OPTIMIZING ALPHA AMYLASE PRODUCTION FROM LOCALLY ISOLATED Aspergillus species USING SELECTED AGRO WASTES AS SUBSTRATES

ABSTRACT

α-Amylase are enzymes that are used in the degradation of starch. This study focuses on optimizing the production of α-Amylase using locally isolated Aspergillus japonicus in defined and undefined medium.

Five grams of cocoyam waste, sweet potato waste, wheat waste and plantain peel were utilized as substrates during fermentation for the production of α-Amylase using 1 ml spore suspension as inoculum. The fermentation media contained in g/l (0.8 NaCl, 0.8 KCl, 0.1 CaCl₂, 2.0 Na₂HPO₄, 0.1 FeSO₄, 8.0 Fructose, 2.0 NH₄Cl).  Temperature, pH, sugar content and amylase activity of the culture filtrates were monitored after every 48 hours. For optimization, mycelia was used as inoculum and different inoculum sizes (5%, 10% and 15%) were used for fermentation in a defined medium containing starch as substrate. The effect of urea as nitrogen source on α -Amylase activity was monitored. Different concentrations (2g and 5g) of cocoyam waste and sweet potato waste were also utilized for optimization using 10% inoculum. The effect of temperature and pH on amylase was determined.

Two grams (2g) of sweet potato waste with NH₄Cl as nitrogen source, using 10% inoculum gave the highest yield of α-Amylase after 96 hours. The optimum pH and temperature for α -amylase production were pH 5 and 50 ^oC respectively.

The ability of the amylase to act within an acidic pH suggests that it is stable within a wide range of acidic pH (2 – 6) and its ability to withstand relatively high temperature (40 ^o C – 60 ^o C) above the optimum growth temperature  of A. japonicus suggests that it is thermo stable. The crude α-Amylase produced from A. japonicus retained 67% of its activity at 60^oC.α-Amylase from this fungus has the potential to be utilized for various biotechnological processes. Agro industrial wastes which are often carelessly discarded into the environment causing health hazards can be utilized as cheap and readily available substrate for the production of alpha amylase hence, it is practicable to rid our environment of these hazardous wastes.

Keywords:α-Amylase, Aspergillus japonicus, Optimization, Agro industrial wastes,                            Environment

Word Count: 332

CHAPTER ONE

INTRODUCTION

1.1       Background to the Study

Amylases are enzymes that are well known for their applications in starch, food, brewing, distilling, textile, paper and pharmaceutical industries (Gupta et al., 2003; Krishna et al., 2011; Pandey et al., 2000). They are currently utilized in various fields e.g. brewing industries, medicinal, analytical chemistry and food processing (Anto et al., 2006; Chimata et al., 2010; Nimkar et al., 2010). This wide range of applications is the reason for the industrial production of amylase (Khan & Yadav, 2011). Amylases are one of the most important and well-known enzymes that can hydrolyse starch or glycogen (Krishna et al., 2011). They hydrolyse α 1-4 glycosidic bonds of glycogen, amylopectin and other related compounds (Lehninger, 1982). It can be produced by submerged fermentation or solid state fermentation (Egas et al., 1998; Khan & Yadav, 2011; Krishna, 2011). The enzyme is one of the mostly sought after, as it has huge importance in biotechnology; comprising a group of industrial enzymes that controls about 25% of the total enzyme market of the world (Rajagopalam & Krishnan, 2008; Reddy et al., 2003).

Amylases are a group of hydrolases that split the O-glycosidic bonds present in starch thereby breaking starch into simple units (Alva et al., 2007; Crabb & Mitchinson, 1997). They have been reported to be produced by microbial, plant and animal sources, although amylase produced by microorganims has been reported to be most effective (Khan & Yadav, 2011). A wide range of microorganisms, such as bacteria and fungi are utilized in the industrial production of amylases (Krishna et al., 2011). The use of microbes for the production of amylases is economical because microorganisms can be easily manipulated to produce metabolites e.g. enzymes (Aiyer, 2005). However, fungi are preferred over bacteria for enzyme production because of their filamentous nature, which helps in its penetration through solid substrate (Ramachandran et al., 2004).

The synthetic media utilized for the production of amylases are costly and this poses a major challenge to researchers especially in developing countries. Hence, researches are now focused on methods to reduce production cost (Khan & Yadav, 2011). Wastes from agro based industries have been reported to be good and readily available substrates for the cost effective production of α-Amylase (Kirankumar et al., 2011; Pandey et al., 2000). Agrarian nations possess inexhaustible supply of wastes annually generated from their breweries, rice mills, yam flour, plantain and banana chips producing outfits, processing units, and other small industries (Adeniran & Abiose, 2009).

The generation of waste materials (e.g. peels) emanating from the utilization of food and other food products pose potentially severe pollution problems and represent a loss of valuable biomass. Some of these wastes are usually carelessly dumped in the environment where they are left to decay. Soil and plant around the heaps of the waste are usually considered unproductive due to chemical and biological reactions that take place between the decomposing wastes, soil and the surrounding vegetation (Ajao et al., 2009; El-Shimi et al., 1987). Apart from their environmental pollution aspects, generally, these wastes may have the potential to be utilized as raw material for other industries or for their use as feed or food after biological treatment (Okolo et al., 1995). Agro industrial wastes include plantain peel, cocoyam waste, sweet potato waste, cocoyam waste, cassava waste, wheat bran, rice husk, banana peel, vegetable waste and citrus waste.

Plantain (Musa spp.) occupies a strategic position for rapid production of food in Nigeria. It is ranked third among starchy staples (IITA, 2014). The “total world production of plantain is estimated to be over 75 million metric tons (John & Marchal, 1995) out of which 12 million metric tons are produced annually in Africa (Fakayode et al., 2011)”. Nigeria is one of the leading producers of plantain worldwide, it is the largest producer in West Africa producing about 2.4 million metric tons yearly (FAO, 2006). However, Nigeria is not an exporter of plantain because production is more for local consumption (Fortaleza, 2012). About 15 million people depend on plantain as their major source of carbohydrate (Adeolu & Enesi, 2013). The high demand for plantain also generates wastes which are often discarded, and sometimes used as animal feeds (Olabanji et al., 2012).

In Nigeria, the ripe fruit are processed into different forms for consumption either by boiling, frying and roasting. Considerable interest has been generated, in the recent years, for value addition to plantain, such as the production of plantain chips, dodoikire (commonly sold along highway in the South Western part of Nigeria) and plantain powder because of improper storage facilities which usually lead to postharvest losses. During the course of producing some of these products, the plantain peel accumulates in bulk posing serious environmental problems.

Over the years, cocoyam has been a major crop in the system of farming in South Eastern and South Western part of Nigeria. It is one of the most important tuber crops grown in this region. The tubers contain starch that serves as dietary fibre, it can be boiled, roasted, fried and eaten with palm oil (Ezejiofor, 2012). Nigeria is the largest producer of cocoyam in the world (Okoye, et al., 2008; Onwueme, 1987). Over 20 million tonnes of cocoyam yields are annually wasted because of improper storage (IITA, 2009). It is consumed as vegetables by many rural inhabitats where they are available in large quantities (Okigbo, 1987). It is not expensive and usually available throughout the year (Braide & Nwaoguikpe, 2011). The high level of carbohydrate in cocoyam has not been completely harnessed in the industries (Nwufo & Fajola, 1998). Nigeria is one of the foremost producers of root crops such as cocoyam, which is one of the most under-utilized crops with huge economic potential (Eneh, 2013; Onwuka & Eneh, 1998).

Sweet potato is the seventh most important food crop in the world (Betiku et al., 2013). It is a vital food crop worldwide (Hasem et al., 2015).  It is one the most important crops on fresh-weight basis in some developing nations after cassava, wheat, rice and maize (Ezeano, 2010). Ezeano (2010) reported that, there seems to be a rise in the growing and usage of sweet potato in Nigeria because of new development employed by farmers.

Wheat is cultivated worldwide; it has been cultivated in Nigeria for some time (Ohiagu et al., 1987; Olugbemi et al., 1979). Olabanji et al. (2007) reported that the cultivated varieties are relatively recent introduction. Wheat is used for the production of bread, semolina and for fermentation to make alcoholic beer (Poehlman, 1959), vodka (Palmer, 2001) or biofuel (Neil 2002). It is also used for the production of pasta and noodles (Li et al., 2014).

1.2       Statement of the Problem

The synthetic media used in the production of α-Amylase is expensive and this poses a major challenge for a developing country like Nigeria. There is a need to explore other cheaper and readily available substrates for the production of this enzyme. Nigeria is the largest producer of cocoyam and one of the leading producers of sweet potato and plantain in the world. However, cocoyam and sweet potato are under-exploited and a significant portion is often wasted. Most of the plantain produced in Nigeria is consumed locally; the epicarp of the plantain (plantain peel) is often considered as waste and is usually carelessly discarded. Additionally, Nigeria is not a major producer of wheat but this does not forestall the importation, processing and consumption of wheat and wheat products. The accumulation of wastes from these crops may contribute to environmental and health hazards in different parts of the country. This study aims at generating additional value to cocoyam, sweet potato, plantain and wheat by utilizing its waste as substrates for the production of α-Amylase.

1.3       Objective of the Study

            The main objective of this study was to utilize different agro wastes for the production of α-Amylase enzyme using locally isolated Aspergillus sp. in submerged fermentation. The specific objectives are to:

1. evaluate the production of α-Amylase by Aspergillus sp. in defined and undefined growth media;
2. determine pH and sugar content in the culture filtrates;
3. extract the crude alpha amylase produced during fermentation;
4. determine the amylase activity;
5. optimize α-Amylase production and
6. determine the effect of pH of temperature on α-Amylase.

1.4       Research Questions

1. Are there differences in amylase production using defined medium and/or undefined medium?
2. Are there variations in the pH, temperature and sugar content in the different culture filtrates?
3. How can the crude alpha amylase produced during fermentation be extracted?
4. How can the α-Amylase activity be determined?
5. How can α-Amylase production be optimized?
6. How will varying temperature and pH affect the crude α-Amylase produced?

1.5       Significance of the Study

This study would provide baseline information about the ability of a locally isolated Aspergillus sp. to utilize pre-treated cocoyam and sweet potato waste for α-Amylase production. It would also add to the existing literature the possibility of utilizing wheat waste and plantain peel as a cheap and readily available substrate for α-Amylase production using locally isolated Aspergillus sp. from pulverise cocoa.

1.6       Justification for the Study

Wastes generated from plants poses serious environmental and health hazard to humans. This study would focus on utilizing agro wastes for α-Amylase production in a view to converting these agro industrial and potentially hazardous “wastes to wealth”.

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