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CONFIDE NTIALAuthors : C . E . Rix October 23, 1968
C . N . EatonJ . G . Jones Notebook Pages : 151501-151550,
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 .
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 .
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- ~
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
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 .
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
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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
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
a A 500 ml . column of wet resin regenerated with 600 ml . of 3N HC1 .b From stainless steel reactor .
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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%
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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 .
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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5 . Whistler, R. L ., and Wolfrom, M . L ., "Methods in CarbohydrateChemistry," Vol . 1, Academic Press, New York, New York, p . 88 (1962) .
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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) .