Growth of Cunninghamella elegans UCP 542 and production of chitin and chitosan using yam bean medium
Christina Montenegro Stamford
Lucia Montenegro Stamford
de Barros Neto
Maria de Campos-Takaki*
Financial support: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the Universidade Católica de Pernambuco (UNICAP).
Keywords: biopolymers, chitin, chitosan, Cunninghamella elegans, Zygomycetes.
processes were used for chitin and chitosan productions by Cunninghamella
elegans (UCP 542) grown in a new economic culture medium. The
assay was carried out to evaluate the growth of C. elegans using
yam bean (Pachyrhizus erosus L. Urban) medium, in different
times of growth (24, 48, 72 and 96 hrs), incubated at
Chitin, the insoluble linear β1,4- linked homopolymer of N-acetyl-D-glucosamine (GlcNAc), is the second most abundant natural polysaccharide (after cellulose). Chitosan is a cationic amino polysaccharide, essentially composed of β-1,4 D-glucosamine (GlcNAc) linked to N-acetyl-D-glucosamine residues (Andrade et al. 2000; Campos-Takaki, 2005), derived from de-N-acetylation of chitin (Tharanathan and Kittur, 2003; Amorim et al. 2005). These polysaccharides are found in a wide range of natural sources, such as crustaceans, insects annelids, molluscs, coelenterates and is a common constituent of fungal cell walls (Andrade et al. 2003; Synowiecki and Al-Khatteb, 2003; Franco et al. 2005).
and chitosan hold a great economic value as due to their versatile
biological activities and chemical applications, mainly in medical
(Murugan and Ramakrishna, 2004; Yadav
and Bhise, 2004) and pharmaceutical (Takeuchi et
al. 2001; Kato et al. 2003) areas. The wide
range of applications of these polymers has been extensively studied
Recent advances in fermentation technologies suggest that the cultivation of selected fungi can provide an alternative source of chitin and chitosan. The amount of these polysaccharides depends of the fungi species and culture conditions (Tan et al. 1996; Pochanavanich and Suntornsuk, 2002; Andrade et al. 2003; Synowiecki and Al-Khatteb, 2003). Filamentous fungi have been considered an attractive source of chitin and chitosan for industrial applications because their specific products can be manufactured under standardized conditions (Synowiecki and Al-Khatteb, 1997; Pochanavanich and Suntornsuk, 2002; Nemtsev et al. 2004). Usually, the Zygomycetes Class has higher amounts of chitin and chitosan in their cell walls when compared to other classes of fungi (Andrade et al. 2000; Campos-Takaki, 2005; Franco et al. 2005).
The use of biomass from fungi has demonstrated great advantages, such as: independence of seasonal factor, wide scale production, simultaneous extraction of chitin and chitosan, extraction process is simple and cheap resulting in reduction in time and cost required for production, and also absence of proteins contamination, mainly the proteins that could cause allergy reactions in individuals with shellfish allergies (Andrade et al. 2000; Amorim et al. 2001; Nadarajah et al. 2001; Andrade et al. 2003; Franco et al. 2005). However, to optimize the production of chitin and chitosan from fungi, it's usually used complex or synthetics cultures media, which are expensive. It’s becomes necessary to obtain economic culture media that promote the growth of fungi and stimulate the production of the polymers.
microbial culture media normally use vegetables components. Yam bean
(Pachyrhizus erosus L. Urban) is a leguminous plant native
from the Amazon region and from
The present paper aims to investigate the chitin and chitosan production using the Mucoralen fungi Cunninghamella elegans (UCP 542), grown by submerse fermentation in economic culture medium, yam bean (Pachyrhizus erosus L. Urban), as substrate, and was compared with four traditional culture media.
elegans UCP 542 (Culture Collection of Catholic University of
elegans was grown, for chitin and chitosan production, in five
different culture media: a) Sabouraud sucrose- (bacteriological peptone
profile. The sporangioles of C. elegans were harvested
from cultures grown for seven days at
and nitrogen consumption and pH determination. The glucose consumption
was determined by the enzymatic colorimetric method (Labtest®
Kit - Glucose oxidase). A standard curve was made by using a range
of glucose solutions (0.5 to 10.0 g/L). Colorimetric method Labtest®
Kit for protein was utilized for the nitrogen consumption determination,
using a spectrophotometer Spectronic Genesys 2. Changes in pH were
measured using a potentiometer (Digital Pontentiometer Quimis Mod.
and chitosan extraction. The process of extraction involved deproteination
with 2% w/v sodium hydroxide solution (30:1 v/w,
Infrared spectroscopy (Deacetylation degree - DD%). The degree of deacetylation for microbial chitin and chitosan were determined using the infrared spectroscopy according to Roberts (1992), using the absorbance ratio A1655/A3450 and calculated according to equation 1:
milligrams sample of fungal chitin and chitosan, which had been dried
weight. The molecular weights of chitin and chitosan were determined
by viscosity, using the procedure described by Dos Santos
et al. (2003). The viscosity of chitosan was determined using
an AVS-350 viscometer (Schott-Geräte), type/capillary: Cannon-Fenskedinside=
data were analyzed for significance using the Student’s t-test and
chi-square test using STATISTICA program version 6.0 of Statsolt Inc.,
profile of growth of C. elegans (UCP 542) and chitin /chitosan
production using yam bean medium and four different culture media,
traditional for Mucorales, are showed in Figure
However, C. elegans (UCP 542) grown in Hesseltine and Anderson medium shows a biomass yield of 10.3 g/L, with 96 hrs of cultivation. The result is in agreement with the growth curve of C. elegans (IFM 46109) established by Andrade et al. (2000) and Franco et al. (2005) using the same culture medium, which referred biomass yield of 11.0 and 11.6 g/L, respectively.
elegans (UCP 542) grown in malt medium with glucose 2% during
96 hrs, shows average biomass production of 4.8 g/L. This result is
similar to the reported by Synowiecki and Al-Khatteb
(1997) which obtained a yield biomass of Mucor rouxii grown
in yeast extract and glucose 2% medium, for 48 hrs, to the
Chitin and chitosan production by C. elegans grown in yam bean medium are studied on the cultivation profile. C. elegans (UCP 542) growth curve for biomass, pH, nitrogen content and glucose consumption is present in Figure 2. Biomass production increase rapidly up to 48 hrs, reached about the maximum of 24.3 g/L of dry weight. The result is superior to the value 10.41 g/L and 11.6 g/L reported by Andrade et al. (2000) and Franco et al. (2004), respectively, for C. elegans (URM 46109) grown during 96 hrs in Mucorales medium.
The results in Figure 2, demonstrate the residual glucose and nitrogen were 1.27 g/L and 0.18 g/L respectively. Similar results were reported by Andrade et al. (2000) and Franco et al. (2004). Amorim et al. (2001) suggested that the remaining glucose and nitrogen as due to nitrogenous compounds like secondary metabolites present at the end of growth of the fungi metabolism and excess of carbon source in the medium.
The pH of yam bean medium oscillates between 7 and 5, during the culture time (Figure 2). The values of pH decrease during the exponential phase, probably, by pyruvic acid formation, as due to the high glucose and starch concentration in yam bean medium. Franco et al. (2004) described constant values of pH during the “lag” phase and pH decrease during the exponential phase. That information of higher amount of the yam bean glucose and starch was previously mentioned by Sarangbin and Watanapokasin (1999), during the citric acid production. Amorim et al. (2001) reported that during growth of C. elegans the pH of the media drops in the first 24 hrs and remained low (between 3 and 4) during the first 96 hrs of cultivation, probably because of the interaction between the medium substrate and the release of ions from the cell.
Figure 3 shows chitin and chitosan yield extracted, to each 24 hrs, from C. elegans (UCP 542) grown in yam beam medium during 96 hrs of cultivation. The best yields of the polysaccharides (mg per gram of dry mycelia biomass) are obtained with 48 hrs of culture for chitosan (66 mg/g or 6.6%) and with 72 hrs for chitin (440 mg/g or 44%). Similar results were reported to Tan et al. (1996), which studied different Zygomycetes strains and observed that Cunninghamella echinulata was the best chitosan producing strain, with a yield of approximately 7.0% of chitosan per mycelia dry weight.
Chitosan production stabilize after 48 hrs of culture, although, chitin production increase up to 72 hrs of culture, and decrease at 96 hrs. The higher chitosan yields in 48 hrs of growth suggest that during initial growth chitin is less crystalline and thus more susceptible to chitin deacetylase, and the chitosan formed by this enzime prevails in acid pH (Figure 3). According to Amorim et al. (2005) optimum pH for chitin deacetylase activity from Zygomycetes is pH 4.5. During C. elegans growth the pH of the yam bean medium drops in the first 48 hrs (Figure 2), stowing high metabolic interchange between the medium substrate uptake and the release of ions from the cells. Amorim et al. (2001) reported higher yields of chitosan were found within 24 hrs of cultivation of M. racemosus and C. elegans at pH 3.5, which seems to be also a stimulating agent for production of this biopolymer. The data in the present work are in agreement with Pochanavanich and Suntornsuk (2002) and Nadarajah et al. (2001) that chitosan production by the microorganisms is strongly dependent of the culture conditions, including cultivation time. Chung et al. (2004) demonstrated that chitin and chitosan content in the cellular wall of fungi change according to the species and these polymers usually show higher values in Zygomycetes.
The characterization of chitin and chitosan obtained from C. elegans in yam bean medium by infrared spectra are similar to those reported in the literature (Andrade et al. 2000; Amorim et al. 2001; Franco et al. 2005). The most significant parts of chitin and chitosan spectra are those showing the amide bands at approximately 1665, 1555 e 1313 cm-1, which could be assigned to the C = O stretching, the N-H deformation in the CONH plane and the CN bond stretching plus CH2 wagging. In a similar way, chitin from C. elegans shows bands in the amide II region, which were 1153, 1378 and 1558 cm-1. The results are in agreement with Shigemasa and Minami (1996). Andrade et al. (2003) and Franco et al. (2005) which reported that chitin structure containing two types of amide group and both form C = O N-H intermolecular bonds, but one is also an acceptor for the CH2OH group.
According to Dos Santos et al. (2003) the deacetylation and the regeneration process, cause disturbance in the initial crystalline reticulum of chitin, inducing a reordering of the hydrogen linking of chitosan. This can be observed in the central band at approximately 3483 cm-1 e 3305 cm-1, in the region of the axial deformation of OH, which appears overlapping the band of axial deformation of NH indicating the intermolecular hydrogen linking formation, and at the displacement of the higher frequency band indicating an increase in the structural order. The data are in accordance with the reported in literature when comparing both chitin and chitosan infrared spectra obtained by microbiological methods (Andrade et al. 2000; Amorim et al. 2001; Pochanavanich and Suntornsuk, 2002; Dos Santos et al. 2003; Franco et al. 2005).
Deacetylation degree (%DD) is an important parameter associated with the physical-chemical properties of chitosan, because it’s linked directly to the chitosan cationic properties (Pochanavanich and Suntornsuk, 2002). In the present study chitin and chitosan from C. elegans grown in yam bean medium present 6,2% DD and 85% DD, respectively. Amorim et al. (2001), Pochanavanich and Suntornsuk (2002) and Franco et al. (2004), reported deacetylation degree of chitosan from fungi between 80 to 90% DD.
The average viscosimetric molecular weight (MV) of chitin and chitosan from C. elegans obtain in the present research are 3.25 x 104 g/mol for chitin, and 2.72 x 104 g/mol for chitosan. The results are in agreement with the results found in the literature, molar weights ranged between 1,0 x 104 to 9,0 x 105 g/mol (Nadarajah et al. 2001; Pochanavanich and Suntornsuk, 2002; Dos Santos et al. 2003).
The results present here suggest a high biotechnological potential of yam bean as an economic medium to chitin and chitosan production by C. elegans, and may be used to reduce the cost price of these polysaccharides production.
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