Nutrient Requirements and Fermentation Conditions for Mycelia and Crude Exo-Polysaccharides Production by Lentinus squarrosulus

Felicia N. Anike¹, Omoanghe S. Isikhuemhen^1*, Dietrich Blum¹, Hitoshi Neda²

¹Mushroom Biology and Fungal Biotechnology Laboratory, North Carolina A&T State University, Greensboro, USA.
²Forestry and Forest Products Research Institute (FFPRI), Tsukuba, Japan.

DOI: 10.4236/abb.2015.68055                

Abstract

Lentinus squarrosulus Mont. is an emerging tropical white rot basidiomycete, with nutritional and medicinal benefits. Low levels of commercial cultivation of the mushrooms limit their availability for use as food and medicine. Mycelia from submerged fermentation are a suitable alternative to the mushroom from L. squarrosulus. Three strains, 340, 339 and 218, were studied to determine optimum growth conditions for mycelia mass and crude exo-polysaccharides (CEPS) production. The experiments were conducted in a completely randomized design (CRD) with a factorial structure. Nutrients involving 8 carbon and 8 nitrogen sources were screened, and concentrations of the best sources were optimized. Optimized nutrients, interaction between strains and other parameters such as agitation and medium volume were investigated to obtain optimum fermentation conditions for biomass and CEPS production. Biomass yield varied among strains depending on carbon or nitrogen nutrient sources. Starch and yeast extract at 30 and 25 g/L were identified as the most important nutrients in mycelia and CEPS production. Nutrient optimization resulted in a 3-fold increase in mycelia mass: 12.8, 10.0 and 15.3 g/L in strains 340, 339 and 218 respectively. There was a significant interaction between strain, agitation, and volume (p < 0.001). Mycelia mass increased with volume under shake conditions, while polysaccharides decreased. There was a weak and negative correlation between mycelia mass and polysaccharides (p = 0.02). Static conditions favored more polysaccharide production. Optimized fermentation conditions resulted in very high increase in biomass: 238.1, 266.9 and 185.0 g/L in strains 340, 339 and 218 respectively. Results obtained could be useful in modeling fermentation systems for large-scale production of mycelia mass, CEPS and other bio-products from L. squarrosulus.

Keywords

Mycelia Mass, Carbon and Nitrogen Requirements, Exopolysaccharides, Lentinus squarrosulus, Submerged Fermentation

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Isikhuenhen, O.S., Adenipekun, C.O. and Ohimain, E. (2010) Preliminary Studies on Mating and Improved Strain Selection in the Tropical Mushroom Lentinus squarrosulus Mont. International Journal of Medicinal Mushrooms, 12, 177-183. http://dx.doi.org/10.1615/IntJMedMushr.v12.i2.80
[2]	Okhuoya, J.A., Akpaja, E.O. and Abot, O. (2005) Cultivation of Lentinus squarrosulus (Mont) Singer on Sawdust of Selected Tropical Tree Species. International Journal of Medicinal Mushrooms, 7, 213-218. http://dx.doi.org/10.1615/IntJMedMushr.v7.i3.790
[3]	Isaka, M., Sappan, M., Rachtawee, P. and Boonpratuang, T. (2011) A Tetrahydrobenzofuran Derivative from the Fermentation Broth of Lentinus squarrosulu BCC 22366. Phytochemistry Letters, 2, 106-108. http://dx.doi.org/10.1016/j.phytol.2010.12.002
[4]	Sudirman, L.I., Lefebvre, G., Kiffer, E. and Botton, B. (1994) Purification of Antibiotics Produced by Lentinus squarrosulus and Preliminary Characterization of a Compound Active against Rigidoporus lignosus. Current Microbiology, 29, 1-6. http://dx.doi.org/10.1007/BF01570183
[5]	Adenikpekun, C.O. and Isikhuemhen, O.S. (2008) Bioremediation of Engine Oil Polluted Soil by the Tropical White Rot Fungus Lentinus squarrosulus Mont. (Singer). Pakistan Journal of Biological Sciences, 11, 1634-1637. http://dx.doi.org/10.3923/pjbs.2008.1634.1637
[6]	Omar, N.A.M., Abdullah, N., Kuppusamy, Abdulla, M.A. and Sabaratnam, V. (2011) Nutritional Composition, Antioxidant Activities, and Antiulcer Potential of Lentinus squarrosulus (Mont.) Mycelial Extract. Evidence-Based Complementary and Alternative Medicine, 2011, Article ID: 539356.
[7]	Bhunia, S.A., Dey, B., Maity, K.K., Patra, S., Maiti, S., Maiti, T., Sikdar, S.R. and Islam, S.S. (2011) Isolation and Characterization of an Immunoenhancing Extracts of an Edible Mushroom, Lentinus squarrosulus (Mont.) Singer. Carbohydrate Research, 346, 2039-2044. http://dx.doi.org/10.1016/j.carres.2011.05.029
[8]	Tang, Y.Z., Zhu, L.W., Li, H.M. and Li, D.S. (2007) Submerged Culture of Mushrooms in Bioreactors—Challenges, Current-State-of-the-Art, and Future. Food Technology, 45, 221-229.
[9]	Castro, J.I. and Taylor, E.R. (2002) Use of Different Nitrogen Sources by the Ectomycorrhizal Mushroom Cantharellus cibarius. Mycorrhiza, 12, 131-137. http://dx.doi.org/10.1007/s00572-002-0160-2
[10]	Chen, W., Zhao, Z. and Li, Y. (2011) Simultaneous Increase of Mycelia Biomass and Intracellular Polysaccharide from Formes formentarius and Its Biological Function of Gastric Cancer Intervention. Carbohydrate Polymers, 65, 369-375. http://dx.doi.org/10.1016/j.carbpol.2011.02.035
[11]	Zhong, J.J. and Tang, Y.J. (2004) Submerged Cultivation of Medicinal Mushrooms for Production of Valuable Bioactive Metabolites. Advances in Biochemical Engineering/Biotechnology, 87, 25-59. http://dx.doi.org/10.1007/b94367
[12]	Wasser, S.P., Elisashvili, V.I. and Tan, K.K. (2003) Effects of Carbon and Nitrogen Sources in the Medium on Tremella mesenterica Retz: Fr. (Heterobasidiomycetes) Growth and Polysaccharide Production. International Journal of Medicinal Mushrooms, 5, 49-56. http://dx.doi.org/10.1615/InterJMedicMush.v5.i1.70
[13]	Manjunathan, J. and Kaviyarasan, V. (2010) Studies on the Growth Requirements of Lentinus tuber-regium (Fr.), An Edible Mushroom. Middle-East Journal of Scientific Research, 5, 81-85.
[14]	Sinha, J., Bae, J.T., Park, J.P., Kim, K.H., Song, C.H. and Yun, J.W. (2001) Changes in Morphology of Paecilomyces japonica and Their Effect on Broth Rheology during Production of Exo-Biopolymers. Applied Microbiology and Biotechnology, 56, 88-92. http://dx.doi.org/10.1007/s002530100606
[15]	Gbolagade, J.S., Fasidi, I.O., Ajayi, E.J. and Sobowale, A.A. (2006) Effect of Physico-Chemical Factors and Semi-Synthetic Media on Vegetative Growth of Lentinus subnudus (Berk.), an Edible Mushroom from Nigeria. Food Chemistry, 99, 742-747. http://dx.doi.org/10.1016/j.foodchem.2005.08.052
[16]	Ahmad, R., Al-Shorgani, N.K.N., Hamid, A.A., Yusoff, W.M.W. and Daud, F. (2013) Optimization of Medium Components Using Response Surface Methodology (RSM) for Mycelium Biomass and Exopolysaccharide Production by Lentinus squarrosulus. Advances in Bioscience and Biotechnology, 4, 1079-1085. http://dx.doi.org/10.4236/abb.2013.412144
[17]	Isikhuenhen, O.S., Mikiashvili, N.A., Adenikpekun, C.O., Ohimain, E. and Shahbazi, G. (2012) The Tropical White Rot Fungus, Lentinus squarrosulus Mont.: Lignocellulolytic Enzymes Activities and Sugar Release from Cornstalks under Solid State Fermentation. World Journal of Microbiology and Biotechnology, 28, 1961-1966. http://dx.doi.org/10.1007/s11274-011-0998-6
[18]	Kwon, J.S., Lee, J.S., Shin, W.C., Lee, K.E. and Hong, E.K. (2009) Optimization of Culture Conditions and Medium Compositions for the Production of Mycelial Biomass and Exo-Polysaccharides with Cordyceps militaris in Liquid Culture. Biotechnology and Bioprocess Engineering, 14, 756-762. http://dx.doi.org/10.1007/s12257-009-0024-0
[19]	Griffin, D.H. (1994) Fungal Physiology. 2nd Edition, John Wiley and Sons, New York.
[20]	Wu, J.Z., Cheung, P.C.K., Wong, K.H. and Huang, N.L. (2003) Studies on the Submerged Fermentation of Pleurotus tuber-regium (Fr.) Singer: 1. Physical and Chemical Factors Affecting the Rate of Mycelial Growth and Bioconversion Efficiency. Food Chemistry, 81, 389-393. http://dx.doi.org/10.1016/S0308-8146(02)00457-0
[21]	Shih, I., Chou, B., Chen, C., Wu, J. and Hsieh, C. (2008) Study of Mycelia Growth and Bioactive Polysaccharide Production in Batch and Feed-Batch Culture of Grifola frondosa. Bioresource Technology, 99, 785-793. http://dx.doi.org/10.1016/j.biortech.2007.01.030
[22]	Solomon, P.S., Water, O.D.C. and Oliver, R.P. (2007) Decoding the Mannitol Enigma in Filamentous Fungi. Trends in Microbiology, 15, 257-262. http://dx.doi.org/10.1016/j.tim.2007.04.002
[23]	Shih, I., Pan, K. and Hsieh, C. (2006) Influence of Nutritional Components and Oxygen Supply on Mycelia Growth and Bioactive Metabolites Production in Culture of Anthrodia cinnamomea. Process Biochemistry, 41, 1129-1135. http://dx.doi.org/10.1016/j.procbio.2005.12.005
[24]	Huang, D., Cui, F., Li, Y., Zhang, Z., Zhao, J., Han, X., Xiao, X., Qian, J., Wu, Q. and Guan, G. (2007) Nutritional Requirement for the Mycelia Biomass and Exopolymer Production by Hericum erinaceus CZ-2. Food Technology and Biotechnology, 45, 389-395.
[25]	Jung, I.C., Kim, S.H., Kwon, Y.I., Kim, S.Y., Lee, J.S., Park, S., Park, K.S. and Lee, J.S. (1997) Cultural Condition for the Mycelial Growth of Phelinus igniarius on Chemically Defined Medium and Grains. The Korean Journal of Mycology, 25, 133-142.
[26]	Jennison, M.W., Newcomb, M.D. and Henderson, R. (1955) Physiology of the Wood-Rotting Basidiomycetes. Growth and Nutrition in Submerged Culture in Synthetic Media. Mycologia, 47, 275-304. http://dx.doi.org/10.2307/3755451
[27]	Xiao, J., Chen, D., Wan, W., Hu, X., Qi, Y. and Liang, Z. (2006) Enhanced Simultaneous Production of Mycelia and Intracellular Polysaccharide in Submerged Cultivation of Cordyceps jiangxiensis Using Desirability Functions. Process Biochemistry, 41, 1887-1893. http://dx.doi.org/10.1016/j.procbio.2006.03.031
[28]	Park, J.P., Kim, Y.M., Kim, S.W., Hwang, H.J., Cho, Y.J., Lee, Y.S., Song, C.H. and Yun, J.W. (2002) Effect of Agitation Intensity on the Exo-Biopolymer Production and Mycelia Morphology in Cordyceps militaris. Enzyme and Microbial Technology, 34, 433-438.
[29]	Babitskaya, V.G., Shcherba, V.V., Puchkova, T.A. and Smirnov, D.A. (2005) Polysaccharides of Ganoderma lucidum: Factors Affecting Their Production. Applied Biochemistry and Microbiology, 41, 169-173. http://dx.doi.org/10.1007/s10438-005-0029-1
[30]	El-Dein, M.M.N., El-Fallal, A.A., Toson, E.S.A. and Hereher, F.E. (2004) Exopolysaccharides Production by Pleurotus pulmonarius: Factors Affecting Formation and Their Structures. Pakistan Journal of Biological Sciences, 7, 1078-1084. http://dx.doi.org/10.3923/pjbs.2004.1078.1084
[31]	Lin, E. and Sung, S. (2006) Cultivation Conditions Influence Exopolysaccharide Production by the Edible Basidiomycete Antrodia cinnamomea in Submerged Culture. International Journal of Food Microbiology, 108, 182-187. http://dx.doi.org/10.1016/j.ijfoodmicro.2005.11.010
[32]	Diamantopoulou, P., Papanikolaou, S., Katsarou, E., Komaitis, M., Aggelis, G. and Philippoussis, A. (2012) Mushroom Polysaccharides and Lipids Synthesized in Liquid Agitated and Static Cultures. Part I: Screening Various Mushroom Species. Applied Biochemistry and Biotechnology, 167, 536-551. http://dx.doi.org/10.1007/s12010-012-9713-9
[33]	Diamantopoulou, P., Papanikolaou, S., Katsarou, E., Komaitis, M., Aggelis, G. and Philippoussis, A. (2012) Mushroom Polysaccharides and Lipids Synthesized in Liquid Agitated and Static Cultures. Part II: Study of Volvariella volvacea. Applied Biochemistry and Biotechnology, 167, 1890-1906. http://dx.doi.org/10.1007/s12010-012-9714-8
[34]	Diamantopoulou, P., Papanikolaou, S., Komaitis, M., Aggelis, G. and Philippoussis, A. (2014) Patterns of Major Metabolites Biosynthesis by Different Mushroom Fungi Grown on Glucose-Based Submerged Cultures. Bioprocess and Biosystems Engineering, 37, 1385-1400. http://dx.doi.org/10.1007/s00449-013-1112-2