Climate change is a significant issue in today’s world. As accepted by the wider scientific, political and social communities, climate change is unequivocally—a greater than 90% chance—due to global warming caused by the activities of humans since the 1750s (IPCC 2007). Thus a project which absorbs CO2, a major contributor
to global warming, would play a significant role in combating climate change. The importance of the microalgal biodiesel production is underlined by the ability of microalgae to absorb carbon dioxide. Carbon capturing occurs in the cultivation phase of the algae biomass, where CO2 fixation occurs through the biological photo­synthesis reaction. This CO2 bio-sequestration has attracted attention due to the pos­sibility of converting this harmful waste into a valuable product.

The carbon and energy audits are focused on Australia (but applicable else­where), and used as a basis for all the discussions in this section. To reach the CO2 reduction targets, the Australian Federal Government has implemented two primary drivers: The National Greenhouse and Emission Reporting Act (NGER Act) which regulates and sets guidelines on how both Scope 1 (activity direct) and Scope 2 (activity dependent) emissions should be reported; and the Carbon Pollution Reduction Scheme (CPRS) which is the “cap-and-trade” scheme where emitters are required to purchase permits for their emissions.

Under CPRS, emitters who exceed certain limits are required to obtain permits for their Scope 1 emissions. For example, if Company A emits 10 tonnes of CO2-e above a certain limit, the company is required to possess 10 permits (each permit is equivalent to 1 tonne of CO2-e) (NGER Guidelines, 2008). The permit is either allo­cated to mitigate costs of the scheme to some key industries or auctioned to the highest bidder. There is a fixed amount of permits sold in line with the national emis­sion cap, with an initial selling price of $25 per permit [37]. Companies would pur­chase permits if their internal costs of abatement are higher than the price of permits, and would directly reduce their emissions if their internal costs of abatement are lower than the price of permits. It is expected that permit prices might rise to between $35 and 50 per permit by 2020 [37]. The microalgal cultivation process which cap­tures CO2 will reduce the overall Scope 1 emissions of an industry and convert this harmful waste into valuable products. By undertaking a complete audit on the pro­cess, the exact capturing ability of the process will be ascertained and analysed.

Joao C. M. Carvalho, Raquel P. Bezerra, Marcelo C. Matsudo, and Sunao Sato

Abstract This chapter comments on fed-batch cultivation of Arthrospiraplatensis under different carbon and nitrogen sources, pH, temperature, light intensity, type of photobioreactor and typical parameters of the fed-batch process, such as feeding time, addition protocol and flow rate. Inexpensive nitrogen sources, such as urea, ammonium salts and nitrogen-rich wastewaters can be used for A. platensis cultiva­tion, with results that can be comparable to those with classical nitrate sources. Closed photobioreactors are useful for preventing ammonia loss. The use of organic carbon sources needs to be carried out under aseptic conditions, and it is necessary to evaluate the best supplying conditions when using fed-batch process. The addi­tion of CO2 ensures the control of pH and, at the same time, supply of the carbon source into the culture medium. The fed-batch process can be useful for the produc­tion of A. platensis using CO2 from industrial plants, particularly from industrial alcoholic fermentation.

1 Introduction

The cultivation of microalgae and cyanobacteria is an important current issue because of the possibility of supplying human needs related to food production and removal of atmospheric or industrial carbon dioxide. Arthrospira (Spirulina) plat­ensis, Dunaliella salina and Chlorella vulgaris are among the most studied photo­synthetic microorganisms, but several other cyanobacteria and microalgae have been investigated lately, mainly for biodiesel production.

J. C.M. Carvalho (H) • R. P. Bezerra • M. C. Matsudo • S. Sato

Department of Biochemical and Pharmaceutical Technology, University of Sao Paulo, Av. Prof. Lineu Prestes 580, Bl. 16, Sao Paulo 05508-900, SP, Brazil e-mail: jcmdcarv@usp. br

J. W. Lee (ed.), Advanced Biofuels and Bioproducts, DOI 10.1007/978-1-4614-3348-4_33, 781

© Springer Science+Business Media New York 2013

The increasing demand for protein sources and other high biological value products, such as polyunsaturated fatty acids and pigments, associated with the need of the development of new technologies that contribute to the mitigation of environmental pollution indicates that the market for microorganisms such as A. platensis is going to increase in the coming years.

The previous uses of photosynthetic microorganisms as food are related to events in China 2,000 years ago, where Nostoc was used in periods of food shortage. Additionally, Spirulina sp. was consumed by the Aztecs in the Mexico Valley and by people living near Chad Lake in Central Africa [54]. They have been consumed by Africans, where French researchers first reported in 1940 the use of Spirulina platensis as food [54] .

Currently, the correct scientific designation for S. platensis is A. platensis [95] . Despite this, in this chapter, it was maintained the denomination given by the authors of the cited works. The genus Arthrospira (family Cyanophyceae) encompasses the photosynthetic cyanobacteria with helically coiled trichomes along the entire length of the multicellular filaments and visible septa (Fig. 1). The last characteristic dif­ferentiates this genus from true Spirulina which has invisible septa [16].

Arthrospira (Spirulina) platensis is one of the most promising microorganisms, among microalgae and cyanobacteria, not only to be used as food but also for other industrial applications because of its composition.

It contains a great amount of polyunsaturated fatty acids and pigments such as phycocyanin and zeaxantine [24]. Palmitic, linoleic, g-linolenic, and oleic acids are the predominant fatty acids in S. platensis. g-Linolenic is only found in significant amounts in breast milk, some fruits, species of fungi and cyanobacteria [25].

S. platensis is also an interesting source of chlorophyll, since this microorganism synthesizes only chlorophyll a, which is more stable than chlorophyll b, very com­mon in vegetables. Moreover the cell wall is composed of mucopolysaccharides and therefore easily digested [44], which is an advantage for the bioavailability of cell components.

This cyanobacterium shows low nucleic acid content in dry biomass (4-6%) in comparison with yeasts (8-12%) and other bacteria (20%) [3], so the daily intake of this biomass would not cause any damage to the human body [73].

Besides the high protein content in dry biomass, S. platensis shows a satisfacto­rily balanced amino acid content, possessing even methionine, which is absent in most microalgae [35]. About 20% of the cellular protein is represented by the main pigments in this microorganism, called phycobilins [82] .

This biomass also contains important vitamins such as cyanocobalamin (B12), pyridoxine (B6), thiamin (B1), tocopherol (E), and phylloquinone or phytonadione (K) [12]. Moreover, recent studies indicate that some trace elements such as chro­mium III [57] and selenium [19] can be accumulated in S. platensis biomass depend­ing on the cultivation conditions.

In fact, Spirulina spp. are noted in the literature as an alternative protein source, due to the high protein content in dry biomass (reaching as high as 70%), good digestibility, low nucleic acid content, and presence of vitamins, polyunsaturated fatty acids, immunomodulatory polysaccharides, pigments, and antioxidants [24] .

S. platensis is mainly used as a food supplement. One of the applications is the use of this microorganism as a source of pigments for food industries [33, 69]. S. platensis was also shown to act as a prebiotic, improving the growth in vitro of lactic acid bacteria such as Lactobacillus lactis, Lactobacillus delbrueckii and Lactobacillus bulgaricus [73]. For application in animal feed, some researchers have studied the use of S. platensis in aquaculture, to feed shrimp larvae, for instance [46].

Another important aspect of this microorganism is the possibility of obtaining bioactive compounds [21]. Since the 1980s, several studies have evaluated the use of S. platensis as a dietary supplement for intestinal disorders [39] , diabetes melli — tus, hyperglycemia [74], hyperlipidemia [67, 85], anemia [17], and hypertension [102). Moreover, it can act as an anti-inflammatory )101]. Recent studies also focused on the isolation of fatty acids, particularly the polyunsaturated ones, and pigments from photosynthetic microorganisms. Such characteristics indicate that this microorganism can be a source of molecules with potential use in pharmaceuti­cal and cosmetic industries as well. Besides, Arthrospira (Spirulina) spp. have been used for the removal of heavy metals from wastewater [27, 45], and it is important to emphasize its potential for CO2 biofixation [114], including CO2 from ethanol production plants [15, 40, 63].

In the large-scale production process, S. platensis can be easily cultivated due to the fact that it grows at high alkalinity and high salinity inorganic medium, with high content of carbonate and bicarbonate. These characteristics make it possible to inhibit or prevent contamination. Large-scale cultivation can thus be carried out in open ponds, which is very common in algae cultivation farms, where 5,000 m2 ponds are employed [93], even though there are several studies about their cultiva­tion in closed bioreactors. A. platensis biomass recovery is also facilitated due to its fi lamentous morphology.

Tacon and Jackson [104] list the following advantages for cultivation of S. platensis: they are able to use both organic and inorganic carbon sources; they exhibit a short generation time under optimum growth conditions; and they are easily cultivated in small areas. Cyanobacterial strains are carefully selected among collections around the world, which are periodically sub-cultured in the laboratory in order to maintain actively growing cells. Major criteria in the selection of strains are growth rate, biochemical composition, and resistance to environmental stress at each production site. It must be emphasized, however, that a strain that shows a good performance in the laboratory does not always display the same behavior in an outdoor open pond operation [93] .

Even though microalgae and cyanobacteria have been used by humans for a long time, microalgal biotechnology has only begun in the middle of the last century. Currently, 5,000 tons of dry microalgal biomass is marketed per year, representing up to US$1.25B [99]. The production of photosynthetic microorganisms consider­ably increased in the world due to the possibility of using this kind of culture for oxygen production and as a source of protein for food in space travels [9].

At the end of the 1970s, Sosa Texcoco Co., in Mexico, was the first responsible for large-scale Spirulina production [23, 93]. Afterward, several countries such as Taiwan, India, USA and Japan also started producing this cyanobacterium in open ponds [93]. Among the different cyanobacterial species, A. platensis stands out due to its characteristics related to cell composition, cell growth and cell recovery.

PLE is another technique that, nowadays, is regarded as an advanced extraction technique, due to the advantages that presents over other traditional extraction mechanism. PLE is based on the use of high temperatures and pressures so that the solvent is maintained in the liquid state during the whole extraction procedure. As a result of the application of these particular conditions, faster extraction processes are obtained in which generally the extraction yield is significantly higher than that obtained using traditional extraction techniques, besides, using lower amounts of organic solvents. Moreover, most of the instruments used for PLE are automated, allowing the development of less labor intensive methods and improving reproducibility.

The principles governing this kind of extraction and providing the above men­tioned characteristics are: (a) the mass transfer rate is improved as a result of the increment on the solubility of the compounds as a consequence of the increase of the extraction temperature; (b) under the PLE experimental conditions, the sur­face tension of the solvent is reduced, allowing a better penetration of the solvent into the sample matrix, increasing likewise the mass transfer; (c) the effect of the pressure theoretically could help to matrix disruption, increasing again the mass transfer rate.

Method development in PLE is by far easier than in SFE, since less parameters influencing the extraction should be considered. Once the solvent has been selected according to the nature of the compounds to be extracted, only two parameters are of significant importance: extraction time and extraction temperature. Although the extraction pressure could help to disrupt the matrix enhancing the mass transfer of the analytes contained on it, as it has been already mentioned, in practice, several reports have shown that the influence of this parameter is not significant once the pressure is high enough to maintain the solvent in the liquid state. The extraction temperature has to be optimized always keeping in mind the possible thermal deg­radation effects that might occur over the interesting extracted compounds. Although generally an increase in the temperature produces the subsequent increase in the extraction yield, for bioactive compounds, too high temperatures might lead to the degradation of these compounds. Therefore, this value should be carefully maxi­mized just to the level in which the interesting compounds start to get degraded. On the other hand, the extraction time has to be minimum enough to have an adequate mass transfer. Longer extraction times would result on slower extraction procedures and could also favor the thermal degradation, once the solvent solution is saturated with analytes from the food matrix. Therefore, quite simple experimental designs, such as full factorial designs with two factors and three levels can be useful to opti­mize the bioactives PLE extraction conditions.

Compared to SFE, the possibility of choosing among a high number of solvents causes PLE to be more versatile in terms of polarity of the bioactive compounds to be extracted and thus, the solvent will be selected depending on their nature. However, this technique is considered by far less selective than SFE. Therefore, it is important to keep in mind, that even if the extraction of the bioactives is attained, it would be possible to find other interfering compounds in the obtained extract. To avoid this problem, other steps can be included. For instance, an extraction step using hexane/acetone as solvent was performed before the PLE of phenolic com­pounds from several algae species using 80% methanol in water at 130°C for 20 min (two 10 min cycles) [132]. Ethanol has been selected to extract antioxidants from different species, such as Synechocystis sp. and Himanthalia elongata [143] or anti­microbial compounds from H. pluvialis [165]. Generally, the best extraction condi­tions in these applications were obtained at mild temperatures, around 100°C.

Moreover, PLE can be applied using a wide variety of extraction solvents, although GRAS extraction solvents, like ethanol, are most commonly used. When the extraction solvent is water, this technique is commonly called subcritical water extraction (SWE). The principles of extraction are the same, but in this case, another parameter has critical importance, the dielectric constant of water. This property of water is greatly modified with the increasing temperature when water is maintained in the liquid state. In fact, the value of dielectric constant of water (e) can vary from 80 at room temperature to values around 25 when is submitted to temperatures of ca. 250°C. This value is similar to the one presented by some organic solvents at room temperature, such as ethanol or methanol, and thus, the use of SWE could be an alternative to the use of this type of solvents in some applications. This technique has been already used to explore the possibility of obtaining antioxidants from dif­ferent microalgae species [52, 55]. However, the wide development of novel appli­cations for the extraction of bioactives from algae by using SWE has not been fully explored so far.

The pH is another important environmental factor for the ADP. Different groups of methanogens have different ranges of optimum pH. The acidogens exhibit maxi­mum activity at pH 5.5-6.5 while the optimum for methanogens is pH 7.8-8.2 [68]. Since the methanogens are more sensitive to pH variation, the pH in anaerobic digesters is usually maintained in the range of 7-8. Rapid inhibition of methanogens at pH higher than 8 can be caused by dissociation of NH4+ to the neutral NH3 form [69]. The presence of alkalinity is an important marker of pH persistence in anaero­bic digesters. The bicarbonate alkalinity buffers the fluctuations in the generation of VFAs and carbon dioxide at pH close to neutral. A stable ADP is characterized by the bicarbonate alkalinity in the range from 1,000 to 5,000 mg/L as CaCO3 [70].

The ratio between VFAs to alkalinity should be in the range of 0.1-0.25. A further increase of the ratio of VFAs to alkalinity indicates possible process deterioration and requires the OLR to decrease in order to lower the VFA formation rate.

The main goals for improving the ADP are increasing the conversion efficiency while simultaneously decreasing capital and operational costs.

5.1.2 Inoculum Source for Anaerobic Digestion of Algae

As discussed earlier, algal biomass has specific biochemical composition and con­tains unique compounds, such as algin, laminarin, and fucoidan. Moreover, marine algal biomass has a high salt concentration that can affect anaerobic microorgan­isms. Isolation and application of microorganisms adapted for digestion of specific algae is labor-intensive but has the potential to improve algal ADP.

Generally, anaerobic sludge from a domestic sewage plant or marine anaerobic sediment is used for startup of the algal ADP. Several authors reported that anaerobic organisms adapt readily to algal biomass as a sole substrate, and the inoculum source has a minor or no effect on the final methane yield and VS reduction [77,159, 368]. On the other hand, addition of an inoculum from marine sediments to anaerobic sewage sludge increased the initial methane production rate (Fig. 13) [77],

and addition of a rumen and sewage sludge inoculum adapted to algal substrate increased biogas production and methane concentration [369]. Application of an inoc­ulum adapted to high ammonia concentration is a possible solution to overcome the problem of ammonia inhibition. The inoculum from a piggery anaerobic pond yielded stable methane production from algae with an added ammonia-N concentration up to 3 g/L [370]. In contrast, a sewage mesophilic digester inoculum showed inhibition in methane production at an added ammonia-N concentration larger than 0.5 g/L.

Table 1 lists several estimates of natural gas, in hydrate form, in the geosphere’s GH stability zone (i. e., the P and T regime within which hydrates are stable). The maxi­mum value (3.053 x 10,8 m3 STP of CH.) of Trofimuk et al. [195] is based on the assumption of GH occurrence wherever a satisfactory P-T regime exists, while the minimum value (2 x 1014 m3 STP) of Soloviev [180] accounts for limiting factors such as CH4 availability, limited organic matter, porosity, regional thermal history, etc.

The Klauda and Sandler [80] estimate in Table 1 has received significant atten­tion, as it is based on a state-of-the-art model that explains most known GH occur­rences and offers plausible reasons for discrepancies from its predictions.

Even the most conservative estimates suggest enormous amounts of gas in hydrated form, the magnitude of which can be appreciated by comparing them to the current rate of 1012 m3 STP of gas-equivalent annual energy consumption in the United States. All estimates are comparatively large relative to estimates of the conventional gas reserves of 1.5 x 1014 m3 of methane [155]. Kvenvolden [97] indi­cated that his estimate of 1.8 x 1016 m3 of CH4 in hydrates may surpass the recover­able conventional CH4 by two orders of magnitude, or be a factor of 2 larger than the CH4 equivalent of the total of all fossil fuel deposits.

These include the following [125]:

• Class 4 deposits: Earlier studies by Moridis and Sloan [140] have indicated the hopelessness of such deposits under any combination of conditions and produc­tion practices.

• Fine sediments (i. e., rich in silts and clays), deformed fractured systems, and hydrates in veins and nodules despite high SH, because of the geomechanical instabilities in such systems under production.

• In Class 2 deposits: Inappropriate well configurations [131].

• In Class 2 deposits: Constant-P production, because it can lead to early break­through and massive water production; however, such an approach can be used in a short-term flow test to determine the HBL properties [55].

• In Class 2 deposits: Deep WZ, and/or permeable overburden and underburden, can drastically reduce gas production [160]. Additionally, the use of multi-well (five — spot) systems involving simultaneous depressurization (at the production well) and thermal stimulation (through warm water injection) appears disappointing [128].

• In all Classes: Permeable upper boundaries drastically reduce production [157,160].

• In all Classes: Pure thermal dissociation methods and/or inhibitor methods have high cost and limited (and continuously eroding) effectiveness [132].

• In all Classes: SH that are so high that the remaining fluids are below their irre­ducible saturation levels. Such hydrates may not be prone to depressurization — induced dissociation.

• In all Classes: Fracturing appears to have limited effect on increasing productivity from hydrate deposits [53,94].

To convert 2-KIV to isobutyraldehyde, a 2-Kivd enzyme is used. The Kivd enzyme from L. lactis is an uncommon enzyme with a traditional industrial role of aldehyde production for aroma development in cheese [75]. Given its activity with branched chain ketoacid substrates, this enzyme is uniquely suited to the task of bridging the BCAA production pathway to Adh to allow for heterologous IBT production [33]. The enzyme is a non-oxidative, thiamine diphosphate dependent, Mg2+-dependent keto acid decarboxylase [75]. The kivd gene can be expressed in an active form in R. eutropha (Sinskey laboratory, data not shown).

A wide range of valuable substances have so far been produced with microalgae. Commercial applications partially aim at high value products, e. g., carotenoids or poly-unsaturated fatty acids (PFUAs) for the pharmaceutical and cosmetics indus­tries. Microalgae biomass, rich in unsaturated fatty acids, is also a valuable source for food supplements and suitable for feed in aquacultures. Recent efforts explore the production of fine chemicals and energetic utilization of microalgae biomass and their products, e. g., biodiesel, ethanol, biogas, and hydrogen.

R. Dillschneider • C. Posten (H)

Institute of Life Science Engineering, Division III: Bioprocess Engineering, Fritz-Haber-Weg 2, 76131 Karlsruhe, Germany e-mail: clemens. posten@kit. edu

J. W. Lee (ed.), Advanced Biofuels and Bioproducts, DOI 10.1007/978-1-4614-3348-4_26, © Springer Science+Business Media New York 2013

One of the most persuasive benefits for the energetic utilization of microalgae is their capability of efficiently converting incident solar radiation to biomass. In terms of efficiency phototrophic organisms and also entire cultivation systems can be evaluated by their photoconversion efficiency (PCE). This value represents the percentage of incident solar radiation which is ultimately stored as chemical energy of the biomass. In theory, microalgae can attain PCE values of 12.6% [51]. However, in practice a PCE value of only 5% is achievable [30]. The difference can be traced to physiological and physical causes. With regard to physiology, high oxygen concentrations induce respiration and therewith loss of biomass. Moreover, excess incident light energy is dissipated as fluorescence and heat. Physical causes com­prise, amongst others, mutual shading of cells and reflectance of radiation at the surface of reactors. However, photobioreactor improvement aims at optimizing conditions, such as gas concentrations and illumination in order to minimize losses and to approach a generally assumed technical upper limit of 9% PCE [26, 30]. In temperate climates, PCE values for terrestrial plants are reported to be in the range of or even below 1% [6, 29].

For each individual application and valorization of products a certain price limit for the biomass is given and fundamentally influences the process design itself. Prices for pharmaceutical products are certainly higher than for biodiesel and there­fore justify expensive processes. In this case, energy balances and process costs are not crucial for the overall profitability. In the aquaculture sector, production costs of dry microalgae biomass range from 50 to 150 US$/kg. Maximal values were even specified to reach 1,000 US$/kg [32]. Prices for biomass targeting the animal feed market need to decline to less than 10€ (circa 13 US$). Production cost for biomass targeting the energy market need to be even below these values [35] .

Cultivation of microalgae for the energy market imposes challenging restraints for bioreactor design. Even though prices on the energy market are expected to continually increase in the next years and even decades, the continuing exploitation of fossil resources confines the upper price limit for alternative and sustainable energy sources. Gross margins earned with low-price products, such as hydrogen or biodiesel, are very small. Learning curve effects in microalgae cultivation and cost reduction of large scale implementation (economies of scale) are not only expected to reduce costs but are also necessary for price-competitive applications [5] . Moreover, an integrated utilization of products serving the energy market and the simultaneous valorization of side-products might be a promising approach to meet the challenge and increase the overall added value.

Nevertheless, price-competitive bioprocesses must be focused on and engineer­ing must aim at providing low-cost bioreactors which attain high productivities. Moreover, a positive net energy balance is crucial for a competitive bioprocess and also fundamentally determines the ecologic benefit of the process. In order to meet both economic and energetic demands, development of novel photobioreactors requires the consideration and permanent assessment of the three indicators produc­tivity, cost, and net energy gain.

E. Adair Johnson, Zhanfei Liu, Elodie Salmon, and Patrick G. Hatcher

Abstract We describe a new procedure for conversion of algal biomass into biodiesel using a single step process through the use of tetramethylammonium hydroxide (TMAH). The dried algae is placed in a laboratory-scale reactor with TMAH reagent (25% in methanol) under a blanket of flowing nitrogen gas and converted to a con­densable gas-phase product (biodiesel) at temperatures ranging from 250 to 550°C. The condensed biodiesel is freed of methanol and analyzed by gas chromatography/ mass spectrometry. Fatty acid methyl esters (FAME) are the main products of the reaction at all temperatures studied. Residues from the one-step conversion exhibit varying levels of transformation which may likely affect their end use.

1 Introduction

Because of increases in crude oil prices, limited resources of fossil fuels, and the growing concern of greenhouse gases, there has been a renewed interest in convert­ing biomass, vegetable oils, and animal fats to biodiesel fuels [5, 9, 19] . There are many forms of biodiesel from biomass, but one form is commonly produced from vegetable, plant, and animal-based oils that are converted to fatty acid methyl esters (FAMEs) by a transesterification process [8, 22]. The traditional biological sources of biomass oils are soybean, sunflower, and rapeseed. A drawback to using these

E. A. Johnson • E. Salmon • P. G. Hatcher (*)

Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA 23529, USA e-mail: PHatcher@odu. edu

Z. Liu

Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA 23529, USA

Marine Science Institute, The University of Texas at Austin,

Port Aransas, TX 78373, USA

J. W. Lee (ed.), Advanced Biofuels and Bioproducts, DOI 10.1007/978-1-4614-3348-4_29, 695

© Springer Science+Business Media New York 2013

biomass sources for producing biofuel is that they compete for land with agricultural crops used as food sources. Algae as a renewable biomass, however, eliminate many of the problems associated with traditional biomass sources.

The use of microalgae as a source of biodiesel is not a new concept and was a focus of the Department of Energy’s “Aquatic Species Program” commenced in 1978 [28]. Although great progress was made, the program was discontinued in 1996 because of decreasing federal budgets and low petroleum costs [25]. With cur­rent higher petroleum costs and overall interest in biodiesel, the interest in algal — derived biofuel has increased significantly. Algae are an attractive form of biomass for biodiesel because they do not compete for land needed for food crops. They are an inexpensive and vast renewable resource, and some species are highly enriched with lipids [5]. The main advantage of microalgae is that they can exhibit doubling rates of once or twice a day, making them among the most efficient organisms at converting sunlight and atmospheric CO2 into biomass. They can grow photosyn­thetically so that no carbon source other than CO2 is required for growth. The com­bustion of any fuel from this biomass source will yield CO2 previously fi xed from existing atmospheric CO2 so that the energy supply will be regarded as CO2 neutral [27]. In comparison with other more traditional biomass fuel sources, algae have the potential to yield more energy per acre per unit time and appear to be the only source of biodiesel that has the potential to replace fossil diesel if used exclusively [5]. Weyer et al. [29] calculated the theoretical maximum for algal oil production to be on the order of 354,000 Lha-1year-1 of unrefined oil, with best cases examined ranging from 40,700 to 53,200 L ha-1year-1 of unrefined oil.

Finding an economical process to convert algae to biodiesel is one of the major challenges of producing algal biofuel on an industrial scale. The transesterification of oils to FAMEs through a base-catalyzed process is a common commercial tech­nique known for about 100 years [4]. This chemical process converts the triglycer­ides from vegetable oils and animal fats into FAMEs via a multistep synthesis at temperatures less than 60°C [22]. There have been recent attempts to develop cata­lysts and processes to perform this conversion of the oil through a single-step pro­cess; however, we are unaware that anyone has demonstrated direct conversion to FAMEs by a one-step treatment of the biomass [4, 24, 30]. We report here a pro­posed method in which the conversion of algae to biodiesel involves only a one-step methylation/transesterification and simultaneous distillation of FAMEs at high temperature (e. g., 250°C) using the alkylation reagent Tetramethylammonium hydroxide (TMAH) and shown in (1) [7, 16, 17].

This reagent has previously been used for conversion of cooking oils to FAMEs at temperatures of less than 60°C as its strongly alkaline properties make it ideal for the conventional transesterification process [1, 4] in the presence of methanol.

The one-step thermal conversion of algae to biodiesel with TMAH was carried out in a reactor we consider only as a prototype for larger, more commercial units. Analysis of the collected liquid product was carried out using GC-MS to confirm the presence of FAMEs and other products. The unconverted residue was analyzed for use as a potential fertilizer by elemental analysis and solid-state 1 3C nuclear magnetic resonance (NMR) to evaluate the extent of the transformation brought about by elevated temperatures in the reactor.