Enzymatic processes

Technology
Technology details
Name:	Enzymatic processes
Category:	Conversion (Biochemical processes and technologies)
Feedstock:	Garden and park waste
Product:	Cellulose, hemicellulose, lignin

Enzymatic processes utilise enzymes (/ˈɛnzaɪmz/) which are proteins that act as biological catalysts (biocatalysts). In terms of lignocellulosic biomass valorisation, enzymes find two main applications: i) biomass pretreatment, and ii) polysaccharides hydrolysis. Biomass enzymatic pre-treatment falls under the category of "biological pre-treatment". Other pre-treatment methods for lignocellulosic biomass includes, physical (e.g., mechanical), chemical (e.g., acid and alkali), physico-chemical (e.g., steam explosion and AFEX), and a combination thereof. polysaccharides hydrolysis is a concept mostly applied in biorefineries as part of the hydrolysis of plant cell wall constituents like cellulose, hemicellulose, and lignin.

Feedstock

Origin and composition

Lignocellulosic biomass (LCB) can be collected as a waste material from forest residues, agricultural, and industrial activities. LCB is mainly characterized by the presence of two carbohydrate polymers, namely cellulose and hemicellulose, as well as an aromatic polymer called lignin. Other components in LCB, found in smaller amounts, are ash, pectin, and proteins. The percentage content of celluloce, hemicelllulose, and lignin are varied among different lignocellulosic materials. In general, the content of cellulose, hemicellulose, and lignin in LCB is in the range of 30-60%, 20-40%, and 15-25%, respectively. The physical appearance and strenght of the biomass depend on the varying concentration of these polymers and therefore greatly influences the type of pre-treatment strategy applied for its deconstruction.

Composition of the plant cell wall

Structural features LCB:

Cellulose

Cellulose is a polysaccharide polymer of glucose disaccharide units, cellobiose, linked tightly by ß-1,4-glycoside bonds. Cellulose molecules are linked by hdyrogen bonds and have different orientations resulting in different levels of crystallinity. Its crystallinity plays a crucial role in the biodegradation of cellulose and, in general, the higher crystallinity level makes it harder to biodegrade the cellulose.

Hemicellulose

Hemicellulose is a random and branched heterogeneous polymer of different polysaccharides including pentoses (xylose and arabinose), hexoses (glucose, galactose, and mannose) and sugar acids. The branched nature of the hemicellulose allows it to form strong bonds with cellulose (through hydrogen bonds) and lignin (through covalent bonds).

Lignin

Lignin is a complex and large compound made out of phenylpropane units linked in a three-dimensional structure. The main monomers of lignin are p-hydroxyphenyl alcohol, coniferyl alcohol, and sinapyl alcohol. Lignin acts as a cementing material that links celluose and hemicellulose to form the rigid three-dimensional structure of the plant cell wall.

Pre-treatment

Schematic of lignocellulosic biomass pretreatment

The following pre-treatments may be considered prior to enzymatic pre-treatment:

• Sizing (e.g., milling, grinding)
• Steam explosion (hybrid pre-treatment; e.g., combined with laccase pretreatment)

The following pre-treatments may be considered prior to enzymatic hydrolysis^,:

• Physical (e.g., milling, grinding, ultrasonication, extrusion)
• Chemical (e.g., acid, alkali, ionic liquid, organosolv)
• Physico-chemical (e.g., steam explosion, hot water, AFEX, wet oxidation)
• Biological (e.g., microbial and enzymatic)

Process and technologies

Enzymatic pre-treatment (biological pre-treatment)

Biological pre-treatment systems rely on biological agents (e.g., enzymes) to delignify lignocellulose and make the process of enzymatic hydrolysis more convenient. The effect of enzymes on the lignocellulosic biomass depends on the type of enzymes as well as the composition of the biomass being treated. This is due to enzyme specificity in terms of the type of the reactions that they catalyze. Laccase (Lac), manganese peroxide (MnP) and versatile peroxide (VP) are enzymes that are used extensively to treat the lignocellulosic substrate. Biological pre-treatment of LCB is often knows as a simple, inexpensive, selective, and environmentally-friendly technology. This is mainly due to the fact that biological pre-treatment does not require high energy inputs or chemicals addition. Furthermore, enzymatic treatment has found success in the removal of toxic inhibitory compounds (i.e., complete removal of phenolic compounds). The limitations asociated with enzymatic pretreatment is its production cost, stability, shelf life, and reusability.

Enzymatic hydrolysis

Enzymatic hydrolysis processes allow to produce monomeric sugars from (ligno)cellulosic biomass by using specific enzymes (e.g., cellulases and hemicellulases) able to break down the chemical bonds in cellulose and hemicellulose polymers. Several factors can affect the efficiency of this process: accessible surface area and crystallinity of the biomass, as well as pH, time and temperatures of the process. Enzymatic hydrolysis is gaining increased attention with respect to acid hydrolysis due to equipment corrosion, energy consumption, non-recyclability of reagents, and fermentation inhibitors production during acid hydrolysis . Different enzymes play different roles in the hydrolysis of lignocellulosic biomass:

Cellulases

Cellulases are one of the key enzymes in biomass hydrolysis. Cellulases are a family of enzymes that synergistically act on cellulose to hydrolyze it to its monomers. Cellulases acts on the ß-1,4-glycosidic linkages in cellulose. The complete hydrolysis of cellulose is mediated by combination of three main cellulases, namely endoglucanases (EC 3.2.14), exoglucanases (EC 3.2.1.91), and glycosidase (EC 3.2.1.21). T. reesei-based cellulases have been the focus of research for the past several years and are widely used in laboratory- as well as pilot-scale studies for bioethanol application. Most commercially accessible enzymes for biomass hydrolysis are actually cocktails of cellulases from Trichoderma or Aspergillus supplemented with ß-glucosidases from other sources.

Hemicellulases

Hemicellulose consists of a mixture of glucose and sugar monomers. Xylan is the most abundant hemicellulose-containing pentose sugars, such as xylose. The enzyme xylanase helps to catalyze the hydrolysis of xylan. Hydrolysis of xylan is mediated by the action of multiple xylanases (i.e., endoxylanases and exoxylanases). Commercially, xylanases are produced from T. reesei, A. niger, humicola insolens, and Bacillus sp.

Pectinases

Pectinase is a complex enzyme that degrades the pectin present in lignocellulosic biomass. Pectin is a polymer of α-1,4-linked D-galacturonic acid. This enzyme breaks down polygalacturonic acid (GalA) into a monomeric unit by opening glycosidic linkages. It helps the softening of biomass and therefore aids in the hydrolysis of biomass.

Other accessory enzymes

The accessory enzymes are also a crucial part in the hydrolysis of LCB and enhance the hydrolysis yield and reduce the enzyme cost and dosages. The breakdown of hemicelluloses is further accompanied by the addition of ß-xylosidases which produces a final product of oligomer with different length as intermediates. Another important accessory enzyme is α-arabinofuranidase for breaking down arabinose into monomers of furanose and pyranose.

Product

Products enzymatic pre-treatment:

• Cellulose
• Hemicellulose
• Lignin

Potential products after fermentation:

• Bioethanol
• Biodiesel
• Biobutanol
• Methane
• Specialty chemicals

Post-treatment

Currently no post-treatment has been identified.

Technology providers

Technology comparison

Company name	Country	Technology category	Technology name	TRL	Capacity [kg/h]	Processable volume [L]	Temperature [°C]	Feedstock: Food waste	Feedstock: Garden & park waste
Company 1	[Country HQ location]	[Technology category (if different sub-categories are defined this has to be specified here, the available categories can be found on each technology page under the chapter Process and technologies)]	[Technology name (the "branded name" or the usual naming from company side)]	[4-9]	[numeric value]			●	●
Company 2	[Country HQ location]	[(if different sub-categories are defined this has to be specified here, the available categories can be found on each technology page under the chapter Process and technologies)]	[Technology name (the "branded name" or the usual naming from company side)]	[4-9]	[numeric value]			●	●

NovelYeast bv

Enzymatic processes provider

General information
Company:	NovelYeast bv
Country:	Belgium
Contact:	johan.thevelein@novelyeast.com
Webpage:	https://www.linkedin.com/in/johan-thevelein-aab60a10/
Technology and process details
Technology name:	Enzymatic saccharification, In-situ enzyme production	Technology category:	Conversion (Biochemical processes and technologies)
TRL:	3-5	Capacity:	Lab scale kg·h-1
Agitator:	Shake flasks, magnetic stirring	Controlled parameters:	Standard parameters
Processable volume:	5 L	Reactor:	Shake flasks, static tubes with magnetic stirring
Reactor material:	Glass	Safety restrictions:	Standard microbiological practice
Temperature:	25-60 °C	Other:	Application of commercial enzyme cocktails, development of in situ enzyme production
Feedstock and product details
Feedstock:	1G and 2G feedstocks	Product:	Fermentable sugars

NovelYeast bv was founded in 2019 by Prof. Johan Thevelein (KU Leuven and VIB) to continue his R&D activities after his retirement in 2020 as emeritus. The company focusses on the development and industrial implementation of yeast cell factories for the production of biofuels, bio-based chemicals as well as specialty sugars and ingredients with first- and second-generation feedstocks. It also develops cell factories for the production of specific proteins for food applications and enzymes for saccharification of lignocellulosic biomass. In addition, it uses yeast as a tool for biomedical and agroindustrial applications, including yeast probiotics and anti-cancer drugs selected by screening in yeast. NovelYeast has several R&D service collaborations with companies world-wide.

University of Naples (Naples, Italy)

Enzymatic processes provider

General information
Company:	University of Naples "Federico II"		200x100px
Country:	Italy
Contact:	Professor Vincenza Faraco, PhD Department of Chemical Sciences Via Cintia, 4 IT-80126 Napoli
Webpage:	https://www.docenti.unina.it/vincenza.faraco
Technology and process details
Technology name:	Enzymatic Hydrolysis	Technology category:	Conversion (Biochemical processes and technologies)
TRL:	2-4	Capacity:	kg·h-1
Agitator:		Controlled parameters:
Processable volume:	L	Reactor:
Reactor material:		Safety restrictions:
Temperature:	°C	Other:
Feedstock and product details
Feedstock:	1G and 2G feedstocks	Product:	Fermentable sugars

The Department of Chemical Sciences of University of Napoli Federico II hosts about 100 researchers and 20 units of technicians and administrative personnel. The main activities of the Department are the Research, the Didactics and the so-called third mission activitities. The Research activities cover several areas of Chemistry, including the design and synthesis of new molecules, from low mass to macromolecules, the purification and the analytic characterization of natural and synthetic molecules, the structural characterization of new molecules through X-ray diffraction, nuclear magnetic resonance, optical and spin electron spectroscopy techniques, mass spectroscopy. The design, the synthesis and the characterization of new molecules are aimed, for example, to the production of innovative molecules with catalytic properties in important chemical and polymerization processes, or to the production of new functional materials, for several applications in a wide range of fields. Research activities also regard the study of biomolecules and biopolymers for applications in biotechnology, from the development of biosensors  to biomedical applications, the study of organic functional molecules and organic polymers for special applications, such as the microelectronics, or the development of new materials with innovative mechanical properties, and the study of nanostructured materials for applications in several fields going from the biology to the medicine, from microelectronics to nanophotonics.

The Department of Chemical Sciences offer the following study programmes:

• Bachelor Degree in Chemistry
• Master Degree in Chemistry
• Bachelor Degree in Industrial Chemistry
• Master Degree in Science and Technology of the Industrial Chemistry
• Bachelor Degree in Molecular and Industrial Biotechnologies
• Master Degree in Molecular and Industrial Biotechnologies

The Department of Chemical Sciences offers the following PhD Courses:

• PhD Course in Chemical Sciences

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Patents

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