THIRTY YEARS OF PROGRESS IN CHITIN AND CHITOSAN
George A. F. Roberts
Bioengineering Research Unit, Nottingham University,
Nottingham NG2 7RD, England
E-mail: [email protected]
1. Introduction
Although Braconnot’s discovery of chitin preceded Payen’s discovery of cellulose by almost
30 years, and the amount of chitin produced annually by biosynthesis is not that much less
than cellulose, the industrial application of cellulose is staggeringly in advance of that of
chitin/chitosan. One possible reason for this is the much greater ease of obtaining industrial
quantities of cellulose and another is the much greater complexity of the chitosan molecule.
We are only beginning to really understand chitosan and so perhaps we are at last on the
verge of its commercial exploitation.
A major boost to chitosan research was given by the 1st International Conference on
Chitin/Chitosan (ICCC), held in Boston in May 1977. It was mainly organised by Mr Vincent
LoCicero of the Massachusetts Science and Technology Foundation, and hosted jointly by
the MIT Sea Grant Program and the Massachusetts Science and Technology Foundation.
At the present time, when there are more chitin/chitosan conferences than anyone can find
time (or funding) to attend, it is perhaps difficult to fully understand the impact that this first
conference had on researchers in the field of chitin/chitosan. There was almost no contact
between scientists working in different areas of chitin/chitosan research, such as the chemical
and the biological areas, and only limited contact even between scientists working in the
same area. The 1st ICCC brought together, for the first time, some 240 scientists working on
any and every aspect of this one molecule, chitin, and its main derivative chitosan.
In this paper I want to consider what our knowledge of chitin and chitosan was at the time
and see how our understanding of it has developed over the succeeding 30 years, and also
indicate areas where our knowledge is still less than satisfactory.
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2. Sources
A critical evaluation of potential sources of chitin and chitosan [1] concluded that shrimp,
prawn and crab waste were the principle source of chitin and chitosan and would remain so
for the immediate future. However it was envisaged that both Antarctic krill and cultured
fungi would become major sources of these raw materials.
Thirty years later shrimp and prawn continue to predominate due to at least two factors.
The first is the growth of aquaculture, which has allowed a more reliable and continuous
supply of waste material and the second is the large increase in consumption of what was
still considered, in 1977, to be luxury food items. Together these two factors, the second of
which was not foreseen in the 1977 paper, have given rise to the tremendous increase in the
quantity of shrimp and prawn being processed, particularly in Asia but also in the Middle
East, and hence in the amount of waste available for chitin/chitosan production. Crab, lobster
and crayfish are generally included together with shrimp and prawn in the general grouping
of shellfish. Although not as important as shrimp and prawn as a raw material source, crabderived chitin/chitosan is commercially available and may become more important with
the westward migration of the Kamchatka crab species and its gradual movement along the
coast of north and northwest Norway. Also there are considerable quantities of lobster shell
as yet not commercially exploited in Newfoundland, and of crayfish in the USA.
Krill has been the subject of considerable study as a potential source of chitosan. initially
in Chile but more extensively in Poland under the leadership of the late Professor Brzeski
Although a pilot plant scale production line was set up and run for some time at the Sea
Fisheries Institute. Gdynia. krill chitosan is currently not a commercial product, despite
the large amounts that are potentially available, In contrast to this. squid pen chitin and
chitosan are produced commercially although the total amounts available are relatively
small compared to those of shrimp and prawn. The comment was made in the 1977 paper
that “squid will not become a major source of chitosan until there is a substantial change in
the eating habits of the Western world.” However, even such a change would not necessarily
increase the amount of squid-derived chitosan available unless the squid were to be processed
centrally in the various regions.
Considerable research has been carried out on using mycelium waste from fermentation
processes as a source of fungal chitin and chitosan- It is argued that this would offer a
stable. non-seasonal source of raw material that would be more consistent in character than
shellfish waste, but so far this route does not appear to have been taken up by chitosanproducing companies. Currently there is only one commercial source of fungal chitosan and
that is me Belgium company Kitozyme. However their raw material is not mycelium waste
from a fermentation process, which is what is normally envisaged when fungal chitosan is
referred to, but actually conventional edible mushrooms grown under contract in France
and shipped to Belgium for processing So mycelium waste still remains a vast and as yet
untapped potential source of chitosan
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3. Production
Demineralisation and deproteinisation - At the time of the 1st ICCC the process of
converting shellfish waste to chitin had changed little from that disclosed in the early
literature and patents, with the particular conditions adopted being based on very little in
me way of detailed comparative studies However recently the two steps of demineralisation
and deprotemisation of a-chitin using the traditional reagents. HCl and NaOH. have been
rigorously investigated [2 - 4]. This work shows that demineralisation of shrimp waste is
complete within 15 minutes using 0.25 M HCl at room temperature and a liquor ratio of
40 : 1. This is considerably milder than any demineralisation treatment in the literature up
to and including the 1st ICCC. and should reduce the amount of acid hydrolysis occurring
in me demineralisation step. Deproteinisation studies were carried out at 70 °C for 24 hours
in 1 M NaOH at a liquor ratio of 15 : 1. which is quite similar to various processes reported
previously in die literature. The chitin obtained from this process had a calcium content of
< 0.01% and a residual protein content of 0.6% and may represent the ultimate in chemical
processing of crustacean-based waste.
It was confirmed that squid pen requires much milder processing conditions [4], there being
no need to use an acidic demineralising step due to the very low mineral content of the pen,
thus avoiding possible acid hydrolysis. It was also found that providing the lipoproteins are
removed by a preliminary treatment in 2 : 1 chlorofom : methanol, the residual protein can
be reduced to 0.4% by a 24 hour treatment in 1 M NaOH at room temperature.
The use of enzymes in the deproteinisation step was first mentioned in the original Rigbv
patent of 1934 but there has been a renewed interest in this approach since 1977. This work
has led to the lactic acid bacterial fermentation process, studied most extensively bv Hall
and co-workers [5, 6]. In this. well ground shellfish waste is inoculated with a lactic acid
culture and a carbohydrate source and the whole mixed thoroughly together Lactic acid is
produced causing a lowering of the pH. so dissolving the CaCO3, while at the same time the
residual protein undergoes proteolysis by the enzymes from the shellfish viscera. However
there are considerable amounts of calcium salts and protein remaining in the chitin at the
end of the process, and the requires to be further purified by standard, but milder, chemical
treatments.
A related approach to this, in which the deproteinisation and demineralisation steps are
separated, has recently been reported [7]. In this method shrimp waste was deproteinised
using Aspergillus niger, washed and dried, then demineralised using acetic or lactic acid
produced by fermentation from low cost biomass such as cheese whey. The calcium,
magnesium and potassium acetates obtained as by-products are suggested as possible deiceing agents, being less corrosive and more environmentally friendly than the chloride salts,
while the calcium and potassium lactates could find applications as food preservatives.
A totally new approach to the extraction of chitin from shellfish waste is the electrochemical
process developed in Russia by Dr Maslova and co-workers [8 - 10]. Details of the process,
the quality and purity of the chitin produced, and the cost of the process do not appear to
be available, at least in English, but it is claimed to produce chitin with better functional
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capacities under milder conditions than with conventional processes. One attractive feature
of the process is that the processing unit can be containerised, as its overall dimensions are
4 m × 2.2 m × 2.5 m, and taken round to areas where shellfish are processed, rather than
having to transport wet shellfish waste into a central chitin producing plant that might be
located quite a distance from the shellfish processing sites. This could considerably increase
the available raw material for chitin production.
Deacetylation - Although the heterogeneous alkaline deacetylation of chitin would appear
to be a simple one, it is still being studied and modified nearly 150 years after Rouget’s
original paper [11]. The conditions of deacetylation reported in presentations at the 1st ICCC
mainly involved the use of 50% (wt./wt.) NaOH and the relatively high temperatures of
100 - 120 °C. Since then there has been a trend towards lower temperatures and reduction in
the amounts of NaOH used, together with a considerable amount of research into enzymatic
deacetylation using chitin deacetylases.
n Lower temperature deacetylation - The use of lower temperatures has been reported by a
number of researchers and some industrial processors also now carry out the deacetylation
step within the temperature range of 60 - 80 °C. One detailed study [12] examined the
deacetylation of chitin at 30 °C using NaOH solutions having concentrations ranging
from 23 wt.-% to 39.5 wt.% and varying liquor ratios. At the lowest liquor ratio, 14 : 1,
acid-soluble products were only obtained with NaOH solutions of 33 wt.-% or higher,
with the time required to obtain them decreasing with increase in NaOH concentration:
6 days for 33 wt.-%, 5 days for 35 wt.-%, and 4 days for 37.5 or 39.5 wt.-%. Surprisingly,
increasing the liquor ratio was found to reduce the reaction time required and with
33 wt.-% NaOH and a liquor ratio of 56 : 1 a fully acid-soluble product was obtained
after only 1 day compared to 6 days at a liquor ratio of 14 : 1. No explanation was offered
for this effect of liquor ratio, which cannot be due to smaller changes in the NaOH
concentration during deacetylation at the higher liquor ratios since even at 14 : 1 the
amount of NaOH present is many times that required for total deacetylation.
n Reduced alkali deacetylation - The conventional laboratory deacetylation process is
carried out at a liquor ratio of from 10 : 1 to 15 : 1, with industrial processes at a slightly
lower ratio, leading to considerable cost in terms of chemicals, effluent disposal, and
corrosion effects. So it is not surprising that attempts have been made to reduce the
amounts of NaOH solution utilised in the deacetylation step. One approach [13] has
been to mix 1 part chitin with 2 - 5 parts NaOH solution then heat, and another similar
process involves steeping chitin in excess NaOH solution, filtering to leave a mixture
containing 0.5 - 2.5 parts NaOH to 1 part chitin, then heating [14]. A second approach
is to use a water-miscible diluent such as IPA, TBA or acetone that acts both as the
reaction medium, enabling the mass of chitin particles to be stirred, and to distribute
the alkali uniformly throughout the chitin [15]. This latter approach can be extended to
allow concurrent deacetylation and derivatisation as demonstrated in the production of
N-,O-carboxymethylchitosan [16].
n Enzymatic deacetylation - There have been a number of studies devoted to trying
to develop an enzyme-based method for converting chitin to chitosan using chitin
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deacetylases. The attractions of such a process are fairly obvious: more environmentally
friendly; lower chemical costs; non-degradative hence higher molecular weight; ambient
temperature for processing. However there are difficulties, particularly the very limited
accessibility of the substrate, crystalline chitin, to large molecules such as an enzyme.
In what appears to be the earliest attempt [17] chitin deacetylases extracted from
Colletotrichum lindemuthianum and Mucor rouxii were used successfully to deacetylate
water-soluble glycol chitin. However when tested on insoluble chitin, either crystalline
or amorphous chitin with the latter produced in the form of colloidal chitin particles by
reprecipitation from solution, the results were not promising, with 0.5% deacetylation
of the unmodified substrate and 5% (Colletotrichum lindemuthianum) and 9.5% (Mucor
rouxii) of the amorphous chitin substrate. It was found however that Mucor rouxii was
very effective in converting partially deacetylated chitosan substrate to almost fully
deacetylated ones. Since the first reports of enzymatic deacetylation there has been little
change and the chitin substrate requires either to be solubilised, through derivatisation,
or rendered amorphous, by a solubilisation/precipitation process, if more than a very
limited deacetylation level is to be achieved.
n Fundamental studies on deacetylation - The above variations on the “traditional
process are empirical in character and have not led to any new understanding of the
actual mechanism of the deacetylation of chitin. Recently a series of papers [18-20]
have reported a very detailed study of the mechanism and kinetics of the heterogeneous
alkaline deacetylation of both a- and b-chitin, treating the process as a multi-step one.
with isolation, washing and drying between the steps.
This work first confirmed the fact that the rate of deacetvlation of p-chitin is considerably
greater at all temperatures than is that of a-chitin. although the activation energy of
deacetylation is approximately the same for both polymorphic forms. The greater rate of
deacetylation of b-chitin arises from die greater of its pre-exponential factor. A, which
is a measure of the frequency of collisions between NaOH molecules and amide groups
that are in the proper orientation for reaction The much higher value of A for b-chitin is
confirmation of the accepted view that the greater reactivity of b-chitin in the first stage of
deacetylation is due to the greater accessibility of the chitin chains in the b-polymorph due
to its less extensive interchain network of H-bonding. Furthermore the b-chitin becomes
fully amorphous early on in the deacetylation process, at Fa = 0.75. whereas the crystalline
regions of a-chitin are not disrupted until a much lower Fa value (0.52) is reached.
Because of the almost completely amorphous character of both partially deacetylated materials after the first stage, the two substrates showed very similar initial rates of deacetylation
m the second stage. Also very little difference was found between either the activation energies of deacetylation or the pre-exponential factors for both samples The state of hvdration
of the sodium hydroxide was also found to be very important in determining the rate and
effectiveness of the deacetylation reaction, with the mono-hydrate and dehydrate being the
most reactive in the deacetylation step, whilst the anhydrous sodium hydroxide is more or
less ineffective.
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Practical developments - Two new technological developments have been reported. In the
first [21] the chitin. prior to deacetylation, is swollen for 65 - 75 hours in aqueous alkali,
preferably 15 M NaOH. at a temperature of below 30 °C then deacetylated by heating to
above 40 °C in the alkaline solution used for swelling the material. The products are claimed
to have enhanced water solubility at physiological or near physiological pH.
The second development is technically more complex and involves freeze-pump out-thaw
(FPT) cycles [22]. In these the chitin and alkali are placed in a reactor which is sealed and
placed in liquid nitrogen. Once frozen the reactor is degassed by means of a vacuum pump,
then the frozen mixture is allowed to thaw at room temperature. Six or seven cycles are
required to completely degas the reaction medium and ensure the absence of oxygen. After
the final cycle deacetylation is carried out by placing the reactor in a preheated oil bath and
the suspension stirred for 5-120 minutes once the required temperature had been reached. It
is claimed that the FPT cycles disrupt the crystalline structure of the chitin, making it more
permeable to the NaOH solution, thereby giving the heterogeneous reaction the benefits of
a homogeneous reaction without the disadvantages. However it is difficult to imagine the
application of this technique in large scale processing of chitin.
Economics of production - Three papers at the 1st ICCC considered the economics of
chitin/chitosan production from shellfish waste as related to the USA [23 - 25]. Taking a
plant capable of producing approximately 5 × 105 kg chitosan/year a spread of possible
production costs were calculated ranging from $1.20/kg [23] to $2.2 I/kg [24] (chitin) and
from $1.82 [23] to $4.42 [24] (chitosan). The third paper [25] only considered chitosan
production and proposed a production cost of $2.21 - $5.53/kg. This gives an average cost of
$3.50/kg, which at today’s prices, based on the Consumer Price Index (1913-present) of the
Federal Reserve Bank of Minneapolis, converts to $11.50/kg at present values. This is very
close to the market price of reasonable quality chitosan from China. Of course this price does
not include the cost of transportation which, for small quantities of less than about 100 kg,
would almost double the price. However the calculations made in 1977 were based on the
assumption that the raw material, that is the waste from the shellfish processing plants,
would be available at very low or even zero cost - indeed in one paper it was suggested that
the shellfish processor might actually pay the chitosan producer to take it away! This is one
factor that has changed considerably and it was obvious from some comments heard at the
ICCC conference in Montpellier in 2006 that chitosan producers are now experiencing some
difficulty in obtaining all the raw material that they require. Competition for the shellfish
waste is now coming in particular from animal feed stuff manufacturers, including those
supplying prawn and shrimp farms. This conflict was foreseen in a paper presented at the
1st ICCC (24) and may become a major obstacle to increasing chitosan production if a large
scale chitosan application was to emerge in the future. As mentioned above, the use of waste
mycelium as a raw material, and use of the electrochemical process, could both contribute
to overcoming this potential shortage should it ever arise.
Although the use of chitosan in medical applications was the subject of a paper in 1977 [26]
there was no suggestion that the price would be so very high as is currently the case, with
‘medical grade’ chitosan being offered by several companies at a cost of approximately
$25,000/kg. It remains to be seen whether such a premium price can be sustained now that
about half-a-dozen companies offer this grade of chitosan.
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4. Structure
A major change in our thinking about chitosan 30 years on from the first conference is
that we now accept that the term ‘chitosan’ does not identify a single unique substance
but rather refers to a continuum of copolymers varying in the ratio of anhydro-N-acetylD-glucosamine to anhydro-D-glucosamine residues, and with the common property of
solubility in dilute organic acids such as acetic acid. Of the 24 papers dealing with ‘chitosan’
in the Proceedings of the 1st ICCC only 4 contained any suggestion that chitosan was not a
single substance with defined characteristics. Nowadays most authors define the particular
chitosan(s) used in their research in terms at least of the Fa (chemical composition) and
molecular weight and would not dream of comparing, for example, the metal ion absorption
capacity of chitosan samples from several sources without determining the concentration of
amine groups in each of the samples This was not the case up to 1977. Indeed in 1977 the
methods of determining the concentration of amine groups in a given sample of chitosan
were both few and very laborious Nowadays there is a whole spectrum of techniques of
varying accuracy and ease of performance with NMR spectroscopy generally regarded as
the ‘Gold Standard’
5. Some remaining problems
1. What is the difference, if any, between materials prepared by heterogeneous and
homogeneous deacetylation of chitin?’
Despite the considerable amount of work carried out on this question, it has not yet
been answered conclusively
2. Is enzymic deacetylation of chitin a reasonable possibility for commercial production
or is it a modem-day ‘Philosopher’s stone’?
Is it possible to devise an inexpensive method of “opening up’’ the chitin structure and
thereby make it accessible to chitin deacetylases?
3. What are the morphologies of chitin and chitosan?
Both a- and b-chitm are two-phase materials, that is they each contain both crystalline
and amorphous regions. However, although the crystal structure of each is known we do
not yet have an overall morphology for how the crystalline and amorphous regions relate
to one another. In view of the overall similarity of the chain structure of chitin to that of
cellulose it is reasonable, in the absence of any evidence to the contrary, to suppose that
chitin exhibits a fringed micelle structure similar to that proposed for cellulose. However
the possibility of chain folding in the crystalline regions cannot, as yet, be ruled out.
4. Can we develop a universally-acceptable nomenclature system for chitosan’’ The
recognition of the variability of the chitosan structure raises other problems, a major one
being nomenclature. I have raised this issue on two other occasions, in the first issue of
the European Chitin Society’s Newsletter and in a paper at the 7th ICCC/EUCHIS ‘97
meeting, but I think that it is a very important issue so raise it again here. Even today, it
is possible to find many terms used in a single conference proceedings to describe what
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G. A. F. Roberts
is, in fact, the mole fraction of N-acetylated units in the chain: degree of deacetylation.
degree of acetylation, % deacetylation. % acetylation. % N-acetylation, degree of
residual N-acetylation and many more. In addition there is no agreed definition of what
the term ‘chitosan’ covers. I again urge that we adopt a unified approach and propose the
following outline of a system for consideration,
1 All b-(l __ 4)-linked copolymers of anhydro-2-acetamido-2-deoxy-D-glucopyranose
and anhydro-2-amino-2-deoxy-D-glucopyranose units should be designated as chitin
or chitosan on the basis of their insolubility or solubility, respectively, in 0.1 M acetic
acid.
2. The confusing multitude of terms currently used in the literature to describe what is
essentially the mole faction of anhydro-2-acetamido-2-deoxy-D-glucopyranose units
should be replaced by the symbol Fa representing the mole fraction of anhydro-2acetamido-2-deoxy-D-glucopyranose units. In cases where it is necessary to express
the mole faction of anhydro-2-amino-2-deoxy-D-glucopyranose units, this can be
either by use of the symbol Fd or. preferably. (I-Fa).
3. The Fa, if known, should be given in brackets after the name e.g. chitin [0.94], chitosan
[0.25],
4. If deacetylation has been carried out under homogeneous conditions this can be
indicated by an italicised ‘h’ after the figure giving the Fa e.g. chitosan [0.5h] which
would be expected to differ markedly m properties from chitosan [0.5].
5. A distinction should be made between material prepared directly from chitin by
deacetylation (chitin or chitosan depending on solubility in 0.1 M acetic acid) and
material prepared by re-N-acetylation of chitosan (N-acetylchitosans). In the case of
the latter material the Fa is given in two parts; the first representing the Fa of the
starting chitosan and the second representing the value of the increase in Fa due to the
N-acetylation process. Thus N-acetylchitosan [0.1/0.4h] would unambiguously identify
a material having a total Fa of 0.5 and which had been prepared by homogeneous
N-acetylation of a chitosan sample previously prepared by heterogeneous deacetylation
of chitin and having an Fa value of 0.1.
References
I. Allan, G. G., Fox, J. R. and Kong, N. (1978) A critical evaluation of the potential sources of chitin
and chitosan. In Proceedings of the First International Conference on Chitin and Chitosan (R.
A. A. Muzzarelli and E. R. Pariser, eds.) pp. 64-78.
2. Percot, A., Viton, C. and Domard, A. (2003) Optimization of chitin extraction from shrimp shells.
Biomacromolecules. 4, pp. 12-18.
3. Percot, A., Viton, C. and Domard, A. (2003) Characterization of shrimp shell deproteinization.
Biomacromolecules. 4, pp. 1380-1385.
4. Chaussard, C. and Domard, A. (2004) New aspects of the extraction of chitin from squid pens.
Biomacromolecules. 5, pp. 559-564.
5. Guerrero Legarreta, I., Zakaria, Z. and Hall, G. M. (1996) Lactic fermentation of prawn waste:
comparison of commercial and isolated starter cultures. In Advances in Chitin Science Vol. I (A.
Domard, C. Jeuniaux, R. Muzzarelli and G. Roberts, eds.) pp. 399-406, Jaques Andre.
6. Cira, L. A., Huerta, S., Guerrero, I., Rosas, R. and Shirai, K. (2000) Scaling up of lactic acid
fermentation of prawn wastes in packed-bed column reactor for chitin recovery. In Advances in
Chitin Science Vol. IV ( M. G. Peter, A, Domard and R. A. A. Muzzarelli, eds.) pp. 2-27, Universitet
Potsdam.
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7. Mahmoud, N. S., Ghaly, A. E. and Arab, F. (2007) Unconventional approach for demineralization
of deproteinized crustacean shells for chitin production. Amer. J. Biochem. Biotech. 3(1), pp. 1-9.
8. Maslova, G. V., Kuprina, E. E., Bogeruk, A. K. and Ezjov. V. G. (1996) Russian Patent
No. 2059390.
9. Maslova, G. V. (2001) Theory and practice of producing chitin by electrochemical method. In
Chitin and Chitosan (T. Uragami, K. Kurita and T. Fukamizo, eds.) pp. 325-327, Kodansha
Scientific Ltd.
10. M aslova, G. V. (2003) Electrochemical method of obtaining chitin: the essence and
physicochemical aspects of electrochemical activation. In Advances in Chitin Science Vol. VI
(K. M. Varum, A. Domard and 0. Smidsred, eds.) pp. 263-264, NTNU Trondheim.
11. Rouget, C. (1859) Des substances amylacees dans Ie tissue des animaux, specialement les
Atricules (Chitine). Comp. Rend. 48, pp. 792-795.
12. Alimuniar, A. and Zainuddin, R. (1992) An economical technique for producing chitosan. In
Advances in Chitin and Chitosan (C. J. Brine, P. A. Sandford and J. P. Zikakis, eds.) pp.627-633,
Elsevier Applied Science
13. Peniston, Q. P. and Johnson, E. L. (1980) U. S. Patent No. 4195175.
14. Yoshiichi, A., Tomoya, T. and Akira, A. (1987) Japanese Patent No. 87179503.
15. Batista, I. and Roberts, G. A. F. (1990) A novel, facile technique for deacetylating chitin. Makromol.
Chemie, 191, pp. 429-434.
16. Hayes, E. R. (1992) Canadian Patent No. 1274507.
17. Tsigos, I., Martinou, A., Varum, K. M. and Bouriotis, V. (1996) Enzymatic deacetylation of chitinous
substrates employing chitin dacetylases. In Advances in Chitin Science Vol. I (A. Domard, C.
Jeuniaux, R. Muzzarelli and G. Roberts, eds.) pp. 59-69, Jaques Andre
18. Lamarque, G., Viton, C. and Domard, A. (2004) Comparative study of the first heterogeneous
deacetylation of a- and p-chitins in a multistep process. Biomacromolecules, 5. pp. 992-1001.
19. Lamarque, G., Viton, C. and Domard, A. (2004) Comparative study of the second and third
heterogeneous deacetylations of a- and p-chitins in a multistep process. Biomacromolecules,
5, pp. 1899-1907.
20. L amarque, G., Chaussard, G. and Domard, A. (2007) Thennodynamic aspects of the
heterogeneous deacetylation of p-chitin: Reaction mechanisms. Biomacromolecules, 8, pp.
1943-1950.
21. Varum, K. M. and Smidsrod, 0. (1994) Chitosan Preparation. World Patent Application No.
W003011912
22. Lamarque, G., Cretenet, M., Viton, C. and Domard, A. (2005) New route of deacetylation of a- and
p-chitins by means of Freeze-Pump Out-Thaw cycles. Biomacromolecules, 6, pp. 1380-1388.
23. Johnson, E. L. and Peniston, Q. P. (1978) The production of chitin and chitosan. In Proceedings
of the First International Conference on Chitin and Chitosan (R. A. A. Muzzarelli and E. R. Pariser,
eds.) pp. 80-87.
24. Perceval, P. M. (1978) The economics of chitin recovery and production. In Proceedings of the
First International Conference on Chitin and Chitosan ( R. A. A. Muzzarelli and E. R. Pariser,
eds.) pp. 45-53.
25. Murray, A. E. and Hattis, D. (1978) Approaches to a practical assessment of supply and demand
for chitin products in the United States. In Proceedings of the First International Conference on
Chitin and Chitosan ( R. A. A. Muzzarelli and E. R. Pariser, eds.) pp. 30-44.
26. Balassa, L. L. and Prudden, J. F. (1978) Applications of chitin and chitosan in wound-healing
acceleration. In Proceedings of the First International Conference on Chitin and Chitosan ( R.
A. A. Muzzarelli and E. R. Pariser, eds.) pp. 296-305.
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