**5. Subcritical water processing of wet algal biomass to produce green fuels**

Recently, many researchers have identified, and studied the importance of byproducts in the production of algal biofuels. Algae are being used to produce various kinds of natural products like protein, polyunsaturated fatty acids (PUFAs), vitamins, carbohydrates, and dietary fibers. Extraction and separation of these compounds is a key factor for the commercialization of algal biofuels [23, 24]. The recovery of these valuable products cannot be achieved using direct conversion methods due to severe processing conditions and degradation of these compounds. New novel methods called hydrothermal extraction and liquefaction (HT E&L) or subcritical water extraction or liquefaction can be used for the recovery of valuable products along with energy dense biocrude oil, and bio-char from wet algal biomass. Water attains these selective extraction and liquefaction properties due to increased temperature and pressure. The dielectric constant of water reduces slowly with increasing temperatures, which provides more solvating power to the water. This solvating power varies with the process parameters which include temperature, pressure, solids loading and extraction time. This technique has been used for many selective extraction applications such as bioactive components from *Haemato‐ coccus pluvialis* microalga[25], nutraceutical compounds from citrus pomaces[26], and oils from coriander seeds[27]. Typical extraction temperature will vary between 120-250o C, and changes along with pressure variation for a particular target compound. Another major advantage of this method is higher extraction efficiencies can be achieved when compared to conventional solvent extraction.

Similar to the above given examples, lipids from wet algal biomass can be extracted. In this process water present in the harvested algal biomass itself acts as the solvent; which also eliminates the drying step in the process and achieves maximum extraction of oil. This selective extraction of lipids is demonstrated with the wet algal biomass of *nannochloropsis salina.* The reaction temperature, solids (biomass) loading and reaction time are studied to extract the maximal amount of lipids from the biomass. The RSM (response surface methodology) analysis of extraction results indicated that the increase in temperature causes increase in the yield of crude extract and lipids with in the crude extract up to 217o C. Due to increased solvating power or reduced dielectric constant; further rise in temperature caused a reduction in crude extract due to secondary reactions which caused redistribution of crude extract into other product fractions like the water phase or gas phase. The biomass loading is another crucial parameter which affects the extraction efficiency. The amount of crude extract is increased with more diluted biomass and a maximum is found to be at 5%; meaning less biomass loading provides more solvent for the extraction of target compounds. But at the same time the energy consumption should also be considered; more water in the biomass requires more energy to reach the target processing conditions. Due to this, optimum biomass loading was increased to 7.5%, which slightly affects the extraction efficiency. The reaction time was found to be very when compared large to solvent extraction techniques, and maximum extraction was achieved within 25 min. Through this subcritical water extraction method nearly 60-70% of the lipids present in the algal biomass can be extracted. Along with the lipids, the water fraction contains sugars, sugar alcohols, and PUFAs in algae oil which have commercial value in the pharmaceutical and food industry; Additionally the bio-char contains proteins (~45% by wt.) and 24.9 MJ/kg of energy making it another valuable byproduct [28]. The biodiesel produced with subcritical water extraction with thermal energy recovery (60%) and utilization of bio-char to produce biogas (methane) consumes only 28.23 MJ of energy for 1kg biodiesel [29]. The process can be used only for algal biomass which has more lipids and for the production of biodiesel. In order to produce biofuels from algal biomass having fewer lipids, higher hydrothermal processing conditions should be used.

**Algal biomass:** *Nannochloropsis sp.*

172 Biofuels - Status and Perspective

solvent extraction.

**Yield (on the basis of total lipids)**

**Table 4.** Reaction conditions for maximum yields of biodiesel with algal biomass

**Reaction temperature**

When compared to biodiesel production using vegetable oils by supercritical alcohol process, nearly 2-3 times more alcohol is needed for algal biomass conversion. More energy is required for the separation of the extra alcohol, making the process more energy intensive. The production of biodiesel directly from the wet algal biomass is possible; but supercritical processing of expensive feedstock like algae demands complex infrastructure and higher energy, making production of biofuels less profitable. During this process valuable byproducts like polyunsaturated fatty acid ethyl esters are lost in order to maintain the fuel properties.

**5. Subcritical water processing of wet algal biomass to produce green fuels**

Recently, many researchers have identified, and studied the importance of byproducts in the production of algal biofuels. Algae are being used to produce various kinds of natural products like protein, polyunsaturated fatty acids (PUFAs), vitamins, carbohydrates, and dietary fibers. Extraction and separation of these compounds is a key factor for the commercialization of algal biofuels [23, 24]. The recovery of these valuable products cannot be achieved using direct conversion methods due to severe processing conditions and degradation of these compounds. New novel methods called hydrothermal extraction and liquefaction (HT E&L) or subcritical water extraction or liquefaction can be used for the recovery of valuable products along with energy dense biocrude oil, and bio-char from wet algal biomass. Water attains these selective extraction and liquefaction properties due to increased temperature and pressure. The dielectric constant of water reduces slowly with increasing temperatures, which provides more solvating power to the water. This solvating power varies with the process parameters which include temperature, pressure, solids loading and extraction time. This technique has been used for many selective extraction applications such as bioactive components from *Haemato‐ coccus pluvialis* microalga[25], nutraceutical compounds from citrus pomaces[26], and oils from

coriander seeds[27]. Typical extraction temperature will vary between 120-250o

along with pressure variation for a particular target compound. Another major advantage of this method is higher extraction efficiencies can be achieved when compared to conventional

Similar to the above given examples, lipids from wet algal biomass can be extracted. In this process water present in the harvested algal biomass itself acts as the solvent; which also eliminates the drying step in the process and achieves maximum extraction of oil. This selective extraction of lipids is demonstrated with the wet algal biomass of *nannochloropsis salina.* The reaction temperature, solids (biomass) loading and reaction time are studied to extract the

**Algae to alcohol ratio**

**Reaction time (min.)**

C, and changes

**(wt./vol.)**

**(oC)**

FAME (methanol)[22] 84% 255 1:9 25 FAEE (ethanol)[21] 67% 265 1:9 20

> Further increase in temperature of the hydrothermal extraction process, results in hydrother‐ mal liquefaction due to the enhanced reaction capabilities of water. The density of water decreases as the temperature and pressure rise towards the critical point and drops drastically after the critical point; here water medium attains gas like densities and liquid like solvent properties. The ability of water in these conditions to provide H+ or OH- ions (varying ion product (Kw) of water) is useful in performing acid or base catalyzed reactions without using external catalysts [30-32]. During the hydrothermal liquefaction, the macromolecules present in the biomass are subjected to hydrolysis, which degrades them into smaller molecules. During this process, the oxygen present in the biomass will be removed by dehydration in the form of water, and by decarboxylation in the form of carbon dioxide. During the liquefaction process energy dense biocrude oil, aqueous phase with water soluble compounds, bio-char and gaseous product will be produced [31, 33]. The subsequent intermediate reactions of the hydrolyzed or extracted compounds determine the yields of product fractions, which are influenced by processing conditions.

> Numerous studies on hydrothermal liquefaction of algae are available in literature. Few examples of these studies are hydrothermal liquefaction of algal biomass *nannochloropsis sp.,* [34], *chaetomorpha linum*[35], *chlorella pyrenoidosa*[36], or *spirulina plantesis*[37]. The typical processing temperature used for hydrothermal liquefaction ranges between 250-370o C. The yields and properties of the products vary with reaction temperature. The yield of energy dense

biocrude oil increases with rise in temperature up to 350o C. Lower yields can be observed at lower temperatures due to a decrease in hydrolysis of the biochemical compounds and repolymerization. Above 350o C, hydrothermal gasification becomes more dominant and contributes to the production of more gaseous products rather than biocrude oil. The typical yields of biocrude oil yields range between 20-60% on the ash free dry weight basis (AFDW). As the temperature rises the water becomes more reactive than an extraction solvent due to its increased ion dissociation constant. The ion dissociation constant at 250o C is 1000 times greater than at the room temperature [38]. Because more protons and hydroxide ions present in these conditions hydrolysis becomes more active reaction. The hydrolysis reaction causes the degradation of basic chemical compounds, and when accompanied by reactions like repolymerization produces bio-crude oil. The yields differ with process conditions, and biochemical composition of the algal biomass. The contribution of lipids, proteins and carbohydrates to the yield of biocrude oil yield are in the order of lipids>proteins>carbohy‐ drates [39]. The biocrude oil yields of commonly used algal biomass are presented in Table 5. The high heating value (H.H.V) of biocrude oil varies in the range of 32-39 MJ/kg. The reaction temperature greatly affects the quality of biocrude oil. At lower processing temperature, lipids contribute more to the yield of biocrude oil which contains more energy or higher H.H.V. At higher temperatures, due to the contribution of protein derived compounds and increased nitrogen in biocrude oil the H.H.Vs of biocrude oil reduce to lower levels. The variation reaction pressure beyond saturation has little to no effect on the product distribution in HTL of algae [36]. Even though most of the studies have used residence times between 20-120 minutes in batch (reactor) mode operation, 2-5 min. of reaction time is sufficient to get better biocrude oil yields in a continuous flow system [40].


**Table 5.** Yields of biocrude oil produced with various strains of algal biomass

The other product fractions are bio-char, aqueous phase and gaseous fraction. The bio-char yield decreases with an increase in temperatures, as the metabolites of the biomass converts into other products. The range of bio-char yields can be found between 10-70% depending on the processing temperature and time. Bio-char produced at lower temperatures tend to have higher H.H.V due to hydrothermal carbonization. The H.H.V of bio-char can be around 18-22 MJ/kg at lower temperatures and reduces to 8-10 MJ/kg at higher processing temperatures above 300o C. Due to extraction or conversion of biochemical compounds present in the biomass at higher temperatures, H.H.Vs of the bio-char are reduced compared to the H.H.V of the original biomass and bio-char produced at lower temperatures [20].

biocrude oil increases with rise in temperature up to 350o

biocrude oil yields in a continuous flow system [40].

*Chlorella sp*.[40] 4 60 25 350o

*Chlorella sp.* [41] 60 9 13 300o

*Nannochloropsis sp.* [41] 14 52.4 5 300o

*Nannochloropsis salina* [42] 12 37 33 310o

*Spirulina platensis* [42] 6 60 19 350o

*Nannochloropsis sp.* [34] 14 59 20 300o

*Nannochloropsis sp.* [43] 28 52 12 350o

*Spirulina platensis* [44] 11 49 31 350o

*Scenedesmus sp.*[45] 13 56 25 300o

*Spirulina sp.*[45] 5 64 20 300o

*Dunaliella tertiolecta* [46] 20 63 15 340o

*Dunaliella tertiolecta* [47] 22 24 46 300o

*Chlorella sorokiniana* [47] 4 30 54 300o

**Table 5.** Yields of biocrude oil produced with various strains of algal biomass

**Algal Biomass**

repolymerization. Above 350o

174 Biofuels - Status and Perspective

lower temperatures due to a decrease in hydrolysis of the biochemical compounds and

contributes to the production of more gaseous products rather than biocrude oil. The typical yields of biocrude oil yields range between 20-60% on the ash free dry weight basis (AFDW). As the temperature rises the water becomes more reactive than an extraction solvent due to

greater than at the room temperature [38]. Because more protons and hydroxide ions present in these conditions hydrolysis becomes more active reaction. The hydrolysis reaction causes the degradation of basic chemical compounds, and when accompanied by reactions like repolymerization produces bio-crude oil. The yields differ with process conditions, and biochemical composition of the algal biomass. The contribution of lipids, proteins and carbohydrates to the yield of biocrude oil yield are in the order of lipids>proteins>carbohy‐ drates [39]. The biocrude oil yields of commonly used algal biomass are presented in Table 5. The high heating value (H.H.V) of biocrude oil varies in the range of 32-39 MJ/kg. The reaction temperature greatly affects the quality of biocrude oil. At lower processing temperature, lipids contribute more to the yield of biocrude oil which contains more energy or higher H.H.V. At higher temperatures, due to the contribution of protein derived compounds and increased nitrogen in biocrude oil the H.H.Vs of biocrude oil reduce to lower levels. The variation reaction pressure beyond saturation has little to no effect on the product distribution in HTL of algae [36]. Even though most of the studies have used residence times between 20-120 minutes in batch (reactor) mode operation, 2-5 min. of reaction time is sufficient to get better

**Biochemical composition Reaction conditions**

Lipids Protein Carbohydrates **(% of AFDW)** Temperature,

solids loading and Reaction time

C, 10% and 3 min. 42

C, 20% and 90 min. 66

C, 15% and 60 min. 48.4

C, 25% and 30 min. 46

C, 25% and 30 min. 38

C, and 10 min. 50

C, 21% and 60 min. 43

C, 20% and 60 min. 39

C, 20% and 30 min. 45

C, 20% and 30 min. 31

C, and 5 min. 41

C, 10% and 60 min. 30

C, 10% and 60 min. 18

its increased ion dissociation constant. The ion dissociation constant at 250o

C. Lower yields can be observed at

C is 1000 times

**Biocrude yield**

C, hydrothermal gasification becomes more dominant and

The aqueous phase of the HTL is another valuable product fraction which contains essential nutrients (NH3-N and PO4 3-), amino acids and carbohydrates depending on the processing temperature. The hydrolysis of proteins results in the formation of amino acids, and the deamination (further hydrolysis) of these amino acids produces the ammoniacal nitrogen. Usually the amount of ammoniacal nitrogen increases with an increase in temperature, because the amino acids decompose rapidly at higher temperatures. At the same time, the concentra‐ tion of amino acids decreases due to rapid conversion with an increase in HTL temperatures. The other valuable nutrient phosphate behaves in a different way; the amount of phosphate in water phase decreases with an increase in temperature and deposits in the bio-char fraction. The concentrations of these nutrients can be as low as 400 ppm for NH3-N, 6 ppm for PO4 3- and as high as 6300 ppm for NH3-N, 3000 ppm for PO4 3-. However, these concentrations vary greatly with the biochemical composition of the biomass and the processing temperature [20, 48, 49]. Recycling these nutrients back to cultivation is a very crucial step as it can save of fresh on addition nutrient supply and reduce overall cost. Along with the nutrients, the aqueous phase also contains valuable carbohydrates including polysaccharides, monosaccharides, sugar alcohols, amino acids and glycerol. At milder temperatures most of the carbohydrates present in the algal biomass are extracted into the aqueous phase. With an increase in HTL temperature, these polysaccharides hydrolyze to yield simple sugars and derivative compounds. At higher processing temperatures these compounds start degrading or converting into other product fractions due to secondary and tertiary reactions. A Typical optimum point for the extraction of polysaccharides is around 160o C [50]. Similar to the extraction of lipids, polysaccharides from algal biomass can be extracted at lower temperatures, and the remaining biomass can be used to produce biocrude oil. The recoverable quantity of polysaccharides and biocrude oil depends on the original biochemical compositions of the biomass.

As mentioned earlier, separation and recovery of these valuable amino acids, nutrients, and carbohydrates is vital for the production of algal biofuels. This concept of algal bio refinery with sequential HTL (SEHTL) or hydrothermal extraction and liquefaction (HT E&L) is shown in Figure 4. The processing strategies for each strain of biomass vary as they possess different biochemical composition. The separation of these compounds provides a much needed economic life line and also improves the quality of biocrude oil. The removal of amino acids and nitrogen based compounds at lower temperatures reduces the quantity of nitrogen in the biocrude oil produced in the second step. When compared to cellulosic ethanol production,

**Figure 5.** Algal biorefinery with HTL process

HTL of algal biomass has better energy return on investment (EROI) and lower emissions can be achieved [41].

The biocrude oil produced in the HTL process contains nitrogen (4-9 wt.%) and oxygen (2-7wt. %). The biocrude oil produced with oils/fats in the CH process is easy to upgrade compared to biocrude oils produced with algae due to the absence of nitrogenous compounds. Due to the presence of nitrogen and oxygen, processing the biocrude oil becomes slightly complex. Two strategies can be followed to produce hydrocarbon fuels from biocrude oil. The first one is, co-processing the biocrude in existing petroleum refineries by diluting the crude petroleum up to the permissible levels of nitrogen and oxygen. The second option is direct processing of biocrude oil with suitable catalysts. These catalysts include metal oxides of Ni, Co, Mo, Pt and W supported on γ-Al2O3, SiO2, zeolites and carbon. Commercialization of these catalysts for processing biocrude oil may take some more time. More research and development is needed to optimize both the HTL of algal biomass and suitable catalysts for biocrude oils with varying properties. Compared to conventional jet fuel, the biojet fuel produced from algal biomass with HTL and upgrading can reduce life cycle greenhouse gas emissions by 76% [51].
