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. 2020 Mar 19;5(12):6684–6696. doi: 10.1021/acsomega.9b04468

Improvement of Bio-Oil and Nitrogen Recovery from Microalgae Using Two-Stage Hydrothermal Liquefaction with Solid Carbon and HCl Acid Catalysis

Ryo Usami 1, Kengo Fujii 1, Chihiro Fushimi 1,*
PMCID: PMC7114750  PMID: 32258904

Abstract

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Bio-oil production from microalgae by using hydrothermal liquefaction (HTL) has been conducted extensively in the last decade. In this work, we conducted two-stage HTL of a microalga (Fistulifera solaris, JPCC DA0580) in the presence of 5.0 g/L carbon solid acid or a 0.02–0.50 M HCl catalyst to increase bio-oil yield and nitrogen recovery into the aqueous phase (AP). The first stage (HTL 1), to hydrolyze proteins, carbohydrates, and lipids and elute nitrogen components into the AP, was conducted at 100–250 °C for 30–120 min. The second stage (HTL 2), to produce the bio-oil, was conducted at 280–320 °C for 0–30 min. The best conditions to obtain a high bio-oil yield and NH4+ recovery in the AP were 200 °C and 30 min of residence time for HTL 1 and 320 °C and 0 min residence time for HTL 2. We found that 0.50 M HCl decreased the bio-oil yield while greatly increasing NH4+ in the AP and decreasing the nitrogen content in the bio-oil. This was probably due to the catalytic effect of HCl promoting hydrolysis of protein and deamination of amino acids during HTL 1. The fractions of water-soluble products were greatly increased by performing HTL 2 in neutral conditions while this maintained low nitrogen content in the bio-oil. From GC–MS analyses of the bio-oil, it was observed that, by using 0.50 M HCl, peak intensities of all the GC peaks decreased and MS spectra of amines decreased. The carbon solid acid had an insignificant influence on bio-oil and NH4+ yields.

1. Introduction

Bio-oil derived from microalgae is an attractive alternative to fossil fuels since microalgae generally contain a large amount of lipids, their growth rate is high, and their cultivation does not compete with food production.1−5 The bio-oil from microalgae is produced via cultivation, harvesting, dehydration, and extraction processes1 using solvents, supercritical CO2, or microwaves. In the solvent extraction method, bio-oil is extracted using nonpolar organic solvents such as hexane or polar organic solvents such as methanol and chloroform.5−9 It is possible to maximize the extraction of lipids by decomposing polar lipid complexes with polar organic solvents and solubilizing intracellular lipids with nonpolar organic solvents.7 However, solvent extraction is usually uneconomic due to consumption of much energy to dry the microalgae as pretreatment and the need to separate bio-oil from solvent after extraction. Tsutsumi et al.10 reported that many environmental hazards and safety problems remain in bio-oil recovery by solvent extraction because a large amount of organic solvent is needed.

Hydrothermal liquefaction (HTL) is another method of extracting bio-oil from biomass. In HTL, microalgae react in subcritical water at 250–350 °C and 5–20 MPa and are decomposed into an oil phase, an aqueous phase (AP), gas-phase products, and char.11−13 Because this method does not require an energy-intensive drying process, energy consumption is considered to be low.14 In addition, the nutrients dissolved in the AP15,16 can be reused in the culturing process.17−20 Moreover, the bio-oil yield is higher than that by other extraction methods since substances (carbohydrates and proteins) other than lipids can also be decomposed and converted to oil components. However, the bio-oil obtained after HTL contains a large amount of nitrogen compared to that obtained by the solvent extraction method. This causes oligomerization of the molecules, an increase in viscosity of the bio-oil, and inhibitory effects in the purification process.21 It is therefore necessary to decrease the nitrogen content in the bio-oil by eluting more NH4+ into the AP after HTL. This also greatly lowers the cost of nutrients for the culturing process.

Shakya et al.22,23 measured yields of products (bio-oil, char, water-soluble products, and gas), total acid number of the products, pH, density, heating value, ash, moisture, and elemental composition of bio-oils produced after HTL of different strains of microalgae at various temperatures in the absence and presence of Na2CO3. They concluded that other than the product yield, the use of Na2CO3 had no significant effect on the properties of the bio-oil,22 and the obtained APs contained a large amount of total organic carbon (12–43 g/L), chemical oxygen demand (35–160 g/L), total nitrogen (1–18 g/L), ammonium (0.34–12 g/L), and phosphate (0.7–12 g/L).23

It has been reported that the reaction mechanism of HTL of algae involves four consecutive steps: (1) hydrolytic depolymerization of the proteins and carbohydrates to form water-soluble monomers such as peptide fragments, amino acids, and carbohydrate derivatives; (2) degradation of the monomers by dehydration, deamination, and decarboxylation; (3) recombination/condensation of reactive fragments between (i) fatty acids and (ii) the decarboxylation products of amino acids and fragments of the carbohydrate derivatives and/or fatty acids to form the bio-oil; and (4) polymerization at a prolonged reaction time to form char.24−28 Chiaberge et al. stated that the most likely origin of fatty acid amides in hydrothermal bio-oils is the condensation reaction between fatty acids and the decarboxylation products of amino acids.25 Matayeva et al. recently investigated the reaction mechanisms of HTL of phenylalanine, leucine, and a mixture of tripalmitine and phenylalanine and stated that phenylethylamine or isopentylamine, which is produced by decarboxylation of phenylalanine or leucine, reacted with other products, forming amides that became nitrogen-containing components of the bio-oil.28 Nitrogen from amino acids can be recovered in the AP mainly as NH4+ following the deamination of amino acids.29−34 Based on these reaction mechanisms, it is very important to elute NH4+ (or NH3) by promoting the deamination of amino acids and to decrease the amount of nitrogen in bio-oil by suppressing production of amides and nitrogen-containing heterocyclic compounds.

A two-stage HTL method has been attracting attention (Table 1).35−38 In the first stage, the proteins in the microalgae are hydrolyzed and solubilized into the AP at ≤250 °C. In the second stage, the bio-oil is produced during decomposition of other components and repolymerization at 250–350 °C.35 Bio-oil with low nitrogen content has been produced using two-stage HTL.35−37 Costanzo et al.36 successfully reduced 0–23 wt % of the nitrogen content in bio-oil with two-stage HTL of UGA Consortium, Nannochloropsis and Spirulina relative to one-stage HTL. Prapaiwatcharapan et al.37 reported that bio-oil with low nitrogen content (approximately 2–4 wt%) was produced from Coelastrum sp. Jazrawi et al.35 reported the bio-oil obtained by direct HTL of Chlorella vulgaris (C. vulgaris) at 300 °C contained 7.5 wt % of nitrogen and the nitrogen content in the bio-oil was decreased to 3.4% by two-stage HTL. Moreover, Sunphorka et al.38 investigated the nitrogen content in the AP after two-stage HTL of Coelastrum sp. and reported that two-stage HTL resulted in higher NH4+ yields than that of conventional one-stage HTL. Regarding the bio-oil yield, it has been reported37 that the two-stage HTL method increased the bio-oil yield compared with one-stage HTL. However, the bio-oil yield decreased from 25 to 11–20 wt % in the two-stage HTL reported by Jazrawi et al.,35 and the nitrogen content in the bio-oil was still high.

Table 1. Research into Two-Stage HTL of Microalgae.

  first HTL conditions
 second HTL conditions
    
alga temp [°C] time [min] catalyst temp [°C] time [min] catalyst comments ref
Fistulifera solaris (F. solaris) 100–250 30–120 5.0 g/L solid acid/0.02–0.50 M HCl 280–320 0–30 0.5 wt % solid acid/0.02–0.50 M HCl   this study
Spirulina, Nannochloropsis, UGA Consortium 125–225 0.5–30   350 60   Nitrogen content in bio-oil was decreased by 0–23% relative to one-stage HTL. (36)
Coelastrum sp. 150, 200 120   280, 320 120   Two-stage HTL gave a higher biocrude yield (36 wt %) and nutrient recovery level in terms of nitrogen-containing compounds than one-stage HTL (38)
Coelastrum sp. 150–225 120   280–360 120   Using a semi-continuous process, a higher bio-oil yield with a lower nitrogen content was obtained with the two-stage HTL than with one-stage HTL. (37)
C. vulgaris 100–200 30–120 5 wt % H2SO4, HCOOH 250–350 10 – In two-stage HTL, a lower bio-oil yield was obtained with a low nitrogen content in the bio-oil. Solubilization of protein under mild conditions (∼100 °C) was enhanced in the presence of acids (35)
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On the basis of the results of these studies, it is believed that two-stage HTL with acid catalysts prevents the polymerization of bio-oil, amino acids, and their derivatives during thermal decomposition in subcritical water, decreases the nitrogen content in the bio-oil, and promotes elution of water-soluble products such as NH4+ into the AP. The objective of this study was to determine the optimum conditions for two-stage HTL and the effect of using acid catalysts on bio-oil quantity and quality and NH4+ recovery from microalgae. It was reported that the addition of acetic acid significantly increases bio-oil yields and N contents in the bio-oil in the case of phenylalanine and leucine HTL though formic acid has an insignificant effect on the bio-oil yields and N contents.28 Thus, in the present study, two-stage HTL was conducted using carbon solid acid (SA) and inorganic acid (HCl) to promote protein hydrolysis and deamination of amino acids at low temperature and bio-oil production.

2. Results and Discussion

2.1. Effects of Reaction Temperature, Residence Time, and Carbon SA on Bio-Oil and NH4+ Yields in One-Stage HTL

The one-stage HTL was conducted at various reaction temperatures (280, 300, and 320 °C) and residence times (0, 15, and 30 min) in the absence and presence of the carbon SA catalyst (0, 5.0 g/L). Note the residence time was defined as the time after the reactor temperature reached the set temperature. Blank experiments were conducted by using the carbon SA catalyst. Table S1 shows the results. At 320 °C, 17%, 23.93%, and 29.1% SA was lost during HTL for 0, 15, and 30 min, respectively. No oil-phase formation was observed. These indicate that some of the SA dissolved into AP or was converted into gas. At 300 and 280 °C, a much smaller amount of SA was lost. Figure 1a shows the bio-oil yield as a function of temperature when the residence time was 30 min. Note hereafter that the error bars show the standard deviations of the results under each condition and the weight of the dry microalga is of a dry basis. In the absence of the carbon SA catalyst, the bio-oil yield was 0.0668 ± 0.00440 g/g-dry microalgae at 280 °C, and it decreased when the temperature was increased to 300 and 320 °C. In the presence of the carbon SA, the bio-oil yield increased as the temperature increased. The bio-oil yield peaked at 0.0747 ± 0.00473 g/g-dry microalgae in the presence of the SA at 320 °C. At 300 and 320 °C, the bio-oil yield was increased by the addition of the carbon SA relative to its absence. This implies that hydrolysates derived from lipids, carbohydrates, and proteins were polymerized and converted into bio-oil components. Figure 1b shows the NH4+ yield as a function of temperature with or without the SA when the residence time was 30 min. The NH4+ yield increased up to 300 °C. No further increase in the NH4+ yield was observed at 320 °C. This implies that the proteins were not completely hydrolyzed and decomposed to NH4+ at 280 °C in 30 min; thus, it was necessary to increase the reaction temperature to ≥300 °C to hydrolyze more proteins and increase the NH4+ yield. No significant effect of carbon SA was observed on the NH4+ yield. From the viewpoints of bio-oil and NH4+ yields, the optimum temperature for the one-stage HTL (corresponding to step HTL 2 in the two-stage HTL) was 320 °C.

Figure 1.

Figure 1

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Effects of reaction temperature on bio-oil and NH4+ yields in the presence/absence of the carbon solid acid catalyst in one-stage HTL when the residence time was 30 min: (a) bio-oil yield and (b) NH4+ yield.

Figure 2a,b shows the bio-oil and NH4+ yields as a function of residence time with or without SA when the temperature in one-stage HTL was 320 °C. It can be seen that the bio-oil yield peaked at 0.0852 ± 0.00144 g/g-microalgae in the absence of SA at a 0 min residence time and decreased with increased residence time. When the residence time was 0 or 15 min, the bio-oil yield was decreased by carbon SA. However, at 30 min, the bio-oil yield was increased by carbon SA. No clear effect of carbon SA and residence time on the NH4+ yield was observed. This implies that deamination of amino acids, which produces NH4+ and organic acids,29−34 was completed when the reaction temperature reached 320 °C.

Figure 2.

Figure 2

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Effects of residence time on bio-oil and NH4+ yields in the presence/absence of the carbon solid acid catalyst in one-stage extraction when the reaction temperature was 320 °C: (a) bio-oil yield and (b) NH4+ yield.

Table 2 shows the yields of char and AP products (total C, NH4+, and total N) and pH of the AP under the same conditions as in Figures 1 and 2. No significant decrease in pH of the AP was observed by the addition of the SA. Yields of char and total C decreased as temperature increased and residence time became longer. In the presence of SA, the char yield increased and the total C yield decreased. The increase in char yield by the addition of SA was in the range of 50.9–83.2 mg/g-microalgae (dry basis) under the same reaction temperature and residence time. As stated in the Experimental Section, 3.0 g of dry microalgae and 5.0 g/L SA were used in 100 cm3 of distilled water. Thus, the increase in the char yield should be 167 mg/g-microalgae (dry basis) (= 5.0 g-SA/L × 0.100 L/3.0 g-microalgae × 1000). This indicates that some of the SA was dissolved during HTL. The total N yield did not increase with increased temperature or residence time. On addition of SA, the total N yield decreased slightly. Thus, the ratio of NH4+ to total N in the AP was increased by the SA. It was found that most of the aqueous N was recovered as NH4+ after HTL. Shakya et al.22 conducted HTL of Nannochloropsis, Pavlova, and Isochrysis at 250, 300, and 350 °C and reported that the yield of bio-oils increased and that of the water-soluble fraction decreased with increasing reaction temperature. This was due to polymerization of water-soluble components in the AP into bio-oil. They reported that the char yield decreased from 250 to 300 °C; however, no significant effect was observed on increasing the temperature to 350 °C.22 In their following study,23 they conducted HTL of Chlorella, Nannochloropsis, Pavlova, and Scenedesmus at 280 and 320 °C and reported similar trends for yields of bio-oil, water-soluble fractions, and char with the reaction temperature. They also reported that, in most of the algal species, NH4+ yields increased with the increase of reaction temperature from 280 to 320 °C. A similar trend for char and total C yields was observed in the present study.

Table 2. Results of One-Stage HTLa.

          aqueous phase
solid acid (SA) concentration [g/L] residence time (RT) [min] temp [°C] pH of the AP char yield [mg/g-microalgae (dry basis)] total C yield [mg/g-microalgae (dry basis)] NH4+ yield [mg/g-microalgae (dry basis)] total N yield [mg/g-microalgae (dry basis)] ratio of NH4+ to total Nb
0 30 280 7.05 ± 0.21 306.4 ± 15.4 123.5 ± 4.4 29.6 ± 0.6 37.0 ± 0.9 0.800
0 30 300 7.32 ± 0.02 311.3 ± 5.2 122.3 ± 1.1 33.5 ± 0.1 37.0 ± 0.5 0.905
0 30 320 7.20 ± 0.12 301.9 ± 3.3 118.9 ± 0.8 32.7 ± 0.4 36.8 ± 0.1 0.889
5.0 30 280 7.15 ± 0.17 389.6 ± 11.2 111.2 ± 1.7 28.8 ± 0.6 35.5 ± 0.5 0.811
5.0 30 300 7.23 ± 0.14 366.4 ± 0.3 104.2 ± 2.0 34.0 ± 0.3 35.0 ± 0.5 0.971
5.0 30 320 7.07 ± 0.11 362.7 ± 8.8 103.7 ± 1.3 31.8 ± 0.8 35.1 ± 0.1 0.906
0 0 320 6.90 ± 0.20 292.7 ± 6.4 123.5 ± 4.4 32.9 ± 1.8 37.0 ± 0.9 0.889
0 15 320 6.97 ± 0.25 283.5 ± 3.2 122.3 ± 1.1 31.8 ± 1.2 37.0 ± 0.5 0.859
5.0 0 320 7.03 ± 0.03 364.8 ± 3.9 111.2 ± 1.7 33.1 ± 2.3 35.5 ± 0.5 0.932
5.0 15 320 6.83 ± 0.28 334.4 ± 23.9 104.2 ± 2.0 34.4 ± 0.1 35.0 ± 0.5 0.983
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a

Errors (±) indicate standard deviation.

b

Ratio of NH4+ to total N (mg-NH4+ in AP/mg-total N in AP).

Based on the above results, to enhance bio-oil and NH4+ yields, the optimum residence time in one-stage extraction (corresponding to HTL 2 in the two-stage HTL) at 320 °C was determined to be 0 min.

2.2. Effects of Reaction Temperature, Residence Time, and Carbon SA in HTL 1 in Two-Stage HTL

The two-stage HTL was performed at various temperatures (100, 150, 200, and 250 °C) for 30 min in HTL 1 with and without SA (0, 5.0 g/L). Figure 3a shows the total bio-oil yield at the end of HTL 2 as a function of temperature in HTL 1 compared with the results of one-stage HTL. The HTL 2 was conducted at 320 °C for the residence time of 0 min. In the absence of SA, the bio-oil yield was slightly increased by the increase of temperature from 100 to 200 °C. However, at 250 °C, the bio-oil yield significantly decreased. No significant influence of the presence of SA on the bio-oil yield was observed at 100 °C. However, above 100 °C, the bio-oil yield was significantly decreased by SA. The bio-oil yields were 0.0820 ± 0.0012 g/g-dry microalgae and 0.0713 ± 0.008 g/g-dry microalgae in the absence and presence of SA at 200 °C during HTL 1, respectively. It was observed that the bio-oil yield was decreased by 7–15 wt % by the addition of SA. Compared with the results of bio-oil yields in one-stage HTL (see Figures 1a and 2a), the addition of SA increased the bio-oil yield only under the conditions of one-stage HTL at 300 and 320 °C for 30 min of the residence time. In other cases at different reaction temperatures and shorter residence times in HTL 1, the bio-oil yields were decreased by the addition of SA. There is a possibility that some of the bio-oil is absorbed by the remaining SA.

Figure 3.

Figure 3

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Effects of reaction temperature on bio-oil and NH4+ yields in the presence/absence of carbon solid acid catalyst in two-stage HTL when the residence time in HTL 1 was 30 min and the residence time of HTL 2 was 0 min: (a) bio-oil yield and (b) NH4+ yield. Note: (−) indicates one-stage HTL 2 without HTL 1 (residence time was 0 min, cf. Figure 2).

Figure 3b shows the NH4+ yield as a function of temperature with or without the SA. As the temperature in HTL 1 rose, the NH4+ yield in the AP after HTL 1 greatly increased. This implies that hydrolysis of proteins and deamination of amino acids were promoted by increasing the temperature in HTL 1, eluting NH4+ into the AP. In the following HTL 2, further NH4+ was eluted into the AP at each temperature. The total NH4+ yield peaked at 29.7 ± 0.1 and 27.3 ± 1.6 mg/g-dry microalgae when HTL 1 was conducted at 200 °C and HTL 2 was then conducted as 320 °C in the absence and presence of SA, respectively. These values were higher than those when one-stage HTL 2 was conducted at 320 °C, indicating that the two-stage HTL is effective for recovery of NH4+. To maximize bio-oil and NH4+ yields, the optimum temperature in HTL 1 was found to be 200 °C. The optimum temperatures of HTL 1 and HTL 2 obtained in this study were similar to those of the two-stage HTL for Coelastrum sp. (i.e., 200 and 320 °C, respectively) reported by Prapaiwatcharapan et al.37

The two-stage HTL was carried out changing the residence time in HTL 1. Figure 4a shows the total bio-oil yield after HTL 1 for 30, 60, and 120 min when the temperature was 200 °C followed by HTL 2 at 320 °C with 0 min residence time. Note that the SA catalyst was not used in this experiment. As the residence time increased, the total bio-oil yield decreased. The amount of carbon components in the AP increased in HTL 1 (Table 2). These results were similar to those of the report of Jazrawi et al.35 After HTL 2, as the residence time used during HTL 1 increased, an insignificant increase of the total C yield was observed, indicating that most of water-soluble carbon components were eluted into the AP. Figure 4b shows the NH4+ yield after HTL 1 and after HTL 2 as a function of residence time during HTL 1. When the residence time in HTL 1 was prolonged, the NH4+ yield increased slightly after HTL 1, implying slight promotion of the hydrolysis of protein and deamination of amino acids. However, changing the residence time had no significant effect on the total NH4+ yield. The total N yield in the AP slightly increased with the increase in residence time in HTL 1, implying promotion of hydrolysis of protein.

Figure 4.

Figure 4

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Effects of residence time of HTL 1 on bio-oil and NH4+ yields in two-stage HTL in the absence of the carbon solid acid when the reaction temperature of HTL 1 was 200 °C: (a) bio-oil yield and (b) NH4+ yield. Note: subsequent HTL 2 was conducted at 320 °C for 0 min.

Table 3a shows the yields of NH4+, total C, and total N in the AP and char after HTL 1 and after HTL 1 and HTL 2. The pH of the AP after HTL is also shown. In most of the experiments, the pH of the AP was slightly larger than 7. No significant influence of the SA on pH was observed. In the case of SA, the char yield increased due to the remaining SA. Total C and N yields were greatly increased with increasing temperature in HTL 1. The total C yield did not significantly increase in HTL 2 at 320 °C after HTL 1 at 200 and 250 °C for 30 min. This indicates that 200 °C and 30 min is sufficient to elute water-soluble carbon components in HTL 1. The total C yield in the AP was slightly decreased by the carbon SA. The total N yield greatly increased in HTL 2 compared with the results in HTL 1 at each temperature, indicating hydrolysis and decomposition of protein and amino acids. However, the total NH4+ and total N yields were almost constant after HTL 1 and HTL 2 were conducted. No significant influence of temperature on the char yield was observed. Table 3b shows elemental analyses of bio-oil and char. No significant influence of the SA addition on bio-oil composition was observed. On the basis of the above results, the optimum conditions for HTL 1 and HTL 2 were found to be HTL 1: 200 °C, 30 min residence time and HTL 2: 320 °C, 0 min residence time.

Table 3. Results of Two-Stage HTL (a) Char Yield and Results of Aqueous Phasea.

              (a) aqueous phase [mg/g-microalgae (dry basis)]
  HTL 1
 HTL 2
     HTL 1
 HTL1 + HTL 2
SA concn [g/L] RT [min] temp [°C] RT [min] temp [°C] pH of the AP char yield [mg/g-microalgae (dry basis)] NH4+ yield total C yield total N yield total NH4+ yield total C yield total N yield
0 30 100 0 320 7.39 ± 0.23 281.2 ± 10.0 1.5 ± 0.1 30.9 ± 1.8 5.0 ± 0.4 (0.30)b 27.3 ± 1.4 118.3 ± 1.0 37.3 ± 0.6 (0.732)b
0 30 150 0 320 7.82 ± 0.11 284.8 ± 3.9 2.6 ± 0.1 39.9 ± 0.0 11.1 ± 0.0 (0.23)b 25.4 ± 0.4 116.5 ± 3.6 35.9 ± 0.8 (0.708)b
0 30 200 0 320 7.63 ± 0.23 289.9 ± 2.0 10.9 ± 0.1 106.0 ± 2.3 27.8 ± 0.4 (0.392)b 29.7 ± 0.1 110.5 ± 0.0 36.4 ± 0.1 (0.816)b
0 30 250 0 320 7.70 ± 0.31 281.1 ± 5.0 17.6 ± 0.2 127.3 ± 4.3 37.4 ± 1.1 (0.203)b 23.6 ± 1.2 104.4 ± 2.5 35.0 ± 0.5 (0.674)b
5.0 30 100 0 320 7.59 ± 0.16 366.1 ± 4.1 1.6 ± 0.1 35.5 ± 2.5 6.9 ± 1.8 (0.23)b 24.2 ± 0.9 104.9 ± 3.4 34.0 ± 1.3 (0.712)b
5.0 30 150 0 320 7.56 ± 0.07 366.3 ± 1.1 2.6 ± 0.1 32.3 ± 2.0 9.4 ± 1.2 (0.28)b 26.4 ± 0.0 106.4 ± 6.4 35.7 ± 1.1 (0.739)b
5.0 30 200 0 320 8.01 ± 0.16 377.9 ± 2.8 10.1 ± 0.4 100.2 ± 1.5 26.8 ± 0.7 (0.377)b 27.3 ± 1.6 103.7 ± 1.1 36.2 ± 1.5 (0.754)b
5.0 30 250 0 320 7.49 ± 0.31 373.1 ± 13.1 17.0 ± 0.0 115.5 ± 2.2 35.8 ± 0.3 (0.475)b 23.1 ± 0.8 95.1 ± 2.4 33.2 ± 0.4 (0.696)b
0 60 200 0 320 6.95 ± 0.20 305.5 ± 6.4 10.0 ± 0.4 114.4 ± 1.4 29.9 ± 0.1 (0.334)b 27.7 ± 0.4 113.1 ± 7.6 37.8 ± 2.0 (0.733)b
0 120 200 0 320 7.13 ± 0.07 281.6 ± 9.1 13.4 ± 0.3 117.9 ± 4.1 33.4 ± 0.2 (0.411)b 27.9 ± 0.5 116.9 ± 2.4 38.7 ± 0.5 (0.721)b
          (b) elemental analysis [wt % (dry basis)]
  HTL 1
 HTL 2
 bio-oil
 char
SA concn [g/L] RT [min] temp [°C] RT [min] temp [°C] C H N O (diff.) C H N O + ash (diff.)
0 30 200 0 320 77.41 ± 0.55 11.29 ± 0.10 2.17 ± 0.01 9.13 ± 0.63 11.94 ± 0.21 2.20 ± 0.04 1.32 ± 0 84.54 ± 0.25
0 30 250 0 320 76.34 ± 1.31 11.21 ± 0.24 2.12 ± 0.08 10.34 ± 1.63 11.92 ± 0.49 2.20 ± 0.10 1.23 ± 0.04 84.65 ± 0.52
5.0 30 200 0 320 76.41 ± 0.24 11.70 ± 0.05 1.97 ± 0.03 9.92 ± 0.33 23.67 ± 1.02 2.51 ± 0.07 1.62 ± 0.09 72.21 ± 1.19
5.0 30 250 0 320 77.40 ± 1.78 11.43 ± 0.27 2.09 ± 0.08 9.08 ± 2.10 25.07 ± 1.78 2.66 ± 0.10 1.67 ± 0.10 70.61 ± 1.96
0c     0 320 76.74 ± 0.04 11.12 ± 0.11 2.21 ± 0.05 9.94 ± 0.02 11.21 ± 0.18 2.23 ± 0.05 1.23 ± 0.03 85.33 ± 0.17
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a

For abbreviations, please refer to Table 2. Errors (±) indicate standard deviation.

b

Ratio of NH4+ to total N (mg-NH4+ in AP/mg-total N in AP).

c

Results of one-stage HTL (at 320 °C for 0 min) for comparison.

2.3. Effect of HCl on the Bio-Oil Yield and Nitrogen Components in the Two-Stage HTL

In this study, no clear effect of SA was observed on bio-oil production and NH4+ recovery in the two-stage HTL. This may be because (i) there was insufficient contact between the microalga/its derivatives and catalyst particles and/or (ii) the acidity was insufficient. Thus, we tried using inorganic acid (HCl) in the two-stage HTL to promote hydrolysis of protein and deamination of amino acids by increasing the contact and acidity of the catalyst.39 Note: we did not use carboxylic acid to avoid formation of amides. Because the concentration of acidic organic functional groups on the surface of the SA was 0.018 M as calculated from eq 1.

2.3. 1

The concentration of HCl solution was set to 0.02, 0.10, and 0.50 M in the following experiments. The diatom sample was mixed with the HCl solutions instead of distilled water, and then the HTL experiments were conducted with the same procedures.

Table 4 shows yields and elemental analysis of bio-oil and char after two-stage HTL (200 °C, 30 min and 320 °C, 0 min) at various HCl concentrations. An insignificant change in the bio-oil yield was observed when using 0.02 M HCl relative to the absence of HCl. However, a further increase in the HCl concentration led to a significant decrease in the bio-oil yield. In particular, 0.50 M HCl caused 42.8 wt % reduction of the bio-oil yield relative to the case without HCl. The same trend can be seen in the char yield. The nitrogen content of bio-oil without the HCl catalyst was 2.17 wt %. However, the nitrogen content with 0.50 M HCl was greatly decreased to 0.41 wt %. Thus, using 0.50 M HCl in the two-stage HTL successfully decreased the nitrogen content in the bio-oil by approximately 80 wt %. In one-stage HTL, it has not been confirmed that acid catalysts significantly decrease the nitrogen content in the bio-oil.16,40,41 Also, decreased nitrogen content in char was observed with 0.50 M HCl. The nitrogen contents of the char and carbon contents of bio-oil and char greatly decreased by using 0.5 M HCl.

Table 4. Yield and Elemental Analysis of Bio-Oil and Char after Two-Stage HTL (HTL 1 at 200 °C for 30 min and HTL 2 at 320 °C for 0 min) at Various HCl Concentrationsa.

  bio-oil (HTL 1 + HTL 2)
 char (HTL 1 + HTL 2)
    elemental analysis on a dry, ash-free basis [wt %]
   elemental analysis on a dry basis [wt %]
HCl [M] amount [mg/g-microalgae (dry basis)] H C N O (dif.) amount [mg/g-microalgae (dry basis)] H C N O + ash (dif.)
0 83.1 ± 1.1 11.29 ± 0.10 77.41 ± 0.55 2.17 ± 0.01 9.13 ± 0.63 293.8 ± 2.3 2.20 ± 0.04 11.94 ± 0.21 1.32 ± 0.00 84.54 ± 0.25
0.02 78.6 ± 3.9 10.87 ± 0.22 75.93 ± 0.87 1.90 ± 0.04 11.30 ± 1.06 298.7 ± 2.4 2.12 ± 0.05 12.08 ± 0.44 1.23 ± 0.05 84.57 ± 0.45
0.10 69.1 ± 1.8 10.85 ± 0.15 79.61 ± 0.60 1.98 ± 0.08 7.55 ± 0.83 243.5 ± 1.1 2.19 ± 0.10 12.84 ± 0.23 1.29 ± 0.02 83.69 ± 0.35
0.50 47.5 ± 2.7 10.11 ± 1.82 69.76 ± 9.74 0.41 ± 0.04 20.32 ± 11.5 163.8 ± 26.9 2.05 ± 0.04 9.32 ± 1.33 0.76 ± 0.04 87.87 ± 2.23
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a

Errors (±) indicate standard deviation.

Table 5 shows the NH4+ yield, total C, total N, and pH in the AP as a function of the concentration of HCl. Higher HCl concentrations (0.50 M) greatly increased the NH4+, total C, and total N yields after HTL 1 and decreased the nitrogen content of the bio-oil and char. This obviously indicates that 0.50 M HCl as the acid catalyst promotes protein hydrolysis in HTL 1 and elution of water-soluble hydrolysates including NH4+ into the AP before the hydrolysates polymerize with the lipid component and enter the bio-oil in HTL 2. Approximately 77 to 82 wt % the nitrogen components in the AP were present as NH4+ after HTL 2. These results are indirectly in accordance with the results of Torri et al. in that protein degradation drastically increased bio-oil yields and the hydrophobic portion of protein or carbohydrate derived materials increased the nitrogen content of the bio-oil.26 Matayeva et al.28 recently conducted HTL of model amino acids (phenylalanine and leucine), glucose, and lipids (tripalmitin) in the presence of homogeneous catalysts (0.02 M CH3COOH, HCOOH, and Na2CO3) and reported that the yields of bio-oil and nitrogen components in the bio-oil increased and nitrogen in the AP was decreased by CH3COOH. However, HCOOH and Na2CO3 did not significantly affect the bio-oil yield and elemental composition.28 Our results show that pH should be lower than 5 to decrease the nitrogen content in bio-oil.

Table 5. Aqueous Phase Products after Two-Stage HTL (HTL 1 at 200 °C for 30 min and HTL 2 at 320 °C for 0 min) at Various HCl Concentrationsa.

  aqueous phase [mg/g-microalgae (dry basis)]
  
  HTL 1
 HTL 1 + HTL 2
  
HCl [M] NH4+ total C total N NH4+ total C total N pH
0 10.9 ± 0.1 106.0 ± 2.3 27.8 ± 0.4 (0.392)b 29.8 ± 0.2 110.5 ± 0.01 36.4 ± 0.1 (0.819)b 7.63 ± 0.23
0.02 9.5 ± 0.2 98.4 ± 2.9 26.1 ± 0.9 (0.36)b 28.5 ± 0.4 107.1 ± 1.7 36.9 ± 0.5 (0.772)b 7.11 ± 0.12
0.10 10.0 ± 0.1 100.5 ± 0.3 27.7 ± 0.9 (0.361)b 32.7 ± 0.5 101.6 ± 2.5 39.9 ± 0.7 (0.820)b 5.01 ± 0.05
0.50 18.3 ± 1.0 159.0 ± 1.9 43.0 ± 1.5 (0.426)b 35.7 ± 2.9 106.8 ± 4.2 46.5 ± 0.7 (0.768)b 1.44 ± 0.16
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a

Errors (±) indicate standard deviation.

b

Ratio of NH4+ to total N (mg-NH4+ in AP/mg-total N in AP).

From the results in Tables 4 and 5, a decrease of the bio-oil yield caused by the presence of HCl is considered to occur in HTL 2. Therefore, HTL 2 was performed with distilled water (i.e., HCl = 0 M) after 0.50 M HCl was added as a catalyst in HTL 1, and the acidic solution was then removed by solid–liquid separation. Figure 5 shows the carbon and nitrogen balances of char, bio-oil, the AP, and gas in one-stage HTL and two-stage HTL with HCl as a catalyst in HTL 1 and HTL 2. The total carbon components in the AP significantly changed from 31.9% in the fifth set of conditions we tested (i.e., condition 5: HTL 1: 0.50 HCl M + HTL 2: 0.50 M HCl) to 58.9% in the sixth set (i.e., condition 6: HTL 1: 0.50 HCl M + HTL 2: distilled water). The carbon and nitrogen components of char and bio-oil were greatly decreased by increasing the HCl concentration in HTL 1. It is likely that the bio-oil and char were decomposed into gases. Biller et al.40 reported that increasing acidity decreased the bio-oil yield and increased gas production. Our data also show that increasing the acidity was disadvantageous in terms of the bio-oil yield. The carbon component of the bio-oil was 10.34% and 5.24% under conditions 5 and 6, respectively. It can be seen that the nitrogen contents in the AP were increased from 76.9% under condition 5 to 80.7% under condition 6 by performing HTL 2 in neutral conditions. The carbon balance in char was 14.36% and 5.50%, and the nitrogen balance in char was 2.04% and 1.08% in conditions 5 and 6, respectively. It can be concluded that the nitrogen content in the bio-oil is decreased by the addition of HCl in HTL 1, which promotes the hydrolysis of proteins and deamination of amino acids while suppressing the formation of amines and amides, and it is better to carry out neutral conditions by using distilled water in HTL 2 to recover a larger amount of nutrients in the AP.

Figure 5.

Figure 5

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(a) Carbon and (b) nitrogen balances of the products after one-stage HTL and two-stage HTL in the presence of HCl. Conditions: HTL 1: 200 °C and 30 min; HTL 2: 320 °C and 0 min. Note: (−) indicates one-stage HTL 2 without HTL 1; 0 M indicates pure water was used.

Figure 6 shows the results of gas chromatography–mass spectrometry (GC–MS) analysis of bio-oil obtained after one-stage and two-stage HTL in the absence/presence of HCl under the same experimental conditions as those in Figure 5. The mass spectra of each GC peak at each retention time and possible chemical composition of each molecular weight (MW) are shown in Figure S1 and Table S2, respectively.

Figure 6.

Figure 6

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Gas chromatography–mass spectrometry (GC–MS) analysis of bio-oil obtained after one-stage and two-stage HTL in the presence of HCl. Conditions are same as those in Figure 5.

In Figure 6, it is observed that GC peaks at the retention time (RT) around 9.2, 9.5, 9.9, 14.9, 17.5–17.8, 18.1–18.5, and 18.8 min increased until the HCl concentration increased up to 0.10 M (condition 4) and decreased in the case of 0.50 M HCl was used (conditions 5 and 6). In particular, the GC peaks around 14.9 and 17.5–17.8 min greatly decreased under conditions 5 and 6. When the HCl concentrations were 0.10 and 0.50 M (conditions 4, 5, and 6), a small GC peak was observed around 17.2 min.

From MS spectra, when the RT was 9.2 min (Figure S1a), the MS spectra of amines (MW = 55, 57, 97, 111, 140), oxygen-containing hydrocarbons (MW = 70, 126, 140, 196), hydrocarbons (MW = 196), and linoleic acid (C18:2, MW = 280) were observed. Similar MS spectra were observed at different HCl concentrations up to 0.10 M (conditions 1–4). Under conditions 5 and 6, the intensities of the MS spectra were under the detection limit.

When the RT was 9.5 min (Figure S1b), amines (MW = 57, 109, 123, 137), nitrile (MW = 82, 95, 123, 137), and linolenic acid (C18:3, MW = 278) were observed. The intensities of the spectra were similar under conditions 1–5. However, under condition 6, peaks of MW = 57, 109, and 137 greatly decreased. When the RT was 9.9 min (Figure S1c), very similar trends of the MS peaks compared with Figure S1b were observed except for the great increase of MW = 109 under condition 5. When the RT was 14.9 min (Figure S1d), the MS spectra of amines (MW = 60, 73, 85, 115, 129, 143, 157, 171, 185) and myristic acid (C14:0, MW = 228) were observed. The intensities of the spectra were similar under all conditions. Under conditions 4, the MS spectra of many peaks (RT = 17.2 min) were observed (see Figure S1e). With 0.50 M HCl, the MS spectra of long-chain amines (MW = 255, 283, 381), long-chain hydrocarbons (MW = 234, 236), and long-chain oxygen-containing hydrocarbons (MW = 396) were greatly reduced. When the RT was 17.5–17.8 min (Figure S1f), the characteristic MS spectra of long-chain oxygen-containing hydrocarbons (MW = 396, 404), amines (MW = 55, 83, 97, 111, 125, 137, 140), nitriles (MS = 83, 137), oxygen-containing hydrocarbons (MW = 180), and hydrocarbons (MW = 166, 180, 222, 236) were observed. The intensities of the spectra were similar under all conditions except an appreciable increase in the spectra of long-chain amines (MW = 255, 283, 381, 396) under condition 5. The peak intensities of these spectra greatly decreased under condition 6. When the RT was 18.1–18.5 min (Figure S1g), the MS spectra of amines (MW = 60, 73, 85, 115, 129, 143, 157, 171, 185, 213, 227) and linoleic acid (C18:2, MW = 256) were observed. The intensities of the spectra were similar under all conditions. In summary, when 0.50 M HCl was used, the peaks of amines and long-chain amines in the bio-oil were decreased.

When the RT was 9.5 min (Figure S1b), amines (MW = 57, 109, 123, 137), nitrile (MW = 82, 95, 123, 137), and linolenic acid (C18:3, MW = 278) were observed. The intensities of the spectra were similar under conditions 1–5. However, under condition 6, peaks of MW = 57, 109, and 137 greatly decreased. When the RT was 9.9 min (Figure S1c), very similar trends of the MS peaks compared with Figure S1b were observed except for the great increase of MW = 109 under condition 5. When the RT was 14.9 min (Figure S1d), the MS spectra of amines (MW = 60, 73, 85, 115, 129, 143, 157, 171, 185) and myristic acid (C14:0, MW = 228) were observed. The intensities of the spectra were similar under all conditions. Under conditions 4, the MS spectra of many peaks (RT = 17.2 min) were observed (see Figure S1e). With 0.50 M HCl, the MS spectra of long-chain amines (MW = 255, 283, 381), long-chain hydrocarbons (MW = 234, 236), and long-chain oxygen-containing hydrocarbons (MW = 396) were greatly reduced. When the RT was 17.5–17.8 min (Figure S1f), the characteristic MS spectra of long-chain oxygen-containing hydrocarbons (MW = 396, 404), amines (MW = 55, 83, 97, 111, 125, 137, 140), nitriles (MS = 83, 137), oxygen-containing hydrocarbons (MW = 180), and hydrocarbons (MW = 166, 180, 222, 236) were observed. The intensities of the spectra were similar under all conditions except an appreciable increase in the spectra of long-chain amines (MW = 255, 283, 381, 396) under condition 5. The peak intensities of these spectra greatly decreased under condition 6. When the RT was 18.1–18.5 min (Figure S1g), the MS spectra of amines (MW = 60, 73, 85, 115, 129, 143, 157, 171, 185, 213, 227) and linoleic acid (C18:2, MW = 256) were observed. The intensities of the spectra were similar under all conditions. In summary, when 0.50 M HCl was used, the peaks of amines and long-chain amines in the bio-oil were decreased.

3. Conclusions

Two-stage HTL of the microalga Fistulifera sp. JPCC DA0580 was conducted in subcritical water in a batch reactor to increase both bio-oil and NH4+ yields. We investigated the optimum experimental conditions (reaction temperature and residence time) of two-stage HTL and the effects of acid catalysts (carbon SA and HCl) on the bio-oil and char yields, components, and AP content. The best conditions were HTL 1: 200 °C, 30 min of residence time and HTL 2: 320 °C, 0 min of residence time. No significant effect of the carbon SA catalyst was observed when its concentration was 5.0 g/L. By introducing 0.50 M HCl in the two-stage HTL, we greatly decreased the nitrogen content of the bio-oil and char and increased NH4+ in the AP. This is probably due to a catalytic effect of HCl, promoting hydrolysis of protein and deamination of amino acids during HTL 1. From the results of GC–MS analyses of the produced bio-oil, it was observed that 0.50 M HCl suppresses the formation of amines. It is also likely that the recombination/polymerization of amines with other water-soluble fractions forms the bio-oil and char. It was found that there is a trade-off between the bio-oil yield and nitrogen content in the bio-oil.

4. Experimental Section

4.1. Materials

4.1.1. Microalga

A marine diatom, F. solaris JPCC DA058042−46 was used for the experiments. The sample was precultured for 7 days and then cultured for 12 days in a bioreactor. The sample was stored in a deep freezer at −77 °C before experiments.45Table 6 shows ultimate analysis of the diatom sample.45 Note: this freeze-dried microalga sample contains a small amount of lipid and a large amount of ash.45 Details of the culturing procedures, 2f medium, the artificial seawater, and the methods of concentration measurements are given in our previous studies.47,48

Table 6. Ultimate Analysis of Diatom Sample (Data Reported in ref (45)).
ultimate analysis (wt %; dry basis)
C H N O (diff.) ash
34.03 5.54 6.04 26.07 28.32
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4.1.2. SA

Sulfated carbon SA powder (CP150H, Futamura Chemical Co. Ltd., Hiroshima, Japan)49,50 was used as a catalyst. The specific surface area of the SA is 852 m2/g. In each experiment, 0.50 g of the carbon SA powder was used, corresponding to a concentration of 5.0 g-SA/L-algal slurry. The total amount of acidic oxygen functional groups (mostly sulfo groups) measured by the Boehm method51 using NaOH (0.02 mol/L) was 3.6 mmol/g-SA. Details of the procedure of the Boehm method are given in our previous study.45

4.2. Bio-Oil Extraction by HTL

4.2.1. One-Stage HTL

A schematic view of the experimental apparatus is shown in Figure 7. An autoclave reactor (TPR-1, 300 mL, Taiatsu Techno. Corporation, Osaka, Japan) was charged with 3.0 g of the microalga and 100 cm3 of distilled water. After the reactor was purged with Ar gas (ca. 2.5 MPa), the microalgal slurry was heated to 280–320 °C while stirring with an impeller at 300 rpm. The time required to heat the reactor to 150, 200, 260, and 300 °C was 11.5, 14.5, 22.0, and 31.0 min, respectively (data not shown). The reaction temperature was then maintained for 0–60 min. Note: in the case that the residence time was 0 min, the reactor was cooled soon after the temperature reached the reaction temperature.

Figure 7.

Figure 7

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Experimental apparatus for hydrothermal liquefaction (HTL).

4.2.2. Two-Stage HTL

Stage 1 (HTL 1): The procedures to charge the sample and purge the reactor were the same as explained in Section 2.2.1. The reaction temperature was set to 100–250 °C, and the residence time was set to 30–120 min. After the reactor was cooled to room temperature, the solution in the reactor (ca. 1 cm3) was collected with a vial syringe. Stage 2 (HTL 2): After the reactor was purged with Ar gas (ca. 2.5 MPa), the remaining 99 cm3 of microalgal slurry was heated to 320 °C while stirring with the impeller at 300 rpm. The reactor was cooled soon after the temperature reached 320 °C (i.e., the residence time was 0 min in HTL 2). Each experiment was conducted two or three times under the same conditions to confirm reproducibility.

4.3. Separation of Products

The procedure for product separation in this study is shown in Figure 8. After the reactor was cooled to room temperature, the products were separated into bio-oil/char and aqueous solution (i.e., AP product) by filtration. The bio-oil and solids, which were strongly attached, were collected from the filter paper and the reactor by using acetone (ca. 100 cm3). The bio-oil was extracted from the solids in acetone for 45 min and then separated with a nylon mesh. Acetone was evaporated in nitrogen at 80 °C, and then the residue was mixed with 9 cm3 of hexane to dissolve the remaining bio-oil. The bio-oil was obtained after complete evaporation of the hexane at 80 °C. Note that the amount of bio-oil was decreased after the extraction with hexane. Details of the bio-oil recovery procedure were described elsewhere.47,48

Figure 8.

Figure 8

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Experimental procedure for separation of liquefaction products.

4.4. Analysis of Products

4.4.1. Bio-Oil and Char

After gravimetry, drying was carried out using a vacuum dryer (SVD30P, Sansyo, Tokyo, Japan). Next, elemental analysis was performed on the oil and char with a CHN analyzer (Micro Coder IM10, J-Science, Kyoto, Japan).

4.4.2. Water-Soluble Fraction

The amount of NH4+ in the AP was determined with a flow injection analyzer (PD202, J-Science). The total amount of nitrogen and carbon in the AP was determined with a TOC analyzer (TOC-VCSH, Shimadzu, Kyoto, Japan).

Acknowledgments

This study was supported by a Grant-in-Aid for Scientific Research B (JSPS Kakenhi Kiban B, 26289302) and a research grant from the JFE 21st Century Foundation in 2016. The authors thank Professor Tsuyoshi Tanaka, Professor Tomoko Yoshino, and Dr. Yoshiaki Maeda at Tokyo University of Agriculture and Technology (TUAT) and Dr. Mitsufumi Matsumoto (J-Power) for providing the microalgal sample; Professor Akihiko Terada, Professor Shohei Riya, Professor Keiichi Noguchi, Dr. Yuka Sakai, Ms. Nozomi Sakamoto, Dr. Yohei Okada, and Ms. Masayo Koyama at TUAT for technical support in sample analyses; Professor Masaru Watanabe (Tohoku University) and Mr. Hirofumi Yamada (Futamura Chemicals Co. Ltd.) for providing the sulfated carbon SA catalyst sample; and James Allen, DPhil from Edanz Group (www.edanzediting.com/ac) for editing the draft of this manuscript.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.9b04468.

  • Table S1: mass change of solid acid during one-stage HTL blank experiment without microalga, Table S2: possible chemical products in the bio-oil, and Figure S1: mass spectra of GC–MS analysis of bio-oil at each retention time (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao9b04468_si_001.pdf (1.5MB, pdf)

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