Biofuel Production from Marine Resources

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stringent feedstock specifications, lower chemical additive demand, reduced waste .... Biofuel is any fuel that is derived from biomass, also consider as a renewable ...... Biobutanol can be utilized in internal combustion engines as both a gasoline .... to convert the disaccharide into glucose and fructose (both C6H12O6).

     

           

                                                                      

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Biofuel Production from Marine Resources By Hassan A.H. Ibrahim &

Khouloud M. Brakat

Marine Microbiology Lab., Division of Marine Environment, National Institute of Oceanography & Fisheries, Alexandria, Egypt. [email protected] , +201009160921 [email protected], +201023347533

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CONTENTS PREFACE͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘ CHAPTER 1: Biofuel Generations͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘.......................................................................... 1.1.Energy crisis͙͙͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘ 1.2. Alternatives of fossil fuel͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘͘͘͘͘͘ 1.3. What is the biofuel? ͙͙͙͙͙͙͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘ 1.4.Generations of biofuel production ͙͙͙͙͙͙͙͙͘͘͘͘͘͘͘͘͘.͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘ i.First generation͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘ ii. Second generation͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘ iii. Third generation͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘ iv. Fourth generation͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘ 1.5.Marine organic feedstocks͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘ 1.6.Bacteria and fungi as a biofuel feedstock͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘͘͘ 1.7. Cyanobacteria as a biofuel feedstock͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘ 1.8. Microalgae as a biofuel feedstock ͙͙͙͙͙͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘............ 1.9. Macroalgae (Seaweeds)͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘ 1.10. Sea grasses as a biofuel feedstock͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙… 1.11. Crustaceans as a biofuel feedstock͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘ 1.12. Types of biofuels͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘.... CHAPTER 2: Bioalcohols͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘ 2.1. Biomethanol͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘ Ϯ͘Ϯ͘Bioethanol͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘͘ 2.3. Biobutanol͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘ Ϯ͘ϰ͘Production process of bioalcohols͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘ 2.4.1. Pretreatment processes of biomass͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘ (i)- Physical pretreatment͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘ (ii)- Chemical pretreatment͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘ a-Alkaline hydrolysis͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘͘͘͘͘͘͘͘͘͘͘ b- Acid hydrolysis ͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘͘͘͘͘͘͘͘͘͘ (iii)- Biological pretreatment͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘ 2.4.2. Saccharification process͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘ 2.4.3. Fermentation process͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘ 2.4.4. Optimization of bioalcohol production͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘ (i) Experimental designs͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘͘͘͘ (ii) Immobilization technique͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘ CHAPTER 3: Biodiesel………………………………………………………………………… 3.1. Biodiesel production……………………………………………………………………… 3.2. Production process of biodiesel …………………………………………………………. 3.3. Microalgae biomass for biodiesel production……..……………………………………..… (i)- Open ponds (raceway)……………………………….................................................... (ii)- Photo-bioreactors͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘ (iii) Algal biomass harvesting͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘ a. Filtration ………………………………………………………………………………… ď͘ Centrifugation͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘ c. Flocculation/Flotation ………………………………………………………………… Ě͘ Electroflocculation͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘ 3.4. Biodiesel extraction from microalgae͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘͘͘

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(i) Transesterification ……………………………….............................................................. (ii) Pyrolysis ……………………………………………………………………………..….… (iii) Long-chain hydrocarbons extraction……………………………………………………… (iv) Fischer-Tropsch ͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘ 3.5. Companies specialized in bio-diesel production from microalgae͙͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘ CHAPTER 4: Biogas͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘ 4.1. Origin & nomenclature of biogas……………………………..……………………………. 4.2. Biogas history……………………..………………………………………………………... 4.3. Biogas characteristic ……………………………………………………………………….. 4.3.1. Biogas component………………………………………………............................... 4.3.2. Biogas parameters………………………………………………………………………. i. Total solid (TS)…...……………………………………………………………………….. ii. Volatile solid (VS) ……………………………………………………………………...… iii. Hydraulic retention time (HRT)………………………………………………………..… iv. Gas production rate (GPS)….…………………………………………………………..… v. Organic space loading rate (OLR)…………………………….... ……………………..… vi. Gas production rate of materials (GPRM)………………………………………………... vii. Biochemical oxygen demand (BOD)……………………….…………………………...… viii. Chemical oxygen demand (COD)………………………………………………………... 4.3.3. The relationship among the biogas parameter͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘ 4.3. Biochemistry and microbiology of biogas fermentation͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘͘ 4.4.1. Three steps for biogas fermentation͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘͘ (i) The first step…………………………………………………………………….......... (ii) The second step ………………………………................................................................ (iii) The third step…………………………………………………………………….……… 4.4.2. Microbes in three-step of biogas fermentation…………………………………….......... 4.4.2.1. Biogas microbes in nature……………………………………………………………. 4.4.3. Groups and actions of non-methane-producing microbes………………………………. 4.4.3.1. Variety of Non-methane-producing microbes……………………………………….. i. Bacteria…………………………………………………………………….…….... ii. Fungi……………………………………………………………………………… iii. Protozoa…………………………………………………………………………... 4.4.3.2. The amount of non-methane-producing bacteria…………………….……………… 4.4.4. Estimation and calculation of the amount of methane…………………….……………… 4.4.4.1. Methane produced from organic compounds………………………………………. 4.4.5. Anaerobic digestive process of complex organic compounds…………………………..… 4.4.5.1. Degradation of carbohydrates…………………………………………………….….. 4.4.5.2. Anaerobic degradation of glucose……………………………………………………. 4.4.5.3. Cellulose after removal of lignin……………………………………………..……… 4.4.5.4. The metabolism of semi-cellulose, pectin-gel, starch, and cellulose under anaerobic conditions…………………………………………………………………………. 4.4.5.5. Metabolism of lipids…………………………………………………………………… 4.4.5.6. Metabolism of protein…………………………………………………………………. 4.4.6. Fermentative bacteria……………………………………………………………………. 4.4.6.1. Methane-producing bacteria…………………………………………………………. 4.4.6.2. Morphology and classification of methane-producing bacteria…………………….. 4.4.6.3. Classification of Methanobacterium…………………………………………………. ϯ

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4.4.7. Metabolic substrates of methane-producing bacteria…………………………………. 4.4.8. Relationships between different biogas microbes……………………………………….. 4.5. Biogas use…………………………………………………………………………………. 4.5.1. Biogas conversion options………………………………………………………….… 4.5.2. Treatment of biogas……………………………………………………………………. 4.5.3. Storage of biogas……………………………………………………………………….. 4.5.4. Compression of biogas………………………………………………………………….. 4.5.5. Biogas utilization…………………………………………………………………..……. 4.5.6. Direct combustion……………………………………………..………………………… 4.5.7. Internal combustion systems……………………………………..…………………… 4.5.8. Vehicular use…………………………………………………………...………………. CHAPTER 5: Biohydrogen……………………………………………………………………... 5.1. Role of hydrogenase in the hydrogen production ………………………………………. 5.2. Expression of Fe-hydrogenase in green algae................................................................... Physiological rules of H2 production in green algae……………………………………………. Two-stage photosynthesis and H2 production in Ch. Reinhardtii…………………………..….. The amount of H2 can be produced by a mass culture of green algae͙͙͙͙͙͙͙͙͙͙͙͙͙͙͘͘͘ CONCLUSION AND RECOMMENDATIONS …………………………………………… GLOSSARY & WEB SITES …..…………............................................................................ REFERENCES ……………………………………………………………………………..

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PREFACE

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orld fossil oil production is struggling to meet demand so it is necessary to develop energy efficient production pathways for transportation biofuels. This book provides insight into four promising pathways for the biological production of the main transportation biofuels: bioethanol, biodiesel, biogas and biohydrogen. These offer higher yields, less stringent feedstock specifications, lower chemical additive demand, reduced waste production and better energy balances than conventional methods. The book reflects not only extended research but also shows some practical experimental results. Liquid, gaseous or solid biofuels hold great promise to deliver an increasing share of the energy required to power a new global green economy. Many in government and the energy industry believe this modern bioenergy can play a significant role in reducing pollution and greenhouse gases, and promoting development through new business opportunities and jobs. Modern bioenergy can be a mechanism for economic development enabling local communities to secure the energy they need, with farmers earning additional income and achieving greater price stability for their production. Biofuels remain a complex and often contentious issue. Over the past few years the risks of competition with food production and potential negative impacts on the atmosphere, biodiversity, soil and water have been highlighted. The way biofuels are made and used is critical: they may either help mitigate or contribute to climate change, reduce or exacerbate impacts on ecosystems and resources. Issues related to biofuels are complex and interconnected: they require solid planning and balancing of objectives and trade-offs. Safeguards are needed and special emphasis should be given to options that help mitigate risks and create positive effects and co-benefits.

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   CHAPTER 1 Biofuel Generations

ϲ 

Energy is an important, if not the most critical factor in developmental activities of any nation. So, no one can deny the massive impact that fossil fuels have had on human development over the past two centuries. They have very high energy density at relatively low weight, making cars convenient and airplanes possible, and much of our way of life depends upon the plentiful supply of petrochemicals (Guzmán et al., 2009). Fossil fuels that can be accessed economically are running out due to stress upon usage of oil, coal, and natural gas for long time and increasing in the population earth. So, new fuel resources have to be created to face the limitations in these traditional fuel sources (5DPDQDWKDQSingh and Olsen, 2011). Marine floras, such as bacteria, actinomycetes, cyanobacteria, fungi, yeasts, microalgae, seaweeds, mangroves and other halophytes are extremely important oceanic resource, constituting over 90% of the oceanic biomass. By the biological processes, the marine biomass has been converted to usable energy forms (Zaldivar et alOlofsson et al Karuppaiya et al., 2009). The microbial lipids contain high fractions of poly-unsaturated fatty acids and have the potential to serve as a source of significant quantities of transportation fuels (Subramaniam et al., 2010). For example, the production of fuel from microalgae provides many advantages when compared to the fuel produced from other sources like agro-based raw materials. Also, algae do not affect fresh water resources because they can be produced using ocean and wastewater (Ferrell and Sarisky-Reed, 2010; Singh and Olsen, 2011). Actually, some algae (microalgae and macroalgae) were reported to have high contents of carbohydrate that can be used as substrates for bioethanol production. They also can provide several different types of renewable biofuels such as; bioalcohols, biodiesel, biohydrogen and biogas (Singh and Olsen, 2011). Bioalcohols production from natural resources will be passed throughout two major processes, one is a saccharification of polysaccharide to monosaccharides and ϳ 

the other is the fermentation process by yeast (Graves et al., 2006). However, the biofuel production has been passed through four major generations (Fortman et al., /DUVRQ Demirbas, 20111LJDPDQG6LQJK DVIROORZV 1. First generation; in which bio-fuels produced using food (corn, soy, palm, sugarcane) which have hydrocarbons in their sugars, starches and oils that are extracted by fermentation or by making transesters from the organic oils. 2. Second generation; in which biofuels produced using cellulosic materials such as non-edible parts of food stocks (corn stalks, husks, etc.) as well as from non-food plants. 3. Third generation; in which biofuels produced using algae that produce oil thirty times as much as any soy or plant based feedstock grown on agricultural land. 4. Fourth generation; in which biofuels produced using microbes, genetically altered, that digest woodchips or wheat straw and the digestive byproduct is hydrocarbon rich. Algae have been investigated for the production of different biofuels including biodiesel, biogas, and biohydrogen. It is possible to produce adequate algal biofuel to satisfy the fast growing energy demand within the restraints of land and water resources. Biologically, the alcohols, most commonly ethanol, are produced by the action of microorganisms and enzymes through the fermentation of sugars or starches (easiest), or cellulose (which is more difficult) (Tijmensen et al., 2002). Biomethanol is mainly produced from natural gas, but biomass can also be gasified to it (Balat et al., 2009). Bioethanol can be derived from biomass with many different varieties of feedstock such as corn, sugarcane, wood, fruits wastes, seaweeds and agriculture wastes (trees and grasses) that are renewable and sustainable resources, which are easily accessible and reliable and can help to clean the environment from the wastes (Shah and Sen, 2011). Nonetheless, the feasibility of using lignocellulosic biomass materials as a feedstock is often limited by the low yield and the high cost of the hydrolysis process based on the current technologies (John et al., 2011). In this perspective, algal ϴ 

biomass is gaining wide attention as an alternative renewable feedstock for the production of bioethanol (Nigam and Singh, 2011). Biobutanol is produced by acetone-butanol-ethanol (ABE) fermentation using several genera of bacteria, particularly clostridia, yielding acetone and ethanol (Jones and Woods, 1986). Recent interest in using oleaginous microalgae as a nonedible biodiesel feedstock has grown considerably, largely on the promise of high oil yields (Levine et al., 2010). Unlike terrestrial oilseeds, microalgae are cultivated in dilute aqueous suspensions that make lipid recovery complicated (Hu et al., 2008). Microalgae grown outdoors in open ponds typically have cell density and productivity ranging from 0.5 to 2 g dry biomass l-1 and 10–40 gm-2d-1, respectively (Benemann and Oswald, 1996). Though higher biomass densities (5–200 gl-1) can be achieved in thin plate photobioreactors (Doucha et al., 2005) and fermentors (Xiong et al., 2008), dewatering and drying remain energy- and cost-intensive processes (Molina et al., 2003). Many companies are currently involved in algae biodiesel research, but according to an article by Public Mechanics, some major players are claiming that they will have large scale algae bio-diesel production soon (Duvall and Fraker, 2009). 1.1. Energy crisis Fossil fuel depletion has become a great concern as the world population expands and the demand for basic human needs increases rapidly. The increasing industrialization and motorization of the world has led to a steep rise for the demand of petroleum-based fuels (Singh and Olsen, 2011). Fossil fuels are running out because of the stress upon their usage for long time and increasing in the population earth advanced technologies have resulted in fast depletion of the naturally occurring conventional fossil fuel reserves (Ramanathan, 2000). ϵ 

Recent soaring oil prices, diminishing world oil reserves, and the environmental deterioration associated with fossil fuel consumption have generated renewed interest in using algae as an alternative and renewable feedstock for fuel production. However, before this concept can become a commercial reality, many fundamental biological questions relating to the biosynthesis and regulation of fatty acids and TAG in algae need to be answered. Clearly, physiological and genetic manipulations of growth and lipid metabolism must be readily implementable, and critical engineering breakthroughs related to algal mass culture and downstream processing are necessary (Hu et al., 2008). Energy used each day per person by country is represented in Figure 1, and expressed by British Thermal Unit (BTU) (Exxon Mobil, 2009).

Figure 1: Energy used each day per person by country (Exxon Mobil, 2009) On the other side, the major disadvantage of using petroleum bades fuels is atmospheric pollution created by the use of petroleum diesel. The petroleum diesel coumbustion is a major source of greenhouse gas (GHG). Apart from these emissions, is also major source of other air contaminants including; NOx, SOx, CO, particulate matter and volatile organic compounds (Klass, 1998).

ϭϬ 

Due to the recent energy crisis and rising concern over climate change rooted in over consumption of fossil fuels, the development of clean alternative energy sources and environmentally friendly is significant interest (Ramanathan, 2000). These non-renewable energy sources are mainly consumed in transport sector. These resources are being depleted at an alarming rate and by the year 2050, these reserves will be nearly consumed (Pratima, 2004). For example, the prices of current energy increasing with demand in all USA sectors of economy; transportation, residential, industrial and commercial; is leading to a potential big problem for the US in the future. The transportation sector demands the most resources from petroleum, but if there were other competitive sources of fuel, prices could be controlled and that, in turn, could help the U.S. economy greatly (Figure 2) (DDzelia et al., 2010).

Figure 2: Energy consumption by source and sector in USA (DDzelia et al., 2010) All these reasons have brought significant attention forward the production of biofuels from biomass (Zverlov et al., 2006). Finding sufficient supplies of clean energy for the future is one of societics most dauting challenges and is intimately ϭϭ 

linked with global stability, economic prosperity and quality of life. Vehicles must be cleaner and more fuel efficient. So, the use of biofuels can play an important role in avoiding the excessive dependence on fossil fuels and ensuring security of supply, in promoting environmental sustainability (Al-Widyan and Al-6K\RXNK$QWROLQ et al., 2002).

1.2. Alternatives of fossil fuel In order to face the limitations in the traditional fuel resources as cool and oil and to overcome the problems caused by fossil fuels and energy crisis, there is an alternative fuel that can replace fossil fuel such as bio-alcohols and bio-diesel (Zaldivar et al. 2001). As well as, the increasing global demand of bio-fuels for energy security and reduction in climate change effects generate the opportunity to explore new biomass sources (Singh and Olsen, 2011). Because of the biomass is renewable, abundant, and limitless, and its use is often regarded as carbon neutral, the consumption of biofuels derived from biomass releases fewer greenhouse gases, such as carbon dioxide, WKDQGRHVIRVVLOIXHOV 'XUUH:DWDQDEH'HPLUEDV). Biofuel is any fuel that is derived from biomass, also consider as a renewable energy source, unlike other natural resources such as petroleum, coal and nuclear fuels (/DUVRQDemirbas, 2009). There are many reports on the potential and bioeconomics of algal biomass to generate fuels and most of these are based on the premise that one would utilize the CO2 emitted from fossil fuelled power stations or other industrial sources of CO2 (Vunjak-Novakovic et al.  GH 0RUDLV DQG Costa, 2007). In order to ensure net societal benefits of biofuel production, governments, researchers, and companies will need to work together to carry out comprehensive assessments, map suitable and unsuitable areas, and define and apply standards relevant to the different circumstances of each country (Phalan, 2009). ϭϮ 

1.3.

What is the biofuel? Biofuel is defined as “the fuel that is formed by microbial activities through the

conversion of organic matter or organic wastes aerobically and/or anaerobically into bioethanol, biomethanol, biobutanol, biodiesel, biogas and biohydrogen” (Figure 3). Marine floras, such as bacteria, actinomycetes, cyanobacteria, fungi, yeasts, microalgae, and other halophytes are extremely important resource for bio-fuel production (Olofsson et al. Karuppaiya et alArai et al., 2010). 

Figure 3: Bioconversion process of biomass (or organic wastes) to produce different energy forms (www.biorefinery.ws) 1.4. Generations of biofuel production Biofuels can be classified into 'four generations', according to the type of technology they rely on and the biomass feedstock's they convert into fuel (Larson, 2008). i. First generation In first generation, biofuels were produced from food crops (sugar or oil crops) and other food based feedstock (e.g. food waste). The most important biofuels of the ϭϯ 

first generation are bioethanol which produced by fermenting sugars from starch and sugar biomass (e.g. cereal crops such as corn or maize, sugarcane, beets, wheat and sorghum), biodiesel that made from vegetable oils of rapeseed, soya, palm fruits or other oil crops via the reaction of triglycerides with methanol (transesterification process), and biogas that is obtained by anaerobic treatment of manure and other humid biomass materials (e.g. in landfills), including food waste, and then upgraded to biogas that can be feed-in into the natural gas grid and e.g. used in natural gas vehicles (Larson, 2008). The utilization of only a small fraction of total plant biomass reduced the land use efficiency. The first generation biofuels have high production cost due to competition with food. The rapid expansion of global biofuel production from grain, sugar, and oil seed crops has raised the cost of certain crops and food stuffs. These limitations favor the search of nonedible biomass for the production of biofuels (Nigam and Singh, 2011).

ii. Second generation: Many problems associated with first generation biofuels can be addressed by the production of biofuels manufactured from agricultural and forest residues and from non-food crop feedstock. Where the lignocellulosic feedstock is to be produced from specialist energy crops grown on arable land, several concerns remain over competing land use, although energy yields are likely to be higher than if crops grown for first generation biofuels are produced on the same land. In addition poorer quality land could possibly be utilized besides rising up the food prices and carbon HPLVVLRQV /DUVRQ  1LJDP DQG 6LQJK  6LPV et al., 2011).So, the main advantage of the second generation biofuels from nonedible feedstock is that it limits the direct food versus fuel competition associated with first generation biofuels (Figure 4). It is believed that the basic characteristics of feedstock holds potential for lower costs, and significant energy and environmental benefits for the majority of ϭϰ 

second generation biofuels (Larson, 2008). However, second, third and fourth generations biofuels are also called advanced biofuels.

Figure 4: An overview of potential feedstock for production of second generation biofuels (www.intechopen.com)

iii. Third generation: The third generation biofuel is a biofuel manufactured from algae. So, it is called algal fuel, algaeoleum biofuel. Algae are low-input, high-yield feedstock to produce biofuels. Based on laboratory experiments, it is claimed that algae can produce up to 30 times more energy per hectare than land crops. At present researches are being conducted by algal culture to produce different fuels for making biodiesel. Taking in review the sustainability and economic factor biofuel from algal culture seems to be most promising fuel for future (Demirbas, 2011). ϭϱ 

iv. Fourth generation: Fourth generation biofuels are produced by using microbes, genetically altered, that digest woodchips or wheat straw and the digestive by-product is hydrocarbon rich (Fortman et al., 2008). Decades of work have produced a considerable knowledge base for the physiology and pathway engineering of microbes, making microbial engineering an ideal strategy for producing biofuel. Although ethanol currently dominates the biofuel market, some of its inherent physical properties make it a less than ideal product (Lee et al., 2008). Occasionally, microbes can be engineered to produce biologically-derived replacements for gasoline, diesel, and aviation fuel. Although much research has focused on ethanol as a biogasoline, there are many other biofuels that offer advantages such as high energy density, low freezing point, and compatibility with the existing fuel storage and distribution infrastructure (Fortman et al., 2008). Fourth generation biofuels, such as long-chain alcohols, fatty acid derived, and isoprenoid derived fuels offer promise as new biofuels and can be synthesized by microbes. These fuels are being developed as either supplements or drop-in replacements for existing petroleum fuels (Lee et al., 2008).

1.5. Marine organic feedstocks The plant and marine biomasses are recently gaining attention as a countermeasure to global warming and as an alternative to petrol as bio-fuel resources. Biomass can be defined as “renewable and organisms originated organic materials excluding fossil resources”. For example, plants, food waste, excretory substance of livestock, woody materials and used paper from agriculture resources and microalgae, seaweeds and other halophytes from marine resources are listed as biomass (Nguyen et al., 2010). Williams and Laurens (2010) concluded that: (i) the biochemical composition of the biomass influences the economics, in particular, increased lipid content reduces other valuable compounds in the biomass; (ii) the “biofuel only” option is ϭϲ 

unlikely to be economically viable; and (iii) among the hardest problems in assessing the economics are the cost of the CO2 supply and uncertain nature of downstream processing.

1.6.

Bacteria and fungi as a biofuel feedstock Microorganisms have been rich sources for natural products, some of which

have found use as fuels, commodity chemicals, specialty chemicals, polymers, and drugs. The recent interest in the production of transportation fuels from renewable microbial systems has been catalyzed (Mukhopadhyay et al., 2008). Marine microbes are both extremely abundant and taxonomically diverse and metabolically complex and the environments they occupy likewise consist of very diverse niches. The microbial world accounts for a tremendous amount of life in the marine environment; just in a single teaspoon of seawater, quantities of microscopic organism reach approximately 100,000 plants, 1000 animals, 1 million bacteria and over 1 billion viruses. Due to the sheer number of microorganisms in the oceans many of these species are scarcely understood or completely new to science (Kennedy et al., 2008). Despite this, only a small proportion of the bioresources of marine microbiota have thus far been examined and an even smaller proportion has been exploited (Kennedy et al., 2008). Marine microbes are both the primary producers of biomass in the oceans, harvesting light and fixing carbon, and the primary recyclers of nutrients. Microbial processes are essential for all the major cycles necessary for the maintenance of ocean life. Marine microbes are also known to be involved in the global cycling of bio-elements such as nitrogen, carbon, oxygen, phosphorous, iron, sulphur and trace elements. However, because of the versatility of their biochemical capabilities and the vast microbial biomass present in marine ecosystems, they are believed to be the main components responsible for the maintenance of these cycles, which help sustain all living things in these ecosystems (Karl, 2007). ϭϳ 

The oleaginous microorganisms are available for substituting conventional oil in biodiesel production. Most of the oleaginous microorganisms like microalgae, bacillus, fungi and yeast are all available for biodiesel production because whome derived from conventional petrol or from oilseeds or animal fat cannot meet realistic need, and can only be used for a small fraction of existing demand for transport fuels. In addition, expensive large acreages for sufficient production of oilseed crops or cost to feed animals are needed for raw oil production (Meng et al., 2009). A number of microorganisms belonging to the genera of bacteria and fungi have ability to accumulate neutral lipids under specific cultivation conditions. The microbial lipids contain high fractions of poly-unsaturated fatty acids and have the potential to serve as a source of significant quantities of transportation fuels (Subramaniam et al., 2010). Bacteria and fungi have many advantages over plants for production of lipids, such as short life cycles, less labor required, less demand on space, venue, season and climate, and ease of scale up. Photosynthetic microorganisms have 100-fold higher yield of lipids per hectare than plants. Lipids produed by oleaginous microorganisms are considred as promising candidates for biodiesel production because fatty acid composition is similar to that of vegetable oils. Microbial lipids are rich in specific polyunsaturated fatty acids also and are often used in dietary supplements and for infant nutrition. In addition, the marine environment is extremely diverse and marine microbes are exposed to extremes in pressure, temperature, salinity and nutrient availability. These distinct marine environmental niches are likely to possess highly diverse bacterial communities, possessing potentially unique biochemistry. Enzymes isolated from microbes from such environments are likely to have a range of quite diverse biochemical and physiological characteristics that have allowed the microbial communities to adapt and ultimately thrive in these conditions. For example bacteria which colonize marine snow are known to produce hydrolytic enzymes, whose function is to degrade proteins and polysaccharides within the snow. Thus the ϭϴ 

potential exists to exploit the enzymes produced by these marine microbes which are likely to possess unique biocatalytic activities capable of functioning under extreme conditions (Azam and Long, 2001). Novel enzymes which have recently been identified from marine environments include a non-specific nuclease isolated from a bacteriophage which predates on the marine thermophile Geobacillus sp. 6K51. This enzyme has been shown to have no known homology to any previously isolated enzymes and a temperature optimum of 60°C (Song and Zhang, 2008). At the other end of the temperature scale are the coldadapted enzymes such as the lipase isolated from the Ȗ-proteobacterium; Pseudoalteromonas haloplanktis TAC125. This lipase is the first member of a new family of lipases, which share homology to the Į/ȕ hydrolases superfamily (de Pascale et al., 2008). Other recently reported enzymes include phospolipases (Nishihara et al., 2008), extracelluar amylotic enzymes (Yoon et al., 2008), agarases (Fu et al., 2008) and endochitinases (Itoi et al., 2007). As well as, a number of cultivable aerobic microbes have recently been isolated from the deep subseafloor sediments from off-shore the Shimokita peninsula in Japan at a water depth of 1180 m. These microbes produced a variety of different enzymatic activities including protease, amylase, lipase, chitinase, deoxyribonuclease and phosphatase activities. Some of these enzymes have a significant role in the breakingdown polysaccharides of marine organic wastes into simple sugars for biofuels production throughout fermentation process. As known, the enzymes extracted from marine microbes are more tolerant against sever conditions and hence they are more effective (Kobayashi et al., 2008). Lactic acid bacteria (LAB) can be found in lactic acid containing products and in decomposing plants, producing lactic acid as the major metabolic product of carbohydrate fermentation. They also isolated from fresh and decomposing sponges, seaweeds, shellfish, crabs and fishes of the marine environment. The marine strains may have better potential in fermentation than their terrestrial counterparts. Their ϭϵ 

special characteristic to show high tolerance to low pH range makes them different from other species of bacteria (Kathiresan and Thiruneelakandan, 2008). LABs follow two fermentation patterns i.e. homo fermentation and hetero fermentation. When glucose is in excess with limited availability of oxygen, homo fermentative LABs convert one molecule of glucose to yield two pyruvate molecules using the Embden Meyerhof pathway (EMP). Hetero fermentative LABs using pentose phosphate pathway, dehydrogenate one molecule of glucose-6-phosphate to 6-phosphogluconate and subsequently decarboxylate it to one molecule of carbon dioxide while reducing pentose-5-phosphate to one molecule of glyceraldehydes phosphate (GAP) and one acetyl phosphate molecule. Glyceraldehyde phosphate is further cleaved into lactate and acetyl phosphate is reduced to ethanol, producing acetyl-CoA and acetaldehyde as intermediates (Ljungh and Wadström, 2009). Scientist presumed that Lactobacillus possesses the properties to affect the industrial ethanol fermentation processes positively (Kandler and Weiss, 1986). It has been observed that the pH of the fermentation process decreases by the addition of lactic acid bacteria to the fermenter containing single yeast species. Some of the Lactobacillus species are adapted to the nutritional and alcoholic conditions of the ethanol fermentation process. It can create the problem of yeast flocculation during alcoholic fermentation. The lactic acid produced by Lactobacilli inhibits yeast metabolism and decreases ethanol yield (Oliva-Neto and Yokoya, 2001). Lactobacillus vini is physiologically versatile, having the ability to ferment pentoses and hexoses to lactic acid, having facultative anaerobic homofermentative metabolism and can grow between 25oC and 45oC (Rodas et al., 2006). Dekkera bruxellensis and Lactobacillus vini may act as potential stable consortium for the industrial production of bioethanol (Dato et al., 2005). Moreover, a novel welldefined starter culture of LAB from marine origin intended for seaweed fermentation for recovery of bioactive molecules (Shobharani et al., 2013). Other bacteria and yeasts such as; Escherichia coli, Saccharomyces cerevisiae, Zymomonas mobilis, and Clostridium acetobutylicum are used to break down the ϮϬ 

biomass converting it into bio-fuel through fermentation or similar processes (Fischer et al., 2008). Recent developments allow for the optimization of this process through manipulation of the genetic makeup of these microorganisms. Bio-fuel production is maximized by focusing the microbe’s metabolic processes on the pathways involved in production and eliminating non-essential competing pathways (Stephanopoulos, 2007). An amiaizing research was conducted by a team of researchers at the Biotechnology Institute at the University of Minnesota - Twin Cities. They have found a method for growing iron-oxidizing bacteria by feeding it electricity. It’s primarily a way to better study a recently-discovered type of bacteria, but it also holds the promise of turning electricity into bio-fuel. This bacterium is ĨĞƌƌŽŽdžLJĚĂŶƐ

DĂƌŝƉƌŽĨƵŶĚƵƐ

PV-1. It’s an aerobic bacterium that was first found in deep ocean volcanic

vents, but has since been discovered in estuarine and marine habitats all over the world. According to the team, the fact that it prefers to live at the interface where an aerobic environment meets an anaerobic one makes it difficult to study, because of the many problems involved in its cultivation. DĂƌŝƉƌŽĨƵŶĚƵƐ is one of a group of bacteria responsible for what is known as “biocorrosion”. The process also occurs in places much closer to home, with bacteria happily munching away in caves and on steel pipelines, bridges, piers, and ships. The interesting thing about

DĂƌŝƉƌŽĨƵŶĚƵƐ

is

that it “breathes” electrons. Normally, to grow and reproduce, it lifts electrons off of a form of dissolved iron called Fe (II), also known as iron (II) oxide. This turns it into a solid precipitate of Fe(III) (iron (III) oxide). Another word for this precipitate is rust. This makes the bacteria very interesting to scientists, but also very hard to cultivate (Szondy, 2013). This team was in developing what they call (electrochemical cultivation). This involves supplying the bacteria with a stream of electrons so it can breathe. It’s a new way to cultivate a microorganism that’s been very difficult to study. But the fact that these organisms can synthesize everything they need using only electricity makes us very interested in their abilities. It’s believed that the bacteria’s electron-swapping Ϯϭ 

breathing takes place on the microbe’s surface. If that’s the case, the team reasoned, electrons could be applied directly to it instead of through iron. They placed the DĂƌŝƉƌŽĨƵŶĚƵƐ

in a nutrient solution that contained no iron. Instead, an electrode was

introduced and an electric current applied. It wasn’t long before the bacteria multiplied and the electrode was coated with a film of them. In other words, the bacteria were feeding off electricity that they combined with carbon dioxide to grow and reproduce (Szondy, 2013).

1.7.

Cyanobacteria as a biofuel feedstock Cyanobacteria also called blue green bacteria, are gram negative photosynthetic

prokaryotes and are one of the most ancient organisms existing on earth; they are apparently the first organism capable of oxygenic photosynthesis, utilizing water as electron source to generate reductant in photosynthesis. The associated release of oxygen was one of the most important events in the history of planet. It has gradually changed the early reducing atmosphere into an oxidizing one, enabling the development of aerobic mode of life in the world. Oxygenic photosynthesis, evolved in cyanobacteria and apparently inherited by green plants is the most important process for capturing the light energy from sun on earth. The chemical energy and reductant produced in the light reactions are used for CO2 fixation. Photosynthesis is main factor in the cyclic transformation of oxygen and carbon and maintaining very important gaseous composition of the atmosphere (Fay, 1992). Oxygenic photosynthetic microalgae and cyanobacteria (for simplicity, algae) represent an extremely diverse, yet highly specialized group of microorganisms that live in diverse ecological habitats such as freshwater, brackish, marine and hypersaline, with a range of temperatures and pH, and unique nutrient availabilities (Falkowski and Raven, 1997). With over 40 000 species already identified and with many more yet to be identified, algae are classified in multiple major groupings as follows: cyanobacteria (Cyanophyceae), green algae (Chlorophyceae), diatoms (Bacillariophyceae), yellowϮϮ 

green

algae

(Xanthophyceae),

golden

algae

(Chrysophyceae),

red

algae

(Rhodophyceae), brown algae (Phaeophyceae), dinoflagellates (Dinophyceae) and ‘pico-plankton’ (Prasinophyceae and Eustigmatophyceae) (Van den Hoek et al., 1995). Basically, a cyanobacterium’s cellular organization is characterized by the presence of massive intracellular membranes, thylakoids, which constitute the photosynthetic apparatus and hold the photosynthetic pigments. Cytoplasm contains different kinds of granular inclusions with diverse functions and compositions cytoplasm. The planktonic form of cyanobacteria contains gas vacuoles in their cells, which provide buoyancy to the cells and enable cyanobacteria to occupy a certain position within the water body (Fay, 1992). The conversion of photosynthetic energy in cyanobacteria involves the action of two different photosystems; photosystem I and II. These two systems are linked in a series

and

interact

through

an

electron

carrier

chain.

Phycobiliproteins

(allophycocyanin, phycocyanin and phycoerythrin) which are the main light harvesting pigments and contribute to the color of cyanobacteria are organized as complexes called phycobilisomes and are attached to the thylakoid’s membranes in regular arrays. Excitation energy is transferred through phycobiliproteins to chlorophyll a in the reaction centers (Fay, 1992). The assimilation of CO2 (photoautotrophy) in light is the main important mode of metabolism in cyanobacteria. The primary path by which carbon is assimilated is the Calvin cycle involving the two most important enzymes phosphoribulokinase and Rubisco (ribulose-1, 5-bisphosphate carboxylase/oxygenase) (Fay, 1992). In order to increase the cyanobacteria capacity to fix CO2, and to increase the yield of the final product i.e. carbohydrate, it has been previously by introducing an additional cassette of specific genes into an engineered cyanobacterium; Synechococcus elongatus (Atsumi, 2009). Cyanobacteria have also been subjected to screening for lipid production (Cobelas and Lechado, 1989; Basova, 2005). Unfortunately, considerable amounts of Ϯϯ 

total lipids have not been found in cyanophycean organisms examined in the laboratory, and the accumulation of neutral lipid triacylglycerols has not been observed in naturally occurring cyanobacteria.

1.8. Microalgae as a biofuel feedstock Microalgae are unicellular eukaryotic photosynthetic organisms that appeared over 3 billion years of evolution, and highly diverse (Falkowski et al., 2004). They are comprised of 28–63% protein, 4–57% carbohydrates, and 2–40% lipids/oils by ZHLJKW 6OXLWHU-RKQet al., 2011). Extensive research has been conducted to investigate the utilization of microalgae as an energy feedstock, with applications being developed for the production of biodiesel, bioethanol, and biohydrogen +XQWOH\DQG5HGDOMH5RVHQEHUJet al., 2008). Microalgae can tolerate and utilize substantially higher levels of CO2 than terrestrial plants; hence they can utilize CO2 emitted from petroleum-based power stations or other industrial sources which in turn can reduce emission of green house gas (Benemann et al., 1982). The whole algal biomass or algal oil extracts can be converted into different fuel forms, such as biogas, liquid and gaseous transportation fuel, kerosene, ethanol and biohydrogen through the implementation of processing technologies such as anaerobic digestion, pyrolysis, gasification, catalytic cracking, and enzymatic or chemical transesterification (Subhadra, 2010). As microalgae grow in aqueous environments, directly passing flue gases through this medium is a very efficient way of capturing the CO2 in those streams (Benemann et al., 1982). The application of CO2 directly to terrestrial crops via enclosures is likely to be prohibitively expensive though indirect stimulation of land species by flue gases is an alternative approach, which may be cost-effective despite being very much less direct and less efficient (Packer, 2009). Microalgae are also very efficient in utilizing the nutrients from industrial effluents and municipal wastewater. Therefore cultivation of algal biomass provide dual benefit, it provides biomass for the production of biofuels and also save our Ϯϰ 

environment from air and water pollution (Singh and Olsen, 2011). Microalgae represent an exceptionally diverse but highly specialized group of microorganisms adapted to various ecological habitats. Many microalgae have the ability to produce substantial amounts (e.g. 20–50% dry cell weight) of triacylglycerols (TAG) as a storage lipid under photo-oxidative stress or other adverse environmental conditions. Fatty acids, the building blocks for TAGs and all other cellular lipids, are synthesized in the chloroplast using a single set of enzymes, of which acetyl CoA carboxylase (ACCase) is key in regulating fatty acid synthesis rates (Hu et al., 2008). The expression of genes involved in fatty acid synthesis is poorly understood in the microalgae. Synthesis and sequestration of TAG into cytosolic lipid bodies appear to be a protective mechanism by which algal cells cope with stress conditions, but little is known about regulation of TAG formation at the molecular and cellular level. While the concept of using microalgae as an alternative and renewable source of lipid-rich biomass feedstock for biofuels has been explored over the past few decades, a scalable, commercially viable system has yet to emerge. Today, the production of algal oil is primarily confined to high-value specialty oils with nutritional value, rather than commodity oils for biofuel (Hu et al., 2008). Different microalgal species (Chlorella vulgaris, Chlorella minutissions, Chlorella

prototheoides,

Phaeodactylum

Chlorella

tricornutum,

sorokiniana,

Botryococcus

Neochloris

braunii,

Navicula

oleoabundas, pelliculosa,

Scenedsmus acutus, Crypthecodinium cohnii, Dunaliella primolecta, Monallanthus salina and Tetraselmis sueica) can accumulate oils and they had varied ability for oil production (Christi, 2007). Some of these species listed in Table 1 with lipid content/% dry cell weight (Li et al., 2008a). The microalgae store starch mainly in the cells and biomass can be harvested at regular intervals from photobioreactors or shallow raceway ponds. The starch can be extracted from the cells with the mechanical tools (e.g., ultrasonic, explosive disintegration, mechanical shear, etc.) or by dissolution of cell walls using enzymes. The starch is then separated by extraction with water or an organic solvent and used Ϯϱ 

for fermentation to yield bioethanol. Both saccharification and fermentation processes can be simultaneously carried out in a single step if an amylase producing strain can be used for ethanol fermentation. Utilization of starch degrading ethanol producers can preclude the cost incurred for acid or enzymatic saccharification of starch (John et al., 2011). Table 1: Oil accumulation produced by different microalgal species (Li et al., 2008a) Microalgal Species Oil content (% dry cell weight) Chlorella vulgaris Chlorella minutissions

40-56 57

Chlorella prototheoides

23

Chlorella sorokiniana

22

Neochloris oleoabundas

54

Phaeodactylum tricornutum

62

Hu et al. (2008) summarized the potential advantages of algae as feedstocks for biofuels include their ability to the followings: 1. Synthesize and accumulate large quantities of neutral lipids/oil (20–50% DCW),  2. Grow at high rates (e.g. 1–3 doublings per day), 3. ௘Thrive in saline/brackish water/coastal seawater for which there are few competing demands, 4. Tolerate marginal lands (e.g. desert, arid- and semi-arid lands) that are not suitable for conventional agriculture, 5. Utilize growth nutrients such as nitrogen and phosphorus from a variety of wastewater sources (e.g. agricultural run-off, concentrated animal feed operations, and industrial and municipal wastewaters), providing the additional benefit of wastewater bio-remediation,

Ϯϲ 

6. Sequester carbon dioxide from flue gases emitted from fossil fuel-fired power plants and other sources, thereby reducing emissions of a major greenhouse gas, 7. Produce value-added co-products or by-products (e.g. biopolymers, proteins, polysaccharides, pigments, animal feed, fertilizer and H2), 8. Grow in suitable culture vessels (photo-bioreactors) throughout the year with annual biomass productivity, on an area basis, exceeding that of terrestrial plants by approximately tenfold. Recently, Harun et al. (2010) investigated the suitability of lipid extracted microalgal debris for fermentation with a yield of bioethanol about 4–10 gl-1 of the substrate. In such manner, several algae, especially green algae can accumulate cellulose besides starch as the cell wall carbohydrate, which can also be used for ethanol production. The biomass from red alga can be de-polymerized to yield mixed monosugars such as glucose and galactose (John et al., 2011). However, Brennan and Owende (2010) has listed the desirable characteristics of algal strains to be considered as candidates for biofuel production, such as: (1) robust and able to survive the shear stresses common in photo-ELRUHDFWRUV   DEOH WR GRPLQDWH ZLOG VWUDLQV LQ RSHQ SRQG SURGXFWLRQ V\VWHPV   KLJK &22 sinking FDSDFLW\   OLPLWHG QXWULHQW UHTXLUHPHQWV   WROHUDQW WR D ZLGH UDQJH LQ WHPSHUDWXUHVUHVXOWLQJIURPWKHGLXUQDOF\FOHDQGVHDVRQDOYDULDWLRQV  SRWHQWLDOWR provide valuable co-SURGXFWV   IDVW SURGXFWLYLW\ F\FOH   KLJK SKRWRV\QWKHWLF efficiency, and (9) display self-flocculation characteristics.

1.9.

Macroalgae (Seaweeds) as a biofuel feedstock Macroalgae, or seaweeds, represent a broad group of eukaryotic photosynthetic

marine organisms. They are almost everywhere. They are seen flourish in both fresh and saline water. Algae grow in almost any aquatic environment and use light and carbon dioxide (CO2) to create biomass (autotrophic algae) (Packer, 2009; Mata et al., 6XEKDGUD, 1LJDPDQG6LQJK  Ϯϳ 

They are evolutionarily diverse and abundant in the world oceans and coastal waters. They have low lipid content as a general rule but are high in carbohydrates that can be converted to various biofuels (Baldauf, 2003). Therefore, they are gaining some attention as an alternative renewable source of biomass for the production of bioethanol, although algal fermentation facilities are relatively expensive to construct and operate, although are known to be reliable and produce high yields with a range of feedstock (Borowitzka, 1992). Compared with terrestrial plants, macroalgae (i.e. seaweeds) have a high water content of approximately 70–90%, a relatively high protein content of approximately 10%, and contain varying levels of carbohydrates (Park et al., 2006). They also contain a low concentration of lignin (Wi et al., 2009) or no lignin at all (Ge et al., 2011). A number of features of macroalgae make them attractive when compared to terrestrial feedstock crops due to their fast growth rate and large biomass yield, with superior productivity to many terrestrial crops (Zhang et al., -RKQet al., 2011) (Table 2). Although their growth requirements are similar to terrestrial plants, they use these resources very efficiently (Briggs, 2004) and therefore have high productivity with comparatively low water use (Chelf et al., 1993). In areas with limited lignocellulosic waste streams, cultivation of dedicated biomass crops would have to sustain industrial-scale production of cellulosic ethanol. The cultivation of a dedicated photosynthetic biomass crop has the additional environmental benefit of a net zero carbon economy when used for energy and, if not used for energy production, has the potential to be traded internationally as carbon credit. To date, marine macroalgae have largely been ignored as a bioenergy crop, since they require ocean access and are thus not widely available. However, macroalgae offer a number of potential advantages for all coastal locales. Macroalgae can be farmed in mariculture facilities because they form complex organized structures that allow controlled spatial containment and exhibit high growth rates3– 6). Offshore mariculture requires neither significant land area nor freshwater, and Ϯϴ 

environmental impacts can be minimized by careful species selection and management (Abbott, 1999). Mainly, seaweeds are classified into three groups: green, brown, and red. They contain various types of glucans, which are polysaccharides composed of glucose, though the concentration of these glucans is known to be relatively low. In fact, the glucans found in green and red seaweeds are cellulose and starch, and brown seaweeds contain cellulose and ȕ-1, 3-glucan. These glucans can be hydrolyzed by saccharification enzymes, and thus, ethanol can be produced from the hydrolysate (Horn et alD*Het al:DQJet al., 2011).

Table 2: Comparisons of yield, hydrolysable carbohydrate, and potential bioethanol production between major terrestrial bioethanol crops and macroalgae (Adams et al., 2009) Wheat (grain) Average world yield (kg ha−1 year−1) Dry weight of hydrolysable carbohydrates (kg ha−1 year−1) Potential volume of bio-ethanol (L ha−1 year−1)

Corn (kernel)

Sugar Beet

SugarCane

Macro -algae

2800

4815

47,070

68,260

730,000

1560

3100

8825

11,600

40,150

1010

2010

5150

6756

23,400

Conversion of biomass from marine algae into ethanol could be economically feasible since some algae hydrolysate can contain more total carbohydrate and hexose sugars than some terrestrial, lignocellulosic biomass feedstock (Sluiter, 2006). Aquatic algal cells are buoyant, avoiding the need for structural biopolymers such as hemicellulose and lignin that are essential for higher plant growth in terrestrial environment. This simplifies the process of bioethanol production by eliminating the chemical and enzymatic pretreatment steps (John et al., 2011). The green macroalgae (chlorophyceae), like Ulva lactuca has been considered as a potential aquatic energy crop as early as in the Aquatic Species Programme in Ϯϵ 

the USA back in 1978–1996, due to its high potential growth rates and high content of carbohydrates (Ryther et al., 1984). The high yield of macroalgae is attributed to their lower energy requirement for the production of supporting tissues than terrestrial plants, in addition to their capability to absorb nutrients over their entire surface area (Zhang et al., 2010), and the energy-savings derived from zero requirements for internal nutrient transport (Wi et al., 2009). Many types of seaweed exhibit a mass productivity of 13.1 kg dry weight m−2 over a seven month growth period, compared to terrestrial plants achieving 0.5–4.4 kg dry weight m−2 over an entire year (Goh et al., 2010). Furthermore, macroalgae generally have a greater hydrolysable carbohydrate content, and potential volume of ethanol than current bioethanol feedstock (John et al., 2011). Ocassionally, the harvest of wild stock accounted for about 1.1 million wet metric tons of the annual world macroalgae production in 2006, while aquaculture accounted for about 15.1 million wet metric tons of the annual production (Table 3), outpacing that of wild harvests by over ten fold. Annual production associated with harvest of wild stock was more evenly distributed worldwide than production from aquaculture. Whereas the top ten countries harvesting wild stocks included countries in Asia, South America, Central America, Europe, and Iceland, plus Russia, and Australia, production from aquaculture was centered in Asia and dominated by China, which accounted for 72% of the total production and 73% of the total value of cultured macroalgae. The other top ten countries for macroalgae aquaculture accounted for 99% of the remaining aquaculture EDVHG SURGXFWLRQ )$2  Roesijadi et al., 2010). Macroalgae with high carbohydrate contents are promising candidates for bioethanol production, including: Sargassum, Gracilaria, Prymnesium parvum, Euglena gracilis, Gelidium amansii (Wi et al., 2009), and Laminaria (Adams et al., 2009). Macroalgae carbohydrate contents vary widely by species and cultivar, and species selection can develop strains with very high contents of carbohydrate for use as an efficient bioethanol feedstock. For example, the brown macroalgae such as ϯϬ 

Laminaria spp. contain up to 55% (dry weight) of carbohydrates laminarin and mannitol (Horn et al., 2000b). Table 3: World production (wet metric ton) of wild stock harvest and cultured macroalgae plus monetary value of cultured (USD) in 2006 by country (FAO, 2008) Source World total

Harvest of wild stock Production (metric ton) 1,143,273

China

323,810

% of Total

Source

Aquaculture Production (metric ton) 7,187,125

% of Total 100.00 72.09

100.00

World total

28.32

China

10,867,410

Chile

305,748

26.74

Philippines

1,468,905

9.74

Norway

145,429

12.72

Indonesia

910,636

6.04 5.08

Japan

113,665

9.94

Korea Rep.

765,595

Russian Fed.

65,554

5.73

Japan

490,062

3.25

Ireland

29,500

2.58

Korea DPRp.

444,300

2.95

Mexico

27,000

2.36

Chile

33,586

0.22

Iceland

20,964

1.83

Malaysia

30,000

0.20

France

19,160

1.68

Vietnam

30,000

0.20

Australia

15,504

1.36

Cambodia

16.000

0.11

Morocco

14,870

1.30

China,Tiwan

5,949

0.04

Korea Rep.

13,754

1.20

India

4,668

0.03 0.03

Canada

11,313

0.99

Kiribati

3,900

Indonesia

9,830

0.86

South Africa

3,000

0.02

South Africa

6,600

0.58

Russian Fed.

818

0.01

USA

6, 238

0.55

Tanzania

320

0.00

Madagascar

5,300

0.46

Solomon Is.

120

0.00

Peru

3,434

0.30

Fiji Islands

119

0.00 0.00

Italy

1,400

0.12

Mali

90

Ukraine

1,121

0.10

Nambia

70

0.00

Portugal

765

0.07

France

45

0.00

Spain

485

0.04

Mozambique

15

0.00

Estonia

394

0.03

Burkina Faso

2

0.00

Tonga

356

0.03

St Lucia

1

0.00

Fiji Islands

350

0.03

Spain

1

0.00

0.03

Philippines

314

New Zealand

225

0.02

China, Tiwan

190

0.02

ϯϭ 

The brown macroalgae carbohydrates consist of primarily cellulose, hemicellulose, free sugars, and also the energy storage molecules laminarin and mannitol. As crude fiber is composed of cellulose and hemicelluloses, the % carbohydrates only constitute the storage products and free sugars. However, macroalgae constituents are not constant throughout the year. As an example, research by Horn et al., (2000b) on the composition of brown algae, Ascophyllum nodosum, describes seasonal component flux, and also includes indicative magnitudes for each component during the year. The brown algae are comprised of around 24–29% alginic acid, a polymer of d-mannuronic and l-guluronic acids covalently linked together in sequence.

1.10. Sea grasses as a biofuel feedstock Sea grasses are flowering plants from one of four plant families (Posidoniaceae, Zosteraceae, Hydrocharitaceae, or Cymodoceaceae), all in the order Alismatales (in the class of monocotyledons), which grow in marine, fully saline environments (Ravikumar et al., 2011). Sea grass roots are colonized by anaerobic bacteria, which not only contribute to the vitality of sea grasses but also to the biogeochemistry of the surrounding sediment. Kusel et al. (2001) isolated an ethanol producing anaerobic Gram positive bacterium, from the root of the sea grass Halodule wrightii, which metabolizes certain substrates via the acetyl-CoA pathway, these bacteria could also tolerate and consume limited amounts of O2, which enhance the production of ethanol, lactate, and H2. It was suggested the ability to cope with limited amounts of O2 might contribute to its survival in a habitat subject to daily gradients of O2. In addition, when the transport of O2 to the sea grass roots is not enough to meet the demand for aerobic respiration, then sea grasses, may switch to a fermentation pathway releasing ethanol for short time periods (Smith et al., 1988). Studies on marine plants as a source for bio-fuels are rarely reported in the literature. At the same time, large amounts of marine plants are deposited on the beaches of many countries every year because of eutrophication. Usually, this ϯϮ 

residue is unused and a calamity for tourism (Morand and Briand, 1996). Among the collectable submerged sea grasses, Zostera marina is one of the more widespread (Viola et al., 2008). However, studies related with sea grass bio-wastes on ethanol production are too limited (Ravikumar et al., 2011). But if marine vegetation sea grasses, macroalgae, microalgae were utilized as bio-fuel feedstock, reduction of competition against food crops and removal of land limitation could be attained (Marquez et al., 2013). Ravikumar et al. (2011) produced the bio-ethanol from sea grass bio-wastes using commercial yeast; Saccharomyces cerevisiae. It reveals that, the maximum production of ethanol (0.047 mlg-1) was recorded from the fresh seagrass leaves in acid pretreatment than the semidecayed leaves. Earlier findings revealed that, the ethanol production was recorded in eel grass (Viola et al., 2008) and switchgrass (Keshwani and Cheng, 2009). Viola et al. (2008) investigated Zostera marina for bio-ethanol production. The examined plant contained 30% of glucan. It was pretreated by steam explosion. Temperature, time of pretreatment and oxalic acid load were selected as experimental factors and analyzed by the response surface regression procedure. The best results (5.06 g of soluble sugars and 52.9 g of glucose, respectively, from 100 g of exploded material and 100 g of insoluble fiber) were attained at pretreatment of 180°C, 5 min and 2 wt% of oxalic acid. Fermentation tests of the hydrolyzed fiber were carried out and the ethanol production was in the best cases 243 g of ethanol was produced per kg of Zostera fiber. However, steam explosion is one of the more used because of its ability to induce auto-hydrolysis and defibration (Kaar et al., 1998). To further improve the substrate digestability and bioconversion, impregnation with acid was sometime reported (Emmel et al., 2003). In the case of eel grass, the use of oxalic acid could activate a synergic mechanism as it is known that the oxalate anion captures the metal ions that are in the fiber, making the carbohydrate structure more available ϯϯ 

(Oyodova et al., 1968). Another sea grass species known as (sea wrack) is dislodged marine vegetation, specifically, seaweeds and sea grasses. These dislodged macroflora could be found along beaches or floating near coasts. In the Philippines, for example, during strong monsoon and typhoon months, sea wrack biomass covers wide areas of coast. Furthermore, increasing water level due to climate change increases the marine area; thus providing wider inward habitat for sea grass (Short et al., 2001) and seaweed communities (Harley et al., 2012). Utilization of sea wrack for biogas production could mitigate the emission of methane, a greenhouse gas, and would economically benefit local island communities (Nettmann et al., 2010). Marquez et al. (2013) used sea wrack in biogas production. Three microbial seeds cow manure (CM), marine sediment (MS), and sea wrack-associated microflora (SWA) were explored for biogas production. The average bio-gas produced were 2172 ± 156 ml (MS), 1223 ± 308 ml (SWA) and 551 ± 126 ml (CM). Though methane potential (396.9 ml CH4 g-1 volatile solid) computed from sea wrack proximate values was comparable to other feedstocks, highest methane yield was low (MS = 94.33 ml CH4 g-1 VS). Among the microbial seeds, MS proved the best microbial source in utilizing sea wrack biomass and seawater.

1.11. Crustaceans as a biofuel feedstock Many living organisms use networks of fibrous and crystalline polysaccharides to maintain structural integrity. Enzymatic conversion of the most recalcitrant of these polysaccharides is of great biological and economic importance and affects processes varying from the interplay between, for example, plants, crustaceans or insects and their pathogens to the production of second generation bio-ethanol. In plants, the major structural polysaccharide is cellulose, whereas non-plants such as insects, crustaceans and fungi employ chitin, which occurs in two major forms, Įchitin and ȕ-chitin. In nature, degradation of cellulosic or chitinous biomass is ϯϰ 

achieved by mixtures of hydrolytic exo- and endo-acting enzymes that act in a synergistic manner 0HULQRDQG&KHUU\:\PDQ07). Recently, new insights into how enzymes accomplish the bioconversion of chitin into bioethanol in the development of second generation have been obtained (Eijsink et al., 2008). Chitin, which is a polymer of ȕ-(1–4) linked N-acetyl-dglucosamine (GlcNAc) residues, is one of the most abundant renewable resources in nature, after cellulose (Inokuma et al., 2013). It is a principal structural component of most fungi, yeasts and algae cell walls, insect exoskeletons, shells of crustaceans and the microfilarial sheath of nematodes (Flach et al., 1992). Recent investigations confirmed the suitability of chitin and its derivatives in a broad range of applications for use in medicines, cosmetics, agriculture, food preservation, textile industry, sorbents and enzyme supports based on their polyelectrolyte properties, the presence of reactive functional groups, gel forming ability, high adsorption capacity, biodegradability, bacteriostatic, fungistatic and antitumor influence (Synowiecki and Al-Khateeb, 2003). An estimated annual production of chitin is in the order of 1010 to 1011 tons on earth (Gooday, 1990). At present, however, only small quantity of shell wastes are being utilized for animal feed or chitin isolation for the purposes mentioned above, and the processing of shellfish leads to environmental pollution. Thus, if chitin and its derivatives can be converted to ethanol efficiently using microorganisms, it will become one of the strong favorites for petroleum substitutes. Wendland et al. (2009) engineered S. cerevisiae strains, which can utilize GlcNAc as a carbon source and produce ethanol by introducing four genes required for a GlcNAc catabolic pathway from Candida albicans, which can utilize GlcNAc as a sole carbon source (Biswas et al., 2007). However, fermentation of GlcNAc using these strains was slow and the obtained ethanol titers were low (3 gl-1 after 11 days). On the other hand, Evvyernie et al. (2001) reported the conversion of chitinous wastes to hydrogen gas by Clostridium paraputrificum M-21. Hydrogen is considered to be a potential source of alternative energy (Evvyernie et al  ϯϱ 

Morimoto et al., 2005). However, the enzymatic hydrolysis of chitin to GlcNAc is carried out by chitinolytic enzyme system, which consists mainly of endochitinase and ȕ-N cetylhexosaminidase (GlcNAcase). Endo-chitinase cleaves glycosidic linkages randomly along the chain to give chitooligomers, and GlcNAcase cleaves chitooligomers from the non-reducing end to give GlcNAc. These enzymes have been detected in a wide variety of microorganisms (Binod et al  6XUHVK DQG Kumar, 2012). Inokuma et al. (2013) found some native Mucor strains, which can use GlcNAc and chitin substrates as carbon sources for growth and bioethanol production. One of these strains, M. circinelloides NBRC 6746 produced 18.6 ± 0.6 g/l of ethanol from 50 g/l of GlcNAc after 72 hr and the maximum ethanol production rate was 0.75 ± 0.1 g1-1hr-1. Furthermore, M. circinelloides NBRC 4572 produced 6.00 ± 0.22 and 0.46 ± 0.04 gl-1 of ethanol; produced from 50 gl-1 colloidal chitin and chitin powder after 16 and 12 days, respectively. They also found an extracellular chitinolytic enzyme producing strain M. ambiguus NBRC 8092, and successfully improved ethanol productivity of NBRC 4572 from colloidal chitin using crude chitinolytic enzyme derived from NBRC 8092. The ethanol titer reached 9.44 ± 0.10 gl-1 after 16 days. They assumed that their results were the first bioethanol production from GlcNAc and chitin substrates by native organisms, and also suggest that these Mucor strains have great potential for the simultaneous saccharification and fermentation (SSF) of chitin biomass. 1.12. Types of biofuels Biofuels, like fossil fuels, come in a number of forms and meet a number of different energy needs. This context will categorize the different biofuels according to their nature into main four types, as follows: 1. Bioalcohols, 2. Biodiesel, 3. Biogas, 4. Biohydrogen. ϯϲ 



 CHAPTER 2 Bioalcohols

ϯϳ 

Bialcohols are a wide range of fuels which are in some way derived from biomass (Tijmensen et al., 2002). Biologically they are produced, most commonly ethanol, and less commonly propanol, methanol and butanol, by the action of microorganisms and enzymes through the fermentation of sugars or starch (easiest), or cellulose (which is more difficult) (Tijmensen et al., 2002).

2.1. Biomethanol Biomethanol (CH3OH) is mainly produced from natural gas, but biomass can also be gasified to it (Balat et al., 2009).Gasification creates synthetic gas (syngas), which is essentially H2 and CO. Syngas has the potential to produce a wide range of commercial fuels and chemicals, including synthetic diesel, methanol and lower carbon alcohols, acetic acid, dimethyl ether, etc. (Demirbas, 2010a). Biomethanol is produced from synthesis gas utilizing conventional gasification of biomass at high temperatures (800-1000ΣC) and, subsequently, catalytic synthesis of the produced mixture of CO2 and H2 with a molar ratio of 1:2, under elevated pressures (4̽10 MPa) (Oliveira and Franca, 2009). On the other hand, bio-methanol can be made with any renewable resource containing carbon such as seaweed, waste wood and garbage. This is a promising alternative, with a diversity of fuel applications with proven environmental, economic and consumer benefits (Demirbas, 2009). The product yield for the conversion process is estimated to be 185 kg of biomethanol per metric ton of solid waste (Keskin and Emiroglu, 2010). Agriculture-based methanol is at present more expensive of methanol from natural gas. The use of biomethanol as a motor fuel received attention during the oil crises of the 1970s due to its availability and low cost (Balat, 2009). In comparison with gasoline, biomethanol is a superior engine fuel. Thermal efficiency values for the engine are higher and there are no emission problems because of a high octane number (Van Swaaij et al., 2004). It is an excellent fuel for high-compression engines (Balat et al., 2009). ϯϴ 

A higher octane rating allows certain engine design parameters, such as compression ratio, and valve timing, to be altered in such ways that fuel economy and power are increased (Demirbas, 2009). On the basis of the mass unit, bio-methanol has a lower energy value than gasoline (Kar and Deveci 2006). As a fuel, biomethanol is most often used as a blend with gasoline called M85 (85% biomethanol and 15% gasoline), although the fuel can also be used in pure form (M100) (Balat et al., 2009). Even on using M100 as a motor gasoline very few carburetor modifications are required. However, the mixture must be preheated and a large tank is necessary. These alterations are a result of bio-methanol increased heat of vaporization ̽ three times greater than gasoline ̽ and the approximately 50% lower energy content (Weissermel and Arpe, 2004). Methanol is produced on a very large scale (35x106 metric tons/year). About 30% is used for formaldehyde production and 30% for MTBE (methyl tertiary butyl ether). Only a few percent is used as fuel. In the future, the role of methanol as liquid fuel may increase considerably, e.g. in fuel cell cars, as intermediate for hydrogen production, etc... (Van Swaaij et al., 2004). The various aspects related to the production of biomethanol from organic waste materials are discussed by Demirbas (2008). He stated that the methanol can be made using wood waste or garbage via partial oxidation reaction into syngas, followed by catalytic conversion into methanol called biomethanol.

2.2. Bioethanol Bioethanol (C2H5OH) is an attractive alternative fuel because it is not only a renewable biobased resource but also is oxygenated, thereby providing the potential to reduce particulate emissions in compression ignition engines (Hansen et al., 2005). It is carbon neutral and free from sulfur and aromatics; therefore it can be considered as not harmful to living organisms. Thus, by using bioethanol as fuel, people can effectively protect the next generation against the upheaval resulting from global warming in the future (Goh et al., 2010). ϯϵ 

Bioethanol is widely used in the USA and in Brazil. It can be derived from biomass with many different varieties of feedstock such as corn, sugarcane, wood, fruits wastes, seaweeds and agriculture wastes (trees and grasses) that are renewable and sustainable resources, which are easily accessible and reliable and can help to clean the environment from the wastes (Shah and Sen, 2011). Many bioethanol production methods have also been developed by the fermentation of different types of biomass such as starch, lignocellulose, or agricultural, forest residues and marine biomass by; Pichia stipitis, Pichia wickerhamii and Candida tropicalis (Dominguez et al., 2000), Corynebacterium glutamicum (Inui et al., 2004), Saccharomyces cerevisiae (Watanabe et al  Yanase et al., 2010), and Zymomonas mobilis (dos Santos Dda et al., 2010). Similarly, the fungus Fusarium sp., Rhizopus sp., and the bacterial genus Clostridium was also able to produce ethanol from xylose (Rahayu and Rahayu, 1988). The yeasts convert six-carbon sugars (mainly glucose) to bio-ethanol. Initially, the sugar of raw materials is separated after that fermentation processes use yeast to convert the glucose into ethanol. The distillation and the dehydration are used as the last steps for reaching the desired concentration (hydrated or anhydrous ethanol) that can be blended with fossil fuels or directly used as fuel. When the used raw materials are grains, usually hydrolysis is used for converting the starches into glucose (Escobar et al., 2009). Scientists still wishing to increase bioethanol production might by evaluating the effects of many different factors (or variables) on the rate of yeast fermentation. Some of the variables known to affect fermentation in yeast cells are; type and concentration of carbohydrate, concentration of NaCl, osmolarity, pH and ethanol concentration. Indeed, during batch fermentation system, several parameters can cause a decrease in specific growth rate of the microbes that caused both by the concentration of substrate and product of ethanol. Therefore, Rakin et al. (2009) produced bioethanol using immobilized yeast cells. They observed that the immobilized cells in fermentation processes have been developed to reduce the ϰϬ 

inhibition caused by high concentrations of substrates and products, thereby increasing productivity and yield of ethanol. Wargacki et al. (2012) used Vibrio splendidus enzymes for alginate transport and metabolism of macroalgae (seaweeds) as feedstocks for bioconversion into bioethanol. When further engineered for ethanol synthesis, this platform enables bioethanol production directly from macroalgae via a consolidated process, achieving a titer of 4.7% volume/volume and a yield of 0.281 weight ethanol/weight dry macroalgae (equivalent to ~80% of the maximum theoretical yield from the sugar composition in macroalgae). Microalgae like Chlorella, Dunaliella, Chlamydomonas, Scenedesmus and Spirulina are known to contain a large amount (>50% of the dry weight) of starch, cellulose and glycogen, which are raw materials for ethanol production (Chen et al., 2009). Oleaginous microalgae generate biomass waste with high starch/cellulose content after oil extraction. This can be hydrolyzed to generate sugary syrup for ethanol production (John et al., 2011). On the other side, there are several companies working in bioethanol production, such as; Seaweed Energy Solutions, Green Gold Algae and Seaweed Sciences, Inc. Butamax Advance Fuels-Dupont-BioArchitecture Lab-Statoil and Holmfjord AS8 (Roesijadi et al., 2010).

2.3. Biobutanol Biobutanol (C4H9OH) (also called biogasoline) is often claimed to provide a direct replacement for gasoline, because it can be used directly in a gasoline engine (Tijmensen et al., 2002). Biobutanol is considered a promising product of biomass fermentations for potential industrial use as a solvent, chemical feedstock, and particularly liquid fuel (Durre, 2008). Therefore, it is very important to optimize the product yield and simultaneously reduce the level of other end products (i.e., H2, CO2, acetate, and butyrate). ϰϭ 

Biobutanol can be utilized in internal combustion engines as both a gasoline additive and or a fuel blend with gasoline. The energy content of biobutanol is 10% less than that of regular gasoline. This is not as bad as energy density of ethanol is 40% lower. Since biobutanol is more chemically similar to gasoline than ethanol, it can be integrated into regular internal combustion engines easier than ethanol. Biobutanol has displayed the potential to reduce the carbon emissions by 85% when compared to gasoline, making it a superior alternative to gasoline and a gasolineethanol blended fuel. Its bio-production route was halted in the 1960s due to high production price with respect to production from petroleum. New technology advancements and an increase in petroleum prices are making bio-production of butanol more competitive and safer (Peter, 2007). Biobutanol is produced by acetone-butanol-ethanol (ABE) fermentation using several genera of bacteria, particularly clostridia, yielding acetone and ethanol (Jones and Woods, 1986). The ABE fermentation was one of the first large-scale industrial fermentation processes to be developed. During the early 20th century through World Wars I and II, ABE fermentation was important for the production of butanol and acetone solvents. However, its use has declined since the 1950s owing to increasing costs of the substrate molasses, and the availability of much cheaper feedstock for chemical solvent synthesis by the petrochemical industry, except in South Africa, the Soviet Union, and China (Durre, 2007). The 1973 oil crisis led to renewed interest in solvent production by ABE fermentation. In particular, butanol produced by ABE fermentation has been an attractive biofuel alternative because of the advantages described above. In the present century, there have been numerous studies on butanol production in various fields, and many companies have declared their plans for the commercial production of biobutanol using biomass (Ni and Sun, 2009). Biobutanol is made via fermentation of biomasses from substrates ranging from corn grain, corn stovers, sugar beets and other feedstock. Microbes, specifically of the Clostridium acetobutylicum, are introduced to the sugars produced from the biomass. These sugars are broken down into various alcohols, which include ethanol ϰϮ 

and butanol. Unfortunately, a rise in alcohol concentration causes the butanol to be toxic to the microorganisms, killing them off after a period of time. This made the fermentation process expensive and unrealistic when compared to the petroleum costs of the late 50’s. Luckily, new technological advances and the discovery of new microbes have improved the efficiency and cost of the fermentation process tremendously. Through genetic engineering, researchers have been able to modify the most efficient microbes to be able to withstand high alcohol concentrations. New modifications are constantly being researched, including the modification to enzymes and genes involved in butanol formation from biomass fermentation (Huang et al., 'RDQet al., 2012). Since 1980s efforts have been made to improve ABE productivity by delineating

the

physiology

of

solventogenic

clostridia,

particularly

C.

acetobutylicum) (Girbal and Soucaille, 1994) and genetics (Inui et al7RPDV et al., 2004). New possibilities for more sustainable solvent production via ABE fermentation with less expensive substrates have been proposed (Yu et al., 2007). For instance, lignocellulosic materials such as domestic organic waste (Claassen et al., 2000) or fibrous corn wastes (Qureshi et al., 2006) can be used for ABE fermentation. Efforts are currently underway to improve the existing microbes used for fermentation. Next major cost hurdle is separation costs of butanol from fermentation broth several membrane based separation methods are under investigation which can reduce costs of biobutanol by 40-50%. Through a mixture of genetic engineering and membrane separation, biobutanol has a promising future (Huang et al., 2010). In addition, the fermentation of Ulva lactuca solution to butanol was performed with butanol ultimately removed by distillation. With the bacterial strains (Clostridium beijerinckii and C. saccharoperbutylacetonicum) and the algal sugar solutions used; ABE fermentation was used to make butanol (Van der Wal et al., 2013). The butanol concentration in the fermentation broth reached 4 gl-1, which is closed to the theoretical value for the sugar concentration obtained (Potts et al., 2012). ϰϯ 

As well as, increased butanol concentrations, productivity, and yields were achieved by the establishment of high cell density continuous cultures by cell immobilization (Lienhardt et al., 2002) or by cell recycling together with cell bleeding (Tashiro et al., 2005), fed-batch culture integrated with in situ butanol recovery system (Ezeji et al., 2004), and butanol production from butyric acid in living cells (Tashiro et al., 2007). Although the history of metabolic engineering and systems biology is shorter than that of microbial technology, in the last 1 or 2 decades, novel and significant findings have been obtained and useful techniques and powerful tools have been developed, in step with the progress in genetics technology and computer technology. Over a dozen companies are focused on developing biobutanol on commercial scale. This is a true biofuel for masses with potential of little or no impact on food supply and ability to compete favorably with $80 bbl oil. Effort is focused on both fermentation of sugars, starch and other biomass and through pyrolysis and reformulation of biomass. The secondary appeal of biobutanol is its variety of commercial uses in an existing market worth over $5 billion dollars (Wei et al., 2013). A promising trend is a slew of recent ethanol fermentation plants purchases by biobutanol companies. These ethanol plants are being retrofitted with advanced separation systems to allow them to produce biobutanol. Since biobutanol has inherently higher value vs. bioethanol, the trend of the plant conversions is likely to continue (Doan et al., 2012).

2.4. Production process of bioalcohols The bioalcohols production involves several processes begans at pretreatments of biomass (physically, chemically and/or biologically) and passing with the saccharification process to obtain reducing sugar then by the fermentation process yields final product. ϰϰ 

2.4.1. Pretreatment processes of biomass One of the concerns for economical production of bioalcohol from biomass is the large volume and high cost of the hydrolytic enzymes used to convert biomass into fermentable sugars (Eckard et al., 2013). Therefore, pretreatment process is required to increase the surface area of exposed cellulose and hemicellulose for microbial and/or enzymatic degradation. To achieve enzymatic degradation in the production of ethanol or in order to improve formation of biofuel, a pretreatment process is necessary. Several methods have been introduced for pretreatment of waste material to enzymatic hydrolysis. These methods are classified into: physical pretreatment, physico-chemical pretreatment and biological pretreatment (Taherzadeh and Karimi, 2008).

(i)- Physical pretreatment Physical pretreatment can increase the accessible surface area and size of pores, and decrease the crystallinity and degrees of polymerization of cellulose. Different types of physical processes such as milling (e.g. ball milling, two-roll milling, hammer milling, colloid milling, and energy milling) and irradiation (e.g. by gamma rays, electron beam or microwaves) can be used to improve the enzymatic hydrolysis or biodegradability of lignocellulosic waste materials (Taherzadeh and Karimi, 2008).

(ii)- Chemical pretreatment c- Alkaline hydrolysis Alkali pretreatment refers to the application of alkaline solutions such as NaOH, Ca(OH)2 (lime) or ammonia to remove lignin and a part of the hemicellulose, and efficiently increase the accessibility of enzyme to the cellulose. The alkali pretreatment can result in a sharp increase in saccharification, with manifold yields. Pretreatment can be performed at low temperatures but with a relatively long time and high concentration of the base. Alkaline pretreatment was shown to be more effective on agricultural residues than on wood materials. The alkaline pretreatment ϰϱ 

was also used as a pretreatment method in biogas production (Taherzadeh and Karimi, 2008).

d- Acid hydrolysis Treatment of lignocellulosic materials with acid at a high temperature can efficiently improve the enzymatic hydrolysis. Sulfuric acid is the most applied acid, while other acids such as HCl and nitric acid were also reported (Yazdani et al., 2011; Borines et al., 2013). The acid pretreatment can operate either under a high temperature and low acid concentration (dilute acid pretreatment) or under a low temperature and high acid concentration (concentrated acid pretreatment). The lower operating temperature in concentrated-acid pretreatment (e.g. 40°C) is a clear advantage compared to dilute acid processes. However, high acid concentration (e.g. 30-70%) in the concentrated acid process makes it extremely corrosive and dangerous. Dilute acid hydrolysis is probably the most commonly applied method among the chemical pretreatment methods. It can be used either as a pretreatment of lignocellulose for enzymatic hydrolysis, or as the actual method of hydrolyzing to fermentable sugars. Dilute acid pretreatment can be performed either in short retention time (e.g. 5 min) at high temperature (e.g. 180°C) or in a relatively long retention time (e.g. 30-90 min) at lower temperatures (e.g. 120°C) (Taherzadeh and Karimi, 2008).

(iii)- Biological pretreatment Microorganisms can also be used to treat the lignocelluloses and enhance enzymatic hydrolysis. The applied microorganisms usually degrade lignin and hemicellulose but very little part of cellulose, since cellulose is more resistance than the other parts of lignocelluloses to the biological attack. Several fungi, e.g. brown, white and soft rot fungi, have been used for this purpose. White-rot fungi are among the most effective microorganisms for biological pretreatment of lignocelluloses. Biological treatments with microorganisms or enzymes are also investigated to ϰϲ 

improve digestion in biogas production. It might be used not only for lignin removal, but also for biological removal of specific components such as antimicrobial substances. In addition, low energy requirement, no chemical requirement, and mild environmental conditions are the main advantages of biological pretreatment. However, the treatment rate is very low in most biological pretreatment processes (Sun and Cheng, 2002). Bacterial pretreatment of wastes involves both anaerobic and aerobic systems. Anaerobic degradation utilizes mainly mesophillic rumen derived bacteria (Hu and Yu, 1HYHVet al+Xet al E-GPRM > P-GPRM (Raposo et al., 2011) The calculating formula of GPRM was as follows: GPRM = Biogas (m3)/ TS (Kg) GPRM = Biogas (m3)/ VS (Kg) GPRM= Biogas (m3)/COD (Kg) (vi)Biochemical oxygen demand (BOD) The BOD was defined as the consuming oxygen per liter waste water which was needed for degrading organic waste by microorganism under the condition of 20oC and aerating. The unit of BOD was ml/L. The more organic waste was in water, the more BOD value was. The BOD value could indicate the organic loading of the waste water indirectly (Lesteur et al., 2011). ϳϵ 

(vii) Chemical oxygen demand (COD) The COD was defined as the consuming oxygen which was needed for chemical reaction between organic matter and potassium dichromate. The unit of COD was mg/L or kg/L. The more chemical oxygen demand was the more organic matter was. Only when the waste water was too poisonous to measure the BOD, it could use chemical oxygen demand. Both COD and BOD were applied to show the quantity of organic matter in "the material. The BOD could indicate the quantity of organic being degraded by microorganism, while the COD value was more than BOD due to some nondegradable matter in the material. The ratio between the BOD and COD was from 0.4 to 0.8 (Lesteur et al., 2011). 4.3.3. The relationship among the biogas parameter There were some principles for the designed parameter in the biogas engineering. The higher organic loading rate was the higher volume gas production rate was. The long hydraulic retention time was for high concentration and high temperature. The hydraulic retention time for manure was 60 days, while for straw 90 days (Rozzi and Remigi, 2004). The relationship among the organic loading rate, feedstock concentration and hydraulic retention time was: TS organic loading rate (kg/m3-day) = V0/V1 4.4. Biochemistry and microbiology of biogas fermentation Biogas generation was the process of catabolic metabolism of organic matter degraded by microorganism under the condition of air absence, certain moisture, and certain temperature and pH value (Chae et al., 2008). The metabolic microorganism was

called

biogas

fermentative

microorganism,

or

anaerobic

digestive

microorganism. The process called biogas fermentation, or anaerobic digestion, or methane fermentation (Ahring, 1995, Lesteur et al., 2011). However, some characteristics of the biogas fermentation were as follows: ϴϬ 

1. The energy consumption of biogas anaerobic microorganism was one twentieth or one thirtieth of that belongs to aerobic microorganism. In the anaerobic digestion, we can obtain from 100 to 300 calorie free energy, while we can obtain 3000 calorie free energy in the aerobic digestion with the degradation of per gram COD. 2. The biogas fermentation can treat organic waste water with its COD value above 10000 milligram/liter, while the aerobic digestion only can treat organic waste water with its COD value below 1000 milligram/Liter. 3. The biogas fermentative microorganism could degrade variety waste due to its low demand for nutrition 4. The biogas fermentation has the most effective temperature. 5. The biogas fermentation residues were separated from broth easily. Compared with ordinary compost, the biogas anaerobic fermentation not only utilized methane, but also conserved nitrogen (Figure 12): Methane fermentation

Composting fermentation

Figure 12: The decomposable process of organic waste 4.4.1. Three steps for biogas fermentation At present, in the study of overall process of biogas fermentation, there is little known in detail. Based on the basic process of the action of microbes in biogas ϴϭ 

fermentation, McInerney (1965) proposed a two-step process: acid-producing and gas-producing periods, which were modified later by Lawrence and McCarty (1965) who divided the process of biogas fermentation into three steps in the same year: hydrolysis, acid-producing and gas-producing, that accomplished by three major classes of microbes. Even others suggested a four step process, but the most generally acceptable are three step process. The biogas fermentation process was that the organic waste was decomposed into methane and carbon dioxide by symbiotic bacteria (Figure 13). Protein

Carbohydrates

Lipids

Amino Acid

Saccharides

Fatty acids

Pyruvic acid +Volatile fatty acid+ H2+CO2+NH3+H2S

,LJĚƌŽůĂƐĞƐ

ĐŝĚWƌŽĚƵĐŝŶŐ  ďĂĐƚĞƌŝĂ

,ϮͲWƌŽĚƵĐŝŶŐĐĞƚŽŐĞŶŝĐďĂĐƚĞƌŝĂ

CH3COOH+H2+CO2+NH3+H2S DĂƚŚĂŶŽŐĞŶƐ

CH4+CO2+NH3+H2S Figure 13: The three-step process of biogas fermentation

(iv) The first step Fermenting bacteria secrete exoenzymes hydrolyzing organics. The variety and amount of these bacteria vary with the variety and quantity of the organics involved. Based on the substrates they act upon they are divided reasonably into cellulosesplitting, fat-splitting and protein-splitting ones. By the actions of them, polysaccharides are hydrolyzed into monosaccharides, proteins into peptides or amino acids and fats into glycerol and fatty acids. (v) The second step The hydrogen-producing acetogenic bacteria, such as Acetobacterium xylinum, some clostridium, can catabolize higher fatty acids to yield hydrogen and acetic acid. ϴϮ 

In addition the long chain fatty acids and aromatic amino acids produced in the 1st step can also be degraded yielding H2; and acetic acid. (vi) The third step Methane-producing bacteria utilize the simple compounds, i.e. acetic acid, hydrogen, formic acid and CO2, to form methane and carbon dioxide. For simplicity, many authors also usually generalize the overall process of biogas fermentation to two steps, in this connection the fermenting microbes involved can be grouped into non-methane-producing and methane-producing ones respectively. There, however exist different ideas is no matter the process of biogas fermentation is grouped into two, three or four steps. Many investigators think that it is not sure to group the process of biogas fermentation to acidifying and aerifying steps, because what is yielded in acidifying step is not only acid while gases produced do not occur only in the aerifying step. Actually, the two steps are not invariably departed, but proceed simultaneously and alternately. Thus, in 1977, Mathematician proposed a diagram for the process of catabolism of complex organics (Figure 14). It is necessary for normally and vitally proceeding biogas fermenting process to have mutually combining action of non--methane-producing and methane-producing bacteria. The excess or lack in amount of any group of the bacteria and functional activeness or inactiveness could lead to destroy the kinetic balance, lead to an abnormality even failure of the process of fermentation (Triolo et al., 2011).

Figure 14: The process of catabolism of complex organics ϴϯ 

4.4.2. Microbes in three-step of biogas fermentation 4.4.2.1. Biogas microbes in nature Biogas fermentation is a common occurrence and typical process in nature and an important component of Mass Cycle of Nature (Figure 15) (Schnürer and Jarvis, 2010). Biogas fermenting microbes are widely distributed in nature, especially in lakes, manure pit, sewage and various organized sludge (Bruni et al., 2010). These are resources for obtaining utilizable biogas by humans. Methane bacteria also found in some higher plants and animals, among which ruminant stomach of ruminates is a typical organ of biogas fermentation. Through a study of microbes in ruminant stomach, men obtained good knowledge concerning bacteria. There are plenty of biogas bacteria in ruminant stomach where methane and carbon dioxide are formed. In the ruminant stomach of a head of milk cow, there are l00 liters of cellulose fermenting material, which by fermentation can give more than 200 liters of methane, breathing out during expiration (Rozzi and Remigi, 2004).

lst. Step: Fermenting Bacteria

2nd. Step: Hydrogen producing acetogenic bacteria 3rd. Step: Methane producing bacteria CH4 + CO2 + H2

Figure 15: Microbes involved in three steps in nature 4.4.3. Groups and actions of non-methane-producing microbes Non-methane-producing microbes are series of microbes converting complex organics into simpler, smaller molecular compounds, i.e. what have been mentioned previously: the fermenting bacteria and hydrogen-producing bacteria (Figure 16). The ϴϰ 

microbes involved in this step include anaerobes and facultative anaerobes, their variety and amount change with the variety and quantity of fermenting materials (Bal and Dhagat, 2001). Although at present a basic understanding of the physiological aspects of various microbes in non-methane-producing step has been achieved. A study of isolation and identification of some pure strains of some bacteria has been performed by some investigators, in brief, the microbiology of non-methane producing step is still less and needs further study later (Angelidaki et al., 2009). Polysaccharides, Amino acids, Fatty acids (long chain) Non-methane-producing bacteria Volatile fatty acids (low MW), Alcohols, Neutral compounds, H2, CO2 Methane-producing bacteria and non-methane-producing bacteria Acetic acid, CH4, CO2 Methane-producing bacteria ± non-methane-producing bacteria CH4+CO2 Figure 16: Catabolism of complex organic compounds

4.4.3.1.Variety of non-methane-producing microbes By classification, the variety of non-methane-producing microbes may be divided into three groups, namely, bacteria, fungi and protozoa, among which bacteria are the most of importance (Weiland, 2010). (i) Bacteria There are many types of non-methane-producing bacteria. But those microbes with hydrolytic activity accounted for a small group of the overall colonies, among of which the obligate anaerobes are 100 to 200 times those of the facultative anaerobes and aerobes. The obligate anaerobes are those involved in non-methane-producing step, that play an important role in the process, including Clostridium butyricum, Bacillus lactam, Gram‘s positive cocci and so on. In the 50's and 60's, a study of isolation and identification of non-methane-producing bacteria was performed by ϴϱ 

many investigators (Amon et al., 2007). The result of a study of anaerobic digesting microbes in beef's waste showed that there were three colonies of anaerobic and facultative anaerobes: the fDPLO\ RI IDFXOWDWLYH FRFFL a WKH IDPLO\ RI %DFWHURLGHV a DQG RWKHU DQDHURELF EDFWHULD PDLQO\ Clostridium, with a small number of curved Gram’ s negative bacillus (VDI 4630, 2006). According to census from a part of the data, the non-methane-producing bacteria isolated and studied have reached 50 species in 18 genera (Table 6). Based on their physiological aspects, non-methane-producing bacteria are again divided into seven groups, namely: cellulose-splitting, semi-cellulose-splitting, protein-splitting, fat-splitting, hydrogen-producing bacteria and other specific microbes, such as thio-vibro and lactic acid-utilizing ones. Certain colony group of bacteria can also be of several functions, for example, some cellulose-splitting bacteria may be capable of catabolizing proteins (Weiland, 2010). Table 6: Some non-methane-producing bacteria in biogas fermentation Genus

Species studied

Aerobacter Aeromonas Alcaligenes Bacillus Bacteroides Clostridium Escherichia Klebsiella Leptospira Micrococcus Neisseria Para-colobacterium Proteus Rhodopseudomonas Sarcina Serratia Streptococcus

1 12 4 12 1 2 3 1 2 5 1 2 9 1 2 1 1

(ii) Fungi In the 50's and 60's, Cooke and his workers found great number of mould and yeasts in anaerobic digestion and isolated 36 genera of mould, among which there ϴϲ 

were plenty of half-known microbes and some zygorhynchus. Through artificial cultivation and recovery detection test, he thought that mould and yeasts might take part in digestion process, from which they got nutrients and grew (Bengelsdorf et al., 2012). (iii) Protozoa Protozoa were also detected during biogas fermentation process. Bengelsdorf et al. (2012) pointed out that detectable protozoan were mainly plasmodium, flagellate and amoeba etc., totally 18 species, being less in amount, so they might play a minor role in the process.

4.4.3.2. The amount of non-methane-producing bacteria In anaerobic digestion, bacteria are among the most of non-methane-producing microbes (Cirne et al., 2007). Bacterial counting was done by many investigators but due to the counting techniques used and some other reasons, the results showed variance (Table 7). Based on a technique of incubation of microbes from the ruminant stomach, a kind of counting non-methane-producing bacteria in biogas fermenting system was developed and the numbers of bacteria in several digesters were 39x107- l5x109/ml for obligate anaerobic non-methane-producing bacteria, 8x105-1x108/ml for aerobes and facultative aerobes; the amount of anaerobic bacteria was often 100 times to that of aerobic ones (Kampmann et al., 2012). This indicates that in biogas fermentation, the species group of non-methane producing bacteria in specific anaerobic conditions is, in amount, the most important part of the overall colonies of microbes. In addition, the physiological amount of some microbes in biogas fermentation was detected separately by many investigators. Data reported by Lahav et al. (2002) were that anaerobic cellulose-splitting bacteria in digesting sludge was 0.82.0x103CFU/ml, and for domestic wastes 4xl05 CFU/ml; the amount of semicellulose-splitting bacteria were similar to that of cellulose-splitting bacteria, being 104 CFU/ml or little bit more. Using dilution technique, the amount of proteinϴϳ 

splitting Gram‘s positive bacteria in a being digested sludge was 7xl04 CFU/ml. Sulfur-reducing bacteria detected from digesting sludge was 3 - 5x104 CFU/ml, lactate-utilizing bacteria in the digested material of Beefy waste were 3xl07 CFU/ml. The total amount of aerobic bacteria in digesters of several laboratories and large scale production units were 3 - 300xl06 CFU/ml; aerobic protein-splitting bacteria l 90xl05 CFU/ml and aerobic fat-splitting bacteria 2 – 16xl04 CFU/ml. Table 7: Amount of non-methane-producing bacteia Amount x 103/ml

Group of Bacteria

Materials fermented

Count totally (aerobic)

fatty acids

200-20000

Count totally (aerobic)

Sugars

1500-350000

Count totally (microscopic)

sludge (underground)

6000

Count totally (aerobic)

sugars and wastes

3000-300000

Count totally (anaerobic)

synthesized substrate

390000-1500000

Count totally (aerobic)

synthesized substrate

800-100000

Protein-splitting bact.(aerobic)

sugars and wastes

100-9000

Fat-splitting bact.(aerobic)

sugars and wastes

20-160

Cellulose-splitting bact.(anaerobic)

underground waste

0.8-2.0

Cellulose-splitting bact.(anaerobic)

underground waste

16-970

Leptospira Sp.

underground waste

1

4.4.4. Estimation and calculation of the amount of methane 4.4.4.1. Methane produced from organic compounds The theoretical value of methane production can be estimated and calculated according to the chemical composition of materials. In 1939, Buswell and Neave, through a series of experiments, found that all organic compounds can undergo anaerobic degradation to yield methane, and the amount and composition of methane generated of their complete degradation are determined by the composition of materials added. The relationship may be expressed by the formula CnHaOb + (n - a/4 - b/2) H2O = (n/2 – a/8 +b/4) CO2 + (n/2 + a/8 – b/4) CH4 When n> a/4 + b/2, water contributes to methane production. If the chemical compositions of the fed materials are known, the theoretical amount of gas generated may be calculated through the formula. The formula is called Buswell Formula. By using this formula, the amount and components ϴϴ 

generated from three major organic compounds was calculated (Table 8). Data listed in Table 4 of various organic compounds vary (Ushida, 2011). It was simply calculated because of the greater variance in chemical compositions of different polysaccharides, proteins and fats used (Laukenmann et al., 2010). Table 8: Gases generated by several organic compounds under completely anaerobic conditions Organics carbohydrates Lipids Proteins

Component (Wt.%) CO2 73 72 50

Amount of gas produced/kg TS (m3)

CH4 27 28 50

Biogas 0.75 l.44 0.98

CH4 0.37 l .04 0.49

4.4.5. Anaerobic digestive process of complex organic compounds 4.4.5.1. Degradation of carbohydrates Polysaccharides are the main stuffs for biogas fermentation. They consist principally of carbon, hydrogen and oxygen, expressed by the formula Cn(H2O)n. According to the extent of their hydrolysis, there are monosaccharides, oligosaccharides and polysaccharides can be further hydrolysed to still smaller ones. Oligosaccharides are those of 2 - 6 monosaccharides after condensation while those of many monosaccharides are known as polysaccharides (Chynoweth, 1978). The variety of polysaccharides is rich, including cellulose, starch, and xylose and lignin. Under anaerobic conditions, polysaccharides can be hydrolyzed by exoenzymes secreted by microbes, giving mainly glucose, which can be further, degraded (Amon, et al., 2007). 4.4.5.2. Anaerobic degradation of glucose Much more work of investigation have been done to anaerobic degradation of glucose and the results showed: glucose is mainly undergone anaerobic glycolysis forming pyruvic acid, still somebody thought that in biogas fermentation glucose may undergo pentose phosphate pathway forming glycerol-phosphate and then to pyruvic acid (Pirc et al., 2010) ϴϵ 

Acetic acid is easily to be convened into methane. Thus, from pyruvic acid through acetic acid and finally to methane is a main pathway in biogas fermentation Figure 17. Each molecule of glucose can produce three molecules of methane and three molecules of carbon dioxide, among which two third of methane is from acetic acid (Pirc et al., 2012). C6H12O6 —>2CH3CH2CH2COOH+2H2 2CH3CH2CH2COOH +2H2O + CO2 —> 4CH3COOH+CH4 4CH3COOH —> 4CH4+4CO2 CO2+4H2 —> CH4+H2O ________________________________________________ C6H12O6 ——> 3CH4 + 3CO2

The overall metabolic pathways of glycolysis are as follows: G1ucose Glucose- 6-phosphate Frucrose 1,6- phosphate CH2OH Triose phosphates

C=O HCOH

3-phospho-glycerate

HCHOP

2-phosphoenol pyruvate

CH3CHOHCOOH Pyruvate

HCOOH CH3CH2OH

CH3COOH CH3CH2CH2COOH CH4+CO2 Figure 17: Anaerobic degradation of cellulose pathway ϵϬ 

4.4.5.3. Cellulose after removal of lignin Cellulose is an important component of composition of rural biogas fermenting resources, which together with semi-cellulose constitutes of about 50-60% of the total solids of straw, and 30- 50% of that of dung resources (Holwerda et al., 2014). Cellulose is one kind of polysaccharides, which is composed of glucose units, linked together through P-D-l.-4- glucosidic bonds. The overweighting bulk of all natural cellulose exists in the form of long unbranched chain and its molecular weight may reach from hundred thousand to millions. A great number of cellulose molecules constitute microprobril, integrated in bundless known as microfibril (Lynd et al., 2005). Microfibril together with lignin and semi-cellulose constitutes complex dense in structure. Pure cellulose is easily degraded by biogas microbes while naturally occurring ones, due to its combination with lignin are not easily splitted by the microbes. Cutting and grinding short chain, and treating remain chain thermochemically may speed up its degradation (Lu et al., 2006). Some anaerobic microbes can combine cellulose to form cellulose-enzyme complex, i. e. "compound enzyme", being composed of C1, C and ß-glucosidase. The order of action is as follows: Through the action of these enzymes, cellulose is hydrolyzed into glucose. The enzymes are of two kinds, one is exoenzymes dissolved in the fermenting fluid and the other is a cell surface bonded enzyme. Taking cellulose as the only carbon resource for biogas fermentation revealed, there were three peaks appeared and the second peak showed the highest production of gas. In the process of fermentation, butyrate-3-utilizmg microbes grow tremendously. When the fermentation is blocked, there is a marked increase in amount of butyric acid and acetic acid-utilizing microbes which may double their number to thousand times (Holwerda et al., 2013). 4.4.5.4. The metabolism of semi-cellulose, pectin-gel, starch, and cellulose under anaerobic conditions Semi-cellulose is a mixture of poly-condensed pentoses and poly-condensed hexoses. Pectin-gel is one kind of poly-condensed pentoses amounting less in ϵϭ 

resources of biogas fermentation. Starch is one kind of high molecular weight compound being composed of -l, 4-glucosidic bonds. Under anaerobic conditions, those three groups of materials all can be easily hydrolyzed into pentose, hexose, thereby undergoing further degradation in the saccharide fermenting process (Olson et al., 2013). Lignin is one kind of shapeless, cyclic polymeric compounds, and usually exits in combination with cellulose and semi-cellulose, which are complex compounds; hardly being degraded by microbes. Some thought that lignin may form fermented plant acid, which has an accelerative affect on the metabolism of biogas microbes (He et al., 2011).The often used rural biogas fermenting resources contain large quantity of lignin, being 21% the total solids of beef dung, 35% of cow dung and l2% of rice straw. To study the action of lignin in biogas fermentation and to search for an effective method of splitting it, therefore, would be helpful for better utilizing biogas resources (Desvaux et al., 2000). 4.4.5.5.Metabolism of lipids Lipid is rather a large kingdom, including fats and oils, waxes, phospholipids, glycolipids and steroids, which are insoluble in water and soluble in solvents at ether and chloroform, etc. Lipids in biogas fermenting materials are mainly fats. They are composed of glycerol and fatty acids. Under anaerobic conditions, fats are easily to be hydrolyzed into glycerol and fatty acids, the glycerol formed can be converted into dicarboxil-phosphoacetone, thereby, through fermenting pathway to form pyruvic acid (Lakaniemi et al., 2011). CH2OH

CH2OH

CH2OH

CHOH

CHOH

CO

CH3

CH2OH

CH2OPO3H2

CH2OPO3H2

COOH

CH4

Fatty acids undergo –oxidation pathway forming acetoacyl ß–coenzyne A(CH3CO-SCoA) and then acetic acid. The hydrogen (2H) released in ß–oxidation can be reduced to form methane (Figure 18). ϵϮ 

Figure 18: ß-oxidation pathway of Fats (Knoop hypothesis)

4.4.5.6. Metabolism of protein There is less protein in amount of rural biogas fermenting resources. In biogas fermentation, proteins are hydrolyzed into peptides or amino acids. Peptides and amino acids can be either utilized by microbes for synthesizing cellular substances or further degraded into lower molecular weight fatty acids, H2S, amines, phenols and ammonium (Figure 19). Low molecular volatile fatty acids and amine can further be converted to form methane, ammonia, on the other hand, can either be utilized as nitrogenous source for synthesizing cellular components or forming ammonium bicarbonate (NH4HCO3), thereby HCO3- ion is increased, raising buffer capacity in the fermenting fluid and favoring methane formation. Hydrogen sulfide can make some heavy metals precipitated, releasing their toxic effect away from the system. As experienced, biogas fermenting fluid often appeared dark in color; which came out formation of hydrogen sulfide. Proteins and their nitrogen-containing component are of importance to microbe‘s nutrition and methane formation (Kovács et al., 2013).

ϵϯ 

4.4.6.

Fermentative bacteria

The organic compounds-carbohydrate, proteins and fats-in biogas fermenting material, by action of liquefaction of non-methane-yielding microbes (exoenzymes) are splitted into soluble simpler compounds after entering bacterial cells, these simpler compounds are undergone various decomposition yielding organic acids, alcohol, ketones and CO3, H3, NH3, H2S etc. The methane-producing bacteria can not directly utilize organic compounds to form methane unless they are degraded to simple compounds of smaller molecular compounds by non-methane-producing bacteria. Non-methane producing microbes, therefore, are of importance in biogas fermentation (Ahring, 2003) As mentioned above, according to their physiology, non-methane-producing bacteria are grouped into seven classes, which are of different functions (Wirth et al., 2012). I. Cellulose-splitting bacteria II. Semi-cellulose-splitting bacteria III. Starch-splitting bacteria IV. Protein-splitting bacteria V. Fat-splitting bacteria VI. The obligate H2 producing acetogenic bacteria Concept was proposed by Bryant and Woln |(1967) through their metabolic coupling analysis of species between ruminant methanobacterium and "S" organism grown on medium containing acetoketones; metabolic relationship between species and methane-producing and non-methane-producing bacteria is expressed mainly in "hydrogen transfer between species" i.e. hydrogen liberated in the anaerobic respiration by non-methane-producing bacteria is utilized by methane-producing bacteria serving as part of substrates to form methane. This fashion of oxidationreduction between two bacteria or more than two of different species is known as interspecies H2-Transfer. For example, as illustrated by Bryant et al (1967), M. onteliansltii was coupled by MOH strain of methanobacterium with "S" organism to ϵϰ 

form “symbiotic body" (intergrowth), within this symbiotic body ethanol was oxidized by "S" organism forming CH3COOH and H2 (this reaction can also be inhibited by self-produced H2) and H2 produced was utilized by MOH strain to synthesize CH4; in this connection, H2 producing was increased by "S" organism. Taking this as presupposition for inter-survival, molecular hydrogen is the intermediate body for coupling of two bacteria (Aubert et al., 2000). That the effect of hydrogen transfer occurs between species leads to degradation and utilization of substrates by microbes under anaerobic conditions (Figure 19). 2CH3CH2OH

4H2

CO2

2H2O

2CH3COOH "S" strain

CH4+2H2 Methanobacterium bryantii

Figure 19: Interspecies H2-transfer There also exists hydrogen transfer between methanobacterium and H2 producing bacteria, such as sulfate-reducing bacteria besides what mentioned above. In addition, some extent of combination of Methanosarcina with non-methaneproducing bacterium of several oblique anaerobes does occur and this kind of nonmethane producing bacteria of these oblique anaerobes is known as satellite organisms. For example, Eubacterium limosum, Bacteroides sp. and one species of Bacteroides totally knew as 3rd isolates. All of them are likely to be hydrogenproducing bacteria and their relationship to Methanobacterium might be that of "hydrogen transfer between species (Calteau et al., 2005) In biogas fermentation, owing to the presence of "hydrogen transfer between species" between Methanobacteria and non-methanobacteria, hydrogen is provided successively by hydrogen-producing bacteria while H; yielded is used continuously by Methanobacteria, which ensures hydrogen production by H2-producing bacteria, ϵϱ 

not inhibited by accumulation of it. It was the dynamic equilibrium that kept the biogas fermentation progressing normally (Chen et al., 2005) (Table 9). Besides what have been discussed above, i.e. cooperative actions between them, there exist mutual inhibiting and mutual restricting actions between different biogas fermenting microbes, including that of metabolites themselves and that of species. Furthermore, the optimum pH and oxidation-reduction potentials between acid-forming bacteria and methanobacteria are markedly different. In a digesting system, it is impossible to accommodate the living demands for both microbes. Eventually there exit contradictions (Finke et al., 2007). In the early stage of biogas fermentation, however, the conditions are favorable to acid-forming microbes that grow more prosperously, being predominant in this stage. As time goes on, ammonium yielded by the action NH3 producing bacteria causes a gradual increase in pH and a fall in oxidation-reduction potentials, which favorite the activity of methanobacteria resulting in an increase in amount of them. And again, the increase of pH resulted by the action of NH3-producmg bacteria restricts the action of acid-forming bacteria and collaborates on methanobacteria, thereby an equilibrium of digestion and biochemical changes in this process from acid formation to methane-production is achieved (Hillesland and Stahl, 2010)

Hydrogen-producing acetogenic bacteria in aqua of biogas fermentation Apart from acetic acid, CO2 and H2 in the products of anaerobic fermentation of carbohydrate and protein which can be directly utilized by methane-producing bacteria, the rest, such as alcohol, volatile saturated organics, including butyric acid and propionic acid cannot be used directly by them which cannot serve as resource of energy and carbon unless they are further converted into acetic acid, CO2 and H2. This group of bacteria, therefore, is of extreme importance in the colony of nonmethane-producing microbes (Kato and Watanabe, 2010).

ϵϲ 

Table 9: Syntrophic relations between hydrogen-producing acetogenic bacteria and methanogenic bacteria Ethanol Butyric

Syntrophomonas wolfei Methanogenic bacteria

Propionic

Syntrophobacter wolinii Methanogenic bacteria

Benzoic

Bacteria "S" organism Methanogenic Bacteria

Syntrophus buswelii Methanogenic bacteria

Acetic

Substrate

Acetate oxidizing bacteria (Clostridium aceticum)

Bioreaction 2Ethanol-+2H2O 2Acetate+2H++4H2 4H2+2HCO3-+H+ 3H2O+CH4 2Ethanol-+2HCO32Acetate+H++H2O+CH4

Total reaction

Total reaction

2Butyrate-+4H2O 2Acetate+H++4H2 4H2+2HCO3-+H+ 3H2O+CH4 2Butyrate-+2HCO32Acetate+H++H2O+CH4 Propionate-+12H2O 4Acetate+4HCO3-+ 4H++12H2 + 12H2+3HCO3 +3H 9H2O+3CH4 Propionate-+3H2O 4Acetate+ HCO3-+H++3CH4

Total reaction

Benzoate-+28H2O 12Acetate+4HCO3-+ 12H++12H2 12H2+3HCO3-+3H+ 9H2O+3CH4 Benzoate-+19H2O 12Acetate+ HCO3-+9H++3CH4

Total reaction

Acetate-+4H2O 4H2+HCO3-+H+ Acetate-+H2O

Methanogenic bacteria Total reaction

2HCO3-+H++4H2 3H2O+CH4 HCO3-+CH4

Front l980 to l982, a study of hydrogen-producing microbes in biogas digester was performed by Chengdu Institute of Biology, Chengdu Branch of Chinese Academy of Science and the result showed that when adding Chinese yam to the abstracting media of biogas sludge, the production of hydrogen was abundant. And 24 strains of hydrogen-producing bacteria were isolated from the fermenting material in a biogas digester which belongs to Enterobacteriaceae and Bacillaceae. There were five species in Enterobacteriaceae while there was only one species in Bacillaceae. The propanotyl-alcohol Clostridium isolated was of stronger hydrogen-producing capability and its starch-utilizing ability is also strong. By combining the media of hydrogen-producing bacteria and methane-producing bacteria for incubation, the production of methane can be raised while CO2 content is reduced markedly (Wudi et al., 2010). Some other specific bacteria were also found in anaerobic digestion: a) Desulphoribric bacteria-reduce sulphates, ϵϳ 

b) Lactic acid utilizing bacteria-mainly utilize lactic acid producing acetic acid and propaonic acid including Streptococcus sp., Bacteroides sp, and Clostridium sp. c) Besides, photosynthesizing bacteria were also isolated from biogas digester, but their actions involving anaerobic digestion still need further study.

4.4.6.1. Methane-producing bacteria The main product of biogas fermentation is methane, which is produced by the action of methane-producing bacteria. Thus, methane-producing bacteria again are the core of biogas fermenting microbes. The study of methane-producing bacteria is not only for investigating the mechanism of fermentation and biogas production but also of importance in studying the origin of life (Batstone et al., 2014).

4.4.6.2. Morphology and classification of methane-producing bacteria 1. Forms of methanobacteria There are four forms of Methanobacterium, Sarcina, Bacillus, globular and spiral (Figure 20) (Bengelsdorf et al., 2012). a) Methanosarcina The cell multiplication of Methanosarcina is regular; its size is similar like sand particles piled together, not only its form but its size is different from that of true Sarcina. Even in the same species of Methanosarcina; their envelops are difference. b) Methanobacillus Methanobacillus is bacillary, usually curved, like a chain or long filament while that of M. arbophilicum is short and straight. No filament-formation in liquid culture. c) Methanococcus The globular cells of them are from round to oval, paired or chained. d) Methanospirillum The cells appear regular, curved and finally form spiral. ϵϴ 

DĞƚŚĂŶŽƐĂƌĐŝŶĂ

 DĞƚŚĂŶŽďĂĐŝůůƵƐ

 DĞƚŚĂŶŽƐƉŝƌŝůůƵŵŚƵŶŐĂƚĞŝ

DĞƚŚĂŶŽĐŽĐĐƵƐ

Figure 20: Morphology of Methanobacteria

4.4.6.3. Classification of Methanobacterium Regarding classification of Methanobacterium at present there is no better integrated idea. It was summed up Methanobacteria as one family, i.e. Methanobacteriaceae, including bacilli and cocci. This family consists of four genera and 8 species (Bodelier et al. 2005). In Bergy's Manual of Determination Bacteriology, Methanobacteriaceae was summed up in one family including 3 genera and 9 species. It was suggested a newer classification system by which methanobacteria were grouped into 3 orders, 9 families, 7 genera and 13 species (Cirne et al., 2007). Now it is added to 5 orders, 10 families, 25 genera and 59 species (Table 10). 4.4.7. Metabolic substrates of methane-producing bacteria The methane-producing bacteria could utilize H2, HCOOH, CH3OH, methylamine, acetic acid etc., as substrate to produce methane. Some special methanogens may use ethanol, propionic acid, iso-butyric acid to produce methane. About two-third methane was originated from acetic acid splitting, while the other ϵϵ 

methane was come from carbon dioxide reduction. Metabolism of methane with pure culture was studied and found that almost methanogens could utilize H2 and CO2 to produce methane (Gerardi, 2003). Table 10: Classification of Methanobacteria Methanobacteriales

ORDER

FA M l LY Methanobacteriaceae

Methanococcales

Methanothermaceae Methanococcaceae Methanocaldoccaceae

Methanomicrobiales

Methanomicrobiaceae

Methanosarcinales

Methanocorpusculaceae Methanospirillaceae Methanosarcinaceae

Methanopyrales 5

Methanosaetaceae Methanopyraceae 10

GENUS Methanobacterium Methanobrevibacter Methanosphaera Methanothermobacter Methanothermus Methanococcus Methanothermus Methanocaldoccus Methanoignis Methanomicrobium Methanolacinia Methanogenium Methanoplanus Methanocalleus Methanofolollis Methanocorpusculum Methanospirillum Methanosarcina Methanolobus Methanococcoides Methanohalophilus Methanohalobium Methanosalsus Methanosaeta Methanopyrus 25

SPFCIES 10 3 2 2 2 4 1 1 1 1 1 3 2 3 1 4 1 5 4 1 2 1 1 2 1 59

Some chemical formula about metabolic substrates of methane-producing bacteria was as follows: H2 reducing CO2: 4H2+CO2= CH4+H2O Formic acid: 4 HCOOH= CH4+3CO2+2H2O ϭϬϬ 

Methanol: 4CH3OH = 3CH4+ CO2+2H2O Acetic acid: CH3COOH= CH4+CO2 Methylamine: 4CH3NH +2H2O = 3CH4+4NH3 +CO2 Dimethylamine: 2(CH3)2NH +2H2O = 3CH4+2NH3 +CO2 Trimethylamine: 4(CH3)3N +6H2O = 9CH4+4NH3 +3CO2 4.4.8. Relationships between different biogas microbes There exist multiple denies of species and at spectrum of complex microbes in biogas fermentation. Methane production is the result of such mutual actions and restrictions between various microbes of this microorganism's spectrum (Hobson and Wheatley, 1993). The mutual actions of biogas fermenting microbes include the actions between non-methane-producing bacteria and methane-producing bacteria, and the actions between non-methane -producing bacteria, among which the former is the most of importance. In a biogas fermentation system, non-methane-producing bacteria and methaneproducing bacteria depend upon each other and serve material basis and optimum conditions for the needs of their life span, but at the same time they restrict each other (Laurent et al., 2009) This mode of the relationship between them exhibits mainly the following feature: (l) Non-methane-producing bacteria provide substances required for the growth and methane production of methane-producing bacteria whilst the later get rid off the feedback inhibition for the former. Non-methane producing bacteria make various complex organic materials undergo anaerobic fermentation and form H2, CO2, NH3, CH3COOH, HCOOH, CH3CH2COOH, CH3CH2CH2COOH, CH3OH,

C2H5OH

and

so

forth;

among

which

CH3CH2COOH,

CH3CH2CH2COOH, C2H5OH etc. can be converted into H2, CO2 and CH3COOH etc. by hydrogen-producing acetogenic bacteria microbes. Thus, non-methane-bacteria through their life processes, provide materials and energy for continuous synthesis of cellular" components, and precursors and energy ϭϬϭ 

needed by methanobacteria. On the other hand, the products themselves yielded by non-methane-bacteria can inhibit process of fermentation: the accumulation of acids can inhibit acid-producing bacteria to yield acid subsequently and accumulation of hydrogen inhibits hydrogen production, but because of the products- acids, H2 and CO2 being successively used by methanobacteria, which enables non-methanobacteria grow and metabolize normally. Owing to these mutual actions between them, a balance of acid-producing and CH4-forming mechanism is established in the process which turns the process out normally. (2) Non-methanobacteria provide an optimum oxidation-reduction environment (anaerobic condition) for methanobacteria in the early stages of biogas fermentation, due to excess of air which gets into the biogas digester accompanying the feeding of materials and water. But because of the action of aerobes and facultative anaerobes of non-methane-producing bacteria along which causes a continuous fall of the oxidation-reduction potentials in the media of fermentation, which gradually provides an anaerobic condition to the growth and methane formation by methane-producing bacteria. (3) Toxic substances released by non-methane-producing bacteria for methaneproducing bacteria. Using waste-water and wastes from industry as resources for fermentation, there usually exist toxic substances harmful to methane-producing bacteria, such as phenols, benzene, cyanides, long-chain fatty acids and heavy metals. Fortunately, those toxic substances can be released and utilized by many of non-methane-producing bacteria, which favorites the process of fermentation. In addition, the toxic effects of heavy metals can be abolished by forming precipitates of metal-sulfuric compounds (heavy metal ions +H2S released by non-methane-producing bacteria), thereby some toxic effects of heavy metals are got rid of. (4) Optimum pH is maintained by the mutual actions of them. In the early stage of biogas fermentation, starch and sugars are first degraded by non-methaneproducing bacteria and large amount of organic acids are produced, and at the ϭϬϮ 

same time CO2 yielded is dissolved in water, which cause a drop of pH of the fermenting media. Ammoniation, however, occurs in the system forming NH3 which neutralizes part of the acids, and furthermore because of CH3COOH, H2 and CO2 being successively used by methane-producing bacteria and formed NH3 resulting in stabilization of pH in a favorable range. 4.5. Biogas use 4.5.1. Biogas conversion options The choice in the final means for utilization of biogas impacts the design and equipment requirements for biogas processing, storage and the economics of the biogas conversion system (Luostarinen, 2011). The biogas may be applied in direct combustion systems (boilers, turbines, or fuel cells) for producing space heating, water heating, drying, absorption cooling, and steam production. The gas used directly in gas turbines and fuel cells may produce electricity. An alternative choice in biogas conversion is the use in stationary or mobile internal combustion engines which may results in shaft horsepower, cogeneration of electricity, and/or vehicular transportation. A final opportunity exists for sale of the biogas through injection into a natural gas pipeline (Colombo et al., 2007).

4.5.2. Treatment of biogas The hydrogen sulfide contained in biogas caused odors, corrosiveness, and sulfur emissions when the gas is burned. High levels of sulfide in biogas may require removal to protect equipment if the gas is to be used in internal combustion engines, turbines, or fuel cells. The concentration of hydrogen sulfide in the gas is a function of the digester feed substrate and inorganic sulfate content. Wastes which are high in proteins containing sulfur based amino acids (methionine and cysteine) can significantly influence biogas hydrogen sulfide levels (Einola et al., 2001). For instance, layer poultry waste containing feathers made of keratin may produce biogas sulfide levels up to 20,000 ppm. Also, sulfate present in the waste, either ϭϬϯ 

from an industrial source (eg. pulping of wood) or from seawater (marine aquiculture) will be reduced by sulfate reducing bacteria in the digester and end up contributing to sulfide levels in the gas (Davidsson et al., 2007). The treatment of biogas may include removal of components including hydrogen sulfide, water, mercaptans, carbon dioxide, trace organics, and particulates. Due to the corrosive nature of hydrogen sulfide, removal processes for this component are well developed and include both dry and wet removal processes. In a wet process the biogas is passed up-flow through a stripping tower where the aqueous solutions are sprayed counter-currently. The tower is generally separated by distribution trays which maximize contact between the biogas and the solution (Kryvoruchko et al., 2009). For small-scale biogas producers, an alternative to the wet absorption systems described above is dry adsorption or chemisorption. Several dry processes are available, using particles of either activated carbon, molecular sieve, iron sponge or other iron-based, granular compounds to remove sulfide from the gas phase to the solid phase. These are sometimes referred to as dry oxidation processes because elemental sulfur or oxides of sulfur are produced (and can be recovered) during oxidative regeneration of the catalyst (Luostarinen et al., 2008). In addition to those aqueous absorbents described for H2S removal in the previous section, there are many chemical solutions commercially available which can be used to remove CO2 and H2S concurrently. In general, these processes employ either solvation solutions where the objective is to dissolve CO2 and H2S in the liquid, or solutions which react chemically to alter the ionic character of these gases and therefore drive them into solution. Solutions of the former category include the solvents and the latter include the alkanolamines and alkaline salts (Lindrofer et al., 2008). There are membrane materials which are specially formulated to selectively separate CO2 from CH4. The permeability of the membrane is a direct function of the chemical solubility of the target compound in the membrane. To separate two compounds such as CO2 and CH4, one gas must have a ϭϬϰ 

high solubility in the membrane while the other is insoluble. Accordingly, rejection (separation) efficiencies are typically quite high when the systems are operated as designed (Masse et al., 2007). 4.5.3. Storage of biogas Biogas is not typically produced at the time or in the quantity needed to satisfy the conversion system load that it serves. When this occurs, storage systems are employed to smooth out variations in gas production, gas quality and gas consumption. The storage component also acts as a reservoir, allowing downstream equipment to operate at a constant pressure (Mata-Alvarez, 2003). A wide variety of materials have been used in making biogas storage vessels. Medium-and highpressure storage vessels are usually constructed of mild steel while low-pressure storage vessels can be made of steel, concrete and plastics. Each material possesses advantages and disadvantages that the system designer must consider. The newest reinforced plastics feature polyester fabric which appears to be suitable for flexible digester covers. The delivery pressure required for the final biogas conversion system affects the choice for biogas storage (Mladenovska et al., 2006).

4.5.4. Compression of biogas The operating gas pressure for most anaerobic digesters rarely exceeds 24 “WC and can be used without some form of compression, only in the simplest direct combustion devices such as flares and simple boilers. In addition the pressure drop along delivery piping and in clean-up processes can entail the need for some type of blower or compressor to overcome these losses. The use of biogas in mobile engines requires compression to high pressures to achieve minimal storage volume (Mladenovska et al., 2006). 4.5.5. Biogas utilization Biogas can be used readily in all applications designed for natural gas such as direct combustion including absorption heating and cooling, cooking, space and ϭϬϱ 

water heating, drying, and gas turbines. It may also be used in fueling internal combustion engines and fuel cells for production of mechanical work and/or electricity. If cleaned up to adequate standards is may be injected into gas pipelines and provide illumination and steam production (Persson et al., 2006). Finally, through a catalytic chemical oxidation methane can be used in the production of methanol production. 4.5.6. Direct combustion Biogas conversion in direct combustion provides the simplest method of direct utilization on-site. Most combustion systems designed for either propane or natural gas may be easily modified for biogas. Care must be taken to consider the heat input rate, the fluid handling capability, the flame stability and the furnace atmosphere when such modifications are made. Due to the lower heating value of biogas equipment may operate at a lower rating and the size of gas inlet piping may need to be increased (Raven and Gregersen, 2007). If cogeneration is employed in the biogas conversion system heat normally wasted may be recovered and used for hot water production. In the gas of gas turbines, the waste heat may be used to make steam and drive an additional steam turbine with the final waste heat going to hot water production and this is termed a combined cycle cogeneration system. Combining hot water recovery with electricity generation, biogas can provide an overall conversion efficiency of 65-85%. Modern gas turbine plants are small, extremely efficient, and visually unobtrusive (Vasen et al., 2004). An additional direct combustion conversion process which should be considered is the use of steam to run adsorption refrigeration systems. Such systems can be employed to provide heating and cooling and can utilize waste heat from a topping cycle. In typical adsorption systems, a fluid is contacted with salt brine and the heat of solution is rejected. Input heat then boils the fluid from the brine; it is condensed and then used as a refrigerant fluid in a standard expansions valve arrangement.

ϭϬϲ 

Multi-staged adsorption systems can be combined to improve the coefficient of performance of the overall system (Öberg et al., 2004). 4.5.7. Internal combustion systems For smaller biogas installations shaft horsepower and electrical generation is most effectively met by the use of a stationary internal combustion engine. Adequate removal of hydrogen sulfide to below 10 ppm is important to reduce engine maintain requirement. Often more frequent changing of engine oil and testing for oil sulfur content can increase engine component life. Some applications have used a dual-fuel carburetor so that propane or natural gas can be employed to startup and shut down the engine system effectively removing trace sulfide from the internal parts. When waste heat from engine cooling and exhaust gases is recovered and used the efficiency of the engine cogeneration system improves. Waste heat may be used for digester heating, space heating, hot water and or refrigeration (Tower, 2003). 4.5.8. Vehicular use Biogas, if compressed for use as an alternative transportation fuel in light and heavy duty vehicles, can use the same existing technique for fueling already being used for compressed natural gas vehicles. In many countries, biogas is viewed as an environmentally attractive alternative to diesel and gasoline for operating buses and other local transit vehicles (Öberg et al., 2004). The sound level generated by methane-powdered engines is generally lower than that generated by diesel engines and the exhaust fume emissions are considered lower than the emission from diesel engines, and the emission of nitrogen oxides is very low. Application of biogas in mobile engines requires compression to high pressure gas (>3000 psig) and may be best applied in fleet vehicles. A refueling station may be required to lower fueling time and provide adequate fuel storage (Vasen et al., 2004).

ϭϬϳ 

 

CHAPTER 5 Biohydrogen

ϭϬϴ 

Hydrogen is a valuable gas as a clean energy source and as feedstock for some industries. Therefore, the demand on hydrogen production has increased considerably in recent years. Electrolysis of water, steam reforming of hydrocarbons and autothermal processes are well-known methods for hydrogen gas production, but not costeffective due to high energy requirements. A challenging problem in establishing H2 as a source of energy for the future is the renewable and environmentally friendly generation of large quantities of H2 gas. Thus, processes those are presently conceptual in nature, or at a developmental stage in the laboratory, need to be encouraged, tested for feasibility, and otherwise applied toward commercialization. Biological production of hydrogen gas has significant advantages over chemical methods. The major biological processes utilized for hydrogen gas production are bio-photolysis of water by algae, dark and photo-fermentation of organic materials, usually carbohydrates by bacteria. Sequential dark and photo-fermentation process is a rather new approach for biohydrogen production. One of the major problems in dark and photo-fermentative hydrogen production is the raw material cost. Carbohydrate rich, nitrogen deficient solid wastes such as cellulose and starch containing agricultural and food industry wastes and some food industry wastewaters such as cheese whey, olive mill and baker’s yeast industry wastewaters can be used for hydrogen production by using suitable bio-process technologies. Utilization of aforementioned wastes for hydrogen production provides inexpensive energy generation with simultaneous waste treatment (Kapdan and Kargi, 2006). The low energy content of solar irradiation dictates that photosynthetic processes operate at high conversion efficiencies and places severe restrictions on photobioreactor economics. Conversion efficiencies for direct biophotolysis are below 1% and indirect biophotolysis remains to be demonstrated. Dark fermentation of biomass or wastes presents an alternative route to biological hydrogen production that has been little studied. In this case the critical factor is the amount of hydrogen that can be produced per mole of substrate. Known pathways and experimental evidence indicates that at most 2–3 mol of hydrogen can be obtained from substrates ϭϬϵ 

such as glucose. Process economics require that means be sought to increase these yields (Hallenbeck and Benemann, 2002). However, there are many eukaryotic and prokaryotic microorganisms capable of photobiological H2 production (Melis et al  'HPLUEDV b). Among a selection of biological systems, algae and cyanobacteria have garnered major interests as potential cell factories for hydrogen production. In conjunction with photosynthesis, these organisms utilize inexpensive inorganic substrates and solar energy for simultaneous biosynthesis and hydrogen evolution. The hydrogen yield associated with these organisms remains far too low to compete with the existing chemical systems (Levinab et alSrirangan et al., 2011). Actually, some microalgae and cyanobacteria can produce H2 according to the following reaction (Melis et al., 2000): 2H2O + light energy

O2 + 4H+ + 4e

O2 + 2H2

Cyanobacteria and microalgae can carry out photo-evolution of hydrogen catalyzed by hydrogenases. The reactions are similar to electrolysis involving splitting of water into oxygen and hydrogen. Microalgae possess the necessary genetic, metabolic and enzymatic characteristics to photoproduce H2 gas (Ghirardi et al., 2000). During photosynthesis, microalgae convert water molecules into hydrogen ions +

(H ) and oxygen; the H+ are then subsequently converted by hydrogenase enzymes into H2 under anaerobic conditions (Cantrell et al., 2008). Due to reversibility of the reaction, H2 is either produced or consumed by the simple conversion of protons to H2 (Clark and Deswarte, 2008). Photosynthetic oxygen production causes rapid inhibition to the hydrogenase enzyme, and the photosynthetic H2 production process LVLPSHGHG 0HOLV&DQWUHOOet al., 2008). Consequently, microalgae cultures for H2 production must be subjected to anaerobic conditions (Levine et al., 2010). There are two fundamental approaches for photosynthetic H2 production from water. The first H2 production process is a two-stage photosynthesis process where photosynthetic oxygen production and H2 gas generation are spatially separated ϭϭϬ 

(Ghirardi et al., 2000). In the first stage, algae are grown photosynthetically in normal conditions. During the second stage, the algae are deprived of sulfur thereby inducing anaerobic conditions and stimulating consistent H2 production (Melis, 2002). This production process becomes limited with time, as H2 yield will begin to level off after 60 hr of production. The use of this production system does not generate toxic or environmentally harmful products but could give value added products as a result of biomass cultivation (Melis and Happe, 2001). The second approach involves the simultaneous production of photosynthetic oxygen and H2 gas. In this approach, electrons that are released upon photosynthetic H2O oxidation are fed directly into the hydrogenase mediated H2-evolution process (Ghirardi et al., 2000). As shown in Figure 18, Srirangan et al., (2011) explained the possible electron flows (dotted arrows) associated with bio-hydrogen production in photosynthetic cyanobacteria and microalgae.

Figure 21: An overview of possible electron flows (dotted arrows) associated with bio-hydrogen production in photosynthetic cyanobacteria and microalgae (Srirangan et al., 2011)

ϭϭϭ 

Anaerobic hydrogen production proceeds photofermentatively as well as without the presence of light. Anaerobic bacteria use organic substances as the sole source of electrons and energy, converting them into hydrogen. The reactions involved in hydrogen production are rapid and do not require solar radiation (Demirbas, 2010b), they are represented in the following equations. (1) Glucose + 2H2O

2Acetate + 2CO2 + 4H2

(2) Glucose + Butyrate

2CO2 + 2H2

The H2 productivity is theoretically superior to the two-stage photosynthetic process, but the simultaneous production process suffers severe hydrogenase inhibition after a very short period due to the photosynthetic production of oxygen (Ghirardi et al., 2000). Melis and Happe (2001) found that using the two-stage photosynthesis process and H2 production, a theoretical maximum yield of hydrogen by green algae could be about 198 kg H2 ha-1 day-1. A new fermentation process that converts valueless organic waste streams into hydrogen-rich gas has been developed by Van Ginkel et al. (2001). The process employs mixed microbial cultures readily available in the nature, such as compost, anaerobic digester sludge, soil etc. to convert organic wastes into hydrogen-rich gas (Demirbas, 2010b). An enriched culture of hydrogen producing bacteria such as Clostridia was obtained by heat treatment, pH control and HRT control of the treatment system. Anaerobic fermentative microorganism, cyanobacteria and algae are suitable in biological production of hydrogen via hydrogenase due to reversible hydrogenases (Adams, 1990). Since the pioneering discovery by Gaffron and coworkers over 60 years ago *DIIURQ  *DIIURQ DQG 5XELQ   WKH DELOLW\ RI XQLFHOOXODU JUHHQ DOJDH WR produce H2 gas upon illumination has been mostly a biological curiosity. They found that with Scenedesmus obliquus, following a period of dark anaerobic incubation, H2 production could be observed. ϭϭϮ 

5.1. Role of hydrogenase in the hydrogen production A hydrogenase enzyme activity in green algae was expressed under anaerobic incubation of the cells in the dark and catalyzed, with high specific activity, a lightmediated H2 evolution. The monomeric form of the enzyme, reported to belong to the class of Fe hydrogenases (Happe et al  6FKXO]   LV HQFRGHG LQ WKH nucleus of the unicellular green algae. However, the mature protein is localized and functions in the chloroplast stroma (Happe et al., 1994). Light absorption by the photosynthetic apparatus is essential for the generation of hydrogen gas because light energy facilitates the oxidation of water molecules, the release of electrons and protons, and the endergonic transport of these electrons to ferredoxin. The photosynthetic ferredoxin (PetF) serves as the physiological electron donor to the Fehydrogenase and, thus, links the Fe hydrogenase to the electron transport chain in the chloroplast of the green algae (Florin et al., 2001). Under these conditions, the activity of the hydrogenase is only transient, that lasts from several seconds to a few minutes, because, in addition to electrons and protons, the light-dependent oxidation of water entails the release of molecular O2. Oxygen is a powerful inhibitor of the Fe hydrogenase (Ghirardi et al., 2000). Current technological developments in this field have not yet succeeded in overcoming this mutually exclusive nature of the O2 and H2 photoproduction reactions. Thus, the physiological significance and role of the Fe hydrogenase in green algae, which normally grow under aerobic photosynthetic conditions, has long been a mystery. Given the O2 sensitivity of the Fe hydrogenase and the prevailing oxidative environmental conditions on earth, questions have been asked as to whether the hydrogenase is anything more than a relic of the evolutionary past of the chloroplast in green algae, and whether this enzyme and the process of photosynthesis can ever be utilized to generate H2 gas for commercial purposes (Zhang et al., 2001). Aside from the above described photosystem II (PSII)-dependent H2 photoevolution, which involves water as a source of electrons and produces 2:1 ϭϭϯ 

stoichiometric amounts of H2:O2, an alternative mechanism has been described in the literature (Gfeller and Gibbs, 1984). Upon a dark anaerobic incubation of the algae and the ensuing induction of the hydrogenase, electrons for the photosynthetic apparatus are derived upon a catabolism of endogenous substrate and the attendant oxidative carbon metabolism in the green algae. Electrons from such endogenous substrate catabolism feed into the photosynthetic electron transport chain between the two photosystems, and probably at the level of the plastoquinone pool. Light absorption by PSI and the ensuing electron transport elevates the redox potential of these electrons to the redox equivalent of ferredoxin and the hydrogenase, thus permitting the generation of molecular H2 (Gibbs et al., 1986). In the presence of the PSII inhibitor 3-(3, 4-dichlorophenyl)-1,1-dimethylurea (DCMU), this process generates 2:1 stoichiometric amounts of H2:CO2. Thus, following a sufficiently long dark anaerobic incubation of the culture, initially high rates of H2 production can be detected upon illumination of the algae in the presence RI'&08D36,,LQKLELWRU +DSSHDQG1DEHU)ORULQet al., 2001). 5.2.

Expression of Fe-hydrogenase in green algae Fe hydrogenases originally were cloned from H2- producing anaerobic

PLFURRUJDQLVPV DQG SURWR]RD %XL DQG -RKQVRQ  9LJQDLV et al., 2001). These enzymes make it possible to sustain a fermentative metabolism under anaerobic conditions by utilizing protons (instead of O2) as the terminal electron acceptor and to sustain the process by releasing H2 gas (Peters, 1999). The Fe hydrogenases are distinguished by their CO sensitivity and a high enzymatic activity that is 100-fold greater than that of the NiFe hydrogenases. The structure of the Fe hydrogenases from Clostridium pasteurianum and Desulfovibrio desulfuricans were elucidated recently by x-ray crystallography (Peters et al  1LFROHW et al., 1999). These proteins have a multidomain structure with numerous [Fe-S] clusters including a novel type of [Fe-S] cluster (H cluster) within the catalytic site of the enzyme. The H cluster comprises a conventional [4Fe-4S] complex bridged by the sulfur atom of a ϭϭϰ 

Cys residue to a unique binuclear iron-sulfur subcluster (Adams and Stiefel, 2000). Highly conserved amino acid residues comprising four Cys ligands and several hydrophobic amino acid residues at the active center are thought to be involved in the formation of H and H2 channels, thus connecting the catalytic site (located deep within the protein matrix) to the protein surface (Vignais et al., 2001). Despite the discovery of hydrogen metabolism in green algae over 60 years ago and the great interest in biological H2 evolution ever since, attempts to clone and characterize the hydrogenase gene from these photosynthetic organisms were unsuccessful. Recently, hydrogenase genes have been isolated from the green algae S. obliquus (Florin et al., 2001), C. reinhardtii (Happe and Kaminski, 2001), and Chlorella fusca. All three genes were shown to belong to the class of Fe hydrogenases. However, they showed novel structural properties and suggested a unique biochemical function. It is interesting that Fe hydrogenase genes could not be found in cyanobacteria, the free-living ancestors of plastids, raising the prospect of a non-cyanobacterial origin for the algal hydrogenases. The Fe hydrogenases from green algae are monomeric proteins of about 45 to 50 kD and have been purified to homogeneity (Happe and Naber, 1993). The nucleusencoded polypeptides are synthesized in the cytosol as precursor proteins but the mature protein is localized in the chloroplast stroma (Happe et al., 1994). A transit peptide domain that routes the Fe hydrogenases from the cytoplasm across the chloroplast envelope and into the chloroplast stroma has been identified in the Nterminal region of the enzyme (Florin et al., 2001). The chloroplast-targeting domain of the protein is probably cleaved by a stroma-localized peptidase at a conserved cleavage site. No accessory genes that might be involved in the biosynthesis and/or assembly of Fe hydrogenases have been identified yet, either in green algae or in other microorganisms that contain Fe hydrogenases. The genetic data on green alga Fe hydrogenases (HydA) reveal unique features in this class of enzymes (Florin et al., 2001). They constitute the smallest known Fe hydrogenase proteins with a significantly shortened N-terminal domain and a ϭϭϱ 

conserved C-terminal domain that contains the catalytic site. The functionally important C terminus of the HydA sequence is very similar to that of other Fe hydrogenases from anaerobic microorganisms. Four highly conserved Cys residues coordinate the special [6Fe- 6S] cluster (H cluster) in the catalytic site. A number of additional amino acid residues define the environment of the active site. It was postulated that 12 mostly hydrophobic amino acid residues might play a role in protecting the H cluster from the surrounding aqueous medium (Peters et al., 1998). Ten residues are strictly conserved, whereas two residues vary within the Fe hydrogenase family (Ser-232 and Ile-268 in C. pasteurianum; Ala-109 and Thr-145 in D. desulfuricans; Ala-38 and Thr-74 in C. reinhardtii; and Ala-44 and Thr-80 in S. obliquus). However, the green algal sequences include an insertion of 16 to 45 amino acids that is absent from the bacterial sequences and that forms an external peptide loop in the fully assembled protein. This additional peptide loop in the green alga hydrogenase might be involved in electrostatic binding of the natural electron donor ferredoxin. In the N-terminal domain of bacterial and other nonalgal Fe-hydrogenases, a number of Cys residues, which are obviously missing from the green algal counterparts, were found to bind accessory ironsulfur clusters. In all nonalgal Fe hydrogenases, a ferredoxin-like domain (F cluster) coordinates two [4Fe-4S] clusters (Peters et al  $GDPV DQG 6WLHIHO   $GGLWLRQDO LURQ VXOIXU FOXVWHUV ZHUH detected within the Fe hydrogenases of C. pasteurianum, Thermotoga maritima (Verhagen et al., 1999), and Nyctotherus ovalis (Akhmanova et al., 1998). The F cluster in these organisms is responsible for electron transfer from the electron donor (mostly ferredoxin) to the H cluster (Nicolet et al., 2000). These accessory [Fe-S] centers are missing from the algal Fe hydrogenases, indicating a novel electron transport pathway from the donor PetF ferredoxin to the hydrogenase H cluster. The absence of such accessory [Fe-S] centers and the correspondingly shorter polypeptide of the green algal Fe-hydrogenase significantly reduce the distance from the ferredoxin electron donation site to the H cluster (Florin et al., 2001). In this respect, ϭϭϲ 

the external peptide loop of the algal hydrogenases might compensate for the missing domains. The positively charged amino acids in the loop structure may serve as a ferredoxin-docking domain. Thus, it may help to orient the negatively charged ferredoxin to facilitate linkage and efficient electron transfer between ferredoxin and hydrogenase. Such interaction is a prerequisite for the meaningful coupling of the enzyme with the electron transport chain in chloroplasts. 5.3. Physiological rules of H2 production in green algae Hans Gaffron made the first observation of hydrogen metabolism in green algae (Gaffron, 1939). Upon exposure to hydrogen of anaerobically adapted cells, he observed uptake of molecular H2 by the algae and a concomitant CO2 reduction in the dark. The reverse reaction, e.g. hydrogen production in the light, was first reported with the green alga Scenedesmus obliquus (Gaffron and Rubin, 1942). High rates of H2 evolution could be measured in the light for short periods of time (from several seconds to a few minutes). Electrons were generated either upon the photochemical oxidation of water by PSII, which results in the simultaneous production of O2 and H2 6SUXLW  *UHHQEDXP et al., 1983), or upon the oxidation of endogenous substrate, feeding electrons into the thylakoid membrane with the simultaneous release of CO2 to the medium (Bamberger et al., 1982). Florin et al. (2001) observed the hydrogen evolution in the green alga Scenedesmus obliquus after a phase of anaerobic adaptation. They reported the biochemical and genetical characterization of a new type of iron hydrogenase (HydA) in this photosynthetic organism. The monomeric enzyme has a molecular mass of 44.5 kDa. The complete hydA cDNA of 2609 base pairs comprises an open reading frame encoding a polypeptide of 448 amino acids. The protein contains a short transit peptide that routes the nucleus encoded hydrogenase to the chloroplast. Antibodies raised against the iron hydrogenase from Chlamydomonas reinhardtii react with both the isolated and in Escherichia coli overexpressed protein of S. obliquus as shown by Western blotting. By analyzing 5 kilobases of the genomic DNA, the transcription ϭϭϳ 

initiation site and five introns within hydA were revealed. Northern experiments suggest that hydA transcription is induced during anaerobic incubation. Alignments of S. obliquus HydA with known iron hydrogenases and sequencing of the N terminus of the purified protein confirm that HydA belongs to the class of iron hydrogenases. The C terminus of the enzyme including the catalytic site (H cluster) reveals a high degree of identity to iron hydrogenases. However, the lack of additional Fe-S clusters in the N-terminal domain indicates a novel pathway of electron transfer. Inhibitor experiments show that the ferredoxin PetF functions as natural electron donor linking the enzyme to the photosynthetic electron transport chain. PetF probably binds to the hydrogenase through electrostatic interactions. It is known that C. reinhardtii can photoproduce hydrogen when PSII is blocked by DCMU, but no H2 evolution occurs after an addition of 2,5-dibromo-3-methyl-6isopropyl-p-benzoquinone (Stuart and Gaffron, 1972), which blocks the function of the cytochrome b-f complex. Under anaerobic conditions in the presence of DCMU, accumulated reducing equivalents from the fermentative catabolism of the algae cannot be oxidized via respiration because the terminal electron acceptor O2 is absent. An NAD(P)H reductase protein complex that feeds electrons into the plastoquinone pool recently has been identified in many vascular plant chloroplasts (Kubicki et al.,  6D]DQRY et al., 1998) but so far only from the green alga Nephroselmis olivacea (Turmel et al., 1999). Nevertheless, inhibitor experiments have yielded evidence in support of a thylakoid membrane-localized NAD(P)H reductase in C. reinhardtii (Godde and Trebst, 1980), suggesting that electrons derived upon the oxidation of endogenous substrate may feed into the plastoquinone pool. Thereafter, electrons are driven upon light absorption by PSI to ferredoxin. The latter is an efficient electron donor to the Fe hydrogenase, which efficiently combines these electrons with protons to generate molecular H2 (Florin et al., 2001). The physiology of H2 production upon S deprivation has many similarities and some distinct differences from the process described above. Sulfur deprived and sealed cultures of C. reinhardtii become anaerobic in the light due to a significant and ϭϭϴ 

specific slowdown in the activity of the O2-evolving PSII, which is followed by automatic induction of the Fe hydrogenase and by photosynthetic H2 production. Biochemical analyses revealed that, concomitant with the H2 production process, starch and protein content of the cells gradually declined (Zhang et al., 2001). Such catabolic pathway(s) could be generating reductant that feeds electrons into the thylakoid membrane, perhaps via a chloroplast NAD(P)Hdependent process (Gfeller and Gibbs, 1984). More important, starch catabolism must also generate substrate for the cell’s mitochondrial respiration. Mitochondrial respiration scavenges the small amounts of O2 that evolve due to the residual activity of photosynthesis and thus ensures the maintenance of anaerobiosis in the culture. Thus, the physiology of H2 production by S deprivation involves a coordinated interaction between: I.

Oxygenic photosynthesis, i.e. the residual PSII activity for the generation of electrons upon oxidation of water. These electrons are transported through the photosynthetic electron transport chain and eventually feed into the Fe hydrogenase, thereby contributing to H2 production.

II.

Mitochondrial respiration scavenges all oxygen generated by the residual photosynthesis and, thus, maintains anaerobiosis in the culture.

III.

Endogenous substrate catabolism, including starch, protein, and probably lipid catabolism, yields substrate suitable for the operation of oxidative phosphorylation in mitochondria, and possibly for an NAD(P)H-dependent electron transport in the chloroplast, both of which contribute to the generation of much-needed ATP.

IV.

Electron transport via the hydrogenase pathway and the ensuing release of H2 gas by the algae sustains a baseline level of photosynthesis and, therefore, of respiratory electron transport for the generation of ATP and thus ensures the survival of the organism under protracted stress conditions.

ϭϭϵ 

5.4. Two-stage photosynthesis and H2 production in Chlamydomonas reinhardtii The green alga Chlamydomonas reinhardtii has been used extensively as a model organism and has also offered valuable information regarding the mechanisms underlying photobiological H2 production (Melis et al.  .UXVH et al., 2005). Chlamydomonas reinhardtii is able to produce H2 using two [Fe-hydrogenases, HydA1 and HydA2 (Happe and Naber, 1993; Forestier et al., 2003). Hydrogenase transcripts and their enzymes have been shown to be up-regulated by the onset of anaerobiosis (Posewitz et al., 2005) and it is under these conditions that H2 production can be observed. Although C. reinhardtii currently produces the highest continual levels of H2 from water of all eukaryotic species tested, there remains the distinct possibility that other green algae may be better suited to perform this task, as the huge biodiversity available is only beginning to be screened. The H2 produced from their early experiments was only minimal and it was not until 2000 that it was reported that, by depriving C. reinhardtii of sulphur, H2 production could be observed for several days (Melis et al., 2000). Sulphur deprivation causes a reduction in photosynthetic O2 evolution and leads to the onset of anaerobiosis. It is under such anaerobic circumstances that H2 can be produced. Photosystem II splits water using energy from light during photosynthesis to provide electrons and protons for H2 production, but at the same time also produces molecular oxygen (O2), which inhibits transcription and function of the hydrogenase enzymes (Ghirardi et al., 1997). For H2 to be continually produced, respiratory O2 uptake must match the rate that it is produced through photosynthesis to keep the culture anaerobic. Endogenous substrates such as starch and protein are consumed during sulphur deprivation and are likely to aid in maintaining an anaerobic environment through their oxidation and consumption by mitochondrial respiration. Such energy reserves may also play a more direct role in H2 production through nonphotochemical reduction of the plastoquinone pool (Stuart and Gaffron, 1972). In this scenario NAD(P)H reduces the plastoquinone pool via a NAD(P)H plastoquinone reductase complex and these electrons are then transferred to PSI, where they are ϭϮϬ 

light energized and eventually donated to the hydrogenase via ferredoxin (Bernard et al., 2006). Timmins et al. (2009) reported a select set of microalgae to be able to catalyze photobiological H2 production from water. They developed a method for the screening of naturally occurring H2-producing microalgae based on the model organism Chlamydomonas reinhardtii. This method was applied by purging algal cultures with N2 in the dark and subsequent illumination; it is possible to rapidly induce photobiological H2 evolution. Using NMR spectroscopy for metabolic profiling in C. reinhardtii, acetate, formate, and ethanol were found to be key compounds contributing to metabolic variance during the assay. They suggested such procedure to be used as a precise tool to test algal species existing as axenic or mixed cultures for their ability to produce H2. Terashima et al. (2010) observed that the versatile metabolism of the green alga Chlamydomonas reinhardtii is reflected in its complex response to anaerobic conditions. The anaerobic response is also remarkable in the context of renewable energy because C. reinhardtii is able to produce hydrogen under anaerobic conditions. They identified the proteins involved during anaerobic acclimation as well as pathways to the powerhouses of the cell, chloroplasts and mitochondria from C. reinhardtii in aerobic and anaerobic (induced by 8 h of argon bubbling). They actually identified a total of 2315 proteins and found clearly that 606 of these proteins localized to the chloroplast, including many proteins of the fermentative metabolism. Recent work has shown that lack of sulfur from the growth medium of Chlamydomonas reinhardtii causes a specific but reversible decline in the rate of oxygenic photosynthesis (Wykoff et al., 1998) but does not affect the rate of mitochondrial respiration (Melis et al., 2000). In sealed cultures, imbalance in the photosynthesis-respiration relationship by S deprivation resulted in net consumption of oxygen by the cells causing anaerobiosis in the growth medium, a condition that automatically elicited H2 production by the algae (Melis et al., 2000). In the course of this recent work, it was shown that expression of the Fe hydrogenase can be induced ϭϮϭ 

in the light, so long as anaerobiosis is maintained within the culture (Ghirardi et al., 0HOLVet al., 2000). Under such conditions, it was possible to photoproduce and to accumulate significant volumes of H2 gas, using the green alga C. reinhardtii, in a sustainable process that could be employed continuously for several days. Thus, progress was achieved by circumventing the sensitivity of the Fe hydrogenase to O2 through a temporal separation of the reactions of O2 and H2 photoproduction, i.e. by the socalled “two-stage photosynthesis and H2 production” process (Melis et al., 2000). The novel application of this two-stage protocol revealed the occurrence of hitherto unknown metabolic, regulatory, and electron transport pathways in the green alga C. reinhardtii (Zhang et al., 2001), leading to the significant and sustainable light dependent release of H2 gas by the cells. An inducible chloroplast gene expression system was developed in C. reinhardtii by taking advantage of the properties of the copper-sensitive cytochrome c6 promoter and of the nucleusencoded Nac2 chloroplast protein. This protein is specifically required for the stable accumulation of the chloroplast psbD RNA and acts on its 5UTR. A construct containing the Nac2 coding sequence fused to the cytochrome c6 promoter was introduced into the nac2-26 mutant strain deficient in Nac2. In this transformant, psbD is expressed in copper-depleted but not in copperreplete medium. Because psbD encodes the D2 reaction center polypeptide of photosystem II (PSII), the repression of psbD leads to the loss of PSII. We have tested this system for hydrogen production. Upon addition of copper to cells pregrown in copper-deficient medium, PSII levels declined to a level at which oxygen consumption by respiration exceeded oxygen evolution by PSII. The resulting anaerobic conditions led to the induction of hydrogenase activity. Because the Cyc6 promoter is also induced under anaerobic conditions, this system opens possibilities for sustained cycling hydrogen production. Moreover, this inducible gene expression system is applicable to any chloroplast gene by replacing its 5UTR with the psbD 5UTR in the same genetic background. To make these strains phototrophic, the ϭϮϮ 

5UTR of the psbD gene was replaced by the petA 5UTR. As an example, we show that the reporter gene aadA driven by the psbD 5UTR confers resistance to spectinomycin in the absence of copper and sensitivity in its presence in the culture medium. As well as, Song et al. (2011) studied hydrogen production using immobilized green alga Chlorella sp. through a two-stage cyclic process where immobilized cells were first incubated in oxygenic photosynthesis followed by anaerobic incubation for H2 production in the absence of sulfur. Chlorella sp. used in this study was capable of generating H2 under immobilized state in agar. The externally added glucose enhanced H2 production rates and total produced volume while shortened the lag time required for cell adaptation prior to H2 evolution. The rate of hydrogen evolution was increased as temperature increased, and the maximum evolution rate under 30 mM glucose was 183 ml/h/L and 238 ml/h/L at 37°C and 40°C, respectively. In order to continue repeated cycles of H2 production, at least two days of photosynthesis stage should be allowed for cells to recover H2 production potential and cell viability before returning to H2 production stage again. 5.5. The amount of H2 can be produced by a mass culture of green algae Application of the two-stage photosynthesis and H2 production protocol to a green alga mass culture could provide a commercially viable method of renewable hydrogen generation. There three specific biological challenges need to be overcome to effect greater actual yields of green alga H2 production, as follows: i. The yield of H2 production currently achieved in the laboratory corresponds to only 15% to 20% of the measured capacity of the photosynthetic apparatus for electron transport (Melis et al., 2000). ii. The optical properties of light absorption by green algae impose a limitation in terms of solar conversion efficiency in the alga chloroplast. This is because wild-type green algae are equipped with a large light-harvesting chlorophyll antenna size to absorb as much sunlight as they can. Under direct and bright ϭϮϯ 

sunlight, they could waste up to 60% of the absorbed irradiance (Melis et al., 1999). This evolutionary trait may be good for survival of the organism in the wild, where light is often limiting, but it is not good for the photosynthetic productivity of a green algal mass culture. This optical property of the cells could further lower the productivity of a commercial H2 production farm. iii. The current necessity to cycle a culture between the two stages (normal photosynthesis in the presence of S alternating with H2 production upon S deprivation) introduces a “down time” as far as H2 production is concerned. It is inevitable that the “down time” would further erode the yield of the H2 production process. Thus, with current technology, it is estimated that the actual yield of H2 production would be lower than that of the theoretical maximum, achieving perhaps a mere 10%, or lower, than the calculated theoretical maximum.

CONCLUSION AND RECOMMENDATIONS The research for biofuel renewable resoureces has become a sizable challeng since energy crisis in the 1970s. Today, the marine microbial resources, and even non-microbial resources, have been proven that they are promising in the production of several types of bio-fuels. Up to this point, there is a global concern for rising up the yield and quality with the reduction of time and cost. So, the current review concludes the following points: 1. The ocean is the mother of life and it is believed that it contains a massive variety of marine biomass. 2. Microalgae represent a potential microbial resource for biodiesel production. Moreover, the residual microalgal biomass generated in the lipid extraction for biodiesel can be appropriately utilized for the production of bioethanol or biogas.

ϭϮϰ 

3. Marine algal biomass (seaweeds) can be utilized for the production of various biofuels such as: bioalcohols, biodiesel, biogas and biohydrogen. 4. Marine microbes have been proved their potentiality in the algal substrate hydrolysis and then production of reducing sugars. 5. Experimental designs and immobilization techniques exhibited obvious frequency in optimizing both saccharification and fermentation processes. Taking into consideration the advances in this field, we should believe that future activities must focus the following principal area: 1.

Countries need much more private and government incentive to make large scale microalgal production, and lipid extraction process, a reality as it holds much promise to improve the economy and the environment, including the multitude of useful products it can put on the market. Also, the advanced approaches for biogas and biohydrogen production must be in focus and developed.

2.

Further studies should be carried out on different types of marine starchy and cellulosic wastes for maximum biofuel %.

3.

Research for superior marine microbial isolates for rising up biofuels % and reducing the cost is a vital demand.

4.

Yeast, E. coli or others, should be genetically engineered to efficiently carry out both saccharification and fermentation processes together saving time and cost.

ϭϮϱ 

GLOSSARY & WEB SITES http://www.biorefinery.ws  http://www.intechopen.com http://www.tutorvista.com Harmonic Arts Botanical harmonicarts.ca GCSE Science Practicalsscienceteacher.org.uk Patients Crossing Ocean semedtravel.wordpress.com http://www.oilgae.com/algae/oil/biod/tra/tra.html http://www.et.byu.edu http://www.oilgae.com http://www.extension.org www.intechopen.com

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