Butyl Alcohol Fermentation
of W o o d Sugar N. 0. SJOLANDER, A. F. LANGLYKKE, AND W. H. PETERSON University of Wisconsin, Madison, Wis.
T
HE conversion of waste wood into sugar has been exliminary treatment of the hydrolyzate was necessary in order to tensively investigated in recent years, especially in remove substances which inhibited the growth of microorganGermany where two processes for the production of isms. As a result of numerous experiments, which need not wood sugar have been developed and are in use a t the present be reported here, the following procedure was adopted: The time. I n the Bergius process (1) superconcentrated hydrowood liquor was neutralized with an excess of calcium carbonchloric acid a t relatively low temperatures is employed for ate, the calcium sulfate was filtered off and the filtrate made hydrolysis. Scholler (8) developed the other process, in alkaline to about p H 10 with lime in order to precipitate iron which dilute sulfuric acid (0.2-1.0 per cent) is percolated and copper. After filtration the liquor was made slightly acid through shredded wood under pressure a t elevated tempera(about pH 6.5), clarified with Korite, and adjusted to pH 6.0. tures (e. g., 160" C.). A dilute solution of sugar is obtained This solution was used for the preparation of media. which has so far been utilized largely for the production of Thirteen batches of wood sugar liquor were studied. They alcohol and yeast. were prepared by slightly different methods or from different The present investigamaterials, with the result tion is concerned with the that there was some variaf e r m e n t a t i o n of d i l u t e tion in amount of impuriwood sugar solutions simities, concentration of reSugars obtained from wood by hylar to those obtained in the ducing sugar, and ratio of drolysis with dilute sulfuric acid at eleScholler process. The pentoses to hexoses. vated temperatures were readily ferhydrolyzates were furnished by the Dow Chemimented by the butyl anaerobes, ClostriSelection of cal Company. Most of dium felsineum and Clostridium butyliCultures the liquors contained cu m. The hydrolyzates were prepared for A preliminary survey about 1 per cent of sulfuric fermentation by treatment with calcium was made to determine acid and from 3 to 6 per the ability of selected culcarbonate, lime, and Norite. A malt cent of reducing sugar, tures from various groups calculated as glucose. The sprouts medium was most satisfactory of microorganisms to utih y d r ol y z a t es prepared for maximum sugar utilization. The lize wood sugar. These from hardwoods contained addition of corn or other supplementary included yeasts, molds, chiefly pentoses; those carbohydrate was unnecessary. and many species of bacfrom softwoods contained teria-via., those forming Wood sugar i n concentrations up to more of the hexose sugars. propionic acid, lactic acid, I n the fermentation of about 5 per cent was almost completely ethyl alcohol, and butyl wood sugar, therefore, it fermented. Thirty to forty per cent of alcohol. Suitable media appeared desirable t o the fermented sugar was converted into for each group were preselect organisms which pared, inoculated with acneutral volatile products. The distribuwere capable of converttively growing cultures, tion of products formed by Cl. felsineum ing both pentoses and and incubated a t the optihexoses into useful prodwas practically the same f r o m wood mum temperatures until ucts. The butyl-alcoholsugar as from glucose. Butyl alcohol f e r m e n t a t i o n ceased. forming bacteria seemed was the principal product ; smaller Wood sugar utilization to be particularly suitable, was determined by analyamounts of ethyl alcohol and acetone and the fermentation of ses, for reducing sugar were formed. Cl. bufylicurn produced, wood sugar by this group (calculated as glucose) acof micro5rganisms was in addition, considerable amounts of cording t o the method of most extensively studied. isopropyl alcohol (more from wood sugar Stiles, Peterson, and Fred than from glucose). I t is shown that this (9). Preliminary TreatTable I summarizes the additional isopropyl alcohol arose from ment of Wood data for some fermentathe acetic acid present in the wood Hydrolyzate tions by bacteria and hydrolyzates. Before the wood sugar yeasts. Propionibaccould be fermented, a preterium pentosaceum 1251
INDUSTRIAL AND ENGINEERING CHEMISTRY
1252
utilized quite large amounts of hemlock wood sugar but only after long incubation. As noted in previous papers, the Lactobacilli fermented this material rapidly and nearly completely. Pseudomonas lindneri, an ethyl-alcohol-producing bacterium, fermented wood sugar very poorly. About 75 per cent of the hemlock wood sugar was fermented by the two strains of yeast in 5 days. These cultures, however, utilized much less of the hardwood sugars which contain greater proportions of pentose sugar. OF HEMLOCK WOODSUGAR BY TABLE I. EERMENTATION VARIOUSMICRO~RQANISMS
Incubatjon Period Days Propionabacterium pentosaceum 5 22 Organism
Reducing Sugar Calcd. as Glucose Original concn. Fermented
%
%
3 08 3.08
38 72
Lactobacillus pentoaceticus
5 22
3.08 3.08
63 64
Lactobacillus pentosus
5 22
3.08 3.08
8s
Pseudomonas lindneri Hungarian yeast Malaga yeast
9 5 5
3.26 3.04 3.04
29 75 75
95
Mold fermentations of wood sugar were carried out with variow strains of Aspergillus niger. Although the molds were able in some cases to grow on the wood sugar media, there was no accumulation of citric acid during the fermentation. Low yields of citric acid from wood sugar have been reported by Palei and Frantzuzova (7). The results of fermentations by some of the butyl-alcoholforming bacteria are recorded in Table 11. The cultures were selected from the stocks a t the laboratory on the basis of their ability to ferment glucose and xylose. They are chiefly butyl alcohol formers and include a strain of Clostridium acetobutylic u m (designated BY). Sugar analyses were made after in-
TABLE 11. FERMENTATION O F WOOD BUTYLANAEROBES 7 -
Culture
--Hemlock Original concn.
SUGAR BY
Reducing Sugar Calcd. as Glucose-lo--Beech 11Original Fermented ooncn. Fermented
No.
%
%
%
8
3.73 3.73 3.24 3.73 3.30 3.73 3.73 3.73 3.73 3.22 3.22 3.22 3.22
83 84 85 79 87 29 83 87 21 29 37 15 20
5.15 5.15 4.63 5.15 4.88 5.15 5.15 5.15 5.15 2.27 2.27 2.27 2.27
22 41 42 64 69 73 77 BY P-A P-B2 P-B3 P-20
% 0
0 79 0 62 0
0 0 18 21 30 53 22
cubation periods of 5 days a t 37 O C. The results show that a number of the butyl anaerobes were capable of readily fermenting the hemlock wood sugar, but most of them were unable to attack the beech wood sugar a t the higher concentrations. Cultures 41, which is Clostridium felsineum (Carbone) Donker, and 64, which was isolated at this laboratory and identified as Clostridium butylicum (Beijerinck) Donker, showed exceptional ability in fermenting both the softwood and hardwood sugars. Since these cultures are also known to form useful products from sugars, the remainder of the work was devoted to a study of the fermentation of wood sugar by C1.felsineum and Cl. butylicum.
VOL. 30, NO. 11
Selection of Base Medium The treated wood sugar solution without added nutrients was unsatisfactory for supporting growth of butyl anaerobes. A number of experiments were therefore carried out in order to find the most suitable base medium for wood sugar utilization. The media were prepared with various batches of treated wood liquors, inoculated with active cultures of Cl. felsineum and C1. butylicum and analyzed for reducing sugar after 5 days a t 37" C. Some of the nutrient materials tested in this way were corn mash, potato mash, malt sprouts, tryptone, liver, corn gluten, and corn steep. Malt sprouts in a concentration of about 2 per cent was found to be superior to any of the other nutrients tried. The addition of small amounts of dried whole, liver and dibasic ammonium phosphate to the malt sprouts medium gave still better utilization of the wood sugar, especially at the higher concentrations, The further addition of asparagine had no apparent effect upon the fermentation. A small amount of calcium carbonate added to the sterile media slightly increased sugar utilization, probably by enabling the organism to overcome the initial acidity and allowing formation of neutral volatile products. On the basis of these results the following medium was adopted: 2 per cent malt sprouts, 0.25 per cent dried whole liver, 0.05 per cent dibasic ammonium phosphate, treated wood sugar solution, and 0.1 per cent calcium carbonate. (The calcium carbonate was sterilized separately and added aseptically to the sterile medium.) This medium was used for the remainder of the work because it apparently supplied sufficient nutrients for maximum wood sugar utilization by the butyl anaerobes. In industrial practice, however, it might be possible to reduce in quantity, replace, or eliminate some of the more expensive constituents such as dried whole liver,
Additional Factors Affecting Fermentability PREPARATION OF INOCULA. Since wood sugar, even a t the lower concentrations, was sometimes poorly fermented by the butyl anaerobes, it appeared that preparation of the inoculum might be a n important factor in the fermentation. A study was therefore made to determine the effectiveness of the inoculum as related to its age. Butyl anaerobes sporulate rapidly in corn mash media, and a culture which on microscopic examination showed a number of spores produced a weak fermentation. Wood sugar utilization by Cl. felsineum was most consistently high when the following procedure was employed in the preparation of the inoculum. A tube of cornliver medium was inoculated with the soil spore stock; after gas production became abundant (about 24 hours), 1 cc. of this culture was transferred to a second tube of corn-liver medium. This culture was used as a source of inoculum after about 10 to 15 hours, a t which time vigorous gassing was observed and almost all of the cells were found to be in the vegetative state. With C1. butylicum the same procedure was followed except that the use of a liver-broth culture for developing the final inoculum gave somewhat better results. I n some cases, especially a t higher concentrations of wood sugar, daily reinoculation of inactive fermentations finally resulted in growth and greater wood sugar utilization than was otherwise attained a t the same concentrations. The amount of wood sugar WOODSUQARCONCENTRATION. that could be fermented was found to depend also upon the original sugar concentration. The data in Table I11 show the effect of the concentration of beech 11 wood sugar upon its utilization by Cl. felsineum. At concentrations up to 3 or 4 per cent reducing sugar there was very little change in fermentability; a t from 4 to 5 per cent sugar utilization began to fall off, and above that it was markedly reduced. Although the actual amount of sugar utilized a t each concentration would be different with other batches of wood sugar, with
NOVEMBER, 1938
INDUSTRIAL AND ENGINEERING CHEMISTRY
other organisms, and under different experimental conditions, the effect of increased concentration would, In general, be the same. This decrease in fermentability a t the higher concentrations might be due to a number of factors. I n addition to increased osmotic pressure and increased amounts of metabolic products, there is the effect of an increased concentration of toxic substances formed in the hydrolysis.
1253
proving the fermentability of wood sugar. Vacuum distillation of the wood liquor without previous extraction was then tried. Batches of hemlock and beech wood liquors were diminished approximately 25 per cent in volume by vacuum distillation a t pH 5.0, then treated, made up into media, and inoculated as usual with cultures of Cl.felsineumand Cl. butylicum. Utilization of the hemlock wood sugar was unaffected by the distillation procedure. I n the case of the beech wood sugar, however, fermentation of the vacuum distilled liquor was somewhat better than that of the undistilled liquor. NeverTABLE111. EFFECTOF CONCENTRATION OF WOODSUGAR theless, it seems questionable whether the additional trouble (BEECH11) UPON ITS UTILIZATION BY CZ. felsineum and expense involved would warrant such a treatment. Sunarconcn.,a% 1.16 2 . 2 7 2 . 4 8 3 . 3 7 4.26 4 . 6 3 5 . 2 5 5 . 4 4 Sugar fermcnted,a The toxicity toward butyl anaerobes of substances removed yo of total 89 85 89 89 83 79 60 10 during vacuum distillation was demonstrated in the following a Reducing sugar calculated as glucose. experiment: Media were prepared from each wood liquor distillate with 5 per cent corn meal and 1 per cent powdered liver in undiluted distillate and in distillate diluted 1 to 1 with distilled water, and inoculated with Cl. felsineum and Cl. butyliAXAEROBICINCUBATION. Ordinarily all fermentations cum. Both cultures were definitely inhibited in the undilutedwere carried out in flasks or tubes plugged with cotton but distillate media, but there was little or no effect a t the 1 to 1 otherwise open to the air. I n this experiment half of the dilution. I n the undiluted distillate, however, the concentratubes were placed in a jar made anaerobic by the addition of tion of the more volatile toxic substances was probably greater moistened oats. The others were incubated aerobicallythan it was in the original wood liquor. It is not surprising, that is, exposed to the air-for controls. The media were pretherefore, that the cultures which were inhibited by the conpared as previously described and inoculated with cultures of centrated distillates can ordinarily ferment most of the wood Cl. felsineum and Cl. butylicum. Sugar analyses, made after sugar in the presence of the toxic substances. 5 days, showed that some slight improvement in wood sugar Qualitative tests indicated that furfural is probably an imutilization could be expected under strictly anaerobic incubaportant constituent of the volatile toxic fraction. A sodium tion. Later experiments showed that yield of products from bisulfite treatment of the hydrolyzate did not improve ferthe fermented sugar was also slightly greater under these conmentability of the wood sugar for Cl. felsineum. ditions. REMOVAL OF TOXIC SUBSTAXCES FROM WOODHYDROLYZATE. The difficulty with which the more concentrated wood W o o d Sugar Utilization by Yeasts and by sugars were fermented led to an investigation of toxic subCl. felsineum stances present in the hydrolyzates. When a natural organic Although most of the butyl anaerobes ferment pentoses in material as complex as wood is heated under pressure with addition to hexoses, yeasts are able to utilize only the hexose sulfuric acid, the formation of a number of decomposition sugars. Table IV presents a summary of data from a number products is to be expected. Hexosans, pentosans, tannins, of fermentations on the various wood sugar solutions by culresins, and numerous other substances are hydrolyzed into tures of yeasts and of Cl. felsineum. The wood hydroIyzates compounds which are often toxic to the growth of microcrganwere treated as previously described and diluted to a concenisms. Luers et al. (6) recently tested the toxicity of some of tration of about 1 per cent reducing sugar (except in the cases these cleavage products toward yeasts. In concentrations of of beech 4 and hemlock 6, in which the concentration was less than 0.01 per cent, menthol and pinene, which are derived about 3 per cent for the yeast fermentation). The medium for from resins, caused 25 per cent inhibition of yeast fermentayeasts was prepared by adding 0.5 per cent dibasic ammonium tion. Furfural, tannin, vanillin, and eucalyptol inhibited phosphate, 0.1 per cent monobasic potassium phosphate, and growth and fermentation a t concentrations of 0.1 per cent or 0.05 per cent magnesium sulfate, and adjusting to p H 5.0. less. Cleavage products of tannins were less toxic. They Three cultures of Saccharomyces species, designated Malaga, also found that furfural occurs in much larger quantities in Hungarian, and Fleischmann single cell, were found to be the the hardwood hydrolyzates, which may in part account for best wood sugar fermenters. The data given in Table I V for the greater difficulty with which the hardwood sugars are sugar utilization by yeasts are averages of a number of ferfermented. Steam distillation was recommended as a means mentations by these cultures. The range of variation on the for reducing the concentration of the more volatile toxic subsame sugar was small. The Cl. felsineum fermentations were stances. carried out as previously described except that the sugar soluEfforts in this connection were directed primarily toward tions were more dilute. the use of two methods of treatment-extraction and distillation. Acid, alkaline, and neutral wood liquors were extracted with a number of organic solvents, including benzene, ethyl ether, isopropyl ether, chloroform, butyl alcohol, and UTILIZATION BY YEASTS AND TABLEIV. WOODSUGAR amyl alcohol. Butyl alcohol extraction of the acid liquor was C1. felsineum the best of the various procedures tried. After the acid % of Sugar (as Glucose) Fermented liquor had been shaken with five successive portions of butyl Wood Sugar Batch BY yeasts By Cl. felsineicm alcohol amd freed of excess solvent by vacuum distillation, Beech 4 15a 87 sugar utilization was, in most cases, slightly improved for Hemlock 6 75" 90 Hemlock 7 76 89 yeasts arid Cl. felsineum. The higher concentrations of the 93 89 Maple 8 78 89 Maple 9 extract were toxic toward yeasts. One disadvantage of this 73 88 Hemlock 10 procedure, however, was that in addition t o removing some of Beech 11 19 89 17 88 Birch 12 the inhibiting substances, the butyl alcohol also extracted a Hardwood sawdust 13 25 89 portion of the sugar. Original concentration of reducing sugar was 3 per cent, in all others I n preliminary experiments steam distillation of the hydrolyabout 1 per cent. sates appeared t o be a rather unsatisfactory method for im0
The results show that C1. felsineum fermented a rather constant amount of sugar at this concentration, regardless of the type of wood from which it was obtained. The yeasts, however, showed great variation in their ability to utilize wood sugar, depending upon the relative proportions of hexose and nonfermentable pentose in the particular wood hydrolyzate. On sugars derived from hemlock (a softwood), yeasts fermented 70 to 80 per cent of the total reducing sugar. Only up to 25 per cent of the sugars from hardwoods, such as beech and birch, were fermented. Sugar utilization depended also upon the procedure followed in the hydrolysis of the wood. The high fermentability of the maple wood hydrolyzates, for example, was due t o previous removal of pentosans by mild initial hydrolyses. Maple 9 hydrolyzate was prepared at 180" C. with 1.3 per cent sulfuric acid on a washed residue from an initial hydrolysis at 160" C. Maple 8 hydrolyzate resulted from a treatment at 180" C. with 1.7 per cent acid on the washed residue from maple 9. The pentose content of the maple 8 batch was only about one-tenth that of maple 9.
TABLE V.
the method of Allgeier and Tatum (6). The distillation residue was made acid and steam-distilled to obtain the volatile acids, which were determined according t o a modification of the Virtanen and Pulkki distillation procedure (10). Typical results from analyscs on a number of fermentations are summarized in Table V. At these particular concentrations, both cultures showed exceptional alility in fermenting hemlock 10 and beech 11wood sugars. Since there are doubtless other nonfermentable reducing substances present in the hydrolyzate, 92 per cent probably approaches the limit of ferrnentablc sugar. The yield of neutral volatile products was also good in every case. Cl. felsineum produced large quantities of butyl alcohol and smaller amounts of ethyl alcohol and acetone. The percentage distribution of these products from hemlock 10 and beech 11wood sugars was similar t o that from glucose. There was only a slight increase in volatile acids during the fcrmentation. Butyl alcohol was also the principal neutral volatile product of Cl. butylicum, but this organism also formed significant
PRODUCTS OF FERMENTATIWS BY Cl. felsineum
Y-A-7
Carbohydrate Source
Culture
el. felsineum
Original concn. Fermented
Hemlock 10 Beech 11 Glucose
Butyl alcohol
Ethyl alcohol
Iso-
proppl Acetone alC(Jt1d
%
%"
%
%
%
%
92 88 97
34.0 31 .O 35.6
59 56 58
l18 7 20
24 26 22
0 0 0
92 90 99 75 98
34.6 33.2 29.8 26.8 28.7
59 5.1 70 68 49
9 8 7 10 1
4 4 3 3 9
28 35 20 19 41
+
+
Neutral Volatile Products
%
Hemlock 10 3.15 Beech 11 2.82 Glucose 2.90 Beech 11 (ether-extd.) 2.99 Glucose acetate 2.95 Based on the apparent sugar fermented. Volatile acids in uninoculated media (in mg. per CC.): Acetic 4.4 9.0 0.4 8.1
Cl. butylicum
2.31 2.82 2.90
61. butglioum
Hemlock 10 Beech 1 1 Beech 11 (ether-extd.) acetate Glucose
AND
Distribution of Neutral Volatile olatile 7 Products 4
Sugar as Glucose
a 6
VOL. 30, NO. 11
INDUSTRIAL AND ENGINEERING CHEMISTRY
1254
-
-
Volatile Acid9b
Acetic Butyric Mg. per cc. 5.1 1.1 9.0 2.1 2.2 0.4 2.4 4.8 1.0 1.1 4.3
1.3 3.3 1.1 1.5 2.7
Butyric 0
0.2
0
0.1
Products of Wood Sugar Fermentation by Cl. felsineum and Cl. butylicum I n previous experiments attempts were made to determine the optimum conditions for wood sugar utilization by C1. felsineum and CE. butylicum. After this information became available, wood sugar fermentations by these organisms were set up and analyzed for products in the following manner: Batches of hemlock 10 and beech 11 wood hydrolyzates were treated with calcium carbonate, lime, and Norite in the usual way. Distilled water was added to the treated liquor in order to reduce the concentration of toxic substances and to ensure more nearly complete sugar utilization. These solutions were adjusted to pH 6.0 and used to prepare media, which also contained 2 per cent malt sprouts, 0-25 per cent dried liver, and 0.05 per cent dibasic ammonium phosphate. After sterilization 0.1 per cent calcium carbonate was added Tubes of each medium were inoculated (5 per cent inoculum) with actively gassing cultures of CZ. felsineum in corn-liver culture and CZ. butylicum in liver broth culture. All tubes were incubated for 5 days a t 37 C, in an anaerobic jar. On the basis of sugar analyses, representative fermentations were selected and analyzed for products by the following procedure: A suitable aliquot of the fermentation liquor was made alkaline, and the neutral volatile products were distilled off. The distillate was analyzed for ethyl and butyl alcohols by Johnson's method (S),for acetone by Goodwin's modification (2) of the Messinger method, and for isopropyl alcohol by
quantities of isopropyl alcohol and little or no ethyl alcohol and acetone. The results in Table V show the extent of variation in the relative amounts of butyl alcohol and isopropyl alcohol formed by C1. butylicum from hemlock 10 and beech 11 wood sugars and glucose. More isopropyl alcohol was formed from the wood sugars than from glucose. I n this connection it is interesting to note that the acetic acid decreased during the fermentation of wood sugar. It seemed reasonable then t o assume that the increase in isopropyl alcohol might be associated with the disappearance of acetic acid. I n the generally accepted mechanism (4) for the dissimilation of glucose by organisms of this type, isopropyl alcohol is believed to arise from glucose through acetic acid by condensation and decarboxylation to form acetone, which' is then reduced to isopropyl alcohol. This possible explanation was tested by two experiments. In the first, some of the beech 11wood hydrolyzate was extracted continuously for 48 hours with ethyl ether, and then treated, made up into media, and inoculated in the usual manner. The medium was practically free from volatile acids before fermentation. The distribution of neutral volatile products was in this case similar to that of the same products from glucose-large quantities of butyl alcohol and small amounts of isopropyl alcohol. I n a second experiment calcium acetate was added t o the glucose-malt sprouts medium in a concentration corresponding t o that of acetic acid in beech 11 wood hydrolyzate. Analyses on this fermentation are also recorded in Table V. Almost half of the added ace-
NOVEMBER, 1938
INDUSTRIAL AND ENGINEERTNG CHEMISTRY
tate was fermented, and the yield of isopropyl alcohol was twice as great as that in a normal glucose fermentation. The yield of butyl alcohol was reduced and the percentage distribution of neutral volatile products corresponded more nearly t o that of a wood sugar fermentation. It appears, therefore, that the increased yield of isopropyl alcohol formed from wood sugar by 61. butylicum is due to the conversion Of acetic acid in the wood hydrolyzate into this alcohol.
Acknowledgment Acknowledgment is made to the Dow Chemical Company for establishing a fellowship which made this work possible, for supplying the wood hydrolyzates, and for coBperation and advice during the progress of the investigation.
12.55
Literature Cited (1) (2) (3) (4) (5)
~ ~F., ~ ~ i 247 (1937). ~ ~ , cHIM., 29, Goodwin, L. F., J. Am. Chem. SOC.,42, 39 (1920). Johnson, M. J., IND. E N O . CHEM.,Anal. Ed., 4, 20 (1932). A. J.i Ergeb* Enzymforsch** 4 1230 Langlykke, A. F., Peterson, W. H., and McCoy, E., J. Bact., K1uyverp
29, 333 (1935). (6) Luers, H., Fries, G . , Hiittinger, W., Morike, E., Enders,
C.,
KBrnbaoh, K., and Wieninger, F., Z . Spiritusind., 60, 7 (1937).
(7) Palei. T. Y . , and Frantzuzova, M. A., Proc. Inst. Sci. Research Food Znj. (U.S. S. R.), 3, 7 (1936). (8) Soholler, H., Chem.-Ztg., 60, 293 (1936). (9) Stiles, H. R . , Peterson, W. H., and Fred, E. B., J. Bact., 12, 4 2 7 (1926).
(10)Virtanen, A. I., and Pulkki. L.,
J. Am. Chem.
SOC.,50, 3138
(1928).
RECEIVED June 6, 1938.
THE ALCHEMIST B y Jan Weiland
y T k g a i n we thank Sir William J. Pope for his kind cooperation in supplying a photograph of one of t h e paintings in his extensive collection, enabling us t o add still another Dutch artist t o the series. Jan Weiland -as born in Rotterdam about 1861 and after completing his studies practiced his art, largely a s a genre painter in Rotterdam where he later became professor at t h e Academy. The original, 23 b y 15l/2 inches in size, is o n canvas. This is No. 95 in t h e Berolzheimer Series of Alchemical and Historical Reproductions. D. D. Berolzheimer 50 E a s t 41st Street New York, N. Y. A list of Reproductions Nos. 1 t o 60 appeared in our issue of January, 1936, page 129; the list of Nos. 61 t o 72 appeared in January, 1937. paKe 74; Nos. 73 to 84 are listed in January 1938, page 70, where also is shown No. 85 with details for ob: taining photographic copies of the originals. No. 86 appears on page 145, February issue; No. 87, page 269. Marrh jssue; No. 85, pare 427. April issue; No. 89, page 500, May issue; No. 90, page 630, June issue; No. 91, page 834 July issue: No. 92, page 033, August issue; Np. 93, pase 9 9 i , September issue; No. 94, pake 1162, October issue. The photographs of these paintings are aupplied in black and white only
