FERMENTATION OF CELLULOSE
V. Omelianski (Compt. Rend., 1897, 125, 1131-1133).
Pure paper was allowed to ferment in the presence of calcium carbonate at a temperature of 35° for 13 months. The products obtained from 3.4743 grams of paper were: acids of the acetic series, 2.2402 grams; carbonic anhydride, 0.9722 grams; and hydrogen, 0.0138 gram. The acids were chiefly acetic and butyric acid, the ratio of the former to the latter being 1.7: 1. Small quantities of valeric acid, higher alcohols, and odorous products were formed.
The absence of methane from the products of fermentation is remarkable, but the formation of this gas seems to be due to a special organism readily distinguishable from the ferment that produces the fatty acids. This organism is at present under investigation.
(p. 75) Constitution of Cellulose.—It may be fairly premised that the problem of the constitution of cellulose cannot be solved independently of that of molecular aggregation. We find in effect that the structural properties of cellulose and its derivatives are directly connected with their constitution. So far we have only a superficial perception of this correlation. We know that a fibrous cellulose treated with acids or alkalis in such a way that only hydrolytic changes can take place is converted into a variety of forms of very different structural characteristics, and these products, while still preserving the main chemical characteristics of the original, show when converted into derivatives by simple synthesis, e.g. esters and sulphocarbonates, a corresponding differentiation of the physical properties of these derivatives, from the normal standard, and therefore that the new reacting unit determines a new physical aggregate. Thus the sulphocarbonate of a 'hydrocellulose' is formed with lower proportions of alkaline hydrate and carbon disulphide, gives solutions of relatively low viscosity, and, when decomposed to give a film or thread of the regenerated cellulose, these are found to be deficient in strength and elasticity. Similarly with the acetate. The normal acetate gives solutions of high viscosity, films of considerable tenacity, and when those are saponified the cellulose is regenerated as an unbroken film. The acetates of hydrolysed celluloses manifest a retrogradation in structural and physical properties, proportioned to the degree of hydrolysis of the original.
We may take this opportunity of pointing out that the celluloses not only suggest with some definiteness the connection of the structural properties of visible aggregates—that is, of matter in the mass—with the configuration of the chemical molecule or reacting unit, but supply unique material for the actual experimental investigation of the problems involved. Of all the 'organic' colloids cellulose is the only one which can be converted into a variety of derivative forms, from each of which a regular solid can be produced in continuous length and of any prescribed dimensions. Thus we can compare the structural properties of cellulose with those of its hydrates, nitrates, acetates, and benzoates, in terms of measurements of breaking strain, extensibility, elasticity. Investigations in this field are being prosecuted, but the results are not as yet sufficiently elaborated for reduction to formulæ. One striking general conclusion is, however, established, and that is that the structural properties of cellulose are but little affected by esterification and appear therefore to be a function of the special arrangement of the carbon atoms, i.e. of the molecular constitution. Also it is established that the molecular aggregate which constitutes a cellulose is of a resistant type, and undoubtedly persists in the solutions of the compounds.
It may be urged that it is superfluous to import these questions of mass-aggregation into the problem of the chemical constitution of cellulose. But we shall find that the point again arises in attempting to define the reacting unit, which is another term for the molecule. In the majority of cases we rely for this upon physical measurements; and in fact the purely chemical determination of such quantities is inferential. Attempts have been made to determine the molecular weights of the cellulose esters in solution, by observations of depression of solidifying and boiling-points. But the numbers have little value. The only other well-defined compound is the sulphocarbonate. It has been pointed out that, by successive precipitations of this compound, there occurs a continual aggregation of the cellulose with dissociation of the alkali and CS residues and it has been found impossible to assign a limit to the dissociation, i.e. to fix a point at which the transition from soluble sulphocarbonate to insoluble cellulose takes place.
On these grounds it will be seen we are reduced to a somewhat speculative treatment of the hypothetical ultimate unit group, which is taken as of C6 dimensions.
As there has been no addition of experimental facts directly contributing to the solution of the problem, the material available for a discussion of the probabilities remains very much as stated in the first edition, pp. 75-77. It is now generally admitted that the tetracetate n [C6H6O.(OAc)4] is a normal cellulose ester; therefore that four of the five O atoms are hydroxylic. The fifth is undoubtedly carbonyl oxygen. The reactions of cellulose certainly indicate that the CO- group is ketonic rather than aldehydic. Even when attacked by strong sulphuric acid the resolution proceeds some considerable way before products are obtained reducing Fehling's solution. This is not easily reconcilable with any polyaldose formula. Nor is the resistance of cellulose to very severe alkaline treatments. The probability may be noted here that under the action of the alkaline hydrates there occurs a change of configuration. Lobry de Bruyn's researches on the change of position of the typical CO- group of the simple hexoses, in presence of alkalis, point very definitely in this direction. It is probable that in the formation of alkali cellulose there is a constitutional change of the cellulose, which may in effect be due to a migration of a CO- position within the unit group. Again also we have the interesting fact that structural changes accompany the chemical reaction. It is surprising that there should have been no investigation of these changes of external form and structure, otherwise than as mass effects. We cannot, therefore, say what may be the molecular interpretation of these effects. It has not yet been determined whether there are any intrinsic volume changes in the cellulose substance itself: and as regards what changes are determined in the reacting unit or molecule, we can only note a fruitful subject for future investigation. A priori our views of the probable changes depend upon the assumed constitution of the unit group. If of the ordinary carbohydrate type, formulated with an open chain, there is little to surmise beyond the change of position of a CO- group. But alternative formulæ have been proposed. Thus the tetracetate is a derivative to be reckoned with in the problem. It is formed under conditions which preclude constitutional changes within the unit groups. The temperature of the main reaction is 30°-40°, the reagents are used but little in excess of the quantitative proportions, and the yields are approximately quantitative. If now the derivative is formed entirely without the hydrolysis the empirical formula C6H6O.(OAc)4 justifies a closed-ring formula for the original viz. CO<[CHOH]4>CH2; and the preference for this formula depends upon the explanation it affords of the aggregation of the groups by way of CO-CH2 synthesis.
The exact relationship of the tetracetate to the original cellulose is somewhat difficult to determine. The starting-point is a cellulose hydrate, since it is the product obtained by decomposition of the sulphocarbonate. The degree of hydrolysis attending the cycle of reactions is indicated by the formula 4 C6H10O5.H2O. It has been already shown that this degree of hydrolysis does not produce molecular disaggregation. If this hydrate survived the acetylation it would of course affect the empirical composition, i.e. chiefly the carbon percentage, of the product. It may be here pointed out that the extreme variation of the carbon in this group of carbohydrate esters is as between C14H20O10 (C = 48.3 p.ct.) and C14H18O9 (C = 50.8 p.ct.) i.e. a tetracetate of C6H12O6 and C6H10O5 respectively. In the fractional intermediate terms it is clear that we come within the range of ordinary experimental errors, and to solve this critical point by way of ultimate analysis must involve an extended series of analyses with precautions for specially minimising and quantifying the error. The determination of the acetyl by saponification is also subject to an error sufficiently large to preclude the results being applied to solve the point. While, therefore, we must defer the final statement as to whether the tetracetate is produced from or contains a partly hydrolysed cellulose molecule, it is clear that at least a large proportion of the unit groups must be acetylated in the proportion C6H6O.(OAc)4.
It has been shown that by the method of Franchimont a higher proportion of acetyl groups can be introduced; but this result involves a destructive hydrolysis of the cellulose: the acetates are not derivatives of cellulose, but of products of hydrolytic decomposition.
It appears, therefore, that with the normal limit of acetylation at the tetracetate the aggregation of the unit groups must depend upon the CO- groups and a ring formula of the general form CO<[CHOH]4>CH2 is consistent with the facts.
Vignon has proposed for cellulose the constitutional formula
with reference to the highest nitrate, and the decomposition of the nitrate by alkalis with formation of hydroxypyruvic acid. While these reactions afford no very sure ground for deductions as to constitutional relationships, it certainly appears that, if the aldose view of the unit group is to be retained, this form of the anhydride contains suggestions of the general tendency of the celluloses on treatment with condensing acids to split off formic acid in relatively large quantity [Ber. 1895, 1940]; the condensation of the oxycelluloses to furfural; the non-formation of the normal hydroxy-dicarboxylic acids by nitric acid oxidations. Indirectly we may point out that any hypothesis which retains the polyaldose view of cellulose, and so fails to differentiate its constitution from that of starch, has little promise of progress. The above formula, moreover, concerns the assumed unit group, with no suggestion as to the mode of aggregation in the cellulose complex. Also there is no suggestion as to how far the formula is applicable to the celluloses considered as a group. In extending this view to the oxycelluloses, Vignon introduces the derived oxidised group
—of which one is apportioned to three or four groups of the cellulose previously formulated: these groups in condensed union together constitute an oxycellulose.
These views are in agreement with the experimental results obtained by Faber and Tollens (p. 71). They regard the oxycelluloses as compounds of 'celloxin' C6H_8{O}6 with 1-4 mols. unaltered cellulose; and the former they particularly refer to as a lactone of glycuronic acid. But on boiling with lime they obtain dioxybutyric and isosaccharinic acids; both of which are not very obviously related to the compounds formulated by Vignon. We revert with preference to a definitely ketonic formula, for which, moreover, some farther grounds remain to be mentioned. In the systematic investigation of the nitric esters of the carbohydrates (p. 41) Will and Lenze have definitely differentiated the ketoses from the aldoses, as showing an internal condensation accompanying the ester reaction. Not only are the OH groups taking part in the latter consequently less by two than in the corresponding aldoses, but the nitrates show a much increased stability. This would give a simple explanation of the well-known facts obtaining in the corresponding esters of the normal cellulose. We may note here that an important item in the quantitative factors of the cellulose nitric ester reaction has been overlooked: that is, the yield calculated to the NO3 groups fixed. The theoretical yields for the higher nitrates are
| Yield p.ct. of cellulose | N p.ct. of nitrate | |
| Pentanitrate | 169 | 12.7 |
| Hexanitrate | 183 | 14.1 |
From such statistics as are recorded the yields are not in accordance with the above. There is a sensible deficiency. Thus Will and Lenze record a yield of 170 p.ct. for a product with 13.8 p.ct. N, indicating a deficiency of about 10 p.ct. As the by-products soluble in the acid mixture are extremely small, the deficiency represents approximately the water split off by an internal reaction. In this important point the celluloses behave as ketoses.
In the lignocelluloses the condensed constituents of the complex are of well-marked ketonic, i.e. quinonic, type. In 'nitrating' the lignocelluloses this phenomenon of internal condensation is much more pronounced (see p. 131). As the reaction is mainly confined to the cellulose of the fibre, we have this additional evidence that the typical carbonyl is of ketonic function. It is still an open question whether the cellulose constituents of the lignocelluloses are progressively condensed—with progress of 'lignification'—to the unsaturated or lignone groups. There is much in favour of this view, the evidence being dealt with in the first edition, p. 180. The transition from a cellulose-ketone to the lignone-ketone involves a simple condensation without rearrangement; from which we may argue back to the greater probability of the ketonic structure of the cellulose. We must note, however, that the celluloses of the lignocelluloses are obtained as residues of various reactions, and are not homogeneous. They yield on boiling with condensing acids from 6 to 9 p.ct. furfural. It is usual to regard furfural as invariably produced from a pentose residue. But this interpretation ignores a number of other probable sources of the aldehyde. It must be particularly remembered that lævulose is readily condensed (a) to a methylhydroxyfurfural
C6H1O6 - 3H2O = C6H6O3 = C5(OH).H2.(CH3)O2
and (b) by HBr, with further loss of OH, as under:
C6H12O6 - 4H2O + HBr = C5H3(CH2Br)O
and generally the ketoses are distinguished from the aldoses by their susceptibility to condensation. Such condensation of lævulose has been effected by two methods: (a) by heating the concentrated aqueous solution with a small proportion of oxalic acid at 3 atm. pressure [Kiermayer, Chem. Ztg. 19, 100]; (b) by the action of hydrobromic acid (gas) in presence of anhydrous ether; the actual compound obtained being the ω-brommethyl derivative [Fenton, J. Chem. Soc. 1899, 423].
This latter method is being extended to the investigation of typical celluloses, and the results appear to confirm the view that cellulose may be of ketonic constitution.
The evidence which is obtainable from the synthetical side of the question rests of course mainly upon the physiological basis. There are two points which may be noted. Since the researches of Brown and Morris (J. Chem. Soc. 1893, 604) have altered our views of the relationships of starch and cane sugar to the assimilation process, and have placed the latter in the position of a primary product with starch as a species of overflow and reserve product, it appears that lævulose must play an important part in the elaboration of cellulose. Moreover, A. J. Brown, in studying the cellulosic cell-collecting envelope produced by the Bacterium xylinum, found that the proportion of this product to the carbohydrate disappearing under the action of the ferment was highest in the case of lævulose. These facts being also taken into consideration there is a concurrence of suggestion that the typical CO group in the celluloses is of ketonic character. That the typical cotton cellulose breaks down finally under the action of sulphuric acid to dextrose cannot be held to prove the aldehydic position of the carbonyls in the unit groups of the actual cellulose molecule or aggregate.
We again are confronted with the problem of the aggregate and as to how far it may affect the constitution of the unit groups. That it modifies the functions or reactivity of the ultimate constituent groups we have seen from the study of the esters. Thus with the direct ester reactions the normal fibrous cellulose (C6H16O5) yields a monoacetate, dibenzoate, and a trinitrate respectively under conditions which determine, with the simple hexoses and anhydrides, the maximum esterification, i.e. all the OH groups reacting. If the OH groups are of variable function, we should expect the CO groups a fortiori to be susceptible of change of function, i.e. of position within the unit groups.
But as to how far this is a problem of the constitution or phases of constitution of the unit groups or of the aggregate under reaction we have as yet no grounds to determine.
The subjoined communication, appearing after the completion of the MS. of the book, and belonging to a date subsequent to the period intended to be covered, is nevertheless included by reason of its exceptional importance and special bearing on the constitutional problem above discussed.
THE ACTION OF HYDROGEN BROMINE ON CARBOHYDRATES.[4]
H. J. H. Fenton and Mildred Gostling (J. Chem. Soc., 1901, 361).
The authors have shown in a previous communication (Trans., 1898, 73, 554) that certain classes of carbohydrates when acted upon at the ordinary temperature with dry hydrogen bromide in ethereal solution give an intense and beautiful purple colour.[5] It was further shown (Trans., 1899, 75, 423) that this purple substance, when neutralised with sodium carbonate and extracted with ether, yields golden-yellow prisms of ω-brommethylfurfural,
This reaction is produced by lævulose, sorbose, cane sugar, and inulin, an intense colour being given within an hour or two. Dextrose, maltose, milk sugar, galactose, and the polyhydric alcohols give, if anything, only insignificant colours, and these only after long standing. The authors therefore suggested that the reaction might be employed as a means of distinguishing these classes of carbohydrates, the rapid production of the purple colour being indicative of ketohexoses, or of substances which produce these by hydrolysis.
By relying only on the production of the purple colour, however, a mistake might possibly arise, owing to the fact that xylose gives a somewhat similar colour after standing for a few hours. Hence, the observations should be confirmed by isolation of the crystals of brommethylfurfural. No trace of this substance is obtained from the xylose product.
In order to identify the substance, the ether extract, after neutralisation, is allowed to evaporate to a syrup, and crystallisation promoted either by rubbing with a glass rod, or by the more certain and highly characteristic method of 'sowing' with the most minute trace of ω-brommethylfurfural, when crystals are almost instantly formed. These are recrystallised from ether, or a mixture of ether and light petroleum, and further identified by the melting-point (59.5-60.5°), and, if considered desirable, by estimation of the bromine.
It is now found, so reactive is the bromine atom in this compound, that the estimation may be accurately made by titration with silver nitrate according to Volhard's process, the crystals for this purpose being dissolved in dilute alcohol:
0.1970 gram required 10.5 c.c. N/10 AgNO3. Br = 42.63 p.ct., calculated 42.32 p.ct.
This method of applying hydrogen bromide in ethereal solution is, of course, unsuitable for investigations where a higher temperature has to be employed, or where long standing is necessary, since, under such circumstances, the ether itself is attacked. Wishing to make investigations under these conditions, the authors have tried several solvents, and, at present, find that chloroform is best suited to the purpose. In each of the following experiments, 10 grms. of the substance were covered with 250 c.c. of chloroform which had been saturated at 0° with dry hydrogen bromide. The mixture was contained in an accurately stoppered bottle, firmly secured with an iron clamp, and heated in a water-bath to about the boiling temperature for two hours. After standing for several hours, the mixture was treated with sodium carbonate (first anhydrous solid, and afterwards a few drops of strong solution), filtered, and the solution dried over calcium chloride. Most of the chloroform was then distilled off, and the remaining solution allowed to evaporate to a thick syrup in a weighed dish.
The product was then tested for ω-brommethylfurfural by 'sowing' with the most minute trace of the substance, as described above. It was then warmed on a water-oven, kept in a vacuum desiccator over solid paraffin, and the weight estimated. When necessary, the product was recrystallised from ether, and further identified by the tests mentioned. The following results were obtained:
| Weight of crude residue. | |||
| Swedish filter paper | 3.0 | crystallised at once | by 'sowing.' |
| Ordinary cotton | 3.3 | " | " |
| Mercerised cotton | 2.1 | " | " |
| Straw cellulose[6] | 2.3 | " | " |
| Lævulose | 2.2 | " | " |
| Inulin | 1.3 | " | " |
| Potato starch | 0.37 | " | " |
| Cane sugar | 0.85 | " | " |
| Dextrose | 0.33 | uncrystallisable. | |
| Milk sugar | 0.37 | " | |
| Glycogen | 0.34 | " | |
| Galactose | 0.34 | " | |
The products from dextrose, milk sugar, and galactose absolutely refused to crystallise even when extracted with ether and again evaporated, or by 'sowing,' stirring, &c.
The glycogen product deposited a very small amount of crystalline matter on standing, but the quantity was too minute for examination; moreover, it refused altogether to crystallise in contact with the aldehyde. It may fairly be stated, therefore, that these last four substances give absolutely negative results as regards the formation of ω-brommethylfurfural; if any is formed, its quantity is altogether too small to be detected.
The specimen of starch examined was freshly prepared from potato, and purified by digestion for twenty-four hours each with N/10 KOH, N/4 HCl, and strong alcohol; it was then washed with water and allowed to dry in the air. It will be seen that this substance gave a positive result, but that the yield was extremely small, and might yet be due to impurity. Considering the importance of the behaviour of starch, for the purpose of drawing general conclusions from these observations, it was thought advisable to make further experiments with specimens which could be relied upon, and also to investigate the behaviour of dextrin. This the authors have been enabled to do upon a series of specimens specially prepared by C. O'Sullivan, and thus described by him:
1. Rice starch, specially purified by the permanganate method.
2. Wheat starch " " "
3. Oat starch, contains traces of oil, washed with dilute KOH and dilute HCl.
4. Pea starch, first crop, washed with alkali, acid (HCl), and strong alcohol.
5. Natural dextrin, D = 3.87, αD = 194.7; K = 0.95, (c 2.628).
6. α-Dextrin, C equation purified without fermentation, 30 precipitations with alcohol (Trans., 1879, 35, 772).
The examination of these specimens was conducted on a smaller scale, but under the same conditions as before, one gram of the substance being treated with 12.5 c.c. of the saturated chloroform solution and heated in sealed tubes for two hours as above. The results were as follows:
| Weight of crude residue. | |||
| 1. Rice starch | 0.046 | crystallised at once | by 'sowing.' |
| 2. Wheat starch | 0.044 | " | " |
| 3. Oat starch | 0.049 | " | " |
| 4. Pea starch | 0.064 | " | " |
| 5. Natural dextrin | 0.088 | " | " |
| 6. α-Dextrin | 0.055 | " | " |
The results may therefore be summarised as follows:—Treated under these particular conditions all forms of cellulose give large yields of ω-brommethylfurfural, some varieties giving as much as 33 per cent. Lævulose, inulin, and cane sugar give yields varying from 22 to 8.5 per cent.; various starches give small yields (average about 4.5 per cent.); and dextrins 5 to 8 per cent., whereas dextrose, milk sugar, and galactose give, apparently, none at all.
The yields represent the solid crystalline residue; this when purified by recrystallisation gives, probably, about three-quarters of its weight of pure crystals. (In the case of dextrose, &c., the yields represent the weight of syrup.)
These numbers, however, by no means represent the maximum yields obtainable, owing to the comparatively slight solubility of hydrogen bromide in chloroform. The process was conducted in the above manner only for the sake of uniform comparison. The ether method previously described gives much larger yields; for example, 12 grms. of inulin treated with only 60 c.c. of the saturated ether gave 2.5 grms. of substance. For the purpose of obtaining larger yields, other methods are being investigated.
The facts recorded above, taken in conjunction with those given in our previous communications, appear to point definitely to the following general conclusions. First, that the various forms of cellulose contain one or more groups or nuclei identical with that contained in lævulose, and that such groups constitute the main or essential part of the molecule. Secondly, that similar groupings are contained in starches and dextrins, but that the proportion of such groupings represents a relatively small part of the whole structure.
The nature of this grouping is, according to the generally accepted constitution of lævulose, the six-carbon chain with a ketonic group:
But the results might, on the other hand, be considered indicative of the anhydride or 'lacton' grouping, which Tollens suggested for lævulose:
The latter very simply represents the formation of ω-brommethylfurfural from lævulose,[7]
giving
although by a little further 'manipulation' of the symbols the change could, of course, be represented by reference to the ketonic formula.