Microwave-Assisted Enzymatic Hydrolysis of Rice Straw Using Effective Microorganisms for Bioethanol Production
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INTRODUCTION
Background of the Study
The evolution of industrialization and mechanization in the 21st century made it possible to transform manual equipment into modern machinery. With this development, global reliance on petroleum-based fuel is constantly increasing, giving rise to a challenge on energy sufficiency. Negative impacts, such as greenhouse gas (GHG) emissions, are associated with the combustion of these petroleum-based fuels (MacLellan, 2010). Major GHG contributors identified by the United States Environmental Protection Agency (EPA) include industry (20%), residential and commercial (11%), and agriculture (8%) (Bogart, 2013). Adverse effects on health and the environment of using petroleum-based fuels, thus, urge scientific community to search and develop alternative renewable fuel to replace the current and existing petroleum-based fuels. Biofuels have been demonstrated by several researches to reduce GHG emissions as compared to gasoline (petrol) (Leen, 2012). Among the alternative biofuels, bioethanol has received considerable attention in transportation sector because of its utility as an octane booster, fuel additive, and even as neat fuel (Mudliar, et. al., 2009). Bioethanol can be derived from organic materials, such as energy crops like corn, wheat, sugar cane, sugar beet, and cassava, among others (Neves, et. al., 2007). However, due to their primary utility as food, these crops cannot provide the global demand for bioethanol production. Among other feedstock for bioethanol production, biomass has been reported ideal and well-suited because of its large-scale availability, low cost, and environmentally benign production (Brodeur, 2011). Feedstock biomass is an organic matter that can be converted into energy from crop residues and agricultural wastes like rice straw (Bracmont, 2012). Rice straw is consists predominantly of cell walls and comprised with cellulose and hemicelluloses, which can be converted into bioethanol, and lignin. Annual generation in the country is 11.3 million tons according to the Philippine Rice Research Institute (PhilRice) (Yap, 2012); while in the whole Asia is 667.6 million tons (Sarkar, et. al., 2012) Nowadays, there is much activity in the area of lignocellulosic bioethanol, where the cellulose component of a biomass is broken down into simple sugars and subsequently to ethanol (http://www.ncsu.edu). Pretreatment has been used to separate the three main components of biomass (cellulose, hemicelluloses, and lignin) to increase access on the surface area and size the pores, decrease its crystallinity and polymerization degree (Maria, Galleti & Antonetti, 2011), and break lignin seal (Binod, et. al., 2009). Common pretreatment technique include chemical, physical and thermal processes like acid crystallinization, steam explosion and liquid hot water (Harmsen, et. al., 2010). Yet, these pretreatment involves hazardous chemicals and typically expensive to operate because of high energy costs and generation of certain types of pollutants (Elsevier, 2008). Thus, the search for efficient pretreatment technique will help address issues on high energy and operation cost (Neves, et.al., 2007). A study made by Li Xian-jun (2010) showed that microwave pretreatment gives internal pressure that causes deformation and damage of the cell walls. The use of microwave oven for pretreatment of biomass has also been reported to make the substrate more susceptible to the subsequent enzymatic hydrolysis (Neves, et.al, 2007), which is a complex procedure that uses enzymes to breakdown cellulose polymers to glucose (MacLellan, 2010). With this information in mind, the researcher decided to find out the efficiency of combined-microwave pretreatment and enzymatic hydrolysis using effective microorganisms (EM-1) in converting rice straw into fermentable sugars for bioethanol production. If found effective, it can address environmental issues related to the use of petroleum-based fuels. Likewise, farmers will be encouraged not to burn rice straw which adds pollutants to our atmosphere and at the same time generating additional income. Generally, this study contributes to the advancement of science for it can provide further information to other researchers in efficient pretreating cellulosic biomass insofar as energy and cost consumption.
Statement of the Problem
Generally, the researcher aims to find out the effectiveness of combined-microwave pretreatment and enzymatic hydrolysis using effective microorganism in converting complex carbohydrates of rice straw into fermentable sugars for subsequent bioethanol production. Specifically, this scientific investigation sought answers to the following questions: 1. Which set of treatments between microwave-treated (MW) and nonmicrowave-treated (NMW), is more effective in pretreating rice straw in terms of: a. Hydrolysis efficiency b. Percentage alcohol yield c. Percentage sugar-to-alcohol conversion 2. Which of the following amounts of EM is optimum in hydrolyzing rice straw and will give the highest fermentable sugars expressed as Total Soluble Solids (TSS): a. 100 mL b. 150 mL c. 200 mL 3. Is there a significant difference between the bioethanol produced from rice straw and the existing one sold in the market, in terms of: a. Cost b. Volume of bioethanol for local production
Hypotheses
1. Microwave-treated (MW) set of treatments is more effective in pretreating rice straw than nonmicrowave-treated (NMW) set in terms of: a. Hydrolysis efficiency b. Percentage alcohol yield c. Percentage sugar-to-alcohol conversion 2. Each amount of EM has the same efficiency in hydrolyzing rice straw which gives similar fermentable sugars expressed as TSS. 3. There is no significant difference between the bioethanol produced in rice straw and the existing one sold in the market, in terms of: a. Cost b. Volume of bioethanol for local production Significance of the Study Generally, findings from this study can be used to help address environmental issues related to the use of petroleum-based fuels. Production of alternative fuels such as bioethanol particularly from lignocellulosic feedstock has been reported to help reduce global reliance on petroleum-based fuels, address spiralling crude oil prices, and reduce GHG emissions caused by the use of gasoline and diesel fuels including issues on the use of food crops for bioethanol production. Specifically, findings from this study can contribute to the advancement of lignocellulosic bioethanol production researches particularly on pre-treatment and hydrolysis of biomass feedstock insofar as energy and production costs, sustainability due to availability of biomass, and environmental issues are concerned. An efficient pre-treatment and hydrolysis technique will help a lot to improve the existing techniques of converting complex into simpler sugars for subsequent conversion into bioethanol. Additionally, farmers will be encouraged not to burn rice straw in their fields which such practice add up pollutants to our atmosphere, and at the same time could generate for them an additional income.
Scope and Limitations of the Study This study was conducted at Brgy. Villa Isidra, Science City of Muñoz, Nueva Ecija from May-August 2013. Sample preparation and physicochemical analyses were undertaken at the Rice Chemistry and Food Science Division, Philippine Rice Research Institute, Maligaya, Science City of Muñoz, Nueva Ecija. Review of Related Literature
The emergence of increasing GHG emissions caused by petroleum-based fuel that made global warming to get in line with environmental problems require an alternative renewable one. On the other hand, the use of bioethanol for several years as a natural fuel showed its promising contribution on reducing GHG emissions. Bioethanol is an alcohol made by fermentation mostly from carbohydrates produced in sugar or starch crops such as corn or sugar cane. It can be used as a fuel in its pure form but it is usually used as a gasoline additive to increase the octane and improve vehicle emissions. Advantages of bioethanol are the following: (1) bioethanol is easily available from common biomass sources, (2) it represents a CO2 cycle in combustion, (3) bioethanol has a considerable environmental-friendly potential, (4) there are many benefits in the environment, economy, and consumers in using bioethanol, and (5) it is biodegradable and contribute to sustainability (Balat, et. al., 2009). It is a type of fuel whose energy is derived from biological carbon fixation, includes fuels derived from biomass conversion, as well as biomass, liquid and various biogases. Non-food sources such as trees and grasses which are cellulose biomass, is also being developed as a feedstock for ethanol production. Biomass is organic matter that can be converted into energy. Over the last few years, the concept of biomass has grown to include such diverse sources as algae, construction debris, municipal solid waste, yard waste, and food waste. Some contend that biomass has seen limited use as an energy source thus far because it is not readily available as a year-round feedstock, is often located at dispersed sites, can be expensive to transport, lacks long-term performance data, requires costly technology to convert to energy, and might not meet quality specifications to reliably fuel electric generators. Common examples of biomass include food crops, crops for energy (e.g., switchgrass or prairie perennials), crop residues (e.g., corn stover), wood waste and by-products (both mill residues and traditionally non-commercial biomass in the woods), and rice straw (Bracmont, 2012). Rice straw is an agricultural by-product. It is the dry stalks after the grain and chaff have been removed. It makes up about half of the yield of cereal crops such as barley, oats, rice, rye and wheat. It is usually gathered and stored in a straw bale, which is a bundle of straw tightly bound with twine or wire. In the Philippines, annual generation of rice straw is 11.3 million tons as reported by Philippine Rice Research Institute (PhilRice) (Yap, 2012). On the other hand, open burning of rice straw after harvesting is a common practice, thus, this activity releases a large amount of air pollutants which can cause serious effects on the ambient air quality, public health and climate. In Thailand, the area of annual harvested rice straw is about 9.8 million hectares and over 90% of it are burned (Tipayarom, et. al., 2007). To compound the problem, farmers used rice as a raw material to produce livelihood products. It has many uses including livestock, bedding and fodder, thatching, basket-making and fuel for it has high cellulose (32-47%) and hemicellulose (19-27%) content that can be readily hydrolyzed into fermentable sugars (Binod, et. al., 2009). Cellulose is a molecule comprised of carbon, hydrogen and oxygen, and is found in the cellular structure of virtually all plant matter. This organic compound, which considered the most abundant on earth, is even excreted by some bacteria. It provides structure and strength to the cell walls of plants and provides fiber in our diets. Although some animals, such as ruminants, can digest cellulose, humans cannot. Cellulose falls into the category of indigestible carbohydrates known as dietary fiber (Vandeman, 2000) Hemicellulose is one of the main components in plant biomass, which is second most plentiful natural resource in the world. It is a complex, branched and heterogeneous polymeric network, based on pentoses such as xylose and arabinose, hexoses such as glucose, mannose and galactose, and sugar acids. It has a lower molecular weight than cellulose and its role is to connect lignin and cellulose fiber (Maria, Galleti and Antonetti, 2011). On the other hand, both cellulose and hemicellulose are complex sugars that cannot be economically broken down into simpler one to produce alcohol through fermentation, thus pretreatment is done before the hydrolysis to make it accessible to further treatment. Pretreatment is a cleaning or treating process used to break down certain material into simpler one. Typical goals of pretreatment include (1) production of highly digestible solids that enhances sugar yields during enzyme hydrolysis, (2) avoiding the degradation of sugars (mainly pentoses) including those derived from hemicellulose, (3) minimizing the formation of inhibitors for subsequent fermentation steps, (4) recovery of lignin for conversion into valuable co-products, and (5) to be cost effective by operating in reactors of moderate size and by minimizing heat and power requirements. The process of pretreatment is considered to be one of the expensive steps in the conversion of lignocellulosic feedstocks to ethanol and accounts for nearly $0.30/gallon of ethanol produced (Brodeur, et. al., 2011). It can be placed into four categories such as biological, chemical, physical and thermal processes. Biological processes use microbial organisms to metabolize organic wastes into carbon dioxide, water, methane gas, simpler organic acids and microbial matter. Thermal processes breakdown the toxic components into simpler toxic forms. Chemical processes alter the chemical structure of a constituent like metal precipitation by pH adjustment. And physical processes separate components of a substance without altering the chemical structure of the constituent (Elsevier, 2008). Among these processes, chemical and physical processes involve hazardous chemicals and high energy and money consumption. The figure below shows how the pretreatment deconstructs lignocellulosic biomass. (Source http://origin-ars.els-cdn.com/content/image/1-s2.0-S0960852404002536-gr1.gif ).
Figure 1. Schematic representation of the matrix of polymers in which cellulose exists. Pretreatment of biomass by different methods removes hemicellulose and lignin from this matrix before hydrolysis. Common chemical pretreatment methods comprise dilute acid, alkaline, ammonia, organic solvent, SO2, CO2 or other chemicals. Acid catalyzed pretreatment of biomass prior to fermentation provide a near-term technology for production of fuel-grade ethanol from cellulosic biomass, but the relatively low yields of sugars from cellulose and hemicellulose (c.a. 50 % to 60 % of the theoretical yield) typical of dilute acid systems still have to be increased somehow, in order to be competitive with existing fuel options in a free market economy. Concentrated acid or halogen acids achieve high yields (essentially, 100 % of theoretical). However, because low-cost acids (such as H2SO4) must be used in large amounts while more potent halogen acids are relatively expensive, recycling of acid by efficient, low-cost recovery operations is essential to achieve economic operation (Neves, 2007). Alkaline processes use bases as NaOH or Ca (OH)2. All lignin and part of the hemicellulose are removed. Cellulose reactivity is sufficiently increased and the reactor costs are lower than those for acid technologies. Alkaline-based methods are generally more effective at solubilising a greater fraction of lignin, while leaving behind much of the hemicellulose in an insoluble, polymeric form. Physical or uncatalyzed processes generally use steam explosion or Liquid Hot Water. Steam explosion is one of the most promising methods to make biomass more accessible to cellulase attack. Basically, the method consists of heating the material using high-pressure steam (20-50 bar, 210 to 290C) for a few minutes; the reaction is then stopped by sudden decompression to atmospheric pressure. Using this method, xylose sugar recoveries between 45 and 64% were reported, revealing steam-explosion pretreatment as economically attractive treatment. The Liquid Hot Water uses compressed hot liquid water (at pressure above saturation point) to hydrolyse the hemicellulose. Xylose recovery is relatively high (88-89%), and no acid or chemical catalyst is required, which makes it environmentally attractive and economically interesting. The use of microwave oven for pretreatment of lignocellulosic has also been reported to make the substrate more susceptible to the subsequent enzymatic hydrolysis (Neves, 2007). One of the most extensively investigated pretreatment processes is the enzymatic hydrolysis (EH), in which fungal cellulolytic enzymes are used to convert the cellulose onto of the biomass to glucose, which is then fermented to ethanol. Basically, three major classes of enzymes may be used for EH pretreatment of lignocellulosic biomass for bioethanol production, as follows: the endo-1, 4-β- glucanases or 1,4-β-D-glucan 4-glucanohydrollases (EC 3.2.1.4), the exo-1, 4-β-D-glucanases, including both the 1,4-β-D-glucan glucohydrolases (EC 3.2.1.74), the “β-D-glucosidases” or β-D-glucoside glucohydrolases (EC 3.2.1.21). The three types of enzymes have been recognized for EH of lignocellulosic material, as they work together synergistically in a complex interplay, resulting in efficient decrystallization and hydrolysis of native cellulose. There are other microorganisms used in hydrolysis that work synergistically, in particular, effective microorganism (EM). Effective Microorganism (EM) is a combination of useful regenerative microorganisms that exist freely in nature and are not manipulated in any way. This mixture increases the natural resistance of soil, plants, water, human and animals. EM considerably improves the quality and fertility of soil as well as the growth and quality of crops. EM consists of 80 different kinds of effective disease-suppressing microorganisms. Each of these effective microorganisms has a specific task. This means that synergy occurs. EM consists of the following five families of microorganisms: (1) lactic acid bacteria, these bacteria are differentiated by their powerful sterilizing properties and they suppress harmful microorganisms and encourage quick breakdown of organic substances; (2) yeast, these manufacture antimicrobial and useful substances for plant growth; (3) actinomycetes, these suppress harmful fungi and bacteria and can live together with photosynthetic bacteria; (4) photosynthetic bacteria, these bacteria play in the leading role in the activity of EM; and (5) fungi that bring about fermentation, these break down the organic substances quickly.
Review of Related Studies
There are several studies that have been conducted about the production of bioethanol. One of the related studies that the researcher had gathered was made by A. M. Roslan, et. al. (2011) of Department of Bioprocess Technology, Bioscience Institute, Universiti Putra Malaysia, entitled, Production of Bioethanol from Rice Straw using Cellulase by Local Aspergillus sp. Cellulase production in situ was considered as one of the alternatives to produce bioethanol production cost. In this study, cellulase enzyme was produced from rice straw by locally isolated Aspergillus sp. in solid state fermentation. The crude cellulase was measured to have activity of 6.3 FPU g-1 rice straw. The straw was pretreated by few cycles of wet disc milling prior saccharification. The saccharified product was subjected to fermentation by yeast. The highest bioethanol yield produced from the fermentation was 0.102 g g-1 rice straw which is equivalent to 62.61% of theoretical bioethanol yield. It was concluded that the use of crude cellulase from rice straw onto rice straw can lead to a good yield of bioethanol, provided an effective pretreatment was used.
In the study made by Fatma H. Abd El-Zaher, et. al. (2010) of Department of Agricultural Microbiology, National Research Center, Cairo, Egypt, entitled, Production of Bioethanol Via Enzymatic Saccharification of Rice Straw by Cellulase Produced by Trichoderma Reesei under Solid State Fermentation, states that alternative substrates to produce useful chemicals such as biofuel have been attractive. Rice straw, one of the most abundant lignocellulosic wastes by-products worldwide can be used for this purpose. In the present study the production of cellulase by Trichoderma reesei F-481 cultivated on alkali treated rice straw cultivated on substance with about 75% (v/w) moisture, pH 4.8 for 5 days incubation at 28 ± 20C, as it gaves 16.2 IU/g substrate. The obtained cellulase luof 1.2 IU/ ml culture filtrate was applied for saccharification (5% w/v) of alkali treated rice straw, in 0.1 M citrate buffer pH 4.8 in shaker water bath of 100rpm. Sugary solution of 1.07% glucose was achieved after 16 hrs. The sugary solution was concentrated to give 10% (w/v) glucose. Ethanolic fermentation was conducted by Saccharomyces cerevisiae SHF-5 under static condition giving 5.1% (v/v) ethanol after 24 hrs. The fermented mash contained 3.6 g/L yeast cell can be utilized as fooder yeast used for animal feeding.
Another study was conducted by Mark Christian O. Luis (March 2009) in Department of Biological Sciences, College of Arts and Sciences, Central Luzon State University, entitled, Production of Bioethanol from Composted Rice Straw using Fungal Decomposers (Trichoderma viride and Aspergillus oryzae) and Rumen Fluid. In this study, four-treatment set-ups were prepared containing fungal decomposers and rumen fluid and subjected for the decomposition of rice straw. Each treatment was replicated thrice and was inoculated with Saccharomyces cerevisiae for alcoholic fermentation. The produced ferments from each treatment were subjected to Rotary Vacuum Evaporator to produce distillate for alcohol content determination. Using AOAC Official Method of Analysis for Alcohol content Determination, the alcohol percentage was determined. The study showed that 1kg of rice straw yielded 1.71% as effected by Aspergillus oryzae, 1.91% as effected by Trichoderma viride + Aspergillus oryzae and 1.95% as effected by Trichoderma viride. This study reveals that rice straw using fungal decomposers and rumen fluid can be used to produce ethanol. Another study was conducted by Jomar Larman Concepcion (April 2009) in Department of Biological Sciences, College of Arts and Sciences, Central Luzon State University, entitled, Cellulosic Ethanol Production using Corn Stover (Zea mays) Biomass. Bioethanol can be produced by fermentation of sugars from various agricultural waste materials. Whichever system for bioethanol production is chosen, the attention must be paid to the overall economics and energy consumption. The aim of the study was to investigate the use of carabao rumen fluid for bioethanol production from corn stover hydrolyzates. For this purpose various concentration of carabao rumen fluid were used. The parameters for bioethanol production, such as alcohol potential and ethanol content of corn stover for the efficient ethanol production were studied. Sugar production curve was done to obtain the optimum period of fermentation for each treatment. Yeast was used to convert sugar into ethanol. Simple distillation was done just to isolate the ethanol present in the yeast fermentation hydrolyzate-rotary evaporator was used to further concentrate the ethanol from the distillates. Results showed that the highest sugar produced was observed at day seven to T1(1% rumen fluid ) while for T2(5% rumen fluid) at day five and day four, respectively. The mean alcohol potentials were found to be 0.43%, 0.57% and 0.77% for T1, T2 and T3, respectively. The conversion efficiency rumen fermentation was 39.69%, 53.36% and 66.61% respectively. The difference among treatments was highly significant (P<0.01). Gas chromatographic analysis of T1, T2 and T3 concentrates revealed the purity of ethanol as 21.68%, 30.30% and 22.37%, respectively. The average alcohol content per 100g of corn stover for T1, T2 and T3, were found to be 3.47g, 4.9g and 5.55g, respectively. Mass Spectroscopy analysis revealed the presence of impurities in the ethanol concentrates which are suspected to be 1—propanol, 1—butanol, 1, 2 propanediol and methyl hydrazine. Results of the study showed that ethanol can be produced from corn stover with hydrolysis process using rumen fluid prior to yeast fermentation of the hydrolyzed sugar without addition of any enhancement. The two stage biological process can be further studied for optimum yield of cellulose ethanol. In the study made by Badal C. Saha et. al. (2005) of Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, USDA-ARS, Peoria, Illinois, entitled, Dilute Acid Pretreatment, Enzymatic Saccharification and Fermentation of Rice Hulls to Ethanol, states that Rice hulls, a complex lignocellulosic material with high lignin (15.38 ± 0.01%) content, contain 35.62 ± 0.12% cellulose and 11.96 ± 0.73% hemicelluloses and has the potential to serve as low-cost feedstock for production of ethanol. Dilute H2SO4 pretreatments at varied temperature (120 - 190 °C) and enzymatic saccharification (45 °C, pH 5.0) were evaluated for conversion of rice hull cellulose and hemicellulose to monomeric sugars. The maximum yield of monomeric sugars from rice hulls (15%, w/v) by dilute H2SO4 (1.0%, v/v) pretreatment and enzymatic saccharification (45 °C, pH 5.0, 72 h) using cellulase, β-glucosidase, xylanase, esterase, and Tween 20 was 287 ± 3 mg/g (60% yield based on total carbohydrate content). Under this condition, no furfural and hydroxymethyl furfural were produced. The yield of ethanol per L by the mixed sugar utilizing recombinant Escherichia coli strain FBR 5 from rice hull hydrolyzate containing 43.6 ± 3.0 g fermentable sugars (glucose 18.2 ± 1.4 g; xylose, 21.4 ± 1.1 g; arabinose, 2.4 ± 0.3 g; galactose, 1.6 ± 0.2 g) was 18.7 ± 0.6 g (0.43 ± 0.2 g/g sugars obtained; 0.13 ± 0.01 g/g rice hulls) at pH 6.5 and 35 °C. Detoxification of the acid- and enzyme-treated rice hull hydrolyzate by overliming (pH 10.5, 90 °C, 30 min) reduced the time required for maximum ethanol production (17 ± 0.2 g from 42.0 ± 0.7 g sugar per L) by the E. coli strain from 64 to 39 h in the case of separate hydrolysis and fermentation and increased the maximum ethanol yield (per L) from 7.1 ± 2.3 g in 140 h to 9.1 ± 0.7 in 112 h in the case simultaneous saccharification and fermentation.
Another study made by Beatriz Palmarola-Adrados et. al. of Department of Chemical Engineering, Lund University, entitled, Ethanol Production from Non-starch Carbohydrates of Wheat Bran. Wheat bran (WB), produced worldwide in large quantities as a by-product of the wheat milling industry, constitutes a significant underutilized source of sugars. This paper describes various methods of hydrolyzing the abundant polysaccharide in bran to yield a sugar feedstock suitable for fermentation into bioethanol. Firstly, the starch in the bran was released using amylolytic enzymes. The fibrous material remaining was further hydrolyzed. Acid hydrolysis, heat pretreatment followed by enzymatic hydrolysis and direct enzymatic hydrolysis were compared in terms of total sugar yield and pentose sugar yield. The maximum total sugar yield was achieved when small amounts of acid were added at the pretreatment step prior to enzymatic hydrolysis. This form of pretreatment released most pentosans and significantly enhanced the hydrolysis of cellulose. The overall sugar yield of this combined hydrolysis method reached 80% of the theoretical and it considered of 13.5 g arabinose, 2.2 8 g xylose and 16.7 g glucose per 100 g starch-free bran.
Definition of Terms Alcohol – is an organic compound in which the hydroxyl functional group (-OH) is bound to a carbon atom Alternative – available as another possibility Anthropogenic – result from the influence of human beings on nature
Carbon – an abundant non-metallic tetravalent element1 occurring in all organic compounds
Cellulose – is a long chain of linked sugar molecules that gives wood its remarkable strength
Cell walls – nonliving structure that surrounds the plant cell and is made up of a tough fiber called cellulose
Composting – producing organic matter that has been decomposed and recycled as a fertilizer and soil amendment
Crystallinity – refers to the degree of structural order in a solid
Degree of Polymerization (DP) – is usually defined as the number of monomeric units is a macromolecule or polymer or oligomer molecule
Distillation – is a method of separating mixtures based on differences in volatility of components in a boiling liquid mixtures
Enzyme - natural proteins produced in tiny quantities by all living organisms (bacteria, plants and animals)
Ethanol- is a volatile, flammable and colorless liquid
Fermentation – refers to the conversion to sugar to acid, gases and/or alcohol using yeast or bacteria
Fuel – is any material that stores potential energy in a form that can be practicably released and used as heat energy
Greenhouse Gas (GHG)- is a gas in an atmosphere that absorbs and emits radiation within the thermal infrared range
Hydrate – is term used in organic and inorganic chemistry to indicate that a substance contains water
Hydrolysis – means the cleavage of chemical bonds by addition of water or a step in the degradation of a substance
Hydrolyzate- product from hydrolysis
Lignocellulose- refers to plant dry matter and is abundantly available on Earth for the production of biofuel, mainly bioethanol
Microwave – are radio waves with wave lengths raging from as long as one meter to a short as one millimetre, or equivalent with frequencies between 300 MHz (0.3 GHz) and 300GHz (8.5-11.5bel Hz)
Microwave-assisted- means with the help of microwave in pretreating the rice straw
Microwave Oven – is a kitchen appliance that heats food by bombarding it with electromagnetic radiation is the microwave spectrum causing polarized molecules in the food to rotate and build up thermal energy in a process as dielectric heating
Octane- is a hydrocarbon and alkane with the chemical formula C8H18
Pasteurization – is any process of heating food which is usually a liquid to a specific temperature to a predefined length of time and then immediately cooling it after it is removed from the heat
Petroleum – is a naturally occurring flammable liquid consisting of complex mixture of hydrocarbons pH- is a measure of the acidity and basicity of an aqueous solution
Pollution – is the introduction of contaminants into the natural environment that adverse change
Pretreatment – any treatment received before some other process
Recalcitrance – resistance of plant cell walls to deconstruction
Starch -a white, odourless, tasteless, amorphous, powdery carbohydrate C6H10O5, insoluble in cold water, alcohol, and other liquids found in seeds, pith or tubers of most plants
Sugar – is a class of chemically related sweet flavoured substances most of which are used as foods
Total Soluble Solids (TSS)- determined by the index of refraction, using refractometer and is referred to as the degree Brix
Yeast- is an eukaryotic microorganisms classified in the kingdom Fungi
* Amount of rice straw per treatment * Amount of water per treatment in the hydration process * Duration of hydration * Duration of fermentation * Yeast a. Saccharomyces cerevisiae b. Pichia stipitis * Environmental factors
Figure 2. Variables of the Study METHODOLOGY
RS Collection and Preparation
Rice straw (RS) was collected from the rice fields of Brgy. Catalanacan and Brgy. Licaong, Science City of Muñoz, Nueva Ecija. Collected RS was scattered in an open space and sun-dried. Dried RS was grinded using a grinder (Cutting mill, 240V/60Hz) at Titanic, Agronomy Soil and Plant Physiology Division of the Philippine Rice Research Institute (PhilRice), Maligaya, Science City of Munoz. Pulverized RS was then placed in a clean plastic container and was set aside, until experimentation.
Evaluation of RS Hydration Duration
Two-hundred fifty kilograms of pulverized RS was placed in a fabricated fermentation tank (Figure 3) and was added with water (1:9) and stored. RS added with water was observed every 24 hours for odor, color and appearance, including changes in physical characteristics. During the initial incubation period, the RS floated after 30 minutes as expected. Then it submerged after five days, which was an indication of hydration duration.
Figure 3. Fabricated Fermentation Tank
Microwave Pretreatment of the Hydrated RS
For each treatment, one kilogram of pulverized RS was added with water (1:9) and each was incubated, based on the findings of the evaluation of hydration duration. After hydration, samples were subjected to microwave treatment for two minutes using commercial microwave oven (American Homes, 800 watts) set at high setting. Fifty milliliters each of MW and NMW sample was then collected in centrifuged tubes and subjected to physicochemical analysis using pH meter (Eutech, Japan) and refractometer (Abbe Leica, Germany) to determine its pH level and sugar level or total soluble solids expressed in °Brix.
Enzymatic Hydrolysis of RS
Microwave-treated (MW) RS was equilibrated prior to the addition of Effective Microorganism (EM). Diffirent amounts of EM were added to MW and NMW RS according to the table of treatments below:
Table 1. Experimental Design MICROWAVE-TREATED (MW) | NONMICROWAVE-TREATED (NMW) | T1(Control) -1kg of RS w/o EM-1 | T1(Control) -1kg of RS w/o EM-1 | T2-1kg of RS + 100ml EM-1 | T2-1kg of RS + 100ml EM-1 | T3-1kg of RS + 150ml EM-1 | T3-1kg of RS + 150ml EM-1 | T4 -1kg of RS + 200ml EM-1 | T4 -1kg of RS + 200ml EM-1 | MW and NMW RS without EM were used as controls. MW and NMW treated RS with EM were incubated for 20 days to allow enzymatic hydrolysis (conversion of cellulose and hemicellulose to fermentable sugars). Sugar (TSS) and pH levels of each sample were monitored every five days during the hydrolysis using Refractometer and pH meter, respectively. Pasteurization of RS After 20 days of enzymatic hydrolysis, liquid fraction of the hydrolyzed RS was obtained by filtration using clean cheese cloth. Filtrate was poured in glass bottles and was pasteurized under steam at 70oC for 20 minutes prior to addition of yeasts. Fermentation of Hydrolyzed RS Pasteurized hydrolyzate was poured in the fermentation setup and fermented using active commercial dry yeast (Saccharomyces cerevisiae) (for 6-carbon sugars) (activated in lukewarm water and sugar) and Pichia stipitis (for 5-carbon sugars) under anaerobic condition. Fermentation lock was used to maintain anaerobic condition and fermentation was conducted for about five days or until no evolution of bubbles are observed.
Distillation of Fermented RS Hydrolyzate
Fifty milliliters of fermented RS hydrolyzate in each samples were collected through decantation. Collected ferments (50 mL) were distilled using Rotary Vacuum Evaporator (Eyela, Japan) at 70oC and 76cm of Hg (pressure) to collect about 50mL distillate (bioethanol). Alcohol content of the distillate was analyzed using Refractometer according to Association of Official Analytical Chemists (AOAC) (1990) method.
Evaluation of Pretreatment and Hydrolysis Efficiency
Percent efficiency and alcohol yield were calculated using the formula below: 1. Hydrolysis Efficiency = Volume of Sugar (mL) Mass of Rice Straw (g)
2. X 100 Alcohol Yield Percentage = Volume of Alcohol (mL) Mass of Rice Straw (g)
3. X 100
Sugar-to-Alcohol = Volume of ethanol recovered (mL) Conversion Percentage Volume of Sugar (mL)
4. Projected Volume Bioethanol = Alcohol yield X 11.3 million tons for local production
Economic Analysis
Production cost of bioethanol from RS and commercial one sold in the market were compared using the formula below: Total Expenses
Total Volume of Bioethanol Produced
Production cost
=
Statistical Design and Data Analysis
All treatments were done in duplicate. Analysis of Variance (ANOVA) was conducted to detect the difference between treatments at p < 0.05 level. When a significant difference was observed, treatment means were separated using Least Significant Difference (LSD) test. Statistical analyses were performed using SAS ver. 9.1 Windows (New York, USA).
Experimental Procedure RS Collection and Preparation
Evaluation of RS Hydration Duration
Hydration of RS
MW Treated
NMW Treated
Enzymatic Hydrolysis
Fermentation of Hydrolyzate
Distillation of Fermented
RS Hydrolyzate
Microwave Treatment Pasteurization of treated RS
Enzymatic Hydrolysis
Pasteurization of treated RS
Economic Analysis
Fermentation of Hydrolyzate
Distillation of Fermented
RS Hydrolyzate
Data Processing and Analysis Data Processing and Analysis
Figure 4. Flow Diagram of the Study
RESULTS AND DISCUSSION
Combined microwave pretreatment and enzymatic hydrolysis using effective microorganisms (EM-1) were tested for their efficiency in converting complex carbohydrates of rice straw (RS) into fermentable sugars for the production of bioethanol. The RS samples were obtained from Brgy. Catalanacan and Brgy. Licaong, Science City of Munoz.
Effect of microwave treatment on pH and sugar levels of hydrated RS
Samples were subjected to microwave pretreatment and 50 mL of MW and NMW RS were collected in centrifuged tubes for phycochemical analysis. Sugar level (TSS) expressed in °Brix and pH level using Refractometer and pH meter according to Association of Official Analytical Chemists (AOAC) (1990) method is shown below:
Table 2. pH and TSS of microwave and nonmicrowave –treated hydrated rice straw (5 days) Sample | pH | Total Soluble Solids (°Brix) | MW Treated | 7.83a | 1.75a | NMW Treated | 5.88b | 1.00b |
*Means within a row with same letters are not significantly different (α=0.05)
Physicochemical analysis of hydrated rice straw for 5 days showed a significant increase in both pH and sugars or total soluble solids (TSS) levels upon microwave treatment. TSS increased from 1.00 °Brix (NMW-treated) to 1.75 °Brix (MW-treated) level, while pH increased from 5.88 (nonmicrowave-treated) to 7.83 (microwave-treated). The increase in both TSS and sugar levels may be attributed to the effect of microwave treatment on the hydrated rice straw. Microwave treatment may have caused partial hydrolysis of the rice straw causing the release of soluble sugars from its complex components. This could then indicate that microwave pre-treatment is an effective technique in pretreating biomass such as RS prior to hydrolysis.
Similar to the effect of microwave pre-treatment on TSS, pH of the samples increased. The change of pH of the hydrated rice straw from acidic (pH 5.88) to nearly neutral (pH 7.83) may have affected by the release of soluble sugars. This may also imply destruction of carboxylic acids and other acidic substances produced through enzymatic or chemical reactions during the course of incubation caused by microwave treatment.
Effectiveness of combined-microwave pre-treatment and enzymatic hydrolysis using effective microorganism
Fifty milliliters of MW and NMW RS hydrolyzates were collected every week for Total Soluble Solids and pH determination. TSS content tests the solid concentration of glucose containing solution; while pH is the measure of acidy and basicity of an aqueous solution. TSS and pH in each week were compared, as seen on the below figures:
Change in TSS during hydrolysis Change in TSS levels of microwave and nonmicrowave –treated rice straw during hydrolysis using effective microorganisms were monitored using a refractometer to evaluate the effectiveness of microwave pre-treatment in helping overcome the lignocellulosic recalcitrance. Results showed no significant change in TSS between microwave and non-microwave treated samples during hydrolysis. Likewise, no significant change in TSS was observed on different samples even with different amounts of effective microorganisms (0 ml to 200 mL) indicative of inactivity in hydrolysing rice straw of the EM that was used. TSS ranged from 0.6 °Brix to 2.0 °Brix (Figure 1). Hence, combined-microwave pretreatment and enzymatic hydrolysis applied to the samples may not be efficient.
Figure 5. Total soluble solids of microwave and nonmicrowave-treated samples during hydrolysis
However, the refractometry method that was used may not be suitable to the type of samples measured. Samples were characterized by a turbid solution with suspended particles of ground rice straw (Figure 6) which is among the limitations of the method. For instance, prism of the refractometer used suffers difficulty in distinguishing refraction of the samples as indicated by blurred appearance of the samples in the optical lenses. Hence, readings may not be representative as to the actual sugar levels in the samples. Thus, it is recommended to use other methods in measurement of sugars for such kind of samples.
Figure 6. Turbid Sample
Similar finding was obtained with the study of Abd El-Zaher and Fadel (2010) when he evaluated the effect of alkaline pretreatment of RS on the release of sugar, from 11.17 IU/g to 7.59 IU/g. Melo, et. al. (2007) also reported that the sugar level declined with prolonged incubation, that could be due to loss of moisture or denaturation of the microorganism used, resulting from variation in pH. In addition, Ojumu, et. al. (2003) and Alam, et,al. (2005) also reported that the decrease of microorganism activity may be due to the accumulative effect of cellulobiose and the time of the highest cellulose activity depends upon the substrate and fungus.
Change in pH during hydrolysis
pH levels were monitored to identify possible physicochemical changes undertaking during hydrolysis. For instance, a decrease in pH may be attributed to the products of physicochemical changes caused by oxidation-reduction reactions and/or microbial activity such as aldehydes, ketones or carboxylic acids. Results showed that microwave-treated samples had higher pH than nonmicrowave-tread RS hydrolyzate throughout the hydrolysis period (Table 3). pH levels of microwave-treated samples ranged from 5.8-7.8, while nonmicrowave-treated samples ranged from 5.4-7.0.
Generally, pH levels of microwave-treated samples decreased starting 2 weeks of incubation, whereas pH of nonmicrowave-treated samples did not change significantly. Decrease in pH based on the difference between the final and initial pH of microwave-treated samples was 1.0 to 1.7 levels; whereas a negligible increase in pH on nonmicrowave-treated samples was noted (0.1 to 0.3 levels).
Hydrolysis efficiency
Combined microwave pre-treatment and enzymatic hydrolysis using effective microorganisms were tested for their efficiency in converting complex carbohydrates of rice straw (RS) into fermentable sugars for the production of bioethanol. Although an increase in TSS levels in all samples was observed immediately upon microwave pretreatment suggestive of its efficacy, hydrolysis efficiency as indicated by an increase in TSS levels upon hydrolysis was observed low in all treatments. This may indicate inactivity of the effective microorganism used as implied by no significant change in TSS after 4 weeks of hydrolysis period. Likewise, a reduction in TSS levels was noted in all treatments, except that of NMW-150 mL, after 4 weeks of hydrolysis.
Several factors may have affected the efficacy or activity of the microorganisms present in the EM used or the problem may be originated from the EM product by itself. For instance, the EM type was commercial with no specified indication of the amount of active (live) effective microorganism present. Another factor may be attributed to the condition of the substrate (pre-treated hydrated RS) in terms of pH and present of other unwanted microorganisms, among others. Collectively, it is therefore important and suggested to utilize specific, identified and confirmed active microorganism for such hydrolysis of lignocellulosic biomass.
Hydrolyzed RS samples were fermented using two yeasts, namely active commercial dry yeast of Saccharomyces cerevisiae and Pichia stipitis to convert 6-carbon and 5-carbon sugars into bioethanol, respectively. Alcohol yield was then evaluated based on alcohol produced and alcohol yield per unit of RS that were used. Results showed that no alcohol was produced on treatments with less than 200 mL of effective microorganism indicating that such amount may be not sufficient in the hydrolysis of RS. Only samples with 200 mL of effective microorganism produced bioethanol (Figure 7). Higher amount of alcohol was obtained for microwave-treated sample at 8.3%, indicating higher availability of simple sugars readily convertible to bioethanol. With the same amount of effective microorganism, this increase in alcohol yield may be attributed to the efficacy of microwave pre-treatment in promoting better hydrolysis of the complex sugars in the RS. This finding is congruence with the initial TSS results where an increase was attributed to microwave pre-treatment of the samples. Figure 7. Alcohol produced from fermented microwave and nonmicrowave-treated RS
Alcohol produced in each treatment was then used to calculate the yield per unit of RS. Results showed that total volume of alcohol produced from microwave-treated RS with 200 mL of effective microorganism was 747.5 mL, while for nonmicrowave-treated was 89.5 mL; which is equivalent to an percentage alcohol yield per unit of RS of 74.7% (0.75 mL alcohol/ 1 g of RS) and 8.9% (0.09 mL alcohol/ 1 g of RS), respectively (Table 5).
Table 5. Alcohol produced and alcohol yield of microwave and nonmicrowave-treated samples after fermentation | | | | | | Treatment | Alcohol, % | Vol. of Alcohol Produced per Treatment* (mL) | Alcohol Yield per unit RS** (mL alcohol/ g RS) | Percent Alcohol Yield per unit RS***(%) | Control | | | | | | MW | 0.0 | 0 | 0 | 0 | | NMW | 0.0 | 0 | 0 | 0 | EM-100 Ml | | | | 0 | | MW | 0.0 | 0 | 0 | 0 | | NMW | 0.0 | 0 | 0 | 0 | EM-150 mL | | | | 0 | | MW | 0.0 | 0 | 0 | 0 | | NMW | 0.0 | 0 | 0 | 0 | EM-200 mL | | | | 0 | | MW | 8.3 | 747 | 0.747 | 74.7 | | NMW | 1.0 | 9000 | 0.009 | 9.0 |
Note: *Vol. of alcohol produced per treatment = % alcohol x 9000 mL (total volume of hydrolyzate); **Alcohol yield per unit RS= Vol of alcohol per treatment/ 1000 g (weight of RS); ***Percent alcohol yield per unit RS= [Vol of alcohol per treatment/ 1000 g (weight of RS)] x 100%
Bioethanol for Local Production
Philippines is one of the country that relies on petroleum-based, consequently, faces its negative effects to be solve by virtue of bioethanol. Bioethanol for local production was calculated by converting volume of alcohol (L) per mass of RS (kg).
Table 6. Projected volume of bioethanol for local production TREATMENTS | % alcohol yield | volume of alcohol (L/kg) | volume of alcohol (L/ton) | volume of bioethnol in 11.3 million ton | MW Treated | 8.3 | 0.747 | 677.529 | 7656077700 | NMW Treated | 1.0 | 0.009 | 8.163 | 92241900 |
It can gleaned from the above table that the volume for local production of bioethanol produced using MW pretreatment using 200 mL (P7656077700) is higher than the commercial local production (P24 million liters)
Economic Analysis The expenses incurred during production of lignocellulosic bioethanol are presented in Table 7. Bioethanol from MW treated RS had total expense of P 274.02 while from NMW treated RS had costs P 22,743.33.
Table 7. Expenses incurred in the production of lignocellulosic bioethanol TREATMENTS | Effective Microorganism | Yeast | Equipment Rental | volume of bioethanol produced (L) | TOTAL EXPENSES | | | | | | | MW Treated | 75 | 6.25 | 123.44 | 0.747 | 274.02 | NMW Treated | 75 | 6.25 | 123.44 | 0.009 | 22, 743.33 |
Economic analysis showed that the production cost of bioethanol using MW pretreatment is lower than the other treatment produced without pretreatment. This implies that the production of lignocellulosic bioethanol using microwave pretreatment with 200mL of EM is lower than the production cost of commercial bioethanol (P394.57).
SUMMARY AND CONCLUSION
Lignocellulosic bioethanol production are gaining much attention worldwide as substitute for petrochemical-based fuels to help address energy security and climate change concerns associated with petroleum fuels. However, available pretreatment techniques to overcome lignocellulosic recalcitrance on biomass possess several disadvantages which significantly increase production and operation costs of bioethanol. Thus, this research aimed to find out the effectiveness of combined-microwave pre-treatment and enzymatic hydrolysis using different amounts of effective microorganism in converting complex carbohydrates in rice straw into fermentable sugars for subsequent bioethanol production.
Results confirmed the efficacy of microwave pretreatment prior to hydrolysis as indicated by higher TSS levels of microwave treated RS compared to nontreated samples. On the other hand, hydrolysis efficiency was observed low in all treatments.
Only samples with 200 mL of effective microorganism produced bioethanol, indicating higher amount may be needed to sufficiently hydrolyze RS. Higher amount of alcohol production and yield was obtained for microwave-treated sample, indicating higher availability of simple sugars readily convertible to bioethanol attributed to microwave pre-treatment. In addition, projected volume of bioethanol for local production was higher than the standard production in the Philippines.
Furthermore, cost analysis revealed that the production of lignocellulosic bioethanol using combined-MW pretreatment and enzymatic hydrolysis using EM was lower than the production cost of commercial bioethanol. This implies that there is significant difference in production cost between bioethanol produced from RS and the existing one sold in the market.
RECOMMENDATIONS
Based on the results, the researcher recommended the following activities for further investigation:
1. Use of microwave pretreatment in other cellulosic feedstock crop biomass such as rice husks, rice bran, corn stover, and corn straw, among others for production of lignocellulosic bioethanol. 2. Utilize identified, specific, and confirmed active microorganisms in hydrolyzing lignocellulosic feedstock. 3. Assess bioconversion of lignin residue for electric power and organic fertilizer production. 4. Use other methods in measurement of sugars.
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APPENDICES
APPENDIX TABLES
Appendix Table 1. Raw Data of pH level in Four Consecutive Weeks
Rice Straw for Hydration
Fabricated Fermentation Tanks
Hydration of Powered Samples
Subjection to Microwave
Microwave Oven
Pretreatment of Hydrated Rice Straw
Application of Microorganism
Effective Microorganism (EM-1) Application of EM
Hydrolyzation of Pretreated Rice Straw
Refractometer
Hydrolyzate
pH meter
Fermentation Set-up
Fermentation Lock
Physicochemical analysis of Hydrolyzate
Fermentation of Hydrolyzate
Collected Distillate
Rotary Vacuum Evaporator
Distillation of Rice Straw Hydrolyzate
Analysis for Refractive Index
Refractometer
Distillate for Analysis
CURRICULUM VITAE
Personal Data
Name: Marc Ralph M. Solomon
Nickname: Marc, Dong
Address: Catalanacan, Science City of Muńoz, N.E.
Date of Birth: August 27, 1997
Father: Rolando Solomon Sr.
Mother: Wilma Solomon
Bother: Rolando Solomon Jr.
Sisters: Mary Rose Ann Solomon Rose Jane Pie Solomon
Favorite Subject/s: Chemistry, Biology, Geometry, Trigonometry
Motto: “Apart from God, we are nothing.”
Ambition: To be a successful Chemist someday.
Educational Background
High School: Muńoz National High School Science City of Muńoz, Nueva Ecija S.Y.: 2010- present Elementary: Muńoz South Central School Science City of Muńoz, Nueva Ecija S.Y.: 2006-2010 San Sebastian School Science City of Muńoz, Nueva Ecija S.Y: 2005-2006 Muńoz South Central School Science City of Muńoz, Nueva Ecija S.Y.: 2004-2005 Pre-elem.: Cadena de Day Care Center Catalanacan, Science City of Muńoz, Nueva Ecija S.Y.: 2002-2003
