Xylooligomers are strong inhibitors of cellulose hydrolysis by enzymes

It has been known that xylooligomers are  the inhibitors to enzymatic hydrolysis. An customized enzyme cocktails have to be developed and applied on specific biomass substrates. Here are two recently published papers reported their research results on this issue.

•  "Supplementation with xylanase and b-xylosidase to reduce xylo-oligomer and xylan inhibition of enzymatic hydrolysis of cellulose and pretreated corn stover".
•  "Hemicellulases and auxiliary enzymes for improved conversion of lignocellulosic biomass to monosaccharides".

The dominating factor for biomass cellulose accessibility to external enzymes:

To improve biomass cellulose accessibility to external enzymes, pretreatment is often required, which seems to plat the following roles depending on the processing conditions:

• lignin or hemicellulose removal
• destroy cellulose crystal structure,
• open channels/cell wall pores
• increase cellulose surface exposure.

Since enzymatic hydrolysis follows the tunneling mechanism: eroding glucose from the inside of cell wall to the outside. Therefore, the pores or channels in cell wall are the dominant factor for the efficiency of enzymatic hydrolysis, i.e. control the rate of diffusion/transport of enzymes and sugar dissolution.

Size reduction by mechanical milling before themo/chemical pretreatment is the worst scenario although the surface area are improved.

Granular Starch Hydrolysis

Granular Starch Hydrolysis with external enzymes or corn with expressed enzymes:


• Use granular starch-hydrolyzing (GSH) enzymes (e.g., Stargen from Genencor or BPX from Novozymes) to convert starch into dextrins at temperatures lower than 48 C and hydrolyze dextrins into fermentable sugars during SSF


• Not require heating of the corn slurry to high temperatures for cooking or liquefaction; therefore, GSH enzymes reduce the overall utility requirements of the dry grind process.


• With GSH enzyme, the liquefaction, saccharification, and fermentation steps can all be combined into one single step. Compared to conventional enzyme treatments,
      o No increase in viscosity of the corn slurry that occurs during gelatinization and cooking; Therefore, higher concentrations of solids can be used in corn slurries, which allows the fermentation to reach increased final ethanol concentrations
     o Glucose concentrations with GSH enzymes are typically lower but the final ethanol concentrations and ethanol yields remain similar.
     o glycerol concentrations are lower for GSH treatment so that yeast cells are thus subjected to a low osmotic stress; thus improving the overall productivity by lowering the production of glycerol


• The cost of GSH enzymes is approximately double that of conventional enzymes; but overall material, capital, and operational cost may be advantageous.


In addition, if using enzyme expressed corn seeds as feedstocks, the cost of enzyme will be signifcantly low.

Structural features lead to two-phase enzymatic hydrolysis for mild pretreated biomass

The limiting factors that affect enzymatic hydrolysis of biomass have been traditionally divided into two groups: substrate-based and enzyme based. The substrate-based factors mainly involve chemical structural features such as the compositions of cellulose, hemicellulose, lignin, and side groups bound to hemicellulose and physical structural features that consist of accessible surface area, crystallinity, the physical distribution of lignin in the biomass matrix, degree of polymerization, pore volume, and biomass particle size.

For mild pretreated biomass, the inital rate of enzymatic hydrolysis will be influenced by the transport of enzymes and the dominant hydrolysis will be on amorphous cellulose. At this phase, the hydrolysis of hemicellulose will correlate with the slow increase of glucose yield. When the hemicellulose removal to some point, say 50% removal, a slight hemicellulose hydrolysis will lead to significant increase in cellulose hydrolysis, i.e. crystal cellulose hydrolysis: A transition point occurs, demonstrating the main or 2nd phase hydrolysis.

At 1st phase, lignin content and side groups in hemicellulose play an important role. Any treatments to remove lignin and peel off side groups and break down hemicellulose will improve enzyme transport though opened channels; At the 2nd phase,cellulose crystallility become the limiting factor, any destruction of cellulose crystallility will speed up the hydrolysis and increase the yield.

Whole slurry hydrolysis: an approach to produce cheap sugar

Almost all of currently reported  biomass pretreatments are those reqyiring post- liquor/solid separation or washing,  or nutralization, or even detoxification before further processing (hydrolysis), which will add additional cost due to the more step unit operations. A cost-effective pretreatment should be low demand of post- pretreatment processing. A "whole slurry" processing or hydrolysis will be ideal case. Therefore, the pretreatment should not be to acidic or too basic, or too harsher that generate too much toxic compounds. I am working on a process that really can realize this goal.

Benefits from Surfactants (Tween and BSA) During Enzymic Hydrolysis

It appears that Tween improves biomass enaymatic hydrolysis through three effects: enzyme stabilizer, lignocellulose disrupter, and enzyme effector. BSA treatment can improve both cellulase and beta-glucosidase activity due to the non-specific competitive, irreversible adsorption of BSA on lignin.

However, all of the research were based on the current leading pretreatment methods. Scince these pretreatments are really cost non-competitive. Any slight improvements in hydrolysis yield and reduction in enzyme loading is negligible compared with the cost of pretreatment and large scale enzyme production.

A cost effective pretreatment must be developed, which does not require harsher conditions (very acidic and very alkaline, and very high temperature). Is it possible? Yes, if we think the chemistry carefully!

When reading the most recently published review paper on pretreatments, nothing new included. I do not understand that there are so many people working in this field, but most of them just copy the ideas and focus on those so-called leading pretreatments.

I think more education on biomass chemistry is really need to train people/students to develop new approches/solve problems before fundemetal understanding of the problems.

Xylooligomers: Inhibitiors of cellulases

During 2010 AIChE meeting, a paper by Qing Qing from UC Riverside presented their research on "The Mechanism of Inhibition on Cellulose Hydrolysis by Different Chain Length Xylooligomers”. Their research suggests that xylooligomers are strong inhibitors of cellulases (more than xylose, xylan, glucose and cellobiose) based on the experimental results: A trend of decreasing glucan conversion with increasing DP xylooligomers, which was believed to be due to either obstruction of the cellulose by the oligomers, non-specific binding of the cellulases to the xylan or inhibition by the xylooligomers. Protein adsorption tests demonstrated that the spezyme CP cellulase, multifect xylanase, and β-xylosidase bound to the xylooligomers.

Their solution to reduce inhibition was to add the hemicellulases before cellulases. However, the effect was not as big as what expected, probably a synergistic effect is also needed.

Hemicellulose removal on enzymatic hydrolysis

It has been reported (Grohmann et al., 1985, 1986) that hemicellulose removal substantially enhances cellulose digestion despite of high lignin content. It is believed  that hemicellulose in biomass blocks the contact of cellulase with cellulose by adsorbing enzyme and by physically blocking access of the cellulase to the cellulose surface. As a result, hemicellulose removal alone can increase the surface area and pore volume and makes cellulose more accessible to cellulase. It is suggested to remove at least 50% of hemicellulose to significantly increase cellulose digestibility.

Lignin, its effect on enzymatic hydrolysis

Biomass lignin is an important factor in enzymatic hydrolysis and sugar yields. Several mechanisms have been suggested about how lignin limits enzymatic hydrolysis:


1. toxic to the enzymes for simple phenolic compounds

2. steric hindrance caused by lignin-polysaccharide linkages that limit access of fibrolytic enzymes to specific carbohydrate moieties. For example, the degradation rate of xylan is said to depend on the number and location of side branches and their lignin associations

3. Lignin as hydrophobic filler that displaces water in the cell wall matrix. As a result of the hydrophobicity of lignin, water cannot enter internal polymers of the cell wall. Hence, the action of hydrophilic enzymes may be limited by this hydrophobic environment

4. Adsorption of enzymes, which increases the loading but decreases the effects

Enzymatic deconstruction of xylan for biofuel production

A review paper by DODD et al titled "Enzymatic deconstruction of xylan for biofuel production" summarizs current understanding of the molecular basis for substrate specificity and catalysis by enzymes involved in xylan deconstruction.

Xylan is the dominant hemicellulose in hardwood and non-woody biomass. Its deconstruction to monomer sugar not only contributes to the source of sugars but also enhances the exposure of cell wall to enzyme/chemical hydrolysis.
production.

Xylan is known a heteropolymeric substrate consisting of a repeating β-1,4-linked xylose backbone branched with acetyl, arabinofuranosyl, and 4-O-methyl glucuronyl groups (Figure 1).In addition, xylan may be cross linked to lignin by aromatic esters. In order to efficiently depolymerize xylan to the component monosaccharides, a mixture of different enzymatic functionalities are required, including endo-1,4-β-xylanases (EC 3.2.1.8), β-D-xylosidases (EC 3.2.1.37), α-L-arabinofuranosidases (AFs) (EC 3.2.1.55), α-glucuronidases (EC 3.2.1.139),acetyl xylan esterases (EC 3.1.1.72), and ferulic/coumaric acid esterases (EC 3.1.1.73).



A better understanding of the structural diversity in xylan and the corresponding enzymatic strategies employed by microbes will be critical to hydrolyze the linkages within this complex heteropolymer.

Inhibitions from oligomers

An intersting research paper with new findings: "xylobiose and higher xylooligomers were shown to inhibit enzymatic hydrolysis of pure glucan, pure xylan, and pretreated corn stover."

"Thus, b-xylosidase alone does not appear to be sufficient to hydrolyze high DP soluble xylooligomers, and supplementation with both xylanase and b-xylosidase appears desirable to realize high monomeric xylose yields, as found for pure xylan and AFEX and SO2 pretreated corn stover."

What about other oligomers such as mannooligomers in softwood?

Cheap sugar: How can it be cheaper?

It is known that feedstock cost account for significant part of overall ethanol production cost. The key is to obtain cheap fermentable monomer sugars from lignocellulosic biomass with the following factors:
· Monomer sugar recovery yield. If we look at biomass, only ~65-70% of cell wall is carbohydrate that is our target to convert into monomer sugars. The question is how to efficiently hydrolyze it with high yield. Chemically or enzymatically? Currently both cannot achieve high sugar yield and high efficiency. The combination of chemical and enzymatical hydrolysis is the direction. However, more factors need to be considered to achieve high sugar yield when talking about a commercialized technology:
o Recoverable/recyclable chemicals that minimize the chemical cost and waste treatment
o The pH of pretreatment that impacts reactor metallurgy
o The temperature of pretreatment that impacts energy usage
o The robust of enzymes that reduce the loading and increase the efficiency

Effects of Cellulose Crystallinity, Hemicellulose, and Lignin on the Enzymatic Hydrolysis

It is known that the efficiency of enzymatic hydrolysis was affected by the degree of cellulose crystallinity, hemicellulose and lignin removal. A recent published paper reported the results on how these factors impacted enzymatic hydrolysis.

Cheap sugar: the key for bioethanol to survive

Bioethanol industry is facing another winter time in its history due to current cheap oil/gas price. Most people believe the price of oil will back up again sooner or later. The question is when? The good news is the incentive policy and stimulus fund from the new government that will bring the spring this industry. However, the long term survival will depend on its own economic viability. The key is the cheap sugar and apparently the renewable source is lignocellulosic biomass. Generally only 2/3 of biomass weight is carbohydrate that can be converted into monomeric sugars for fermentation. The question is how to obtain them with a high yield in a cheap way. The current hydrolysis technology is still not good enough to realize this.

Enzymatic hydrolysis is a direction for bioethanol production from lignocellulosic biomass. Ideally a or a combination of chemicals are used to remove both hemicelluloses and delignification simultaneously, the resulting solid is mostly cellulose with more exposed and accessible surface and pores, free chain ends, leading to lower enzyme loading, high enzyme selectivity, and fast rate of hydrolysis.

The cheap sugar is calling on advanced enzyme!

Integration may lower the overall hydrolysis cost

Before the cost of enzyme is down to an economical level, it may be not enough to increase the efficiency and rate of enzymatic hydrolysis of biomass just via a pretreatment.

Because of the nature of hemicelluloses (branched, amorphous, and variety), the severity of pretreatment should be low to avoid hemicelluloses degradation. However the mild pretreatment will not damage cell wall enough and remove part of recalcitrant lignin (the physical barrier and competitive sites for enzyme adsorption). As a result, the efficiency and rate of enzymatic hydrolysis cannot reach the level of what we expect. Ideally, the process should be integrated with several units: pretreatment without washing to pull out hemicelluloses; followed by delignification to remove lignin; enzymatic hydrolysis of delignified biomass with very limited dosage of enzyme to achieve target and high sugar recovery yield. The additional unit may increase capital cost. Considering the saving of enzyme dosage and time, the overall operation and material cost may be lower. If increasing the rate of hydrolysis, the size of equipment can be smaller. Therefore, the integration of process may lead to a overall efficiency.

We plant trees is to have a forest!

The factors on enzyme transport

Due to the different morphology of cellulase, the rate of transport will be regulated by the following factors:
1. The substrate pore size and shape: the specific surface area accessible to the protein (SSAP)
2. product concentration
3. Substrate adsorption preference
4. Physical barriers

The effect of available cellulose chain ends on the rate of enzymatic hydrolysis

It has been found that available cellulose chain ends directly relate to the rate of enzymatic hydrolysis of biomass, which is controlled by the amount of amorphous regions, pretreatment, and the action of endoglucanases. If a pretreatment just increases the available cellulose surface areas without significant change of cellulose crstallinity, it is possible that the rate usually does not show significant increase. If there is enough cellulose chain ends or not generated fast enough, the increase in enzyme loading often does not show the hydrolysis rate increase.

Enzyme torwards breakdown lingin in biomass hydrolysis

The traditional enzymatic hydrolysis of biomass is to hydrolyze pretreated biomass to release monomer sugars for further processing. A new different approach has been reported recently on the research at MSU: it focused the white-rot fungus that is often found on rotting wood and used for biopulping. The fungus contains the peroxidase enzyme that initiates lignin breakdown. After isolation from the fungus, the enzyme-producing gene was reproduced by introducing it into E. coli. The idea was to to isolate the gene, slice out the DNA and basically have the bacteria eat the lignin.

Integration of pretreatment and enzymatic hydrolysis of biomass

This a new concept: The biomass is first pretreated with water only or with other chemicals. After pretreatment, the treated biomass and slurry is enzymatically hydrolyzed without separating the solid and liquid.

Here is one of the research projects conducted at The BioEnergy Science Center.