Research Article Particulate Size of Microalgal Biomass...

Research ArticleParticulate Size of Microalgal Biomass AffectsHydrolysate Properties and Bioethanol Concentration

Razif Harun12 Michael K Danquah3 and Selvakumar Thiruvenkadam2

1 Department of Chemical Engineering Monash University Clayton VIC 3800 Australia2 Department of Chemical and Environmental Engineering Universiti Putra Malaysia 43400 Serdang Malaysia3 Department of Chemical and Petroleum Engineering Curtin University of Technology 98009 Sarawak Malaysia

Correspondence should be addressed to Razif Harun mh razifupmedumy

Received 28 February 2014 Revised 3 May 2014 Accepted 6 May 2014 Published 29 May 2014

Academic Editor Rajeev Kumar

Copyright copy 2014 Razif Harun et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Effective optimization of microalgae-to-bioethanol process systems hinges on an in-depth characterization of key processparameters relevant to the overall bioprocess engineering One of the such important variables is the biomass particle sizedistribution and the effects on saccharification levels and bioethanol titres This study examined the effects of three differentmicroalgal biomass particle size ranges 35 120583m le 119909 le 90 120583m 125120583m le 119909 le 180120583m and 295 120583m le 119909 le 425 120583m on the degree ofenzymatic hydrolysis and bioethanol production Two scenarios were investigated single enzyme hydrolysis (cellulase) and doubleenzyme hydrolysis (cellulase and cellobiase)The glucose yield from biomass in the smallest particle size range (35 120583mle 119909 le 90 120583m)was the highest 13473mg glucoseg algae while the yield from biomass in the larger particle size range (295 120583m le 119909 le 425 120583m)was 7545mg glucoseg algae A similar trend was observed for bioethanol yield with the highest yield of 047 g EtOHg glucoseobtained from biomass in the smallest particle size range The results have shown that the microalgal biomass particle size has asignificant effect on enzymatic hydrolysis and bioethanol yield

1 Introduction

Theutilization of microalgae to produce a variety of productssuch as fine organic chemicals food animal feed and foodsupplements have been discovered in the past [1ndash3] Currentinterest has been on the development of biofuels such asbioethanol from microalgae as a nonedible feedstock Asidefrom its renewable and sustainable benefits the high carbo-hydrate composition of microalgal biomass can be convertedto fermentable sugars for microbial conversion to bioethanol[4 5] One of such biomass saccharification methods is viaenzymatic hydrolysis

Enzymatic hydrolysis is a well-established process andprovides mild operating conditions high sugar yields highselectivity and minimal by-products formation [6 7] hencea more preferred method of hydrolyzing fermentation sub-strates However process conditions and parameters duringenzymatic hydrolysis require detailed optimization for max-imum product conversion One of the important parameters

that influence the effectiveness of enzymatic hydrolysis isbiomass particle size Fundamentally smaller particle sizebiomass presents a large specific surface area thus increasingthe contact areas between the enzymes and the interparticlebonding of the material during the hydrolysis process [8]

Previous attention has been focused on the effect ofparticle size on enzymatic hydrolysis of either cellulosic(such as cotton plant and fibers) or lignocellulosic biomass(such as corn sugarcane and wheat) Pedersen and Meyer[9] reported that smaller biomass particle size (53ndash149 120583m)increased glucose release up to 90 after 24 h hydrolysis ofwheat straw biomass The finding was in accordance withthose reported by Dasari and Eric Berson [10] and Carvalhoet al [11] who used sawdust and lemon respectively ashydrolysis substrates Biomass particulate size reduction alsoresults in enhancing the hydrolysis rate [10 12] This can beexplained by the easy access to enzyme active sites by smallerbiomass particles Contrary to this Ballesteros et al [13 14]have reported that larger particle size biomass significantly

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 435631 8 pageshttpdxdoiorg1011552014435631

2 BioMed Research International

increases hydrolysis rates and sugar recoveries (particularlyglucose) compared to smaller particle size biomass Theseconflicting views call for further studies on the characteristiceffects of biomass particle size on the degree and effectivenessof enzymatic hydrolysis To the best of our knowledge nosimilar work has been performed on the carbohydrates ofmicroalgae biomass and the concomitant effect on bioethanolyields Therefore this study aims to investigate the effect ofparticle size on enzymatic hydrolysis of microalgal biomassThe glucose yields and the physical properties of the substrateduring the hydrolysis process are examined and discussedAlso the kinetic investigation of enzyme hydrolysis and theeffects on glucose and bioethanol yields are presented

2 Materials and Methods

21 Substrate Preparation Culture samples of Chlorococcuminfusionum obtained from Bio-fuels Pty Ltd (Victoria Aus-tralia) were centrifuged (Heraeus Multifuge 3 S-R Germany)at 4500times g for 10mins and the supernatant was discardedThemicroalgal cakewas dried in a laboratory oven at 60∘C for24 h (Model 400 Memmert Germany) The dried biomasswas pulverized for 1min using a hammer mill (NV TemaGermany) The different particle sizes were separated bypassing the milled sample through a series of cascadedstainless steel sieves (until the desired biomass sizes werepartitioned in the following ranges 35 120583m le 119909 le 90 120583m125 120583m le 119909 le 180 120583m and 295 120583m le 119909 le 425 120583m)The samples were stored at room temperature before furtheranalysis

22 Enzyme Activity The enzymes used in this study werecellulase from Trichoderma reesei (ATCC 26921) and cellobi-ase from Aspergillus niger (Novozyme 188) purchased fromSigma Aldrich Australia The activity of cellulase measuredat 10 unitsmg solid means that one unit of cellulase liberates10 120583mol of glucose from cellulose in 1 h at pH 50 Thecellobiase activity was determined as 250 unitsmg

23 Enzymatic Hydrolysis Varying quantities of microalgalbiomass in powder form (02ndash10 g)within three different par-ticle size ranges 35 120583m le 119909 le 90 120583m 125 120583m le 119909 le 180 120583mand 295 120583m le 119909 le 425 120583m were loaded with a constantcellulase mass of 20mg and a cellobiase volume of 10mLThe samples were hydrolysed in shake flasks with 10mMof 100mL sodium acetate buffer at pH 48 and were placedin an incubator (LH Fermentation Ltd BuckinghamshireEngland) at 40∘C for 48 h with 200RPM agitation Sampleswere taken at 5 h intervals and the enzymatic hydrolysisprocess was halted by heating the hydrolysate to sim90∘C for10min The samples were then cooled to room temperatureand stored in a freezer at minus75∘C (Ultraflow freezer PlymouthUSA) for further analysis

24 Bioethanol Production Saccharomyces cerevisiae pur-chased from Lalvin Winequip Products Pty Ltd (VictoriaAustralia) was used in the microbial fermentation processfor bioethanol production The culture was prepared by

dissolving 50 g of dry yeast powder in 50mL sterile warmwater (sim40∘C) and the pH was adjusted to 7 by 1M NaOHaddition The yeast was cultured in YDP medium withcomposition in gL given as follows 10 g yeast extract 20 gpeptone and 20 g glucose The yeast was harvested after24 h and washed to eliminate the sugars then transferredinto 500mL Erlenmeyer flask containing 100mL of thesugar-containing liquidmediumobtained after the hydrolysisprocess The flasks were tightly sealed and nitrogen gas wasbubbled through to create an oxygen-free environment forbioethanol production The flasks were incubated at 30∘Cunder 200 RPM shaking The pH was maintained at 7 byadding 1M NaOH solution The fermentation continued for50 h and samples for analysis were taken after every 4 h

25 Chemical Analysis The biomass was pretreated usinga sonicator to break down the cell walls Phenol-sulphuricacid method was used to quantify the total carbohydratein the biomass Note that Table 1 is a presaccharificationdata presenting the existence of different carbohydrate formsentrapped in the microalgae system Microalgal biomass andthe hydrolysate compositions were analyzed by HPLC usinga 250mm times 46mm Prevail Carbohydrate ES Column TheHPLC system consists of the following accessory instru-ments a detector (ELSD Alltech 3300) quaternary gradientpump (Model 726 Alltech) degasser (Model 591500M Elitedegassing system Alltech) autosampler (Model 570 All-tech) and system controller (Model 726300M Alltech) Themobile phase was a mixture of acetonitrile and water (85 15)and the operating flow rate was 1mLmin 30 120583L sample wasinjected at 50∘C The sample was filtered through a 13mmmembrane filter prior to injection The sugar concentrationswere evaluated using a calibration curve generated fromHPLC-grade sugars

The ethanol concentration was analyzed using gas chro-matography (GC) (Model 7890A Agilent USA) The GCunit consists of an autosampler flame ion detector (FID)and HP-FFAP column (50m times 020mm times 033 120583m) Theinjector detector and oven temperatures were maintainedat 150∘C 200∘C and 120∘C respectively Nitrogen gaswas used as the carrier gas The bioethanol concentra-tion was quantified using a calibration curve preparedby injecting different concentrations of a standard ethanol(01ndash10 vv)

26 Fourier Transform Infrared Spectroscopy (FTIR) Thepolymorphs of the resulting hydrolysate from the hydrol-ysis process were determined by FTIR FTIR spectra ofhydrolysed samples were recorded on a Nicolet 6700 FTIR(Fischer Scientific Australia) equipped with Thermo Scien-tific iD3 ATR accessory (Fischer Scientific Australia) andthe spectra were run and processed with OMNIC software(Version 70 ThermoNicolet) The dried hydrolysis sampleswere loaded on the sample holder and the spectrum wasrecorded at an average of 32 scans with a spectral reso-lution of 4 cmminus1 from 400 to 4000 cmminus1 Sample spectrawere recorded as absorbance values at each data point intriplicates

BioMed Research International 3

Table 1 Biomass composition of the microalgal species

Component Composition ( ww)Total carbohydrate 3252

Xylose 954Mannose 487Glucose 1522Galactose 289

Starch 1132Otherslowast 5616lowastLipids protein and ash

27 Viscosity Measurement The hydrolysate viscosities weredetermined using a modular advanced rheometer system(HaakeMarsThermo Electron Corp Germany)The systemis equipped with a stainless steel measuring plate (MP 66060mm) and a rotor (PP60H 60mm) The temperature wasset to 30∘C the frequency was maintained at 15Hz andthe gap between the parallel plates was kept at 1mm Thehydrolysed samples were measured for 5min at differentshear rates ranging from 50 to 500 sminus1

3 Results and Discussion

31 Substrate Carbohydrate Composition According toTable 1 carbohydrate constitutes up to 32 of the dryweight of C infusionum biomass with the major fermentablesugar component being glucose (152) followed byxylose (95) mannose (49) and galactose (29) Thisstrain also contains starch at 113 dry weight The totalcarbohydrates present in the biomass could bemade availablefor bioethanol production under optimal saccharificationand microbial fermentation conditions The remainingbiomass composition could represent lipids protein andash that is available in microalgal strain Unlike both redand brown algae the cell wall of most green algae has highcellulose content ranging up to 70 of the dry weight[15 16] The composition of the carbohydrate content inthe unicellular microalgal specie per unit mass does notvary greatly among fractions of different particle sizeFor intact microalgae cells the carbohydrates are welldistributed within the cell membrane and this gives auniform carbohydrate composition in the membrane

32 FTIR Analysis The spectra of hydrolyzed biomasswith different particle sizes were examined using FTIRtechniques and the results are shown in Figure 1 Twotypes of hydrolysates were compared in this study singleenzyme hydrolysate with only cellulase and double enzymehydrolysate with both cellulase and cellobiase These twoscenarios are denoted by Case 1 and Case 2 respectivelyThe FTIR spectra represent samples taken at the end of thehydrolysis processThe spectrum of nonpretreated powderedmicroalgae within the size range of 295 120583m le 119909 le 425120583m was analyzed for comparison According to Murdockand Wetzel [17] the reference absorption peaks for major

01000200030004000

(a)

(b)

(c)(d)(e)(f)(g)

Wavenumber (cmminus1)

Figure 1 FTIR spectra formicroalgal biomasswith different particlesizes under different Cases (a) Nonpretreated microalgal biomass(original powdered sample) Case 1 (cellulase only) (b) 35 120583m le 119909 le90 120583m (d) 125 120583mle 119909 le 180120583m and (f) 295 120583mle 119909 le 425 120583mCase2 (cellulase + cellobiase) (c) 35 120583m le 119909 le 90120583m (e) 125120583m le 119909 le180 120583m and (g) 295120583m le 119909 le 425 120583m

microalgal compositions are sim1100ndash900 cmminus1 for polysaccha-rides (cellulose and starch) sim2970ndash2850 cmminus1 for lipids and1750ndash1500 cmminus1 for proteins and carboxylic groups Since wewish to convert complex carbohydrates in the biomass toproduce fermentable sugars for bioethanol production onlypolysaccharide peaks are of interest The microalgal biomassused in this study showed a relatively high amount of polysac-charides since a strong absorption peak was recorded around1100 cmminus1 to 1000 cmminus1 in the powdered microalgal sampleas summarized in Figure 1 It was observed that the degree ofpolysaccharides absorption decreased as the biomass particlesize decreasedThis indicates that more polysaccharides wereconverted to fermentable sugars in the case of biomass withsmaller particle size during the hydrolysis process Based onthe individual spectrum sugar conversions were calculatedby referring to the peak heights of nonpretreated samplesThe hydrolysis of cellulose with the addition of cellobiase(Case 2) generated hydrolysis conversion of 90 78 and64 of the biomass in the particle size ranges 35120583m le119909 le 90 120583m 125 120583m le 119909 le 180 120583m and 295 120583m le119909 le 425 120583m respectively A lower degree of hydrolysiswas observed without cellobiase addition (Case 1) of 41 29and 18 for biomass in the particle size ranges 35 120583m le119909 le 90 120583m 125 120583m le 119909 le 180 120583m and 295 120583m le 119909 le425 120583m respectively Cellulase contains cellobiohydrolasesendoglucanases and120573-glucosidase that function to efficientlyhydrolyse cellulose The hydrolysis of cellulose to cellobioseis the rate-limiting step and this limitation is resolved by cel-lobiohydrolases which hydrolyse cellulose to cellobiose andcellotriose However the small amount of 120573-glucosidase incellulase hinders the cellulolysis process hence the additionof 120573-glucosidase helps cellulase to hydrolyse the intermediateproduct cellobiose to form glucose in a faster reaction timewhile minimizing product inhibition during the cellulolyticprocess [18ndash25] Furthermore the kinetics of molecularactivation drawdown is faster in the double enzyme case


and this favors forward production of fermentable subunitsduring the hydrolysis process The total crystallinity index(TCI) of the hydrolyzed biomass was calculated as reportedby Nelson and OrsquoConnor [19] From the calculations the TCIof all the hydrolysed samples decreased when compared withthe nonhydrolysed biomass Decreasing biomass crystallinityhas been reported to increase enzymatic hydrolysis rates[26] Although the polysaccharides were degraded duringhydrolysis FTIR spectra analysis showed that the structureof the hydrolysed monomers remained intact for bioethanolproduction

33 Glucose Yield Table 2 shows the yield of glucose fordifferent assays The rate of glucose release was rapid atthe beginning of hydrolysis and slowed down until theend of the hydrolysis process This profile is typical ofbatch hydrolysis [9] Note that the enzymes involved inthe study are not hydrolysing starch composition thusnot accounted for potential glucose for the fermenta-tion process It was found that biomass with smallerparticle size generated higher glucose yields and thisobservation was the same for both Case 1 and Case 2The highest glucose yields were 7545mgg biomass and13473mgg biomass for Cases 1 and 2 respectively forbiomass in the smallest particle size range of 35 120583m le119909 le 90 120583mThe lowest glucose yieldswere 2601mgg biomassand 6155mgg biomass for Cases 1 and 2 respectively forbiomass in the largest particle size range of 295 120583m le 119909 le425 120583m Smaller biomass particle size increases the inter-actions with the enzymes during hydrolysis due to thepresence of a large exposed surface area [12] Hence smallermicroalgal biomass particle size is required to achieve higherglucose yield The amount of microalgal biomass loadedin the hydrolysis process also showed a significant effecton glucose yields Although the same microalgal biomassparticle size was used in assay numbers 1 2 and 3 differentglucose yields of 11108mgg biomass 12577mgg biomassand 13473mgg biomasswere achievedWhen examining theeffect of different substrate concentrations on glucose yieldwithin the same particle size range in Case 2 it was foundthat the glucose yield increased with increasing substrateconcentrationThis trendwas however not observed in Case 1containing cellulose enzymeTherefore high yield of glucosefrom increasing substrate concentration is dependent on thebalanced composition of cellulosic enzyme components tominimize product inhibition [27] Furthermore a significantincrease in glucose yield was observed when the secondenzyme (cellobiase) was introduced to the assays (Case 2)The glucose yields inCase 2were almost the double comparedto those obtained in Case 1 From the collision theoryperspective the kinetics of molecular activation drawdownis faster in the double enzyme case and this favors forwardproduction of fermentable subunits The kinetics of thisdouble enzyme effect is demonstrated with the scheme below

Mechanism of enzymatic hydrolysis of cellulase Case 1

119878 + 1198641198961

lArrrArr119896minus1

SE1198962

997888rarr 119875 + 119864 (1)

where 119878 is the substrate concentration 119864 is the enzymeconcentration SE is the concentration of substrate-enzymecomplex 119875 is the product concentration and 119896

1 119896minus1 1198962are

rate constantsThe rates of change in SE concentration and product

formation are

119889SE119889119905= 1198961119878 times 119864 minus 119896

minus1SE minus 119896

2SE (2)

119889119875

119889119905= 1198962SE (3)

For substrate mass balance the substrate concentration (119878) iswritten as

119878 = 119878119900minus SE minus 119875 (4)

Substituting (4) into (2) gives

119889SE119889119905= 1198961(1198780minus SE minus 119875) times 119864 minus 119896


2SE (5)

Applying equilibrium and steady state conditions to (5) gives

SE =(1198780minus 119875) 119864

119870119890+ 119864 (6)

where119870119890is the equilibrium constant

119870119890=119896minus1+ 1198962

1198961

(7)

The simplified equation (6)may bewritten as follows at initialconditions

(119889119875

119889119905)

1198750

=119896211987801198640

119870119890+ 1198640

(8)

where (119889119875119889119905)1198750

denotes the product formation at the initialconditions

The mechanism of the enzymatic hydrolysis of cellulase(1198641) and cellobiase (119864

2) Case 2 is

119878119862+ 1198641

1198961


1198781198621198641

1198962

997888rarr 119878CB + 1198641

119878CB + 1198642119896

1015840

1

lArrrArr1198961015840

minus1

119878CB1198642119896

1015840

2

997888rarr 119875 + 1198642

(9)

where 119878119862 119878CB and 119875 are concentrations of cellulose cel-

lobiose and glucose respectivelyBy following the same procedure as in Case 1 simplified

equation for Case 2 at the initial conditions is

(119889119875

119889119905)

1198750

=1198961015840

2

1198642(1198781198620

minus 11987811986211986410

minus 1198781198621198610

)

1198701015840119890

+ 11986420

(10)

1198701015840

119890

=1198961015840

2

+ 1198961015840

minus1

11989610158401

(11)


Table 2 Yield of glucose released after 48 h of hydrolysis

Assay number Particle size 120583m Algae loading gL mg glucoseg algal biomassCellulase (Case 1) Cellulase + cellobiase (Case 2)

1 35 le 119909 le 90 25 5421 111082 35 le 119909 le 90 50 4448 125773 35 le 119909 le 90 100 7545 134734 125 le 119909 le 180 25 3024 68795 125 le 119909 le 180 50 2796 85886 125 le 119909 le 180 100 2763 114547 295 le 119909 le 425 25 2601 68798 295 le 119909 le 425 50 2643 92619 295 le 119909 le 425 100 3024 10229

where 11989610158401

1198961015840minus1

and 11989610158402

are rate constants and1198701015840119890

is the equilib-rium constant

Equations (8) and (10) can be rewritten as followsFor Case 1

1

V=119870119890

1198810

[1

119878119900

] +1

1198810

(12)

where

1198810= 1198962119864 (13)

For Case 2

1

V=1198701015840

119890

11988110158400

[1

119878CB] +1

11988110158400

(14)

where

1198811015840

0

= 1198961015840

2

(119864119879minus 1198781198621198641) (15)

From the mathematical derivation 119870119898

which is the equi-librium constant is 119870

119890for Case 1 and 1198701015840

119890

for Case 2 Also119881max which is the maximum forward velocity is 1119881

0for

Case 1 and 111988110158400

for Case 2 where 1198810and 1198811015840

0

occur at theirrespective initial enzyme concentration As can be seen inTable 3 Lineweaver-Burk plot analysis of (12) and (14) showsthat the119870

119898value for Case 1 is higher thanCase 2 and the119881max

value for Case 1 is lower than Case 2 The lower value of 119870119898

and the higher value of 119881max obtained from Case 2 confirmthat the introduction of cellobiase significantly increases thecombined enzyme-substrate affinity and the hydrolysis rate

34 Ethanol Yield The produced hydrolysates were usedas substrates in Saccharomyces cerevisiae fermentations forbioethanol production This yeast strain has widely beenutilized for bioethanol production because it is easy toculture and has a high ethanol tolerance This could allowfermentation to continue under 16-17 vv ethanol con-centrations [28] Figure 2 shows the bioethanol yields forboth Cases 1 and 2 using biomass with different particlesizes The trend in bioethanol yield for the different particlesize biomass was in agreement with the glucose yields

Table 3 119870119898

and 119881max for hydrolysis of cellulose by cellulase (Case1) and cellulase + cellobiase (Case 2)

Case number Enzyme Hydrolysis of cellulose119870119898

119881max

1 Cellulase 1881 35052 Cellulase + cellobiase 1823 13583

biomass with smaller particle size displayed higher glucoseconcentrations to generate higher bioethanol yields It canbe observed that available glucose in the hydrolysate wascompletely consumed after 48 h of fermentationThe highestbioethanol yield of 047 g ethanolg glucose was obtainedwhen hydrolysed under Case 2 with the smallest particlesize biomass (35 120583m le 119909 le 90120583m) at 100 gL microalgaeconcentration whereas the lowest bioethanol yield of 005 gethanolg glucose was obtained when hydrolysed under Case1 with the largest particle size biomass (295120583mle 119909 le 425 120583m)at 25 gL microalgae concentration Assays in Case 2 pro-duced up to 50 more bioethanol yields than the assays inCase 1 reaching a maximum bioethanol yield of 047 ggglucose compared to 019 gg glucose as represented byassay number 3 in both cases Hydrolysate produced in thepresence of cellobiase generated higher bioethanol yields dueto the presence of high glucose concentrations

35 Viscosity Analysis The purpose of the viscosity study isto understand the influence of the rheological properties ofthe hydrolysate during hydrolysis and how this affects thefermentation process for bioethanol production The viscos-ity measurements were performed under different shear rates(50ndash500 sminus1) using hydrolysates obtained from biomass withdifferent particle sizes for an equivalent substrate concentra-tion of 100 gL Figure 3 shows the viscosity data of the differ-ent particle size biomass for both Cases 1 and 2 with samplestaken after the hydrolysis process A decreasing trend ofviscositywas observedwith increasing shear rate and biomasswith smaller particle sizes displaying higher viscositiesFor biomass in the same particle size range Case 2 showed


0

01

02

03

04

05

0 10 20 30 40 50Time (h)

25 gLlowast25 gL^

50gLlowast50gL^

100 gLlowast100 gL^

Etha

nol y

ield

(g Et

OH

g gl

ucos

e)

(a)

Time (h)

0

01

02

03

04

0 10 20 30 40 50

25 gLlowast25 gL^

50gLlowast50gL^

100 gLlowast100 gL^

Etha

nol y

ield

(g Et

OH

g gl

ucos

e)

(b)

Time (h)

0

01

02

03

04

0 10 20 30 40 50

25 gLlowast25 gL^

50gLlowast50gL^

100 gLlowast100 gL^

Etha

nol y

ield

(g Et

OH

g gl

ucos

e)

(c)

Figure 2 Yield of bioethanol after 48 h fermentation of microalgal biomass with different particle sizes for both cases (a) 35 120583m le 119909 le 90 120583m(b) 125120583m le 119909 le 180 120583m and (c) 295 120583m le 119909 le 425 120583m ( lowastCase 1 cellulase andCase 2 cellulase + cellobiase)

900950

100010501100115012001250130013501400

0 100 200 300 400 500

Visc

osity

(mPamiddots)

35 lt x lt 90lowast

125 lt x lt 180lowast


295 lt x lt 425^

125 lt x lt 180^

35 lt x lt 90^

Shear rate (sminus1)

Figure 3 Viscosity versus shear rate for the different particle sizebiomass A decreasing trend in viscosity was observed with increas-ing shear rate ( lowastCase 1 cellulase andCase 2 cellulase + cellobiasesubstrate concentration 100 gL)

a slightly higher viscosity than Case 1 The effective enzyme-substrate interactions associated with smaller particle sizebiomass result in a more viscous hydrolysate than largesize particles as more water molecules are consumed perunit volume exceeding the reduction of total solids concen-tration [29]

We also studied the viscosity profile of the hydrolysatesover the time course of hydrolysis and the data is presentedin Figure 4 The hydrolysate viscosities for both Cases 1 and2 reduced with hydrolysis time with a significant decreasewhich was observed at the initial stage of hydrolysis between4 and 24 h This is probably due to the faster initial kinet-ics structural changes andor the release of intercalatingmolecules in the cell wall Decreasing viscosity during hydrol-ysis is caused by cellulose degradation as the structure andsolid concentration change during the cellulolytic activitycaused by the enzymes [10]

The profiles of viscosity and bioethanol production weresuperimposed to understand their relationship during theenzymatic hydrolysis process as shown in Figure 5 It can beseen that bioethanol yield increases with lower viscosities


90010001100120013001400150016001700

0 10 20 30 40 50Hydrolysis time (h)

Case 1Case 2

Visc

osity

(mPamiddots)

Figure 4Hydrolysate viscosity profiles during hydrolysis for single-enzyme (Case 1 cellulase) and double-enzyme (Case 2 cellulase+ cellobiase) conditions The viscosity of the hydrolysates in bothcases decreased with hydrolysis timeThe data presented is for assaynumber 1 in both cases (substrate concentration 25 gL)

This trend also matches glucose yields as higher releasedglucose produces higher bioethanol yields The results showthat less viscous slurry is required to produce high glucoseyields under effective mixing

4 Conclusion

This paper is the premier study on the effect of particlesize of microalgal biomass on enzymatic hydrolysis andbioethanol production The results show that the highestglucose and bioethanol yields were obtained using biomasswith smaller particle size (35120583m le 119909 le 90 120583m) at asubstrate concentration of 100 gL The addition of the sec-ond enzyme cellobiase increases the glucose yield thusincreasing the bioethanol yield This was confirmed by akinetic investigation of the double enzyme process using therapid equilibriummodelThe viscosity of the hydrolysate alsoinfluences glucose yield Lower viscosities result in higherglucose yields Overall microalgal biomass particle size hasa significant effect on enzymatic hydrolysis and bioethanolproduction

Abbreviations

119878 Substrate concentration gL119864 Enzyme concentration gLSE Concentration of

substrate-enzyme complex119875 Product concentration gL1198961 119896minus1 1198962 11989610158401

1198961015840minus1

and 11989610158402

Rate constants gL119870119890 1198701015840119890

Equilibrium constant gL(119889119875119889119905)

1198750

Product formation at theinitial conditions

1198641 Enzyme I (cellulase)

concentration gL1198642 Enzyme II (cellobiase)

concentration gL

0

01

02

03

04

05

400

800

1200

1600

2000

4 8 24 48Time (h)

ViscosityEthanol yield

Visc

osity

(mPamiddots)

Etha

nol y

ield

(g E

tOH

lg g

luco

se)

Figure 5 Relationship between hydrolysate viscosity andbioethanol yield Bioethanol yield increases with lower hydrolysateviscosities The data presented is for assay number 1 of Case 2(substrate concentration 25 gL)

119878119862 Cellulose concentration gL119878CB Cellobiose concentration gL1198781198621198641 Concentration of cellulose-cellulase complex

119878CB1198642 Concentration of cellobiose-cellobiase complex119870119898 Michaelis constant gL119881max Maximum rate of hydrolysis gLsdotmin

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work has been supported by the Department of Chem-ical Engineering Monash University Australia and theMinistry of Higher Education Malaysia

References

[1] R Harun M Singh G M Forde andM K Danquah ldquoBiopro-cess engineering ofmicroalgae to produce a variety of consumerproductsrdquo Renewable and Sustainable Energy Reviews vol 14no 3 pp 1037ndash1047 2010

[2] V KDhargalkar andXNVerlecar ldquoSouthernOcean seaweedsa resource for exploration in food and drugsrdquo Aquaculture vol287 no 3-4 pp 229ndash242 2009

[3] J L Fortman S Chhabra A Mukhopadhyay et al ldquoBiofuelalternatives to ethanol pumping the microbial wellrdquo Trends inBiotechnology vol 26 no 7 pp 375ndash381 2008

[4] S P ChoiM T Nguyen and S J Sim ldquoEnzymatic pretreatmentofChlamydomonas reinhardtii biomass for ethanol productionrdquoBioresource Technology vol 101 no 14 pp 5330ndash5336 2010


[5] R Harun M K Danquah and G M Forde ldquoMicroalgal bio-mass as a fermentation feedstock for bioethanol productionrdquoJournal of Chemical Technology and Biotechnology vol 85 no2 pp 199ndash203 2010

[6] Y Zheng Z Pan R Zhang and B M Jenkins ldquoKineticmodeling for enzymatic hydrolysis of pretreated creeping wildryegrassrdquo Biotechnology and Bioengineering vol 102 no 6 pp1558ndash1569 2009

[7] Y Sun and J J Cheng ldquoDilute acid pretreatment of rye straw andbermudagrass for ethanol productionrdquo Bioresource Technologyvol 96 no 14 pp 1599ndash1606 2005

[8] L T Fan Y H Lee and D H Beardmore ldquoMechanism of theenzymatic hydrolysis of cellulose effects of major structuralfeatures of cellulose on enzymatic hydrolysisrdquo Biotechnologyand Bioengineering vol 22 pp 177ndash199 1980

[9] M Pedersen and A S Meyer ldquoInfluence of substrate particlesize and wet oxidation on physical surface structures andenzymatic hydrolysis of wheat strawrdquo Biotechnology Progressvol 25 no 2 pp 399ndash408 2009

[10] R K Dasari and R Eric Berson ldquoThe effect of particle size onhydrolysis reaction rates and rheological properties in cellulosicslurriesrdquo Applied Biochemistry and Biotechnology vol 137ndash140no 1ndash12 pp 289ndash299 2007

[11] L M J Carvalho R Borchetta and E M M da Silva ldquoEffect ofenzymatic hydrolysis on particle size reduction in lemon juice(Citrus limon L)rdquo Brazilian Journal of Food Technology vol 4pp 277ndash282 2006

[12] A-I Yeh Y-C Huang and S H Chen ldquoEffect of particle sizeon the rate of enzymatic hydrolysis of celluloserdquo CarbohydratePolymers vol 79 no 1 pp 192ndash199 2010

[13] I Ballesteros J M Oliva A A Navarro A Gonzalez JCarrasco and M Ballesteros ldquoEffect of chip size on steamexplosion pretreatment of softwoodrdquo Applied Biochemistry andBiotechnology A Enzyme Engineering and Biotechnology vol84ndash86 pp 97ndash110 2000

[14] I Ballesteros J M Oliva M J Negro P Manzanares and MBallesteros ldquoEnzymic hydrolysis of steam exploded herbaceousagricultural waste (Brassica carinata) at different particulesizesrdquo Process Biochemistry vol 38 no 2 pp 187ndash192 2002

[15] B Baldan P Andolfo L Navazio C Tolomio and P MarianildquoCellulose in algal cell wall an ldquoin siturdquo localizationrdquo EuropeanJournal of Histochemistry vol 45 no 1 pp 51ndash56 2001

[16] K Sander and G S Murthy ldquoEnzymatic degradation of micro-algal cell wallsrdquo in Proceedings of the American Society of Agri-cultural and Biological Engineers Annual International Meetingpp 2489ndash2500 Reno Nev USA June 2009

[17] J N Murdock and D L Wetzel ldquoFT-IR microspectroscopyenhances biological and ecological analysis of algaerdquo AppliedSpectroscopy Reviews vol 44 no 4 pp 335ndash361 2009

[18] D S Domozych M Ciancia J U Fangel M D MikkelsenP Ulvskov and W G Willats ldquoThe cell walls of green algaea journey through evolution and diversityrdquo Frontiers in PlantScience vol 3 article 82 2012

[19] M L Nelson and R T OrsquoConnor ldquoRelation of certain infraredbands to cellulose crystallinity and crystal latticed type Part ISpectra of lattice types I II III and of amorphous celluloserdquoJournal of Applied Polymer Science vol 8 pp 1311ndash1324 1964

[20] R Sposina Sobral Teixeira A SantrsquoAna da Silva H-W Kimet al ldquoUse of cellobiohydrolase-free cellulase blends for the

hydrolysis of microcrystalline cellulose and sugarcane bagassepretreated by either ball milling or ionic liquid [Emim][Ac]rdquoBioresource Technology vol 149 pp 551ndash555 2013

[21] C Divne J Stahlberg T Reinikainen et al ldquoThe three-dimen-sional crystal structure of the catalytic core of cellobiohydrolaseI from Trichoderma reeseirdquo Science vol 265 no 5171 pp 524ndash528 1994

[22] Y-S Liu J O Baker Y Zeng M E Himmel T Haas and S-Y Ding ldquoCellobiohydrolase hydrolyzes crystalline cellulose onhydrophobic facesrdquo Journal of Biological Chemistry vol 286 no13 pp 11195ndash11201 2011

[23] J Jalak and P Valjamae ldquoMechanism of initial rapid rate retar-dation in cellobiohydrolase catalyzed cellulose hydrolysisrdquoBiotechnology and Bioengineering vol 106 no 6 pp 871ndash8832010

[24] S Al-Zuhair ldquoThe effect of crystallinity of cellulose on therate of reducing sugars production by heterogeneous enzymatichydrolysisrdquo Bioresource Technology vol 99 no 10 pp 4078ndash4085 2008

[25] M Chauve H Mathis D Huc D Casanave F Monot andN L Ferreira ldquoComparative kinetic analysis of two fungal 120573-glucosidasesrdquo Biotechnology for Biofuels vol 3 article 3 2010

[26] L Liu andHChen ldquoEnzymatic hydrolysis of cellulosematerialstreated with ionic liquid [BMIM]Clrdquo Chinese Science Bulletinvol 51 no 20 pp 2432ndash2436 2006

[27] Q Gan S J Allen and G Taylor ldquoKinetic dynamics in het-erogeneous enzymatic hydrolysis of cellulose an overviewan experimental study and mathematical modellingrdquo ProcessBiochemistry vol 38 no 7 pp 1003ndash1018 2003

[28] G P Casey and W M Ingledew ldquoEthanol tolerance in yeastsrdquoCritical Reviews inMicrobiology vol 13 no 3 pp 219ndash280 1986

[29] KW Dunaway R K Dasari N G Bennett and R Eric BersonldquoCharacterization of changes in viscosity and insoluble solidscontent during enzymatic saccharification of pretreated cornstover slurriesrdquoBioresource Technology vol 101 no 10 pp 3575ndash3582 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


increases hydrolysis rates and sugar recoveries (particularlyglucose) compared to smaller particle size biomass Theseconflicting views call for further studies on the characteristiceffects of biomass particle size on the degree and effectivenessof enzymatic hydrolysis To the best of our knowledge nosimilar work has been performed on the carbohydrates ofmicroalgae biomass and the concomitant effect on bioethanolyields Therefore this study aims to investigate the effect ofparticle size on enzymatic hydrolysis of microalgal biomassThe glucose yields and the physical properties of the substrateduring the hydrolysis process are examined and discussedAlso the kinetic investigation of enzyme hydrolysis and theeffects on glucose and bioethanol yields are presented

2 Materials and Methods

21 Substrate Preparation Culture samples of Chlorococcuminfusionum obtained from Bio-fuels Pty Ltd (Victoria Aus-tralia) were centrifuged (Heraeus Multifuge 3 S-R Germany)at 4500times g for 10mins and the supernatant was discardedThemicroalgal cakewas dried in a laboratory oven at 60∘C for24 h (Model 400 Memmert Germany) The dried biomasswas pulverized for 1min using a hammer mill (NV TemaGermany) The different particle sizes were separated bypassing the milled sample through a series of cascadedstainless steel sieves (until the desired biomass sizes werepartitioned in the following ranges 35 120583m le 119909 le 90 120583m125 120583m le 119909 le 180 120583m and 295 120583m le 119909 le 425 120583m)The samples were stored at room temperature before furtheranalysis

22 Enzyme Activity The enzymes used in this study werecellulase from Trichoderma reesei (ATCC 26921) and cellobi-ase from Aspergillus niger (Novozyme 188) purchased fromSigma Aldrich Australia The activity of cellulase measuredat 10 unitsmg solid means that one unit of cellulase liberates10 120583mol of glucose from cellulose in 1 h at pH 50 Thecellobiase activity was determined as 250 unitsmg

23 Enzymatic Hydrolysis Varying quantities of microalgalbiomass in powder form (02ndash10 g)within three different par-ticle size ranges 35 120583m le 119909 le 90 120583m 125 120583m le 119909 le 180 120583mand 295 120583m le 119909 le 425 120583m were loaded with a constantcellulase mass of 20mg and a cellobiase volume of 10mLThe samples were hydrolysed in shake flasks with 10mMof 100mL sodium acetate buffer at pH 48 and were placedin an incubator (LH Fermentation Ltd BuckinghamshireEngland) at 40∘C for 48 h with 200RPM agitation Sampleswere taken at 5 h intervals and the enzymatic hydrolysisprocess was halted by heating the hydrolysate to sim90∘C for10min The samples were then cooled to room temperatureand stored in a freezer at minus75∘C (Ultraflow freezer PlymouthUSA) for further analysis

24 Bioethanol Production Saccharomyces cerevisiae pur-chased from Lalvin Winequip Products Pty Ltd (VictoriaAustralia) was used in the microbial fermentation processfor bioethanol production The culture was prepared by

dissolving 50 g of dry yeast powder in 50mL sterile warmwater (sim40∘C) and the pH was adjusted to 7 by 1M NaOHaddition The yeast was cultured in YDP medium withcomposition in gL given as follows 10 g yeast extract 20 gpeptone and 20 g glucose The yeast was harvested after24 h and washed to eliminate the sugars then transferredinto 500mL Erlenmeyer flask containing 100mL of thesugar-containing liquidmediumobtained after the hydrolysisprocess The flasks were tightly sealed and nitrogen gas wasbubbled through to create an oxygen-free environment forbioethanol production The flasks were incubated at 30∘Cunder 200 RPM shaking The pH was maintained at 7 byadding 1M NaOH solution The fermentation continued for50 h and samples for analysis were taken after every 4 h

25 Chemical Analysis The biomass was pretreated usinga sonicator to break down the cell walls Phenol-sulphuricacid method was used to quantify the total carbohydratein the biomass Note that Table 1 is a presaccharificationdata presenting the existence of different carbohydrate formsentrapped in the microalgae system Microalgal biomass andthe hydrolysate compositions were analyzed by HPLC usinga 250mm times 46mm Prevail Carbohydrate ES Column TheHPLC system consists of the following accessory instru-ments a detector (ELSD Alltech 3300) quaternary gradientpump (Model 726 Alltech) degasser (Model 591500M Elitedegassing system Alltech) autosampler (Model 570 All-tech) and system controller (Model 726300M Alltech) Themobile phase was a mixture of acetonitrile and water (85 15)and the operating flow rate was 1mLmin 30 120583L sample wasinjected at 50∘C The sample was filtered through a 13mmmembrane filter prior to injection The sugar concentrationswere evaluated using a calibration curve generated fromHPLC-grade sugars

The ethanol concentration was analyzed using gas chro-matography (GC) (Model 7890A Agilent USA) The GCunit consists of an autosampler flame ion detector (FID)and HP-FFAP column (50m times 020mm times 033 120583m) Theinjector detector and oven temperatures were maintainedat 150∘C 200∘C and 120∘C respectively Nitrogen gaswas used as the carrier gas The bioethanol concentra-tion was quantified using a calibration curve preparedby injecting different concentrations of a standard ethanol(01ndash10 vv)

26 Fourier Transform Infrared Spectroscopy (FTIR) Thepolymorphs of the resulting hydrolysate from the hydrol-ysis process were determined by FTIR FTIR spectra ofhydrolysed samples were recorded on a Nicolet 6700 FTIR(Fischer Scientific Australia) equipped with Thermo Scien-tific iD3 ATR accessory (Fischer Scientific Australia) andthe spectra were run and processed with OMNIC software(Version 70 ThermoNicolet) The dried hydrolysis sampleswere loaded on the sample holder and the spectrum wasrecorded at an average of 32 scans with a spectral reso-lution of 4 cmminus1 from 400 to 4000 cmminus1 Sample spectrawere recorded as absorbance values at each data point intriplicates










01000200030004000

(a)

(b)

(c)(d)(e)(f)(g)








119878 + 1198641198961


SE1198962

997888rarr 119875 + 119864 (1)


1 119896minus1 1198962are


formation are

119889SE119889119905= 1198961119878 times 119864 minus 119896


2SE (2)

119889119875

119889119905= 1198962SE (3)


119878 = 119878119900minus SE minus 119875 (4)




2SE (5)


SE =(1198780minus 119875) 119864

119870119890+ 119864 (6)


119870119890=119896minus1+ 1198962

1198961

(7)


(119889119875

119889119905)

1198750

=119896211987801198640

119870119890+ 1198640

(8)

where (119889119875119889119905)1198750



2) Case 2 is

119878119862+ 1198641

1198961


1198781198621198641

1198962

997888rarr 119878CB + 1198641

119878CB + 1198642119896

1015840

1

lArrrArr1198961015840

minus1

119878CB1198642119896

1015840

2

997888rarr 119875 + 1198642

(9)




(119889119875

119889119905)

1198750

=1198961015840

2

1198642(1198781198620

minus 11987811986211986410

minus 1198781198621198610

)

1198701015840119890

+ 11986420

(10)

1198701015840

119890

=1198961015840

2

+ 1198961015840

minus1

11989610158401

(11)





where 11989610158401

1198961015840minus1

and 11989610158402




1

V=119870119890

1198810

[1

119878119900

] +1

1198810

(12)

where

1198810= 1198962119864 (13)

For Case 2

1

V=1198701015840

119890

11988110158400

[1

119878CB] +1

11988110158400

(14)

where

1198811015840

0

= 1198961015840

2

(119864119879minus 1198781198621198641) (15)



119890for Case 1 and 1198701015840

119890


0for

Case 1 and 111988110158400


0






Table 3 119870119898



119881max





0

01

02

03

04

05

0 10 20 30 40 50Time (h)

25 gLlowast25 gL^

50gLlowast50gL^

100 gLlowast100 gL^

Etha

nol y

ield

(g Et

OH

g gl

ucos

e)

(a)

Time (h)

0

01

02

03

04

0 10 20 30 40 50

25 gLlowast25 gL^

50gLlowast50gL^

100 gLlowast100 gL^

Etha

nol y

ield

(g Et

OH

g gl

ucos

e)

(b)

Time (h)

0

01

02

03

04

0 10 20 30 40 50

25 gLlowast25 gL^

50gLlowast50gL^

100 gLlowast100 gL^

Etha

nol y

ield

(g Et

OH

g gl

ucos

e)

(c)


900950

100010501100115012001250130013501400

0 100 200 300 400 500

Visc

osity

(mPamiddots)

35 lt x lt 90lowast



295 lt x lt 425^

125 lt x lt 180^

35 lt x lt 90^







90010001100120013001400150016001700


Case 1Case 2

Visc

osity

(mPamiddots)



4 Conclusion


Abbreviations



1198961015840minus1

and 11989610158402



1198750




concentration gL

0

01

02

03

04

05

400

800

1200

1600

2000

4 8 24 48Time (h)


Visc

osity

(mPamiddots)

Etha

nol y

ield

(g E

tOH

lg g

luco

se)






Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










01000200030004000

(a)

(b)

(c)(d)(e)(f)(g)








119878 + 1198641198961


SE1198962

997888rarr 119875 + 119864 (1)


1 119896minus1 1198962are


formation are

119889SE119889119905= 1198961119878 times 119864 minus 119896


2SE (2)

119889119875

119889119905= 1198962SE (3)


119878 = 119878119900minus SE minus 119875 (4)




2SE (5)


SE =(1198780minus 119875) 119864

119870119890+ 119864 (6)


119870119890=119896minus1+ 1198962

1198961

(7)


(119889119875

119889119905)

1198750

=119896211987801198640

119870119890+ 1198640

(8)

where (119889119875119889119905)1198750



2) Case 2 is

119878119862+ 1198641

1198961


1198781198621198641

1198962

997888rarr 119878CB + 1198641

119878CB + 1198642119896

1015840

1

lArrrArr1198961015840

minus1

119878CB1198642119896

1015840

2

997888rarr 119875 + 1198642

(9)




(119889119875

119889119905)

1198750

=1198961015840

2

1198642(1198781198620

minus 11987811986211986410

minus 1198781198621198610

)

1198701015840119890

+ 11986420

(10)

1198701015840

119890

=1198961015840

2

+ 1198961015840

minus1

11989610158401

(11)





where 11989610158401

1198961015840minus1

and 11989610158402




1

V=119870119890

1198810

[1

119878119900

] +1

1198810

(12)

where

1198810= 1198962119864 (13)

For Case 2

1

V=1198701015840

119890

11988110158400

[1

119878CB] +1

11988110158400

(14)

where

1198811015840

0

= 1198961015840

2

(119864119879minus 1198781198621198641) (15)



119890for Case 1 and 1198701015840

119890


0for

Case 1 and 111988110158400


0






Table 3 119870119898



119881max





0

01

02

03

04

05

0 10 20 30 40 50Time (h)

25 gLlowast25 gL^

50gLlowast50gL^

100 gLlowast100 gL^

Etha

nol y

ield

(g Et

OH

g gl

ucos

e)

(a)

Time (h)

0

01

02

03

04

0 10 20 30 40 50

25 gLlowast25 gL^

50gLlowast50gL^

100 gLlowast100 gL^

Etha

nol y

ield

(g Et

OH

g gl

ucos

e)

(b)

Time (h)

0

01

02

03

04

0 10 20 30 40 50

25 gLlowast25 gL^

50gLlowast50gL^

100 gLlowast100 gL^

Etha

nol y

ield

(g Et

OH

g gl

ucos

e)

(c)


900950

100010501100115012001250130013501400

0 100 200 300 400 500

Visc

osity

(mPamiddots)

35 lt x lt 90lowast



295 lt x lt 425^

125 lt x lt 180^

35 lt x lt 90^







90010001100120013001400150016001700


Case 1Case 2

Visc

osity

(mPamiddots)



4 Conclusion


Abbreviations



1198961015840minus1

and 11989610158402



1198750




concentration gL

0

01

02

03

04

05

400

800

1200

1600

2000

4 8 24 48Time (h)


Visc

osity

(mPamiddots)

Etha

nol y

ield

(g E

tOH

lg g

luco

se)






Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





119878 + 1198641198961


SE1198962

997888rarr 119875 + 119864 (1)


1 119896minus1 1198962are


formation are

119889SE119889119905= 1198961119878 times 119864 minus 119896


2SE (2)

119889119875

119889119905= 1198962SE (3)


119878 = 119878119900minus SE minus 119875 (4)




2SE (5)


SE =(1198780minus 119875) 119864

119870119890+ 119864 (6)


119870119890=119896minus1+ 1198962

1198961

(7)


(119889119875

119889119905)

1198750

=119896211987801198640

119870119890+ 1198640

(8)

where (119889119875119889119905)1198750



2) Case 2 is

119878119862+ 1198641

1198961


1198781198621198641

1198962

997888rarr 119878CB + 1198641

119878CB + 1198642119896

1015840

1

lArrrArr1198961015840

minus1

119878CB1198642119896

1015840

2

997888rarr 119875 + 1198642

(9)




(119889119875

119889119905)

1198750

=1198961015840

2

1198642(1198781198620

minus 11987811986211986410

minus 1198781198621198610

)

1198701015840119890

+ 11986420

(10)

1198701015840

119890

=1198961015840

2

+ 1198961015840

minus1

11989610158401

(11)





where 11989610158401

1198961015840minus1

and 11989610158402




1

V=119870119890

1198810

[1

119878119900

] +1

1198810

(12)

where

1198810= 1198962119864 (13)

For Case 2

1

V=1198701015840

119890

11988110158400

[1

119878CB] +1

11988110158400

(14)

where

1198811015840

0

= 1198961015840

2

(119864119879minus 1198781198621198641) (15)



119890for Case 1 and 1198701015840

119890


0for

Case 1 and 111988110158400


0






Table 3 119870119898



119881max





0

01

02

03

04

05

0 10 20 30 40 50Time (h)

25 gLlowast25 gL^

50gLlowast50gL^

100 gLlowast100 gL^

Etha

nol y

ield

(g Et

OH

g gl

ucos

e)

(a)

Time (h)

0

01

02

03

04

0 10 20 30 40 50

25 gLlowast25 gL^

50gLlowast50gL^

100 gLlowast100 gL^

Etha

nol y

ield

(g Et

OH

g gl

ucos

e)

(b)

Time (h)

0

01

02

03

04

0 10 20 30 40 50

25 gLlowast25 gL^

50gLlowast50gL^

100 gLlowast100 gL^

Etha

nol y

ield

(g Et

OH

g gl

ucos

e)

(c)


900950

100010501100115012001250130013501400

0 100 200 300 400 500

Visc

osity

(mPamiddots)

35 lt x lt 90lowast



295 lt x lt 425^

125 lt x lt 180^

35 lt x lt 90^







90010001100120013001400150016001700


Case 1Case 2

Visc

osity

(mPamiddots)



4 Conclusion


Abbreviations



1198961015840minus1

and 11989610158402



1198750




concentration gL

0

01

02

03

04

05

400

800

1200

1600

2000

4 8 24 48Time (h)


Visc

osity

(mPamiddots)

Etha

nol y

ield

(g E

tOH

lg g

luco

se)






Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





where 11989610158401

1198961015840minus1

and 11989610158402




1

V=119870119890

1198810

[1

119878119900

] +1

1198810

(12)

where

1198810= 1198962119864 (13)

For Case 2

1

V=1198701015840

119890

11988110158400

[1

119878CB] +1

11988110158400

(14)

where

1198811015840

0

= 1198961015840

2

(119864119879minus 1198781198621198641) (15)



119890for Case 1 and 1198701015840

119890


0for

Case 1 and 111988110158400


0






Table 3 119870119898



119881max





0

01

02

03

04

05

0 10 20 30 40 50Time (h)

25 gLlowast25 gL^

50gLlowast50gL^

100 gLlowast100 gL^

Etha

nol y

ield

(g Et

OH

g gl

ucos

e)

(a)

Time (h)

0

01

02

03

04

0 10 20 30 40 50

25 gLlowast25 gL^

50gLlowast50gL^

100 gLlowast100 gL^

Etha

nol y

ield

(g Et

OH

g gl

ucos

e)

(b)

Time (h)

0

01

02

03

04

0 10 20 30 40 50

25 gLlowast25 gL^

50gLlowast50gL^

100 gLlowast100 gL^

Etha

nol y

ield

(g Et

OH

g gl

ucos

e)

(c)


900950

100010501100115012001250130013501400

0 100 200 300 400 500

Visc

osity

(mPamiddots)

35 lt x lt 90lowast



295 lt x lt 425^

125 lt x lt 180^

35 lt x lt 90^







90010001100120013001400150016001700


Case 1Case 2

Visc

osity

(mPamiddots)



4 Conclusion


Abbreviations



1198961015840minus1

and 11989610158402



1198750




concentration gL

0

01

02

03

04

05

400

800

1200

1600

2000

4 8 24 48Time (h)


Visc

osity

(mPamiddots)

Etha

nol y

ield

(g E

tOH

lg g

luco

se)






Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

01

02

03

04

05

0 10 20 30 40 50Time (h)

25 gLlowast25 gL^

50gLlowast50gL^

100 gLlowast100 gL^

Etha

nol y

ield

(g Et

OH

g gl

ucos

e)

(a)

Time (h)

0

01

02

03

04

0 10 20 30 40 50

25 gLlowast25 gL^

50gLlowast50gL^

100 gLlowast100 gL^

Etha

nol y

ield

(g Et

OH

g gl

ucos

e)

(b)

Time (h)

0

01

02

03

04

0 10 20 30 40 50

25 gLlowast25 gL^

50gLlowast50gL^

100 gLlowast100 gL^

Etha

nol y

ield

(g Et

OH

g gl

ucos

e)

(c)


900950

100010501100115012001250130013501400

0 100 200 300 400 500

Visc

osity

(mPamiddots)

35 lt x lt 90lowast



295 lt x lt 425^

125 lt x lt 180^

35 lt x lt 90^







90010001100120013001400150016001700


Case 1Case 2

Visc

osity

(mPamiddots)



4 Conclusion


Abbreviations



1198961015840minus1

and 11989610158402



1198750




concentration gL

0

01

02

03

04

05

400

800

1200

1600

2000

4 8 24 48Time (h)


Visc

osity

(mPamiddots)

Etha

nol y

ield

(g E

tOH

lg g

luco

se)






Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


90010001100120013001400150016001700


Case 1Case 2

Visc

osity

(mPamiddots)



4 Conclusion


Abbreviations



1198961015840minus1

and 11989610158402



1198750




concentration gL

0

01

02

03

04

05

400

800

1200

1600

2000

4 8 24 48Time (h)


Visc

osity

(mPamiddots)

Etha

nol y

ield

(g E

tOH

lg g

luco

se)






Acknowledgments


References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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