Evaluation of Elephant Grass (Pennisetum Purpureum) as Substrate for Bioethanol Production Using Co-cultures of Aspergillus Niger and Saccharomyces Cerevisiae

Evaluation of Elephant Grass (Pennisetum Purpureum) as Substrate for Bioethanol Production Using Co-cultures of Aspergillus Niger and Saccharomyces Cerevisiae

Chapter One

AIM OF THE STUDY

The aim of this research work was to evaluate the potential of Elephant grass as substrate for bioethanol production by simultaneous saccharification and fermentation process using co-cultures of selected Saccharomyces cerevisiae and Aspergillus niger.

SPECIFIC OBJECTIVES

To isolate, characterize and screen Saccharomyces cerevisiae and Aspergillus niger from various sources.
To carry out proximate analysis of Elephant grass sample.
To produce bioethanol using Elephant grass as substrate and co-cultures of selected cerevisiae and A. niger strains.
To determine the physico-chemical quality of the bioethanol produced.
To optimize the environmental factors for bioethanol production by the selected organisms in co-cultures.

CHAPTER TWO

LITERATURE REVIEW

Ethanol

Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colourless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a solvent, and as a fuel. In common usage, it is often referred to simply as alcohol or spirits. Ethanol is a straight-chain alcohol, and its molecular formula is C₂H₅OH. Its empirical formula is C₂H₆O(NCBI, 2012). Ethanol is the systematic name defined by the IUPAC nomenclature of organic chemistry for a molecule with two carbon atoms (prefix “eth-“), having a single bond between them (suffix “-ane”), and an attached – OH group (suffix “-ol”) (Myers and Myers, 2007).

The fermentation of sugar into ethanol is one of the earliest organic reactions employed by humanity and the intoxicating effects of ethanol consumption have been known since ancient times. In modern times, ethanol intended for industrial use is also produced from ethylene (Ballinger and Long, 1960). Ethanol has widespread use as a solvent of substances intended for human contact or consumption, including scents, flavourings, colourings, and medicines. In chemistry, it is both an essential solvent and a feedstock for the synthesis of other products. It has a long history as a fuel for heat and light, and more recently as a fuel for internal combustion engines.

History

Humans have used ethanol since prehistory as the intoxicating ingredient of alcoholic beverages. Dried residues on 9,000-year-old pottery found in China imply that Neolithic people consumed alcoholic beverages (Roach, 2005). Although distillation was well known by the early Greeks and Arabs, the first recorded production of alcohol from distilled wine was by the School of Salerno alchemists in the 12th century. The first to mention absolute alcohol, in contrast with alcohol-water mixtures, was Raymond Lull (Forbes, 1948). In 1796, Johann Tobias Lowitz obtained pure ethanol by filtering distilled ethanol through activated charcoal. Antoine Lavoisier described ethanol as a compound of carbon, hydrogen, and oxygen, and in 1808, Nicolas-Théodore de Saussure determined ethanol‟s chemical formula. Fifty years later, Archibald Scott Couper published the structural formula of ethanol. It is one of the first structural formulas determined (AEOL, 2008).

Ethanol was first prepared synthetically in 1826 through the independent efforts of Henry Hennel in Great Britain and S.G. Sérullas in France. In 1828, Michael Faraday prepared ethanol by acid-catalyzed hydration of ethylene, a process similar to current industrial ethanol synthesis (Lide, 2000). Ethanol was used as lamp fuel in the United States as early as 1840, but a tax levied on industrial alcohol during the civil war made its use uneconomical. The tax was however repealed in 1906 (Windholz, 1976). Original Ford Model T automobiles ran on ethanol until 1908. With the advent of Prohibition in 1920, ethanol fuel sellers were accused of being allied with moonshiners and ethanol fuel fell into disuse until late in the 20th century (Morrison and Boyd, 1972; Windholz, 1976).

Physical Properties

Ethanol is a volatile, colourless liquid that has a slight odour. It burns with a smokeless blue flame that is not always visible in normal light. The physical properties of ethanol stem primarily from the presence of its hydroxyl group and the shortness of its carbon chain. Ethanol‟s hydroxyl group is able to participate in hydrogen bonding, rendering it more viscous and less volatile than less polar organic compounds of similar molecular weight (Dahlmann and Schneider, 1989).

CHAPTER THREE

MATERIALS AND METHODS

Collection of samples

Sample collection for Aspergillus niger

A hundred grams of soil sample was gotten from the lawn in the Department of Microbiology, Ahmadu Bello University, Zaria. Bread sample purchased from a local retail shop was moistened and kept in a cupboard for 7 days to allow the growth of moulds.

Sample Collection for Saccharomyces cerevisiae

Fermented palm wine sample was purchased and collected into a pre-washed and sterile bottle from a palm wine dealer along Nupe Street, Sabon-Gari Zaria. “Burukutu” (local beverage) was also purchased and collected into a pre-washed and sterile bottle from a local beer parlour in Palladan, Zaria.

Sample Collection and Identification of Elephant Grass

Elephant grass sample was harvested along the bank of Galma River, Jos road, Zaria, Kaduna state. The plant sample was taken to the herbarium section in the Department of Biological Sciences, Ahmadu Bello University, Zaria for identification and was given the voucher number 475. A voucher specimen was also deposited.

Media for Isolation

Malt extract agar (MEA) (Biotech)was used for the isolation of both A. niger and S. cerevisiae. The medium was prepared according to manufacturer‟s instruction and sterilized at 121^oC for 15 minutes.

CHAPTER FOUR

RESULTS

Characterisation of Aspergillus nigerIsolates

Aspergillus niger presented a woolly growth at first which progressed to dense black sporulation with a pale yellow reverse on MEA plates (Plate I). The microscopic structures were long smooth-walled conidiophores terminating in globose swellings (Plate II) as presented on Table 4.1.

CHAPTER FIVE

DISCUSSION

Ten isolates of A. niger isolated from spoilt bread (3) and soil samples (7) were identified. The isolates showed characteristic woolly growth between 1-3 days of inoculation after which there was dense black sporulation on the plate typical of A. niger. The high frequency of isolation from soil sample is because of their ubiquity in such environments where they play an important role in many essential processes such as organic matter decomposition and elemental release by mineralisation (Christensen, 1989). This agrees with the work of Rabah et al. (2010) who also reported a high frequency of A. niger among fungi isolated from soil sample.

Ten yeast isolates were also obtained from palm wine (4) and burukutu (6). The reason for isolation of yeasts from palmwine is because the palm sap of a palm tree is a rich medium capable of supporting the growth of several types of microorganisms including the yeasts (Santiago-Urbina and Ruiz-Teran, 2014). Also the isolation of the yeast from burukutu agrees with the findings of Eze et al.(2011) who reported that S. cerevisiae found in burukutu serves as an agent of fermentation of the beverage.The ability of the palmwine isolates to assimilate more sugars than those from burukutu could be attributed to the fact that palm wine contains a host of different sugars including glucose, raffinose, saccharose (sucrose) and galactose as reported by Okafor (1972).

Qualitative screening test showed clear zones around the mould growth which is indicative of cellulase production and it also showed that all the mould isolates were capable ofproducing the cellulase enzyme as observed generally for most Aspergillus species (Ong et al., 2004). Quantitative enzyme assay for the total saccharifying cellulase

CHAPTER SIX

CONCLUSION AND RECOMMENDATIONS

Conclusion

Recently, there is a growing interest in biofuel production in most countries because of the increasing concerns about hydrocarbon fuel shortage and global climate changes, also for enhancing agricultural economy and producing local needs for transportation fuel. Ethanol can be produced from biomass by the hydrolysis and sugar fermentation processes. In this study, ethanol was produced without using expensive commercial enzymes instead cellulase was produced by Aspergillus niger. The findings from this study shows maximum ethanol yield of 1.68 g/100ml from 6% pretreated elephant grass substrate. Though much less than that from 6% glucose (8.38 g/100ml), it proves that with good environmental conditions, microorganisms of desired characteristics and genetic stability, ethanol is attainable from this lignocellulose waste hence, converting waste to wealth. The result of this study further shows that simultaneous saccharification and fermentation of elephant grass substrate is feasible. It equally revealed the fact that optimization of culture condition could enhance ethanol production from elephant grass using co-culture technique, thereby increasing the economy, in terms of percentage of cellulose fermentation to ethanol. It also showed that increasing temperature, pH, agitation rate and length of fermentation beyond certain level will not increase ethanol yield.

Recommendations

From the findings of this study, it is hereby recommended that:

Other pretreatment processes such as steam explosion, should be employed to ascertain their efficiency in disrupting the lignocellulose structure of Elephant grass hence, the release of the cellulose fraction for fermentation into ethanol.
It is also of utmost importance that the hemicellulose fraction of the plant sample which also contains fermentable sugars be also harnessed for its ethanol production potential.
A system of simultaneous saccharification and co-fermentation should be employed in the fermentation of plant sample to enable the simultaneous conversion of both hexoses and pentoses into bioethanol.
Finally, Elephant grass should also be considered as substrate for bioconversion into other beneficial value added products such as enzymes which include phytases, cellulases and hemicellulases.

REFERENCES

Abouzeid, M. M. and Steinkraus, K. H. (1983). Some factors affecting rapid ethanol production. Journal of Agricultural Research.7:245-249.
Abouzeid, M. M. and Reddy, A. (1986). Direct fermentation of potato starch to ethanol by co-cultures of Aspergillus niger and Saccharomyces cerevisiae. Journal of Applied and Environmental Microbiology,52:1055-1059.
Aden, A., Ruth, M., Ibsen, K., Jechura, J., Neeves, K., Sheehan, J. and Wallace, B. (2002). Lignocellulosic biomass to ethanol process design and economics utilizing co-current dilute acid prehydrolysis and enzymatic hydrolysis for corn stover. Technical Report: NREL/TP- 510-32438. National Renewable Energy Laboratory. Golden, Colorado, USA., pp. 1-154.
Ado, S.A., Kachalla, G.U., Tijanni, M.B. and Aliyu, M.S. (2009). Ethanol production from corn cobs by co-cultures of Saccharomyces cerevisiae and Aspergillus niger. Bayero Journal of Pure and Applied Sciences, 2(2): 99-101.
Ado, S. A., Olukotun, G. B., Ameh, J. B. and Yabaya, A. (2010). Bioconversion of starch to ethanol in simultaneous saccharification and fermentation process by co-cultures of Aspergillus niger and Saccharomyces cerevisiae. Journal of Pure and Applied Microbiology, 4(1): 55-61.
Agboola, P. O., Agboola, M. O. and Egelioglu, F. (2011). Bio-ethanol derivation from energy crop in Nigeria: A path to food scarcity or bio-fuel advancement. In: Proceedings of the World Congress on Engineering, London, U.K.
Akin-Osanaiye, B. C., Nzelibe, H. C. and Agbaji, A. S. (2008). Ethanol production from
Carica papaya (Pawpaw) fruit waste. Asian Journal of Biochemistry,3(3):188-193.
Akmar, P. F. and Kennedy, J. F. (2001). The potential of oil and sago palm trunk wastes as carbohydrate resources. Journal of Wood Science and Technology, 35(5): 467-473.
Alexpoulous, C. J. and Mims, C. W. (1979). Introductory mycology (3rd ed.). John Wiley and sons Inc., New York. pp. 290-295.

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