Abstract. Introduction

Transcription

1 Development of Efficient Heterologous Expression System For Production Of Enzymes To Convert Industrial Organic Residues To Value Added Products Or Energy Abstract In Today s society, there is a great demand for appropriate nutritional standards, characterized by rising costs and often decreasing availability of raw materials together with much concern about environmental pollution. Consequently there is a considerable emphasis on the recovery, recycling and upgrading of wastes. The energy and environmental crises which the world is experiencing is forcing us, among other things, to re-evaluate the efficient utilization or finding alternative uses for natural, renewable resources, especially organic waste, using clean technologies (Pothiraj, 2006). Developing countries are still grappling with socio-economic issues including meeting the massive energy-shortage demands, food security and developing technological solutions in the agriculture, agro-processing and other related manufacturing sectors. Industrial biotechnology offers significant opportunities to developing countries for addressing some of the issues highlighted since most of the technology is based on the utilization of readily available residual organic residues from industries, plant biomass considered as waste to produce numerous value-added products. By this research proposal we analyse the state of the art of these industrial activities in relation to the possibility of developing biotechnological products and processes for the conversion of wastes into energy, with the aim of reducing the cost of production and the environmental impact, and generating value-added products through anaerobic fermentation: biogas and organic fertilizer. Introduction Bioenergy is a promising, inexhaustible, sustainable source to combat the rising environmental, economic, and technological issues related to depleting fossil fuels. The most important aspect for sustainable production and supply of bioenergy is the availability of feedstock. Among various substrates, production from wastes has received a special acceptance for maintaining environmental integrity. Wastes are generated in many forms and at various stages, ranging from domestic to industrial levels, and their improper disposal has detrimental effects on the environment as well as human life. The human population across the globe is increasingly getting oriented toward healthy and processed food products. It has been observed that of the enormous supply of food for human consumption, about one-third gets wasted globally (FAO,2011). Utilizations of these wastes generated at different levels of delivery starting from the agricultural farm, post-harvest handling, storage, processing, and from distribution to

2 consumption would be economically highly beneficial (Bhardwaj, 2012). Such wastes can either be used directly as an untreated material for microbial growth or be used by appropriate treatment for extraction of useful molecules and bioconversion with enzymes into value-added products or for bioenergy production. This last option seems to be convenient when the waste has a significant cost of disposal and a relevant intrinsic energy content. The products generated from perishable wastes can be in liquid or gaseous forms of biofuels. Since the process is rather specific and varies for conversion into different types of biofuels, it is necessary that the quality, quantity, and characteristics of the feedstock are known or determined beforehand. Based on initial screening and biochemical characterization of the wastes, treatment processes can be designed to recover energy from waste nutrients. Utilization of microbes, often in combination with enzymes, for digestion of discarded biomass into a desired form of fuel is presently one of the most accepted waste management strategies. The strategic application of bioconversion processes on wastes can be used to develop environmentally friendly and low-cost operating systems for production of biofuel and value added products. Wastes coming from industries can be used to both produce biogas and bio-fertilizers: this process allows optimizing the investments and improving the process s efficiency and sustainability. This procedure depends on Anaerobic Digestion, which allows the conversion of complex organic matter into methane and carbon dioxide. This process consists of many steps, but the hydrolysis of complex polymers into simple compounds is the most limiting one. Hence, making the hydrolysis reaction faster using biotechnological tools would significantly enhance this rate limiting step and improve the efficiency of the whole process, making it competitive on the market: in one step wastes would be disposed, a new source of energy would be created and potential material that could be used as a fertilizer would be made. Therefore there is a strong interest in pretreatment methods of complex polymers. Here we focus on the biotechnological tools; however physical and chemical technologies have also been developed. Different approaches have been studied, that include the use of recombinant enzymes. In this we suggest the production of recombinant organisms as the method of choice to express the cocktail of enzymes necessary to complete the hydrolysis process, with an eye on Pichia pastoris, whose advantages in terms of large scale productions are well known: high yields and high secretion levels of more soluble glycosylated proteins, which make it easier to purify the enzymes. Waste from different industries Vegetable and fruit waste. The wastes like peel and pomace of fruits and vegetables, cocoa pod husk, wheat straw, wood chips, pea pods, pea nut skin, wallnut shells, seeds, membranes and cores so generated pose an environmental threat (Laufenberg, 2003). Generation of renewable energy can be achieved by

3 bioconversion of vegetable. In this specific case the technology is waste to fuel to overcome the disposal problems. Poultry waste. Poultry litter is an economically viable fertilizer for many crops and also is used for animal feed, bioenergy production. Poultry litter has a gross energy value close to that of wood and about half that of coal. Therefore, Poultry litter has potential as a fuel to produce energy (Narrod,1994). Leather industry waste. Various industrial animal wastes (skin trimming waste, wool, bristle, horns, feathers, hoofs, keratin waste, fleshing waste, buffing waste) can be used to convert them into a low cost high value biodegradable end products based on biotechnological procedure such as hydrolysis (Sundar, 2011). Waste leather hydrolysates prove to be a valuable protein resource possible to be converted to added value commercial products as soil fertilizers, biodegradable polymers and additives for cosmetic industry, building materials, biofuel. Biodiesel can be produced from pre-fleshing waste. This fuel is an environmentally friendly energy source, which is non-toxic and biodegradable, and which has low emission values. (Ozgunay et al. 2007) Agroindustrial and paper industry waste. Lignocellulosic materials are the most abundant renewable organic biomass. This biomass is emerging as an energy source among many kinds of new energy including wind energy, hydroenergy, solar energy, nuclear energy, etc. The most important applications of lignocellulose biotechnology are in Bioenergy with the bioethanol, biogas, and biohydrogen production (da Silva, 2016). Proposed Work plan 1) Isolation of cellulase, xylanase producing microorganism: Samples from wastes will be collected from different sources. For isolation of bacteria, suspension of samples will be prepared in sterile distilled water, which will then be plated on modified agar medium. Isolates will be purified by repeated plating. 2) Screening of isolates for efficient cellulase and xylanase producers: isolates will be tested qualitatively by growing the culture on modified agar medium followed by observing zone of hydrolysis around hydrolysing enzymes-producing colonies. Cellulase and xylanase producing isolates will be selected and maintained on nutrient agar for further studies. Characterization of selected isolates: Morphological, cultural and bio-chemical characteristics of the

4 selected isolates will be studied according to standard techniques. (a) Parametric optimization of different conditions for cellulase and xylanase production: Physico-chemical parameters (initial ph, temperature, incubation period, agitation, inoculum age and size, carbon and nitrogen source) will be optimized for enhanced cellulase and xylanase production by selected isolates. During these studies one parameter will be varied at a time. (b) Production, purification and characterization of cellulase and xylanase enzymes. Enzymes will be produced by growing the isolates in the production medium. Enzyme will be extracted and purified from culture filtrate of isolate using standard enzyme purification technique such as ammonium sulphate precipitation, hydrophobic interactions, ion exchange chromatography, gel filtration technique. The purified enzyme will be characterized for its ph stability, ph optima, temperature optima and thermal stability, determination of km and Vmax and molecular weight. (c) Analysis of the purified enzymes and identification of encoding genes. The purified enzymes will be analysed by both N-terminal amino acid sequence and MALDI TOF TOF. This information will be used to try to identify the genetic determinant through the analysis of proteins databases. 3) Heterologous expression of hemicellulolytic enzymes in a suitable and efficient heterologous expression system. Some bacteria with known hemicellulolutic activity have been already studied and characterized and are available as a source of genes to be expressed in another system. The heterologous expression systems will be developed for production of the following enzymes: (i) - endocellulase from Bacillus pumilus (ii) - cellobiohydrolase from Xanthomonas axonopodis pv glycines (iii) - beta-glucosidase from Bacillus amyloliquefaciens (iv)- xylanase from Bacillus pumilus Enzymes from (i) to (iii) are known to depolymerize cellulose in three steps: (i) cellulose polymer cleavage and oligomers formation; (ii) removal of dimers (cellobiose) from the cellulose oligomers; (iii) release of glucose from cellobiose dimers. Xylanase is the key emzyme in the depolimerization of hemicellolose, although in cooperation with some so called accessory enzymes (Degrassi, 2000).

5 The genes encoding the above mentioned enzymes will be amplified by PCR, cloned in ptopo, sequenced to verify the correct amplification, then cloned in pqe, an expression vector giving 6xHis tagged proteins. E. coli M15 will be the first expression system to be tested. The proteins will be then purified by a single step-affinity chromatography, thanks to the histidine tag, and used in the experiments of cellulose digestion. An alternative heterologous expression system that will be taken into consideration for the production of the enzymes is the yeast Pichia pastoris. It is known that many enzymes when expressed in E. coli result in insoluble protein production (inclusion bodies), therefore P. pastoris, an efficient eucaryotic expression system, could help overcoming the problem, thanks to the ability of both producing and secreting more soluble glycosylated protein. 4) Hydrolysis. During hydrolysis, the first stage in the multistep-biogas production performed by consortia of microorganisms, bacteria transform the particulate organic substrate into liquefied monomers and polymers i.e. proteins, carbohydrates and fats are transformed into amino acids, monosaccharides and fatty acids respectively. Equation 1 shows an example of a hydrolysis reaction where organic waste is broken down into a simple sugar, in this case, glucose (Ostrem, 2004). Equation 1: C 6H 10O 5 + 2H 2O C 6H 12O 6 + 2H 2 5) Acidogenesis. In the second stage, acidogenic bacteria transform the products of the first reaction into short chain volatile acids, ketones, alcohols, hydrogen and carbon dioxide. The principal acidogenesis stage products are propionic acid (CH 3CH 2COOH), butyrric acid (CH 3CH 2CH 2COOH), acetic acid (CH 3COOH), formic acid (HCOOH), lactic acid (C 3H 6O 3), ethanol (C 2H 5OH) and methanol (CH 3OH), among other. From these products, the hydrogen, carbon dioxide and acetic acid will skip the third stage, acetogenesis, and be utilized directly by the methanogenic bacteria in the final stage. Equations 2, 3 (Ostrem, 2004) and 4 (Bilitewski et al., 1997) represent three typical acidogenesis reactions where glucose is converted to ethanol, propionate and acetic acid, respectively. Equation 2: C 6H 12O 6 2CH 3CH 2OH + 2CO 2 Equation 3: C 6H 12O 6 + 2H 2 2CH 3CH 2COOH + 2H 2O Equation 4: C 6H 12O 6 3CH 3COOH 6) Acetogenesis. Acetogenesis consists in the creation of acetate, a derivative of acetic acid, from carbon and energy sources by acetogens. These microorganisms catabolize many of the products created in acidogenesis into acetic acid, CO 2 and H 2. Acetogens break down the Biomass to a point to which Methanogens can utilize much of the remaining material to create Methane as a Biofuel.

6 7) Methanogenesis. The fourth and final stage is called methanogenesis. During this stage, microorganisms convert the hydrogen and acetic acid formed by the acid formers to methane gas and carbon dioxide (Verma, 2002). The bacteria responsible for this conversion are called methanogens and are strict anaerobes. Waste stabilization is accomplished when methane gas and carbon dioxide are produced. CO 2 + 4H 2 CH 4 + 2H 2O 2C 2H 5OH + CO 2 CH 4 + 2CH 3COOH CH 3COOH CH 4 + CO 2 The proposed study will be carried out during the course of fellowship. References : 1. Bilitewski B, Härdtle G, Marek K (1997): Waste Management. Springer, Berlin, ISBN: Bhardwaj V, Garg N, (2012). Environmental pollution control by utilization of industrial waste. J Earth Sci Climate Change, Volume 3 Issue Pothiraj C. et al. (2006). Bioconversion of Lignocellulose Materials. Mycobiology 34(4):

7 4. Da Silva LL (2016). Adding Value to Agro-Industrial Wastes. Ind Chem 2: e103. doi: / e Degrassi G et al (2000). The acetyl xylan esterase of Bacillus pumilus belongs to a family of esterases with broad substrate specificity. Microbiology, 146, FAO (2011) Global food losses and food waste: extent, causes and prevention. FAO, Rome 7. Laufenberg G et al. (2003) Transformation of vegetable waste into value added products: (A) the upgrading concept; (B) practical implementations. Bioresource Technology 87 (2003) Narrod, C.A., R. Reynells, and H. Wells. (1994). Potential options for poultry waste utilization: A focus on the Delmarva Peninsula. In Environmentally Sound Agriculture. ASAE. St. Joseph, MI. 9. Ostrem, K. 2004: Greening Waste: Anaerobic Digestion For Treating The Organic Fraction Of Municipal Solid Wastes. Earth Engineering Center Columbia University. 10. Ozgunay H, Colak S, Zengin G, Sari O, Sarikahya H, Yuceer L (2007) Performance and emission study of biodiesel from leather industry pre fleshings. Waste Manage 27: Verma, S., 2002: Anaerobic Digestion Of Biodegradable Organics In Municipal Solid Wastes. Department of Earth & Environmental Engineering (Henry Krumb School of Mines) Fu Foundation School of Engineering & Applied Science Columbia University 12. Sundar V. J. et al (2011). Recovery and utilization of proteinous wastes of leather making: a review. Rev Environ Sci Biotechnol (2011) 10: