,. 4

10

CONFIDE NTIALAuthors : C . E . Rix October 23, 1968

C . N . EatonJ . G . Jones Notebook Pages : 151501-151550,

158951-159000, 162501-162550,163201-163250, 163301-163350,163601-163650, 164301-164314,

Division : Chemical Rese arch 167901-167950, 168201-168222,4175551-175553 /6 sr 90/-/ a,7A s

RDR, 1968, No . 37 Dated : February 8, 1967 toOctober 9, 1967

No . of Pages : 19 Previous Reports : None

XYLOSE PRODUCTION FROM CORN HULLS~TND SUGAR L~AND 13~GASSE

OBJECT :

This report is concerned with optimizing the production of xylose-rich syrup from corn hulls, a by-product of the corn wet milling industry,and to a lesser extent with xylose production from sugar cane bagasse .The xylose was to be used as a carbon source in the preparation of luq coseisomerase, an enzyme which converts glucose to fructose .

SUMMARY :

In order to prepare xylose, which is used as a carbon source inlucose isomerase production, destarched corn hulls and sugar cane bagasse

were ydro yzl ed with dilute sulfuric acid at temperatures from 100-150° C .,with reaction times of 1 .0 to 4 .0 hr . The reaction mixtures were filtered,the residue washed with water and the combined filtrates neutralized topH 4 .5 with calcium hydroxide, filtered, decolorized with charcoal anddeionized by passing through Amberlite IR-120 and Duolite A6 ion exchangers .The deionized solutions were then concentrated under reduced pressure .Corn hulls gave a 45% yield of a dry sugar syrup which contained approximately30% arabinose, 52% xylose, 7% galactose, and 7% glucose for an overallxylose yield of 23% . Sugar cane bagasse yielded 16% dry sugar syrupcontaining 6% arabinose, 81% xylose and 9% glucose'for an overall xyloseyield of 13% .

However, in cell growth and lg ucose isomerase production, the xylosesamples, even after crystallization, had only 50-75% of the activity ofthe xylose standard . Due to the low activity and the development of analkaline isomerization process for converting glucose to fructose thisproject was terminated .

M5

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TABLE OF CONTENTSPage

OBJECT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . 3

LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . 3

A . INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . 4

B . EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . . . . . 4

I . Destarchi ng of Corn Hul l s . . . . . . . . . . . . . . 4

II . Acid Hydrolysis of Destarching Liquor . . . . . . . . 5

III . Hydrolysis of Corn Hulls Using AutoclaveConditions . . . . . . . . . . . . . . . . . . . . . 6

IV . Pilot Plant Hydrolysis of Corn Hulls . . . . . . . . . 6

V . Atmospheric Hydrolysis of Sugar Cane Bagasse . . . . 6

VI . Atmospheric Hydrolysis of Corn Hulls . . . . . . . . 10

VII . Neutralization Conditions for Corn HullHydrolyzate . . . . . . . . . . . . . . . . . . . . . 10

VIII . Optimizing Sugar Solution Decolorizationwith Charcoal . . . . . . . . . . . . . . . . . . . . 10

IX . Amberlite IR-120 Ion Exchange Capacity . . . . . . . 13

X . Xylose Crystallization . . . . . . . . . . . . . . . 14

XI . Decomposition of Pure Xylose . . . . . . . . . . . . 14

C . DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . 15

D . CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . 17

E . RECOMMENDATIONS . . . . . . . . . . . . . . . . . . . . . . 17

I . Future Work . . . . . . . . . . . . . . . . . . . . . . 17

II . Patentability . . . . . . . . . . . . . . . . . . . . . 17

BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . 19

3

LIST OF TABLE S

Table No. Title Page

I Destarching of Water-washed ,Air-dried Corn Hulls . . . . . . . . . . . . 4

II Destarching of Wet, Sulfur Dioxid eContaining Corn Hulls . . . . . . . . . . . 5

III Autoclave Hydrolysis of Destarche dCorn Hulls with H2SO4 . . . . . . . . . . . 7

IV Pilot Plant Hydrolysis of Corn Hulls . . . . 9

V Atmospheric Hydrolyses : Corn Hull sand Bagasse . . . . . . . . . . . . . . . . 9

VI Corn Hull Hydrolyzate Neutralizatio nwith Calcium Hydroxide . . . . . . . . . . . 10

VII Determining Color Absorbed per Uni tWeight of Carbon (X/M)Co forNuchar C-190-N . . . . . . . . . . . . . . . 1 1

VIII Charcoal Efficiency in Suga rSolution Decolorization . . . . . . . . . . 13

IX Amberlite IR-120 Depletion withCalcium Hydroxide Neutralized CornHull Hydrolyzate . . . . . . . . . . . . . . 13

X Crystallization of Bagasse XyloseSyrups . . . . . . . . . . . . . . . . . . . 1 4

XI Decomposition of Pure Xylose . . . . . . . . 14

LIST OF FIGURE S

Figure No. Title Page

1 Determination of (X/M)Co forNuchar C-190-N . . . . . . . . . . . . . . . 12

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A . INTRODUCTION

It had been found that xylose was the most desirable carbon sourcefor the production of lug cose isomerase, an enzyme that converts glucoseto fructose . Over the years, many agricultural by-products have servedas sources of x lose, some of which were : corn cobs (1-6), corn stalks (7),oat hulls (6, 8~, cottonseed-hull bran (6, 9, 10), and sugar cane bagasse(6, 9-11) . A promising material in this investigation appeared to be cornhulls, a by-product of the corn wet-milling industry and readily availablefrom Penick & Ford . Analysis indicated that destarched corn hulls contained38-41% pentosans . These pentosans are present in the hemicellulosic portionof the hull and are in the form of a L-arabino-D-xyloglycan . The moleculeis primarily a xylan backbone with short side branches containing arabinose,galactose, and D-glucuronic acid in a terminal position . Another xylosesource, sugar cane bagasse, is relatively inexpensive but the xylan contentis only about 20% . However, this was 80-90% xylose and purification wasgreatly enhanced .

B . EXPERIMENTAL

I . Destarching of Corn Hulls

In a typical reaction, 100 g . of dried corn hulls (4 .4% H20, 8 .37%starch, 38 .47% pentosan) was mixed with 2 .0 liters of water and heated atreflux for 3 .0 hr ., filtered, washed with hot water, a nd air dried .Analysis :and Table

weight loss 19 .9%, starch 0 .98%, pentosan 40 .57% . Table III give destarching conditions and results .

TABLE I

DESTARCHING OF WATER-WASHED,AIR-D IED COWNHULLSy

ReactionTemperature Time

°C. Hr. Wt . Loss, %% StarchRemaining

% PentosanRemaining

Products ofHydrolysisd

UntreatedHulls - - - 8 .4 38 .5

1 100 1 .0 20 .8 1 .1 38 .82 100 2 .0 17 .6 0 .7 39 .33 100 3 .0 22 .9 0 .0 45 .34b 100 2 .0 19 .3 1 .3 52 .35 80-90 0 .5 8 .7 2 .1 39 .3 5% Arabinosee

6 100 3 .0 18 .6 1 .3 39 .621% Glucose

7 100 3 .0 19 .9 1 .0 40 .68c 25 1 .0 11 .8 7 .5 39 .0

Liquid :hulls = 20 :1Ground to 20 mesh2 .0 liters of 0 .25N H2SO4 :100 g . dry hullsIdentifiable products in syrup from hydrolysis of destarching liquorDestarching liquor from 100 g . hulls yields 10 .5 g . of syrup on hydrolysis

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TABLE II

DESTARCHING OF WET, SULFUR DIOXIDECONTAIN VG CORN HUL SB- ~

ReactionTemperature

°C .TimeHr . % Wt . Loss

% StarchRemaining

% PentosanRemaining

Products otHydrolysis

UntreatedHulls - - - 14 .9 31 .2

PilotPlant 1210 2 .0 - 1 .0 39 .8

PilotPlant 135° 1 .0 - 0 .1 33 .3 19% Xylose

PilotPlant 1000 2 .0 0 .5 38 .3

34% Arabinose

17% Maltose

lc 80-90° 0 .5 19 .8 2 .4 38 .636% Glucose7% Arabinose

2 100° 1 .0 10 .0 1 .0 37 .36% Glucose

3 100° 2 .0 12 .5 0 .8 39 .34 100° 3 .0 24 .1 1 .0 39 :75 100° 4 .0 52 .4 1 .0 38 .76d 1000 2 .0 23 .3 1 .6 38 .17d 100° 1 .0 20 .6 2 .7 37 .88d 100° 1 .0 20 .3 2 .9 37 .59 100° 1 .0 21 .6 2 .2 39 .5l0e 25° 1 .0 10 .1 12 .1 36 .0lle 50-60° 1 .0 9 .6 11 .0 37 .4

Liquid : hull ratio = 20 :1Identifiable products in syrup from hydrolysis of destarching liquorOn hydrolysis of destarching liquor from 100 g . of hulls 12 .5 g . ofsyrup obtainedGround to 20 mesh2 .0 liters of 0 .25N H2SO4 :lOO g . dry hulls

abc

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II . Acid Hydrolysis of Destarching Liquor

Sulfur dioxide treated corn hulls were destarched, and an aliquottaken of the destarching liquor . The solution was acidified to pH 2 .0with con . H2SO4, refluxed for 6 .0 hr ., neutralized to pH 5 .0 with Ca(OH)2,filtered, deionized with cationic Amberlite IR-120 and anionic DuoliteA-6 ion exchange resins and concentrated under reduced pressure . A 12 .5%yield of brown syrup was obtained which contained 36% glucose and 17%maltose . The results of other destarching liquor hydrolyses are presentedin Table I and Table II .

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III . Hydrolysis of Corn Hulls Using Autoclave Conditions

Destarched corn hulls (112 .8 g . wet, 100 g . dry basis) were placedin a 4-1 . Erlenmeyer flask and acidified with 2000 ml . of 0 .12N H2SO4(pH 1 .35) . The flask was placed in an autoclave set at 20 psi, 128° C .,heated for 20 min . to reach equilibrium, and then heated for an additional1 .0 hr . The mixture was filtered, and the residue slurried with 500 ml .of water, filtered, and the filtrates combined . The combined hydrolyzatesolution was neutralized to pH 4 .5 with calcium hydroxide, boiled for15 min . and filtered hot . The solution was then passed through a 400 ml .granular charcoal column, 500 ml . of Amberlite IR-120, 500 ml . of DuoliteA-6 and the final pH was adjusted to 5 .0-5 .5 with small amounts of IR-120 .The solution was concentrated under reduced pressure at 60° C . to yield52 .3 g . of dry syrup containing 33-34% arabinose, 54% xylose, and 9-10%glucose and galactose . The total yield of xylose (based on weight of dryhulls) was 28 .2% . Various reaction conditions and yields are presentedin Table III .

IV . Pilot Plant Hydrolysis of Corn Hulls

In the pilot plant studies a horizontal, rotating, stainless steelreactor coated internally with polyvinyl chloride was used . The cylindricalreactor was steam jacketed and possessed internal baffles . The steampressure and internal temperature could be measured directly . The reactorwas charged with 2 .27 kg . (5 .0 lb .) of dry destarched corn hulls and27 .3 kg . of 0 .1 N H2S04 and was heated to 148° C . (50 psi . steam pressure)and maintained at this temperature for an additional 0 .5 hr . The reactionmixture was centrifuged, and the filter cake washed with 6 .0 liters ofwater, yielding 29 .8 kg . of hydrolyzate and 1 .15 kg . of dry residue (7%pentosan remaining) . A 1 .32 kg . aliquot of the hydrolyzate was neutralizedto pH 4 .5 with calcium hydroxide, boiled 0 .25 hr . and filtered hot . Thefiltrate was passed through 400 ml . of granular charcoal, 500 ml . ofAmberlite IR-120, 500 ml . of Duolite A-6, and the final pH was adjustedto ti5 .0 with small amounts of Amberlite IR-120 . On evaporation at 60° C .and reduced pressure, 39 .4 g . of dry syrup was obtained . Analysis showed26% arabinose, 55% xylose, 7% galactose and 8% glucose for a total yieldof xylose based on dry hulls of 21 .5% . Various reaction conditions andyields are presented in Table IV .

V . Atmospheric Hydrolysis of Sugar Cane Bagasse

Sugar cane bagasse (460 g . wet, 200 g . dry basis) was placed in a5 liter round bottomed flask containing 2000 g . of 3 .75% H2SO4 and heatedat reflux with stirring for 3 .0 hr . The purification technique was asabove, yielding 33 .1 g . of dry syrup containing 6% arabinose, 81% xylose,2% galactose, and 7% glucose . The total yield of xylose, based on dryweight of bagasse,was 13 .2% . Various reaction conditions and yields arepresented in Table V .

TABLE III

AUTOCLAVE HYDROLYSIS OF DESTARCHED CORN HULLS WITH H2SO4

ExperimentNo .

LiauidHulls

AcidConc . N Time/Hr .

% DryResidue

% DrySyrup

%Arabinose

%Xylose

% Glucoseand Galactose

Total %Xylose

163302 20 :1 0 .07 2 .0 45 23 .5b 18 60 12 14 .1163303 " 0 .12 2 .0 41 15 .7b 17 50 11 7 .9163304 " 0 .03 3 .0 46 32 .9b 20 50 11 16 .4163305 " 0 .07 3 .0 42 39 .Ob 18 48 13 18 .6163306 " 0 .12 3 .0 43 23 .0b 23 64 16 14 .6163307 " 0 .03 4 .0 53 19 .0b 19 52 13 9 .8163308 " 0 .07 4 .0 45 47 .7b 21 59 17 28 .4163309 " 0 .12 4 .0 42 .1 22 57 17 24 .0163311a " 0 .07 3 .0 42 32 .3 20 40 8 12 .9163312a " 0 .12 3 .0 44 31 .6 18 42 10 13 .3163314a " 0 .03 4 .0 51 23 .4 19 25 6 5 .9163315a " 0 .07 4 .0 45 36 .8 19 40 9 14 .7163316a " 0 .12 4 .0 53 30 .6 19 47 9 14 .4163317 " 0 .07 4 .0 47 26 .6 14 62 22 16 .5163320 " 0 .07 4 .0 40 32 .0 18 49 14 16 .0163323 " 0 .20 2 .0 41 43 .4 23 60 15 26 .0163324 " 0 .30 2 .0 42 30.8 20 51 16 15 .8163325 " 0 .40 2 .0 40 20 .9 21 56 18 11 .7163326 " 0 .20 1 .0 33 39 .6 31 54 13 21 .5163327 " 0 .30 1 .0 42 32 .6 29 52 13 17 .0163328 " 0 .40 1 .0 48 46.5 27 49 14 22 .0163329 3 :1 0 .20 2 .0 37 15 .0 26 46 7 6 .9163330 3 :1 0 .30 2 .0 37 6 .5 25 44 12 2 .8163331 3 :1 0 .40 2 .0 50 50 .0 25 44 8 2 .2163334 20 :1 0 .20 1 .5 33 30 .4 27 47 11 14 .4163335 " 0 .30 1 .5 34 45 .7 29 51 12 23 .3163336 " 0 .40 1 .5 29 47 .7 31 54 14 25 .8163337 " 0 .12 1 .0 34 43 .4 30 55 10 23 .8163338 " 0 .16 1 .0 27 22 .8 32 59 13 13 .4163339 " 0 .25 1 .0 28 23 .4 30 54 9 12 .7163340 " 0 .20 2 .0 37 45 .9 27-31 47-54 8-13 21 .6-24 .8

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TABLE III Cont'd

AUTOCLAVE HYDROLYSIS OF DESTARCHED CORN HULLS WITH H2SO4

ExperimentNo .

LiquidI-1 ,j 11 S

AcidConc . ,f~ Ti,melNr,

% DryRasi,duo

% Dry$ ru n

%Arahi,nogo

%Xylose

% GlucoseGalactose

Total %X ~y,ose

163341 20 :1 0 .20 2 .0 30 51 .4 29 57 14 28 .6163342 m 0 .20 2 .0 34 36 .7 29 55 13 20 .0163345 m 0 .12 1 .0 33 52 .4 33-34 54 9-10 28 .2163346 0 .20 1 .0 37 43 .7 31-33 50-53 11-12 22 .0163347 0 .30 1 .0 32 39 .8 34 55 12 22 .0167901 0 .20 2 .0 37 44 .2 34 57 13 25 .0167902 0 .20 2 .0 32 43 .2 32 56 14 24 .1167903 0 .20 2 .0 37 43 .8 32 52 13 22 .8167908 3 :1 0 .20 4 .0 38 38.8 30 52 14 20 .6167909 3 :1 0 .40 4 .0 38 38 .4 30 51 16 19 .6167910 20 :1 0 .20 4.0 32 46 .0 31 51 15 23 .4167911 20 :1 0 .40 4.0 33 46.5 29 46 17 21 .4

aH3P04 used

bwet

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TABLE IV

PILOT PLANT HYDROLYSIS OF CORN HULLS

ExperimentN uM lber

Liquid :Hul l s

H2SO4Conc . N T i:^e/Hr . Temn •r °C .

%Residue

% DrySyrup

%Arahi-se

%Xylose

% Glucoseand Galactose

Total %Xylose

163343 10 :1 0 .40 1 .5 128 40 35 .7 35 47 8 16 .8167904 10 :1 0 .20 1 .33 142 35 39 .4 31 47 13 18 .5167906 10 :1 0 .20 1 .5 147 32 36 .6 29 42 13 15 .4167919 3 :1 0 .10 0 .5 126 61 17 .3 45 35 0 6 .1167938 12 :1 0 .10 0 .5 148 49 39 .1 26 55 15 21 .5167921 3 :1 0 .30 0 .5 148 42 20 .0 29 51 8 10 .2167945 12 :1 0 .30 0 .5 126 34 35 .9 30 46 21 16 .5167924 3 :1 0 .30 3 .0 148 39 15 .5 26 35 14 5 .4167925 12 .1 0 .30 3 .0 126 28 35 .6 29 48 8 17 .1167926 3 :1 0 .30 3 .0 126 38 34 .9 26 48 7 16 .7167927 12 :1 0 .30 3 .0 148 36 22 .6 24 28 15 6 .4167929 3 :1 0 .10 3 .0 148 34 9 .1 29 46 14 4 .2167930 12 :1 0 .10 3 .0 126 42 37 .6 30 52 14 19 .6167932 7 .5 :1 0 .20 1 .75 138 39 33 .5 27 48 14 16 .0167933 7 .5 :1 0 .20 1 .75 138 36 32 .6 27 48 12 15 .6

TABLE V

ATMOSPHERIC HYDROLYSES : CORN HULLS AND BAGASSE

H2SO4 % % Dry % % % Glucose Total %Material Liquid :Hulls Conc . % Time/Hr . Residue Syrup Arabinose Xylose and Galactose X ly oseHulls 10 :1 4.2 3 .25 38 19 .4 30 50 12 9 .7Hulls 7 .5 :1 5 .6 2 .00 36 45 .2 26 51 11 23 .1Bagasse 10 :1 3 .75 3 .00 78 16 .6 6 81 9 13 .2Bagasse 12 :1 4 .0 3 .00 74 11 .8 2 90 4 10 .6Bagasse 16.7 :1 4 .4 0 .83 77 12 .3 3 87 4 10 .7

9hi6 96009

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VI . Atmospheric Hydrolysis of Corn Hulls

In a 5 liter round-bottomed flask were placed 453 .5 g . of wet cornhulls (11 .8% H20, 400 g . dry basis) and 3000 g . of 5 .6% H2SO4 . After 0 .5hr . the mixture had reached 100° C . and was then refluxed with stirringfor 2 .0 hr . The purification technique was as above, yielding 181 .2 g .of dry syrup containing 26% arabinose, 51% xylose, 6% galactose, and 5%glucose . The total yield of xylose, based on dry weight of hulls, was23 .1% . Various reaction conditions and yields are given in Table V .

VII . Neutralization Conditions for Corn Hull Hydrolyzate

The hydrolyzate had an initial pH of 1 .22 . The transmittance (T)equaled 75 .5% at a wavelength (x) of 520 mu . The theoretical sulfateconcentration was 0 .79% . Calcium hydroxide was added to 100 ml . aliquots,the pH change measured, the solution boiled 15 minutes and filtered hot .The resulting pH, transmittance, [Ca+2], and [SO4-2] were measured andit was found that a pH range of 3 .5 to 4 .5 optimized clarity and sulfateion removal without undue buildup of calcium . The results are recordedin Table VI .

TABLE VI

CORN HULL HYDROLYZATE NEUTRALIZATIONWITH CALCIUM HYDROXIDE

pHWt . Ca(OH)2

Added% T

a=520 mu [Ca+2] M . [SO4-2] M .1 .23 0 .0 75 0 .000 0 .0822 .95 0 .555 g . 61 0 .021 0 .0284 .15 0 .618 72 0 .025 0 .0254 .92 0 .669 64 0 .027 0 .0295 .90 0 .687 56 0 .031 0 .022

VIII . Optimizing Sugar Solution Decolorization with Charcoal

A neutralized (pH 4 .5) corn hull hydrolyzate with initial color(Co) = 0 .95 at 420 mu as measured by a Bauch and Lomb Spectronic 20was used in all experiments . The pulverized carbon to be tested wasadded in increasingly larger dosages (M) to five 100-ml . portions ofsugar solution, stirred for 1 .0 hr . and filtered twice . The residualcolor (C) was measured, and the color absorbed (X = Co -C) was calculated .The color absorbed per unit weight of carbon (X/M) was also calculated andplotted versus residual color (C) on log-log paper . If a vertical lineis erected from the point on the horizontal scale corresponding to theinfluent concentration (Co), and the isotherm is extrapolated to intersectthat line, the (X/M) value at the point of intersection can be read fromthe vertical scale . This value, termed (X/M)co, represents the amountof impurity absorbed per unit weight of carbon when that carbon is in

11

equilibrium with the influent concentration . It represents the ultimatecapacity of the carbon and is used as the standard of comparison . Typicaldata for (X/M)Co calculations are recorded in Table VII and Figure 1 .The comparison of charcoal decolorizing efficiencies shown in Table VIIIindicates that Nuchar C-190-N is the most effective decolorizing agentboth at 23° C . and 50° C .

TABLE VII

DETERMINING COLOR ABSORBED PER UNIT WEIGHTOF CARBON X/M)Co FOR NUCHAR C-190-N

Wt . Carbon (MInitialColor C)

ResidualColor C

Color AbsorbedCO-C = X

Color Absorbedper Unit Wt .

of Carbon (X/M)0 .95

.1 .55 .40 4 .00

.2 .34 .61 3 .05

.3 .21 .74 2 .47

.4 .14 .81 2 .02

.5 .07 .88 1 .76

(X/M)co = 5 .1

12

FIGURE 1

DETERMINATION OF (X/M)CO FOR NUCHAR C-190-N

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x

(D RESIDUAL SOLUTION COLOR (C)

11

13

TABLE VIII

CHARCOAL EFFICIENCY IN SUGAR SOLUTION DECOLORIZATION

Carbon Sample (X/M)co 23° C . (X/M)co 500 C .Duo S-51 4 .3 5 .7Nuchar CE:E-N 3 .5 4 .1

" C-190-N 5 .1 6 .0" C-115-N 3 .83 4 .30" Aqua A 3 .0 2 .46" CE:E A 4 .9 -

Pittsburg CAL pulverized 3 .88 3 .90" CPG pulverized 3 .10 3 .20" SGL pulverized 3 .0 2 .05

IX . Amberlite IR-120 Ion Exchange Capacity

A 500 ml . column of wet Amberlite IR-120 was regenerated with 600 ml .of 3N HC1 and exhausted with a CaSO4 solution from a neutralized corn hullhydrolyzate . The results in Table IX indicate effective ion removal froma 0 .035 M . Ca+2 solution whose volume was sixteen times greater than thewet resin bed .

TABLE IX

AMBERLITE IR-120 DEPLETIONa WITH CALCIUMHYD I EIITWIZED COlt~}TULT~RI'DT'btYZA'f E b

VolumeCollected 0 ml . 500 3300 8000 9000 11,500 13,000

pHEffluent 2 .0 2 .0 2 .0 2 .2 2 .3 4 .3 4 .4[Cr]mg ./1 . 0 .2 0 .08 0 .25 0 .16 0 .20 17 .25 20 .84

Ca 1384 .0 0 .36 0 .29 7 .70 293 .9 564 .0 1054 .0Fe 2 .67 0 .41 0 .52 2 .70 12 .12 20 .2 52 .2Ni 0 .07 0 .10 <0 .1 0 .10 1 .43 6 .0 10 .25Cu 0 .11 0 .02 <0 .02 <0 .02 0 .09 0 .02 0 .03K 31 .73 0 .05 3 .53 3 .50 4 .22 97 .3 42 .3

Na 39 .18 4 .95 4 .66 5 .5 11 .20 195 .0 30 .0Zn 9 .43 <0 .03 - 0 .42 11 .56 26 .3 20 .45Mn 39 .52 <0 .02 - 2 .50 34 .40 70 .8 67 .90Mg 7 .57 0 .49 0 .11 12 .00 86 .70 183 .0 158 .9

a A 500 ml . column of wet resin regenerated with 600 ml . of 3N HC1 .b From stainless steel reactor .

14

X . Xylose Crystallization

Because of the larger xylose content, syrups from sugar cane bagasseshowed a greater tendency to crystallize than those syrups obtained fromcorn hulls . The samples (5% arabinose, 77% xylose, and 8% glucose) wereheated to dissolve the sugar and cooled slowly with shaking to 24° C .Syrups containing 80% solids yielded 63% crystalline material . SeeTable X .

TABLE X

CRYSTALLIZATION OF BAGASSE XYLOSE SYRUPSa

Initial, % Solids After Cr stallizationb~ ~ % o i ater a

<70 100 Trace of fine crystals70 82 .2 17 .875 55 .7 44 .380 36 .7 63 .385 Solidification rather than crystallization

a 5% arabinose, 77% xylose, 8% glucoseb Sugar analysis on crystalline material 0% arabinose, 96% xylose, 2% glucose

XI . Decomposition of Pure Xylose

A 1% xylose solution in 0 .20 N H2SO4 was heated for 4 .0 hr . at 128° C .(20 psi steam pressure) . Ultraviolet analysis and gas chromatographyshowed the formation of furfural, crotonaldehyde and a small amount ofwater-insoluble brown polymer . Table XI gives a breakdown of the decom-position products .

TABLE XI

DECOMPOSITION OF PURE XYLOSE

% ofStarting Material

Initial wt : . xylose 1 .877 g .Xylose as furfural in condensate 0 .052 g .

`Xylose as furfural and crotonaldehyde ~ 10.5%in solution 0 .145 g. _

Unidentified polysaccharide .32 g . 17 .0%Recovered xylose

Polymeric material1 .300trace

69 .5%

97 .0%

15

C . DISCUSSION

In the production of xylose from corn hulls, three general stepsare involved : (1) removing residual starch from the corn hull, (2) acidhydrol sis of the destarched hull to produce a dilute sugar solution,and (3~ purification of the hydrolyzate to yield an acceptable xyloseproduct . Xylose production from bagasse is similar ; however, the initialdestarching is unnecessary .

Any residual starch on the hulls will be hydrolyzed to glucose, andthe glucose in turn will inhibit lucose isomerase production . Thestarch may be removed from the hulls by hot water solubilization . Theinitial starch content of 8-15% was reduced to 1% by refluxing an aqueoussuspension of the hulls for 3 .0 hr ., or heating at 121° C . (15 psi . steampressure) for 2 .0 hr . See Table I and Table II . In addition to starchremoval, there was a weight loss of approximately 20% due to dissolutionof plant gums . Cold dilute acid treatments appeared less efficaciousfor starch removal than boiling water .

When the corn hulls were heated with dilute sulfuric acid, thehemicellulosic xylan chains were broken down in preference to the glucoselinks in the cellulose . Xylose and arabinose were the principal productsof mild hydrolysis, though galactose and glucose were also found . Ifreaction conditions became too severe the xylose formed initially wasdecomposed to furfural, crotonaldehyde, and polymeric material . SeeTable XI .

On a laboratory scale a 7 .5 :1 ratio of 5 .6% sulfuric acid to dry,destarched hulls heated at reflux for 2 .0 hr . produced a 45% yield of drysugar syrup . The syrup contained 26% arabinose, 51% xylose, and 11%galactose and glucose for an overall xylose yield of 23% . See Table V .Using an autoclave set a 20 psi . steam pressure (128° C .), overall yieldsof xylose as high as 28% have been attained . See Table III .

In the pilot plant, reactions were carried out in a horizontal,polyvinyl chloride coated reactor . On treating 2 .27 kg . (5 .0 lbs .) ofdry hulls with 27 .3 kg . (60 lbs .) of 0 .1 N H2SO4 and heating at 148° C .for 0 .5 hr ., a 21 .5% overall yield of xylose was obtained . This accountsfor 90-95% of the xylose initially present in the hull . Statisticalanalysis performed on the data from pilot plant reactions (See Table IV)have produced equations for xylose yield estimation . One polynomialhaving all first degree terms is given below . On filling in values forthe variables an estimate of the total percent yield of xylose will be given .

X10 = .76X1 - 3 .39X2 - .85X3 - .lOX4 + 38 .7 (See 5 .4)

where X1 3 :1 < ratio liquid to hulls < 12 :1

X2 0.1N < [H2SO4] < 0 .3N

X3 0.5 hr . < time < 3 .0 hr .

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X4 258° F . < temperature < 298° F .

X10 0% < total % xylose < 28%

The term See 5 .4 indicates that the answer given will be within ± 5 .4%absolute value of the predicted yield 65% of the time . In the variableranges given above an increase in liquid to hull ratio should producelarger yields, as would a temperature decrease . Reaction time has verylittle effect on yield and acid strength none at all .

After the various sugars in the corn hulls or sugar cane bagassehad been liberated by acid hydrolysis various purification steps werenecessary . The degree of purity determined thoroughness of the reactionworkup .

To neutralize the solution and remove sulfate ions, the pH was adjustedto 4 .5 with calcium hydroxide, boiled 15 minutes and filtered . The decreasedsolubility of calcium sulfate at high temperature permitted removal ofalmost all of the sulfate ions, leaving only a small amount of calciumsulfate in solution ([ CaSO4] = .025M ti3 .0 g ./l . - see Table VI) .

The neutralized sugar solution was then passed through a column ofgranular charcoal to remove color bodies and high molecular weight impuritieswhich caused severe frothing when vacuum evaporation of the solution wasattempted . The water white effluent had a pH of approximately 6 .0 afterpassing through charcoal .

The last traces of ionic m~terial were removed with ion exchangeresins . Cations, primarily Ca+ , were removed with Amberlite IR-120 .The resin was quite effective and total metal ion concentration was reducedto less than 10 mg ./1 . for a 2% xylose solution (See Table IX) . At thislow ionic concentration Na+ and K+ were the most prevalent species . Asthe resin approached exhaustion, loosely held ions were displaced byincoming polyvalent species . This undesirable situation was carefullyavoided in sample preparation . Anions, primarily S04=, and some colorbodies were removed with an anionic Duolite A-6 resin . The effluentshowed no S04= leakage when tested with BaC12 . Ash analysis indicatedno inorganic material was present .

The bagasse syrup (5% arabinose, 77% xylose, 8% glucose) containinga higher percentage of xylose than the corn hull syrup (33% arabinose,54% xylose, 9% glucose and galactose) crystallized much more readily andafforded a method of purification . Upon crystallization, bagasse syrupsyielded a product containing 96% xylose and 2% glucose . However, 30%of the total xylose remained in the mother liquor (See Table X) . Syrupsobtained from corn hulls will crystallize but usually require severalweeks to do so .

After the xylose syrups had been obtained, they were submitted forbiological testing, and their ability to produce glucose isomerase wasmeasured against a standard xylose solution . Crysta ize samp es containing

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96-98% xylose showed a cell growth activity at best only 75% of that ofthe standard . Chemical analyses were not able to isolate any trace materialsresponsible for the difference in cell growth activity .

D . CONCLUSIONS

The corn hulls, readily available from the corn wet milling industry,have a pentosan content of approximately 40% . The pentosans present asarabinose and xylose were liberated by acid hydrolysis and purificationyielded colorless syrups containing 30% arabinose, 52% xylose, 7% galactoseand 7% glucose . However, the xylose in the syrup was only 45% as effectiveas the standard Eastern Chemical xylose for glucose isomerase production .Although xylose rich syrups were produced easily and n ood yields, theadditional arabinose, glucose and trace materials present markedly decreasedtheir ability to function as a carbohydrate source for g~l_~u~~_c~ose isomeraseproduction . Sugar cane bagasse contains ti20% pentosan, a- n~fc upon ac~i3~hydrolysis yielded water white syrups containing 6% arabinose, 81% xylose,2% galactose, and 7% glucose . On crystallization, a product containing96% xylose was obtained . Even this product, however, was only 50-75%as effective as Eastern Chemical Company xylose in the production of9-lu-co-se- isomerase . This lessened cell growth efficiency coupled with theadvent of an economical alkaline isomerization process for convertingglucose to fructose led to termination of this project .

E . RECOMMENDATIONS

I . Future Work

At this time no further work is anticipated .

II . Patentability

The production of xylose from corn hulls has not been previouslyreported in the literature . However, the general methods employed aresimilar to those used in xylose production from other hemicellulosicmaterials . Therefore, acid hydrolysis of corn hulls to yield xylosewould probably not be patentable .

~ aton

~- .-V ~ ____ 0__J. G . Jones

Approved :

(See next page for distribution .

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Distribution :

Dr. Murray Senkus Mr. M . R . HaxtonDr . R . E. Farrar Mr. L . A . Willson, Jr .Mr . E . H . Harwood Dr. H . J . BluhmDr . C . E . Teague, Jr .Dr . C . E . RixMiss C . N . EatonMr . J . G . Jones

,Xibrary (2)Dr . Edward Bernasek

Submitted : October 23, 1968

Completed : October 25, 1968From manuscript :bjv ;kti

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BIBLIOGRAPHY

1 . Hudson, C . S ., and Harding, T . S ., J . AM . CHEM . SOC . 39, 1038 (1917) .

2 . Hudson, C . S ., and Harding, T . S ., Ibid . 40, 1601 (1918) .

3 . Monroe, K . P ., Ibid . 41, 1023 (1919) .

4 . Ling, A . R ., and Nanji, D . R ., J . CHEM . SOC ., 123 (pt . 1) 620 (1923) .

5 . Whistler, R. L ., and Wolfrom, M . L ., "Methods in CarbohydrateChemistry," Vol . 1, Academic Press, New York, New York, p . 88 (1962) .

6 . Dunning, J . W ., and Lathrop, E . C ., IND . ENG . CHEM . 37, 24 (1945) .

7 . Firstenberger, B . G ., Iowa State Jour . Sci ., 18, 27 (1943) .

8 . Bryner, L . C ., Christensen, L . M ., and Fulmer, E . I ., IND . ENG . CHEM .28, 206 (1936) .

9 . Schreiber, W . T ., Geib, N . Y ., Wingfield, B ., and Acree, S . F .,Ibid . 22, 497 (1930) .

10 . Scherrard, E . C ., and Blanco, G . W ., Ibid ., 12, 1160 (1920) .

11 . Ledoga S . p . A ., Brit . Pat . 922,684, April 3, 1963 .

12 . Whistler, R . L ., Ed ., "Industrial Gums," Academic Press, New York,New York, p . 301 (1959) .