Full utilization of marine microalgal hydrothermal liquefaction liquid products through a closed-loop route: towards enhanced bio-oil production and zero-waste approach

Abstract

The present study aimed to evaluate the potential of aqueous phase after hydrothermal liquefaction of microalgae (Aq-P), enriched with seawater, as a growth medium coupled with crude bio-oil production by the halophyte Dunaliella salina. Results showed that Aq-P showed higher content of total organic carbon (TOC) and total nitrogen (10.24, and 5.11 g L⁻¹, respectively), while seawater showed higher anions and cations content. At the 12th day of microalgae incubation, the Aq-P growth medium showed 15.9% higher dry weight than the control (f/2 medium), with enhanced lipid content by 21.2% over the control, and 5.7% significant reduction in carbohydrates. The bio-oil yields of microalgal biomass cultivated in f/2 and Aq-P were 28.74% and 29.54%, respectively. Using Aq-P enhanced the fatty acids/esters and hydrocarbons in the crude bio-oil by 12.6% and 1.7 times, respectively, comparing to f/2-derived bio-oil. However, nitrogen-containing compounds in the Aq-P-derived bio-oil reduced by 60.7% comparing to f/2 medium. Interestingly, diesel carbon-range represented the majority of the products in both f/2- and Aq-P-derived bio-oil (69.1% and 78.3%, respectively). The findings of the present study provide a new approach for development of sustainable microalgal cultivation system for crude bio-oil production through a closed-loop route using Aq-P and seawater.

Introduction

The increasing energy demand due to the growing world's population and modernization has caused a strong reliance on fossil fuels, which resulted in many crucial challenges such as rising petroleum prices and worsening the global warming (Valizadeh et al. 2022). In addition, a circular bioeconomy platform should be established to provide a finer and zero-waste promising technology to combat the environmental crises while promoting ecological stability and clean energy that tallies with the aims of Sustainable Development Goals (ElFar et al. 2022). Over the last decades, biofuels have received increasing attention as alternative energy source to fossil fuels due to many advantages such as sustainability, renewability, and carbon neutrality (Abomohra et al. 2020b). Many feedstocks have been discussed for production of different biofuels, categorized basically into three groups namely liquid, solid, and gas fuels. Amongst, liquid biofuels represent a potential energy source for many applications and, currently, are attracting much investment. The crude bio-oil produced from thermochemical conversion, including pyrolysis and hydrothermal liquefaction (HTL), of biomass and waste can be refined into different forms of fuels suitable for direct application in existing engines with little or no modifications. Pyrolysis requires dry biomass, which needs intensive energy during the drying step. Comparatively, HTL can be used to convert biomass with high water content (Ong et al. 2019), due to the need of compressed water under subcritical conditions to complete the reactions (Wang et al. 2020b), avoiding the energy-intensive drying step.

During thermal conversion, all macromolecules such as lipids, carbohydrates, and proteins are degraded to form different products through polymerization, depolymerization and condensation reactions (Chen et al. 2017). Pyrolysis results in three main products namely bio-oil, syngas, and biochar, while those of HTL include an aqueous phase (Aq-P) as additional byproduct. The liquid products (bio-oil and Aq-P) contain most of the reformed organic compounds present initially in the feedstock (López Barreiro et al. 2015). Although crude bio-oil is the main energy product, biochar and syngas have many industrial applications. However, Aq-P is a waste byproduct that requires a special treatment before disposal to avoid the negative environmental impacts and for full nutrient recovery. About 50% of the feedstock organic carbon are dissolved in the Aq-P, leading to relatively high total organic carbon (TOC) content in the HTL effluents, resulting in loss of energy resource or high-cost treatment (Leng et al. 2020). In that context, treatment of organics in the Aq-P was estimated to account more than 90% of the total HTL-waste disposal cost (Zhu et al. 2014). Considering other technologies that depend on Aq-P production such a recently suggested liquid biphasic flotation (LBF) technology for biomolecules extraction (Mat Aron et al. 2022), finding cost-effective technology for efficient Aq-P recovery and treatment is of great importance towards industrialization of microalgal biofuels.

Among different feedstocks that have been extensively studied for crude bio-oil production, microalgae are considered as one of the most promising biomasses due to competitive advantages over other feedstocks (Sharma et al. 2021). Biomass productivity of microalgae was reported to be 10–20 times higher than any other biofuel crop, and they don’t compete for arable land or freshwater as they are able to grow in saline and wastewater (Narala et al. 2016). The most common methods to cultivate microalgae are open-pond systems (OPSs) and controlled closed photobioreactors (PBRs). Comparatively, PBRs can be designed and controlled according to the cultivated strain, require little space, has lower contamination risk, and provide higher biomass productivity (Molina Grima et al. 1999). However, the elevated production cost of microalgal biomass is the main challenge for biofuel-based industry (Wang et al. 2022). Different microalgal biofuel conversion technologies, including lipid transesterification (Almarashi et al. 2020), fermentation (Xu et al. 2019), pyrolysis (Wang et al. 2020a), anaerobic co-digestion (Kabir et al. 2022), and HTL (Ellersdorfer 2020) are in the research hotspots in recent years. Despite the potential of microalgae as a biofuel feedstock, the economic feasibility of the process still far away from commercial application, mainly due to the high cost of the growth medium (Wang et al. 2022). Recently, many new approaches have been suggested to reduce the overall cost of microalgae cultivation and enhance the feasibility of different applications. For instance, microalgal biorefinery via thermochemical or biochemical pathways was suggested to produce a variety of marketable value-added compounds (Chandrasekhar et al. 2022). Sequential biofuel production was also suggested to enhance the energy recovery from the biomass (Elsayed and Abomohra 2022). In addition, sequential hydrothermal liquefaction of high protein microalgae was suggested to simultaneously enhance nutrient recovery at higher bio-oil yield (Chen et al. 2022). Byproducts recovery such as waste glycerol and lipid-free biomass after biodiesel production showed high positive impact on the total energy output of microalgae (Abomohra et al. 2018). Beside waste streams utilization, microalgae-based carbon capture and utilization (CCU) was suggested as a commercial emerging technology with high potential of net-zero or negative emissions (Shao et al. 2019), which provides an additional advantage for microalgal technology. Microalgae have high moisture content after biomass harvest, which represents the main challenge to be used in pyrolysis. Therefore, HTL of microalgal sludge can provide an efficient route to save the high cost and energy required for dewatering process.

In recent years, new concepts of HTL have been suggested to fully utilize the high-value compounds of microalgal biomass and to enhance the economics of the algal biofuel. For instance, a two-step sequential HTL was employed to separate the value-added polysaccharides from the biomass at relatively low temperature before HTL to enrich the lipid proportion in the biomass residue and enhance the oil quality (Chen et al. 2016). Although the aforementioned technology enhanced the economic feasibility of microalgal HTL, to some extent, the negative environmental impacts and nutrients load in the aqueous phase remains a big challenge. Nutrients in the Aq-P can be recycled for microalgae cultivation. Despite the recent abundant studies on HTL of freshwater microalgae, few studies evaluated the re-utilization of Aq-P for microalgae cultivation, which showed relatively lower growth in many cases, mainly due to the deficiency of micronutrients in the Aq-P-derived growth medium. In that regard, utilization of Aq-P diluted with natural seawater for marine microalgae cultivation was not previously evaluated. Such approach could provide the essential micronutrients from seawater and organics from Aq-P, providing higher growth and biomass productivity. Therefore, the present study aimed to evaluate the utilization of Aq-P from HTL by the marine microalga Dunaliella salina as a growth medium. The HTL of microalgal biomass was performed and the Aq-P biochemical composition was further analyzed. The growth and biochemical characteristics of the isolate on typical synthetic growth medium (f/2) were compared with that grown in diluted Aq-P as a growth medium, then the changes in bio-oil yield and composition were evaluated.

Materials and methods

Materials

In the present study, all chemicals and solvents including: chloroform, methanol, hexane, acetonitrile, ethanol, sodium nitrate, sodium dihydrogenphosphate monohydrate, sodium carbonate, ferric chloride hexahydrate, disodium EDTA dihydrate, copper sulphate pentahydrate, sodium molybdate dihydrate, zinc sulphate heptahydrate, cobalt chloride hexahydrate, manganese chloride tetrahydrate, Coomassie brilliant blue G-250, bovine serum albumin, phosphoric acid, sulphonic acid, glucose, and phenol were of analytical grade and purchased from Merck, Singapore.

Isolation and identification

Water samples were collected in March 2019 from the coastal area of Jeddah city (around 21.924855, 38.946611), Saudi Arabia. Water characteristics were measured (Table 1) and samples were transferred to the laboratory in sterilized bottles. Water samples were enriched by incubation of 50 mL at 28 °C under light intensity of 100 µmol photons m⁻² s⁻¹ for 7 days. After enrichment, 50 µL of the water sample were spread on f/2 agar medium (Guillard 1975) and incubated at the aforementioned conditions. Relatively large colony, referring to faster growth, was picked up into a new agar plate, and subculturing was repeated till complete purification. The isolate was cultivated in 50 mL f/2 liquid medium and incubated at the aforementioned conditions.

Table 1.

The main chemical characteristics of seawater and the aqueous phase produced from HTL (Aq-P) of Dunaliella salina KAU19 biomass grown in f/2 medium for 14 days

Parameters	Seawater	Aq-P
Salinity (ppt)	38.37 ± 0.08	na
Temperature (°C)	28.45 ± 0.11	na
TC (g L−1)	0.62 ± 0.06	13.36 ± 0.56*
TOC (g L−1)	0.46 ± 0.03	10.24 ± 1.09*
TIC (g L−1)	0.16 ± 0.04	3.12 ± 0.53*
TN (g L−1)	64.72 ± 4.27 × 10–3	5.11 ± 0.72*
NH4− (g L−1)	14.65 ± 2.51 × 10–3	3.04 ± 0.59*
NO3− (g L−1)	34.94 ± 4.8 × 10–3	0.94 ± 0.22*
TP (g L−1)	7.12 ± 0.25 × 10–3	1.51 ± 0.17*
Cl− (g L−1)	20.17 ± 1.97	3.59 ± 0.51*
K (g L−1)	5.89 ± 0.44	1.94 ± 0.18*
Na (g L−1)	10.95 ± 1.32	3.12 ± 0.42*
SO4− (g L−1)	1.24 ± 0.08	0.84 ± 0.11 ns
Ca (mg L−1)	54.72 ± 4.58	4.87 ± 0.34*
Mg (mg L−1)	248.51 ± 14.09	3.12 ± 0.22*
Fe (mg L−1)	0.18 ± 0.03	0.64 ± 0.10*

na Not applicable

Values of Aq-P with ^* and ^ns refer to significant and insignificat differences, respectively, with the corresponding value of seawater (at P ≤ 0.05)

Exponentially grown cells were harvested by centrifugation at 3000× g for 10 min and the cellular DNA of the isolate KAU19 was extracted using Qiagen Dneasy Kit (Valencia, USA). The 18S rRNA fragments were amplified using MA1/MA2 universal primer as previously described (Abomohra et al. 2020a). The DNA sequence was compared with those available in the NCBI databases. MEGA 11 was used to align the sequences and the phylogenetic tree was constructed using the Neighbor-Joining method (Saitou and Nei 1987).

Cultivation and growth

To study the growth profile and biochemical composition, the isolate KAU19 was initially cultivated in f/2 medium using 2 L capacity glass reactors at initial optical density of 0.152 ± 0.035. Filtered-sterile CO₂ enriched air (1.5%, v/v) was supplemented to the bottom of the reactors at 0.2 vvm (Han et al. 2016). Cultures were incubated for 18 days at 100 µmol photons m⁻² s⁻¹ (12:12 h light:dark cycle) and 28 °C. To study the efficiency of Aq-P as a growth medium, natural filtered seawater was used to dilute the Aq-P at initial nitrogen content of 13 mg L⁻¹ (similar to f/2 medium) and initial salinity of 38.4 ppt (similar to the seawater at the collection site). Figure 1 shows the experimental routes for hydrothermal liquefaction of Dunaliella salina KAU19 cultivated in f/2 medium as a control comparing to the aqueous phase obtained from hydrothermal liquefaction diluted with seawater (HTL-med).

Fig. 1.

Schematic diagram showing the routes of hydrothermal liquefaction of Dunaliella salina KAU19 cultivated in f/2 medium as a control comparing to the aqueous phase obtained from hydrothermal liquefaction diluted with seawater

Growth and biochemical characteristics

The growth of microalga was recorded by monitoring the optical cell density at wavelength 860 nm (OD₆₈₀) at two days interval using spectrophotometer (UV 2401 PC, Shimadzu, Japan). At the start and end of the exponential growth phase, dry weight (dw) was measured and daily biomass productivity was calculated as g L⁻¹ d⁻¹ using Eq. (1) (Abomohra et al. 2013);

$$\text{BP}\left({\text{g L}}^{-1}{\text{day}}^{-1}\right)=\left({\text{dw}}_2-{\text{dw}}_1\right)/\mathrm{\Delta }\text{t}$$ 1

where dw₁ and dw₂ refer to the dry weight at the start and the end of exponential phase at a time interval ∆t.

Lipids, proteins and carbohydrates were determined according to Bligh and Dyer (1959), Bradford (1976) and Kochert (1978) methods, respectively, as described in the previous study (Abomohra and Almutairi 2020). In addition, element analysis was done using Vario EL/micro cube elemental analyzer (Elementar, Germany). Oxygen content was calculated by difference using Eq. 2 (Tang et al. 2020). In addition, higher heating value (HHV) was calculated from the elements proportions (Mahari et al. 2018) using Eq. 3;

$$\text{Oxygen content}\left(\text{\%}\right)=100-\left(\text{C}+\text{H}+\text{S}+\text{N}+\text{Ash}\right)$$ 2

$$\text{HHV}\left({\text{MJ kg}}^{-1}\right)=\left(34\text{C}+6.3\text{N}+124.3\text{H}+19.3\text{S}-9.8\text{O}\right)/100$$ 3

Hydrothermal liquefaction

Hydrothermal liquefaction was done in 250 mL reactor at 300 °C as described previously (Li et al. 2021). Briefly, 5 g dw of microalgal biomass were suspended in 15 mL distilled water and loaded into the reactor, then 45 mL of 50% ethanol were added with stirring at 300 rpm. After heating up the reactor to the desired temperature, the reaction was performed for 45 min. After colling down the reactor in a water bath for fast quenching, syngas was released and the reactor was opened. In the present study, bio-oil recovery was performed without using organic solvents to avoid extraction of organics from the aqueous phase (López Barreiro et al. 2015). The HTL slurry was poured out and centrifuged at 1000× g to separate the main three phases. The upper oil phase was siphoned into a clean tube, while the remaining portion was filtered using a Whatman nylon membrane (47 mm, 0.45 μm pore size). The aqueous by-products were collected as a filtrate, while solid phase was retained in the upper filter side as biochar. The yields of the different HTL products were calculated using Eq. 4–7;

$$\text{Bio}-\text{oil yield}\left({\text{Y}}_{\text{oil}}\right)=\left(\frac{{\text{M}}_{\text{oil}}}{{\text{W}}_{\text{feed}}}\right)\times 100$$ 4

$$\text{Biochar yield}\left({\text{Y}}_{\text{char}}\right)=\left(\frac{{\text{M}}_{\text{char}}}{{\text{W}}_{\text{feed}}}\right)\times 100$$ 5

$$\text{Aqueous yield}\left({\text{Y}}_{\text{Aq}}\right)=\left(\frac{{\text{M}}_{\text{Aq}}}{{\text{W}}_{\text{feed}}}\right)\times 100$$ 6

$$\text{Gas yield}=100-\left({\text{Y}}_{\text{oil}}{\text{+ Y}}_{\text{char}}{\text{+ Y}}_{\text{Aq}}\right)$$ 7

where M_oil, M_char, and M_Aq refer to the mass of crude bio-oil, biochar, and aqueous phase, respectively, while W_feed refers to the weight of the total inputs (biomass and/or liquids).

Analytical methods

The chemical composition of the crude bio-oil was analyzed using GC–MS (Agilent Technologies, USA) equipped with Agilent 5975C mass spectrometer. The GC oven temperature program started at 70 °C and then increased to 290 °C at a heating rate of 5 °C/min (held for 10 min). Argon was used as the carrier gas at a flow rate of 1 mL min⁻¹. Water salinity and temperature were measured onsite using portable Oakton conductivity meter (Eutech Instruments, Singapore) and temperature meter (HI991001, Hanna, France), respectively. The cations and anions in the liquid samples were determined using ion chromatography. Total nitrogen (TN), total inorganic carbon (TIC), and total carbon (TC) were measured by infrared spectrometry (DIMA-TOC 100, Dimatec, Germany). The total organic carbon (TOC) was represented as the difference between TC and TIC.

Statistical analysis

The experiments were conducted in three replicates, where results are represented as the mean and standard deviation. One-way analysis of variance (ANOVA) followed by Tukey test was done using SPSS (v.20, IBM) at probability level P ≤ 0.05.

Results and discussion

Growth and biochemical composition

In the present study, the isolate KAU19 showed 91.59% similarity with Dunaliella salina (Abomohra et al. 2020a) previously isolated from a hypersaline lagoon at the Red Sea coastal area in Saudi Arabia (Fig. 2A). The relative low similarity might be attributed to the variation in salinity and environmental conditions at different isolation sites, which was reported to significantly influence the microbial genotype as well as phenotype (Li et al. 2022). Dunaliella is a genus of Chlorophyta belongs to Dunaliellaceae, Chlamydomonadales. Thus, the present isolate was identified as D. salina KAU19. Different species of Dunaliella are unicellular green microalgae reported as the key factor for most of the primary production in hypersaline environments worldwide (Oren 2005). In recent years, Dunaliella have received increasing attention as a feedstock for different biofuels including biohydrogen (Chen et al. 2020), biodiesel (Abomohra et al. 2020a), crude bio-oil (Yang et al. 2011), bioethanol (Lee et al. 2013), and biogas (González-González et al. 2019) coupled with seawater desalination and production of value-added compounds (Goswami et al. 2022).

Fig. 2.

Phylogenetic tree of the isolated microalgal species KAU19 showing the highest similarity with Dunaliella salina KSA-HS022 (A) and growth pattern of Dunaliella salina KAU19 in f/2 medium for 18 days showing the yield of different macromolecules at the end of exponential phase (B). The values of the dry weight with the same letter showed insignificant difference (one-way ANOVA followed by Tukey test at P ≤ 0.05)

To identify the different growth phases and biochemical composition of D. salina KAU19, the growth was monitored in f/2 medium for 18 days (Fig. 2B). The isolate showed a short lag phase period of 2 days which indicates the fast adaptation of microalgal cells to the surrounding and, therefore, confirms the suitability of f/2 as a growth medium for KAU19 isolate. The exponential growth phase was extended up to day 14 with the highest significant biomass yield of 1.96 g L⁻¹ (P 0.034 compared to that at day 12), followed by growth stability till the end of cultivation period. The stability of growth after 14 days is attributed to the equal rate of cell division and cell death due to nutrients depletion from the growth medium (Liu 2017). At the end of exponential growth, carbohydrates, lipids, proteins and ash represented 0.805, 0.459, 0.508, and 0.179 g L⁻¹, respectively (Fig. 2B). The recorded biomass productivity of 0.133 g L⁻¹ day⁻¹ was 43.0% higher than that recorded for D. salina KSA-HS022 isolated from the hypersaline Shuaiba Lagoon in Saudi Arabia (Abomohra et al. 2020a) and 9.0% higher than that of the marine microalga Tetraselmis elliptica isolated from the hypersaline Bardawil Lagoon in Egypt (Abomohra et al. 2017). The recorded differences in biochemical composition of KAU19 are attributed to seasonal variation and environmental conditions where the same microalgal species grows, as well as the genetic variation between different species (Cheregi et al. 2021). The biomass harvested at the 14th day of growth was used further for HTL experiments and evaluation of Aq-P.

Aqueous phase composition

After performing HTL for D. salina, the main characteristics of the Aq-P were analyzed (Table 1). Organic solvents (mostly dichloromethane) are typically used in most studies to recover the bio-oil after HTL (Li et al. 2021). However, utilization of organic solvents for crude bio-oil recovery has the potential to extract other dissolved organics from the Aq-P (López Barreiro et al. 2015). In addition, the residues of the organic solvent in the Aq-P might have negative impact on microalgal growth. Therefore, Aq-P was recovered in the present study by filtration and centrifugation, which explains the relatively high content of TOC in the aqueous phase (Table 1). It can be noted that both inorganic and organic carbon can be detected in the Aq-P after HTL (3.12 and 10.24 g L⁻¹, respectively) with relatively high ammonia concentration (3.04 g L⁻¹), indicating that organic carbon and nitrogen-containing compounds are degraded to inorganic carbon/nitrogen during the supercritical water liquefaction. In addition to formation of inorganic nitrogen in the Aq-P as ammonia and nitrate, nitrogen can be deposited in other HTL products including biocrude oil and solid residue (Garcia Alba et al. 2013). The present results confirm the findings of other reports where inorganic nitrogen content in the Aq-P showed high proportion, while no nitrogen was recorded in the gas phase of HTL at temperature lower than 600 °C (Killilea et al. 1992). In that context, a recent study confirmed that utilization of marine resources with terrestrial resources could provide complementary action towards enhanced biological conversion (Abomohra et al. 2022). Therefore, results in Table 1 indicate that seawater has rich content of anions and cations, which could provide synergistic and complimentary action by mixing with organics-rich Aq-P for microalgal growth.

Growth and biochemcial composition in Aq-P

Based on the TN content of the Aq-P, 0.25% (v/v) were added to the filtered seawater and used as HTL-medium. Growth pattern and biomasss productivity of D. salina on f/2 medium as a control and on diluted Aq-P are presented in Fig. 3. Although algal culture density showed similar pattern in both media, cells in the HTL-medium reached the end of the exponential phase earlier (12 day) comparing to 14 days in the control. At the 12th day, HTL-medium culture showed 15.9% significantly higher dry weight (1.97 g L⁻¹, P 0.012) than the control, even though both cultures showed insignificant difference (P 0.348) at the 14th day (Fig. 3). Consequently, biomass productivity of the HTL-medium culture at 12^th day was 16.4% higher than the control. The higher recorded growth might be attributed to the presence of organic compounds in the Aq-P which was reported to enhance the microalgal growth in mixotrophic cultivation modes (Gu et al. 2015). In that context, algal excessive natural growth was reported to be linked with the eutrophication due to excessive nutrients from fertilizers and other organic nutrients that enter the waterbodies forming natural algal blooms (ElFar et al. 2022). In addition, mixotrophic cultivation was reported as a better strategy to enhance biomass production than both heterotrophic and autotrophic cultivations due to integration of both heterotrophic and autotrophic components during the 24 h cycle (Shandilya and Pattarkine 2019). Interestingly, a previous study evaluated the growth of the freshwater Desmodesmus sp. with diluted Aq-P and reported no changes comparing to the standard medium (Garcia Alba et al. 2013), which confirms that supplementation of seawater in the present study provides additional micronutrients and elements required for the microalgal growth, leading to faster growth rate.

Fig. 3.

Growth pattern of Dunaliella salina KAU19 in f/2 medium (Cont) and on the diluted aqueous phase obtained from hydrothermal liquefaction (HTL). The same letter on the dry weight and biomass productivity series represents insignificant differences (one-way ANOVA followed by Tukey test at P ≤ 0.05)

Feedstock characteristics

Based on the growth pattern and biomass productivity, microalgal cells in both cultures were harvested at the 12th day of cultivation, and the variations in biomass characteristics were evaluated (Table 2). Carbon and oxygen contents of the biomass showed insignificant differences using f/2 medium or HTL-medium (P 0.393 and 0.281, respectively). Therefore, the HHV also showed insignificant difference (P 0.202) between the two feedstocks. However, using Aq-P resulted in significant reduction of nitrogen (P 0.000) and sulphur (P 0.001), with simultaneous increase of hydrogen (P 0.027). Reduction of sulphur and nitrogen in the biomass might be attributed to the growth in mixotrophic mode due to the organic compounds in the Aq-P. Regarding macromolecules, utilization of Aq-P significantly enhanced the lipid content by 21.2% over the control (P 0.006), with 5.7% and 7.3% reduction in carbohydrates and protein, respectively. It might also be attributed in the presence of organic compounds in the Aq-P which were reported to enhance the lipid accumulation in microbial cells (Almutairi et al. 2021). High biomass productivity coupled with improved lipid accumulation were reported as the crucial parameters to ensure the feasibility of biofuel production by microalgae through mixotrophic cultivation (Meng et al. 2020). Previous study confirmed enhancement of lipids up to 57% for mixotrophic cultivation of the oleaginous green microalga Chlorella protothecoides (Wang et al. 2013). The effectiveness of mixotrophic cultivation was further evaluated and was attributed to the metabolic changes and subsequently led to more lipid synthesis and accumulation in the microalgal cells (Meng et al. 2020). Higher lipid content in the feedstock could stimulate hydrocarbons and esters proportions in the bio-oil. In addition, lower nitrogen and sulphur content in the biomass are desirable features for emission reduction by reducing the N- and S-containing compounds in the produced bio-oil.

Table 2.

The main chemical composition of Dunaliella salina KAU19 after 12 days of cultivation in f/2 medium (Contol) and on the diluted aqueous phase obtained from hydrothermal liquefaction (HTL-med)

Parameters	Control	HTL-med
C (%dw)	49.52 ± 1.73	50.96 ± 1.95 ns
H (%dw)	6.54 ± 0.47	7.65 ± 0.31*
N (%dw)	6.99 ± 0.13	4.12 ± 0.38*
S (%dw)	0.95 ± 0.04	0.34 ± 0.11*
O (%dw)c	27.02 ± 2.35	29.48 ± 2.49 ns
Carbohydrates (%dw)	46.64 ± 1.68	43.96 ± 1.31*
Lipids (%dw)	21.42 ± 1.14	25.96 ± 0.91*
Proteins (%dw)	23.14 ± 0.87	21.45 ± 1.02 ns
Ash (%dw)	8.98 ± 0.02	7.45 ± 0.58*
HHV (MJ kg−1)c	22.94 ± 1.42	24.27 ± 0.52 ns

^cCalculated values as mentioned in Eqs. 2–3

^ns and ^* mean insignificant or significant differences, respectively, with the corresponding control value (at P ≤ 0.05)

Crude bio-oil production

HTL of the two studied feedstocks showed insignificant differences in all products (P > 0.05), with bio-oil yields of 28.74% and 29.54% from microalgal biomass cultivated in f/2 and Aq-P, respectively (Fig. 4). The aqueous phase of both treatments showed also insignificant differences (23.15% and 21.05%, respectively, P 0.553). The relatively high yield of the aqueous phase among all other products confirms the necessity to establish an efficient platform for its recycling for further nutrients/energy recovery and to ensure sustainability. Despite the insignificant changes in bio-oil yields of the two treatments, using Aq-P showed a pronounced impact on the bio-oil chemical composition as shown in Table 3. The produced bio-oil showed a total of 43 compounds in f/2-derived bio-oil and 41 compounds in Aq-P-derived bio-oil. The recorded compounds can be classified into 6 main groups, where fatty acids/esters and hydrocarbons represented the majority in both f/2 and Aq-P, due to the dominance of hexadecenoic acid and 2-hexadecene 3,7,11,15-tetramethyl-, respectively. Using Aq-P significantly enhanced the fatty acids/esters and hydrocarbons by 12.6% and 1.7 times, respectively, higher than that of f/2-derived bio-oil. Interestingly, nitrogen-containing compounds in the Aq-P showed reduction by 60.7% comparing to f/2 which is in agreement with the element analysis of the biomass (Feedstock characteristics). Distribution and composition of pyrolysis products depend mainly on the composition of biomass feedstock. Due to reduction of protein content under Aq-P application, nitrogen-containing compounds were reduced in the produced bio-oil. In agreement with the present study, mixotrophic cultivation of Spirulina platensis using molasse supplementation showed lower protein content compared to the autotrophic growth, which resulted in less nitrogenated compounds in the pyrolysis products (Chagas et al. 2021). In addition, higher protein content under autotrophic growth of microalgae favored hydrocarbons formation during pyrolysis (Chagas et al. 2016), which confirms the positive impact of mixotrophic growth using Aq-P in the present study.

Fig. 4.

Products yields of hydrothermal liquefaction of Dunaliella salina KAU19 after 12 days of cultivation in f/2 medium (Control) and on the diluted aqueous phase obtained from hydrothermal liquefaction (HTL-med). The same capital letter for the same product in different growth media refers to insignificant difference, while the same small letter for different products in the same growth medium refers to insignificant difference (one-way ANOVA followed by Tukey test at P ≤ 0.05)

Table 3.

Chemical composition of the bio-oil produced from hydrothermal liquefaction of Dunaliella salina KAU19 after 12 days of cultivation f/2 medium (Control) and on the diluted aqueous phase obtained from hydrothermal liquefaction (HTL-med)

Groups/Compounds	Formula	Control	HTL-med
Fatty Acids/Esters
Methyl hexadec-9-enoate	C17H32O2	1.84 ± 0.12	2.15 ± 0.15*
1,2-Benzenedicarboxylic acid, ditridecyl ester	C34H58O4	0.69 ± 0.09	1.52 ± 0.21*
Hexadecanoic acid	C16H32O2	16.24 ± 0.85	20.15 ± 1.63*
Hexadecanoic acid, methyl ester	C17H34O2	3.94 ± 0.65	4.45 ± 0.65ns
Heptadecanoic acid, methyl ester	C18H36O2	1.25 ± 0.41	2.96 ± 0.48*
9,12-Octadecadienoic acid (Z,Z)-, methyl ester	C19H34O2	1.36 ± 0.21	nd*
2,3-Dimethyl-2-cyclopenten-1-one	C7H10O	1.74 ± 0.11	nd*
1,2-Cyclopentanedione, 3-methyl	C6H8O2	3.40 ± 0.36	3.45 ± 0.22ns
Octadecanoic acid	C18H36O2	2.36 ± 0.15	1.94 ± 0.16*
Octadecanoic acid, methyl ester	C19H38O2	1.02 ± 0.21	0.54 ± 0.08*
Pentadecanoic acid	C15H30O2	1.02 ± 0.41	1.89 ± 0.32ns
Pentadecanoic acid, ethyl ester	C17H34O2	4.15 ± 0.65	4.69 ± 0.36ns
Dichloroacetic acid, 2-octyl ester	C10H18Cl2O2	2.24 ± 0.11	2.69 ± 0.41 ns
Phenols
Phenol	C6H5OH	3.78 ± 0.21	2.15 ± 0.54*
Phenol, 4-methyl-	C7H8O	1.97 ± 0.15	0.54 ± 0.12*
Hydrocarbons
4,5-Diphenylocta-1,7-diene(dl)	C20H22	1.45 ± 0.21	2.25 ± 0.19*
2-Hexadecene, 3,7,11,15-tetramethyl-,	C20H40	13.25 ± 1.06	15.24 ± 0.95*
2-Methyl-E-7-octadecene	C19H38	1.05 ± 0.41	0.95 ± 0.08ns
6-Isopropyl-1,4-dimethylnaphthalene	C15H18	1.32 ± 0.31	2.69 ± 0.15*
Heptadecane	C17H36	2.54 ± 0.22	3.65 ± 0.39*
Tetracosane	C24H50	1.74 ± 0.30	1.54 ± 0.18ns
Aromatic hydrocarbons
Naphthalene, 6-methoxy-2-(1-buten-3-yl)-	C15H16O	0.41 ± 0.12	0.25 ± 0.09ns
2(1H)-Naphthalenone, octahydro-4a-phenyl-, cis-	C16H20O	0.16 ± 0.07	0.23 ± 0.08ns
Naphthalene, 2,6-bis(1,1-dimethylethyl)-	C18H24	1.29 ± 0.41	1.69 ± 0.32ns
Naphthalene, 1,2,3,4-tetramethyl-	C14H16	0.94 ± 0.12	1.65 ± 0.06*
Naphthalene, 1,4,5-trimethyl-	C13H14	nd	2.04 ± 0.18*
Azulene, 7-ethyl-1,4-dimethyl-	C14H16	0.22 ± 0.06	1.11 ± 0.11*
Azulene, 1,4-dimethyl-7-(1-methylethyl)-	C15H18	nd	0.54 ± 0.08*
N-contaning compounds
Pyrazine, methyl	C5H6N2	1.62 ± 0.21	0.75 ± 0.10*
2-Pyrrolidinone	C4H7NO	2.58 ± 0.33	0.87 ± 0.21*
2,5-Pyrrolidinedione, 1-methyl-	C5H7NO2	2.74 ± 0.48	0.16 ± 0.07*
1-Butanamine, N,N-dimethyl-	C6H15N	1.25 ± 0.19	0.39 ± 0.05*
Benzenamine, 2,4,6-trimethyl-	C9H13N	0.69 ± 0.11	nd*
Benzaldehyde, 4-(dimethylamino)-	C9H11NO	nd	0.42 ± 0.06*
Beta-carboline, 6-methoxy-1,2-dimethyl-	C14H14N2O	0.78 ± 0.12	0.17 ± 0.04*
Dopamine, N,N-dimethyl-, dimethyl ether	C12H19NO2	1.05 ± 0.17	0.25 ± 0.08*
Dodecanamide, N,N-diethyl-	C16H33NO	1.12 ± 0.19	0.33 ± 0.02*
1 ,3-Oxazolidine, 4-methyl-5-trans-phenyl-2-(4-methoxyphenyl)	C17H19NO2	1.22 ± 0.42	0.47 ± 0.01*
Uracil, 6-amino-1,3-diethyl-	C8H13N3O2	0.33 ± 0.12	0.11 ± 0.08ns
Hexadecanamide	C16H33NO	6.54 ± 0.69	4.19 ± 0.12*
N-Methyldodecanamide	C13H27NO	0.69 ± 0.17	nd*
O-containing compounds
2-Cyclopenten-1-one, 3-methyl-	C6H8O	4.48 ± 0.28	2.95 ± 0.14*
Z-2-Tridecen-1-ol	C13H26O	1.15 ± 0.16	0.56 ± 0.05*
2-Ethyl-4-methylanisole	C10H14O	0.25 ± 0.08	1.08 ± 0.06*
Cyclohexanone, 2,2,6-trimethyl-	C9H16O	nd	1.45 ± 0.15*
Others		2.14 ± 0.24	2.90 ± 0.33ns

nd Not detectable and it was considered as zero in statistical analysis

^ns and ^* mean insignificant or significant differences, respectively, with the corresponding control value (at P ≤ 0.05)

All recorded compounds were within the carbon range C4–C34, while majority of compounds were within 5–21 carbon chain length (Fig. 5A). In that context, carbon number in the bio-oil can be used to evaluate its suitability for different fuel distillation products (Abomohra et al. 2021). The organic components of the bio-oil can be divided into main four fuel categories based on the carbon chain length, namely heavy oil (more than C24), diesel (C13–C24), gasoline (C5–C12), and light fuels (less than C5). It is noteworthy to mention that this is a rough estimation based on the carbon chain length and further analysis on the distilled products is needed to validate the results. According to EIA (2022), products known as No. 1, No. 2, and No. 4 diesel fuel have wide applications and are used in many engines including on-highway diesel engines as well as off-highway engines. However, other fuel oils are used primarily for heating and electric power generation. The current evaluation showed that diesel C-range represents the majority of the products in both f/2- and Aq-P-derived bio-oil (69.1% and 78.3%, respectively), followed by gasoline category (Fig. 5B). Although cultivation in Aq-P showed slight changes in the bio-oil yield, the characteristics of the bio-oil were enhanced with respect to the lower nitrogen-containing compound, higher hydrocarbons, and desired carbon-chain range. Considering the higher biomass productivity using Aq-P (Growth and biochemcial composition in Aq-P), the overall volumetric bio-oil yield could be enhanced as well in the large-scale application.

Fig. 5.

Relative proportions of carbon chains (A), and predicted fuel products (B) from the crude bio-oil produced by hydrothermal liquefaction of Dunaliella salina KAU19 after 12 days of cultivation in f/2 medium (Control) and on the diluted aqueous phase obtainesd from hydrothermal liquefaction (HTL-med)

Conclusions

The present study evaluated, for the first time, the application of seawater mixed with the liquid products of HTL of the marine microalga D. salina for dual use as energy (bio-oil) and growth medium. Utilization of Aq-P mixed with seawater enhanced the biomass productivity by 16.7% over the control, which is attributed to mixotrophic growth due to the organics in Aq-P. In addition, Aq-P enhanced the lipid content by 21.2% over the control, and showed 5.7% significant reduction in carbohydrates. Although utilization of Aq-P showed insignificant difference in the bio-oil yield, its characteristics were enhanced due to reduction of N-containing compounds (by 60.7%) and stimulation of hydrocarbons and fatty acids/esters (by 1.7 times and 12.6%, respectively). The suggested approach brings many advantages in terms of water saving, waste recycling, and renewable energy production. Future studies are needed to evaluate the economic feasibility of the suggested approach in large-scale cultivation.

Funding

King Abdulaziz University,G: 170-662-1443,ADEL W Almutairi

Declarations

Conflict of interest

Authors confirm that there is no conflict of interest.