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OPTIMIZATION OF LIPASE OR CELLULASE PRODUCTION BY BACTERIA ISOLATED FROM AGRO-WASTE IN EBONYI STATE FOR BIODEGRADATION APPLICATIONS.
CHAPTER ONE
INTRODUCTION
1.1 Background to the Study
The increasing world-scale issue of agro-waste and environmental sustainability has heightened attention on the biotechnological utilization of microbial enzymes, notably the hydrolases (lipases and cellulases). Agro-industrial wastes such as rice husks, cassava peels, corn cobs, palm press fibre, sugarcane bagasse, vegetable and fruit wastes, ground nut shells, livestock manure are some of the largest streams of organic waste produced in tropical agricultural economies. Nigeria, with one of the largest agricultural outputs in sub-Saharan Africa, produces millions of tonnes of agro-waste each year with most of it being either burnt in the field or deposited in uncontrolled waste locations and contributing to the level of greenhouse gas emission, soil erosion, and pollution of surface waters (Nweze et al., 2021; FAO, 2023).
Ebonyi State, a southeastern Nigeria state, is mainly agrarian, and is distinguished by significant production of rice (especially in the Afikpo zone), cassava, yam, and different types of vegetables. A large amount of agro-waste is also produced by the state such as waste produced by rice mill, cassava by-products, palm kernel effluents, and waste in the market of vegetables. Such agro-waste matrices contain abundant lignocellulosic biomass, lipids, starch, and protein, which provide nutrient-rich habitats that selectively enrich microbial communities that hydrolyze lignocellulose and lipids via extracellular hydrolytic enzyme(s) strategies (Ezugwu et al., 2021). An extensive collection of well-characterized terragenic bacteria native to these environments containing Bacillus, Pseudomonas, Streptomyces, Aspergillus (associated bacteria), and Serratia have been proven to be active in production of industrially relevant lipases and cellulases (Srivastava et al., 2022; Ibrahim et al., 2023).
Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are enzymes that catalyze the breakdown of the ester bonds in triacylglycerols to produce fatty acids and glycerol. They are used in a variety of industries, such as biodiesel production, detergent production, food and dairy processing, pharmaceutical production, textile processing, leather de-greasing, and bioremediation of lipid-rich food processing industry and restaurant wastewaters (Srivastava et al., 2022; Angural et al., 2021). Cellulases (endo-glucanases, exo-glucanases and 2-glucosidases; EC 3.2.1.4, EC 3.2.1.91, EC 3.2.1.21) are used to degrade cellulose, the most common polysaccharide on earth, into fermentable glucose units. They are used in the production of bioethanol, bio-finishing of textiles, animal feed supplement, bleaching of paper, saccharification of agricultural residue to biogas and bio-chemical production (Ejaz et al., 2021; Ibrahim et al., 2023).
The growth medium, fermentation conditions and genetic and physiological traits of the strain generating an enzyme all have an inherent effect on microbial enzyme production. These parameters such as type and concentration of carbon source, type of nitrogen source, temperature, pH, inoculum size, agitation speed, and incubation period are important to optimize to get commercially viable enzyme yields (Srivastava et al., 2022; Ezugwu et al., 2021). Old-fashioned one-variable-at-a-time (OVAT) forms of optimization were not very efficient and no longer takes into consideration the interaction effect of variables, so they need to adopt statistical optimization methods (Plackett-Burman design, response surface methodology (RSM) based on Central Composite Design (CCD) or Box-Behnken Design (BBD)) to achieve systematic and efficient optimization (Ejaz et
The development of enzymes using agro-waste as a low-cost substrate in solid-state fermentation (SSF) and submerged fermentation (SmF) is of special interest to developing nations such as Nigeria when agro-waste is utilized as a low-cost medium, and the enzyme production cost can be lowered significantly by taking advantage of agro-waste (Nweze et al., 20 Liquidation of bacteria in agro-waste contexts of Ebonyi State to achieve ideal lipase or cellulase production is thus a strategic intersection of waste valorization, industrial biotechnology and environmental management. However, there is paucity of systematic research on this issue in Ebonyi State, thus the study is novel and practical as well.
1.2 Statement of the Problem
The increasing agro-waste load of Ebonyi State which is propelled by the swelling rice milling, cassava processing, and palm oil sectors is a notable environmental management issue. Although these wastes are reported to contain enzyme-producing bacteria, systemate isolation, characterization, and optimization of enzyme production by these bacteria have not been carried out with Ebonyi State agro-waste substrates (Ezugwu et al., 2021; Nweze et al., 2021). Research bodies within the state and its local industries are therefore not able to harness this bioresource in the development of low-cost enzyme products that could be used in waste treatment, biodegradation and in the agro-industrial processing.
Also, the optimizations data is missing, thus, any bacterial isolates producing enzymes in this area are being tested under non-optimal conditions, resulting in an underestimation of their actual enzymatic potential. The practicability of the scaled-up production of enzymes out of Ebonyi agro-waste bacteria to be used in biodegradation or industrial purposes, cannot be established reliably without statistically effective optimization with the application of modern experimental designs. This research aims to fill these gaps by isolating lipase producing bacteria and /or cellulase inventing bacteria in Ebonyi State agro-wastes, to optimize the enzyme production conditions and comparison of the optimized enzymes potential to be utilized in biodegradation processes.
The aim of this study is to optimize lipase and/or cellulase production by bacteria isolated from selected agro-waste sites in Ebonyi State and evaluate the biodegradation potential of the produced enzyme(s).
The specific objectives are to:
- Isolate and characterize lipase- and/or cellulase-producing bacteria from agro-waste substrates (rice husk waste, cassava peel, and palm press fibre) in Ebonyi State.
- Determine the best enzyme producer(s) through primary and secondary screening on indicator media.
- Optimize the physicochemical and nutritional conditions for maximum enzyme production using one-variable-at-a-time (OVAT) and response surface methodology (RSM).
- Characterize the partially purified enzyme for thermostability, pH stability, and substrate specificity.
- Evaluate the efficacy of the optimized enzyme in the biodegradation of selected lipid-rich or cellulosic agro-waste substrates.
1.4 Research Questions
- Which bacterial species from Ebonyi State agro-waste substrates exhibit the highest lipase and/or cellulase activity?
- What physicochemical and nutritional parameters most significantly influence enzyme production?
- What are the optimal conditions for maximum enzyme yield?
- What are the biochemical properties (thermostability, pH stability, substrate specificity) of the partially purified enzyme?
- How effective is the optimized enzyme in degrading lipid-rich or cellulosic waste substrates?
1.5 Research Hypotheses
Ho1: There is no significant difference in lipase and/or cellulase activity among bacteria isolated from different agro-waste substrates in Ebonyi State.
Ho2: Optimization of production conditions has no significant effect on enzyme yield.
Ho3: The optimized enzyme has no significant biodegradation efficacy on the selected agro-waste substrate.
1.6 Significance of the Study
The research will be revealing the Ebonyi State agro-waste indigenous bacteria that have the strongest lipase and/or cellulase production capacity and will be used in the bio-technological characterization of the Nigerian agro-waste microbial diversity. The resulting optimized enzyme systems will form a basis in the formulation of the low-cost, locally sourced, biological agents to agro-waste biodegradation direct applications will be in the bioremediation of soils contaminated by lipids, lignocellulosic biomass saccharification to bioethanol, and food-processing effluents mitigation in Ebonyi State and other states at large Biotechnologists and industrial microbiologists who may want to scale up the production of enzyme using agro-waste-derived bacterial isolates will also be interested in the statistical optimization data.
1.7 Scope of the Study
The research will be based on the gathering of agro-waste (rice husk waste, cassava peel and palm press fibre) at the processing locations in Afikpo, Abakaliki and Ezza-Ohu in Ebonyi State. The isolation, screening, identification of bacteria will be in the lab. OVAT will be used to optimize enzyme after which Plackett-Burman and Box-Behnken RSM designs will be used. The target substrate (lipid-rich or cellulosic waste) will be the sole carbon source and also be used as the test substrate in the experiment on biodegradation using enzymes.
1.8 Operational Definition of Terms
Lipase: An enzyme (triacylglycerol acylhydrolase, EC 3.1.1.3) that catalyzes the hydrolysis of ester bonds in triacylglycerols to produce fatty acids and glycerol.
Cellulase: A group of enzymes (including endoglucanase, exoglucanase, and β-glucosidase) that collectively degrade crystalline and amorphous cellulose to glucose.
Agro-waste: Organic residues generated from agricultural production, harvesting, processing, and marketing activities.
Solid-state fermentation (SSF): A fermentation process in which microorganisms grow on a moist solid substrate in the absence or near-absence of free water.
Response surface methodology (RSM): A collection of statistical and mathematical techniques used for developing, improving, and optimizing processes, particularly useful for identifying optimal factor settings for maximum response.
Biodegradation: The breakdown of organic substances by living organisms, particularly microorganisms and their enzymes, into simpler compounds, water, and carbon dioxide.
References
Angural, S., Kumar, V., Rana, N., Bala, M., Kumari, A., Bhatt, A. K., & Bhatia, R. K. (2021). Screening, isolation and characterization of lipase producing bacteria from mustard oil contaminated soil. Biocatalysis and Agricultural Biotechnology, 29, 101783. https://doi.org/10.1016/j.bcab.2020.101783
Ejaz, U., Sohail, M., & Ghanemi, A. (2021). Cellulases: From bioactivity to a variety of industrial and biotechnological applications. Biology, 10(6), 411. https://doi.org/10.3390/biology10060411
Ezugwu, A. L., Eze, S. O. O., & Chilaka, F. C. (2021). Cellulase production from rice husk by bacteria isolated from agro-waste dumpsites in Enugu State, Nigeria. Brazilian Archives of Biology and Technology, 64, e21190509. https://doi.org/10.1590/1678-4324-2021190509
Food and Agriculture Organization (FAO). (2023). The state of food and agriculture 2023: Revealing the true cost of food. FAO.
Ibrahim, A. S. S., Al-Salamah, A. A., El-Toni, A. M., Ibrahim, S. S. S., & El-Tayeb, M. A. (2023). Detergent-compatible alkaline serine protease from alkaliphilic Bacillus sp.: Production, biochemical characterization, and industrial applications. Electronic Journal of Biotechnology, 54, 1–14. https://doi.org/10.1016/j.ejbt.2021.09.001
Nweze, J. A., Nweze, E. I., & Unah, U. V. (2021). Metagenomics: An evolving tool in the characterization of soil microbiomes and enzyme production potentials in Nigerian agro-ecosystems. Frontiers in Sustainable Food Systems, 5, 639. https://doi.org/10.3389/fsufs.2021.639542
Srivastava, N., Srivastava, M., Ramteke, P. W., & Mishra, P. K. (2022). Synthetic biology approach for improving the efficiency of lignocellulose degrading enzymes. Bioresource Technology Reports, 11, 100471. https://doi.org/10.1016/j.biteb.2020.100471