~ ~ O Y I ' R K R IIIIO Y l;ffGY
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Fermentation of Cellulose and Cellulose Humic Acid and Lignin and Lignin Humic Acid Kr- E. BERLA N D W. KOERBER
Those scientists who believe that lignin is the parent material of bituminous coal, as well as of lignites, base their belief on the assumption that cellulose, and other carbohydrates which form the bulk of plants, are decomposed by bacteria much more quickly and more completely than lignin. This assumption has found friends and opponents. Cllsoii and Petersoil found that very small anourits of lignin ( 1 ('o) iuhibit tlie fermentation of pulp by cellulose fermenting bacteria. One of us has expressed the opinictn that lignin and its derivatives arc not tlie exclusive parent material for bituminous coals. I t has bccii showii that under conditions which may have existed geochemically (high temperature and pressure) ant1 with PH values above 7, bituminous coals could be obtained by artificial iiicoalification of carbohydrates. 'I'hose artificial carbohydrate coals shorn exactly the same properties as natural bituminous coals. This result never could be obed with lignin and its derivatives, like lignin During the first stage of their conversion, with the pH above 7 , cellulose and other carbohydrates forni partly sacchariiiic acids which, upon further iticoalification (with the formation of carbon dioxide, water, aiid lower hydrocarhoris!, pivc carbohydrate jcel1ulr)se)11u111ic acids I'ntil iiow all thosc alhali-solublc. materials preseiit in peat aiitl lignites resiiltiiig from thc decomposition of plaiiti ha\ c' beeii coiisidcwrl as ltgniii humic acitii. .niti i l o t as n i i ~ t u i - n~li c ~ l l u losc. humic acids and l~g11nihumic acids *Isyet iio method of determiiiiiig the amouiit of cellulose humic acids in thesc niiaed acids has been foutid Wc know that cellulose hiimic acids, 111 tlieir chemical compositmi arid i ti their behavior toward further incoalificatioti, are fundamentally different from lignin humic acids Furthermore, as may be shown iii another publ,catioii, their heat stability is complete! iffererit from the heat . t i i t i o f I i q n i i i , atid ligstability of carbohi tlr nin humic acids I t was of great iiiterest to iiiid out how certaiii anaerobic and aerobic bacteria which decompose pi
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cellulose2s'i would behave toward cellulose (carbohydrate) humic acid, lignin humic acid, and lignin. These experiments, which are described below, have been carried out and show a remarkable result. Anaerobic and aerobic bacteria which decompose cellulose do not attack cellulose humic acids, lignin, and lignin humic acids a t all. They do not attack natural humic acids isolated from peat. It has been shown that rather little cellulose, as such, may be present in peat and lignites and that under certain conditions the carbohydrate content of peat and lignites decreases with age. 'l'his does uot lead to the conclusion that cellulose aiid lower Carbohydrates are completely converted into gases and water-soluble compounds. They are very likely transformed into carbohydrate humic acids which resist the action of bacteria because of their phenolic structure. That cellulose humic acids may be present in those alkali-soluble materials obtained from peat can he seen by the fact that the peat humic acid used in our experiments contains only a few per cent. of methoxyl compared with 10.Syo of methoxyl found in the lignin humic acid produced under very drastic conditions a t 250' i n a rotating closed vessel. .I certain amouiit of rnethoxyl may be split off by tlie actioii o f IJXt eria (sec Waksmati
Experimental Part Ccllulosc, cclluloac humic acid, ligniii huiuic acid, and hiittiic acid froni peat wrrc u i e d for t h e frrmentation cxprriinents. -I he preparation of thew inaterials was carried out in the following way (a', Cellulose was used in the form of wadding (b) Cellulose Humic Acids.--Linters were heated at 230" for ten houri with S/sodiuin hydroxide in a revolving bomb The product was filtered and the dark brown filtrate obtained way acidified with hydrochloric acid, the precipitated humic acids filtered and dried. The finely pulverized material was washed with water until the chloride reaction disappeared and then dried over calcium chloride 'I'he dark brown humic acids were dissolved in dcLtoiie, t h t solvents evaporated, dnd the residue dried zlt . f 2 ) W' Omehan~ky. Z c n f r BakL, 11, 36, 472 (1913) 11, 1 2 . 33 (1904), 11, 11, 36Y (1904) 1 1 , 8, 193 (1902) 3 1 Van l'ieghem. Bull SIX botan Ftance, 1, 24, 128 (1877) 14) i 9 Wak5man arid IT W Smith, IH I ? J O U R & * I , 56, 1 2 2 5 I
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FERMENTATION OF CELLULOSE AND LIGNINA N D DERIVATIVES
July, 1038
TABLE I AEROBICFERMENTATION OF CELLULOSE, bH 7.4, FIVE DAYS,55"
PIdSk
Fermented cellulose Cell. dec.
Cell. c . / 2 0 0 CC.
G.
2.92 2.80 3.10 3.00 2.98 2.96
2.00 2.24 2.19 2.34 1.81 2.12
30 31 32 33 34 Av.
% 67.4 80.1 70.6 78.2 G0.7 71.4
Total acid
Yo of
G
1.11 1.46 1.30 1.45 0.82 1.23
cell dec.
55.8 65.3 59.1 61.8 45.2 57.4
Volatile acid Calcd. as acetic % of cell. G. dec.
0.97 1.26 1.11 1.32 0.73 1.08
48.3 b6.2 50.6 56.5 40.2 50.3
GAS
Non-vol acid % of cell. G. dec.
0.14 .22 .18 .12 .09 .15
7.5 9.1 8.5 5.3 5.0 7.1
Alcohol
% of cell
G.
0.050 ,067 ,044 .05l ,029 ,048
% of
dec.
cell. dec. Culture
2.5 3.0 2.0 2.2 1.6 2.2
9.1 11.8 9.5 14.2 13.9 11.8
A A
c
C F
TABLEI1 AEROBIC FERMENTATION OF CELLULOSE pH 6.0, FIVEDAYS,55' Cell Flask
35 36 37 38 39 Av.
g
/ZOO cc.
2.83 3.10 3.00 2.95 2.98 2.97
Fermented cellulose Cell. dec. G. %
0.62 .56 .71 .74 .50 .63
22.0 18.1 23.8 25.2 16.9 20.9
Total acid 70of eell. G. dec.
0.12 .08 -14 .17 .06 .11
19.8 15.2 20 3 23.4 12.7 18.1
Volatile acid Calcd. as acetic % of cell. G. dec.
0.12 .08 .14 .17 .06 .ll
z'acuo over phosphorus pentoxide: C, 68.2; H, 6.1; 0,
25.7; C&O, 0. (c) Lipin.-Lignin was prepared from sawdust according to the hydrochloric acid method of Willstatter. The product obtained was refluxed with 5% sulfuric acid for five hours, filtered, and washed. A treatment with copper ammonium hydroxide a t room temperature for twenty-four hours was added. The residue was thoroughly washed with hot acidified water and dried over phosphorus pentoxide: C, 62.4; H, 5.8; 0, 27.8; CH80, 14.6. (d) Lignin HUmic Acids.-The lignin as described under c was treated with iV sodium hydroxide a t 250" for three hours in a revolving bomb. The further preparation was carried out as described under b: C, 63.8; H, 6.7; 0, 29.5; CHaO, 10.5. from Wisconsin (e) Humic Acids from Peat.-Peat was finely pulverized and extracted with '%1 sodium hydroxide on a shaking machine for twenty-four hours. The alkali-soluble part was separated by filtration, acidified, and the humic acids isolated as described under b: C, 63.4; H, 4.8; 0, 31.8; C&O, 1.9. Isolation of Aerobic Cellulose Fermenting Bacteria.A cellulose fermenting bacillus was isolated from horsedung according to the method described by Snieszko.6 Control experiments were carried out with cellulose in salt media of pH i.4 and 0. Conical flasks of 300 cc. were each charged with 200 cc. of a salt medium containing 0.1% KN03, 0.1% MgSOd, 0.1% K2HP04, 0.5% peptone, exactly 2 g. of calcium carbonate and 3 g. of cellulose (latter dried over phosphorus pentoxide), PH 7.4. After sterilization the flasks were inoculated with the above-mentioned cellulose fermenting culture and incubated for five days a t 5,5". After this time the flasks were.removed from the incubator and each of them titrated with N hydrochloric acid. From the titrated calcium carbonate the total acid formed was calculated. More acid was added and the contents of the ( 5 ) S. Snieszko, Ze~zLr.Bok1 , 11, 88, S . , 403 (l!I331
19.8 15.2 20.3 23.4 12.7 18.1
Non-vol. acid % of cell. G. dec.
None None None None None None
Alcohol
% of cell.
G.
dec.
None None None None None None
Gas
% of
cell. dec. Culture
2.2 2.9 3.5 1.8 4.2 2.8
A A
c c 1'
flasks filtered and washed. The residue was unattackcd cellulose; 25 cc. of 80% &.PO4 was added to the filtrate, which was then steam distilled. The distillate was titrated with N sodium hydroxide (volatile acids expressed as acetic acid). In order to determine the alcohol, the neutral distillate was ether-extracted three times, the ether evaporated, and the extract refluxed with acetyl chloride for two hours. After neutralization the solution was ether-extracted again, hydrolyzed with 10 ml. of 0.5 N sodium hydroxide, and back-titrated with 0.5 N hydrochloric acid (Tables I, 11). Aerobic Fermentation Experiments on Cellulose Humic Acids, Lignin, Lignin Humic Acids, and Humic Acids from Peat.-The above materials were exposed to the action of the cellulose-fermenting bacteria under the sanie conditions as were used in the control experiments on cellulose. None of these products could be fermented by those cultures which were active on cellulose. Evcn after four weeks no fermentation could be obtained with pH 7.4 and 6. Isolation of Anaerobic Cellulose Fermenting Bacteria.Amylobacter naticula was isolated from human feces under anaerobic conditions as described by Clausen.6 Control experiments were performed with cellulose in a salt medium with PH 7.4 and ti. Tubes were each charged with 150 ml. of a salt medium containing 0.1% (NH&S04, 0.1 % MgHP04, 0.05% NaC1, and 1 g. of calcium carbonate; 1.5 g. of cellulose (dried over phosphorus pentoxide) accurately weighed, was added, pH 7.4. After sterilization the tubes were inoculated with an active cellulose fermenting culture of amylobacler nuaicula and incubated a t 37' for five days under anaerobic conditions. After this time the tubes were removed from the incubator, their contents filtered, the residue washed and dried over cakium chloride in vacuo. The only fermentation products being gases, no other determinations were carried out than the weighing of the undecomposed cellii lost,.
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( 6 ) P. Clausen. ibid., 11. 84, A , , 20-60 (1!)31).
zko a decomposition of approximately 707; of the cellulose used. Fermentation products are rolaj PA\ L .i; tile acids, lion-volatile acids, and alcohol in medium Cellulose in medirini rrrmentcd 5c cellulo~c of pH 7.4. (b) The fermentation in medium nf F*pt i: /I50 CI dt~compowd pH t i by the same bacteria sliowed a decomposi1 1 32 fL?
