Comparison and Optimization of Saccharification Conditions of Alkaline Pre-Treated Triticale Straw

24 Pages·2017·6.09 MB·English

energies
Article
Comparison and Optimization of Saccharification
Conditions of Alkaline Pre-Treated Triticale Straw
for Acid and Enzymatic Hydrolysis Followed by
Ethanol Fermentation
RafałŁukajtis*,KarolinaKucharska,IwonaHołowacz,PiotrRybarczyk,KatarzynaWychodnik,
EdytaSłupek,PaulinaNowakandMarianKamin´ski
FacultyofChemistry,DepartmentofChemicalandProcessEngineering,Gdan´skUniversityofTechnology,
Narutowicza11/12Street,80-233Gdan´sk,Poland;[email protected](K.K.);
[email protected](I.H.);[email protected](P.R.);[email protected](K.W.);
[email protected](E.S.);[email protected](P.N.);[email protected](M.K.)
* Correspondence:[email protected];Tel.:[phone]
Received:19February2018;Accepted:8March2018;Published:13March2018
Abstract: Thispaperconcernsthecomparisonoftheefficiencyoftwo-stagehydrolysisprocesses,
i.e., alkaline pre-treatment and acid hydrolysis, as well as alkaline pre-treatment followed by
enzymatichydrolysis,carriedoutinordertoobtainreducingsugarsfromtriticalestraw. Foreachof
theanalyzedsystems,theoptimizationoftheprocessingconditionswascarriedoutwithrespectto
theglucoseyield. Forthealkalinepre-treatment,anoptimalcatalystconcentrationwasselectedfor
constantvaluesoftemperatureandpre-treatmenttime. Forenzymatichydrolysis,optimalprocess
time and concentration of the enzyme preparation were determined. For the acidic hydrolysis,
performedwith85%phosphoricacid,theoptimumtemperatureandhydrolysistimeweredetermined.
In the hydrolysates obtained after the two-stage treatment, the concentration of reducing sugars
wasdeterminedusingHPLC.Theobtainedhydrolysatesweresubjectedtoethanolfermentation.
Theconcentrationsoffermentationinhibitorsaregivenandtheireffectsonthealcoholicfermentation
efficiencyarediscussed.
Keywords: lignocellulosicbiomass;enzymatichydrolysis;acidhydrolysis;alkalinepre-treatment;
optimization;Box-Behnkendesign;bioethanolfermentation
1. Introduction
Fermentation is an anaerobic respiration process carried out by many types of bacteria and
fungi. Fermentation,bydefinition,isadisproportionationreaction. Ethanolfermentationiscarried
outbyyeasts,includingSaccharomycescerevisiae[1–5]. Yeastsareabletofermenthexosesandsome
oligosaccharides, e.g., sucrose, maltose, or inulin, at specific conditions. A decreased pH value
or the presence of inhibiting fermentation products, i.e., ethanol, influences the growthabilities
of Saccharomyces cerevisiae. At ethanol concentrations up to 120 g/L, Saccharomyces are capable of
performingfermentationandtheyareabletogrowatethanolconcentrationsupto200g/L[6–8].
Thecurrentglobaldemandforbiofuelsislargelycoveredbytheproductionoffirst-generation
bioethanol from sucrose or starch contained in such raw materials as sugar cane, wheat, or corn.
An interesting alternative is the production of the second-generation bioethanol as a result of
fermentation processes from lignocellulosic waste. Lignocellulose, the most abundant renewable
carbohydrate-richbiomass,isaccumulatedinlargequantitiesfromagricultural,forestry,ormunicipal
activitiesanddonotcompetewithfoodcrops. Itisalsolessexpensivethanconventionalagricultural
feedstocks. However,accordingtothecomplexityofthechemicalstructure,lignocellulosicbiomassis
Energies 2018,11,639;doi:10.3390/en11030639 www.mdpi.com/journal/energies
Energies 2018,11,639 2of24
resistanttobiodegradation. Cellulose,hemicellulose,andlignin,maincomponentsoflignocellulosic
complex,arestronglyintermeshedandbondedtroughcovalentandnon-covalentbonds. Ligninis
the major barrier to make the carbohydrate components (cellulose and hemicellulose) suitable for
bioconversion[9,10]. Therefore,thepre-treatmentstephasalargeimpactonsubsequentenzymatic
hydrolysis and fermentation steps [11]. It should improve the formation of sugars, be effective in
the removal of lignin, and produce no, or limited amounts of, products that inhibit the action of
thehydroliticenzymesorthegrowthoffermentativemicroorganisms. Pre-treatmentmethodscan
beclasssifiedasbiological, physical, chemical, andphysico-chemicalprocesses. Acombinationof
differentpre-treatmentmethodshavealsobeenconsidered[12].
Themaingoalofalkalinetreatment,oneofthemostcommonchemicalpre-treatmentmethods,
istoremoveligninfromthebiomass[13]. Theactionofalkalinereagentsleadstothedegradation
of side chains of esters and glycosides and then to the structural modification of lignin, cellulose
swelling,cellulosedecrystallization,andhemicellulosesolvation. Ca(OH) alsoremovesacetylgroups
2
fromhemicellulose,reducingsterichindranceandincreasingtheavailabilityoftheenzymetothe
surfaceofhemicelluloseandcellulose. Thesemethodsaremoreeffectiveforcellulosedigestibility
andforligninsolubilization,exhibitingminorcelluloseandhemicellulosedissolutionthanacidor
hydrothermalprocesses[9]. Theadvantageofalkalinemethodsistheirlowcost, especiallywhen
usingCa(OH) . Thelimitationisthatalkaliareconvertedintoun-saltedorintroducedasbiomass
2
saltsafterthepre-treatmentand,therefore,thewaterconsumptionforsaltremovalfrombiomassis
high. Theligninremovalstepisnessesarytoavoidtheformationofinhibitors(salts,furfural,phenolic
acid,aldehydes). Abudietal.[14]investigatedtheeffectsofalkalineandperoxidepre-treatmentof
ricestraw(RS).TheresultsindicatethatNaOHandH O pre-treatmentsaffectedthelignocellulose
2 2
compositiondifferentlyandareparticularlyeffectiveintermsofsolubilizationofRShemicellulosemore
thanofcelluloseandlignin. NaOHpre-treatmentledtoapproximately11%,32%,and22%reductions
inhemicellulose,cellulose,andlignincontent,respectively. InthecaseofH O pre-treatment,the
2 2
correspondingreductionswere16%,41%,and7%,respectively.
After the pre-treatment of lignocellulosic biomass, the hydrolysates are further processed
by biochemical methods.During the enzymatic hydrolysis, crystalline cellulose and hemicellulose
not decomposed during pretreatment, and are degraded to reducing sugars, fermentable by
microorganisms. The conversion of cellulose and hemicellulose is catalysed by cellulases and
hemicellulases,respectively.Cellulasesareusuallyamixtureofseveralenzymes.Thethreepredominant
onesare:endo-1,4-β-glucanase,whichhydrolyzestheinnerβ-1,4-glycosidicbonds;exo-1,4-β-glucanase,
which removes mono- and dimers from the free chain-ends; and β-glucosidase (cellobiase), which
hydrolyzesglucosedimers[15–17].Cellulosehydrolysisstartswithadsorptionofcellulaseenzymes
onto the surface of cellulose. Afterwards, cellulose isbiodegraded to the fermentable sugars and
cellulaseisdesorbedfromthebiomasssurface.Cellulosemicrofibersaresurroundedbyhemicellulose
polysaccharides. Therefore, auxiliary enzymes that attack hemicellulose are used. In the first
step, endo-1,4-β-xylanase depolimerizes xylan into xylooligosaccharides.Further, xylanases, such
as β-glucuronidase, α-arabinofuranosidase, and acetyl xylan esterase, cleave side chains and side
groupsofheteroxylan,whilegalactomannanaseandglucomannanasehydrolyzeglucomannan[18].
Theoptimalbalancedcombinationofenzymesisneededtoeffectivelymodifythecomplexstructure
oflignocellulosicmaterials. Inadditiontothequalityofthehydrolyzingenzymes,thedigestibility
ofcelluloseandhemicelluloseisaffectedbypH,temperature,processtimeandporosity,degreeof
crystallization,andcellulosecontent[19].
Saccharificationoflignocellulosicbiomasswithanacidcatalystisalsopossible.Themainobjective
ofacidtreatmentisthesolubilizationofhemicelluloseandmakingcelluloseaccessibletoenzymes.
Acidhydrolysisisanalternativeforenzymatichydrolysis,asacidhydrolyzesbiomasstofermentable
sugars. However,intensivewashingisrequiredtoremovetheacidbeforefermentationisperformed.
Theadvantageofusingacidsistheirlowpriceandavailability. Additionally,theuseoforganicacids
(formic, acetic, maleic) makes the process environmentally friendly [20]. The disadvantage is the
Energies 2018,11,639 3of24
need to neutralize the used acid by alkali, which generates a stream of reagents, or by vacuum
distillation,Enienrgieos 2r0d18e, r11, txo FOrRe PuEsEeR RtEhVeIEWa c id. Another disadvantage of using acid hydro3l yofs 2i5s is is the
possibility of the formation of fermentation inhibitors, especially when high acid concentrations,
disadvantage is the need to neutralize the used acid by alkali, which generates a stream of reagents,
high tempeorra tbuy revsa,cuaunmd dloisntilglatrieoan,c tiino nortdiemr etos areruesea pthpel ieacdid[. 2A1,n2o2t]h.er Dduisraidnvganatacgide ohfy udsriongly sacisid, the acid
catalyzesthheydbrroelyaskisdiso isw thne opfosgsilbyicliotys oidf tihceb foornmdastiobne otwf feeremnenstuatgioanr irnehsibiditoures,s e,sfpoelclioalwly ewdhebny htighhe abcirde akdown
concentrations, high temperatures, and long reaction times are applied [21,22]. During acid
of released glucose to form hydroxymethylfurfural (HMF), formic acid (FA), and levulinic acid
hydrolysis, the acid catalyzes the breakdown of glycosidic bonds between sugar residues, followed
(LA) (Figure 1) [23]. Compounds, such as furfural, 5-HMF, and phenolic compoundsaffect the
by the breakdown of released glucose to form hydroxymethylfurfural (HMF), formic acid (FA), and
microorganleisvmulinmice taacbido l(iLsAm) in(Fitghueref e1r)m [e23n]t. atCioomnpsotuenpd.s,D siulucht eaasc ifdurpfurrea-l,t re5-aHtmMFe,n tanisd mpoherneofliacv ourable
forindustricaolmappopunlidcsaatffieocnt stdheu emtoicrtohoergraendisumc timonetaobfotlihsme opine rathteio nfearlmaenntdatmiona instteepn. anDcileutceo satcsi(dc orrosion
pre-treatment is more favourable for industrial applications due to the reduction of the operational
problems). Additionally,thiskindofpre-treatmentgeneratesfewerdegradationcompoundsthanthe
and maintenance costs(corrosion problems). Additionally, this kind of pre-treatment generates
concentratedacidpre-treatment[18,24,25].
fewerdegradation compounds than the concentrated acid pre-treatment [18,24,25].

Figure1.FFoigrumrea t1i. oFnoromfatsieolne ocft esedlefceterdm feernmteanttiaotnionin inhhibibiittoorrss frforomm glugclousceo dsueridngu arcinidg haydcirdolyhsyisd. rolysis.
The lignocellulosic hydrolysates are very attractive as a substrate for fermentative conversion
Theligpnroocceesslelus ltoo sbiicoehthyadnrool.l yTshae treessualrtse ovf etrhye alatttersat crteisveaercahs oan stuheb sptrroadtuectfioonr foef rbmioeenthtaantoivl efrocmo nversion
processestowbasitoee ltighnaonceolllu.lToshice brieosmualstss aoref pthreeselnatteeds itnr Teasbelae r1c. hontheproductionofbioethanolfromwaste
lignocellulosicbiomassarepresentedinTable1.
Table 1. The efficiency of bioethanol production related to the pre-treatment method and used
microorganisms.
Table1.Theefficiencyofbioethanolproductionrelatedtothepre-treatmentmethodandusedmicroorganisms.
Type of
Type of Ethanol Yield
Lignocellulosic Used Microorganism Processes Involved References
Pretreatment (g/g)
TypeoMfaterial
Typeof Used Ethanol Processes
LignocelluCloosttiocn Hydrolysis and References
Pretreatment Microorganism Yield(g/g) Involved
Materreisaidlues,corn - Saccharomyces cerevisae 0.027 fermentation realized [21]
stover separately
Hydrolysisand
Cottonresidues, Saccharomyces Simultaneous
- Kluyveromyces 0.027 fermentation [21]
cornstWovheeart straw Steam explosion cerevisae 0.27 saccharification and [26]
marxianus reafelrimzeedntsaetipona rately
WheatsStrubaagwgaarcsasne e SteasAmtecaiedmx d peixlgopelssotiisooinonn, KPlmuicyhaviraex sritoaimpniutyissc es 0.039.2 7 ssaaccScfhSecifmraheimmrruaimferluitnicafielatntantcaeitaotionoatnueint osaio onnundsa nd [27] [26]
Simultaneous
Sugarcane Aciddigestion, 0.29 sacchSaimrifuiclattaionne aonuds
bagasSsuegarcane steameMxpillloinsgi on PPiicchhiiaa ssttipipitiitsi s 0.39 sacfecrhmaernifitactaiotino nand [28] [27]
bagasse Hyfderromlyesnist aantido n
0.27 fermentation realized
Simultaneous
separately
0.29 saccharificationand
Hydrolysis and
SugarcRaincee husks AlMkaillinlien dgigestion EPsicchheiraicshtiaip ciotliis 0.21 fermefnetramtioenn rteaatliiozend [29] [28]
bagasse Hsyedparroaltyelsyi sand
0.27 fermentation
realizedseparately
Hydrolysisand
0.21 fermentation
realizedseparately
Ricehusks Alkalinedigestion Escherichiacoli [29]
Simultaneous
0.20 saccharificationand
fermentation
Energies 2018,11,639 4of24
Table1.Cont.
Typeof
Typeof Used Ethanol Processes
Lignocellulosic References
Pretreatment Microorganism Yield(g/g) Involved
Material
Aciddigestion,
Maizecob Zymomonasmobilis 0.42 Batchreactor [30]
overliming
Drying Clostridium 0.36
thermocellum;
Aciddigestion 0.40
Clostridium
Alkalinedigestion thermosaccharolyticum 0.42
Bananaremnants Batchreactor [31]
Drying 0.33
Clostridium
Aciddigestion thermocellum; 0.36
Clostridiumethanolicus
Alkalinedigestion 0.39
Currently, lignocellulosic biomass is regarded as the forward-looking, large-scale available
non-fossil carbon source for biofuels production. Therefore, there is a strong motivation for
research into industrially and economically viable processes for conversion of biomass to fuels,
including bioethanol. However, successful and effective conversion of lignocellulosic biomass
to monosaccharides in high yields is still a challenge. Acid or enzymatic hydrolysis of biomass
polysaccharides after a pre-treatment stage are the main currently-investigated saccharification
methods [32–36]. Therefore, the primary objective of the present study is the comparison of the
efficiencyoftwo-stagehydrolysisprocesses,i.e.,alkalinepre-treatmentfollowedbyacidhydrolysis,
as well as alkaline pre-treatment followed by enzymatic hydrolysis, carried out in order to obtain
reducingsugarsfromtriticalestraw. Thecomparisonistoconcernnotonlyresultsobtainedformono
sugars,butforbioethanolaswell.
Theaimoftheworkisalsotoexaminethecourseandefficiencyoftheenzymatichydrolysisusing
cellulolyticenzymesimmobilizedondiatomite. Thisprocedureisintendedtoreduceprocesscosts
throughthepossibilityofenzymeregenerationandreuse.
Theoptimizationoftheconditionsofthetwo-stagehydrolysisandtheevaluationoftheinfluence
of process parameters on the glucose yield were performed according to the Box-Behnken design
forthefollowingvariables: concentrationofNaOH,durationtimeofacidhydrolysis,temperature
ofacidhydrolysis,thecontentofViscozymeL,andthedurationtimeoftheenzymatichydrolysis
process. Finally, the parameters’ significance on the performance of sugars were optimized using
the response surface methodology. The effect of acid hydrolysis conditions on the content of
typicalfermentationinhibitors,including5-HMF,furfural,andthetotalphenoliccompoundswas
investigated. Additionally, pre-treatment and hydrolysis effects were evaluated by performing
bioethanolfermentationonpreviously-obtainedhydrolysates.
2. ResultsandDiscussion
2.1. BiomassCharacterisation
Milledandmincedlignocellulosicbiomass(triticalestrawTriticumsp.,originatedfromWejherowo,
Poland) was used in this study. The determined composition of the Triticum sp. was found to be:
43.6% cellulose, 23% hemicellulose, 21.5% lignin, 8.3% water extractives, 8.6% ethanol extractives,
1.2%ash,andamoisturecontentof6.2%(ondryweightbasis). Hemicellulosecontentwasfoundas
follows: xylose(18.1%),galactose(4.1%),andmannosewitharabinose(0.8%).
2.2. InfluenceofAlkalinePre-TreatmentConditionsoftheChemicalCompositionofTriticaleStraw
Theoptimizationofthetwo-stagehydrolysisconditionsandtheevaluationoftheinfluenceof
processparametersontheglucoseyieldweremadefortwotestsystems. Inthefirstsystem,alkaline
pre-treatmentandacidhydrolysiswerecarriedout,while,inthesecondsystem,alkalinepre-treatment
Energies 2018,11,639 5of24
followedbyenzymatichydrolysis. Inbothcases,thealkalinepre-treatmentwascarriedoutforsix
hoursataconstanttemperatureof65◦C.Thetreatmentwascarriedoutforthecatalystconcentrations
(NaOH) of 1%, 6%, and 11%. In the first stage of the research, the effect of alkaline pre-treatment
onthechangeoftriticalestrawcompositionwasdetermined. Forthispurpose,thecompositionof
biomass(reducingsugarsandlignincontents),aswellasthedegreeofligninremoval,thedegreeof
celluloseandhemicelluloserecoveriesweredeterminedintheresidueremainingafterthealkaline
treatment. The results (Table 2) indicate that an increase in the concentration of NaOH causes an
increase in the biomass loss of 37%, 49%, and 52%, respectively, for catalyst concentrations of 1%,
6%,and11%. Asimilardependencewasobtainedin[12,37,38]. Theresearchresultsindicatethatthe
useof1%NaOHalreadysignificantlylowersthelignincontent. Afurtherincreaseinthecatalyst
concentrationabove1%doesnotsignificantlyaffecttheligninremoval. Thealkalinepre-treatment
of the material influences the change in the proportion of the sugar polymer content. The use of
1%NaOHallowsthecontentofallreducingsugarstoincrease. ForNaOHconcentrationsgreaterthan
1%,adecreaseinreducingsugarscomingfromhemicellulosewithasimultaneousincreaseinglucose
concentrationisobserved.
Table2.Compositionofrawbiomassandobtainedhydrolysates.
Glucose Xylose Galactose Mannose, Lignin Cellulose Hemicellulose Lignin
Content Content Content ArabinoseContent Content Recovery Recovery Removal
(mg/gbiomass) (mg/gbiomass) (mg/gbiomass) (mg/gbiomass) (%) (%) (%) (%)
Untreated 436 181 41 8 21.5 - - -
1%NaOH 548 230 58 13 9.5 79.3 82.4 72.1
6%NaOH 644 171 43 0 8.4 75.4 47.4 75.7
11%
701 141 22 0 8.4 77.2 33.9 75.2
NaOH
2.3. OptimisationofAlkalinePre-TreatmentFollowedbyAcidHydrolysis
For the analysis of the influence of input variables on the result of two-stage hydrolysis,
i.e., alkaline treatment and acid hydrolysis, experiments (Table 3) were performed according to
theBox-Behnkendesignforthreevariables. Thefollowingboundaryvalueswereassumedforthe
investigatedvariables: concentrationofNaOH1–11%ataconstanttemperatureof65◦Candalkaline
pre-treatmenttimeof6h,durationofacidhydrolysisof0.5–8h,andthetemperatureofacidhydrolysis
of60–120◦C.Asanoutputparameter,glucoseyield(G )wasselectedasaparameterdescribingthe
y
efficiencyoftheprocess.Additionally,Table3showstheobtainedvaluesofthesumofreducingsugars.
Table3.Box-Behnkendesignforalkalinepre-treatmentandacidhydrolysisofTriticumsp.
No. CNaOH(%) Temperature(◦C) Time(h) Gy(mg/gbiomass) SumofSugars(mg/gbiomass)
1 1 90 0.5 334.5 397.3
2 11 90 0.5 293.9 360.8
3 1 90 8 104.6 122.3
4 11 90 8 106.4 118.5
5 1 60 4.25 229.6 314.6
6 11 60 4.25 204.9 285.0
7 1 120 4.25 7.3 13.2
8 11 120 4.25 7.6 8.2
9 6 60 0.5 56.4 147.9
10 6 60 8 301.7 369.3
11 6 120 0.5 271.4 325.3
12 6 120 8 14.3 14.3
13 6 90 4.25 265.6 314.8
14 6 90 4.25 260.7 312.8
15 6 90 4.25 269.8 326.1
Energies 2018,11,639 6of24
Aninfluenceoftheprocessparameters(NaOHconcentration,temperatureanddurationofacid
hydrolysis)ontheglucoseyieldwasinvestigated. Onthebasisofmultipleregression,theEquation(1)
wasproposed,describingtherelationshipsoccurringduringthetwo-stagehydrolysis.
EnergYies[ %20]18=, 1−1, 1x4 F2O6R− PE3E.R9 8RXE1VI+EW34 . 45X2+177.70X3 −0.16X22 −3.64X32+0.57X1·X3 −1.97X2·X36 of (215)
IInn EEqquuaattiioonn ((11)),, XX11,, XX22, ,aanndd XX33 rreepprreesseenntt ssooddiiuumm hhyyddrrooxxiiddee ccoonncceennttrraattiioonn,, tteemmppeerraattuurree,, aanndd
ttiimmee ooff aacciidd hhyyddrroollyyssiiss,, rreessppeeccttiivveellyy.. YY ddeennootteess gglluuccoossee eeffffiicciieennccyy.. EEqquuaattiioonn ((11)) eennaabblleess ccaallccuullaattiinngg
tthhee tthheeoorreettiiccaall gglluuccoossee yyiieelldd wwiitthhiinn tthhee rraannggee ooff vvaarriiaabblleess aaccccoorrddiinngg ttoo tthhee bboouunnddaarryy ccoonnddiittioionnss. .
TThhee ppaarraammeetteerrss ooffE qEuqautaiotinon(1 )(a1r) eagriev egnivinenT aibnl eT4a.bIlne o4rd. eIrnt oorddeeter rmtoi ndeettheermefifneec ttohfet heeffpeacrt aomf ettehres
psiagrnamifiectaenrcse siognnitfhiceanpceer foonrm thaen cpeerofofrsmugaanrcse, oafn suangaalrys,s iasno afnvaalyrisaisn coef fvoarritahneces uforfra tcheeo sfutrhfaecem oofd tehle's
mreosdpoeln'ss erewspasonmsaed we.aTs hmeafdorem. TohfeE fqourmati oonf E(1q)uiasttihoen r(e1s)u ilst othfer erjeescutilnt gotfh reejleecatsitnsgi gthneifi lceaanstt psiagrnamifiectaenrts
pinartahmeneteexrtsi tiner tahtiev neesxtet pitse.rTahtiivsep srtoecpesd. uTrheissh porwoceeddtuhraet sthheowleaedst tihmapt othrtea nletapsat riammpeotretrasnitn psaurcahmaestyersste imn
siuscthhe ai nsytesrtaecmti oisn tbheet wineteenraccotinocne nbtertawtieoenno cfocnacteanlytsrattainodn toefm captearlaytsut raen(dX 1t·eXm2)p.eFroartutrhei s(Xin1t·Xer2a).c tFioorn ,ththise
ipn-tvearalucetiowna, sthme upc-hvahluigeh weratsh manucthhe haigsshuemr tehdansi gthneifi acsasnucmeeldev seilgpni<fic0a.1n,cwe lheivcehl ips w< 0h.y1,i twwhaicshr eisje wctheyd .
iBt awseads orenjeacntaeldy.s iBsaosfevda orina nacnea,ltyhseiso botfa vinaerdiavnacleu, ethoef tohbetdaienteerdm vinalautieo nofc otheeffi dcieetnetrm(Ri2na=ti0o.9n9 6co,Fefigfiucireen2t)
(iRn2d =ic 0a.t9e9s6a, vFeigryurgeo 2o)d incodrircealtaetsi oan vbeertyw geoeondt hceorerfefilcaiteionncy bceatwlcueelant etdheo enfftihceiebnacsyi scoalfcEuqluaatetido non(1 t)haen bdatshise
oafcEtuqaulagtilounc o(1se) aenffidc itehnec aycwtuiatlh ginlutchoesien evfefsictiigenatceyd wraitnhgine othfev ianrviaebslteigs.ated range of variables.
Table 4. Model parameters for Box-Behnken design for alkaline pre-treatment and acid hydrolysis
Table4.ModelparametersforBox-Behnkendesignforalkalinepre-treatmentandacidhydrolysisof
of Triticum sp.
Triticumsp.
Parameter Value Std. Error t-Value p-Value
Parameter Value Std.Error t-Value p-Value
Intercept −1426 53.340 −26.746 1.81 × 10−7
IntercepXt1 −1−432.968 513.2.33440 −−32.62.2774 6 0.10.18810× 10−7
X −3.98 1.234 −3.227 0.0180
1 X2 34.45 1.118 30.824 7.74 × 10−8
X 34.45 1.118 30.824 7.74×10−8
X2 X3 17177.77.070 66..448855 2277.3.39977 1.561 .×5 610×−7 10−7
3
X 2 X22 −−00.1.616 00..000066 −−2288.0.08877 1.351 .×3 510×−7 10−7
2
X32 X32 −−33.6.464 00..337766 −−99.6.66633 7.047 .×0 410×−5 10−5
X1·X3X1·X3 0.05.757 00..224466 22.2.29955 0.06105.0 615
X2·X3X2·X3 −−11.9.797 00..005555 −−3355.6.63311 3.263 .×2 610×−8 10−8
400
300
R2= 0,996
Experimental
200
G , mg/mL
y
100
0
0 100 200 300 400
Model-predicted G , mg/mL
y

Figure2.Analysisofvarianceforthetheoretical(model-predicted)andexperimentalglucoseyieldfor
Failgkualrien e2.p Aren-tarleyastims oefn tvaanridanacceid fohry tdhreo ltyhseios.retical (model-predicted) and experimental glucose yield
for alkaline pre-treatment and acid hydrolysis.
Theattachedfiguresshowmodelsurfaceresponsesandaretheeffectoftheinteractionsbetween
The attached figures show model surface responses and are the effect of the interactions
twoselectedvariableparameters,whilemaintainingthethirdparameterataconstantlevel. Themodel
between two selected variable parameters, while maintaining the third parameter at a constant
responseistheconcentrationofglucose(glucoseyield)afterthetwo-stagehydrolysis,whereinthe
level. The model response is the concentration of glucose (glucose yield) after the two-stage
hydrolysis, where in the second stage the obtained solid fraction after the alkaline pre-treatment
was used. The analysis of the surface response was useful for the determination of the individual
effects of each parameter and the impact of interactive effects of independent variables on the
glucose yield. This approach allowed to determine the optimal conditions with respect to each
investigated parameter.
Figure 3 presents the effect of NaOH concentration and acid hydrolysis temperature on
glucose yield (acid hydrolysis time 1h). The results indicate that the glucose yield reaches a
Energies 2018,11,639 7of24
secondstagetheobtainedsolidfractionafterthealkalinepre-treatmentwasused. Theanalysisofthe
surfaceresponsewasusefulforthedeterminationoftheindividualeffectsofeachparameterandthe
impactofinteractiveeffectsofindependentvariablesontheglucoseyield. Thisapproachallowedto
determinetheoptimalconditionswithrespecttoeachinvestigatedparameter.
Energies 2018, 11, x FOR PEER REVIEW 7 of 25
Figure3presentstheeffectofNaOHconcentrationandacidhydrolysistemperatureonglucose
yield (acid hydrolysis time 1h). The results indicate that the glucose yield reaches a maximum at
maximum at about 100 °C. Increasing the temperature of acid hydrolysis above 100 °C results in a
about 100 ◦C. Increasing the temperature of acid hydrolysis above 100 ◦C results in a decrease of
decrease of glucose yield due to the breakdown of simple sugars. The increase in NaOH
glucoseyieldduetothebreakdownofsimplesugars. TheincreaseinNaOHconcentrationduringthe
concentration during the pre-treatment causes a decrease in a glucose yield. This is caused by a
pre-treatmentcausesadecreaseinaglucoseyield. Thisiscausedbyasignificantlossofmassduring
significant loss of mass during alkaline pre-treatment. Biomass loss is related to the catalyst
alkalinepre-treatment. Biomasslossisrelatedtothecatalystconcentrationanditisequalto38%for
concentration and it is equal to 38% for the treatment of 1% NaOH and 52% when 11% NaOH is used.
thetreatmentof1%NaOHand52%when11%NaOHisused.
350
G , mg/g
y
300
300-350
250
250-300
200
200-250
150-200 150
11
100-150 100 9
50 7
50-100
0 5
0-50
C , %
60 3 NaOH
70
80
90
Time = 1h Temp, oC 100 1
110
120

FFiigguurree 33.. TThhee ssuurrffaaccee rreessppoonnssee pplloottss ooff tthhee eeffffeeccttss ooff NNaaOOHH ccoonncceennttrraattiioonn aanndd tteemmppeerraattuurree aacciidd
hhyyddrroollyyssiiss oonn tthhee gglluuccoossee yyiieelldd..
Figure 4 shows the effect of temperature and duration time of acid hydrolysis on the glucose
Figure4showstheeffectoftemperatureanddurationtimeofacidhydrolysisontheglucose
yield for a constant sodium base concentration (6%). Glucose efficiency increases with increasing
yield for a constant sodium base concentration (6%). Glucose efficiency increases with increasing
tteemmppeerraattuurree,, hhoowweevveerr,, wwhheenn tthhee ooppttiimmaall tteemmppeerraattuurree ((110000 °◦CC)) iiss rreeaacchheedd,, aa ssiiggnniiffiiccaanntt ddeeccrreeaassee iinn
glucose efficiency is observed if the temperature is further increased. For temperaturesin the range
glucoseefficiencyisobservedifthetemperatureisfurtherincreased. Fortemperaturesintherange
ooff 9900––110000 °◦CC,, aanndd ffoorr aa sshhoorrtt ttiimmee ooff aacciidd hhyyddrroollyyssiiss ((00..55 hh)),, tthhee hhiigghheesstt gglluuccoossee yyiieellddss aarree oobbttaaiinneedd..
An increase in time over 0.5 h results in a decrease in the hydrolysis efficiency. Only for the
Anincreaseintimeover0.5hresultsinadecreaseinthehydrolysisefficiency.Onlyforthetemperature
toefm60pe◦rCa,tuanrei nocfr e6a0s e°iCn, galunc oisnecreefafisceie ninc ygalluocnogsew ietfhfitchieendcuy raatlioonngt imwietho ftahceid dhuyrdartoiolyns itsimiseo bosfe ravceidd.
hydrolysis is observed.
Figure 5a,b present an influence of the duration time of acid hydrolysis and NaOH
Figure 5a,b present an influence of the duration time of acid hydrolysis and NaOH
concentration(alkalinetreatment)ontheglucoseyieldataconstanttemperatureofacidichydrolysis.
concentration (alkaline treatment) on the glucose yield at a constant temperature of acidic
Twopossibilitiesofinterperationoftwo-stagehydrolysisshouldbeconsideredhere. Inonevariant,
hwyhdernoltyhseisa.c Tidwhoy pdoroslsyibsiislittieems poef rianttuerrepeisra1t0i0on◦ Cof( Ftwigou-rseta5gbe), hhyigdhroyliyeslids oshforeudldu cbineg cosungsiadresrceadn hoenrley. bIne
one variant, when the acid hydrolysis temperature is 100 °C (Figure 5b), high yield of reducing
achievedforashortdurationofacidichydrolysis(0.5h). Theincreaseinthedurationoftreatment
sugars can only be achieved for a short duration of acidic hydrolysis (0.5 h). The increase in the
results in a decrease of the glucoseyield due to the degradation of simple sugars. When a lower
dteumrapteiorant uorfe troefa6t0m◦eCnti sreaspupltlsie idn (oa thdeercrveaarsiea notf, Fthige ugrleu5cao)s,etyhieelhdi gdhuees ttoy itehlde idseagcrhaideavteiodna fotfe rsi8mhpolef
sugars. When a lower temperature of 60 °C is applied (other variant, Figure 5a), the highest yield is
acidichydrolysis. Thechoiceofprocesstemperature,besidestheefficiencyofreducingsugars,should
achieved after 8 h of acidic hydrolysis. The choice of process temperature, besides the efficiency of
alsobesupprtedbytheconcentrationoffermentationinhibitorsintheresultinghydrolysate.
reducing sugars, should also be supprted by the concentration of fermentation inhibitors in the
resulting hydrolysate.
Energies 2018,11,639 8of24
Energies 2018, 11, x FOR PEER REVIEW 8 of 25
G, mg/g 350
y
300
300-350
250
250-300
200
200-250
150-200 150
100-150
100
50-100
0-50 50
0
C = 6%
NaOH
0,5
2
3,5
5
120
Time, h 6,5 80 100
680 Temp, oC
Figure4.Thesurfaceresponseplotsoftheeffectsoftemperatureandtimeontheglucoseyield.
Figure 4. The surface response plots of the effects of temperature and time on the glucose yield.
G , mg/g
y
300
250-300
200-250 250
150-200
200
100-150
150
50-100
100 9
0-50 CNaOH, %
50 5
Temp= 60oC
0 1
0,5 1,5 2,5 3,5 4,5 5,5 6,5 7,5
Time, h
(a)
Gy, mg/g 400
300-400 300
200-300
200
100-200
0-100
100
1

0 10
7
0,5
1,5
Temp= 100oC 2,5 3,5 4 CNaOH, %
4,5
5,5
6,5 1
Time, h 7,5

(b)
Figure 5. The surface response plots of the effects of NaOH concentration and time acid hydrolysis
Figure5.ThesurfaceresponseplotsoftheeffectsofNaOHconcentrationandtimeacidhydrolysison
on the glucose yield: (a) at a constant temperature 60 °C; (b) at a constant temperature 100 °C.
theglucoseyield:(a)ataconstanttemperature60◦C;(b)ataconstanttemperature100◦C.
Energies 2018,11,639 9of24
The analysis of the surface response areas (Figures 3–5) allowed to determine the optimal
conditions for a two-stage process involving alkaline pre-treatment (6% NaOH, 6 h, 65 ◦C) and
acidhydrolysis(100◦C,0.5hand85%H PO ). Fortheseconditions,hydrolysisofthetriticalestraw
3 4
wasperformedtoobtain,from1gofrawmaterial,320mgofglucose,42.8mgofxylose,19mgof
galactoseand1.5mgofarabinose,respectively. Asimilarresultoftheyieldofsugarswasobtained
for acid hydrolysis with sulfuric acid (70% H SO , 30 ◦C, 2 h) of Saccharum spontaneum, also after
2 4
treatmentwithsodiumbase(7%, 48h,30◦C).Fortheseconditions,amaximumyieldofreducing
sugarswasobtained369.6mg/g[14]. Theacidhydrolysiswascarriedoutonstrawwhichhadnot
beensubjectedtothepreviousalkalinepre-treatment,obtaining,from1gofrawmaterial,420mgof
glucose,150mgofxylose,38mgofgalactoseand4.7mgofarabinose,respectively. Itisclearthatthe
efficiencyofreducingsugarsforacidhydrolysiswiththeomissionofthealkalinepre-treatmentis
thehighest.
2.4. OptimisationofAlkalinePre-TreatmentFollowedbyEnzymaticHydrolysis
The system consists of 15 experiments planned according to the Box-Behnken design.
Experiments include two-stage hydrolysis of biomass, i.e., alkaline pre-treatment followed by
enzymatic hydrolysis. During the alkaline pre-treatment, NaOH concentration in the first step of
hydrolysis is optimized at a constant process temperature of 65 ◦C, pre-treatment time of 6h, and
granulation of 0.75 mm. In the second step, enzymatic hydrolysis values of the time and ratio of
cellulolyticenzymesimmobilizedonasolidsupportareoptimized. Astheboundaryconditions,on
thebasisofpreviousexperiments,NaOHconcentrationsof1%,6%,and11%wereassumed,andfor
enzymatichydrolysistime8,16,and24handthecontentofViscozymeLinamixtureof0.9,0.95,and
1wereselected. Box-Behnkendesignestimationallowedthedeterminationoftheresponsesurface
basedonthequadraticequation. Astheinitialparameterrepresentingtheefficiencyoftheprocess,the
massoftheobtainedglucosewasselected(glucoseyield,G ). Table5showstheobtainedvaluesofthe
y
glucoseandthesumofreducingsugars.
Table5.Box-Behnkendesignforalkalinepre-treatmentandenzymatichydrolysisofTriticumsp.
No. CNaOH(%) ViscozymeL Time(h) Gy(mg/gbiomass) SumofSugars(mg/gbiomass)
1 1 0.90 16 234.7 278.8
2 11 0.90 16 250.1 292.8
3 1 1.00 16 186.2 245.3
4 11 1.00 16 207.6 289.4
5 1 0.95 8 143.9 184.0
6 11 0.95 8 189.4 249.7
7 1 0.95 24 219.8 268.5
8 11 0.95 24 159.7 195.4
9 6 0.90 8 208.4 289.0
10 6 1.00 8 163.1 204.2
11 6 0.90 24 234.8 275.9
12 6 1.00 24 218.3 257.2
13 6 0.95 16 231.5 302.0
14 6 0.95 16 235.6 296.8
15 6 0.95 16 227.4 281.4
Theinfluenceoftheinputparameters,i.e.,theNaOHconcentration,theViscozymeLcontent,and
thedurationtimeoftheenzymatichydrolysisprocesshavebeenexaminedconsideringtheglucose
yieldasthemodeloutput. Theequationdescribingtherelationshipsoccurringduringthetwo-stage
hydrolysiswasdeterminedbymultipleregression. Theobtainedregressionequationdescribingthe
surfaceresponsetakestheform:
Y[%]=700.18−20.89X −763.82X −0,82X 2 −0.52X 2 −0.66X ·X +23.86X ·X (2)
1 4 1 5 1 5 4 5
Energies 2018,11,639 10of24
EneIrgnieEs 2q0u18a, t1i1o, nx F(O2)R XPE1E,RX R4,EaVnIEdWX 5representsodiumhydroxideconcentration,ViscozymeL10c oofn 2t5e nt,
andthetimeofenzymatichydrolysis,respectively. Ydenotesaglucoseyield. Equation(2)enables
thethcea clcaulclualtaiotinono foft htheet htheeoorreetticicaall gglluuccoossee yyiieelldd wwiitthhiinn tthhee rraanngge eoof fvvarairaibalbelse ascaccocrodrindgin tgo tboobuonudanrdya ry
cocnodnidtiiotinosn.s. InIn oorrddeerr ttoo oobbttaaiinn EEqquuaattiioonn ((22)),, tthhee lleeaasst tssigignnifiificacnatn tpapraarmameteertse rwsewree rreejreecjteecdt eidn itnhet he
consecutive iterative steps. It turned out that the least important parameters are: NaOH
consecutiveiterativesteps. Itturnedoutthattheleastimportantparametersare: NaOHconcentration,
concentration, duration of enzymatic hydrolysis, and the ratio of enzymes raised to the square.
durationofenzymatichydrolysis,andtheratioofenzymesraisedtothesquare.Equation2isdescribed
Equation 2 is described by the model parameters contained in Table 6. Based on the analysis of
bythemodelparameterscontainedinTable6. Basedontheanalysisofvariance,theobtainedvalueof
variance, the obtained value of the determination coefficient (R2 = 0.934) indicates a good correlation
thedeterminationcoefficient(R2=0.934)indicatesagoodcorrelationoftheresultscalculatedfromthe
of the results calculated from the equation with the experimental results of glucose efficiency
equationwiththeexperimentalresultsofglucoseefficiency(Figure6).
(Figure 6).
TaTbalebl6e. 6M. Modoedlelp apraarmameeteterrss ffoorr tthhee BBooxx--BBeehhnnkkeenn ddeessiiggnn ffoor raalklkaalilnine epprer-etr-teraetamtmenet natnadn edneznymzyamtica tic
hyhdyrdorlyolsyissios foTf rTitriictuicmums psp..
Parameter Value Std. Error t-Value p-Value
Parameter Value Std.Error t-Value p-Value
Intercept 700.177 75.480 9.276 1.48 × 10−5
IntercepXt1 70200.1.87877 753..448630 69.0.23716 0.010.04381×2 10−5
X1 X4 2−07.6838.7822 38.84.67737 −86..600341 2.58 ×0 .1000−05 312
X4 X12 −7−603..881292 880..727207 −−38.7.6250 4 0.020.55883×0 10−5
X 2 −0.819 0.220 −3.725 0.005830
1 X52 −0.523 0.084 −6.233 0.000250
X 2 −0.523 0.084 −6.233 0.000250
5 X1·X5 −0.656 0.132 −4.964 0.001102
X ·X −0.656 0.132 −4.964 0.001102
X14·XX554·X5 232.38.68464 22.9.97755 88.0.02222 4.2841.2 ×8 110×−5 10−5
270
250
230
Experimental R2= 0,934
210
G , mg/mL
y
190
170
150
150 200 250 300
Model-predicted G , mg/mL
y

Figure6.Analysisofvarianceforthetheoretical(model-predicted)andexperimentalglucoseyieldfor
Figure 6. Analysis of variance for the theoretical (model-predicted) and experimental glucose yield
alkalinepre-treatmentandenzymatichydrolysis.
for alkaline pre-treatment and enzymatic hydrolysis.
Theanalysisofreactionmixturesamplesshowsthatthemainproductoflignocellulosicbiomass
The analysis of reaction mixture samples shows that the main product of lignocellulosic
hybdioromlyassiss hisydmroaliynsliys gislu mcoasien.lyT agbluleco5sep. rTesaebnlet s5t hpereysieenldts otfheth iysiemldo noof stuhgisa rms oinnotshuegharysd rino lythsaet es.
Thheyodbrtoalyinseadtesr.e sTuhlet wobastacinoemdp raerseudltw witahs thcoemsupmareodf awllitrhe dtuhec insugmsu ogfa rasl.l Prhedyusiccianlgs tsuudgiaerss.h Pavheyssihcaolw n
lowstucodnietse nhtaovfec sehlloowbino lsoewin choyndternotl yosfa cteelsl,otbhieorseef oinre h,yitdwroalsycsrautecsia, lthtoeruesfeoraes, mit awllaasd cdruitcioianl otof βus-ge lau csomsaidlla se
inaadmdiitxitounr eowf βit-hglVuicsocsoizdyamsee inL .aC mellioxtbuiores ewisitahn Vinishciobziytomreo fLc. eClleullloolbyitoicsee nisz yanm ienshainbditoitrs opfr ecesellnucloellyotwic ers
theenezfyfimcieesn cayndof ietns zpyrmesaetnicceh yldowroelryss isth.eF iegfufirceie7ncpyr eosfe netnszeyxmematipcl ahryydcrhoalynsgise.s Finiggulrue co7s epereffsiecnietsn cy
forexeenmzypmlarayti cchhayndgreos liyns igsluccoonsdeu ecftfeicdiefnocry1 f6ohr einnztyhmearatinc gheydofrovlayrsiias bcloensdduecstcerdi bfoerd 1i6nhT ianb tlhee5 r.aBnagsee odf on
thevasrhiaabpleeso fdethscerirbeesdp oinn sTeasbuler f5a.c eBaitsecda nonb ethseta sthedapteh aotf ftohre trheespcohnasneg seuirnfaNcea Oit Hcanco bnec esntattreadti othna,ta folor cal
mathxeim chuamngaer oinu nNdaO9%H icsoonbcesenrtrvaetdio,na, bao vloecaaln mdabxeilmowumw ahriocuhntdh e9%ob itsa ionbesdergvleudc, oasbeoyviee ladnsda breellooww er.
Thwerheifcohr eth,e9 %obNtaainOeHd gcolunccoesnet ryaietilodns iasrec hloowseenr. aTshtehreefoopreti,m 9%al vNaalOueH. Fcoignucreent7raatlisoon sihs ocwhosstehna tast htehree is
aloinpetiamrarle lvaatiluone.s hFiigpubree t7w aelesno tshheowyise ltdhaotf tghleurceo isse aa lnindetahr ereralattiioonosfhβip-g bluectwoseiedna stheei nyitehled mofi xgtluurceoswe ith
and the ratio of β-glucosidase in the mixture with Viscozyme L during the enzymatic hydrolysis,
ViscozymeLduringtheenzymatichydrolysis,whereanincreaseintheratioofβ-glucosidase(orthe
where an increase in the ratio of β-glucosidase (or the decrease of Viscozyme L) corresponds with
decreaseofViscozymeL)correspondswithanincreaseintheyieldofglucoseafterenzymatichydrolysis.
an increase in the yield of glucose after enzymatic hydrolysis. The mentioned changes are small