CA1192541A - Solubilisation and hydrolysis of carbohydrates - Google Patents

Abstract

Abstract A process for the modification, solubilisation and/or hydrolysis of a glycosidically linked carbohydrate having reduc-ing groups using a mixture comprising water, an inorganic acid and a halide of lithium, magnesium or calcium. The process is particularly useful for converting cellulose (derived for example from waste-paper, wood or sawdust) or starch to glucose.
When cellulose is the starting material the preferred halide is a lithium halide. When starch is the starting material a mag-nesium halide is preferred.

Description

;1420 ~ his invention relates to the solu~ilisation and hydro-lysis of glycosidically liDked carbohydrates having reducing groups and ~n particular to the solubilisation of cell~lose or starch and hydrolysis of cellulose or starch to soluble oligo-saccharides and/Qr glucose.
Cellulose is a polysaccharide which forms the principal component of the cell walls of most plants4 It is a polymer of ~-D-glucose units which are linked togetheT with el;~;nation of water to fo~m chains of 2000 4000 units. In plants it occurs together with polysaccharides and hemicelluloses derived from other su~ars such as xylose, arabinose and man~ose~ I~ the woody g ~,i parts of plants cellulose is intima-tely mixed and somet'mes co~al~
ently linked with lignin. Wood, for instance, normally conta~
15 40 5C% cellulose, 20 - ~/0 lignin and 10 - 3~0 hemicelluloses together with mineral salts, protelns and other biochemical compounds~
Degradation of cellulose may be brough-t abou-t by various treatments, including trea-tment with acids and with en~ymes present in certa~n bacteria, fungi and protozoa, and results prLmarily in the cleavage of the cellulose chain molecules and consequently in a reduction of` molecular weigh-t. Partial hydrolysis with acids proauces a variety of products, often terned "hydrocelluloses"~
whose pr~perties are de-termined by the hydrolysis conditions em-ployedO Complete acid hydrolysis of cellulose produces glucoseO
Treatment with acid by solution and reprecipitation often increases the accessibility and susceptibility of cellulose to attack by

2 ~ 31420 enzy~es, microbes and chemical reagents. Degradation of cellulose by enzymes leads to various i~termediate products depending upon the enzyme employed, the final products of enzymic degradation of cellulose being generally glucose but with microbes ma~ pro-ceed to mainly ethanol, carbon dioxide and water.
A number of s-tudies have been made of the effects of cellulase en~ymes upon celluloseO It is recognised that cellulases degrade the more accessible amorphous regions of cellulose but are unable to atta~k the less accessible crystalline regions. ~ Sasaki et al (~iotechnolO and Bioeng~, 1979~ 21, 1031 - 1042~ have shown that cellulose dissolves in 6~/o sulphuric acid and that when it is reprecipitated its crystalline structure has disappeared. ~he bio-logical susceptibility to cellulase of the thus treated cellulose is markedly increased and it can be solubilised to an exte~t of about 95% and saccharified to an extent of 94% in 43 hours. The reported results with an untreated cellulose control are poor, only 2~/o saccharification being achieved after 48 hours~
~ Girard (~nn~ ChimO Phys., 1881, ~ , 337 - 384) has shown that anhydrous hydrogen chloride gas has no effect upon cellulose, a finding ~onfirmed recently by T P ~evell and ~r R
~pton (~arb. ResD, I976~ ~, 163 - 174)~ l'hese latter workers however stress the important effectæ of the presence o~ small amounts of moisture.
~ number of industrial processes ha~e been developed 25 or proposed for the pxodustion of glucose by acid hydrolysis of cell~lose. r~hese include-- ;
1. r~he 3ergious F Process (described in Ind. Eng. ChemO~
1937, ~, 247 and in ~oI~ o Report ~o. 499, 14 ~ovember 1945, pages 10 and 11) in which EC1 is employed and is recovered by vacuum stripping~ An improved version of this process i6 des-cribed by J Schoenemann (Chem~ Ind. (Paris), 1958~ 80, 140) who cla7ms a high glucose yield (in the order of 90% of the poten-tial glucose) in a total reaction time of the order of 7 hours~
2~ I~e l~oguchi-Chisso Process which uses -the effect of small amounts of moistu~e and w~ich re~uires 5% ~Cl at a temperature

3 ~3 ~51420 of 100C ~or 3 hours9 by stagewise contercurren-t contact of cel-lulose with ECl gas at temperatures in the range -5 to 125C.
This process is described by M R Ladisch (Process 3iochem., Ja~.
1979, p 21) who claims conversions of 95% on cellulose and 2~o on hemicel]uloseO
Processes for the treatment of cellulose containing materials such as wood pulp and paper with acids or cellulose enzymes to produce simpler products such as g]ucose have to date had limited commercial significance for a number of reas~ns, their principal disadvantages being -the relatively slow ra-te at which acids alld cellulose enzymes attack cellulose and a requirement in most instances for a prior de-lignification of the cellulose con-taining material before treatment with acid or enzyme can be carried out successfully.
According to the present invention we provide a proces~
for the modification, solubilisation and/or hydrolysis of a gly-cosidically linked carbohydrate having reducing groups to produce one or more of the effects (A) modification of the carbohydrate to induce increased accessibility and susceptibility to enzymes microbes and chemicals5 (~) solubilisation of the carbohydrate, and (C) solubilisa-tion and hydrolysis of one or more glycosidic li~kages in the carbohydrate to produce soluble oligosaccharides and/or glucose wherein the carbohydrate is contacted with a mix-ture comprising an aqueous inorganic acid and a halide of-lithium, mE~esium and/or calcium or a precursor of said halideO
Products of solubilisation and/or hydrolysis include higher saccharides tri-, di-saccharides a~d monosaccharidesO
Specifically the products from cellulose include cellodex-trins, cellotriose, cellobiose and glucose. When the process is used to produce carbohydrate of enhanced susceptibility, the suscept~
ible carbohydrate may be further treated to produce solubilisation and/or degradation products. ~or instance the suscep-tible car~o hydrate may be treated with an enzyme in which case the exact nature of the products will depend upon the enzyme employed and the reaction conditions. In the case of cellulose treatment with pf~
~ 3 ~1~20 - cellulase enzymes ~rill lead under appropriate conditions to the production of glucoseO
rrhe glycosidically linked carbohydrate can be presen-t in any suitable s-tate. r~hus it can be present as free or combined carbohydrate, in its natural s-tate or in the form of a manufactured article. 1rhe process is particularly advantageous in its applic-ation to insoluble or otherwise immobilised carbohydrates such as cellulose alone or ad~ixed with other constituents in e.g. wood, straw, mechanical pulp, chemical pulp, newspaper, cardboard, bagasse~
corn stover, cotton, other natural sources, agricultural products, waste products, by products or manufactured productsO rrhe process is also applicable to carbohydrates which exist in highly oriented forms such as crystalline cellulose a~d other ordered structures which are normally highly inaccessible to enzymes and other cat-alysts. Such inaccessibility may be compounded by the occu~Tenceof a polysaccharide with other polymers such as the cellulose with ligm n. ~he proces~ of the invention is applicable to the modific~
ation or sol~bilisation of` cellulose without prior delignificationO
~he process is applicable to all glycosidically linkea carbohydrates whe-ther the glyco~idic linkage is a ~ linkage as in cellulose9 yeast gluc3n or laminarin, or a ~-linkage as in starch, glycogen, dextran or ~igeran~ Whilst those me~tioned are naturally occurring polymers o~ D~glucose, the process is also applicable to glycosidically linked cal~ohydrates with other constituen-t pentoses, hexoses, heptoses, amino sugars or uronic acids. Such polymers having industrial significance include wood hemicelluloses, yeast mannan, bacteri~l and ~eaweed alginates, industrial gums and mucilages and chitin. Carbohydrates containing 0-sulphate~ ~-sulphate, ~-acetyl, 0-acetyl and pyruvate groups ca~ also be treated by the process of the invention as can carbohydrates de-rived by carboxymethylation~ acylation, hydxoxyethylation and other substitution processes, provided that such carbohydrates con-tain glycosidic linkages. ~cid labile substituents on carbohydrates may be lost during the process of -the invention.
Preferred acids are hydrochloric, hydrobromic and ~ 31420 hydriodic acids, hydrochloric acid being most economical and especially preferred. ~he acid can be used to dissolve the li-thium or ma~nesium halide or a precursor thereof. ~Ihen sul-phuric acid is used, it is preferably used in combination with a halide rather than a precursor thereof particularly a sulphate precursor.
In the mix-ture used in -the process of the invention lithium halides are preferred for the solubilisation of cel-lulose, lithium chloride being especially preferredO Mag~esium halides are preferred for the solubilisa-tion a~d hydrolysis to D-glucose of starchg magnesium chloride bein~ especially preferred.
Other metal salts, particularly higher alkali metal halides such as sodium chloride and potassium chloride, may be pxesent in addition to the lithium magnesium and/or calcium halides. Suit-able halide precursors include carbonates, bicarbonates, andhydroxides, particularly lithium carbonate, lithium hydroxide, magnesium carbonate and magnesium hydroxide. When halogen-con-taining acids are used the halide of the acid is preferably -the same as that of the lithium, magnesium and/or calcium halide, e~g. hydrochloric acid is used, for preference, with lithium chloride. ~he treatment may take place in -two stages, e.g. in the -treatment of cellulose a lithium halide followed by a magnesium halide may be used.
~he concentration of the acid used mayva~y within a ~ide range up to 10 molar. When the process is used -to render the carbo~
hydrate more accessible and susceptible to enzymes, microbes and chemicals with limited or selecti~e carbohydrate solubilisation the pre~erred concentration is 1 molar ox ]ess. When complete solubilisation of -the carbohydrate is desired, the preferred con~
centration is up to 4 molar, partlcularly 1 - 4 molar, but can be higher, i.e. up to 10 molar, in certain cases for example when treating polysaccharides such as chiten.
Preferred lithium, magnesium and/or calcium halides are the chlorides7 bromides and iodides, chlorides being most economical are especially preferred. Preferably the concentration of these 3~ f.~.~

6 ~ 31420 halides in the acid is ~lM, saturated solutions being particularly suitable. Effective concentrations of >8M of lithium halides in appropria-te acids can be achieved at ambient -temperature or at temperatures suitable for the limited objective of increasing the accessibility and susceptibility of the carboh,ydrate to sub-se~uent enzyme a-ttack. In general the higher the concentration of a halogen acid emplo,yed in the process the lower the concen-tration of the lithium~ magnesium or calcium halide in saturation at room temperature. ~he salts lithium chlo~ide, lithium bromide and llthium iodide all have good solubility in a~ueous solu-tions of their corresponding halogen halides at room temperature~ This is not the case ho~-ever with lithium fluoride in hydrofluoric acid.
Lithium halides can also be used together with other acids, such as sulphuric acid, in which they dissolve (although total solubility of lithium salt in sulphuric acid is limited), or trifluoroacetic acid in ~hich two layers form~ However lithium halides in halogen acids are preferred. ~agnesium halides have more limited solubility than lithium halides in halogen acids. A saturated solution (12.65 M) of lithium chloride in 1~05 M hydrochloric acid at 25, contains 20 54O64 g LiCls A saturated solution (llo~ M) of lithium chloride in 4 M hydrochloric acid a-t 20C contains an estimated 47O9 g I~Cl.
~ he temperature of eontacting the earbohydrate with the mixture may be varied within a wide range from -5C -to 125C~- If the objective is to render the carbohydrate more accessible and susceptible to enzymes, microbes or chemicals with limited or selective solubilisation of carbohydrate then the temperature is preferably in the range from 0 - 50C, particularly between 4 -22C9 When complete solubilisation of the earbohydrate is required the temperature range is suitably from 4 - 100C with a preference between 50 - 90C. ~or h~drolysis of the glyeosidie li~kages in the carbohydrate althou~h the rate is appreciable at ambient temper~
atures the preferred ra~ge is 50 - 100C, particularly 50 - 90C~
~ he particularly ad~ntageous part of the process is -the short duration of the carbohydrate contacting process with the mixture to achie~e ~odifying effects much greater than those 7 ~ 31420 produced b~r a~r one or two of the components of the contacting mixture alone. ~rom e~perience it is evident that the pretreat-ment to improve accessibility and susceptibility to enz~mes, microbes and chemicals can be shor-tened to 1 - 24 hours at room temperature or below. Complete solubilisation of the carbohydrate is generally achieved within one hour at 50C but is a few minutes only at 90 - 100C particularly if the concentration of the undis-solved carboh~r~rate is low, the amount remaining undissolved is low or the carbohydrate has been previously contac-ted at 50C or below~ While a carbohydrate, particularly one originally insoluble in the modifying mixture, ma~r be alread~ nearly 5G% hydrolysed at the -time solubilisation is achievedg it appears advantageous to await such solubilisation at 50C or below before heating for the few further minutes required at 90 - 100C to complete the hurdro-lysis to its highest extent without undue degradation During the hydrolysis stage, some of the water in thecontacting mixture is consumed and this becomes important in the presence of high concentxations of soluble carbohydrate. ~hus 162 g of cellulose when completely hydrolysed to glucose will have consumed 18 g of water. Since this will both increase the concen-tration of the acid employed and denude the lithium/magnesiu~/
calciu~ halide of wate~, app~op~iate steps are p~eferably taken to remedy this at high carbohydrate concentrations~
In practice the amount of carbohydrate s~spended origin-ally in the mixture varies according to the nature of the carbo-hydrate, the physical state in which it occurs, its accessibility in that state, and the degree of polymerisation of the carbohydrate~
With cellulose, where suspension presents some difficulties, 5 - lC/o concentrations are easily achievable and 15% concentration with care~
In general the limiting factor becomes mainly one of viscosity bring-ing attendant problems of heat tr2nsfer and effective mixing~ If hydrolysis is allowed to proceed -then further amo~mts of the carbo-hydrate can be solv.bilised. ~he addition of water consvmed in the hydrolysis also becomes important in this respect as does -the effec~
tive concentration of the acid~ Starch, even in the intact starch ~r~
8 ~ 31~2~

grain, can be ~olubilised by a mild treatme~t with the contacting mixture often below its gel point. This is illustrated with the solubilisation and hydrolysis of starch (Amylum maydis) with hydro chloric acid (2~0 M) saturated with Mg C12 w~ere tre~t~e~-t at 50 for 3 hours followed by 90 for 12 minutes gives most effective conversion to D~glucose. This comb-nes -the effect of the added Mg C12 in facilitating th~ solubilisatlon of starch at low -tem~er-at~res wi-th an accelerated rate of hydrolysis to D-glucose at a higher termperature.
Carbohydrates present in micro-organismsl mammalian tissues, plant tissues, and other nat~ral sources can be effectively extracted even if chemically attached therein to proteins or lipids~
Pretreatme~t o~ such tissues or even the isolated carbohydratss, u~der milder conditio~s that avoid exces6ive solubilisatio~ enables enzymes and microbes to atta~k their substrates in a subsequent stage faster and more effec-tively than untreated tissues, c æ~o~
hydrates or carbohydrate containing materi~ls.
M2jor savings in the amount of e~yme or other catalyst ca~ be achieved amountLn~ to a factor of at least ten over a typical process havi~ no such pretreatme~t steps. ~e contacting mi~ture employed is available for recyclLng for reuse~
~ ~iCl-HCl-H2O mixture differed from ~aCl ~ 1 ~ O in its behavior on a ~iogel*P2 column. ~he LiC1-~Cl is excluded from the packing matrix when the mixture is injected whereas sodium chloride is includedO
Most importantly the process of the inventio~ is l~qed in the production of glucose from cellulose or st~rch. Other products which can be produced include glucsse, yeast gluca~, glucosamine from chit-n, hexuronic acids from polyuronides, ~ylose from xylan and hemicellulose~ sugars from their glycosides and the disruption, sclbilisation and hydrolysis of carbohydrates i~ the cell walls of tissues and microbes. ~ltex~atively the proces~
may be used to produce a modified polysaccharide or cellulose which can be u ed in that form to spi~ fibres, non-woven fabric6 or other articles such as films or membranes by conti~uous injectio~
* Trade Mark ,.. .
- ii, 9 ~ 31420 into a liquid im~iscible with the reaction mixture bu-t from which the modified polysaccharide or cellulose is precipitated.
The process of the invention has a number of advantages as a~plied -to cellulose viz:
5 lo ~ prior delignifica-tion step is not required~
2s Pretreatment may be chosen to mLnimise solubility whilst retaining subsequent accessibility to enzyme ac-tion~
3~ Pxetreatment renders all the cellulose accessible to subsequent enzyme action, rather than merely a fraction thereo.

4. The pretreatment can be applied to a variety of polymers alone ox as mixtures e.g~ cellulose a~d hemicellulose to provide ready accessibility to subsequen-t hydrolysis.

5~ Enhanc2d rate of attack by cellulase and hence lowex enzyme requirement for complete reaction.

6~ A versatile, aqueous based, solubilising agent giving control over solubilisation and hydrolysis.

7~ A mode of action that is rapid in both the heterogeneous and homogeneous phases.
20 8. Acceleration of the rate of hydrolysis l.~ith respect to an aqueous acid of the same solution molarity enabling a given rate of hydrolysis to be achieved at a lower te~perature than with an aqueous acid of the same solution molarityO
25 9. ~he ability to deal with high concentrations of cellulose Ln particularly the heterogeneous phase due to the measure of control that can be exerted~
In the application of the process to other members of the wide range of naturally occurring a~d synthetic carbohydrates 3 containing one or more glycosidic linkages and having a spectr~lm of solu~ilities and susceptibility to the reagents of the process, optimisation of conditions along the l:ines given more particularly for cellulose are with~l the competence of workeræ skilled in the artO In the detailed designing of particular processes for pal~ic-ular polysaccharides based on the reagents of the invention -two :L0 ~ 31420 features can be clearly delineated. ~he first is the original accessibility and susceptibili-ty to the reagents of the invention of the polysaccharide in the material in which it occurs which will differ for the same polysaccharide in different e~vironments, and different physical fo~ms. ~he second featwre is the accessib-ility and susceptibility of the glycosidic linkages in the par-tic-ular polysaccharide to the reagents of the invention once the carbohydrate is solubilisedO
~ere the process offers further advantages applied to both cellulose and other carbohydrates containing glycosidic link-ages since the reagents of the invention can be further manipulated during the process -to attain the desired objectives of that process.
~he following are a lis-t of parameters that are not exclusive within the te~ms of the invention but indicate the factors over and above those already mentioned that fall within the claims of the inven-tio~ and which would be applied by those skilled in the art.
1. ~ddition of water over and above that consumed by the hydrolysis of the glycosidic linkages in the car~o-hydrates~ Such water may be added at any stage of the process but preferably once solubisation of the carbohydrate has been achieved. It is intended that steam is included among the fo~ms in which water is added.
2. Addition of an alkali, carbonate or bicarbona-te once carbohydrate solubilisation has been achieved to decrease the overall acid conce~tration of the re-action mixture used in the process.
3. Removal of hydrogen halide from the reagents of the reaction mixture during the course of the process by application of reduced pressure.
4. ~he reduction of the metal halide concentration d~ring the course of the process by addition of aqueous acid~
5. Simultaneous addition of both further carbohydrate and ~Jater during the course of the process.
35 60 ~se of some or all of the acid component of the reagents in the fo~ largely insoluble in or immiscible with the rest of -the reagentsO
7. ~he use of a closed syste~ in ~7~ich the carbohydrate is ~ontacted with the mixture at a pressure -that may be above or below that of atmospheric pressure~

8~ ~he removal of a product of the reaction during the course of the reaction either continuously or discon-tinuously.

9. ~he introduc-tion of a second phase i~miscible with thè
first that can be either gas, liquid or solid tha-t performs one or more f~ctions of agitation of the reaction mixture9 specific or selective partitian of a product or reactant, heat transfer, or modifies the reaction to preven-t undue production of unwanted by-products.
~he in~e~tion is illustrated by the E~amples given belowO
In these Examples the analytical methods and the compositions of the materials used were as follows:-(a) ~
~he cysteine~sulphuric acid reagent (700 mg of L,cysteine hydrochloride monohydrate in 1 litre 8~/o sulphuric acid) was added to a portion of the sample/standard such that the ratio of reagent to sample/standard was 5:1 (normally 5 cm3:1 cm~ he reagent was added -to sample in tubes immersed in an ice bath. ~he tu~es were then placed in a boi].ing water ba-th for 3 minutes, a~ter which time they were removed and allowed to cool to room temper-atureO The absorbance of each solution was measured at 420 ~m and the carboh~drate concentration obtained~ by reference to appropriate standards, to give the results quvted in the Examples~
(b) D^t na-tion of res~
3uffer: Sodium acetate-acetic acid; 0.05M, pH 4.80 Reagent: Potassium fe~ric~Janide (0.117g) and Sodium carbonate (1095 ~) were dissolved in distilled w~ter and di-luted to 100 cm3~ qhis solution was freshly pre-~5 pared each mo~ning~

~ ~D~

Standard solutions (0-600 ~g cm 3 of D-glucose; 0.4 cm3) or sample solutions (0.4 cm3) were added -to test-tubes, cooled in an ice bath, containing reagent (2.0 cm3) and buffer (105 cm3).
After mi~ing, -the test-tubes were held in a boiling water bath for 5 minutes, and thereafter cooled to room temperature. The re action mixtures were d;luted by addition of water (4,0 cm3) and the absorbance of each solution measured at 420 nm. ~he differ-ence in absorba~ce between standard or sample and a bla~ (pre-pared by replacement of ~mple with wate~) enabled calculation of reducing sugar content expressed with respect to D-glucose.
(C) D~
3uffer: 2-~mino-2-(hydroxymethyl)-propane-1,2-diol (~RIS), 0.5 M, pH 7~0 Reage~t ~:Glucose Oxidase (19,500 units per g.g 50 mg.) dis-solved in buffer (50 cm3) Reagent ~Peroxidase (ex horse radish, 90 units per mg., lO mg.) and 2,21 Azino-di-(3-ethyl benzthiazoline sulphonic acid (AB~S, 53 mg.) dissolved in buffer (lOO cm3)~
Standard solutions of D-gluco~e or u~known solutions containing D-glucose (O to O.l mg per cm3, 0.2 cm3) were miged with reagent ~ (0,5 cm3) and reagent ~ (l.O cm3). ~fter 30 minutes at 37C, the absorbance of each solution was measured at 420 nm. and -the D~glucose concen-tration of the u~k~own solu--tions determined by reference to the calibration with D-glucose standard solutions.
(d) ~
Chromatography was performed on ~iogel P~2 (~iclad ~aboratories L;m;ted). Two sizes of column were employed depend-ent on the analytical technique used for determination of material in the column eluate.
Method ~-Chromatography wa~ performed on ~iogel P-2 in a glass column (425 cm3 volume, 150 cm i~ length) wi-th a water jacket maintained at 60C. The column was pumped at 0,8 cm3 min ~ ~he colu-mn eluate was split and analyaed by (i) differential re~ractom~y f~

(Waters Qssociates Model R401) operating at 0.32 cm3 m.in and/
or (ii) an automated c~steine-sulphuric acid method for total hexose determination (S A ~arker, M J ~ow, P V Peplow and P J
Somers, Anal. ~iochem., 26, (1968), 219) operating at 0.1 cm3 min 1 sample flow rate. The volume of sample applied to the ~iogel P-2 column was 0 to 0.1 cm3 containing 0 to 5 mg of carbo-hydrate D
Method ~:
Chromatography was performed as in Method A except that a colu~n (145 cm x oO6 cm internal diameter) was employed operat~
ing at a flow rata of 0015 cm3 min 1. Analysis of the column eluate was by the cysteine-sulphllric acid method for total hexose determination as in method Q. The sample volume employed was 0 to 0.01 cm3 containing 0 to 0.5 mg of carboh~drate.
The area under each peak of carbohydrate material was i~tegrated and compared with the a~ea produced by a standard of D-glucose. ~he results were expressed as a percen-tage of the total carbohydrate deteDmined in the eluate. Where the products were an oligomeric series -the nomenclature G1~ G2 --~ Gn is used to indicate the number of sugar units in each oligomer.
(e) ~ s Analytical results presented are based on the weights taken for analysis and do ~ot allow for moisture u~less stated othe~ise.
Moisture contents observed, on dry.~ng at 55 in vacuo 2 5?
Cellulose fibres, Whatman Chromedia CF11 307%
Mechanical pulp 8.1%
~ewsp~i~t 7- æ/~
30 (f) ~

Duplicate samples ~ 25 mg) were accurately weighed into stoppered test-tubes and sulph~ric acid (98/D, 1 cm3 M~R
grade) added. The temperature of these suspensions was maintaLned below 0C by means of an ice/salt bath (-10C). ~f-ter 48 hours at l~ ~ 31420 4 distilled water (8,0 c~3) was added and the tubes heated for 22 hours in a boiling water bathO ~fter cooling to room temper-ature the D-glucose a~d total carbohydrate contents were determined.
~ne results obtained by this procedure are set o~t in ~able 1a.
~ able 1a Com~osition of materials used expressed as weight percentage with respect to cellulose on a dry weight basis.

Sample D-glucose content Total carbohydrate content _ _ __.
Cellulose fibres l 96-5 97-5 2 97-~ 88.0 15 Mechanical pulp 1 41.0 ¦ 41.0 2 41.0 1 41.0 ~ewsprint l 56.o 55-0 2 63.0 6 D ;
. ~ . _ .
(ii) ~
_~.
hemicellulose) 4 Samples (50 - 60 mg) of dried material were weighed accurately into test-tubes and trifluoroac0tic acid (2.0 M3 2.0 cm3) added~ ~he tubes were sealed and heated in a boiling water bath for 6 hours. After cooling, and opening of -the tubes, trifluoro acetic acid was removed by evaporation. ~he residue was taken up in borate buffer (0.13 M, pH 7.59 l.O cm3) and analysed using borate anion exchange chromatography (JEOL
carbohydrate analysis system), ~he results obtained by this procedure are set out in ~able lb.

5 ~ ~.

~ 3142 Tab].e lb expressed as a weight percentage of dry weight _ _ Cellulose fibres Mech~nical pulp ~ewsprint __ _ _ _ _ _ Component (or time of elution

10 if unidentified) 30 mIn 0.03 0.9 o.45 35 min 0005 _ rhamnose . 0~13 OolO
92 mi~ _ 0012 0015 1514~ min _ 0.2~ 0~17 mannose trace 7027 3~9o ~rabinose (or fructose) _ 0~92 o~5 galactose trace lo 60 Oo 86 xylose 0~14 2~73 1~89 _ _ _ _.
~otal no~-glucose neutral carbohydrates 0.22 13~91 8~06 . . __ __ ._ _ _.. ____ 25gluGose 3~ 32 3.49 2026 oelloboise 0.03 0~11 0.10 ~XaMPLE 1 ~ 0 Prelimina~y work established that pretreatmen-t of cel-lulose fibres with saturated solutions of lithium chloride or lithium iodide for 24 hours gave a signlficant increase in the initial rate of ~ydrolysis of the water washed, pretreated7 cel-~5 lulose by cellulase ove~ periods of 60 minutes at 50 C~

t ~ '~ D ~

16 ~ 31~20 Sampl~s (100 mg) of cellulose fibres were treated withsolutions conta~ning lithium chloride or lithium iodide respect-ively for 24 hours at room temperature~ The fibres were allowed to ~ettle and the supernata~t liquor removed by decantation. ~he fibres were washed with distilled wa-ter (2 x 10 cm3) and resus-pended in aceta-te buffer (0.05 M, pE 4.8). Cellulase (Maxazym~
CL2000, GIS~, 1/~ ~/v in acetate buffer, 0.05 M, p~ 4.8, 4~0 cm3) was added. ~he digestion was carried out at 50 C and aliquots (0.4 cm3) removed at 10 minute intervals. ~he content.of reduc-ing FUga~ was dete~mined. The results obtained are set out in~able 2.
Table 2 with solutions of lithium h~lides _ _ _ Pretrea-tment H20 Lil (Sat) LiCl (Sat) _~,..... . _ ~
Rate of production of reduci~g su OE 7.6 10.4 10.4 (with respect to glucose) ~g cm min ~ _ _ .
~X~MPLæ 2 ~
cellulase~ ;
Samples (100 _g) of cellulose fibres wexe pretreated with saturated aqueous solutions of lithium chloride or lithium iodide, and distilled water as a control, for 24 hours at room temperature. ~he fibres were allowed to settle and the supe~
natant liquid removed by deca~tation~ ~he fibres were washed with di tilled water (2 x 10 cm3) and suspended in buffer (10 cm3)0 After stirring at 50 C for 10 m;~utes, cellulase solution (1% /v in b~Lffer as in Exa_ple 1, 5O0 cm3) was added and dige~tion allowed to proceed at 50C. Samples ~095 cm3) were removed after 19 2, 49 * Trade Mark 17 ~ 31420 6, 24, 48, 96 and 100 hours, immediately diluted to 5.0 cm3 and stored at 4C. When all samples had been collected analysis for reducing sugars were performed, using dilution where appropriate for high concentrations of reducing su~ars7 and ~or to-tal carbo-hydrate~ ~he molecl~ar distribution was examined by gel ferment-ation chromatography. ~he results obtained are set out in Table 3~ It can be seen from this data that the pretreatment with saturated lithium chloride solu-tions provides a greater rate of production of reducing sugar by cellulase and 95% conversion -to available glucose after 24 hol~s. Saturated ]ithium iodide pre-treatment afforded an increased rate of olubilisation and hydro-lysis over that observed with water pretreatment (after 24 hours 77% conversion as compared to 7~/0 with water) but was not as effective as the pretreatment wit.h saturated lithium chloride solution. Total carbohydrate analysis and gel permeation chroma-tography confi~m the reducing sugar analysis and indicate the predominant product to be glucose with small a~ounts of cellobiose and other oligomers. ~11 three materials reached essentially complete hydrolysis after 100 hours.

~L~ f~L

chloride lithi~un iodide or water.
5 ~ _ -~ ____________________ Pretreatment ~ime of ~ r~ ~
Cellulase Saturated Saturated action Distilled li-thium iodide lithium chloride wa-ter solution solutio~
_ _ _ . ,, % conversion as expressed by reducing sugar analysis 96 98 97 g6 100 97 97 . 99 % conversion as expressea by total æugar a~alysis Relati~e proportion of oligomers by gel permeation chromatography Gl 98 ~ C% 1 96 ~ 0% 98.7%
100 G2 2.0/o ¦ 1.0% 0 O 8% ;
G>2 0 ¦ 3.~/o oo5%
_ _ _ 30 ~
~, (i) sodium azide ___ Materials which inhibit microbial growth are ~sually added to enzy~e solutions to pre~ent microbial growth and inhibit 35 production of unwanted material, ~he effect of sodium azide on r-~
19 ~ 31420 the ra-te of production of reducing sugar from cellulose using cellulase was determined. Duplicate samples of cellulose fibres (lO0 mg) were pretreated, for 73 hours, with distilled water at room temperature. After the fibres had settled the supernatant liquid was removed by decantation and buffer (lO cm3) added.
~ollowing the procedure of Example 2 the suspensions were digested with cellulase or cellulase contai~ing sodium aæide (150 mg). ~he results of the analysis are set ou-t in ~able 4. ~he digestion in the presence of sodium azide gives little difference in rate of production of reducing sugar compared with the corresponding con-trol without sodium azide. With sodium azide there is a higher proportion of cellobiose in the final solution than is the case with the control~ ~lhis may be due to inhibition of a cellobiase by sodium azide.

~ _ . _ % conversion as expressed by red~cin~ sugar analysis Time of I
20digestion Cellulase Cellulase and sodium azide 24 78 70 ;
Relative proportion of oligomers by gel permeation chromatography 24 Gl 95% 8~/o 3~2 5% 20/~

(ii) lithium ohloride In previous examples the cellulose fibres were washe-1 with distilled water to remove residual pretreatment solution~
~he effect of residual lithium chloride on the rate of productio~
of reducing sugar and final product composition was determined.

~ ~1420 A sa~ple (100 mg) of cellulose fibres W2S pretreated with a sol-ution of lithium chloride (satura-ted). ~'he fibres were allowed to settle and the supexnatant liq~d removed by decantation. I'he fibres were not ~ashed, buffer (10 cm3) was added and the diges-tion with cellulase and analysis for reducLng sugars were per~formed as in Example 2. ~ control of cellulose pretrea-ted with distilled watex was employed~ ~he results are given in ~able 50 Analysis by gel permeation chromatography show Gl a~d G2 i~ the propor+ion 95% o 50/0 respectivelyO
If the results obtained using unwashed, lithi~n chloride pretreated, cellulose fibres are compared with those using a wash-ing stage (Example 2, ~able 3) it can be seen that the initial rate for the unwashed sample exceeds that for the washed sa~ple, but that the concentratio~ of reducing sugax after 24 hours is higher for the washed sample. ~his may result from the washing procedure removing the lithium chloxide from between the fibres and hence removing the swelling effect, i.e. where the swelling effect is maintained, the initial rate of attack may be enhanced.
~hus removal of the pretreatment solution without was~;ng allowed 73% hydrolysis after 6 hours compared with 57/ after 6 hours with a wasbing step after pretreatmentO

~.
25 l _ _ _ ~ime of Pretreatment ;
cellulase ~
action Distilled ~ater Lithium cbloride (s~turated) __ 3o % Conversion as expressed by reduci~g sugar an31ysis 2 32 ~ 71 21 ~ 31420 r3~,~^r.
Samples of cellulose fibres (lOO mg) were placed in re-action ~essels and soluti.ons of lithium chloride (saturated, lO cm3) added. ~he vessels were heated a-t either 50 or 100C for 1 hour.
Control experiments were performed using distilled water. Af-ter the one hour pretreatment the fibres were washed with distilled water (2 x lO cm3) and digested with cellulase for 24 hours as in Example 2. ~he xesults are set out in ~able 6. ~he results show that no effecti~e improvement is achie~ed by the use of saturated lithium chloride at 50 or 100C compared with pretreatment wi-th water at the same temperatures.
~able 6 ._ ~
~ime of Pretxeatment Cellulase . _ _ _ 20action Distilled water Saturated lithium chloride /0 Oonversion as expressed by reducing sugar analysis ..

12 2l 14 213 ll 4 29 19 32 21 ;
6 36 28 34 3o 24 74 63 ~7 58 ~2~

Samples (lOO mg) of mechanical pulp and newsprInt (chop-ped in a blender) were pretreated with a satuxated solution of ~5 lithium chloride (lO cm3) for thxee weeks at room temperature.

3~

22 ~ 31420 Control, pretrea-ted with distilled water~ was also prepared. ~he superna-tant liquids were removed, wi-th addition of distilled water (5 cm7) to aid ~ettling of the fibres, a~d the fibres washed with distilled water (2 x lO cm3)0 3uffer solution (lO cm3) was added a~d digestion with cellulase carried ou-t as in Example 2.
~he results are set out in 'rable 7. q`he results show -that pro-l.onged -treatment wi.th saturated lithium chloride, of mechanical pulp or newsprint, achieved no improvement over water alone under these conditions.
~
Effect of ~retreatment with lithium chloride solution on -the _.~
,_ ., ~___ Mechanical pulp~ewsprin-t 15~ime of . _ cellulase Pretxeatment with~Pretreatment with:
action I I . _ _ Water ¦ ~ithium chloride Water I ~ithium chloride % Conversion as expressed by reducing sugar analysis 20 l 14 14 20 22 6 17 18 3 3o EX~WPLE 6 :nin~ = -e-i~lsL~ or ~ onl Samples (lO mg) of cellulose fibres, mechanical pulp and newsprin-t were pretreated with a solution (lO cm3) of hydro-chloric acia(l.O M) saturated wlth lithium chloride at ~oom temperature for 24 hoursO ~fter pretreatment the fibres were allowed to settle out 75 (i) An aliquot (5 cm3) of the s~pernatan-t liquid was re~oved ~3 ~ 31~20 and subjected to centrifuga-tion to ensure clarificationO Aliquots (0~1 cm3) were removed and diluted to 10 cm3. Standard solutions of D-glucose were likewise prepared and analysed for total carbo-hydrate and fox D-glucose. ~he results are set out in l1able 8 (ii) The residual fibres were washed with distilled water (2 x 10 cm3) and resuspended ~n buffer (10 cm3). Cellulase di-~estion was performed as in ~xa~le 2. Analysis for reducing sugar9 total carbohydrate and ~-glucose were performed at -the five intervals tabulated, and analysis by gel permeation chroma-tograplly was conducted at the termina-tion of cellulase digestion.
The results are set out in Tables 8 and 9.
As can be seen from the da-ta in Tables 8 and ~, pretreat-ment gives rise -to significant solubilisation, but with limited hydrolysis, and greatly facilitates attack by cellulase on the residual celluloseO

24 13 31~20 Table 8 ~c ~ ~

~ ~ __ Cellulose Mechanical Material fibres pulp~ewsprint . __ . .__ % solubilised 10 dur~g pre-treatment total carbohydrate 18.8 16~7 15~7 l~glucose 5~5 2.5 1.4 _ _ __ % solubilised after 15 pre-treatment and cellulase action Reducing sugar 88.0 33O0 47-o ~otal carbohydrate 92.0 34.0 43.0 D-glucose 93^ 19.0 44oo _ __ Relative molecular distribution after cellulase action (%) Gl 97-5 44O0 9g.o G2 205 52.0 0~5 G~2 __ _ . ~ 0.5 ., ~ 31420 with lithium chloride _ s _ _ _ ~ _ _~
Cellulose fi~res Mechanical pu'p ~ewsprint Ti~e of pretreated with pretreated with pretreated with cellulase Analysis _ action Water ECl/~iCl Water EC1/~iC1 Water HCl/LiC1 10 _ _ .. _ _ ._ __ ,_ .
1 Reducing .
su OE 14 66 6 17 14 ~2 Total carbo-hydrate ~ 67 ~ 18 _ 34 D-glucose ~ 68 ~ 15 ~ 31 _ _ _ ._ ~ __ 2 Reducing sug~r 19 7o 4 20 22 38 _ _ ~ _ ~ .
4 Reducing sugar 3 6410 25 26 36 _ ~ _ _ _ ~ . _.
6 Reducing ~uga~ 44 67 9 25 26 39 ;
__ _ _ _ 24 Reducing ~ugar 7 75 15 28 33 41 ~otal 3o carbo--hydrate ~ 78 ~ 29 _ ~7 D~glucos~ _ 79 _ 16 _ 38 Results are expressed as % co~version 26 ~ 31420 of water hvdrochloric acid and lithium chlorid~ and subseauent ~L~
Samples (100 mg) of cellulose fibres were pretreated for 24 hours at room temperature wi-th aliquots ~10 cm~) of distilled water, hydrochloric acid (140 M) saturated with lithium chloride~
distilled water saturated with lithium chloride, or hydrochloric acid (1.0 M). The supernatants were analysed for solubilised carbohydrate, and the residual fibres for susceptability to cel-lulase digestion~ as described in EYample 6. ~he results are set out in Table 10.
~ rom the data in Table 10 it can be seen that:
(ij Hydrochloric acid (1.0 M) alone does not improve the rate of cellulase action or increase the yield of soluble carbohydrate when compared with a water pretreatment~
(ii) ~oth lithium chlo~ide (saturated) and ~ydrochloric acid (luO M) saturated with lithium chloride improve the ra-te of cellulase ae-tion and the overall yield of soluble carbohyarate and D-glucose.
(iii) Only nydrochloric acid (1~0 M) saturated with lithium chloride results in appreciable solubilisation of aYailable carbo-hydrate in the pretreatment.
(iv) After cellulase action for 1 hour, the cell~lose fibres pre-treated with hydrochloric acid (1.0 M) saturated with lithium chloride, provides 95% of the a~ailable ca~bohydxate in solution.
In the same time scale lithium chloride pretreatment permits only 64% and water pretreatment only 21% of the available carbohydrate to be solubilisedO

27 ~ 31~20 Table 10 oellulase Values are corrected for moisture content of original cellulose fibres9 __ _ _ _ _ . % solubilised ~l~ng action of cellulase Pretreatment AnalysiY % Solu~ilised .~or ~otal %
solution method _ _ solubilised 1 hr 2 hr 4 hr 6 hr _ ~ _ _ _ ~
Distilled Reducing 15water sugar ~.d~ 15 17 24 41 41 ~o-tal 24 carbo-hours hydrate ~.d. 21 26 35 47 47 D-glucos~ nOd~ 17 17 25 31 31 20 _ _ ~ _. _ _ _ _ ECl(l.OM) Reducing saturated sugar 13 7o 7374 74 87 with LiCl ~otal carbo-2524 hydrate 15 80 8182 82 97 .
hours D-gl~cose 11 5o 5360 60 71 , . _ _ . _ _ _ _ ~_ Reducing LiCl sugar ~ do 66 74 78 82 82 3o saturated Total 24 carbo-hours hydrate ~0~1 64 7o 79 82 82 D-glucose nOd, 46 55 57 64 64 ~ly ~

2~ 3 31~20 ~able 10 (co~tinued) . ~
% solubilised dur~
action of cellulase 5 Pretreatment ~naly~is % Sclubilised for ~otal %
solution method _ _ solu~ilised 1 hr 2 hr 4 hr 6 hr _ _ _ _ _ . ~
Reducing 10HCl(l.OM) ~otal nOd. 15 22 29 33 33 24 carbo-hours hydrate nOdo 12 19 27 33 33 D-~uccse n.d~ 17 19 24 31 31 _ _ _ n.d. _ not detectable In view of the enhanced rate of cellulose action obserY~
able after pretreatment with hydrochloric acid (loOM) saturated with lithium chloride a further comparison was made using xeduced pretreatment times and reduced cellulase levels, Samples (100 m~) of cellulose fibres were pretreated ~th either distilled -~ater (10 cm3) or h~drochloric acid (l.OM) saturated with lithium chloride (10 cm3) for ~arious times at room temperature as specified in ~able 11. ~he residu~l f'ibres were analysed for cellulase susceptability as in Example 6, usLng solutions of cellulase at either 1~ or 0.1% W/v concentration.
lhe results obtained are set out in ~able 11. ~he results further demonstrate the e~hanced effective~ess of cell~lase on residual f`ibres after pretreatme~t with hydrochloric acid (l~OM) saturated with lithium chloride as compared with pretreatment with water. Ihis enhanced ef`fectiveness is obtainable after pre-treatme~t times of one hour.

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~ 31420 lithiu ~ e ~ .
~o test solutions were prepared by placing portions (50 mg) of cellulose fibres in two test-tubes and adding there~
to in one instance a saturated solution of lithium chloride (5.0 cm3) and in the other a solution of hydrochloric acid (0~5 M) saturated with lithium chloride. ~he tubes were sealed, kept in a refrigerator ove~ight, and then placed ~n a boiling water bath. ~fte~ 5 mu~utes the tube co~taining HCl/LiCl was removed, as the cellulose had essentially dissolved, and cooled in an ice bath4 ~he tube contain ng ~i~l solution was kep-t in the boiling water bath for 12 hours. ~he solution and super-natant ~espectively were analysed for to-tal carboh~drate employ-ing standard sol~tions o~ D-glucose in saturated lithi~m chloride solution~ ~he res~lts are set out in ~1able 12. ~hese results demonstrate that treatment with hydrochloric acid (0.5 M) satur-ated with lithium chloride gives a high degree of solubilisation (ca 54%)O ~he carbohydrate solubilised was shown by gel ~ermeation chroma-tography to be largely glucose (5.0 mg cm 3 out of 6.o mg cm3 solubilised) with the remainder mainly as a disacchariae.
~able 12 ~~t~ ~ i1 ;iC
_ Solution Concentration of total carbohydrate in supe~natant _ _ _ ~ _ ~iCl ~Cl - 6~o mg cm 3 _ ~iCl 2.4 mg cm 3 31 ~ 31~20 ~L.
Samples (50 m~) of cellulose fibres were placed in test-tubes to each of ~hich was added a solution (5.0 cm3) of hydxo-chloric acid(O.l, 0.5, 1.0,-2.0, 3;G or 4~0 M) sa-turated with lithium chloride. The tubes were sealed and placed in a boiling water bath. Tubes were removed as soon as solubilisation was obser~-ed visu31ly~ or when significant discolouration was apparentO
On removal the tubes were cooled in an ice bath and stored in a refrigerator until analysis for total carbohydrate in solution as in ~xample 8. The results obtained are set out in lable 13~
~he data in ~able 13 demonstrates that hydroc~loric acid (4~0 M) sa-turated with lithium chloride had achieved essentially 100~/o solubilisation~

:~t ~ ~ oO ~

20 _ _ ~ / solubilised HCl con- ~o-tal carbo~ydrate on basis of centration ~ime concentration in total in solution in solution carbohydrate 25(M) heatins ~ath _ . _ analysis ;

4~0 . 55 sec~ 11.2 105 3.0 55 sec~ 8.9 83 2.0 2 m;n 57 sec~ 3~4 32 1.0 5 min~ 7-3 68 3o 0~5 5 min~ 1.4 13 . . _ -30 mIn. ~ D ~ b ] i~e ~:

3~ ~ 31420 hXAMPIE L0 concentrations of lithlum ohloride.
~he method of Example 9 was repeated using a fixed ECl concentration (4O0 M) bu-t va~ying li-thi~um chloride concentrationsO
~he lithium chloride concéntrations usedwe~e 1.0, 2.0, 4.0, 8.0 M
and sa-turated. ~he results are set Ol~t in ~able 14.

10 ~ ~
~_ _. .
~otal carbohyd~ate LiCl con- ~ime in concentration in y0 solubilised on centration heati~gsolutionbasis of total carbo-15 in 3~1 (4.0 M)bathmg cm3hydrate analysis _ ~ ~_~
1.0M 30 min~ 1.9 18 2.0~ 30 min~ 4.2 39 4.0~ 30 mi~O 3.1 29 8~0M 9 m;nO 8.0 76 saturated ~5 sec.10.9 102 EWlIP~ 11 _3~9bl~

te~Deratu~e~
~ he method of Example 9 was repeated save that the hydro~
chloric acid solutions of molarity 0019 0~5 a~d 1.07 saturated with lithium chloride, were employed and that the test solutions were allowed to stand for 60 hours at room temperature before hea-tingO
~he results are se-t out in ~able 15 and -the data thereing when compared ~Jith Table 13, indicates that pretrea-tment increases cellulose solu'~ilisation.

~.
5 ~ _ _ _ _ ~Cl con- ~otal carbohy~rate cen-tration ~ime in concentration in % solubilised on in solution heating solution basis of total carbo-10~M) bath mg cm hydrate analysis 1.0 73 sec 9-7 91 0.5 162 sec 906 90 _ _ _ 25 min 10.2 95 __.__ ~.
~ he materials examined were cellulose fibres, mechanical pulp, newsprint 1 (Daily Mirror), newsprint 2 (Observer, no ink) and a yeast glucan. Sa~ples (50 mg) of each material were sus~
pended in a sol~tion ~5 cm3) of hydrochloric aGid (1.0 M) satur-ated with lithium chloride and treated as in Example 11~ The solutions obtai~ed were clarified by centrifugation prior to analysis for total carbohydrate and for molecular distribution by gel permeation chromatograph~. ~he results obtained are set Ollt in ~able 160 ~he data presented ia ~able 16 indicates that the cellulose fibres have been completely solubilised (within experi-mental error) and that the solubilised carbohydra-te for the mechanical pulp and newspri~t compares favourably with that available therein.

34 ~ 31420 ~able 16 ~ _.
I _ . . __ _ _ -Conce~tration of Relative molecular ~ime of ~otal carbohydrate distribution (%) Material heating in so],ution _ _ _ -3 C-l G2 G3 10 Cellulose fibres 3-5 m'n 10~2 94~2 5~o 0.8 Mech~nical pulp 4.5 min 6.5 92~9 5~3 1.8 ~ewsprint 15~5 _in 7.1 96~ 7 3.3 O
~ewsprint 24~75 m;n 6.1 92.0 2v4 5.6 Yeast glucan3 mi~ 6.6 _ _ _ _ ~
.~
~ ~ .
~_~.
~he materials e~ami~ed were cellulose fibres, mechanical 20 pulp~ newsprint 1 (Daily Mixror), newsprint 2 (Observer, no ink) and as controls glucose and cell'obiose~ Samples (50 mg) of each _aterial were suspended in a solution (500 cm3) of h~drochloric acid (4.0 M) saturated with lithium cblorideO ~he suspensions were sealed in ~lass tubes and placed in a boiling water bath~
~he tubes were then treated a~d analysed as in h~a~le 8 for total carbohydrate and for molecular distribution by gel perme-a-tion chromatagraphyO ~he results obtained are set out in ~able 17. lhe data indicates complete solubilisation of cellulose fibresO

3~.3~ ~

~ 31420 ~ _O

~ ~ _ _ _ _ ~aterialheatingconcentration in (minutes)solution (mg cm~3) Cellulose 1~33 10.5*
Mechanical pulp 1075 5c2 ~ewsprint 1 1 ~75 5~7 ~ewsprint 2 1~75 5.2 Cellobiose 1.0 11.0 Glucose 1~0 9.8 _ *Relati~e molecular distributio~ (%): Gl (28.9), G2 (17.0)9 G3 (13.3 G4 (1107),G5 (8~8)5 G6 (7.1), G7 (4~5),G8 (3~1), G9 (2.43, G10 (1.3), Gll (1.0), G12 (0~8)~ -~

Samples (50 mg3 of cellulose were suspended i~ ~arioussolutions (500 cm3) as specified in ~able 18. ~he suspen~ions were either stored at 4 C for 20 hours before placing in a boil-25 ing water ba-th or placed in a boil1ng water bath immediately, following the procedures described in ~xample 80 All tubes were kept in an ice bath after heatin~ until ready for analysis for total carbohydrate. ~he results obtai~ed are set GUt in ~able 13(a) a~d ~able 18 (b).

5~

36 ~ 31420 combinations 5_ _ ~ Eeating Acid Salt Pretreatment time % solubilisation at 4 C(mLn)Of cellulose HCl(1.OM)LiCl(sa^t) 20 hrs2~0 100 10~HBr(4-0~)LiLr(sat) O 1.33 100 +HBr(1.OM)LiBx(sat) 20 hrs2.5 100 E2S04(2~OM)Li2S04(Sat) O 30 3~5 E2S04( 0 5M)Li2S04( sat) 20 hrs30 14.5 EC1(4.0M)~aCl(sat) O 30 22 15HCl(4.0M)~MgC12(sat) O 30 3o HCl(4~0~)*M$C12(sat) 20 hrs30 49 H2504(0~5M)L.iCl(sat) 20 hrs240 11 T~A(l.OM)LiCl(sat) 20 hrs240 31 ~CA(loO~)LiCl(sat) 20 hxs. 9 6 20L~03(l-0M)LiCl(sat) 20 hrs240 O
E~OOH(l~OM)~iCl(sat) 20 hrs240 O
o~COOH(l.OM)LlCl(sat) 20 hrs90 ~ Derived from a solution of HBr (45% W/v) in glacial acetic acid~
* Derived fro~ MgC126E20.
~ ~orms two phases, upper phase anal~sedO
.

Solubilisation ~L.
5 ~ ~ _ _ _ _ _ Pretreatme~t Heatin~ time % solubili3ation Solution at 4 C(min) of cellulose . _ . . _. _ _ _ _~
HCl (4.0M) satura-ted w th LiCl, 1 part, none 30 24 ECl (4.0M) saturated with MgC126E20, - - - - ~' ~

Samples (50 mg) of cellulose fibres were placed in test-tubes to each of which was added hydrochloric acid (305M, 5~0 cm3)~
~he tubes were sealed and placed in a boiling water bath. ~ubes were removed after 2, 4, 8 and 12 hoursO Solutions after 8 and 12 hours were yellow9 and the residual cellulose blackened, whereas those at 2 and 4 hours we~e colouxless and the resi~ cellulose white. Anal~sis of the superna-tant solution was carried out for total carbohydrate. ~he resul-ts ob-tained are ~et out in ~able 19 ~he data therein, when compared with Example 9 ~able 13, demon-strates the effectiveness of the h~drochloric acid in comb~ation with lithium chloride~ ~hus 17% solubilisation is achieved with HCl (3.5M) in 720 m;nutes as co~pared with complete solubisation in 55 seconds with H~l (4vOM) saturated with lithium chloride or 83/o solubilisation in 55 ~econds with HCl (3~0M) saturated with lithium chloride.

38 ~ 31420 ~ _ .
Heating time /0 solubllised as e~pressed by total (min) carbohydrate in solution __ .. , .. . _ .. . " .. . .

2~0 5 ~ ~ ~ , ~
EXANPIE l6 15 ~ .
Samples of cellulose fibres were placed L~ screw cap bottles and the appropriate test solution ~10 cm3), as specified in ~able 20, was added. ~he bottles were placed in a water bath at 50 arld the contents stirred by means of a magnetic follower~
Samples (0~1 cm3) were removed at specified time intervals, diluted with water (to 10 cm3) and stored at 4C until analysis.
~nalyses for total carbohydrate a~d D-gLucose were perfo~med with appropriate dilution of samples at the higher cell~lose concen~
trations. The results obtained are set out in ~able 20. ~he 25- data contained therein demonstrate the effectiveness of hydro-chloric acid (4.0M) saturated with lithium chloride at solu~ilis-ing cellulose fibres at 1, 5 or 10~/o; complete solubilisation be-ing observed at 50 C with;n one hour~ with~Ln the limits of experimental error~

39 3 31~20 ~able 20 Solubilisation of cellulose f ~ .
. __ _ . . ~ _ _ ~otal Cellulose carbohydrate I~glucose ~o-tal con- ~eat.~g concentra-tion concentration car-bohydra-te centration Solution time in solu-tion in solution solubilised w/employed (hours) mg cm 3 mg cm 3 (%) _ __ _ ~
0.5 9- 3-3 E~1(4.0M 1.0 10~4 7~O
1.Osaturate 1~5 10~5 8.8 97 with ~iC 2.0 10. 5 lOo 2 6.o 10O5 10~3 __~ 1.,0 0-1 0.-0 2.~0 o~5 0~0 1~0 HCl(4.0M) 3~0 1.0 0.0 16 4~0 107 0.0 ECl( l o OM, 1.0 400 1~0 saturate 2.0 7.6 3.4 1.0with LiC 3~o 80 4 5~3 80 pret~eate 4~o 8 ~ 5 6~ 3 at 4C ~ 5~ O 8. 5 7~ O
20 hou~ 6~0 8~ 6 7 ~ 4 _ _ _ . _ . _ _ A _ _ _~
ECl(400M) 1.0 58.0 29~5 5.0 saturated 2.0 55 a 5 ~4~ 5 104 with LiCl 3.0 55.5 45.1 __ 5-5 51.0 35~5 ECl(4.0M) 1.0 107.6 42-4 10~0 saturated 2.0 106 ~ 0 6L 9 100 with LiCl 3 0 104. ( ~ -~5 ~ 3 _ _ _ 5~5 100.3 68.5 __ + ~nalysis of the relative molecular distribution of this sample indicated the following relative percentage composition~ G1(57 1), G2(23.5), G3(7.7), G4(2.5), G5(1.2), G6(0.4), G7(0~2), CT8( 0-1), Uniaentifi~d (7 ~ 4) ~

Solubilisation and hydrolysis of cellulose fibres by hydrochloric acid (4.OM) saturated with lithium chloride by treatment at 50°C
followed by an elevated temperature.
Samples (0.5 or 1.0 g) of cellulose fibres were place in screw cap bottles to each of which was added hydrochloric acid (4.OM) saturated with lithium chloride (10.0 cm3). These bottles were placed in a bath at 50°C for eith 1 or 2 hours, the contents being stirred with the aid of a magnetic follower. At the end of this first stage, aliquots (1.0cm3) were removed and placed in smaller bottles. These bottles were then immersed in a water bath at 80°C or a boiling water bath. Bottles were removed at the specified time intervals, cooled and dept at 4°C until analysed.
The samples were diluted (0.1cm3 to 100 cm3) prior to analysis for total carbohydrate, D-glucose and, where indicated, relative molecular distribution by gel permeation chromatography. The results obtained are set out in Tables 21 and 22. The solutions of hydrochloric acid (4.OM) saturated with lithium chloride were characterised by measurement of refractive index at 20°C using the sodium D line. Solution of various lithium chloride concen-trations were also measured. These results are shown in Table 23.
From this data, and the measured density, a solution of hydrochloric acid (4.OM) saturated with lithium chloride was estimated to contain:
HC1 146.0 g 1-1 LiC1 479.0 g 1-1 H2O 640.7 g 1-1 Unable to recognize this page.

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4~

44 ~ 31~20 ~able 22 . ~ ~ , Cell~lose Relative molecular distribution (%) concentra-tion ~emperature _ _ ~
(% ~/v) coIlditions + Gl G2 G3 G~ G5 ~nidentified . ~ _. _ _ _ 10.0 50C 60 m:i~ 65-9 19.8 3-9 0.8 0.2 9.4 100C 3 m~
_ _ _ 10. o 50U( 60 mi~l 60. 3 21. 6 4~ 4 07 9 0~2 12. 6 100C 7 m n __ ~_ ____ _ _ 10. 050 C 120min 65~ 3 23.0 43 3 0.8 0~8 S~ 3 100C 3 ~ _ ___ _ 5.0 50C 60 min 81~8 lo.6 --1. 0<,2 _ 6.1 100C 2min ~ _ _ _ _ _ _ + 100C nominal, immersion in a boiling wate~ bath~

- ~ ~~ 20 Solution n ~
_ _ _ _ _ _ _ ~
E[Cl ( 4 . OI~) ~ hiCl ( 9 ~ 0M) 1~. 4180 HCl (4.0M), LiCl (lO.0~) 1.4251 ECl (400M), hiCl (ll~OM) 1.4300 E[Cl (400M) ~ I.iCl (~at) 1~4319 LiCl (12M) 1~ 4202 LiCl (l~M) 1.4262 I~iCl (14~I) 1.4322 LiCl (sat) l. 4343 ____ ~ ~

~ 31~20 h~drochloric acid (2.0 M) saturated w th - _ Samples (2~0 g) of starch (~lum maydis) were placed in screw capped containers to each of which was added a solution (20.0 cm3) of hydrochloric acid (2.0 M) satwrated with magnesium chlori~e 6~20. ~he containers were immersed in a constant temper-ature bath at 50 for 30 to 180 minutes -the contents beLng stirred by means of a magnetic follower. After appropriate time inter-vals certain containers were transferred to a bath at 90 for up to twenty minutes~ After oooling the total carbohydrate and D-glucose contents of the solutions were determined. ~he results are set out in ~a~le 24, Control solutions of hydrochloric acid (1~0 M and 4.0 M) were also employed as a solubilisation and hydrolysis medium. It can be seen that under these conditions hydrolysis to glucose is negligable in the absence of the magnesium chloride and that the ready solubilisation achieved in the presence of magnesium chloride is obtained at higher levels of hydrochloric acid.

t~

46 ~ 31420 . .. _ ~ ~. . _ ~ime at Time at D-glucose ~ime at ~ime at - D-glucose 50 90 % 50 90 %
(min) (~Ln) (min) (mi~) _ 5.6 60 0 3708 _ 21~9 60 ¦ 2 38 D 1 _ 38.8 60 1 4 39O9 9 _ 56-5 60 6 51-7 120 _ 6900 60 8 65~1 150 _ 73-6 60 10 73-3 180 ~ 75~8 60 12 76.1 ~0 14 8206 __ _ . _ .. .. __ 3 0 13~ 180 0 65.1 2 114~2 180 2 6702 3 4 50.2 180 4 73.1 6 7-5 180 6 73.6 8 75~6 180 8 77~4 12 78.6 180 12 87-3 _ _ 14 79~9 L 180 14 82.3 ;

D 7 ~ r 1~14~

47 3 31~20 _ I _ .
Time at 50 Solubilisation D-glucose ECl concentration (min) (/) (%) (M) _ . 2~406 l 51-3 0.01 4.0 9-4 .

4 172 2 0.01 1.0 15 . 60 24.6 _ _ a~a~

~ _O
The procedure of Example 18 was followed using starch (1.5 g) in hydrochloric acid (2.0 M) satur~ted with mag~esium chloride 6~ 0 (10 cm3). After three ho~rs at 50 water (0.15 cm3) was added to one set of solutions and hydrolysis continued at 50.
~he D-glucose content of the solutions after various tLmes a~e set out in ~able 250 i3~

48~ 31420 Table ~

_ _ . _ _ __ ~o water additionWater added after 3.0 hours Time at 50 D-Glocose . Time at 50 D-gluoose . ,, (min) (%) . ~m~) (%) 16/7 30 19,4 47.2 60 5.3 1~0 76-5 120 793 180 80~3 180 85~5 210 80~ 210 88.1 . ~ _ 81.7 . ~0 92.4 PA/J~ ~
15 June 1981

Claims (8)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for the modification, solubilisation and/or hydrolysis of a glycosidically linked carbohydrate having reducing groups to produce one or more of the following effects:
(A) modification of the carbohydrate to induce increased accessibility and susceptibility to enzyme, microbes and chemicals, (B) solubilisation of the carbohydrate, and (C) solubilisation and hydrolysis of one or more glycosidic linkages in the carbohydrate to produce soluble oligosa-ccharides and/or glucose, wherein the carbohydrate is contacted at a temperature within the range -5°C to 125°C with a mixture comprising an aqueous inorganic acid at a concentration within the range 1 to 10 molar and a halide of a metal selected from the group consisting of lithium, magnesium and calcium or a precursor of said halide; said halide being present at a concentration within the range 1 molar to saturation.

2. A process according to Claim 1 for the solubilisation and/or hydrolysis of cellulose to produce a product selected from the group consisting of cellodextrin, cellotriose, cellobiose and glucose wherein the halide is a halide of lithium.

3. A process according to Claim 1 for the solubilisation and/or hydrolysis of starch to D-glucose or a mixture of sugars containing D-glucose wherein the halide is a halide of a metal selected from the group consisting of magnesium and calcium.

4. A process according to Claim 1 wherein the halide is a halide of magnesium.

5. A process according to Claim 1 wherein the halide is a chloride.

6. A process according to Claim 1 wherein the inorganic acid is hydrochloric acid.

7. A process according to Claim 1 wherein an additional quantity of water is added during the process.

8. A process according to Claim 1 wherein said halide is a halide of calcium.