Studies on the Suitability of Medicinal and Other Plant Extracts to Formulate Cost Effective Microbiological Culture Media for the Growth of Pathogenic and Saprohytic Fungi

Abstract

Fungi are the diversified group of eukaryotic microorganisms, most of which are free living, and occur as unicellular (yeast) or multicellular filamentous (moulds or mycelial) forms. The fungi are most versatile in their nutritional requirement which may vary from simple inorganic source to complex organic compounds, but all fungi share some common nutritional requirements with reference to nitrogen, carbon, minerals, growth promoting substances and water. The fungi are blessed with an array of enzymes which can degrade complex cross-linked polymeric compounds hence, play an important role in the production of many beneficial substances and metabolites. In order to utilize the maximum potential of fungi, they are required to be grown in an appropriate medium which can fulfill their essential nutritional requirements. Nowadays a variety of dehydrated culture media are commercially available for the growth and cultivation of fungi which are routinely used for teaching, research and diagnostic purposes. Nevertheless, the high cost of these media is becoming a major and serious issue, especially with reference to Pakistan. Therefore, it is a strong urge to develop cost effective microbiological media prepared from simple and easily available constituents/components. The culture media prepared form plant extracts may provide an alternative to expensive culture media and resolve the current problem. In Microbiology, the plant extracts are mostly and generally evaluated for their antimicrobial properties for a number of bacteria and fungi. Consequently, a wealth of information is available in the literature on this aspect. Nevertheless, the plant extracts have also been extensively studied for their phytochemical composition and were found to contain secondary metabolites such as tannins, saponins, alkaloids, steroids and other compounds, known for their antifungal property. However, the choice of a solvent, the selection of the method used for the preparation of the extract and the concentration at which it is used, have great impact on its biological activity. In the present study, water was used as a solvent and decoction as a method of extraction for the preparation of extracts. It is noteworthy that antifungal compounds are extracted by using water as a solvent; but the fungi have enzymes that either transform or catabolize these compounds hence rendering them ineffective. Once these compounds become ineffective or transformed into other xx compounds, they may serve as nutrients for the growth of fungi. Therefore, the initial and specified studies on various plant extracts conducted in our laboratory have shown the growth supporting potential for the fungi. In this regard, the leftovers of bougainvillea, coconut, date, eucalyptus and neem (Azadirachta indica) were used in the formulation of rich and valuable extracts. The extracts were further supplemented with additional sources of carbon, nitrogen (sugarcane peel extract; SPEX and yeast extract; YEX) and mineral salt solution (MSS) in the preparation of low cost culture media. Various fungi; including yeasts and molds were allowed to grow on these media and their growth, end products, metabolites and extracellular enzyme production were determined and compared with commercially available reference media (potato dextrose ; PD, Sabouraud dextrose; SD and YPD medium), both qualitatively and quantitatively. The study is comprised of initial experiments using various combinations of generalized plant extract (GPEX) and specified media to determine the radial growth assays and spore counts of filamentous fungi Aspergillus spp. and percent change ( increase or decrease) in the growth of the yeast spp. The combinations of GPEX supplemented with SPEX, YEX and MSS overall proved to be better than potato dextrose agar for supporting the growth of fungi. The combinations of specified media specifically NBS (neem bark extract supplemented with sugarcane peel extract) supported the growth of various fungi used in this study. The other combinations of neem, coconut and date bark were also found to be growth supportive for fungi. The combinations of bougainvillea branches, coconut, date and neem bark extracts were used to determine the dry cell weight (DCW), pH, protein and reducing sugar content for Aspergillus niger. The maximum DCW of A. niger was observed in coconut bark extract + SPEX + MSS, (CBSM), NBS and date bark extract + SPEX + MSS (DBSM) with 10, 20 and 30 % SPEX in comparison to the DCW in PDB on day 6. The overall pH of extract media with various concentrations of SPEX was found to be acidic in comparison with the pH of PDB with the lowest pH (2.81) in NBS with 30% (v/v) SPEX on day 4 in the stationary cultures. The reducing sugars were undetectable in the extract media combinations with 10% SPEX (with exception of NBS), detectable in 20 and 30% (v/v) SPEX on day 4 in both, stationary and shaking cultures. The overall protein content (extracellular) was more in NBS with various SPEX concentrations followed by DBSM and CBSM in the cultures grown under xxi both conditions. The potential of the filamentous fungi was evaluated for the production of amylase, cellulase and laccase. All the three Aspergillus spp were found to produce the amylase and cellulase but not the laccase. The quantitative estimation of amylase and cellulase by the Aspergillus niger was performed in submerged fermentation (SmF) technique in neem bark extract (NBEX) and date bark extract (DBEX) media. The maximum amylase activity 0.2 IU/ml and 0.1 IU/ml, and cellulase activity 0.028 IU/ml were observed in DBEX + MSS and NBEX+ MSS, respectively. The results for the citric acid production by A. niger were found to be lowest (3.83 g/l) in NBEX with 10% (v/v) SPEX whereas highest (7.33 g/l) in NBEX with 30 % (v/v) SPEX. The potential of the fungus was also evaluated for the growth on raw substrate (various components of the mentioned plants). The better growth of the fungus was observed when grown on the substrate with MSS. These extract media combinations were also found to have growth supporting potential for dermatophytes. The various extract media combinations were used to study the growth kinetics of the yeast spp. The biomass, protein (intracellular), reducing sugar content, pH and DCW estimation was found to be comparable or in some cases, even better than the reference media. The effects of the supplementation of NBEX with various concentrations of SPEX and CBS with various types of nitrogen sources was observed on the growth kinetics, biomass, protein, reducing sugar content, pH and DCW of Saccharomyces cerevisiae . The biomass, intracellular protein, reducing sugar content, pH and DCW was found higher in NBEX with 25% (v/v) SPEX on day 2. Furthermore, it was observed that the increasing sugar concentration had positive impact on the biomass, intracellular protein, reducing sugar content, pH and DCW, however, the supplementation of various nitrogen sources did not have a remarkable effect on various parameters of the yeast. The NBEX with various concentrations of SPEX were used to determine the potential of the Saccharomyces cerevisiae for the ethanol production. The maximum ethanol, 5.74 % (v/v) was found on NBEX with 25% (v/v) SPEX which was even higher than that of PDB 4.94 % (v/v). The productivity ratio and the absolute growth index of the yeast spp. on selected media combinations were found to be better than that of PDA. The selected extract media combinations (liquid and solid) were found to be stable when stored on the temperatures at 42-45 °C and the solid extract media combinations provided reproducible results after storage under ambient temperature for 3-4 months. The other xxii physical characteristics were also found to be similar as that of the freshly prepared media. The development of low cost media formulation will not only help reduce the overall cost but microbiology may also be introduced at high school and graduation levels thus the basic awareness of the subject may be imparted earlier in the career of the students. There are quite a lot uses of these media in various industries for either large volumes of microbial enzymes and/or inoculums production.