ANALYTICAL CHEMISTRY
876 peroxide” error of the reductor was applied as determined by the passage of comparable volumes of 2 F perchloric acid through the micro-Jones reductor and subsequent addition of indicator and perchloratocerate solution. The results from these titrations are given in Table IV. Colorimetric Determination of Iron in Ferric Perchlorate Solution. For the purpose of comparing the accuracy of this type iron determination following perchloratocerat oxidation, a colorimetric determination of iron by the 1,lO-phenanthroline reaction was carried out. The preparation of the solutions of iron for spectrophotometric analysis was as follows: I
Samples of the standard iron solution (0.05010 mg. per gram of solution) were weighed out of the weight buret into 100-ml. beakers. A small excess of sulfuric acid was added and the solutions were evaporated to fumes of sulfuric acid to remove perchloric acid. The solutions thus obtained were diluted with conductivity water and the ferric iron was reduced by the addition of hydroxylamine hydrochloride. The solutions xere then neutralized (Congo red paper) by the addition of dilute ammonia and the ferroin reaction was produced by the addition of 5 ml. of 0.337, aqueous solution of 1,lO-phenanthroline. The solutions thus obtained were transferred to 100-ml. volumetric flasks and diluted to the mark with conductivity water. Spectrophotometric transmittance curves for these solutions were obtained using the G.E. recording spectrophotometer and the per cent transmittancy was read at the point of maximum absorption wave length 512 mp. From a previously accurately determined calibration curve the weight of iron present was determined. A
Table V.
Spectrophotometric Determination of Iron in Ferric Perchlorate Reference Solution
Iron Taken
Transmittance
Iron Found
Mo.
%
.WO.
MO.
0.239 0.339 0.149 0.245 0.246 0.45a 0.249
33.9 21.3 49.7 32.4 32.1 12.7 31.3
0.236 0.340
-0.003 t0.001 f0.002 -0.001 -0.001 -0.002
Error
Fe
0.151
0.244 0.247 0.456 0.251 AV. % error
+o.ooz
0.0017 =
0.61
blink correction to account for traces of iron in the reagents employed was applied. The results are given in Table V. Examination of Tables IV and T- shows that the coloiimetric determination of iron and the volumetric determination by the perchloratocerate titrational procedpre are of comparable accuracy and precision. LITERATURE CITED
(1) Ellis, IA-D.EXG.CHEM.,h s a t . ED., 10, 112 (1938). (2) Kirk and Tompkins, Ibid., 13, 277 (1941). (3) Salomon, Gabrio, and Smith, Arch. Biochem., 11, 433 (1946). (4) Smith, Ind.Eng. Chem., 18, 1216 (1926). RECEIVED November 17, 1947.
Quantitative Determination of Hemicellulose Constituents by Fermentation *
A. H. AUERMHEIMER, L. J. WICKERHAM,
AND L. E. SCHNIEPP Northern Regional Research Laboratory,’ Peoria, Ill.
The D-xylose and L-arabinose in hydroIyzates of hemiceIIuIoses from agricultural residues can be quantitatively determined by fermenting solutions of the uronic acid-free sugars with H . suareolens (NRRL No. 838) and C. guilliermondii (NRRL No. 488). H . suaveolens selectively utilizes D-xylose and C. guilliermondii utilizes both sugars completely. By determination of copper reducing values of unfermented and fermented samples percentage of each sugar may be calculated.
I
N T H E course of investigations of the hemicellulosesfound in agricultural residues, it was desired to determine quantitatively the amounts of xylose and arabinose present in their hydrolysates. Methods had previously been reported for the determination of D-XylOSe and L-arabinose by the use of bacteria which selectively utilized these sugars (1) and for the estimation of D-galactose and D-XylOSe by the use of yeasts (6, 7 ) . Behause the bacterial fermentations required special media and longer times for complete utilization of the sugars, the yeast method was chosen for further investigation and adaptation to the problem. NTiseand Appling ( 7 ) found that Hansenula suaveolens (NRRL No. 838) would selectively ferment D-xylose in the presence of L-arabinose and certain other sugars. However, in a study of the fermentation of wood-sugar stillage, Kurth and Cheldelin (B) claim that H . suaveolens (NRRL No. 838) and Candida lipolytica (NRRL No. 1094) utilize arabinose, though more slowly than xylose. They obtained evidence for this conclusion by ferrnentation of wood-hydrolyzate stillage to which D-arabinose had been added. Studies in this laboratory have confirmed the specificity of both yeasts for D-XylOSe and have shown, contrary to the findings of Kurth and Cheldelin, that neither the D- nor L-forms of arabinose were utilized (Table I). Because of the appreciable difference in the reducing values of D-arabinose and D-Xylose, Kurth and Cheldelin’s calculation of
all reducing values as xylose led them t o erroneous conclusions. Analysis of these sugars with use of Somogyi’sreagent ( 5 ) ,which contains D-tartrate, s h o w that D-arabinose reduces only 70yoas much copper as does D-xylose. This is in accord with the work of Richtmyer and Hudson ( 4 ) , who showed that the optical isomers of some sugars have different reducing values when analyzed with Fehling’s solutions containing optically active tartrates. In an effort to establish a direct method for the determination of L-arabinose, several yeasts were tested on L-arabinose and n-xylose and known mixtures of the two. Although some yeasts utilized L-arabinose more rapidly than n-xylose, none was completely selective for L-arabinose. Three yeasts, Candida krusoides (NRRL No. 305), Candida guilliermondii (NRRL No. 488),and Debaryomyces matruchoti (NRRL KO.833), completely utilized L-arabinose, D-xylose, or mixtures of the two in 48 hours. Because C . guilliermondii gave complete fermentation of both pentoses in 24 hours it was selected as being the most useful for the present work. As the specificity of H. suaveolens (KRRL No. 838) for xylose has been confirmed and C . guilliermondii (KRRL KO.488) found to utilize both D-XylOSe and L-arabinose completely, it is believed that both sugars can be quantitatively determined by fermenting separate portions of a mixture of these sugars with these two
V O L U M E 2 0 , NO. 9, S E P T E M B E R 1 9 4 8
877
WHEATSTRAW XYLAK.Wheat straw xylan was extracted by circulating 5 % sodium hydroxide solution through the straw at room temperature for 25 hours. The hemicelluloses in the filtered extract were precipitated by excess alcohol. The precipitate was reslurried four times with dilute alkali (about 2%) in 50% alcohol and washed repeatedly with 60% alcohol. The color was much less readily removed than from the cob products. The precipitate was finally acidified with acetic acid and dehydrated as above. The product was light tan. Analysis (moisture-free basis): ash 11.57,; lignin 4%; pentosan 82.4% (furfural 57.4%).
yeasts. The decrease in reducing value effected by H . suaveolens is calculated as n-xylose, and the additional decrease brought about by C. guilliermondii is calculated as L-arabinose. Hemicelluloses isolated from cereal straws and corncobs have been found to consist essentially of xylan. Careful examination of their hydrolysis products indicates the presence of L-arabinose and n-glucuronic acid in addition to the expected n-xylose. Fermentation of such hydrolyzates with Saccharomyces carlsbergcnsis (NRRL Yo. 379), a selective glucose fermenter, shows only slight utilization of reducing sugars corresponding to the apparent slight attack b r this yeast on n-xylose, as noted by Wise and Appling ( 7 ) . I t was concluded, therefore, that the authors’ hemicellulose hydrolyzates contained no glucose. Hence, removal of uronic acid constituents, as their alcohol-insoluble barium salts, should leave an essentially pure mixture of n-xylose and L-arabinose which could then be analyzed by the selective fermentation method proposed above. The uronic acid content of the original hemicellulose is determined by other methods. The selective fermentation procedure as applied to hemicellulose hydrolyzates from corncobs and wheat straw is demonstrated by the results shown in Table 11.
Hydrolysis of Hemicelluloses. One-gram samples were dissolved in 45 ml. of 3oj, sulfuric acid (0.62 N ) and refluxed for 2 hours. The cooled solutions were filtered, neutralized with barium hydroxide solution t o approximately pH 7.0, and filtered to remove the barium sulfate. The filtrates were evaporated to dryness under reduced pressure, and the residues extracted repeatedly with hot absolute alcohol t o dissolve the sugars. The alcoholic extracts were evaporated to dryness in vacuo, the residues dissolved in water, and aliquots of these solutions used for the fermentation tests. This procedure gives sugar solutions free of uronic acids. Fermentation Procedure. The fermentation procedure used for both yeasts was essentially the same as that described by Wise and .Ippling ( 7 ) . The baker’s yeast extract used as nutrient n-as prepared by mixing 150 grams of starch-free baker’s yeast with vater and making up t o 1-liter volume. This mixture was autoclaved a t 15 pounds’ pressure for 30 minutes, cooled, and filtered. The cultures of both H . suaveolens ( S R R L S o . 838) and C. guillzernondia ( S R R L S o . 488) were prepared b> the procedure recommended by Kise and Appling (6, 7 ) . The yeast suspensions were made up to approximately 35,000,000 cells per ml. b r photometer. Samples of sugar solutions, 25 ml., containing 50 to 100 mg. of total reducing sugais, n-ere placed in 300-ml. Erlenmeyer flasks and 15 ml. of the baker’s yeast extract nere added. This mixture v a s sterilized in an autoclave at 15 pounds’ pressure for 15 minutes. After cooling, the sterile media ere inoculated Rith 10 ml. of the standardized suspension of either S R R L KO 838 or NRRL No. 488. The flasks were shaken on a reciprocating platform shaker at 30” C. for 24 hours. The fermented samples were diluted to a volume of 100 ml. and centrifuged. The supernatant liquor was decanted from the reast and aliquot samples taken for analysis. All fermentations were conducte in duplicate ivith uninoculated controls.
EXPERlhI ENTA L
Isolation of Hemicelluloses. CORNCOB XYLAX,SAMPLE 1. Ground corncobs (4-mesh) were extracted at room temperature with 8% sodium hydroxide solution. To the dark-colored extract was added an equal volume of 95% ethanol, and the precipitated “xylan” was separated by decantation and filtration. I t was then reslurried successively with the folloTving solutions (filtered between treatments): alcohol 50%; sodium hydroxide 2% in 50% alcohol; alcohol 50 to 6674 (three times); alcohol 95%; absolute alcohol; and dry ether. Nearly all color was removed by this treatment. The air-dried product was nearly white. Analysis (moisture-free basis): ash 5.01 %; lignin 0.79%; pentosans 94.37, (furfural 65.7y0). CORSCOBXYLAN, SAMPLE 2. The hemicellulose sample Tras eytracted arid precipitated by the procedure described above except that the xylan precipitate v a s reslurried one additional time with 2mc alkali in 5 0 7 alcohol and then acidified to pH 2.5 xith hydrochloric acid before the final dehydration. -4n-hite product was obtained. Analysis (moisture-free basis): ash 0.80%; lignin 0 . 9 2 7 ; pentosans 98.1% (furfural 68.37).
.
Table I. Utilization of D- and L-Arabinose and D-Xylose b y H . suaceolens (NRRL No. 838) and C . Zipolytica (NRRL No. 1094) Substrate D-arabinose D-arabinose L-srabino-e L-arabinose D-xylose L-arabinose D-xylose A L-arabinose D-xylose D-xylose
+
Table 11.
Weight, I f g. 100 100 100 100 50 51 50
838 838 838 838 838
Hours 24 72 24 72 48
Pento-e Recovered, Alg. 97 n-arabinose 97 D-arabinose 100 L-arabinose 100 L-arabinose 49 L-arabinose
lo94 838 838
72 24 72
50 L-arabinose 1 D-xylose 1 D-sylose
Yeast KO.
i
51
100 100
1
Reducing Sugars. -411 sugar analyses were made by the procedure of Somogyi (5), with the modification of heating the samples for 15 minutes, a time sufficiently long for the slower reacting arabinose. The following reducing values were found (mg. of sugar per ml. of 0.005 JV sodium thiosulfate): n-xylose 190) 0.129; n-glucose (Bureau of (Eastman Kodak Co. CYD Standards) 0.130: L-arabinose (Eastman Kodak Co.; OID 102.30) 0.146; and n-arabinose (Eastman Kodak Co.; 0133 - 103.50) 0.178.
+
+
LITERATURE CITED
Determination of Pentose Sugars by F e r m e n t a t i o n of Hydrolyzed Hemicelluloses
Before Fermentatlon
Reducing Values a s 111. of 0.005 S Thiosulfate .ifter Fermentation Difference by Due t o KRRL NRRL Arabi838 488 Xylose noqe
Hemicellulose Corncob, sample 1 795 Corncob, sample 2 867 Wheat straw 691 a A diphenylhydrazone value
Xylose, 3Zg.
Arahinoie R~K. R
88
11
11.1a
772 56 100 85 38 606 47 78 (3,8 ) of 12.4% was found for this sample.
8
7.4 8.2
116 95
40 39
679
76
7
(1) Hopkiiis, E. W.,P e t e r s o n , W. H., and F r e d , E. B., J . Am. Chem. SOC.,52,3669 (1930). (2) K u r t h , E. F., a n d Cheldelin, V. H., Ind. Eng. Chem., 38, 617 (1946). (3) N e u b e r g , C., and W o h l g e m u t h , J., 2. physiol. Chem., 35, 31 (1902). (4) R i c h t n i y e r , N . K., and H u d s o n , C. S.,J . Am. Chem. SOC.,58, 2640 (1936). (6) Sornogyi, M., J . Bid. Chem., 160, 61 (1945). (6) W i s e , L. E., and Appling, J. TV., IXD. ENG. CHEM.,ANAL.ED.,16,28 (1944). 17) Ibid.. 17. 182 (19451. (8) Wise’, L. E.,and P e t e r s o n , F. C., Ind. Eng. Chem., 22, 362 (1930). RECEIVED March 1, 1948.
