Screening Cellulase Producing Fungi
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The Fungi strain are relatively high potential source for cellulase production technology because they show the higher diameter of clear zone by 8.2cm, 8.1cm, 7.9cm respectively. The greater the diameter of clear zone, the larger amount of carboxymethylcellulose (CMC) can be degraded. To screen the best suitable of cellulase-producing fungi for the industrial uses, three parameters will be considered: (1) High titer and good enzymatic activity; (2) low production cost, and (3) feasible mass production (Miyamoto, 1997). In the above cases, the diameter clear zone of B19, D19, P19 is available to achieve the goal except for the production cost as we need the more data of different substrates concentration to investigate by considering the price of carbon source.
(b) Carboxymethylcellulose (CMC) is a substrate for endoglucanase to give an enzyme activity, so it can be used as a test for endoglucanase activity. The use of CMC rather than nature cellulose can be explained by three reasons as listed below:
- Endoglucanase can produce large titres by fungi (Cai, Buswell & Chang, 1994).
- Many fungi are not able to degrade cellulose in nature such as wood to produce a detectable cellbiohydrolase (Eaton & Hale, 1993).
- The incubation time in CMC agar is faster than in normal cellulose. The production of nature cellulose may be required many weeks to determine whether test cultures produce cellulose (Hankin & Anagnostakis, 1977).
(c) In general, owing to the low price, easy availability and purity of microbes, microbial enzymes are preferred for selection rather than plant- or animal-derived enzymes.
The primary reason is the production costs. Animal enzymes that are harvested from animal organs and glands are proved to be expensive than the plant-derived and microbial enzymes. The limited amount and kinds of enzyme strains derived from animals like at a time is also a critical factor, particularly the examples of protease, lipase and amylase. Microbial enzymes are derived from micro-organisms (TakeBackYourHealth.com, 2008).
Purity means there is no other substances beyond enzymes that interfere to the production process. Unlike plant and animal tissues, microbial cells are less probably to produce harmful chemical substances, including phenolic compounds from plant or endogenous enzyme inhibitors and proteases found in animals. The selection may include attempts or trials to perform an experiment until the results can be controllable, predictable and repeatable, both plant and animal cell cultures are needed to be made whilst microbial cell cultures do not have concern (Chaplin, 2012).
Another most important reason for microbial enzymes prior to animal enzymes is the unique characteristics of enzymes. Microbes can be fermented and used as enzymes because they exhibit broad range of pH values, temperatures and substrate specificities. Unlike microbial enzymes, animal-derived enzymes have narrow range of pH for utmost activity, for example, enzymes are difficult resist at low pH condition where gastric secretion within the stomach.
Thus, microbes are more ideal source for various enzyme production because of being relatively "safer" and highly productive (Amit et. al., 2007).
(d) To solve the question of "how the inoculum of different fungal strains can be standardized for a quantitative bioassay",
Inoculum size is a factor that affects the production of cellulase by the fungi and it is a possibly non-standardized error when measuring the enzymatic activity of cellulase (Azzaz et.al. 2012). To tend to be standardized the error; the quantitative method is needed for the inoculum of different fungal strains.
In our experiment, the results of zone area was representing that the amount of CMC was degraded, but it cannot measured quantitative bioassay. The inoculum of different fungal strains can be standardized for a quantitative bioassay by Solid- state fermentation (SSF), which is one of the quantitative measurements methods.
The procedure of SSF methods as follow:
In SSF, fermentation tube was used to strains culture.
The SSF substrate contained sugarcane bagasse and wheat bran within 1:1, and the initial moisture content of the SSF substrate was adjusted to 80% using the nutrient medium.
Then adding 20mL of the nutrient medium to each plate and expellent the spores into it by gentle pipetting, which is conidial suspensions of sample strains.
All of them were placeed at 30°C for 8 days in an incubator equipped with a forced air recirculation system and a container filled with water inside the chamber.
After added 100mL of distilled water to each flask and agitated at 120rpm for 40min at 30°C, the extraction would be carried out.
The mixture was then filtered and centrifuged. The clear upper layer was collected from each tube and tested for enzyme activity. The enzymatic activity in the extracts was quantified which was expressed as activity units per gram of dry substrate.
The cellulolytic activity of endoglucanase was determined by the CMC method. 0.5mL of the samples enzyme complexes were incubated together with 0.5mL of the 4% CMC substrate at 50°C for 10min. After incubation, 1.0mL of DNS was added to the tubes for quantification of the reducing sugars.
These results demonstrate the existence of a linear correlation between the two methodologies. Screening on plates employing Congo red dye, followed by a solid-state fermentation analysis, is, therefore, an effective method for selection of cellulase-producing strains.
It was possible to verify this study the correlation between the qualitative methodology in plates using Congo red and quantitative methodology by the process solid-state fermentation. The results obtained showed that the qualitative and quantitative methods are valid and important to selection strains of an extensive collection of the same species of microorganisms (Florencio et al. 2012).
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