West Visayas State UniversityCollege of Arts and SciencesIloilo City

Chapter 1Chapter One consists of seven parts: (1) Background and of the Study; (2) Theoretical Framework; (3) Research Paradigm; (4) Statement of the Problem; (5) Significance of the Study; (6) Definition of Terms; and (7) Delimitation of the Study.Part one, Background of the Study, gives the basis and explanation for choice of the problem.Part two, Conceptual/Theoretical Framework, gives the basis and the variables considered in conducting the study.Part three, Research Design, illustrates the Independent and Dependent Variables of the study and their correlation.Part four, Statement of the Problem, reveals the general and specific statements of the problem sought after for the major objectives of the study.Part Five, Significance of the Study, discusses the benefits that may be derived from the results of the study and the people who would benefit from them.Part Six, Definition of Terms, deals with the conceptual and operational meaning of the important terms used.Part Seven, Delimitation of the Study, sets the limits and scopes of the study.Background of the StudyBiofuels are gaining increased public and scientific attention, driven by factors such as oil price spikes, the need for increased energy security, and concern over greenhouse gas emissions from fossil fuels. It can be obtained from renewable sources containing starch, sugar, or cellulose, such as potatoes, corn, corn cobs and stalks, grains, and wood. One of main problems with using crops or woods as feedstock is that they will affect directly crop prices and will result in destruction of forests. Therefore, seaweed or macroalgae as a solution for this problem has been introduced recently. Some of advantages in using seaweed as feedstock include simple cultivation and possible productiveness. It also has easier manufacturing process (No lignin removal) with a higher CO2 fixation ability.Algae are considered as the only alternative to current bioethanol crops such as corn and soybean as they do not require arable land (Chisti, 2007; Hu et al., 2008; Singh et al., 2010c). The arable land could be used efficiently to grow food crops rather than biomass and oil seed crops for the production of biofuels. Water filled areas that are not suitable for growing food crops and industrial waste water can be used for the cultivation of algal biomass without any compromise with land and water resource for the production of bioethanol that will also not adversely affect the food cost (Singh et al., 2011).In addition, algae can be converted directly into energy, such as biodiesel, bioethanol and bioethanol and therefore can be a source of renewable energy. Given the premises, the researchers want to determine if the brown algae, Mermaid fan seaweed can be an alternative source of fuel which is locally available in the area.Theoretical FrameworkCertain species of microalgae have the ability of producing high levels of carbohydrates instead of lipids as reserve polymers. These species are ideal candidates for the production of bioethanol as carbohydrates from microalgae can be extracted to produce fermentable sugars. It has been estimated that approximately 500015,000 gal of ethanol/acre/year (46,760140,290 L/ha) can be produced from microalgae. This yield is several orders of magnitude larger than yields obtained for other feedstocks. Blue-green algae including Spirogyra species and Chlorococum sp. have been shown to accumulate high levels of polysaccharides both in their complex cell walls and as starch. This starch accumulation can be used in the production of bioethanol (Harun et al., 2010; Eshaq et al., 2011). Harun et al. have shown that the blue-green algae Chlorococum sp. produces 60% higher ethanol concentrations for samples that are pre-extracted for lipids versus those that remain as dried intact cells. This indicates that microalgae can be used for the production of both lipid- based biofuels and for ethanol biofuels from the same biomass as a means to increase their overall economic value (Jones and Mayfield, 2012).With these, the study intends to investigate the potential of the macroalgae named Mermaid Fan Seaweed (sci. name)in producing bioethanol using two types of chemical pretreatment procedure: acid hydrolysis and enzymatic hydrolysis.

Research ParadigmIndependent VariableDependent VariablesMermaid Fan Seaweed using pretreatment procedures:a. Acid hydrolysisb. Enzymatic hydrolysisBioethanol actual yield

Physical Characteristic of bioethanol:a. pHb. methanol contentc. specific gravityd. densitye. water contentf. copper contentg. flash point for flammabilityh. Electrical conductivityi. Visual appearance

Standard for Bioethanol based on ASTM (American International Testing and Materials, 2011)

Fig 1. Showing the relationship between the Independent variable and the dependent variables.

Statement of the ProblemThis study aims to determine the potential of bioethanol production from mermaid fan seaweed, Padina sp (complete sci name with genus and species). The study specifically aims to answer the following questions:1. What is the actual yield of bioethanol produced from Mermaid Fan seaweed (sci name) using the following chemical pretreatment procedures?a. Acid hydrolysisb. Enzymatic hydrolysis2. What are the physical characteristics of bioethanol produced from different pretreatment procedure using the following parameters?3. Does the bioethanol produced is in compliance with standard set by ASTM?4. Is there significant difference on the physical characteristics of bioethanol produced from different pretreatment?HypothesisFor the purpose of the study, the following hypothesis will be advanced:1. There is no significant difference on the physical characteristics of bioethanol produced from different pretreatment.Significance of the studyThe following will benefit the Filipino researchers, students and those who are interested in seaweeds will have more information about the bioethanol production of Mermaid Fan seaweed, Padina sp. in Miagao, Iloilo, Philippines. The fisherfolk will be equipped with idea about possible source of livelihood by engaging in seaweed culture. The policy makers (like barangay, municipal and provincial local government units) of the coastal communities will also benefit from this study in prioritizing the livelihood opportunities in their respective areas. The Local Government Unit will consider the culture of mermaid fan seaweeds thus increasing livelihood in the locality if big companies will establish investments.The students and teachers who are interested to conduct similar topic would use the result of this study as a future reference. This study could serve as their baseline information on bioethanol production of Mermaid Fan seaweed, Padina sp. Companies that are interested in bioethanol production will find this study as an important basis. And also the samples used will be considered as a possible raw material for their production of bioethanol and culture of the samples will be made possible.Definition of TermsFor a clearer understanding of this study, all important words used were given their conceptual and functional meanings.Bioethanol - a renewable energy source made by fermenting the sugar and starch components of plants by-products mainly sugarcane and crops like grain, using yeast (Ecosmartfire, 2014).In this study, "bioethanol" refers to the alcohol product produced through fermentation of Laminaria Pallida subjected to different chemical pretreatment. Laminaria Pallida refers to the species of brown algae with bunch of funnel-shaped structures. Each funnel about 3-5cm in diameter, with concentric circles of tiny hairs and a rolled edge. The funnel is often torn at the edges. The bunch is usually attached to a hard surface and spreads out like a beautiful bouquet when submerged. The white tinge is from the calcium carbonate incorporated in the blade(Seaweed Industry, 2014).In this study, "Laminaria Pallida" refers to the species of brown algae tested for bioethanol production. Delimitation of the StudyThis study aims to determine the potential of Mermaid fan seaweed in producing bioethanol. Fresh samples will be collected along the shores of Miag-ao, Iloilo.The samples will be subjected to physical pretreatment procedure by heating. The solution will be divided into two and will be separately subjected into chemical pretreatement: acid hydrolysis and enzymatic hydrolysis. Both hydrosilate will be subjected into anaerobic fermentation to produce bioethanol. The bioethanol will be subjected to distillation process to determine the actual yield produced and will be compared which chemical pretreatment yields more bioethanol. The products will also be tested for physical characteristics using the following parameters:j. pHk. methanol contentl. specific gravitym. densityn. water contento. copper contentp. flash point for flammabilityq. Electrical conductivityr. Visual appearanceThe physical and chemical pretreatment and fermentation as well as testing for physical characteristics will be done on UPV-CFOS, Miag-ao, Iloilo.

Chapter 2Review of the Related LiteratureChapter Two, consists of the Review of Related Literature. the concepts and studies related to the current study that will give better understanding of the nature of the research, It includes a) Seewed; b) Culture and Harvesting of Seeweed; c) Carbohydrates and its Composition and other important substances in Seaweeds; d) Problems with fuels; e) Previous research on macroalgae biofuels production; f) Potential of macroalgae for biofuels; g)Potential for Bio-ethanol Production in the Philippines.

SeaweedAny of a vast group of simple multicellular plant forms belonging to the algae group, and found growing in the sea, brackish estuaries, and salt marshes, from near the high-tide mark to depths of 100200 m/300600 ft. Many seaweeds have holdfasts (attaching them to rocks or other surfaces), stalks, and fronds, sometimes with air bladders to keep them afloat, and are green, blue-green, red, or brown (Manivannan et al., 2008).Worldwide, there are approximately 2,000 species of brown macroalgae (Phaecophyceae), 5,500 reds (Rhodophyceae), and 1,200 greens (Chlorophyceae), most of which are marine species. Some have traditionally been gathered for food, such as purple laver (Porphyra umbilicalis, which is used by the Japanese to make sushi), green laver (Ulva lactuca), and carrageenan (Chondrus crispus). From the 1960s, seaweeds have been farmed, and the alginates (salts) that are extracted are used in convenience foods, ice cream, and animal feeds, as well as in toothpaste, soap, and the manufacture of iodine and glass (Manivannan et al., 2008).The vast majority of seaweed is collected for human consumption and for hydrocolloid production. Seaweed exploitation in the Philippines is currently restricted to manual harvesting of natural stocks. The majority of Asian seaweed resources are cultivated.The traditional markets for seaweed products sustain a much higher price for raw material than that likely for biofuel production. Thus, seaweed species have to be explored which do not compete for human consumption so that biofuel production becomes more competitive (Huang, 2009).

Seaweed as MacroalgaeMarine macroalgae or seaweeds are plants adapted to the marine environment, generally in coastal areas. Seaweed has long been priced as an excellent source of minerals, which are essential for good health (Manivannan et al., 2008).Seaweeds are also called macroalgae. This distinguishes them from microalgae (Cyanophyceae), which are microscopic in size, often unicellular, and are best known by the blue-green algae that sometimes bloom and contaminate rivers and streams (McHugh, 2003). Seaweeds are any of a large number of marine plants and protists in the category of benthic algae. They are macroscopic, multicellular, and macrothallic (McHugh, 2003)The FAO Guide to the Seaweed Industry provides an excellent overview of the seaweed resource and markets worldwide (McHugh, 2003). It enumerates a worldwide survey performed in 1994/5, listed 221 species of seaweed collected for human applications (145 for food and 101 for hydrocolloid extraction).Types and Classification of SeaweedAccording to McHugh (2003), seaweeds can be classified into three broad groups based on pigmentation: brown, red and green. Botanists refer to these broad groups as Phaeophyceae, Rhodophyceae and Chlorophyceae, respectively. Brown seaweeds are usually large, and range from the giant kelp that is often 20 m long, to thick, leather-like seaweeds from 2-4 m long, to smaller species 30-60 cm long. Red seaweeds are usually smaller, generally ranging from a few centimeters to about a metre in length; however, red seaweeds are not always red: they are sometimes purple, even brownish red, but they are still classified by botanists as Rhodophyceae because of other characteristics. Green seaweeds are also small, with a similar size range to the red seaweeds.There are a very large number of species around the world, belonging to several phylogenic groups. Broadly, three types of seaweeds are defined according to their pigments e.g. the brown seaweeds (e.g.: Laminaria,Fucus, Sargassum), the red seaweeds (e.g. Gelidium, Palmaria, Porphyra) and the green seaweeds (e.g. Ulva,Codium). With the exception of green seaweed, terrestrial and marine plants have little in common. This partly explains the unique chemical composition observed in seaweeds. The marine environment also induces the production of unique chemicals to resist the environmental stresses plants are subjected to (Pons et., 1981)Green SeaweedGreen seaweeds, in particular Ulva spp are being researched as potential renewable fuel feedstock (McHugh, 2003; Bruton et al., 2009). According to the study of Bruton et al. (2009) the moisture content of green seaweed is even higher than that of brown seaweeds, and it has similarly high ash content. The species is attracting interest as an energy resource due to the comparatively high level of accessible sugars, specifically starch. It also has high cellulose content. In these respects it resembles some of the properties of terrestrial plants, suggesting it is compatible with a cellulosic and starch fermentation process. There is some potential for manipulation of the components in favor of energy production. The high sulphate content will cause high yields of H2S during fermentation, which is a fermentation inhibitor.Brown SeaweedThe various brown seaweeds have since the early 20th century, been used for industrial applications, and now attention is turned in many regions with brown seaweed resources to the production of energy (Bruton et al., 2009).There are several species of brown seaweed and one of them is the Mermaid fan seaweed, Padina species. This is a bunch of funnel-shaped structures. Each funnel is about 3-5cm in diameter, with concentric circles of tiny hairs and a rolled edge. The funnel is often torn at the edges.The bunch of Mermaid fan seaweed (Appendix H, Plate 1) is usually attached to a hard surface and spreads out like a beautiful bouquet when submerged. The color is visibly golden brown, sometimes with a bluish or whitish tinge. The white tinge is from the calcium carbonate incorporated in the blade.Padinais the only brown seaweed known to incorporate calcium.The same study stated that the composition of brown seaweeds varies according to species, location, salinity and season so it is usual to give either an average or range of values. Brown seaweeds have high moisture content, typically around 85%, and high ash content, typically around 25% (Bruton et al., 2009).Red SeaweedThe main uses of red seaweeds are as food and as sources of two hydrocolloids: agar and carrageenan. Useful red seaweeds are found in cold waters such as Nova Scotia (Canada) and southern Chile; in more temperate waters, such as the coasts of Morocco and Portugal; and in tropical waters, such as Indonesia and the Philippines. Red seaweed is not of prime interest to biofuel production at the present times.

Culture and Harvesting of SeaweedThe most common system in the Philippines to obtain seaweed biomass is by harvesting natural stocks in coastal areas with rocky shores and a tidal system. The natural population of seaweed is a significant resource. Depending on water temperature, some groups will dominate, like brown seaweeds in cold waters and reds in warmer waters.In 1995 about 3.6 million tons wet weight were collected globally from natural stocks (Lithothamnion not included). This was about 48% of the total global seaweed biomass harvested with the balance produced by aquaculture. More recent numbers (FAO, 2006) give about 1 million tons harvested annually from natural stocks, making up only 6% of the global resource, with over 15 million tons produced by aquaculture.CultureNaturally growing seaweeds are often referred to as wild seaweeds, in contrast to seaweeds that are cultivated or farmed.There is possibility for seaweed biomass generation through cultivation (Richmond, 2009). Only a few genera have been commonly cultivated for many years. The main genera cultivated include: Laminaria, Porphyra, Undaria, Gracilaria, Euchema, Ulva and Chondrus. The seaweed harvested from natural stocks has decreased significantly, while cultivated seaweed has sharply increased. The overall amount of seaweed harvested has almost doubled in the last 10 years to 15 million wet tons (FAO, 2006). Over half of cultured seaweeds, or 7.4 million wet tons, are brown Laminaria sp, mainly L. japonica. The global industry turnover also increased from US $6.2 billion in 1994 to US $7.2 billion in 2006. Values have not been inflation-adjusted, but the trend is of increased volume and static turnover. It reflects the significant cost-reduction brought about by cultivation practices. There is a much larger amount of seaweed available and mechanized operations have improved productivity allowing lower market prices (Edwards et a;., 2008)There are potential economic advantages and opportunities for developing aquaculture facilities in conjunction with off shore wind farms (Yokoyama, 2007). Anchorage of long-lines, ropes and rafts has been a major problem for pilot seaweed cultivation projects with numerous reports of structures being swept away by tides and currents. Sharing infrastructure with a wind farm or other offshore enterprise would seem to make economic sense from planning, design and operation points of view. The right conditions for cultivation of seaweed would need to be present. Previous studies outline this concept in depth.HarvestingProducing significant amounts of biofuel from natural stocks involves the harvesting and processing of large volumes (millions of tons) of seaweeds. To harvest higher amounts may not be sustainable. If for any reason hydrocolloid production decreases or ceases, part of the unused seaweed resource could be redirected towards biofuel production and extraction of other marine natural products from the feed stock or highly abundant seaweed species.Manual harvesting has been used since the preindustrial age. This is still used for harvesting natural stocks of Ascophyllum nodosum and Fucus species, as they are located in the intertidal zone on the shore. At low tide, terrestrial vehicles can access the shore and seaweeds are accessible for manual harvesting. This method of harvesting was also used for Laminaria, but the emergence of large scale application for hydrocolloids, stimulated the development of mechanical systems. Trawlers are used in Norway to cut the large size adult canopy, leaving the small size seaweed attached to the rocks. Re-growth is stimulated by the increased light reaching the small size seaweeds. The trawler system is operated from a boat. Using either a dredge or the Scoubidou allows one man in a boat to collect several tons of seaweed per day. This is a significant improvement over manual harvesting. These two examples are the most widely known mechanized harvesting techniques currently used for industrial applications (FAO, 2006).Another primarily natural source is the drift seaweeds. Some reports suggest as much as 20% of L. hyperborean stocks are washed up on shore every year in Ireland. The location and seasonal availability of these resources are unpredictable. It has traditionally been collected by coastal communities on a small scale to use as fertilizer or soil-conditioner. When collected on the foreshore drift seaweeds are considered a waste product. Developing an application for a waste has very positive connotations.The green seaweed found in the shore of Boracay although this is not a large quantity can contribute to local energy production. However, it can be difficult to build a local enterprise based on wastes which are desirable to eradicate pollution but culturing the species for possible carbohydrates production is one good option. The drift seaweed biomass provides an opportunity, as and when it is available, to be integrated in a broader process using other type of biomass raw materials (Palligarnai et al., 2008)Problems with fuelOver the last few decades, the negative impacts of fossil fuel on the environment and consequent global warming, progressive demand for energy, inevitable depletion of the worlds energy supply, and the unstable oil market (such as the energy crisis of the 1970s) have renewed the interest of society in searching for alternative fuels (1; 2). The alternative fuels are expected to satisfy several requirements including substantial reduction of greenhouse gas emission, worldwide availability of raw materials, and capability of being produced from renewable feedstocks (3). Production of fuel ethanol from biomass seems to be an interesting alternative to traditional fossil fuel, which can be utilized as a sole fuel in cars with dedicated engines or in fuel blends (Pelagiaresearchlibrary.com, 2014)Although CO2 is the most important greenhouse gas (GHG), several studies show that it is important to consider other GHGs as well. The continued use of fossil fuels to meet the majority of the worlds energy demand is threatened by increasing concentrations of CO2 in the atmosphere and concerns over global warming. The combustion of fossil fuels is responsible for 73% of the CO2 production.The heightened awareness of the global warming issue has increased interest in the development of methods to mitigate GHG emissions. Much of the current effort to control such emissions focuses on advancing technologies that: (i) reduce energy consumption, (ii) increase the efciency of energy conversion or utilization, (iii) switch to lower carbon content fuels, (iv) enhance natural sinks for CO2, and (v) capture and store CO2. Reducing use of fossil fuels would considerably reduce the amount of CO2 produced, as well as reduce the levels of pollutants. As concern about global warming and dependence on fossil fuels grows, the search for renewable energy sources that reduce CO2 emissions becomes a matter of widespread attention. To reduce the net contribution of GHGs to the atmosphere, bioethanol has been recognized as a potential alternative to petroleum-derived transportation fuels (Biotek.lu.se, 2014)Previous research on macroalgae biofuels productionThe interest in macroalgae for biofuel lies in their high carbohydrate (e.g. polysaccharide) content. Macroalgae were first proposed as a possible source of energy by Howard Wilcox in the late 1960s, who presciently considered their production not only as a solution to the energy crisis but also for global warming (Wilcox, 1975). Much research was carried out by the US during the 1970s and early 1980s to develop open ocean macroalgae farms to produce a substitute for natural gas, an energy source then considered in the USS to be approaching depletion. The Marine Biomass Program, supported between 1979 and 1985 with over $50 million by the U.S. Dept. of Energy (about twice the budget of the 1980- 1996 U.S. DOEmicroalgae Aquatic Species Program) had as its ultimate objective to replace the entire U.S.Potential of macroalgae for biofuelsMacroalgae are again being considered as a biofuel feedstock for similar reasons as thirty years ago: because they are thought to have very high biomass yields (though still this remains to be established) and, perhaps most importantly, because they dont compete with agricultural crops for land, water resources, and, potentially, fertilizers. Herein we neglect the land-based cultivation of seaweeds, which suffers from several inherent problems, mainly the very high water exchange and/or mixing required for high productivity cultivation, in addition to CO2 supplementation. However, land-based cultivation is already a commercial process for some seaweed species, and may be of interest for biofuels production in some locations, though undoubtedly their greatest potential is in open ocean cultivation. Compared to microalgae, macroalgae have a major advantage: their macroscopic nature allows for ready and low costharvesting. Against these advantages must be placed the difficulties of working in the sea, even near-shore, which imposes significant costs and risks, as already experienced by the earlier U.S. Marine Biomass Program. The three most commonly mentioned fuels that could be derived from macroalgae are methane, ethanol, and butanol.The interest in macroalgae for biofuels was recently re-initiated, mainly in Japan, Korea and Europe, but at a relatively low level of funding initially. In Japan, Tokyo Gas studied the production of biogas from seaweed that was collected from biomass naturally deposited on beaches after storms and high tides (Huesemann et al., 2010). However, several factors - the small amounts and sporadic nature of such harvests, the sand and dirt collected along with such biomass, and the transportation costs make such schemes impractical. Thus most attention has focused on off-shore cultivation of local seaweeds, with a popular candidate species being Laminaria japonica, the most common seaweed currently used for food and chemical production, and already considered thirty years ago for such applications (Tseng, 1981; Chynoweth, 2002). In Ireland, Laminairia ssp and Ulva ssp, are being considered because of their relatively high carbohydrate content (Bruton et al. 2009). Ulva can be readily digested to methane gas and seem to lack epiphytes, e.g. microalgae growing on the surface of the seaweed leaves, which can interfere with their production (Chynoweth 2002). Other species also evaluated for fuel production in the 1980s, include Gracilaria tikvahiae (a red algae species), notable for its high yields in on-shore cultivation tests (Hanisak 1987).Another interesting macroalga is Sargassum, notable because it is one of the few species that is found free floating in the open ocean (Chynoweth 2002). The possibility of growing Sargassum in the open ocean is intriguing, but there is presently no reasonably plausible approach to its mass cultivation. Their cultivation was already discussed over forty years ago, and the concept proposed (but not published) was to release propagules of Sargassum into a marine current, near an upwelling zone, and then harvest the plants downstream, after a few weeks of growing in the ocean current. Of course, this intriguing concept is at this point entirely hypothetical.Because of limited suitable near-shore areas, many already being used extensively for commercial macroalgae cultivation, the key concept for cultivation of macroalgae for biofuels remains, as before, some type of open ocean cultivation technology. However, the design of such systems also remains, as before, mainly hypothetical, with no design apparent at present that could be scaled-up or deployed beyond a near-shore environment. An earlier analysis for the U.S. Biomass Program of such systems pointed out the many inherent essentially insurmountable engineering difficulties of such concepts (Ashare et al., 1978). In what follows we thus must per-force assume that an open ocean cultivation technology will eventually prove to be technically and economically feasible, and that the macroalgae productivities will be high enough to justify such efforts. The remaining issues are then harvesting and conversion to fuels.Algae are considered as the only alternative to current bioethanol crops such as corn and soybean as they do not require arable land (Chisti, 2007; Hu et al., 2008; Singh et al., 2010c). The arable land could be used efficiently to grow food crops rather than biomass and oil seed crops for the production of biofuels. Water filled areas that are not suitable for growing food crops and industrial waste water can be used for the cultivation of algal biomass without any compromise with land and water resource for the production of bioethanol that will also not adversely affect the food cost (Singh et al., 2011). In addition, algae can be converted directly into energy, such as biodiesel, bioethanol and biomethanol and therefore can be a source of renewable energy.Bioethanol from algae holds significant potential due to their low percentage of lignin and hemicellulose as com- pared to other lignocellulosic plants (Harun et al., 2010). While having low lignin content, macroalgae contain significant amount of sugars (at least 50%) that could be used in fermentation for bioethanol production (Wi et al., 2009). However, in certain marine algae such as red algae the carbohydrate content is influenced by the presence of agar, a polymer of galactose and galactopyranose. Current research seeks to develop methods of saccharification to unlock galactose from the agar and further release glucose from cellulose leading to higher ethanol yields during fermentation (Wi et al., 2009; Yoon et al., 2010).Seaweeds are classified into three groups: green, brown, and red, and they contain various types of glucans which are polysaccharides composed of glucose, though the concentration of these glucans is known to be relatively low. Seaweed was proposed as one of the most promising biomass materials that can be easily converted to ethanol, since seaweeds are known to contain a low concentration of lignin or no lignin at all. Three types of seaweed in- cluding sea lettuce, chigaiso, and agar weed were used as representatives of green, brown, and red seaweeds, re- spectively, and methods for obtaining high concentrations of ethanol (bioethanol) from these seaweeds were investi- gated (Yanagisawa et al., 2011). An ethanol yield of more than 3% was obtained from these seaweeds.Bioethanol from algae holds significant potential due to their low percentage of lignin and hemicellulose as com- pared to other lignocellulosic plants (Harun et al., 2010). While having low lignin content, macroalgae contain significant amount of sugars (at least 50%) that could be used in fermentation for bioethanol production (Wi et al., 2009). However, in certain marine algae such as red algae the carbohydrate content is influenced by the presence of agar, a polymer of galactose and galactopyranose. Current research seeks to develop methods of saccharification to unlock galactose from the agar and further release glucose from cellulose leading to higher ethanol yields during fermentation (Wi et al., 2009; Yoon et al., 2010).Brown algae as a seaweed is evolutionarily diverse and abundant in the worlds oceans and coastal waters. The seaweed industry has an estimated total annual value of 5.5 to 6 billion US$, with 7.5 to 8 million tons of naturally grown and cultivated seaweed harvested worldwide. Seaweed is mainly used in food products for human consumption, which generates approximately 5 billion US$ per year, with the remainder used for production of extracted hydrocolloids, fertilizers, and animal feed additives (Adams et al., 2009;McHugh et al., 2003).Brown seaweed has a high content of easily degradable carbohydrates, making it a potential substrate for the production of liquid fuels. The carbohydrates of brown seaweed are mainly composed of alginate, laminaran, mannitol, fucoidan and cellulose in small amounts (Horn et al., 2000)Potential for Bio-ethanol Production in the PhilippinesBiogas production is a long-established technology and previous trials have indicated that anaerobic digestion (AD) of seaweed is technically viable (Bruton et al., 2009). It should initially be possible to incorporate seaweed resources into existing AD plant to allow for smaller quantities and seasonal availability. This is the closest process to commercialization for conversion of macroalgae to energy, though there is still a need to reduce the cost of the raw material by at least 75% over current levels.Alcoholic fermentation is more difficult. The lack of easily fermented sugar polymers such as starch, glucose or sucrose means there is little point in pursuing standard sugar fermentation processes. The polysaccharides that are present will require a new commercial process to break them down into their constituent monomers prior to fermentation, or else a direct fermentation process will have to be developed. Promising work has been initiated in Ireland and elsewhere to isolate marine lyases which would do this efficiently. Theoretically up to 60% of the dry biomass in Laminaria sp. could be fermented with the right process. Ulva spp are also of interest due to their starch content (Pons et al., 1981).The competitiveness of macroalgal biomass for alcohol fermentation must be viewed in the context of other available cellulosic biomass such as wood, straw and dry organic waste which are also potential ethanol feedstock. There is much speculation that integrated bio-refinery solutions would allow sufficient scale to enable economic production of fuel from macroalgae. The only industrial product of significance from macroalgae is hydrocolloids. Extraction of energy from waste streams is a valid commercial bio-refinery concept. If the cost of seaweed permits, a dual production of ethanol and biogas is also possible. There are many other opportunities for extraction of high-value niche products from seaweeds. Each would have to be assessed on commercial terms and demonstrate the feasibility for co-production of energy alongside the higher-value product, with particular attention to whether the scale of operation is appropriate (Pons et al., 1981).

Chapter 3Research Design and MethodologyChapter Three consists of three parts; namely, (1) Purpose of the Study and Research Design; (2) Methodology; and (3) Data Analysis ProcedurePart One, Purpose of the Study and Research Design, presents the research design used, restates the purpose of the study, and describes the plan employed in the conduct of the study.Part Two, Method, presents the subject of the study and sampling techniques. It also describes the data-gathering instruments; the material used, and delineates the procedures followed in the study.Part three, Statistical Analysis Procedure, reports on the procedure for the statistical analysis used in the study.

Research DesignThis quantitative study will investigate the bioethanol produced from Mermaid fan seaweed using two chemical pretreatment procedure: acid hydrolysis and enzymatic hydrolysis.The independent variable employed in the study is the Mermaid fan seaweed subjected to acid hydrolysis and enzymatic hydrolysis. The dependent variables will be actual yield and physical characteristics of bioethanol.Each testing will be done in three trials with triplicates.MethodologyPreliminary ActivitiesGathering of Raw Materials. Mermaid fan seaweed samples will gathered during the month of March, 2015 along the shores of Miagao, Iloilo. An approximately 10 kg. of wet samples will be gathered and will be identified preliminarily using Simpsons (2012). The algae will be further confirmed by an algae expert from UPV-CFOS. The material will be stored in a room temperature prior to experiment.Physical pretreatment. Following the procedure by Sahu et al. (2011), the algae will be soaked in water overnight followed by placing the sample in a pressure cooker raising to high pressure for 30 seconds to 1 minute and reducing it to normal. The sample will be allowed to keep boiling for 2 hours. This is to physically destroy the components of the cell wall of material. The solution will be divided into two flasks and will be subjected separately to chemical pretreatments.Experiment properChemical Pretreatmentscid hydrolysis. 25 mL of 5% H2SO4 (sulfuric acid) will be poured into a bottle glass containing 300 mL seaweed solution, which will boiled using stirrer hotplate for 30 to 120 minutes. The hydrolysis time will be measured after the acid boiled. The hydrolysis temperature will be maintained at 100 degrees Celsius. The solution will be then adjusted to pH 5 by adding dropwise of 0.1 M NaOH (Sudarmadji et al., 1984). Enzymatic hydrolysis. A separate 300 mL of seaweed solution which had undergone physical pretreatment will be obtained and will be subjected to enzymatic hydrolysis. The solution will be added with cellulase of about 5 mL per 100 mL of solution. The pH of the solution will be adjusted by adding 50mM phosphate buffer to pH 5. The set-up will be incubated at 30C for 4 days with occasional shaking through an incubator shaker about 100 RPM.Fermentation of HydrolisateThe hydrolisate from acid hydrolysis and enzymatic hydrolysis will be separately sieved and placed separately in an improvised container designed for anaerobic fermentation. Overnight precultured yeast of about 10% (v/v) will be added as a starter. The fermentation will be conducted at room temperature (28-30C) for 7-15 days. Test for reducing sugars. Monitoring of reduced sugar on the fermentation set-up will be done through DNS method. Liquefied slurry will be transferred into falcon tube and centrifuged at 10,000 rpm for 10 min. Supernatant will be transferred to fresh tube and reducing sugars will be estimated by DNSA reagent (Dinitrosalicylic acid) as described by Miller (1959). DNSA reagent will be prepared by dissolving 1g of dinitrosalicylic acid, 200 mg crystalline phenol and 50 mg of sodium sulfite in 100 ml of 1% NaOH. After addition of DNSA reagent to the standard solution and test solution, whole reaction mixture will be incubated in boiling water bath for 15 min and will be followed by addition of 40% of Rochelle salt in solution to stabilize color of reaction mixture. Subsequently observance will be measured at 510 nm. Data gathering procedureBioethanol actual yield through DistillationAbout 100 ml of ethanol will be placed in a volumetric flask. Add sodium chloride powder so that the solution becomes almost saturated with NaCl. About 50 ml of ether will be added and will be shaken for 2-3 min. It will be settled and the lower layer will be transferred to the distillation flask. About 20-30 ml of saturated sodium chloride solution will be added to the petroleum ether layer and gently shake. Mixture will be allowed again to settle and transfer the aqueous layer to the distillation flask. It will be mixed gently and make the solution just alkaline with NaOH solution. The distillation assembly will be connected via condenser to the volumetric flask. It will be distilled gently. The distillate will be collected in the volumetric flask almost to the mark. The contents will be placed to room temperature. The specific gravity of the alcohol will be confirmed. The percent by volume will be determined (volume/gram) to identify the actual yield through distillation.Physical Characteristics of BioethanolpH. The pHe is a measure of the acid strength of a fuel ethanol and is a predictor of the fuel ethanols corrosion potential. Ethanol obtained from fermentation procedure utilizing acid hydrolysis and from enzymatic hydrolysis will be tested for pH using pH meter. The test will be done in three trials with triplicates. The value will be recorded and will be compared to the standard pH for bioethanol.Standard requirement: pHe 6.5 9.0.Methanol content. Ethanol must contain a minimum concentration of methanol to protect the properties of ethanol/gasoline blends. The method will make use of spectrophotometric method. About 50 ml of sample in a simple still and distil will be obtained, collecting about 40ml of distillate. Then, 1 ml of distillate will be diluted to 5ml of distilled water and will be shaken well. 1 ml of the solution will be obtaained, 1 ml of distilled water (for blank) and 1 ml of each of the methanol standards in to 50 ml stoppered test tubes and will be kept in an ice-cold water bath. To each test tube, 2 ml of KmnO4 reagent will be added and be kept aside for 30 min. The solution will be decolorized by adding a little sodium bisulphite and adding 1 ml of chromotropic acid solution. Mixture will be mixed well and be added with 15ml of sulphuric acid slowly with swirling and place in hot water bath maintaining 800C for 20 min. Color development from violet to red will be noted. The mixture will be cooled and the absorbance be measured at 575 nm. The values will be recorded and will be compared to the standard.Standard requirement: a maximum methanol concentration of 0.5% by volume.Specific gravity. Specific Gravity is required for the conversion of measured volumes to volumes at 15C (the standard temperature). The specific gravity of an ethanol product may be an indicator of contamination.A clean and dry pyknometer will be weighed along with the stopper at room temperature (W). It will be filled with the ethanol to the brim and insert the stopper gently. The sample will be weighed again (W1). Next, the ethanol will be removed and will be filled with water in the same manner as described above. The weight will recorded (W2). The specific gravity will be solved as follows:Specific gravity = W1 W2 W2 WStandard for the specific gravity of ethanol is 0.787.Density. Density is a fundamental physical property. It is a required measurement to convert measurements from mass % to volume %. Standard for the density of ethanol is 0.789 g/cc.Copper content. To the ethanol, 15 ml concentrated nitric acid and 5 ml concentrated sulphuric acid will be added. About 10 ml of water is first added to the sample before adding nitric acid. The mixture will be mixed well and the set-up will be connected to the extractor and water condenser. The mixture will allowed to stand at room temperature overnight in order to prevent foaming. The mixture will then be heated, first by means of a soft flame. The flame will be removed from digestion apparatus as necessary to minimize escape of nitrogen oxides from the top of condenser. Maintaining full heat, the tap will be turned through 900 so that liquid distils into the receiver. Temperature of the vapour in the digestion flask at this stage will not exceed 120C. Then the tap will be turned through further to 900 (what is this?) so that the distillate (mainly water) drain off through another receiver. Turn the tap in such a way that liquid distils into the receiver B. Heating will be intensified in such a way that nitric acid distills into the receiver. If the solution begins to darken add a few millilitres of nitric acid from the receiver with the help of the double bore stopcock. The digest will be transferred quantitatively with the help of redistilled water into a beaker of 100 ml. Adjust the pH of the solution with ammonium hydroxide (25% m/v) to approximately 3. The solution will be transferred and obtained into a separation funnel of 50 ml with the help of redistilled water. The solution should not exceed 30 ml. Pipette I ml of ammonium pyrrolidine dithiocarbamate and 5 ml of methylisobutylketone. Shake for 1 minute. The solution will be subjected to spectrophotometer.Standard copper content is 0.10 mg/kg.Water content. About 20 mL methanol and 10 mL formamide are placed in the titration vessel, warmed to 50C and titrated volumetrically to dryness. The comminuted sample is then added and its water content titrated volumetrically at the same temperature. The water content will be measured using Coulometric Karl Fischer Titration technique.Standard water content is 1.0 0.5% by volumeFlash Point for flammability. This test will be carried out using flash point apparatus. The cup in the apparatus will be dried. About 50ml of each sample (ethanol produced) will be transferred into the flash point cup. The cup will be fixed into the position in the apparatus assembled with thermometer, and the apparatus will be switched on; the heat is controlled by a steadily stirring the ethanol to maintain a uniform temperature while passing a small flame across the material every five seconds. The temperature at which the vapour first flashes with a blue flame will be recorded as the flash point of the sample, after each test the cup will be washed and dried before subsequent test.Standard for flash point for bioethanol is 15-25 Celcius.Electrical Conductivity Test. This test will be done to identify ion content on the ethanol. Digital electrical conductivity meter will be used. About 20 mL of ethanol produced from fermentation using different chemical pretreatment procedure will be used for the test. The electrode will immersed to the censor market point. The meter will be turned on to the required calibrated units and the reading will be taken for all the samples.Standard electrical conductivity is 300 microsiemens/meter (us/m).Visual Inspection. Test for visual appearance of bioethanol will also be used through observation. About 20 mL of ethanol will be placed in a test tube. Ocular observation will be used to check on the clarity of the ethanol. Below is the scoring to be used for visual inspection:ScoreDescription1blurry, not clear2 moderately clear with occasional floating particles3bright and clearData AnalysisDescriptive Data Analysis. Mean and standard deviation will be used to determine the actual yield and physical characteristics of bioethanol produced.Inferential data analysis. t-Test will be used to compare the means of actual yield and physical characteristics of bioethanol produced using acid hydrolysis pretreatment and enzymatic hydrolysis pretreatment.

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