Abstract: Some biochemical parameters during growth of Mucor rouxii and chitosan production in molasses-salt medium were determined. Growth of the organism started with an initial lag of 3 h; maximum growth and chitosan production were obtained at 32 h of fermentation. Fall in sugar concentration from 40.05 to 3.4 g L-1 was noted during the course of fermentation. Degree of acetylation of chitosan was more or less the same whereas increase in molecular weight was noted with the time of fermentation. Maximum 0.61 g chitosan of molecular weight 2.48 x 104 with ~13.0% acetylation could be harvested from mycelia of M. rouxii grown in 1 L of molasses salt medium.
Introduction
Polymer of β-1,4-linked-D-glucosamine, commonly known as chitosan, is the deacetylated product of chitin - a major constituent of exoskeleton of arthropoda. Chitosan is widely used in food (Bégin and Calsteren, 1999; Chatterjee et al., 2004; Roller and Covill, 1999; Tsai et al., 2000) and pharmaceutical industries (Fernández-urrusuno et al., 1999; Shu et al., 2000; Kabbaj and Philips, 2001; Hu et al., 2004). It also finds application in environmental biotechnology (Chatterjee et al. 2005b; Chatterjee et al., 2002, Chiou and Li, 2003; Dambies et al., 2001; Dantas et al., 2001; Jin and Bai, 2002).
Chitosan is generally produced by deacetylation of chitin isolated from the exoskeleton of arthropoda with alkali (Chatterjee et al., 2003). However, the process is not eco-friendly as it requires large volume of concentrated alkali. Moreover, chitosan prepared by this method lacks consistent physico-chemical properties, which are essential for specific application. With the advent of fermentation technology, chitosan having uniform physico-chemical properties can be prepared in an eco-friendly way from fungi belonging mainly to the zygomycetes group (Tan et al., 1996). Organisms of this group contain chitosan as a major constituent of the cell wall; however, depending on the growth conditions, the amount of chitosan may vary. We have already reported the fermentative production and characterization of chitosan from Mucor rouxii grown in a cheap medium containing molasses, a byproduct of the cane sugar industry supplemented with inorganic salts (Chatterjee et al., 2005a). However, details of the fermentative process with molasses are required for large-scale production of M. rouxii mycelia as well as chitosan on the same. In this direction, we describe in this paper the changes, in respect of utilization of sugar, different forms of nitrogen, phosphorous in relation to mycelial growth and chitosan production for better understanding of the fermentation process. This study was conducted in the Biological Chemistry Department of the institute during the past one year.
Materials and Methods
Materials
All chemicals and biochemicals used in this experiment were purchased from
E. Merck, Germany. Molasses was procured from local market.
Microorganism
Mucor rouxii (MTCC 386) used in this study was obtained from the Institute
of Microbial Technology, Chandigarh, India and maintained on potato-dextrose
agar slants.
Fermentation Medium
The medium contains 0.2% NaNO3, 0.1% K2HPO4,
0.001% FeSO4,7H2O; 0.001% MgSO4,7H2O;
0.2% yeast extract and cane molasses. Molasses was added to the medium to attain
the sucrose concentration at 4.0%. pH of the medium was adjusted to 5.1. The
medium was distributed in 50 mL aliquots in 250 mL Erlenmeyer flask and autoclaved
at 121°C for 15 min.
Inoculum and Fermentation
Each Erlenmeyer flask containing medium was inoculated with 1 mL spore suspension
(~6.4x106 spores) prepared by growing the organism in potato dextrose
agar plates at 30°C for 3 days. Flasks were incubated under shaking condition
(120 rpm) at 30°C up to 60 h. At the end of desired incubation period, mycelia
were separated by filtration, washed with water and weights taken after drying
by lyophilization. Both mycelia and culture filtrate (fermented broth) were
kept at –20°C till used for
analysis.
Analytical Techniques
Sugar content (expressed as sucrose) of the culture filtrate was determined
by phenol sulphuric acid method as described by Dubois et al. (1956).
Total and amino nitrogen were estimated by micro Kjeldahl (AOAC, 2000) and Sörensen’s
formol titration (Hawk et al., 1954) method, respectively. Total phosphorus
content of the broth was determined by the process described in AOAC (2000).
pH of the broth was measured with a pH meter (Orion model 420A) while free acids
were estimated by titration with 0.01 (N) NaOH. Chitosan was isolated from the
mycelia (biomass) of M. rouxii by the method as described earlier (Chatterjee
et al., 2005a).
Characterization of Chitosan
Degree of acetylation was determined by the first derivative of UV absorbance
spectrum (Muzzrelli et al., 1997). Weight average molecular weight
Results and Discussion
The growth of M. rouxii as well as chitosan production was dependent on the nutrients used and the chemico-physical environment of the fermentation medium (Chatterjee et al., 2005a). In this study, molasses was used since it is a cheap carbon source obtained as a byproduct of the cane sugar industries. As per objective of this study, the optimum conditions of growth and chitosan production by M. rouxii on molasses are presented with respect to sugar utilization, pH and titratable acidity of the broth at different stages of fermentation (Fig. 1). The mycelial growth started with an initial lag of 3 h and reached maximum at 32 h of incubation, beyond which fall in biomass production was noted indicating beginning of the death phase. Like growth, chitosan production also reached maximum after 32 h of fermentation and then decreased slowly. The change in pH of the medium should be taken into consideration as it greatly influences microbial growth. pH of the medium increased from 5.1 to 6.3 after 30 h of incubation then it started decreasing.
| Fig. 1: | Changes in sugar and acidity of molasses salt medium in relation to biomass and chitosan production by M. rouxii |
| Fig. 2: | Nitrogen and phosphorus nutrition during fermentation and chitosan production by M. rouxii in molasses salt medium |
Initial increase in medium pH may be due to the utilization of nitrate by the organism and low buffering capacity of the medium. As expected titratable acidity of the broth corroborated to the findings of pH change. Steady fall in sugar concentration from 40.5 to 4 g L-1 was noted during the course of fermentation. This is due to the bioconversion of sugar for biomass production. The results suggest that chitosan production varies directly with the biomass as expected because it is a natural constituent of M. rouxii mycelia.
The change in concentration of the total and amino nitrogen along with residual phosphorus of the fermented broth in relation to mycelial growth of M rouxii and chitosan production at different phases of growth are presented in Fig. 2. Phosphorus concentration of the medium decreased from 0.23 to 0.11 g L-1 till maximum biomass production was obtained. This is apparently due to uptake of phosphorous from the medium by the cells to synthesize cellular organic phosphorus compounds. Slight increase in phosphorous concentration of the broth was noted after 32 h of incubation, probably due to release of phosphorus upon death of the cells. Amino nitrogen concentration in the broth was initially low (below detection limit) as the medium contained only a small amount of this nitrogen from the yeast extract and molasses, which was utilized during growth of the organism. The amino nitrogen content could be detected in the broth after 39 h of fermentation with an increasing trend, which may be attributed to the release of protein and peptides due to lysis of the cells. Total nitrogen of the medium declined from 0.36 to 0.14 g L-1 until maximum mycelial growth was obtained, which was probably due to incorporation of nitrogen in the cells. Similarly, lysis of the cells contributed to the rise in nitrogen of the culture filtrate after 32 h of fermentation. The results of phosphorus and nitrogen utilization from the culture medium corroborated excellently with the growth phase of M. rouxii.
Maximum 0.61 g of chitosan could be obtained from the mycelia grown in one
litre of molasses-salt medium after 32 h of incubation. The
Conclusions
Production of chitosan by M. rouxii depends on the time of fermentation and the highest amount of chitosan could be obtained when the myclial growth was maximum. Molecular weight but not degree of acetylation of chitosan depends on the phase of growth of M. rouxii. 0.61 g chitosan having molecular weight 2.48x104 and degree of acetylation ~13% could be harvested from the mycelia of M. rouxii grown in one litre of molasses salt medium after 32 h of fermentation.
Acknowledgements
Authors are thankful to Dr. P.C. Banerjee, Scientist, Indian Institute of Chemical Biology, Kolkata for his valuable suggestions. Sandipan Chatterjee wishes to acknowledge Council of Scientific and Industrial Research Govt. of India, for providing Senior Research Fellowship.