Elsevier

Biotechnology Advances

Volume 30, Issue 6, November–December 2012, Pages 1458-1480
Biotechnology Advances

Research review paper
A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes—Factors affecting enzymes, conversion and synergy

https://doi.org/10.1016/j.biotechadv.2012.03.002Get rights and content

Abstract

Lignocellulose is a complex substrate which requires a variety of enzymes, acting in synergy, for its complete hydrolysis. These synergistic interactions between different enzymes have been investigated in order to design optimal combinations and ratios of enzymes for different lignocellulosic substrates that have been subjected to different pretreatments. This review examines the enzymes required to degrade various components of lignocellulose and the impact of pretreatments on the lignocellulose components and the enzymes required for degradation. Many factors affect the enzymes and the optimisation of the hydrolysis process, such as enzyme ratios, substrate loadings, enzyme loadings, inhibitors, adsorption and surfactants. Consideration is also given to the calculation of degrees of synergy and yield. A model is further proposed for the optimisation of enzyme combinations based on a selection of individual or commercial enzyme mixtures. The main area for further study is the effect of and interaction between different hemicellulases on complex substrates.

Introduction

In 2001, about 97% of the world's liquid transportation fuels were derived from petroleum (Mielenz, 2001). To reduce the reliance on fossil fuels amidst price hikes and unrest in the Middle East, governments have initiated extensive research into the large scale production of alternative liquid transportation fuels from renewable resources. The US Department of Energy has set a target for biofuel production in the US to reach 60 billion gallons per year by 2030 while the EU target aims to supply 25% of transportation fuel requirements through biofuel production by 2030 (Himmel et al., 2007). Production of biofuel from sugarcane (in Brazil) and corn (in the US) has limited capacity to supply such volumes. In many countries there is also ongoing debate about the use of food crops for biofuel production. As a result, lignocellulose biomass has been identified as the most suitable feedstock for biofuel production since it consists of approximately 75% polysaccharide sugars (Bayer et al., 2007, Lynd et al., 1991). Sources of lignocellulose include agricultural waste such as corn stover, bagasse, wood, grass, municipal waste and dedicated energy crops such as miscanthus and switchgrass (Gomez et al., 2008).

Enzymatic hydrolysis of plant carbohydrates has emerged as the most prominent technology for the conversion of biomass into monomer sugars for subsequent fermentation into bioethanol. The biological degradation of the carbohydrates within the biomass is achieved using multiple enzymes in defined ratios to convert the carbohydrates to their monomer sugars. This is followed by the fermentation of these sugars into bioethanol. The enzymes cooperate in a synergistic fashion to degrade the substrate, meaning that the activity of enzymes working together is higher than the addition of their individual activities.

The initial focus has been on the conversion of cellulose into glucose monomers, but research is now focusing on the utilisation of both hexoses and pentoses in fermentation as it increases the theoretical yield and can substantially improve the economics of the process (Merino and Cherry, 2007). This has had an impact on aspects of the process such as the type of pretreatment used and the enzymes required for hydrolysis.

Different types of processes may be used for bioconversion and fermentation of lignocellulose. Separate hydrolysis and fermentation (SHF) refers to the physical separation of these two processes, whereas simultaneous saccharification and fermentation (SSF) refers to these processes taking place within the same bioreactor. Consolidated bioprocessing (CBP) refers to the use of a single organism to produce the enzymes required and to perform both the hydrolysis and fermentation (Lynd et al., 2005, Xu et al., 2009). CBP is potentially considered the most cost-effective process as it has lower capital costs and may achieve enhanced synergy due to microbe/enzyme interactions (Elkins et al., 2010, Lynd et al., 2002). Various processes, namely pretreatment, hydrolysis and fermentation may have an impact on the enzymes involved in hydrolysis. For example, in SHF, higher temperatures (50 °C) can be used for hydrolysis, while in SSF, temperatures have to be lower (30–32 °C) to accommodate the optimal temperature for the fermenting organism (Andric et al., 2010).

This review focuses on the evaluation of synergy studies as reported in literature and various factors that have an impact on synergy, yield and hydrolysis.

Section snippets

Structure of lignocellulose

Lignocellulose consists of lignin, carbohydrates such as cellulose and hemicellulose, pectin, proteins, ash, salt and minerals. Lignin consists of phenylpropane units such as p-coumaryl, coniferyl, guaiacyl, syringyl and sinapyl alcohol and is very resistant to degradation (Hendriks and Zeeman, 2009). Lignin composition is variable between hardwoods and softwoods, although the specific three-dimensional structure of lignin is unknown (Eriksson and Bermek, 2009). Older and more woody plants

Obstacles for hydrolysis of lignocellulose and the role of pretreatments

Pretreatment of lignocellulose biomass is crucial for achieving effective hydrolysis of substrates as enzymatic hydrolysis of native lignocellulose produces less than 20% glucose from the cellulose fraction (Zhang and Lynd, 2004). Although pretreatment is costly, the cost of not pretreating is even larger (Eggeman and Elander, 2005). Depending on the specific pretreatment, different effects may be observed on the substrate that can all contribute to improved hydrolysis. Some of these effects

Enzymes required to degrade lignocellulose

A large variety of enzymes with different specificities are required to degrade all components of lignocellulose. Many reviews are available on this subject (Banerjee et al., 2010b, Gilbert, 2010, Gilbert et al., 2008, Lynd et al., 2002, Saha, 2003, Zhang and Lynd, 2004). Table 2 gives a brief overview of the types of enzymes that are required to degrade complex lignocellulose substrates. However, there are indications that many other proteins may contribute to lignocellulose degradation in

Synergy studies

The degree of synergy or synergism is defined as “the ratio of the rate or yield of product released by enzymes when used together to the sum of the rate or yield of these products when the enzymes are used separately in the same amounts as they were employed in the mixture” (Kumar and Wyman, 2009a). Synergy depends on the ratio of the enzymes involved (Nidetzky et al., 1994), as well as the specific characteristics of the enzymes and the characteristics of the substrate.

Synergy studies have

Measurement of synergism—degree of synergy

The degree of synergy between enzymes is the ratio between the activity of the mixture and the sum of the individual activities on the same substrate (Andersen et al., 2008). It is occasionally also reported as a percentage enhancement of activity (Gottschalk et al., 2010). The degree of synergy is a quantification of the ability of two or more enzymes to cooperate in their action on a substrate. According to Andersen et al. (2008), the degree of synergy can be based on product formation,

Model for enzyme synergy approach to bioconversion

The first step in bioconversion of a lignocellulose substrate is an accurate analysis of the composition of the pretreated substrate, particularly the specific sugars in the hemicellulose fraction which would identify the presence of arabinoxylan, mannan or arabinan which could impact on the selection of enzymes for its degradation. Two models are outlined in Fig. 1, Fig. 2, Fig. 1 based on the use of individual combinations of enzymes whereas Fig. 2 is based on the use of commercial enzymes

Future perspectives

Bioconversion using enzyme synergy has generally opted for two approaches, individual enzyme combinations or combinations of commercial mixtures. Either option can be useful. Use of individual enzymes can lead to a greater understanding of synergy and cooperation between enzymes to degrade a complex substrate, whereas the use of commercial enzymes may be a quicker route to commercialisation.

With respect to the use of individual enzymes, a shortcoming is the lack of commerical availability of

Acknowledgements

JS van Dyk acknowledges the Claude Leon Foundation for funding.

References (246)

  • A. Berlin et al.

    Inhibition of cellulase, xylanase and β-glucosidase activities by softwood lignin preparations

    J Biotechnol

    (2006)
  • N. Beukes et al.

    Effect of lime pre-treatment on the synergistic hydrolysis of sugarcane bagasse by hemicellulases

    Bioresour Technol

    (2010)
  • N. Beukes et al.

    Effect of alkaline pre-treatment on enzyme synergy for efficient hemicellulose hydrolysis in sugarcane bagasse

    Bioresour Technol

    (2011)
  • N. Beukes et al.

    Synergistic associations between Clostridium cellulovorans enzymes XynA, ManA and EngE against sugarcane bagasse

    Enzyme Microb Technol

    (2008)
  • E. Bonnin et al.

    Pectin acetylesterases from Aspergillus are able to deacetylate homogalacturonan as well as rhamnogalacturonan

    Carbohydr Polym

    (2008)
  • M.K. Bothwell et al.

    Synergism between pure Thermomonospora fusca and Trichoderma reesei cellulases

    Biomass Bioenergy

    (1993)
  • A. Boussaid et al.

    Adsorption and activity profiles of cellulases during the hydrolysis of two Douglas fir pulps

    Enzyme Microb Technol

    (1999)
  • T.G. Bridgeman et al.

    Torrefaction of reed canary grass, wheat straw and willow to enhance solid fuel qualities and combustion properties

    Fuel

    (2008)
  • P. Capek et al.

    Enzymatic degradation of cell walls of apples and characterization of solubilized products

    Int J Biol Macromol

    (1995)
  • R. Carapito et al.

    Efficient hydrolysis of hemicellulose by a Fusarium graminearum xylanase blend produced at high levels in Escherichia coli

    Bioresour Technol

    (2009)
  • P. Champagne et al.

    Enzymatic hydrolysis of cellulosic municipal wastewater treatment process residuals as feedstocks for the recovery of simple sugars

    Bioresour Technol

    (2009)
  • M. Chen et al.

    Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate

    Int Biodeter Biodegrad

    (2007)
  • M. Chen et al.

    Enzymatic hydrolysis of maize straw polysaccharides for the production of reducing sugars

    Carbohydr Polym

    (2008)
  • L. Da Costa Sousa et al.

    ‘Cradle-to-grave’ assessment of existing lignocellulose pretreatment technologies

    Curr Opin Biotechnol

    (2009)
  • R.P. De Vries et al.

    Synergy between enzymes from Aspergillus involved in the degradation of plant cell wall polysaccharides

    Carbohydr Res

    (2000)
  • J.P. Delgenes et al.

    Effects of lignocellulose degradation products on ethanol fermentations of glucose and xylose by Saccharomyces cerevisiae, Zymomonas mobilis, Pichia stipitis, and Candida shehatae

    Enzyme Microb Technol

    (1996)
  • B.S. Dien et al.

    Enzyme characterization for hydrolysis of AFEX and liquid hot-water pretreated distillers' grains and their conversion to ethanol

    Bioresour Technol

    (2008)
  • R. Dijkerman et al.

    Degradation of structural polysaccharides by the plant cell-wall degrading enzyme system from anaerobic fungi: and application study

    Enzyme Microb Technol

    (1997)
  • S.-Y. Ding et al.

    A biophysical perspective on the cellulosome: new opportunities for biomass conversion

    Curr Opin Biotechnol

    (2008)
  • S.M. Duncan et al.

    Carbohydrate-hydrolyzing enzyme ratios during fungal degradation of woody and non-woody lignocellulose substrates

    Enzyme Microb Technol

    (2010)
  • T. Eggeman et al.

    Process and economic analysis of pretreatment technologies

    Bioresour Technol

    (2005)
  • J.G. Elkins et al.

    Engineered microbial systems for enhanced conversion of lignocellulosic biomass

    Curr Opin Biotechnol

    (2010)
  • K.-E.L. Eriksson et al.

    Lignin, lignocellulose, ligninase

    Appl Microbiol Ind

    (2009)
  • T. Eriksson et al.

    Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose

    Enzyme Microb Technol

    (2002)
  • C.B. Faulds et al.

    Specificity of feruroyl esterases for water-extractable and water-unextractable feruroylated polysaccharides: influence of xylanase

    J Cereal Sci

    (2003)
  • H.-P. Fierobe et al.

    Design and production of active cellulosome chimeras

    J Biol Chem

    (2001)
  • H.-P. Fierobe et al.

    Degradation of cellulose substrates by cellulose chimeras

    J Biol Chem

    (2002)
  • H.-P. Fierobe et al.

    Action of designer cellulosomes on homogeneous versus complex substrates

    J Biol Chem

    (2005)
  • M. Fitzpatrick et al.

    A biorefinery processing perspective: treatment of lignocellulosic materials for the production of value-added products

    Bioresour Technol

    (2010)
  • T. Foyle et al.

    Compositional analysis of lignocellulosic materials: evaluation of methods used for sugar analysis of waste paper and straw

    Bioresour Technol

    (2007)
  • D. Gao et al.

    Mixture optimization of six core glycosyl hydrolases for maximizing saccharification of ammonia fiber expansion (AFEX) pretreated corn stover

    Bioresour Technol

    (2010)
  • H.J. Gilbert et al.

    How the walls come crumbling down: recent structural biochemistry of plant polysaccharide degradation

    Curr Opin Plant Biol

    (2008)
  • F.M. Girio et al.

    Hemicelluloses for fuel ethanol: a review

    Bioresour Technol

    (2010)
  • L.M. Gottschalk et al.

    Cellulases, xylanases, β-glucosidase and ferulic acid esterase produced by Trichoderma and Aspergillus act synergistically in the hydrolysis of sugarcane bagasse

    Biochem Eng J

    (2010)
  • G.M. Gubitz et al.

    Mannan-degrading enzymes from Sclerotium rolfsii: characterisation and synergism of two endo β-mannanases and a β-mannosidase

    Bioresour Technol

    (1996)
  • Y. Han et al.

    Synergism between corn stover protein and cellulase

    Enzyme Microb Technol

    (2007)
  • T. Hashimoto et al.

    Synergistic degradation of arabinoxylan with α-l-arabinofuranosidase, xylanase and β-xylosidase from soy sauce koji mold, Aspergillus oryzae, in high salt condition

    J Biosci Bioeng

    (2003)
  • A.W. Hendriks et al.

    Pretreatments to enhance the digestibility of lignocellulosic biomass

    Bioresour Technol

    (2009)
  • E. Hoshino et al.

    Synergistic actions of exo-type cellulases in the hydrolysis of cellulose with different crystallinities

    J Ferment Bioeng

    (1997)
  • T.-C. Hsu et al.

    Effect of dilute acid pretreatment of rice straw on structural properties and enzymatic hydrolysis

    Bioresour Technol

    (2010)
  • Cited by (811)

    View all citing articles on Scopus
    1

    Claude Leon Postdoctoral Fellow.

    View full text