Decomposition of mycelia matrix and extraction of chitosan from Gongronella butleri USDB 0201 and Absidia coerulea ATCC 14076
Nitar Nwe1,2,3, Willem F. Stevens3,4, Didier Montet3,5, Seiichi Tokura1, Hiroshi Tamura1*
1Faculty of Chemistry, Materials and Bioengineering and HRC, Kansai University, Suita, Osaka 564-8680, Japan; 2Japan Society for Promotion of Science (JSPS), Japan; 3Bioprocess Technology, Asian Institute of Technology, Bangkok, Thailand; 4Centre of Excellence for Shrimp Biotechnology, Mahidol University, Bangkok, Thailand; 5CIRAD-AMIS, UR Food Quality TA 40/16, 34398 Montpellier Cedex 5, France
*Correspondence author: Tel: +81-6-6368-0871, Email:email@example.com
Free chitosan, 2 g/100 g mycelia from G. butleri and 6.5 g/100g mycelia from A. coerulea were isolated by 1 M NaOH at 45 0C for 13 h and 0.35 M acetic acid at 95 0C for 5 h. Both mycelia matrixes did not break down under these conditions. However mycelia matrix could be decomposed by treatment with 11 M NaOH at 45 0C for 13 h and 0.35 M acetic acid at 95 0C for 5 h and then extracted the total chitosan, 8-9 g/100 g mycelia from both fungi. According to these results, G. butleri has higher amount of complexed chitosan and A. coerulea has higher amount in free chitosan.
Keywords: Acetic acid treatment, Chitosan, Decomposition, Mycelia matrix, NaOH treatment
Chitin is a natural polysaccharide, that consists of a co-polymer of N-acetyl-D-glucosamine and D-glucosamine residues, linked by b-1,4 glucosidic bonds. The deacetylated form of chitin is chitosan. Chitosan with a molecular weight of 5-50 kDa is effective in cholesterol absorption , for production of semipermeable membrane , as antifungal agent and as elicitor of plant growth  and in enhancing protocorm like body formation in orchid tissue culture . The isolation of chitosan from fungal source is attractive to obtain a low molecular weight chitosan (10-50 kDa with high polydispersity, 3-5). This fungal chitosan will be applicable in agriculture and especially in medical applications, where the absence of allergenic shellfish protein is dictated.
Several methods have been developed for the extraction of chitosan from fungal mycelia [6, 7, 8, 9, 10, 11]. The yield of chitosan produced from the fungal mycelia treated with 1 M NaOH is very low. A bottleneck for isolation of larger amounts of chitosan is the binding of chitosan to cell wall 1,3-b glucan . In 1994, Muzzarelli et al., developed a single alkaline extraction method to isolate a chitosan-glucan complex . Nwe and Stevens obtained a high yield of chitosan by applying 11 M NaOH, followed by enzymatic treatment with α-amylase . In this paper crucial factors have been studied involved in the liberation of chitin/chitosan from its anchorage in the fungal cell wall matrix.
The fungi Gongronella butleri USDB 0201 and Absidia coerulea ATCC 14076 were grown by solid substrate fermentation and fed-batch fermentation, respectively. Fungal mycelia were harvested at the end of fermentations and treated with NaOH and acetic acid under different conditions to decompose the mycelia matrix. Information on the nature of mycelia matrix from both fungi was obtained by extraction of chitosan under various conditions, enzyme treatment and microstructural observation. Finally, a comparison was made of the yield of chitosan extracted in different ways.
Materials and methods
Gongronella butleri USDB 0201 and Absidia coerulea ATCC 14076 belonging to the class of Zygomycetes, was obtained from the Department of Biological Sciences, National University of Singapore and the American Type Culture Collection, Rockville, MD, USA respectively. The strains were maintained on 3.9 percent potato dextrose agar (PDA) slants at 4oC. The spore suspension for inoculation was prepared from a 10-day culture of G. bulteri grown on PDA plates at 30oC and from a 7-day culture of A. coerulea grown on PDA plates at 25oC. G. butleri and A. coerulea were grown under solid substrate fermentation  and fed-batch fermentation respectively.
Effect of acetic acid treatment conditions on the decomposition of mycelia matrix of G.butleri and chitosan yield
Chitosan was extracted using a method proposed by Tan et al., modified from Shimahara et al., [12, 13]. Dried mycelia powder (1 g) was treated with 40 ml of 1 M NaOH and sodium borohydride 0.05 g to prevent oxidation. The mixture was autoclaved at 121oC for 15 min and centrifuged at 16000 g for 5 min to sediment the alkali insoluble material (AIM). The AIM was washed twice with distilled water, washed once with 95 % ethanol and dried. Chitosan was extracted from AIM with 200 ml of 0.35 M acetic acid per g dried AIM at various temperatures for 5 h (Table 1). The mixture was centrifuged at 16000 g for 5 min. The supernatant was collected and adjusted to pH 8 -9 with 2 M NaOH to precipitate the chitosan. The suspension was centrifuged at 16000 g for 5 min. The chitosan pellet was collected and washed twice with distilled water, once with 95 % ethanol and freeze-dried.
Effect of NaOH treatment conditions on the decomposition of myceliar matrix of G.butleri
Dried mycelia, 1 g was treated with 40 ml of NaOH solution under various treatment conditions (Table 2). The AIM was collected, washed with distilled water until neutral pH, and dried. Dried AIM, 1 g was treated with 200 ml of 0.35 M acetic acid at 950C for 5 h and observed the decomposition of mycelia matrix.
Extraction of free chitosan
Free non-complex-bound chitosan was isolated by treatment of 1 g mycelia with 40 ml of 1 M NaOH at 45 0C for 13 h. The resultant AIM, 1 g was treated with 200 ml of 0.35 M acetic acid at 95 0C for 5 h. The extracted solution was collected and adjusted the pH of the solution to 9. The chitosan precipitate was washed with distilled water up to neutral pH and freeze-dried.
Extraction of total chitosan
Dried mycelia, 1 g was treated with 40 ml of 11 M NaOH at 45 0C for 13 h. The AIM was collected, washed with distilled water until neutral pH, and dried. Dried AIM, 1g was treated with 200 ml of 0.35 M acetic acid at 95 0C for 5 h. Total chitosan was extracted from the AIM suspension according to the method II described Nwe and Stevens, 2002 .
Characterization of chitosan
The degree of deacetylation was determined by first derivative ultraviolet spectrophotometry  with the modification of Tan et al., . Relative molecular weight was determined by gel permeation chromatography (GPC) in a Waters HPLC equipped with Ultrahydrogel 2000, 1000 and 500 columns and a Waters 410 Differential Refractometer Detector.
Results and discussions
In fungal mycelia, chitin/chitosan consists in 3 forms: free chitosan, free precursor chitin and chitin/chitosan covalently bound to β-glucan. In Zygomycetes, chitosan is synthesized by deacetylation of chitin with the action of the enzyme chitin deacetylase enzyme [16, 17]. Free chitosan could be extracted easily from the fungal mycelia using 1 M NaOH . The individual chains of chitin/chitosan are aggregated into microfibrils by hydrogen bonds. These chitin/chitosan microfibrils are cross-linked to the b-glucan and form a rigid network, the major component of the cell walls of most fungi [18, 19]. For the extraction of chitosan from this network, the cell wall matrix has to be weakened and the chitin microfibrils have to be deacetylated to make the chitosan-β-glucan complex soluble in acetic acid. Thereafter the chitosan-glucan complex has to be split by breaking the covalent bond between the chitosan and glucan. These steps are necessary for the extraction of chitosan anchored in the fungal cell wall.
Effect of acid treatment conditions on the decomposition of mycelia matrix of G. butleri and chitosan yield
The fungal mycelia treated with 1M NaOH at 121oC for 15 min generate very rigid alkaline insoluble materials. The duration and temperature of the acetic acid extraction were varied to optimize the 0.35 M acetic acid extraction condition. The treatment at 95oC for 5h gave a high chitosan yield but the AIM did not break down (Table 1). Extraction at 50oC yielded 1.44 g chitosan per 100 g mycelia. Extraction at 95oC resulted in 3.14 g chitosan per 100 g mycelia. The yield of chitosan decreased to 1.71 g/100g of mycelia when the incubation at 95oC was extended to total 14 h. The decrease amount of chitosan might be the extracted chitosan degradation in hot acetic acid beyond the peak incubation time. Hu et al. reported that acetic acid extraction at 121oC resulted in chitosan chain degradation . Therefore the condition of acetic acid treatment at 95oC for 5 h was selected to study the decomposition of mycelia matrix by NaOH treatment.
Table 1: Effect of acetic acid treatment temperature on the extraction of fungal chitosan.
|Chitosan yield (g/100 g of mycelia)|
Effect of NaOH treatment conditions on the decomposition of mycelia matrix of G.butleri
Mycelia powders were treated with various concentrations of NaOH in the range of 1 to 12 M NaOH various time intervals at various temperatures (Table 2). AIM was collected and treated with 0.35 M acetic acid solution at 95oC for 5 h. Most of the residual acid-alkaline insoluble material obtained from treatments in the range of 1 to 7 M NaOH was very rigid and sedimented quickly on standing (Fig.1A). However breakdown of the matrix was observed after acid extraction of AIM obtained by 11 M NaOH treatments (Table 2). The suspension became very turbid and did not form a pellet on standing (Fig. 1B). Of the conditions tested, the best decomposition condition was observed using 11.7 M NaOH at 46 oC during 13.5 h.
Table 2: Effect of NaOH treatment conditions on the decomposition of alkaline insoluble material.
|Decomposition of AIM in acetic acid solution|
Figure 1: The nature of chitosan-glucan complex obtained from the fungal mycelia treated with 1M (A) and 11M NaOH (B) at 45oC for 13 hr and treated with 0.35 M acetic acid at 950C for 5 h (a, b) followed by Termamyl treatment (c and d)
Muzzarelli et al., reported that the suspension obtained after treatment of Aspergillus niger mycelia with 10 M NaOH contains a chitosan-glucan complex . It is very plausible that a significant part of the turbidity in the suspension of AIM obtained by 11 M NaOH (Fig. 1B) is due to the presence of the chitosan-β-glucan complex. Termamyl enzyme, a commercial alpha-amylase has been used at pH 4.5 to split the chitosan-glucan complex in its components chitosan and β glucan . It appeared to be very effective on the acid extract of AIM obtained by 11 M NaOH. The stable heavy suspension (Fig. 1 B) turned into a clear supernatant and a fast sedimentating pellet (Fig. 1D). The supernatant contains the chitosan and the pellet consists mainly of β-glucan, in agreement with earlier studies . However the alpha-amylase enzyme could not act on the acid extract of AIM obtained by 1 M NaOH to cleave the bond between the chitosan and glucan. The rigid material (Fig. 1A) did not change during alpha-amylase treatment (Fig 1C). It is concluded that the matrix structure has to be destructed first by 11M NaOH in order to successfully extract the chitosan-β-glucan complex.
Effect of NaOH treatment conditions on the yield of chitosan from G.butleri
The yield of free chitosan 2 g/100 g mycelia and total chitosan 8 g/100 g mycelia was obtained when the mycelia was treated according to the free and total chitosan extraction methods. In the treatment with 1 M NaOH, chitin is not deacetylated and not extracted. Only chitosan present as free chitosan in the fungal cell wall was extracted. The yield of total chitosan obtained from the fungal mycelia was increased about 4 times after treatment with 11 M NaOH. In this condition, chitin present in the mycelia cell wall is converted into chitosan. In the pH 4.5 condition used, this free or complexed chitosan will be protonated and become amenable for acetic acid extraction. However the yield of free chitosan and total chitosan decreased to 0.61 g/100 g mycelia and 7.6 g/100 g mycelia respectively when the dried mycelia was treated with 1 and 11 M NaOH at 95 0C for 5 h, acetic acid treatment at 65 0C for 5 h and alpha-amylase enzyme treatment . The similar result has been obtained in above, acetic acid treatment at 95 0C for 14 h. These decrease amount of free chitosans might be the degradation of free chitosan during prolong incubation in hot acid and alkaline conditions. The decrease amount of total chitosan was less than the amount of free chitosan lost. The increase amount of total chitosan might be from complexed chitosan. The microfiber of glucan and chitosan polymers in the mycelia matrix would be more open after treatment with 11 M NaOH at 95 0C for 5 h than 11 M NaOH at 45 0C for 13h. In this case, alpha amylase enzyme could access more on the covalent bond between the chitosan and glucan chains.
In conclusion, the best condition for total chitosan extraction is 11 M NaOH at 45 0C for 13 h for alkaline treatment and 0.35 M acetic acid at 95 0C for 5 h for acid extraction. The degree of deacetylation of fungal chitosan was about 87 % and number average molecular weight was about 55 kDa. The IR spectrum of the fungal chitosan is shown in Fig. 2.
Effect of NaOH treatment conditions on the nature of mycelia matrix of A. coerulea and yield of chitosan
In order to confirm the differences in effectiveness of 1 and 11 M NaOH to disrupt the fungal myceliar matrix and the consequences for the yield of the chitosan extraction, a study was carried out in another fungus, Absidia coerulea ATCC 14076 grown differently in fed-batch fermentation. The dried fungal mycelia were treated according to the free and total chitosan extraction methods. Surprisely, the mycelia did not break down into molecular level and suspended as separated hyphae in the acetic acid solution. Therefore the residual materials obtained from each treatment were observed under confocal laser microscope. After treatment with 11 M NaOH (Fig. 3 A) the mycelia matrix was not broken but after subsequent extraction with acetic acid (0.35 M at 95 0C, 5 h) many separately visible tubular hyphae were present (Fig. 3B). Alpha-amylase clearly affected the morphology of these separate hyphae (Fig. 3C). They became soft and thin. In comparison, mycelia treated with 1 M NaOH (Fig. 4A), extracted with acetic acid (Fig. 4B) and alpha-amylase (Fig. 4C) did not show much changes. These observations clearly point out that 11 M NaOH and hot acetic acid treatments lead to breakdown of the crosslinked points in the mycelia matrix and many hyphae in the mycelia became separated from the mycelia matrix. These observations coincide with the morphology of mycelia obtained from both fungi. The mycelium of G. butleri on solid substrate fermentation was a homogeneous smooth layer and A. coerulea in fed-batch fermentation was spherical aggregated hyphae mass. Individual hyphae of G. butleri on solid substrate crosslink together in several points and results a smooth mycelia layer. This might be the reason; the mycelia matrix of G. butleri was broken down into molecular level in the hot acetic acid after treatment with 11 M NaOH. Therefore mycelia matrix of G. butleri on solid substrate has higher crosslinked points than A. coerulea in submerged fermentation.
The yields of free chitosan and total chitosan extracted from the cell wall of A. coerulea were 6.5 g/100 g of mycelia and 9.0 g/100 g of mycelia respectively. The amount of complexed chitosan in the A. coerulea was lower than G. butleri. The crosslinked points in the fungal mycelia are constructed with chitin/chitosan-glucan complex [18, 19]. Due to it limited amount of crosslinked points, the free chitosan level in A.coerulea was higher than G. butleri.
The choice of the proper chitosan extraction procedure is important for high yield production of fungal chitosan. It is essential to free the chitin/chitosan from its anchorage in the membrane and to the β-glucan. High concentration of NaOH is required in the first step of this solubilization, alpha-amylase enzyme has to be applied to separate the chitosan from the glucan fraction. Using these improved treatments a better quality of fungal chitosan can be produced. Fungal chitosan has a high degree of deacetylation, low viscosity, low molecular weight, high solubility and can not contain shrimp allergenic protein. With these properties, this fungal chitosan will find its way in the agriculture but especially in pharmaceutical industry.
The authors are grateful for the encouragement and generous financial support received from the late Prince Leo de Lignac, Netherlands, “High-Tech Research Center” Project for Private Universities: matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology, Japan), 2005-2009 and Japan Society for the Promotion of Science (JSPS), Japan (FY 2007-2009). They thank Novo Nordisk Co. Ltd (Denmark, Thailand and Japan) and Siam Modified Starch Co. Ltd (Thailand) for the supply of enzyme and Dr. Ng Chuen How for valuable help in measurement of chitosan molecular weight.
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