I f biomass were grown for energy to an amount equal to that consumed during their any given production period, there would be no net buildup of COo in the atmosphere (Gao and Mickinley 19940. Microalgae are particularly promising biomass species because of the high growth rate and high CO2 fixation ability compared to plants (Tsukahara and Sawayama 2005 ).

The earliest idea focused on producing methane gas from microalgae. The concept of producing fuel by using microalgae as a source was reported by Meier (1955). Golueke and Oswald (1959) presented the concept of using microalgae as a substrate for anaerobic diges­tion, and the reuse of the digester effluent as a source of nutrients. They realized these con­cepts by using a large pond (40 pa) to grow microalgae, then the microalgae was digested to methane gas for producing electricity. The gas production by the digester averaged about 10 ft3 per lb of volatile matter introduced. The methane content of the gas varied from 68% to 74%. The maximal efficiency attained by the algal culture was 3%, whereas the maximal overall efficiency of the entire conversion unit was approximately 2% (Golueke and Oswald 1959). From the 1970s, the National Science Foundation-Research Applied to National Needs Program (NSF-RANN) started to support laboratory studies of microalgae fermentations to methane gas (Uziel et al. 1975). Six microalgae species were studied, approximately 60% of microalgae biomass energy could be converted to methane gas. It was found that the rate of biogenic methane gas production by the marine strain methanogenic bacteria at 50% wet algal thalli amendment was greater by 33.4% in comparison with results of the freshwater cattle manure strain methanogenic bacteria under similar experimental conditions. The pro­portion of methane gas content in this biofuel gas was 58%, while the remaining gases are CO2 (major portion), H2S, NH3, N2, and O2 (Silvalingam 1982).

Many microalgae, in particular species classified as “green algae,” produce hydrogen after a period of anaerobic conditions in the dark, during which the hydrogenase enzyme was activated and synthesized, and small amounts of hydrogen production were observed (Das and Veziro lu 2001). Green algae are probably better for hydrogen production than cyano­bacteria (blue-green algae) whereas the latter uses more energyiintensive enzymes, ATPi requiring nitrogenase for the production of H2 (Lee and Greenbaum 1997).

Methane production from microalgae became the basis and motivation of the U. S. DOE’s program to develop renewable transportation fuels from microalgae, which started in 1978 and ended in 1998. This program mainly focused on the production of biodiesel from high lipid-content microalgae grown in ponds, utilizing waste CO2 from coal fired power plants. Biodiesel is an alternative fuel produced from triglycerides and fatty acids present in naturally occurring fats and oils. Traditional oil crops such as corn, soybeans, canola, coconut, and oil palm cannot adequately contribute to replacing petroleum derived from liquid fuels in the foreseeable future due to their relatively low oil yield per hectare compared with microalgae. For example, 30% oil (by wt) of microalgae has an oil yield of 58,700 l/ha and 70% oil (by wt) of microalgae has an oil yield of 136,900 l/ha. By comparison, corn and soybeans have only an oil yield of 172 l/ha and 446 l/ha, respectively (Chisti 2007). Hu et al. (2008) reported that based upon the photosynthetic efficiency and growth potential of microalgae, theoretical calculations indicated that annual oil production of larger than 30,000 L or about 200 barrels of algal oil per hectare of land may be achievable in mass culture of oleaginous algae (algal species have been found to grow rapidly and produce substantial amounts of triacylglycerols or oil). This value was 100-fold greater than that of soybeans, a major feedstock currently being used for biodiesel in the United States.

Another unique benefit of using microalgae to produce biodiesel is that it will not com­promise production of food, fiber, and other products derived from crops. Xu et al. (2006) used n-hexane to extract large amounts of microalgal oil from Chlorella, which the crude lipid content is about 55.2%. Then the microalgal oil was transformed into biodiesel by acidic transesterification. The biodiesel was characterized by a high heating value of 41MJ/kg, a density of 0.864 kg/L, and a viscosity of 5.2 x 10-4Pa s at 40°C (Xu et al. 2006).

Pyrolysis with different solvents and re-agents were conducted on algae-protein (Goldman et al. 1980). The reactions yielded rather low conversions in the presence of water in spite of the existence of carbonates and catalysts, for example, nickel sulfate. The presence of benzene improves the yield and the presence of a mixture of K-Mg-Mn salts was beneficial for such a reaction. The nitrogen content of liquid oil decreased in the presence of carbonates and other catalysts. The maximum amount of protein converted into liquid oil was 27% by weight for algae-proteins containing 5.7 wt % nitrogen.

Lipid content in microalgae was considered as the most important component for yielding biofuel, such as biodiesel and other forms of oil. Peng et al. (2001a) studied the pyrolytic characteristics of Chlorella protothecoides. Chlorella protothecoides were pyrolyzed at the heating rates of 15, 40, 60, and 80°C/min up to 800°C. The pyrolysis reactions mainly took place between 160-520°C with a volatile yield of about 80%. The devolatilization stage consisted of two main temperature zones (I and II) with a transition at 300-320°C. The researcher found that crude lipid in cells decomposed at Zone II while other main components at Zone I, which might indicate that more energy input for lipid pyrolysis seems needed in comparison with other main components (Peng et al. 2001b). In another study, two kinds of high protein and lipid contents microalgae, Cyanobacterium Spirulina platensis (SP) and green alga C. protothecoides (CP) were pyrolyzed at the heating rates of 15, 40, 60, and 80°C/min up to 800°C in the thermogravimetric analyzer to investigate their pyrolytic char­acteristics. The results showed the value of activation energy for CP pyrolysis was lower than that of SP, and the char in final residue of CP was 2%-3% less than that of SP, which indi­cated CP was preferable for pyrolysis over SP (Peng et al. 2001).

Fast pyrolysis (a sweep gas [N2] flow rate of 0.4m3/h, and a vapor residence time of 2-3 s) was used to treat C. protothecoides to produce bio-oil. The highest yield of bio-oil is 57.9%, at an operating temperature of 450°C. This yield is 3.4 times higher than that from autotrophic cells also treated by fast pyrolysis. After reaction, the total liquid products were composed of an aqueous and an oil phase. The oil was fractioned using column liquid chromatography, and separated into n-hexane soluble and n-hexane insoluble compounds. The bio-oil was characterized by a much lower oxygen content, with a higher heating value (41MJ/kg), a lower density (0.92kg/L), and lower viscosity (0.02Pa • s) compared to those of bio — oil from autotrophic cells and wood. These properties are comparable to fossil oil (Miao and Wu 2004).

Algal lipid or even the whole algae could be pyrolized to a similar, high-octane, aromatic gasoline product slate when passed over HZSM — 5, a medium-pore, shaper/ selective, acid catalyst. The first of these results was reported by Milne and Evans (1987). The same authors carried out exploratory studies of the pyrolysis and zeolite conversion of whole algae and their major components. Four species of microalgae were pyrolized: Chaetoceros muelleri var. subsalsum, Monoraphidium minutum, Naviculus Saprophilla — and Nannocloropsis sp. However, the results of whole algae were ambiguous due to very high mineral matter content (10%-50%) and unknown proportions of water, lipids, and other organic components of the exact sample used (Milne et al. 1990).

One of the shortcomings of using pyrolysis to convert microalgae is due to the high moisture content of microalgae. The large amount of energy consumed to vaporize the water during the pyrolysis process was considered as a negative effect of this method (Minowa et al. 1995a. Dote et al. (1994) performed a liquefaction of Botryococcus braunii, with and without sodium carbonate as a catalyst. High-quality oil was obtained, which was more than the content of hydrocarbons in B. braunii (50 wt % db), in a yield of 57-64 wt % at 300°C. The oil was equivalent in quality to petroleum oil (Dote et al., 1994). The properties of the oil obtained from B. braunii were clarified by the same research group (Inoue et al. 1994). The oil was fractionated into three fractions by silica gel column chromatography and analyzed to deter­mine its composition. The yields of the three fractions based on organics were 5% of lower molecular weight hydrocarbons (MW = 197-281), 27.2% of botryococcenes (MW = 438­572), and 22.2% of polar substances (MW = 867-2209). The maximum recovery (78%) of botryococcenes in the liquefied oil was achieved at 200°C with the use of a catalyst.

Minowa et al. (1995b) used HTL to convert Dunaliella tertiolecta (78.4% moisture content) into oil at around 300°C and 10 MPa. The D. tertiolecta was cultured batchwise in an open tank of 10-L capacity at continuous light of 20,000 1ux, temperature of 27°C, and bubbling air with 3% CO2. Then the algal cells grown for HTL were harvested by a centrifugal sepa­rator. Some Na2CO3 (0-5 wt % of the dry solid in the algal cells) was used as additive. However, the results showed it had no catalytic effect on either the oil yield or its properties. Nitrogen was introduced to purge the residual air in the autoclave. To prevent water from vaporizing, additional nitrogen was added to 3 MPa. Several reaction temperatures were tried: 250, 300, and 340°C. The retention time (holding time) was from 5 minutes to 60 minutes.

The gas phase was primarily CO2 . Besides the gas phase, the reaction mixture consisted of a tar-like material and a water phase. The tar-like material floated on the surface of the water phase in all experiments and was easily separated. The oil was extracted from the reaction mixture by dichloromethane. Then the dichloromethane was evaporated from the extract at 35°C under reduced pressure, yielding a dark-brown viscous material, which was referred to as the oil. The oil was obtained in the range of 31%-44% (average 37%) on an organic basis. The oil yield exceeded the algal cells crude fat content (20.5%). The reaction param­eters, including reaction temperature, holding time, and sodium carbonate addition, had no significant effect on the oil yield. However, the author thought the properties of oil strongly depended on the reaction temperature. The viscosity decreased and the heating value increased slightly with a rise in temperature. The carbon and hydrogen content tended to increase with temperature increases. The oil obtained at a reaction temperature of 340°C and holding time of 60 minutes had a viscosity of 150-330mPa • s and a calorific value of 36MJ/kg. These values were comparable to those of No. 3 fuel oil in JIS (50-1000mPa • s, about 40MJ/kg). The results of the energy consumption ratio (ECR) indicated the liquefaction was a net energy producer (Minowa et al. 1995b).

Metal catalysts had been used in microalgae liquefaction. Matsui et al. (1997) investigated the liquefaction of Spirulina, a high-protein microalgae in various organic solvents or water under hydrogen, nitrogen, or carbon monoxide in the temperature range 300-425°C, using Fe(CO)5-S catalyst. Among the solvents of tetralin, 1-methylnaphthalene, toluene, and water, it seemed more favorable for liquefaction of Spirulina to take place in water. After reaction, tetrahydrofuran (THF) was used to extract the production. Then the THF-soluble fraction was further separated into hexane-insoluble and hexane — soluble fractions by precipitation into hexane. The hexane — soluble fraction was denoted as oil in this research. Liquefaction of Spirulina at 300-425°C under hydrogen gave more than 90 wt % conversion and 60 wt % oil yield. Addition of the Fe(CO)5-S catalyst increased the oil yield from 52.3 wt % to 66.9 wt % at 350°C for 60 minutes in tetralin. Liquefaction in water gave an oil yield as high as 78.3 wt % at 350°C even under nitrogen without a catalyst. Liquefaction in toluene gave oil fractions having a heating value of 32-33 MJ/kg, but products obtained in water, containing large amounts of oxygen, were estimated to have a lower heating value of 26 MJ/kg (Matsui et al. 1997).

The microalgae and coal were co-liquefied with the presence of coal liquefaction catalysts (Ikenaga et al. 2001- . Dried samples of Chlorella, Spirulina. and Littorale were used as microalgae and Australian Yallourn brown coal and Illinois No. 6 bituminous coal were employed. Commercial iron pentacarbonyl (Fe[CO]5), trisruthenium dodecacarbonyl (Ru3[CO]i2- , and molybdenum hexacarbonyl (Mo[CO]— were employed as catalysts. The co-liquefaction was carried out under pressurized H2 in 1-methylnaphthalene at 350-400°C for 60 minutes. Co-l iquefaction of Chlorella with Yallourn coal was successfully achieved with excess sulfur to iron (S/Fe = 4), The conversion and the yield of the hexane-soluble fraction were close to the values calculated from the additivity of the product yields of the respective homo reactions. All three catalysts were effective for the co-liquefaction of micro­algae with coal. Some 99.8% of conversion and 65.5% of hexane soluble fraction were obtained at 400°C with Fe(CO)5 at S/Fe = 4, when the 1:1 Chlorella and Yallourn coal were co-liquefied. In the co—iquefaction of Chlorella with Illinois No. 6 coal, the oil yield was close to the additivity of the respective reaction with Fe(CO)5-S, even at S/Fe = 2. Mo(CO)6 catalyst (S/Mo = 4) was the most effective for the respective homo-liquefactions of Chlorella and Yallourn coal. When Littorale and Spirulina were used as microalgae, a similar tendency was observed with the iron catalyst.

Microalgae culturing conditions and the fixation of CO2 during the microalgae growth were studied in order to find the optimal condition to harvest large amounts of algae. It was also useful to investigate the energy balance and CO2 mitigating effect of a liquid fuel production process from microalgae using HTL. Kishimoto et al. 2 1994) studied the CO2 fixation during the microalgae growth and oil production of microalgae by using thermo­chemical liquefaction. The green microalga D. tertiolecta AHTP 30929, which contained 10% glycerol, was used throughout the study. The effects of saline, light intensity, and CO2 concentration on microalgae growth were investigated. The details of the liquefaction were not mentioned in this paper, but the authors concluded that the heavy oil yield from the liquefaction was 35.6%. The contents of the chemical elements were as follows: carbon 73%, hydrogen 9%, nitrogen 5%, and oxygen 13%. The heating value of the heavy oil was 34.7 MJ/kg, which was almost the same as C heavy oil. The viscosity of the oil was 860 mPa • s, the same as that of castor oil. The nitrogen content was higher than that of ordinary petroleum.

Continuous culturing of the B. bruunii Berkeley strain in secondarily treated sewage (STS) was conducted and then liquefied by Sawayama et al. (1995) • B. bruunii grew continuously for a period of over 1 month at a growth rate of 200 mg dry weight per liter water per week and the algal cells fed on STS containing 49% oil as the hexane soluble fraction. Liquefaction of microalgae cells was conducted in a 300 mL autoclave. Wet cells (30g wet weight, 92% moisture content) were charged to the autoclave with 5 wt % or without sodium carbonate. N2 was used as the initial gas for preventing water from vaporizing. The autoclave was heated to 200, 300, and 340°C, with a retention time of 1 hour. Oil was extracted with dichloro — methane from the reaction mixture. The maximum yield of oil obtained by liquefaction was 64 wt % on a dry basis at 300°C with a sodium carbonate catalyst. The yield of the hexane soluble fraction was 97 wt % compared with that in the feedstock algal cells. The heating value of the liquefied oil obtained from this reaction was 49 MJ/kg and the viscosity was 64mPa s at 50°C.

Different microalgae are not the same in producing oil through liquefaction. The data of liquefaction of microalgae B. braunii and D. tertiolecta were collected and compared, and then the energy balance of the reactions was calculated by Sawayama et al. (1995). Liquefaction was performed using a stainless steel autoclave with 100 or 300 mL capacity using 0-5 wt % and Na2CO3 as a catalyst. After purging with nitrogen, the autoclave was charged with nitro­gen at 2-3 MPa. The reaction temperature was 300°C. The reaction mixture included a gas mainly composed of CO2, a tar-like material that sank to the bottom, a water phase, and an oil-like material that floated on the surface of the water phase. Solvent extraction with CH2C12 or acetone was performed to separate the oil from the reaction mixture. The yield of oil was determined as a percentage by weight of the organics in the original material. Based on the energy calculation and comparison, the yield of liquid fuel produced from B. braunii and its lower heating value were high compared with those of D. tertiolecta; therefore, the energy inputs for cultivation and separation of B. braunii were calculated to be smaller than those of D. tertiolecta. The energy input for fertilizers of B. braunii was also smaller than that of D. tertiolecta. Therefore, the hydrocarbon-rich microalga, B. braunii, could be more suitable for liquid fuel production using thermochemical liquefaction compared with D. tertiolecta. The authors also concluded: if a 100-MW thermal plant using coal would be replaced by liquid fuel produced from B. braunii, the quantity of CO2 mitigation could be 1.5 x 105t/yr and 8.4 x 103 ha of microalgal cultivation area could be necessary (Sawayama et al. 1999).

To find the most suitable operational conditions of HTL of microalgae, Yang et al. (2004) used the Microcystis viridis strain as feedstock. The series of experiments were conducted under the following various conditions: catalyst (Na2 CO3) loading rate of 0 and 5 wt %; reaction temperature of 300 and 340°C; and holding times of 30 and 60 minutes. The initial operational pressure was designed at 3 MPa of nitrogen, and the maximum pressure of the autoclave was 10-20MPa in order to decrease water evaporation. The reaction mixture was extracted with chloroform to recover the oil by evaporating chloroform at 40°C. The aqueous phase and insoluble mixture were separated by filtration. The chloroform insoluble fraction remaining on the filter paper was dried at 105°C for one day to obtain a solid residue. The evolved gas was transferred to a sampling bag and composition was determined by gas chromatography. The oil yield was defined as the ratio of the weight of oil products after liquefaction to the weight of organic matter in feedstock. The energy yield was defined as the ratio of the weight of C and H in oil products after liquefaction to the weight of C and H in feedstock. After investigating the effects of the reaction parameters, such as retention time, reaction temperature, and load of the catalyst, the maximum 33% oil yield (energy yield of around 40%) was obtained with the 30-minute holding time, reaction temperature of 340°C, and alkali catalyst dosage of 5 wt %. The heating value of the obtained oil was 31 MJ/kg, less than that of heavy oil (40 Mj/kg). The elemental composition of liquefied oil was composed of 62% carbon, 8% hydrogen, 8% nitrogen, and 2% sulfur. The liquefied oil contained n-alkane of C17-C18 hydrocarbon as a main component of the saturated compounds, so typical aromatic compounds of heavy oil, such as n-naphthalene and n-dibenzothiophene were found in liquefied oil, and it was considered that the liquefied oil should be classified as heavy oil. The gas consisted primarily of CO) and methane. The total nitrogen in the aqueous phase ranged from 998 to 1157mg/L, and half of the total nitrogen was detected as ammonia nitrogen. The total phosphate in the aqueous phase ranged from 2.47 to 5.38mg/l.

After microalgae liquefaction, other forms of biofuel—such as biodiesel—could be obtained by further extraction. Based on the efficiency of biodiesel extraction, Aresta et al. (2005) compared HTL of algae with the extraction using supercritical carbon dioxide (sc-CO2). Green alga Chaetomorpha linum was used as the feedstock. Almost 20 g of fresh, washed thalli of C. linum were placed into the glass reactor which was then put into the autoclave. The latter was closed under N2 atmosphere to purge the residual air and 3.0 MPa of N) . The autoclave was then heated to the desired temperature (250, 300, 350, and 395°C) for 1 hour. The reaction mixture was recovered and treated with CH2 Cl2. Then an organic and an aqueous/solid suspension were separated. From the organic solu­tion, after evaporation of the solvent under controlled conditions, an amber-yellow oily liquid was obtained. The aqueous solution was separated and a solid was recovered by centrifugation. For the sc-CO2 extraction, the thalli of alga were dried for 5-8 days at room temperature, 3-5 g of dried algae were milled in liquid nitrogen (5 mL) to break the cellular wall in order to increase the extraction yield. A 0.5-1 mL of methanol was used as a co-solvent to improve the efficiency of the extraction. In both liquefaction and sc-CO2 extraction, the oils obtained were treated to convert all fatty acid (FA) components into the mono-methyl-esters (Biodiesel). Then the extracted material was analyzed quantitatively by GC and qualitatively by GC-MS. For liquefaction, increasing the temperature for the total amount of oil extracted from the algae increased reaching a plateau between 350- 395°C (about 80 mg/g dry wt). Increasing the temperature affected the amount of fatty acid extracted. However, at higher temperatures, some oil decomposed, resulting in a decrease of the recovered amount of extracted fatty acid. After comparing the results from the sc — CO2 extraction, the authors implied that HTL seemed to be more efficient from the quantitative point of view.