Cellulases in the Biofuel Industry

259 Pages·2022·12.261 MB·English

by  Pratima Bajpai

Cellulases in the Biofuel Industry
Cellulases in the
Biofuel Industry
Pratima Bajpai
Consultant-Pulp and Paper, Kanpur, Uttar Pradesh, India
Preface
There is a major international effort to develop renewable alternatives to fos-
silfuels.Oneapproachistoproducealiquidfuelbyenzymaticallyhydrolyz-
ing carbohydrate polymers in biomass to sugars and fermenting them to
ethanol. Cellulose is the main polymer in biomass and cellulases can hydro-
lyze it to cellobiose, which can be converted to glucose by β-glucosidases.
Extensive research is being carried out to obtain cellulases with higher
activity on pretreated biomass substrates by screening and sequencing new
organisms, engineering cellulases with improved properties, and identifying
proteins that can stimulate cellulases. Despite extensive research on cellu-
lases, there are major gaps in our understanding of how they hydrolyze
crystalline cellulose, act synergistically, and for which role they act in carbo-
hydrate binding modules.
This book presents cost-effective and current scenarios for cellulase pro-
duction in the biofuel industry, including the most recent advancements for
obtaining cellulases with higher activity on pretreated biomass substrates
by screening and sequencing new organisms, engineering cellulases with
improved properties, and identifying proteins that can stimulate cellulases.
The mechanism and efficiency of the cellulase enzyme system on cellulose
are discussed with the specific classification of each cellulase enzyme, as
well as explanations of the limitations of cellulases in terms of their produc-
tion processes, efficiency, and practical applications to biofuels. Various
approaches to improve the production and efficiency of the cellulase enzyme
system have been evaluated along with the current limitations that are
hampering cost-effective production of cellulase and guidance on how these
limitations might be resolved.
Pratima Bajpai
xv
List of tables
Table1.1 Mostcommonbiomassfeedstocks. 3
Table1.2 Benefitsofbiofuels. 4
Table1.3 Greenhousegasemissionsfromvariousbiofuelscomparedto 5
gasoline.
Table1.4 Classificationofcellulaseenzymes. 12
Table1.5 Fungihavingcellulolyticabilities. 16
Table1.6 Bacteriahavingcellulolyticabilities. 17
Table1.7 Actinomyceteshavingcellulolyticabilities. 18
Table2.1 Biofuelyieldsbyfeedstock. 26
Table2.2 Annualworldfuelethanolproduction(Mil.Gal.). 27
Table2.3 Historicalbiorefinerycountandproductioncapacity. 28
Table2.4 AnnualUSfuelethanolproduction. 29
Table2.5 USfuelethanolplantproductioncapacityasofJanuary1,2021. 29
Table2.6 USbiodieselplantproductioncapacityasofJanuary1,2021. 30
Table2.7 Globalcommercialscalecellulosicethanolplants. 31
Table2.8 ThestatusoftheUScommerciallignocellulosicethanolfacilities. 33
Table2.9 OperatingcellulosicethanolplantsintheUS. 34
Table2.10 Commercial-scalebioethanolplantsinAustralia. 43
Table2.11 Biofuelkeyfigures. 49
Table3.1 Yieldofbiofuelsfromdifferentfeedstocksofsecond-generation 60
biofuels.
Table4.1 Processesforthepretreatmentoflignocellulosicbiomass. 72
Table4.2 Costareadistributionofagenerallignocellulosicgreenfieldfacility. 75
Table5.1 Advantagesanddisadvantagesofsolid-stateandsubmerged 99
fermentation.
Table5.2 Cellulaseproductionundersubmergedandsolid-statefermentation. 100
Table6.1 Commercialcellulases,theirsourcesandsuppliers. 113
Table7.1 Modulararchitecturesofcellulasesfromdifferentbacteria. 122
Table8.1 Advantagesanddrawbacksofpotentialorganismsinlignocellulosic- 151
basedbioethanolfermentation.
Table8.2 ComparisonbetweenSimultaneoussaccharificationandfermentation 154
andSeparatehydrolysisandfermentation.
Table9.1 Benefitsofthermostablecellulases. 181
Table10.1 Cellulaseorcellulaseencodinggenespresentinmicroorganisms. 206
Table11.1 Thermostablecellulasesfromvariousthermophilicmicroorganisms 217
andtheircharacteristics.
Table11.2 Cellulasesfromvarioussourcesimmobilizedondifferent 223
nanosupportsandtheirimprovedproperties/applications.
xiii
List of figures
Figure1.1 Structureoflignocellulosicbiomassanditsbiopolymers;cellulose, 6
hemicellulose,andlignin.
Figure1.2 Lignocellulosicbiomassalongwiththeirproducts. 7
Figure1.3 Thecellulosepolymerchainstructure. 9
Figure1.4 Schematicrepresentationforthemacromolecularstructureoflignin 10
(Majormonolignolunitsarecoloredassinapylalcohol-red,guaiacyl
alcohol-blue,p-coumarylalcohol-green).
Figure1.5 Structureofhemicellulose. 10
Figure1.6 Schematicrepresentationofthesynergisticactionof 14
cellobiohydrolases(CBHI,CBHII),endoglucanases(EG),and
β-glucosidases(βG).
Figure1.7 Overviewofthetwostrategies(freeorcell-boundcellulasesystems) 15
fordegradingcellulose.Infreeextracellularsystems,endoglucanases
andexoglucanasesactsynergistically,withtheendoglucanase
cuttingamorphouscelluloseprovidingchainendsforexoglucanases
toreleasecellobiose.Then,β-glucosidaescompletetheprocessof
cellulosehydrolysisbyreleasingglucose.Also,cellodextrinsreleased
byendoglucanasescanbefurtherhydrolyzedbycellodextrinases.
Thecarbohydratebindingdomaindirectstheenzymestotheir
specificsubstrates.Inthecellulosomesystem,allcellulasesare
anchoredtoacommonscaffoldbutaregenerallythoughttofollow
thesamesynergicmodeofaction.Thescaffoldingisboundtothe
cellmembranethroughthesurfacelayerhomologydomain,whilea
networkofdockerinandcohesindomainsamplifiesthenumberof
cellulasesboundtothesamescaffoldingunit.Lastly,acarbohydrate
bindingdomainisresponsibleforthetargetingofthewholecomplex
tothesubstrate.
Figure1.8 Variousstrategiesadoptedforimprovingbioprocessofcellulase 18
production.
Figure2.1 Renewableethanolproductionbyenduse(https://www.clariant. 27
com).
Figure3.1 Biofuelsgenerations. 54
Figure3.2 Differentgenerationsofbiofuelswiththeircharacteristics. 55
Figure3.3 Differentfeedstocksusedinthefirstandsecondgenerationbiorefinery 55
forproducingbiofuels,biochemicals,food,andfeed.
Figure3.4 Firstgenerationbiofuels. 56
Figure3.5 Typesofrawfeedstockforsecond-generationbiofuel. 57
Figure3.6 Biochemicalconversionsofsecond-generationfeedstockstobiofuels. 59
Figure3.7 Biofuelgenerationfrommicroalgae. 62
Figure3.8 Fourthgenerationbiofuelproduction. 63
ix
x Listoffigures
Figure3.9 Aschematicdiagramofbioethanolproductionbasedondifferent 64
generations.
Figure4.1 Chartofvariousstepsinvolvedinabiomasssupplychain. 70
Figure4.2 Schematicrepresentationofbiologicalconversionoflignocellulosic 79
componentsintovariouschemicalsandbiofuels.
Figure5.1 DiagramofdifferentschemesforsequentialSSFandSmF.Thecolor 101
ofthearrowsindicatesthesystemtowhichtheybelong(greenforA,
redforB,andblueforC).
Figure5.2 Differentproductionstrategiesforcommercialproductionof 102
cellulases(A,Off-site;B,On-site;C,Integrated;D,Consolidated).
Figure7.1 Enzymesinvolvedincellulosedegradation. 120
Figure7.2 Schematicrepresentationofthehydrolysisofcelluloseby 123
noncomplexed(A)andcomplexed(B)cellulasesystems.a,cellulose;
b,glucose;c,cellobiose;d,oligosaccharides;e,endoglucanasewith
carbohydrate-bindingmodule(CBM);f,exoglucanase(actingon
reducingends)withCBM;g,exoglucanase(actingonnonreducing
ends)withCBM;h,β-glucosidase;i,cellobiose/cellodextrin
phosphorylase;j,S-layerhomologymodule;k,CBM;l,type-I
dockerin(cid:1)cohesionpair;m,type-IIdockerin(cid:1)cohesinpair.The
figureisnotdrawntoscale.
Figure7.3 Crystalstructuresoffamily6endoglucanaseandexoglucanase.(A) 125
ThestructureofendoglucanaseCel6AofThermobifidafusca(PDB
code:1TML),whichexhibitsadeepcleftattheactivesite.(B)The
structureofexoglucanaseCel6AofHumicolainsolens(PDBcode:
1BVW),inwhichtheactivesiteofitbearsanextendedloopthat
formsatunnel.
Figure7.4 Thesimulationofthesynergybetweenendoglucanaseand 131
exoglucanasesintermsofsubstratecharacteristics(degreeof
polymerization,A;andaccessibility,B)andexperimentalconditions
(enzymeloading,C;andreactiontime,D).
Figure8.1 Schematicdiagramofthesynergicactionofcellulasesoncellulosic 140
biomasshydrolysis.
Figure8.2 Roleofcellulasesduringthecompletebiofuelproductionprocess. 141
Figure8.3 Schematicdiagramforconversionoflignocellulosicbiomassinto 141
ethanol.
Figure8.4 Processingrouteforbioethanolproduction. 145
Figure8.5 Schematicofpretreatmenteffectonlignocellulosicbiomass. 146
Figure8.6 Separationprocessofbioethanolbyextractivedistillation. 157
Figure8.7 ClariantcompletesconstructionoffirstcommercialSunliquid 161
cellulosicethanolplantinPodari,Romania(https://www.clariant.
com).
Figure8.8 Clariant’sflagshipcommercialSunliquidcellulosicethanolplantin 161
Romania.(https://www.clariant.com).
Figure8.9 SunliquidplantPodari(https://www.clariant.com). 162
Figure8.10 TheSunliquidprocessfortheproductionofcellulosicethanolfrom 163
agriculturalresidues(https://www.clariant.com).
Figure8.11 Sunliquid—afullyintegratedprocessdesign(https://www.clariant. 163
com).Reproducedwithpermission.
Figure8.12 Sunliquidtechnologyplatformfortheproductionofsustainable 165
biobasedproducts.
Figure8.13 Sunliquidtechnology:fivelicensessoldglobally(https://www. 165
clariant.com).Reproducedwithpermission.
Listoffigures xi
Figure8.14 ReductionofCarbondioxideemissions(https://www.clariant.com). 166
Figure8.15 Sunliquid:fullyintegratedprocesssaves~95%GHGemissions 166
(https://www.clariant.com).
Figure8.16 ClariantBioethanolPilotPlant,Straubing,Germany(https://www. 167
clariant.com).
Figure9.1 Cellulosomestructureandassembly. 188
Figure10.1 Schematicdiagramoftheactionsofcellulasesandsynergistic 207
proteinsinhydrolysisofcellulose.TheC1-Cxlignocellulose
degradationmodelforcellulosedegradation.First,C1factors,such
asexpansin,andlyticpolysaccharidemonooxygenases,which
efficientlycatalyzeoxidativecleavageofglycosidicbondsinthe
recalcitrantpolysaccharidesofcrystallinecelluloseusingmolecular
oxygenandtheexternalelectrondonor,suchascellobiose
dehydrogenase,ascorbicacidorgallicacid,creatingnicking,
swollenanddisintegratedcellulosicstructure,formingnewinitiation
sitesforconventionalcellulases,namely,Cxfactors.Second,the
amorphouscellulosewashydrolyzedintomonosaccharideby
cellulasesystem.Theendoglucanaseactsontheamorphous
(internal)regionofthefibrilsbycleavageoftheβ-glucosidiclinkage,
thenthecellobiohydrolasereleasescellobiosefromtheendofthe
polysaccharidechain,finally,β-glucosidasecompletesthe
degradationprocessbyhydrolyzingcellobioseandother
cellodextrinstoglucoseunits.BGL,β-glucosidase;CBHI,
cellobiohydrolase1;CBHII,cellobiohydrolase2;CDH,cellobiose
dehydrogenase;C1factor,cellulosehingdomains(CBMs),plant
expansins,bacterialexpansins,andlyticpolysaccharide
monoxygenases;Cxfactor,endoglucanase,cellobiohydrolase,and
β-glucosidase;EG,endoglucanase;LPMO,lyticpolysaccharide
monooxygenase.
Figure11.1 Advantagesofenzymatichydrolysiscarriedoutatelevated 215
temperature.
Figure11.2 Strategiestoincreasecellulaseefficiencyandcharacteristics. 226
Figure11.3 Geneticmanipulationstrategiesinfungiforimprovedcellulase 227
production.
Figure12.1 Biomass-to-biofuelssupplychain. 244
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Contents
Listoffigures ix
Listoftables xiii
Preface xv
Acknowledgments xvii
1. Background 1
1.1 Introduction 1
1.2 Bioenergyandbiofuels 2
1.3 Biomass 5
1.4 Cellulaseenzyme 11
References 19
2. Worldwide scenario of biofuel production 25
2.1 Introduction 25
2.2 Statusofbiofuelproduction 28
2.2.1 UnitedStates 28
2.2.2 Brazil 33
2.2.3 EuropeanUnion 37
2.2.4 Argentina 39
2.2.5 China 39
2.2.6 Australia 41
2.2.7 Canada 43
2.2.8 Thailand 45
2.2.9 Japan 46
2.2.10 India 47
References 50
3. Generations of biofuels 53
3.1 Introduction 53
3.2 Firstgenerationbiofuels 56
3.3 Secondgenerationbiofuels 58
3.4 Thirdgenerationbiofuels 61
3.5 Fourthgenerationbiofuels 62
References 64
v