Hydrolysis of Pectin by Various Micoörganisms - American Chemical

in a muslin cloth. This was done to remove excess of tan liquor. Each powder was then dried in the air and analyzed for water, fat (chloroform extract...
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INDUSTRIAL A N D ENGINEERING CHEMISTRY

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After tanning, each hide powder sample was washed five times by alternate suspension in water and squeezing by hand in a muslin cloth. This was done to remove excess of tan liquor. Each powder was then dried in the air and analyzed for water, fat (chloroform extraction), hide substance, total sulfur as SOs, and water-soluble matter. Total sulfur was determined by oxidation with fuming nitric acid, followed by precipitation as barium sulfate. Water-soluble matter was determined by extraction of a weighed sample with running distilled water in a Wilson-Kern extractor for 24 hours. The insoluble residue was collected quantitatively, dried first a t 50" C. and then a t 105" C., and weighed. The percentage of matter soluble in water mas taken as the difference between 100 per cent and the sum of the percentages of water and fat (in the tanned powder) and insoluble matter. Insoluble ash and insoluble sulfur were determined in portions of the waterinsoluble residue and calculated as percentages of the original tanned powder. Fixed tannin was taken as the difference between 100 per cent and the sum of the percentages of water, fat, hide substance, water-soluble matter, and insoluble ash. The results of these experiments are given in Table 11. Table 11-Sulfur i n Hide Powders T a n n e d with Spruce Extract so3 IN

FIXED TREATMENTTANNIN^

WATER- TOTAI. S SOLUBLE AS MATTER'

INSOLU- TOTAL MABLE S TERIAL ABAS

so&'

sosu

SORBED B Y

SKIN

Per cent OTIEBRACHO AND S P R U C E EXTRACTS

Tanned for 24 hours at: 3 . 5 pH 28.24 22.13 4 . 5 pH 6 . 5 pH 21.90 Tanned for 96 hours a t : 3 . 5 pH 30.00 26.16 4 . 5 pH 26.04 5 . 5 pH

12.09 12.05 10.20

2.99 1.62 1.75

2.30 1.37 0.89

7 5 5

10.12 10.45 8.90

2.43 2.00 2.00

1.96 1.81 1.74

6 5 6

skin from spruce extract is very resistant to hydrolysis, particularly in the case of leathers tanned with spruce extract only. Second, in any one mixture of tanning materials the ratio of total material absorbed (tans plus water-solubles) to sulfur compound absorbed is practically constant, being independent both of the tanning period and of the pH value. Third, the ratio of total material absorbed to sulfur compounds absorbed is practically the same as the ratio of total tannin in the liquor to total sulfur compound in the liquor. This is shown by comparing the results obtained with spruce alone and those obtained with mixtures of spruce and quebracho. The spruce-quebracho liquors contained about half as much spruce. and therefore half as much sulfate, per unit of tannin as the straight spruce liquors, and the leathers tanned with spruce-quebracho liquors took up about half as much spruce per unit of material fixed as did the leathers tanned with spruce only. From these facts we conclude that the sulfur-containing compound of spruce extract must be regarded as an integral part of the tannin molecule. The sulfur-containing radical is not removable by long washing and appears to be no more hydrolyzable than the tanning material as a whole. The determination of mineral acid on leathers tanned with spruce extract is of no significance, as the results are a measure only of the amount of sulfur that is oxidized to sulfate during the ignition with sodium carbonate, which depends wholly upon the conditions of ignition. It appears probable that a method based on the determination of the p H value of an aqueous extract of the leather, as has been described by Thompson and Atkin (I, S), is the only one that will give significant results for acid in such leathers. Literature Cited

S P R U C E EX'rRACT

Tanned for 24 hours at: 3.35 3.42 8.22 3 . 5 pH 20.3s 3.09 2.76 6.87 4 . 5 pH 19.00 2.17 10.72 2.30 5 . 5 DH 19.14 a Expressed as grams per 100 grams of hide substance.

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(1) Atkin and Thompson, J . Infern. SOC.Leather Trades' Chem., 13, 301 (1929). (2) Innes,Ibid., 12, 256 (1928). (3) Thompson and Atkin, Ibid., 13, 297 (1929). (4) Wilson, "Chemistry of Leather Manufacture," Vol. 11, Chemical Catalog C o . , S e w York, 1929.

8

Three facts disclosed by the results in Table I1 deserve special comment. First, the sulfur compound absorbed by

Hydrolysis of Pectin b y Various Microorganisms' A Comparative Study Gilbert A. Pitman and W. V. Cruess UNIVERSITY OF CALIFORNIA,

D

URIKG the last few years a great deal of research has been conducted on the chemistry, manufacture, and industrial utilization of pectin. Relatively little attention, however, has been given to the action of the common fermentation and fruit-decay microorganisms on pectin. It is of industrial importance as well as of scientific interest to have accurate information on the hydrolytic effect of the more important of these organisms. The investigations here reported were undertaken for the purpose of obtaining this information. The effect of Sclerotinia and Monilia on pectin was studied by Willaman (IO) and by Davison and Willaman, (2) who state that Sclerotinia secretes an enzyme, pectase, which attacks pectin. Davison and Willaman follow the recommendation of the pectin nomenclature committee of the American Chemical Society in recognizing three enzymes that attack pectin compounds. These are protopectinase, which 1

Received July 3, 1929.

BERKELEY, CALIFORNIA

liberates pectin from insoluble protopectin; pectase, which hydrolyzes pectin to pectic acid, splitting off the methyl groups; and pectinase, which hydrolyzes pectin into its simpler constituents, galactose, galacturonic acid, etc. The action of Bacillus carotovorus on pectin is well known. According to Jones ( h ) , it dissolves the middle lamellae so completely as to leave the individual cells floating free. Willaman (10) also states the Sclerotinia will not attack the pectin so long as simple sugars are available. Coles (1) used the difference in hydrolytic effect of various microorganisms on pectin as a means of distinguishing between strains. He studied nearly 500 strains of organisms, almost all of which were bacteria, but found only 15 strains that gave any indication of hydrolytic activity. As no definite chemical analysis was made in any case, however, his work was not of much assistance in the present investigation except to indicate in a general way that bacteria as a group do not usually attack pectin.

December, 1929

INDUSTRIAL A N D ESGILVEERING CHEMISTRY Plan of Investigation

T o apple juice was added commercial powdered apple pectin to give approximately a 1 per cent solution. Equal volumes of the solution were placed in quart bottles, which were then plugged with cotton and sterilized a t 100" C. for 60 minutes. Inoculations with the organisms were made from agar slants. The bottles were stored a t room temperature for nearly four months. Pectin content, viscosity, and jellying power of the samples were determined (Table I). As these organisins must penetrate a layer of pectin in the middle lamellae of many plant tissues, several were chosen because of their known ability t o attack living plant tissue. Method of Analysis

Study of the available methods of pectin analysis indicated that for our purpose Wichmann's pectic acid method (-9) was the most suitable. As a check on the pectin analyses, the viscosity of the liquid from each culture was determined with the aid of a Stormer viscometer. Ohn 16) and Myers and Baker ( 5 )have shown that a definite relationship exists between the jellying power or' pure pectin solutions and the viscosity. Accordingly, on several of the samples, the jellying power of the pectin was tested by the method of Singh ( 7 ) . This method consists of finding the number of grams of sugar a given quantity (2,j cc.) of the sample would support in a jelly. Using this method, 25 cc. of the juice was added to a weighed amount of sugar and the sample boiled to a constant percentage of sugar (70 per cent). The sugar contained in the sample was added to the weighed sugar in calculating the first weight of the jelly. The samples were weighed from time to time during boiling until the calculated weight was reached. The samples were then poured into small jelly glasses and allowed t o stand 24 hours before examination. Before analysis all samples were tested for total acid, expressed as malic, and for density in degrees Rrix, and the p H value of each was determined by a Cenco-Wendt hydrogen-ion apparatus. The results of these tests were considered in all of the analyses, and those factors that might have a bearing on any one analysis were brought t o a constant value before the determination. Three sets of supplementary experiments were made, one to determine the effect of Saccharomiices ceremiiae and the natural enzymes of the fruit on the pectin and protopectin. The results of the first experiment shorn conclusively that Saccharomyces does not attack pectin or protopectin. I n this experiment, four 2000-gram lots of ground apple pomace were diluted with 6000 grams of water and treated as follows: LOT 1 (CHECKOR BLAiiK)-This lot was boiled for 30 minutes, made to the original weight (8000 grams) by addition of water, and pressed. The expressed juice was preserved with sodium benzoate and stored a t -18" C. until analyzed. LOT 2 (EFFECT O F BOTH YEAST AKD XATUIiAL PECTIC Eszk-vEs)-This lot was inoculated n i t h a culture of S. cerezksiae without previously boiling the fruit and water mixture in order to allow both the yeast and the natural fruit enzymes to attack the pectin. The culture was stored at room temperature during fermentation. When the fermentation mas complete the sample n-as boiled for 30 minutes, brought t o original weight, and pressed. Pectin n a s determined by the Wichmann method. LOT 3 (EFFECT O F SATGR.4L FRUIT EKZYIIES 0KLY)This lot was preserved with sodium benzoate and was not boiled until the fruit enzymes had had an opportunity to attack the pectin present. It was stored a t room temperature for the same period as Lot 2. It was then boiled for 30

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minutes and brought to original weight, when the juice was expressed and the pectin determined by the Wichmann method. LOT4 (EFFECTOF YEASTOmY)-This lot was boiled for 30 minutes to destroy the fruit enzymes, cooled, and fermented with yeast for the same period as Lot 2. When the fermentation was complete it was brought to original weight and pressed and the pectin was determined. Another set of experiments m-as conducted to determine the rates of hydrolysis of pectin a t various pH values by Penicillium glaucum. A third set mas designed to determine the protective effect of dextrose on hydrolysis of pectin in solution by P. glaucum in a synthetic medium. Results

Table I gives the results of the analyses of liquids from cultures a t the end of the incubation periods. Table I-Analyses

ORGASISMU

of Liquids from Cultures a t End of I n c u b a t i o n Period PECSWAR VISTOTAL JELLIED COMETIN ACID BY TER HYA5 D E N - 25 CC. READ- PECUROPH MALIC SITY J U I C E 1 s G TIN LYZEDb Per cent O Brix Grams Seconds Per cent Per cent

Series I : P.glaucum ( 2 ) 3.4 0.421 10 c 2.7 0,217 S. ellipsaideus-Burw n d y (3) 3.5 0,437 0 35+ 4 6 0 889 Mycoderma (4) 3.0 0.418 13.5 30f 4.4 0.889 Blank (1) 3.5 0,503 15.5 35+ 4.6 0.918 Series 11: B . a c e t i (1) 2.4 2.03 19.0 30 3.8 0.710 Torula (3) 3.3 0,704 14.0 25 3 . 1 0.698 Penicilliumsp. (4) 3 . 4 0,509 17.5 15 3.7 0.624 Penicilliumsp. (5) 3 . 5 0.335 17 25 2.7 0.571 Mucor (7) 2.4 0,670 13.5 E 3.0 0.625 Blank (8) 2.1 0,604 18.5 20 4.0 0.741 Series 111: 0.715 0.535 0 40 5 0 3.5 S. cerevzsiae (1) 11.5 30 5 6 0.794 3.6 0.456 F . nivium (6) 18 4 0 0.552 1.9 2.1 30 1'. albo-atrum (7) 2 8 0,544 17 25 R. nigricans ( 8 ) 2.8 1.0 0.491 16.5 15 2 9 3.4 0.67 Botrytis sp. (9) 14.7 0,628 0.50 30 3.4 3 9 P . digitatus (10) C 2 s 0.379 0.415 16 Cytospora sp. (11) 3 . 5 25 4 1 0.626 16.5 0.335 2.9 C. roseum (12) 25 4 1 0.651 12 0,489 F . moniliform (13) 3 . 6 c 15 3 6 0.564 0.50 Colletotrichum (14;1 3 . 5 Helmint hosporium 4 6 0,687 0.37 17 35 3.0 sp. (15) 25 0.622 16 3 s B . amylovorous (16)' 3 . 5 0 . 3 6 2 c 0,028 0,885 15 2 7 Pythium sp. (17) 3 . 1 4 5 0.690 0.51 17 17 Rhizoctonia (18) 3 . 5 0.50 0.618 3 8 3.5 17 35 Torula (19) 16 .. 0.673 4 1 3.2 0.835 Mycoderma (20) 0.755 0.463 16 4 3 Mycoderma (21) 3 . 6 0,770 4 2 15 .. 3.5 0.59 Mycoderma (22) 16 0.678 4 6 35 3.4 0.50 Blank (24) a S u m b e r s in parentheses indicate number of organisms. b T h e significance of a percentage less t h a n 5 is doubtful c No jelly.

..

76.3 2 35 3.16 0

4.18

5,SO 15.8

22.9

15.8 0

0 0 18.6 19.8 27.6 7.36 44.1 7.68 3.99 16.8 0 8.26 95.7 0 8.85 0.73 0 0 0

The data from the experiments made to determine the effect of yeast fermentation on the pectin of apple pomace are given in Table 11. Table 11-Effect

of Yeast F e r m e n t a t i o n a n d Fruit P e c t i n a s e on Pectin

DESSITY PECTIN AFTER

SAMPLE 1 2 3 4

TREATMEKT

Check h-ot heated; fermented with yeast

AFTER

STORAGE STORAGE Brix 4 0 1.5

Preserved with sodium benzoate; not heated or fermented 3 3 2.0 Boiled and fermented with yeast

Per r e n t 0 680 0.501

0 410 0 790

This experiment was repeated by Marsh of the ITiii.i.ersity of California, with even more striking results than those given in Table 11. Alarsli found that the yeast did not destroy a measurable amount of pectin, whercns the fruit pectinase during about 10 days' incubation destroyed approximately 50 per cent of the pectin. TWOsimilar sets of experiments n-ere made on the pectin in lemon rind, with practically the same results, indicating

INDUSTRIAL A N D EIYGIXEERING CHEMISTRY

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clearly that sugar may be removed from boiled pectin stock by fermentation without serious loss of pectin. The data showing the effect of p H on the hydrolysis of pectin by P . glaucum are given graphically in Figure 1. The results of the third experiment indicate that as long as such a sugar is present P. glaucum will not attack the pectin as rapidly as it will in the absence of sugar. As soon as the supply of sugar is exhausted, however, the rate of pectin hydrolysis is materially increased. Discussion of Results

The organisms tested fall into three general classes according to the quantities of pectin hydrolyzed-those hydrolyzed 0-10 per cent, those that hydrolyzed 10-50 per cent, and those that hydrolyzed 50-100 per cent of the pectin.

VOl. 21, No. 12

This method of testing the jellying strength, that is, the Singh test, was made only as check on the other methods of examination. It must be pointed out, however, that the results of the Wichmann determination do not check very closely with the viscosity. Myers and Baker (5) have shown that all of the gravimetric methods of analysis, including the Wichmann method, are open to the objection that, while they give the quantity of pectin in a sample, they give no measure of its quality. It was found that a sample treated with Colletotrichum contained 0.564 per cent “pectin” but would not give a jelly, while a sample treated with Rhyzopus nigricans that showed 0.544 per cent pectin by the Wichmann method jellied a 1 to 1 mixture of sugar and juice, a point that bears out the criticism of gravimetric pectin methods by hlyers and Baker. From Table I1 it is evident that no appreciable decrease of pectin took place in the sample of boiled pomace fermented dry with yeast. I n sample 4 fermentation disintegrated the pomace considerably, a fact that probably rendered pectin extraction more effective and accounts for the high indicated pectin content of this sample. The increase of density during storage was probably caused by diffusion of sugars from the pomace. There is evidence t h a t in the raw pomace the enzymes of the apple hydrolyzed some of the pectin. Marsh’s results show even a more striking hydrolytic effect of the apple enzymes than that shown in Table 11. Conclusions

I

E 4

2 I’ 0

/-

I

I

1

1

I

I

I

I



2

Figure 1-Rate

I

I

I

I

8 1 0 1 2 1 4 DAY 5 of Hydrolysis of Pectin by P. glaucum 4

6

These differences probably represent species differences in the production of pectinase, as this is the only one of the three pectic enzymes that affects the pectic acid nucleus of pectin. The method used for determining pectin in these experiments involved estimation of the total pectic substances, including pectic acid precipitated by alcohol. The samples were all filtered clear before precipitation by alcohol, however, and, according to Wichmann (personal communication), pectic acid is only very slightly soluble a t the pH values existing in the samples used. Thus, while the pectic acid i n solution a t the time of alcohol addition would appear as pectin in the determination, it is probable that the quantities of pectic acid in solution were small. Where qualitative differences in the pectin were observed the enzyme pectase is probably involved, as the removal of the methoxyl groups annuls the jellying power of the pectin. The Pythium sp. and P. glaucum were particularly destructive. During the analysis of the samples from various organisms it was interesting to note the marked differences in the texture of the pectin precipitates. Those samples that had lost considerable pectin yielded a precipitate that was very finely divided and almost granular. This may be further evidence that the splitting of pectin by microorganisms is a process that is accomplished in steps. The agreement between the viscosity and the maximum weight of sugar that could be added to each sample without failure to jelly was satisfactory and appears to confirm in a general way the work of Hardy (3) and Sucharipa (8).

The pectinase activity of representatives of the more important groups of microorganisms occurring on fruits was determined for one set of conditions-namely, aerobic growth of the organisms a t room temperature in or on apple juice containing added apple pectin. Of the microdrganisms tested, two molds, Penicillium glaucum ( P . erpansum) and a Pythium sp. exerted the greatest hydrolytic action. I n fact, the Pythium sp. destroyed practically all of the pectin in 12 weeks’ incubation a t approximately 20” C. The bacterial cultures, B. aceti and B. amylocorus, had little effect under the experimental conditions. Yeasts, Saccharomyces cerevisiw, several strains of Mycoderma, and S.ellipsoideus from grapes had no noticeable effect on the pectin. The effect of several organisms on the pectin was not only quantitative but qualitative. The appearance and texture of the pectin precipitate varied greatly, indicating that the degradation of the pectin had occurred in more or less definite stages. One organism had, apparently not greatly reduced the quantity of pectin as shown by thewichmann method, but it had changed its character so markedly that the “pectin” refused to form a jelly under conditions where even lower concentrations of pectin from other cultures gave a firm jelly. The enzyme involved in the latter case was apparently pectase rather than pectinase. The viscosities of the liquids from most of the cultures followed in only a general way the apparent pectin content as determined by the Wichmann method. I n the presence of calcium salts pectic acid as well as pectin affects the viscosity. The natural enzymes of apple tissue did not greatly reduce the pectin content during the period required for complete alcoholic fermentation of the sugars by S. cerevisiae a t 35” C. This finding confirms earlier observations made in the Fruit Products Laboratory and indicates a method of reducing the cost of extraction of pectin commercially from apple waste ; that is, the alcohol could be recovered by distillation and used in the precipitation of pectin from the concentrated water extract of the waste, or it would be used in the manufacture of distilled vinegar and thus provide a revenue t h a t would cover part of the cost of recovery of the pectin. Also,

I S D C S T R I B L AAYD EJVGIA~EERISGCHEIIIISTRY

December, 1929

fermentation rids the pomace of sugar, so that pectin extracts of high pectin content and low sugar content can be prepared. Hydrolysis of pectin by P. glaucum was more rapid in media of p H 6 and 5 than in one of p H 3. This is in keeping with the finding of ivilson (11) and others that pectin iS more stable in solutions of moderately low p H value than in t'hose of high p H value. stock solution, is not Dextrose, when present in a altogether objectionable, as it has a protective effect against the hydrolysis of pectin by P. glauc?on. This effect would probably apply to other organisms.

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Literature Cited Physiol., 12 379 (1926). Davison and Willaman, Botan. Gaz., 83, 329 (1927). Hardy, Biochem, J , , 18, 283 (1921), Jones, x, Y.Agr. Expt. Sta., Tech. B U L L 11 (1909). Myers and Baker, Delaware Agr. Expt. Sta., Bull. 149 ( 1 9 2 i ) . Ohn, IXD. END.CHEJI.,18, 1295 (1926). Sin&, Master's Thesis, University of California, 1922. ( 8 ) Sucharipa, J . Assocn. O f i c i o l Agv. Chem., 7, 57 (1923). (9) Wichmann, Ibid,, 8 , 123 (192j), (10) Willaman, ~ o t u n Gaz., . 70, 221 (1920). (11) Wilson, IND. E x . CHEM.,17,1065 (1923).

(l)

(2) (3) (4) (5) (6) (7)

Benzene-Pressure Extraction of Coal' J. I). Davis and D. A. Reynolds PITTEBL-RCH EXPBRIJIBNT STATION, U. S. R U R B A U O F MINBS, PITTSBURGH, PA.

T"

'REE years ago the writers investigated the coking

properties of two coals, following in general the method of benzene-pressure extraction developed by Fischer (6) and his associates. Our conclusions (S),drawn from this investigation, were not in agreement with those of Fischer, and, because our study had included only tmo coals, further study of additional coals was necessary to support those conclusions. The later investigation, herein described, includes the study of six coals, each representati1.e of a distinct type as regards industrial carbonization. Nature of Coals Tested

Table I gives proximate and ultimate analyses of the coals tested. a n d U l t i m a t e Analyses of Coals Extracted on As-Received Basis

Table I-Proximate

1 c0.4L

I

PROXIMATE

'

la- Fixed iMois-'t?k car- Ash ture matter bon

1%

70

%

1

ULTIMATE

H

701%

C

N

0

S

70

%

%

70

Ash I3.t.u.

R

Mesa Verda 3 . 4 4 2 . 9 4 6 . 2 7 . 5 5 . 6 69.9 1 . 4 1 4 . 8 0 . 8 7 . 6 12,790 IllinoisNo. 6 7 . 4 3 5 . 5 52.fi 4 . 5 5 . 8 7 1 . 5 1 . 6 l 5 , 2 1 . 4 4 . 5 12,760 Lower Kittanning 1 . 6 2 5 . 3 63.R 9 . 6 4 . 9 7 7 . 5 1 . 4 4 . 6 2 . 0 9 . 6 13,730 Pratt Pittsburgh

1 . 0 2 9 . 2 5 9 . 6 1 0 . 2 5 . 1 7 6 . 3 1 . 6 6 . 0 0 . 8 1 0 . 2 13,640 1 . 4 3 4 . 2 5 8 . 3 6.1 5 . 4 7 5 . 8 1 . 7 6 . 8 1 . 2 6 . 1 14,160

The Mesa Verda coal is weakly coking and not adaptable to by-product coking. Illinois No. 6 coal is a moderately coking coal which may be coked under suitable conditions. Lower Kittanning coal represents about the lower limit in volatile content in coking coals, as charged to by-product ovens, in the United States. Ruhr is a coking coal and was used by Fischer in his study of this problem. Pratt coal is representative of the medium volatile coals yielding good metallurgical coke. Pittsburgh coal represents the high-volatile coking coals; this sample comes from a portion of the Pittsburgh bed characterized by a lower oxygen content than that sampled for our previous investigation. Experimental Method

The method of extraction with benzene under prcssure and separation of the extract into oily and solid bitumens was the same as that described by Fischer and used by us in the previous work. Briefly, the procedure is as follows: Presented before the Dixicion of Gas and Fuel Chemistry at the 78th Meeting of the .4merican Chemical Society, Minneapolis, 3Iinn September 9 to 13, 1929 J

Dry 400 grams of 6- t o 10-mesh coal "as received" by distilling from it 600 cc. of benzene. Charge this sample with 1000 cc. of benzene in a cylindrical autoclave placed in a swinging, electrically heated furnace. Raise the temperature to 285" in one hour and continue heating 4 hours a t that temperature. After cooling, filter off the benzene solution, add fresh benzene, and repeat the extraction. Free insoluble residue and extract of solvent by warming in vmuo. fieparate the extract into oily and solid bitumens by pouring a concentrated benzene solution of the extract into a large e x c ~ s sof petroleum ether (b. p. 30-60" C.), whereupon the solid is precipitated and the oily bitumen remains in solution. An important point not mentioned by other investigators and noted early in this work was the increase in hardness of the extract with successive extractions. For this reason the total extract from each sample TTas combined and the separation into oily and solid bitumens was made on R sample representative of the whole Separation of the individual extracts obtained from repeated extraction of the same sample showed that the proportion of solid bitumen increased with the duration of extraction. I n other words, oily bitumen is more readily removed from the coal by benzene-pressure extraction. Ruhr coal extracted the first time yielded a n extract containing 51.7 per cent of solid bitumen, whereas the third estract contained 82 3 per cent. Lower Kittanning coal yielded nearly pure solid bitumen from the seventh extraction. This increase in the ratio of solid to oily bitumen with continued extraction readily accounts for the increase in the hardness of the extract noted. I n addition to the three fractions (insoluble residue, oily bitumen, and solid bitumen) obtained by the method outlined, a fourth fraction not mentioned by other investigators was noted in this work. Upon cooling the autoclave, the substance precipitated upon the granules of coal and the autoclave walls. This substance, a brown powder similar t o solid bitumen, will be referred t o as "insoluble" bitumen. The writers modified the previously outlined procedure by filtering the solution of extract from the coal before cooling, thereby permitting the insoluble bitumen to precipitate out of contact with the coal. I n this manner it was readily recovered. Results of Extractions

Extractions were continued until the yield of soluble substance obtained during a 4-hour extraction did not exceed 0.5 per cent of the coal (moisture- and ash-free basis). Table I1 gives the yields of the extracts obtained, together Tvith other extraction data.