Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
;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
11 3 31420
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~
12 3 31420
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~
13 B 31420
(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
18 3 31420
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
1 10 11 12
2 24 26 33
4 31 34 39
6 56 57 57
24 70 77 95
48 91 94 97
96 98 97 g6
100 97 97 . 99
% conversion as expressea by total æugar a~alysis
190 98 1 97 1 100
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
4 372 28
25 6 35 36
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
6 35 73
24 78 85
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
50C I 100C50C I 100C
/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
2 15 15 25 24
4 16 18 26 27
6 17 18 3 3o
2524 24 25 34 36
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.
Unable to recognize this page.
~ 31420
EXAMPIE 8
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.
33 3 31420
~.
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
EXAMPLE 12
__.__
~.
~ 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
.
37 3 31420
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
__ .. , .. . _ .. . " .. . .
120 2
2~0 5
480 14
- 10 1 720 17
~ ~ ~ , ~
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) ~
EXAMPLE 17
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.
Unable to recognize this page.
Unable to recognize this page.
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
79-6 180 10 80 D 1
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
71 69
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
