermentation of hexose and pentose. In this process, the enzymatic hydrolysis continuously releases hexose sugars, which increases the rate of glycolysis such that the pentose sugars are fermented faster and with higher yield.

The big advantage of SSCF is, that the process can be done in the same tank, which results in lower costs.

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