SiO2 is one generally used structure promoter for precipitated Fe catalyst. It can not only increase the specific surface area of Fe catalyst, but also bind iron species to prevent its loss from catalyst particle during reaction. Several methods are used to introduce SiO2 into precipitated Fe catalysts with potassium silicate, silica sol, and so on as silica source. Sasol impregnated washed precipitate with potassium water — glass solution [15]. Dlamini et al. added silica sol at different stages during catalyst preparation (before precipitation, after precipitation, after drying at 397 K, and after calcination at 723 K) [57] . This method was also used in the study of Yang et al. [58]. Some researchers introduced SiO2 with hydrolyzed tetraethyl orthosilicate and added it to the iron(III) nitrate solution to give the desired level of silicon [59].

In this work, potassium silicate was selected as silica source and it was intro­duced before precipitation. The influences of Fe-Si interaction on precipitated Fe catalyst structure and activity had been partially reported [45]. Here, the influence of SiO2 on precipitated Fe catalyst is reported in view of CO2 hydrogenation.

1.3 Experimental

Catalysts were prepared in two methods. Method I was to precipitate a mixed solution of Fe(NO.)3 and potassium silicate with (NH.)2CO3 solution. Method II was to precipitate Fe(NO3)3 solution with the mixed solution of (NH4)2CO3 and potassium silicate. Then, the precipitate was washed with distilled water and centrifuged for

ten times. Promoters of Zn, K and Cu were usually impregnated onto the precipitate with Zn(NO3)2, KNO3, and Cu(NO3)2 solution. After the precipitate was dried and calcined, it was shaped into desired particle size for activity test. The obtained cata­lysts are expressed as ZlKmCn/FSr-I or ZlKmCn/FSr-II according to the method to introduce SiO2. In the above abbreviation of catalysts, Z, K, C, F, and S represent Zn, K, Cu, Fe, and SiO2, respectively. L, m, n, and r are the nominal mass percent of corresponding materials relative to Fe2O3.

The activity of catalysts was tested in a stainless steel fixed bed reactor. A 1.0 g catalyst (80-150 mm) was mixed with 4.0 g quartz particles and filled into the reactor. After the catalysts were reduced in CO of 50 mL min-1 at 573 K for 6 h, it was cooled to room temperature. Then, the feedgas was changed into reactants of

1.6 MPa. The catalyst was heated to 503 K in about 3 h for activity evaluation. The detail parameters for activity testing are given with the experimental results.

At its foundation, this approach uses a microorganism that is very adept at utilizing cellulose as a growth substrate, and attempts to instill the ability in the organism to produce high yields and titers of ethanol in the presence of a biomass hydrolysate environment. This approach benefits from having the extremely complex process of deconstructing plant cell walls already programmed within the organism’s metabo­lism. In this particular case, suites of cellulase enzymes do not need to be heterolo­gously expressed thereby allowing metabolic engineering and strain adaptation approaches to focus solely on increasing ethanol production and on product and hydrolysate tolerance mechanisms, while reducing organic acid production. However, this is no trivial matter as most candidate cellulolytic organisms have only very minimal capabilities of producing ethanol anywhere near the yields and titers that would be required for a viable cellulosic ethanol production process. Further complicating the native cellulophile approach is that, generally speaking, the molec­ular biology tools for engineering candidate cellulolytic organisms have not been as well established relative to other commonly used model organisms. This makes the process of creating a robust fermentation organism from a cellulolytic candidate challenging to say the least. With that said, some progress has been made along this front and several cellulolytic candidate organisms have made it to the forefront as potential CBP host organisms including Clostridium thermocellum, Clostridium phytofermentans, Clostridium cellulolyticum, Thermoanaerbacterium saccharolyti — cum, and recently, Trichoderma reesei. We discuss the potential of several of these candidate microorganisms in further detail below.

Because of the importance of enzymes in industrial degradation and modification of cellulosic materials, a large number of cellulases have been identified and character­ized. As with many areas of biochemical research, early studies involved isolation, purification, and characterization of activities of cellulose-degrading enzymes from natural sources (bacteria, fungi), with gene sequencing following these isolation procedures in a one-to-one pace. Results from these early studies set the stage to characterize a large number of cellulases, with different structures, sequences, and activities.

With the emergence of large-scale genome sequencing, and substantial application (supported by the US Department of Energy) to the genomes of biomass-degrading microbes, the number of putative cellulase sequences has grown much more quickly than biochemical characterization studies. Thus, there is a rich database of sequences that may have potentially useful activities. Thanks to the early (and ongoing) studies that connect biochemical activity to sequence and structure, many of these putative cellulase sequences can be grouped, based on primary sequence, into “families” and “clans” with similar folds and evolutionary origin. However, as will be discussed, this grouping is not sufficient to predict activity and substrate specificity. Here, dif­ferent levels of classification will be discussed, along with their relative merits and/ or shortcomings. Although we cannot yet predict function in full detail from pri­mary sequence, structure, and/or taxonomy, the wealth of data and analysis in all three of these areas provides a good starting point.

James Weifu Lee

Abstract This chapter describes an invention on photosynthetic ethanol production through application of synthetic biology. The designer plants, designer algae, and designer plant cells are created such that the endogenous photosynthesis regulation mechanism is tamed, and the reducing power (NADPH) and energy (ATP) acquired from the photosynthetic water splitting and proton gradient-coupled electron trans­port process are used for immediate synthesis of ethanol (CH3CH2OH) directly from carbon dioxide (CO2 ) and water (H2 O). This photobiological ethanol-production method eliminates the problem of recalcitrant lignocellulosics by bypassing the bottleneck problem of the conventional biomass technology. The photosynthetic ethanol-production technology is expected to have a much higher solar-to-ethanol energy-conversion efficiency than the conventional technology. Furthermore, this approach enables the use of seawater for photobiological production of ethanol without requiring freshwater or agricultural soil, since the designer photosynthetic organisms can be created from certain marine algae that can use seawater.

1 Introduction

Ethanol (CH3CH2OH) can be used as a liquid fuel to run engines such as cars. A significant market for ethanol as a liquid fuel already exists in the current transpor­tation and energy systems. In the United States, currently, ethanol is generated primar­ily from corn starch using a yeast-fermentation process. Therefore, the “cornstarch

J. W. Lee (*)

Department of Chemistry & Biochemistry, Old Dominion University,

Physical Sciences Building, Room 3100B, 4402 Elkhorn Avenue, Norfolk, VA 23529, USA

Johns Hopkins University, Whiting School of Engineering, 118 Latrobe Hall,

Baltimore, MD 21218, USA

e-mail: jwlee@ODU. edu; JLee349@JHU. edu

J. W. Lee (ed.), Advanced Biofuels and Bioproducts, DOI 10.1007/978-1-4614-3348-4_21, 405

© Springer Science+Business Media New York 2013

ethanol-production” process requires a number of energy-consuming steps including agricultural corn-crop cultivation, corn-grain harvesting, corn-grain starch process­ing, and starch-to-sugar-to-ethanol fermentation. Independent studies have recently shown that the net energy efficiency of the “cornstarch ethanol-production” process is actually negative in some cases. That is, the “cornstarch ethanol-production” process costs more energy than the energy value of its product ethanol. This is not surprising, understandably because the cornstarch that the current technology can use represents only a small fraction of the corn-crop biomass that includes the corn stalks, leaves, and roots. The cornstovers are commonly discarded in the agricultural fields where they slowly decompose back to CO2 , because they represent largely lignocellulosic biomass materials that the current biorefinery industry cannot efficiently use for ethanol production. There are research efforts in trying to make ethanol from lignocellulosic plant biomass materials—a concept called “cellulosic ethanol.” However, plant biomass has evolved effective mechanisms for resisting assault on its cell-wall structural sugars from the microbial and animal kingdoms. This property underlies a natural recalcitrance, creating roadblocks to the cost-effective transfor­mation of lignocellulosic biomass to fermentable sugars. Therefore, one of its problems known as the “lignocellulosic recalcitrance” represents a formidable technical barrier to the cost-effective conversion of plant biomass to fermentable sugars. That is, because of the recalcitrance problem, lignocellulosic biomasses (such as cornstover, switchgrass, and woody plant materials) could not be readily converted to fermentable sugars to make ethanol without certain pretreatment, which is often associated with high processing cost. Despite more than 25 years of R&D efforts in lignocellulosic biomass pretreatment and fermentative ethanol-production processing, the problem of recalcitrant lignocellulosics still remains as a formidable technical barrier that has not yet been eliminated so far. Furthermore, the steps of lignocellulosic biomass cultivation, harvesting, pretreatment processing, and cellulose-to-sugar-to-ethanol fermentation all cost energy. Therefore, any new technology that could bypass these bottleneck problems of the biomass technology would be useful.

Algae (such as Chlamydomonas reinhardtii, Platymonas subcordiformis, Chlorella fusca, Dunaliella salina, Ankistrodesmus braunii, and Scenedesmus obliquus), which can perform photosynthetic assimilation of CO2 with O2 evolution from water in a liquid culture medium with a maximal theoretical solar-to-biomass energy conversion of about 10%, have tremendous potential to be a clean and renew­able energy resource. However, the wild-type oxygenic photosynthetic green plants, such as eukaryotic algae, do not possess the ability to produce ethanol directly from CO2 and H2O. As shown in Fig. 1, the wild-type photosynthesis uses the reducing power (NADPH) and energy (ATP) from the photosynthetic water splitting and proton gradient-coupled electron transport process through the algal thylakoid membrane system to reduce CO2 into carbohydrates (CH2O)n such as starch with a series of enzymes collectively called the “Calvin cycle” at the stroma region in an algal or green-plant chloroplast. The net result of the wild-type photosynthetic process is the conversion of CO2 and H2O into carbohydrates (CH2O)n and O2 using sunlight energy according to the following process reaction:

The carbohydrates (CH2O)n are then further converted to all kinds of complicated cellular (biomass) materials including proteins, lipids, and cellulose and other cell- wall materials during cell metabolism and growth.

2n certain algae such as C. reinhardtii2 some of the organic reserves such as starch could be slowly metabolized to ethanol through a secondary fermentative metabolic pathway. The algal fermentative metabolic pathway is similar to the yeast-fermentation process, by which starch is breakdown to smaller sugars such as glucose that is, in turn, transformed into pyruvate by a glycolysis process. Pyruvate may then be converted to formate, acetate, and ethanol by a number of additional metabolic steps 2 12 • The efficiency of this secondary metabolic process is quite limited, probably because it could use only a small fraction of the limited organic reserve such as starch in an algal cell. The maximal concentration of ethanol that can be generated by the fermentative algal metabolic process is only about 1% or less, which is not high enough to become a viable technology for energy production. To be an economically viable technology, the ethanol concentration in a bioreactor medium is preferred to reach as high as about 3-5% before an ethanol-distillation process may be profitably applied. Therefore, a new ethanol-producing mechanism with a high solar-to-ethanol energy efficiency is needed.

This chapter reports an invention (U. S. Patent No. 7,973,214 B2) that can provide revolutionary designer organisms, which are capable of directly synthesizing ethanol from CO2 and H2O. The ethanol-production system provided by the present invention could bypass the bottleneck problems of the biomass technology mentioned above.

Table 2 shows the fatty acid composition of Jatropha oil containing of 23.6% of saturated fatty acids mainly from palmitic, stearic, and myristic acid and 76.4% of unsaturated fatty acids which consist of mainly oleic, linoleic acid, and palmitoleic. The physicochemical properties of Jatropha oil which is extracted from the seeds of different origin viz., Malaysia, Indonesia, Thailand, Nigeria, Brazil, are given in Table 3.

The ability of fluid to pump and flow within an engine is determined by its vis­cosity. The desired viscosity of diesel fuel ranges from 1.9 to 4.1 cSt. Transesterification is one of the recognized and efficient methods to reduce the viscosity of the vegetable oil to make it suitable as a biodiesel [25] .

The iodine value is a measurement for the unsaturation level in fats and oils; a high iodine value is an indication of the presence of high unsaturation levels in the oils [43]. The high iodine value of Jatropha oil is due to the presence of high amounts of unsaturated fatty acids such as oleic and linoleic acid (Table 3).

The peroxide value determines the formation of hydro peroxides (primary oxida­tion products) [30]. This can be associated with the presence of higher amounts of polyunsaturated fatty acids such as linoleic acid (Table 3). The instability of any oil is directly related to the level of unsaturation.

Acid value (“acid number” or “acidity”) is the mass of potassium hydroxide (KOH) in milligrams that is required to neutralize 1 g of chemical substance.

The acid value of edible oils or their corresponding esters indicates the quantity of free fatty acids (FFA) and mineral acids (negligible) present in the sample.

Fatty acids can be bound or attached to other molecules such as triglycerides or phospholipids. When they are not attached to other molecules, they are known as “free” fatty acids (FFA). FFA is detrimental to the biodiesel-making process. For biodiesel production purposes, FFA content of Jatropha oil indicates two types of Jatropha oil: low FFA oil (FFA < 2.5%) and high FFA oil (FFA > 2.5%) [61]. A high saponification value indicates that Jatropha oil possesses normal triglycerides and may be useful in the production of liquid soap and shampoo [30].

Liam Brennan and Philip Owende

Abstract Continued reliance on fossil fuel reserves as the primary energy resource is increasingly becoming unsustainable, owing to the need for: minimal exposure to the associate price volatility, reduction of greenhouse gas emissions by energy conserva­tion, and deployment of cleaner and locally produced energy feedstock (including recovery from waste). Based on current knowledge and technology projections, third — generation biofuels (low input-high yielding feedstock) specifically derived from microalgae are considered to be viable alternative energy resource. They are devoid of the major drawbacks associated with first-generation biofuels (mainly terrestrial crops, e. g. sugarcane, sugar beet, maize and rapeseed) and second-generation biofuels (derived from lignocellulosic energy crops and agricultural and forest biomass residues). This chapter focuses on technologies underpinning microalgae-to-biofuels production systems, and evaluates the scale-up and commercial potential of biofuel production, including benchmarking of fuel standards. It articulates the importance of integrating biofuels production with the production of high-value biomass fractions in a biorefinery concept. It also addresses sustainability of resource deployment through the synergistic coupling of microalgae propagation techniques with CO2 sequestration and bioremediation of wastewater treatment potential for mitigation of environmental impacts associated with energy conversion and utilisation.

L. Brennan (*)

School of Agriculture, Food Science and Veterinary Medicine,

Bioresources Research Centre, Charles Parsons Energy Research Programme, University College Dublin, Belfield, Dublin 4, Ireland e-mail: liam. brennan@ucd. ie

P. Owende

School of Agriculture, Food Science and Veterinary Medicine,

Bioresources Research Centre, Charles Parsons Energy Research Programme, University College Dublin, Bel fi eld, Dublin 4 , Ireland

School of Informatics and Engineering, Institute of Technology Blanchardstown, Blanchardstown Road North, Dublin 15, Ireland e-mail: philip. owende@itb. ie

J. W. Lee (ed.), Advanced Biofuels and Bioproducts, DOI 10.1007/978-1-4614-3348-4_24, 553

© Springer Science+Business Media New York 2013

1 Introduction

The organic solvent extraction described above is generally performed as a batch process. Even though lipid extraction in a batch mode is limited by the establish­ment of lipid partition equilibrium, a continuous organic solvent extraction which overcomes this limitation is not a viable option due to the high cost associated with supplying the increased solvent volume, even with an integrated solvent recovery system. As such, a system that is somehow able to continuously replenish cells with fresh solvent (hence avoiding equilibrium limitation) while simultaneously mini­mizing solvent consumption is desirable. The Soxhlet apparatus achieves this dual objective through its ingenious cycles of solvent evaporation and condensation [35, 62]. The apparatus has three compartments as shown in Fig. 8: the continuously heated solvent flask to store the extracting organic solvent, the Soxhlet extractor to hold the microalgal cells (existing as either a wet paste or dried biomass), and the continuously cooled condenser. The evaporated organic solvent from the heated

Fig. 8 The Soxhlet apparatus, extracted from Wang and Weller [62]

Soxhlet

extractor

Solvent flask

solvent flask enters the condenser. As the recondensing solvent is channelled into the Soxhlet extractor, it comes in contact with the microalgal cells to extract lipids. Once the rising solvent in the extractor reaches the overflow level, a syphon unloads the lipid-saturated solvent in the extractor back into the solvent flask. The solvent evaporates again while the extracted lipids remain in the solvent flask, and the cycle repeats. The operation continues until no more lipids are isolated in the Soxhlet extractor. Even though the semi-continuous Soxhlet apparatus solves the equilib­rium limitation of a batch organic solvent extraction without any increase in solvent consumption, its high energy requirement for continuous distillation prevents its large-scale application [35, 62].

Three other modifications of organic solvent extraction have been reported: ultra­sound-assisted, microwave-assisted and accelerated organic solvent extraction. Each of these modifications makes use of an auxiliary process to enhance lipid extraction by organic solvents [35, 62]. Ultrasound-assisted organic solvent extraction obtains higher extraction kinetics by using mechanical vibrations from sound waves not only to disrupt cellulosic cell walls but also to induce greater penetration of solvent mole­cules into the cellular structures. Microwave-assisted organic solvent extraction uses electromagnetic radiations within specific frequency to impart large amount of ther­mal energy into the cells, thereby disrupting their cellulosic cell walls and promoting lipid extraction. During accelerated organic solvent extraction, lipid extraction is

Fig. 9 Pressure (P)-temperature (T) phase diagram for carbon dioxide, showing the supercritical region performed at elevated pressures and temperatures, but below critical conditions, in order to accelerate extraction kinetics. Even though all three of these modifications seem to show some promises, they have not been successfully transferred to the industrial scale due to their high energy requirements. Their effectiveness in extract­ing lipids from microalgal cells still needs further demonstration [35, 62].

CEC analysis was performed using the following method: The ground char sample was thoroughly mixed and 2 g was placed in a 250-mL Erlenmeyer flask. Hundred milliliters of 0.5 N HCl was added, the flask was covered with parafilm and shaken vigorously periodically for 2 h. Sample was filtered using a glass fiber fi. ter in a Buchner funnel, washing with 100 mL portions of H2O until wash shows no precipitate with AgNO3. Filtrate was discarded. Moist char was immediately transferred to a clean 250-mL Erlenmeyer flask and a total of 100 mL 0.5 N Ba(OAc)2 was added and a stopper placed on the flask. The mixture was shaken vigorously periodically for 1 h and was filtered, washing with three 100 mL portions H2O. The char was discarded, and the filtrate was titrated with 0.0714 N NaOH using phenolphthalein to first pink. The following equation was used to calculate the CEC value:

milliequivalent mL x normality NaOH x 100 (1)

100g air — dried char (g) sample

The United Nations 2008 Revision of World Population Prospects estimates the world population, which stood at 6.8 billion in 2009, is projected to reach 9 billion in 2050 [50]. Most of the additional people expected by 2050 will be concentrated in developing countries, whose population is projected to rise from 5.6 billion in 2009 to 7.9 billion in 2050. This high growth in the rate of urbanization and development will drive significant increases in demand for energy production while generating ever-expanding volumes of centralized organic waste in urban centers. Organic wastes typically from urban centers are from parks and gardens, food waste, and wastewater solids from sewage treatment plants. The increasing dissociation of this organic waste resource from farming production areas significantly challenges the ongoing sustainability of rural crop production that relies on the effective cycling of carbon and nutrients [ 2] . Urban centers are also challenged with the lack of appropriate area for landfilling wastes, with the transporting of wet, bulky, and often odorous waste to landfills increasing in costs and social pressure (Fig. 4).

Slow-pyrolysis technology applied to municipal organic wastes may help in addressing these challenges experienced currently in urban centers that are expected to be exacerbated by the predicted growth in urban populations. The environmental and economic benefits of utilizing urban waste water sludges in thermal conver­sion processes has been demonstrated in the literature [12, 42]. Slow-pyrolysis processing of organic wastes could provide not only a renewable source of elec­tricity, but it also fills in the missing link between soil carbon, nutrient cycling, and urban food consumption through the production of biochar. The nutrients and carbon contained in the organic wastes are concentrated into a greatly reduced mass and volume of biochar that is therefore more cost-effectively transported back to agricultural land.

The pyrolysis process effectively sterilizes the wastes so that biosecurity risk (human health, animal disease risk, plant pathogen, plant propagule, etc.) is greatly

diminished. It should be noted, however, that there is potential for contamination in waste streams and therefore an evaluation, monitoring, and verification plan should be adopted to ensure the risk of applying contaminated biochar to land is mitigated [17] .

Benefits for local governments of urban centers:

• Job creation.

• Renewable energy production.

• Increased resource recovery of waste organics.

• Decreased need for landfill.

• Value adding of wastes to marketable product.

• Decreased mass and volume of product—less to transport.

• Concentration of carbon and nutrients into biochar.

• Odor reduction.

• Improved biosecurity through pathogen destruction.

• Decreased greenhouse gases from landfill.

• Carbon offsets generated to contribute to achieving targets.

• Stabilization of carbon for sequestration.

• Enhanced energy security.

• Enhanced food security.

The production of biochar also presents some opportunities unique to urban uses. For example, the incorporation of gardens into the landscape of urban build­ing development provides many environmental and social benefits. The concept of retrofitting existing roof areas with gardens, know as “green roofs,” is becoming increasingly popular. One of the challenges of this practice is that existing roofs have load ratings that greatly limit the amount of heavy soil and water that they can support. Biochar has been demonstrated to have a low bulk density [18] and good water holding capacity [13] which potentially make it an ideal substrate for soil mixes which need to be light weight and retain moisture. Another advantage of biochar for this application is that it is recalcitrant and therefore breaks down slowly in the environment. This means that it will need to be replaced a lot less frequently than other low-bulk density substrates that are made from more labile carbon components. This becomes important when access to roof areas for bulk goods is difficult.

The use of slow-pyrolysis for producing thermal energy, in the form of high pressure hot water, from urban waste organics for district heating also presents a unique resource recovery opportunity. Local governments overseeing the delivery of both waste management and district heating services to the community are in position to implement such projects without the need for complicated counterparty agreements.

The lignin-derived phenolic compounds (monomeric phenolic compounds and pyrolytic lignins) can be isolated from crude bio-oils by different methods [1, 7, 20], and they are known to be used as phenol replacement in production of phenol — formaldehyde resins [19, 40, 42, 80].

As indicated above, the monomeric phenolic compounds are usually formed in much lower yield than the pyrolytic lignins. Since the monomers are more reactive than the oligomers for resin production, and thus, it will be attractive to promote the production of monomeric phenolic compounds. According to some previous studies, fast pyrolysis of biomass impregnated with some alkaline compounds (NaOH, KOH) could increase the yield of monomeric phenolic compounds [23, 24, 63].

In another study, Pd/SBA-15 catalysts were employed to catalytic crack biomass fast pyrolysis vapors that contained a lot of pyrolytic lignins, and the results indi­cated that the Pd/SBA-15 catalysts were able to promote the conversion the pyro­lytic lignins to monomeric phenolic compounds, and meanwhile to crack and decrease holocellulose-derived products. Hence, the content of the total monomeric phenolic compounds reached 55% (peak area% on the GC/MS ion chromatograms) in the catalytic pyrolytic products [51]. Some other catalysts were also confirmed to possess the catalytic capability to increase the yield of monomeric phenolic com­pounds or their content in the catalytic bio-oils [3, 8, 9, 62].

Furthermore, if single phenolic compounds could be produced and recovered, they should be much more valuable than the mixed phenolic compounds. According to a previous study done by Murwanashyaka et al. [59] , the evolution of major monomeric phenolic compounds took place in the following order: methyl — guaiacol, ethylguaiacol, guaiacol, propenylsyringol, phenol, and catechol, which suggested that the production of specific phenolic compounds might be achieved by stepwise pyrolysis of biomass. Catalytic pyrolysis is another promising way. For example, during the catalytic cracking of biomass fast pyrolysis vapors with Pd/ SBA-15 catalysts, the 4-ethyl-2-methoxy-phenol was increased greatly, with the content up to 10% (peak area% on the GC/MS ion chromatograms) in the pyrolytic products [51]. Some subsequent studies have also been reported for the recovery of pure single phenolic compounds [60] .