Hydrothermal liquefaction of Gracilaria gracilis and Cladophora glomerata macro-algae for biocrude production

Accepted Manuscript Hydrothermal Liquefaction of Gracilaria gracilis and Cladophora glomerata macro-algae for biocrude production Mehran Parsa, Hamoon Jalilzadeh, Maryam Pazoki, Reza Ghasemzadeh, MohammadAli Abduli PII: DOI: Reference:

S0960-8524(17)31888-6 https://doi.org/10.1016/j.biortech.2017.10.059 BITE 19100

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

16 August 2017 9 October 2017 13 October 2017

Please cite this article as: Parsa, M., Jalilzadeh, H., Pazoki, M., Ghasemzadeh, R., Abduli, M., Hydrothermal Liquefaction of Gracilaria gracilis and Cladophora glomerata macro-algae for biocrude production, Bioresource Technology (2017), doi: https://doi.org/10.1016/j.biortech.2017.10.059

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Hydrothermal Liquefaction of Gracilaria gracilis and Cladophora glomerata macro-algae for biocrude production Mehran Parsa1, Hamoon Jalilzadeh2, Maryam Pazoki3, *, Reza Ghasemzadeh4, MohammadAli Abduli5 1

Solid waste Engineering Group, Department of Environmental Engineering, Graduated Faculty of Environment, University of Tehran, Tehran, Iran. Email: [email protected].

2

Department of Environmental Engineering, Graduated Faculty of Environment, University of Tehran, Tehran, Iran. Email: [email protected].

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Department of Environmental Engineering, Graduated Faculty of Environment, University of Tehran, Tehran, Iran. E-mail: [email protected].

4

Department of Environmental Engineering, Graduated Faculty of Environment, University of Tehran, Tehran, Iran. Email: [email protected].

5

Professor of Environmental Engineering, Graduate Faculty of Environment, University of Tehran, Tehran, Iran. E-mail: mabdoli ut.ac.ir.

*Corresponding author Email: [email protected], Tel: (+98) (21) 66479764, P.O.Box: 14155-6135 (Maryam Pazoki). Abstract The potential of Gracilaria gracilis (G.gracilis) and Cladophora glomerata (C.glomerata) macro-algae species harvested from Caspian Sea for biocrude oil production under Hydrothermal Liquefaction (HTL) reaction at 350 ºC and 15 minutes has been investigated. Furthermore, the effect of using recycled aqueous phase as the HTL reaction solvent was studied. The biocrude yield for G.gracilis and C.glomerata was 15.7 and 16.9 wt%, respectively with higher heating value (HHV) of 36.01 and 33.06 MJ/Kg. The sources of each existing component in bio-oil were identified by GC-MS based on their suggested reaction pathways. Moreover, after two series of aqueous solution recycling, experiments

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showed that the bio-oil yield significantly incresead compared with the initial condition. This increasing directly relates with recovery of carbon content from the aqueous solution residue. Key words: Gracilaria gracilis; Cladophora glomerata; Macro-algae; Hydrothermal Liquefaction (HTL); Biocrude 1. Introduction Most researches agree that the main cause of climate change and global warming is carbon dioxide emissions from fossil fuels. These environmental issues have significant effects on the quality of human life and economy (Heidari and Pearce, 2016; Höök and Tang, 2013). Biofuels, obtained entirely from natural sources, are known as alternative, renewable source for energy. Most species of algae are great sources for sustainable biofuel production due to their fast growth rate and the ability to grow widely in oceans and sea without artificial nutrients (Baicha et al., 2016; Najafi et al., 2011). The third generation of biofuels utilized micro-algae and macro-algae or seaweed as wet biomass. The advantage of sustainable algae biofuels is not exclusively in decreasing air pollution by less emission of greenhouse gasses (i.e. NOX, SOX and CO2) (Pegallapati and Frank, 2016), CO2 capturing (Hernández-Calderón et al., 2016) and reduction of waterways pollution during growth (Lu et al., 2016); it is one of the most effective approaches for creating jobs and economic prosperity in rural regions through sea-farming concept (Fernand et al., 2017). Algae biofuel is not fully compatible with vehicles yet (Wallington et al., 2016) which

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indicates more researches are needed concerning the use of new feedstock for production of biofuel by thermal conversion process. In recent decades, most researches have concentrated on micro-algae as a suitable feedstock for bio-oil production (Barreiro et al., 2013). The algae feedstock is provided for biofuel production in two steps: algae growth program and algae harvest plan. The main cost of micro-algae bio-oil production is related to harvesting steps (Draaisma et al., 2013). However, the alternative choice is using macro-algae for biofuel feedstock. Macro-algae harvesting in comparison with micro-algae is cheap and simple (Barreiro et al., 2015; Lawton et al., 2016). There are three main types of macro-algae: green (Chlorophyta), brown (Phaeophyta) and red (Rhodophyta). Furthermore, macro-algae can be utilized as feedstock in numerous industries such as chemicals, animal food, cosmetics and health-care products (Griffiths et al., 2016; Wei et al., 2013). The large amount of water in macro-algae means that hydrothermal conversion of biomass can be a suitable and eco-friendly option for production of biofuel (Barreiro et al., 2015). HTL is carried out under a moderate to high temperature (200–374 ºC), sufficient pressure (10–25 Mpa) and in subcritical conditions (Guo et al., 2015). This fast process has been used with or without the presence of catalysts for a range of biomasses. During HTL process, the macromolecules in feedstock degrade to low molecular weight compounds through a series of complex reactions such as: hydrolysis, fragmentation, aromatization, dehydration and deoxygenation (Toor et al., 2011).

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Recent researches about macro-algae HTL process for production of bio-oil are summarized in Table 1. Most previous researches have focused on HTL of green and brown macro-algae, and to the best of our knowledge the HTL of red macro-algae has not been reported. Zhou et al. (Zhou et al., 2010) studied HTL of macro-algae in 2010, the first report of its kind. They found that the biocrude oil yield was 23% from Enteromorpha prolifera via HTL with HHV 30 MJ/Kg. In the literature review it is demonstrated that the optimal operational parameters for macro-algae HTL process are a range of conditions such as mass/algae ratio (5–50%), holding temperature (200–380 ˚C) and retention time (5–120 min) (Anastasakis and Ross, 2015; Barreiro et al., 2015). The optimal parameters for HTL process relate directly with physical and chemical properties of raw material. Yield range and HHV of the bio-oil produced under normal HTL process was reported to be between 9.8-35.3 % and 26-36.5 MJ/Kg, respectively (Anastasakis and Ross, 2015; Neveux et al., 2014). To date, the maximum reported yield of biocrude oil is about 79%: partly employed fast HTL of Laminaria saccharina under the HTL condition at 350 ˚C, 15 min, and heating rate of approximately 585 ° C /minute (Bach et al., 2014). In this study, two low lipid macro-algae (Gracilaria gracilis and Cladophora glomerata) that can be commonly found in Caspian Sea, Iran are examined under HTL. Macro-algae biomass was collected from natural environments and then chemical, thermal and physical properties of macro-algae feedstock was analysed. The yields and energy recovery content of the three phases of HTL products (bio-oil, bio-char and aqueous phase) are calculated and compared with each other. The macro-algae HTL products were determined by several analysis in order to investigate the effect of thermal process on chemical and physical

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properties of feedstock. Furthermore, the suggested reaction pathways were studied to identify each existing component in bio-oil. Moreover, the effect of using aqueous phase after consecutive recycling as the reaction solvent has been examined. 2. Materials and methods 2.1. Raw Materials The feedstock is prepared from red (Gracilaria gracilis) and green macro-algae (Cladophora glomerata). The macro-algae samples were collected at the end of June 2016 from south coast of Caspian Sea, Iran. After being dried in open air and being in oven at 60 °C for 6 hours, the feedstock was ground to a mean particle size of <150µm. 2.1.1.Macro-algae characterization Proximate, ultimate and biochemical analysis were performed for both macro-algae feedstocks and the results are shown in Table 2. Other researches reported the same results about low lipid content in these macro-algae species which can directly affect bio-oil yield under HTL process (Francavilla et al., 2013). High nitrogen content in macro-algae samples relate with high value of protein in their chemical composition which can produce undesirable N-contained fraction in bio-oil. The amount of metals in feedstocks were determinated by ICP-OES and the results are listed in Table 3. The results show high level of alkali and alkaline metals such as Ca, Mg, Na and K which can have catalytic effect on the liquefaction process (Schumacher et al., 2011). 2.2. Hydrothermal procedure

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The experiments were carried out in a batch custom-built stainless steel reactor with 75 ml volume. In a typical test, 3 g of dried macro-algae was introduced into the reactor followed by adding 30 ml of deionized water. Inside the reactor was purged by N2 gas to remove the remaining air present in the vacant volume of reactor and then the solution mixture was stirred. The reactor was kept at 350 °C for 15 minutes after being heated up from 100 to 350 °C under approximately 20 ºC/min heating rate. This optimal condition was reported for reaching the maximum bio-oil yield in literature studies (Anastasakis and Ross, 2015, 2011; Li et al., 2012; Neveux et al., 2014). After completion of reactions, the reactor was put in water/ice and was allowed to reach the room temperature. Moreover, the test with consecutive recycling of the aqueous phase was investigated in the same condition at 350 ºC and 15 min. All the experiments were carried out at least in triplicate, and the results show their mean and standard deviation values. 2.3.Product separation and yield analysis After opening the reactor, the gaseous product was vented and another product was separated using dichloromethane (DCM) and water. Reactor mixture phase was washed twice with 25 mL DCM and solid by-product (biochar) was filtered by 1 µm pore size filter paper. The separated aqueous phase was filtered to eliminate undissolved materials, then the solvent (DCM) was evaporated under reduced pressure at 40 ˚C and 451 mbar and vacuum dried at 50 ˚C and 23 mbar in a rotary evaporator to separate bio-oil. Product yields were calculated by the following equation (Eq. (1)) (Barreiro et al., 2015):

Y PRODUCT =

W PRODUCT ×100% W FEEDSTOCK

(1)

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Where YPRODUCT is yield of biocrude or biochar (wt. %) on a dry weight basis, WPRODUCT is the mass of product (g) and WFEEDSTOCK is the mass of biomass feedstock used (g). 2.4.Analysis of material and product Ash content, moisture content (MC), volatile matter (VM) and fixed carbon (FC) of macro-algae were calculated by TGA analysis. The samples were analyzed for metals by inductively coupled plasma spectrometry (ICP) with optical emission spectroscopy (OES) on a Perkin Elmer Optima 5300DV. The protein, carbohydrate and lipid contents were analyzed in the Nanotechnology Research Center of Graduate Faculty of Environment, University of Tehran. Fourier transform infrared (FTIR) spectra of samples were observed using a Bruker FT-IR spectrometer. KBr disks for FTIR analysis were prepared using the crushed and sieved (<0.018 mm) macroalgea and biochar samples that were vacuum dried (at 30oC for 24 h). FTIR spectra were analyzed with the samples mixed with KBr at a sample/KBr mass ratio of 1:100 at 4 1/cm resolution in the range from 400 to 4000 1/cm. The surface properties of samples were analyzed by the HITACHI S-4160 scanning electron microscope (SEM). The carbon, hydrogen, oxygen, nitrogen and sulfur contents on a dry weight basis of samples were analyzed by Thermo Finnegan, FlashEA 1112 series. The total organic carbon (TOC) and total nitrogen (TN) in the aqueous phase were subsequently quantified (ZarAzma Mineral Studies Company, Iran). The Higher Heating Value (HHV) was calculated by using elemental composition in Boie's formula (Eq. (2)) (Annamalai et al., 1987):

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HHV Boie ( MJ.Kg -1 ) = 0.3516 C +1.16225 H - 0.1109O + 0.0628 N

(2)

Using Agilent 5973 mass spectrometer with 6890 Plus Gas Chromatograph, a variety of chemical compounds in biocrude were examined. The Derivative compounds were separated by an Rtx-5MS column, 30 m length, 0.25 mm Diameter and 0.25 µm thickness. Also Helium with 99.999% purity was utilized as the carrier gas with 1 mL/min flow rate. 1 µg of samples were analysed in the initial temperature of 40 ºC for 15 min, then the temperature was ramped up to 250ºC at 10 ºC/min and was kept in this condition for 10 minutes with 63.9 kPa as the column head pressure. Considering the molecules with a match quality above 90%, chemical compounds identification is supported by the NIST (National Institute of Standards and Technology) library of mass spectra. 2.5. Energy recovery and mass balance The energy recovery (ER) of the liquefaction products was calculated according to Eq. (3):

ER=

HHV PRODUCT × W PRODUCT

(3)

×100%

HHV FEEDSTOCK × W FEEDSTOCK

where ER is the energy recovery of macro-algae HTL (%), HHVPRODUCT is HTL bio-oil and biochar higher heating value (MJ/kg), WPRODUCT is the mass of HTL bio-oil and biochar (g), HHVFEEDSTOCK is the feedstock macro-algae higher heating value (MJ/kg) and WFEEDSTOCK is the mass of feedstock macro-algae used (g).

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The same equation was used to calculate the recovery of N and C in each product of macro-algae liquefaction by replacing HTL produced phases and feedstock HHV with the respective carbon or nitrogen amounts. 3. Results and discussion 3.1.Thermal characteristics Thermal Gravimetric (TG) and derivative mass loss (DTG) analysis of macro-algae species and HTL solid residue samples were preformed. Dehydration, decomposition of carbohydrate and protein, and thermal degradation of lipid are the three major peaks observed in the DTG curves of both samples. The temperature ranges exhibiting major thermal degradation zone were 220-400 ºC, 175-300 ºC for C.glomerata and G.gracilis, respectively. The maximum mass loss temperatures (Tmml) were 360 and 260 ºC for C.glomerata and G.gracilis, respectively. Higher lipid content in C.glomerata chemical composition is the reason for the wider range of degradation temperature zone and Tmml. The solid residual mass or ash content after the TGA of macro-algae was about 26.1 and 36 wt%, for C.glomerata and G.gracilis, respectively. High ash value in both samples can have negative effect on reactor's operation and increase the formation of gas and char in the process due to catalytic effect of ash in raw material (Li et al., 2014). 3.2.Surface property analysis The functional groups present on feedstocks and biochar samples surfaces are provided by FTIR analysis. The OH bands (3200-3600 cm−1) in samples results are related to the potential presence of alcohols (Pokorna et al., 2009). The broad bands 3000 and 3100 cm−1

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were assigned to C–H stretching vibrations and revealed the presence of alkanes and alkenes in the samples. The peak between 2800-3000 cm−1 is attributed to aliphatic CH2CH3 stretching or methylene group of lipid (Gao et al., 2012). The strong peaks around 1650-1840 cm−1 were attributed to the amide group associated with proteins. The peak around 1400-1600 cm−1 was attributed to C=C aromatic. The peaks between 900-1200 cm−1 represent C-O, C-C, and C-O-C stretching vibrations of polysaccharides in materials (Lijian Leng et al., 2015; Li-jian Leng et al., 2015). Moreover, between 800 and 1000 cm−1, out of plane aromatic C-H functional group was recognized as well (Lijian Leng et al., 2015). The peaks from 2800 to 2900 cm−1 in G.gracilis show the decomposition of aromatic groups which increases the aliphatic groups absorption. Moreover, the decomposition of C=C aromatic bonds and the decrease in their peaks indicate that components such as carbohydrates and protein are also decomposed in G.gracilis. Furtheremore, OH peaks (3400, 1150 and 1126 cm−1) have increased. Oxygen functional groups (1790 and 1760 cm−1) in biochar sample have decreased compared with the raw material. Another firm indicator for aromatic decomposition is the decrease in 1362 cm−1 peak of CH3 aromatic and CH3-CH2 or C=C aromatic. For C.glomerata samples, the aromatic functional groups (3000-3100, 1630, 1360 and 1420 cm−1) in biochar sample have increased compared with the raw material and The C-O (1000-1150 cm−1) and the O-H (3400 cm−1) bonds peaks have also increased. The FTIR results have shown that HTL process significantly led to degradation of complex aromatic groups on the surface of raw materials to the aliphatic side groups on biochar surface. Moreover, the amount of oxygen-containing groups clearly increased in samples.

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The results of SEM analysis show that The HTL has significant effects on morphology of the macro-algae. After thermal conversion, the smooth surfaces of both macro-algae samples with a sheet-like structure were converted to small and porous elements with rough surface. 3.3.Product analysis 3.3.1. Product yield Yields from three phases of products obtained from hydrothermal liquefaction of G.gracilis and C.glomerata at 350 ºC and 15 min resident time are shown in Table 4. The value of bio-oil yield is related to the values of inorganic content (ash + moisture) and biochemical composition in feedstocks. As it was predicted, the yield of bio-oil from C.glomerata (16.9 ± 1.0 % dw) is more than the yield of bio-oil from G.gracilis (15.7 ± 0.9 % dw). In both samples the yield value is not relatively high because of the high value of ash in raw material. Moreover, ash has direct effect on slagging and fouling problems in continuous flow reactors (Liu and Balasubramanian, 2014). Furthermore, the difference in value of biocrude yield between G.gracilis and C.glomerata is related to biochemical contents (protein, lipid, carbohydrate) in algae feedstock. In HTL process, the yield of biocrude is achieved by two steps. First, in low temperature (200-250 ºC) lipid, some short chain algaenans, and some hydrophobic protein fragments extract from macro-algae. Then as the temperature of reactor increases to near supercritical point (300-375 ºC), biocrude yield increases due to proteins and cellulose ongoing degradation (Torri et al., 2012). Generally, typical biocrude yield of the macro-algae HTL process is between 9-30 percent (Neveux et al., 2014). Also, these values depend on operating conditions. However, the

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biocrude yields obtained from macro-algae in this study compares favorably with yields reported in previous studies. The biochar yield of C.glomerata and G.gracilis are 15.1 ± 2.2 and 15.0 ± 1.8 wt% respectively. Carbohydrates in macro-algae have a positive effect on the yield of biochar production under HTL process (Biller and Ross, 2011). Carbohydrate contents in macroalgae which include saccharides, glucose, fructose, xylose and starch have higher amounts compared to micro-algae species. Due to this reason, macro-algae have generally more yield of char formation than micro-algaes. Also high ash content is another reason for the increased amount of solid residue during the HTL process. Comparing two macro-algae used in this research, C.glomerata with higher amount of ash and carbohydrate has more biochar yield. Moreover, biochar yield values for both macro-algae are comparable to biochar yields reported for the HTL of other species of macro-algae which is lower than 25 wt% (Anastasakis and Ross, 2011; Zhou et al., 2010). The largest fractions of the products were distributed in aqueous phase, which had an aqueous yield of 69.2 ± 1.3 and 68.1 ± 0.8 wt% for G.gracilis and C.glomerata, respectively. These results are supported by previous studies on HTL of macro-algae where most of the feedstock was recovered in aqueous phase (Biller and Ross, 2011; Torri et al., 2012).The produced gas from HTL is typically composed of CO2, CO, CH4, H2 (Barreiro et al., 2013). The yield of gaseous phase in this study is calculated to be 9.1 and 5.3 wt% for G.gracilis and C.glomerata which is in range with other reports (Barreiro et al., 2015; Neveux et al., 2014).

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3.3.2. Element analysis of products The results from ultimate analysis of all the product phases following HTL of the macro-algae samples are shown in Table 5. The biocrude oil is a dark-brown viscose liquid which is formed from macro-algae samples under HTL process. The biocrude HHV values of C.glomerata (33.06 MJ/Kg) and G.gracilis (36.01 MJ/kg) fall in the range of other macro-algae biocrude reported from other studies which can be seen in Table 1. Compared with raw material C, H, O, N and S values increased under HTL process which subsequently resulted in higher HHV values for biocrude. The increase of carbon content in biocrude indicates that the oxygenated compounds such as protein and carbohydrate in the macro-algae feedstock degrade by decarboxylation and hydration reactions under liquefaction process (Kruse and Dahmen, 2015). The existing nitrogen and sulfur in biocrude oil is the critical character for using these products as a fuel. Unfortunately, these impurities in combustion lead to undesired emissions such as NOx and SOx. The accumulation of N and S in produced biocrude is high which is related to protein and inorganic contents in composition of feedstocks. Moreover, the content of O in biocrude oil is relatively high which causes HHV values to decrease. However, using this biocrude oil demands post-refining and upgrading. As investigated in recent researches, hydro-treating and hydro-cracking approaches could effectively decrease impurities in biocrude (Biller et al., 2015; Cole et al., 2016). The biochar samples are rich-organic materials. The element compositions of biochar are listed in Table 5. The HHV of the macro-algae biochar is 13.1 and 10.1 MJ/Kg for

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C.glomerata and G.gracilis, respectively. The relatively high carbon content in the biochar can be recovered as feedstock or catalyst on other thermal biomass conversions (i.e. HTL, Hydrothermal gasification and pyrolysis) (Ren et al., 2014). Moreover, high ranges of organic and inorganics materials in biochars can justify its use as direct soil nutrient, organic fertilizer and soil conditioner (Wu et al., 2016). In addition, porous materials like biochar have adsorbent properties which can be utilized in wastewater treatment for removing pollution from aqueous solutions (Zheng et al., 2017). 3.3.3. Energy recovery and mass balance Energy recovery potential from macro-algae HTL that is shown in Table 5 depends on the HHV and yield of the feedstock and the HTL products (biochar and biocrude) (Eq. (3)). High energy recovery in biocrude directly relates with optimized operational parameters of HTL process (i.e. ratio of water/biomass, temperature and residential time). The energy recovery of biocrude oil produced by HTL process is 48.3 and 40.8% for C.glomerata and G.gracilis, respectively. Also the energy recovery of biochar samples is 14.3 and 13.0 % for C.glomerata and G.gracilis, respectively. The higher energy recovery of C.glomerata compared to G.gracilis can be attributed to the higher yield of biocrude and biochar in C.glomerata. The combined aqueous and gas products (including eventual losses) form a significant proportion of biomass energy ( 37.4 % for C.glomerata and 46.2 % for G.gracilis). The results of carbon and nitrogen conversion to the new product were investigated by results of CHNS analysis of biocrude and biochar, and from total organic carbon (TOC)

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and total nitrogen (TN) analysis of the aqueous solution which can be found in Fig 1. Having calculated the carbon and nitrogen content in other phases previously, the remaining carbon and nitrogen in gaseous phase were calculated with a simple subtraction. As it can be seen in Fig 1, most of the carbon in raw material has converted to gaseous phase. Carbon content in gaseous phase is mostly in CO2 form which is the main product of HTL reaction. After gaseous phase most of the carbon was recovered in biocrude (38 wt% for C.glomerata and 31 wt% for G.gracilis), biochar (17 wt% for C.glomerata and 13 wt% for G.gracilis) and aqueous phase (17 wt% for C.glomerata and 13 wt% for G.gracilis), respectively. The recovered amount of C in solid phase can be used as feedstock resource for other thermal processes via post-conversion which would increase the total energy recovery. The main source of HTL product is the protein in macro-algae which causes nitrogen to be produced in each phase. After process, N accumulation in aqueous phases provide potential of using it in algae culturing medium. Although, recovery of N in biocrude has negative effect on oil quality. In this study, the most percentage of nitrogen has been recovered in aqueous solution (47 and 45 % for C.glomerata and G.gracilis, respectively). The distribution of N in biocrude is 14 and 40 % for C.glomerata and G.gracilis, respectively. The nitrogen balance results show that liquefaction of G.gracilis has led to more nitrogen recovery than C.glomerata in biocrude oil. This nitrogen has mostly been identified in form of N-contained compounds such as Pyrazine derivatives. Moreover, the nitrogen recovery from biochar samples were 17 and 8 % of nitrogen for C.glomerata and

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G.gracilis, respectively. The recovered amount of N and C in aqueous phase could be used as nutrient for growing microorganisms. 3.3.4. GC−MS Analysis of Bio-oils Bio-oil produced by liquefaction of Gracilaria gracilis and Cladophora glomerata is a very complex compound and it is characterized by GC–MS. Table 6 shows the compounds identified in the bio-oil obtained from the Gracilaria gracilis and Cladophora glomerata, respectively. The contents of identified compounds take up about 70% of the total area for each bio-oil. Both bio-oil samples obtained from biomass were composed of alcohols, ketones, aldehydes, fatty acids, esters, aromatics, amino acids, nitrogen-containing heterocyclic compounds and chlorinated/ fluorinated compounds. The formation of compounds under HTL process was very complex and their reaction was often unknown. The biochemical in macro-algae as feedstock have the main effect on the nature of produced bio-oil. In addition, other factors such as inorganic material in the feedstock (catalytic effect), HTL holding time and temperature, the heating rate of reactor and the solvent which was used for liquefaction have direct effects on the characteristics of biocrude. However, six probable pathways were suggested for recognized compounds by GC-MS analysis in this study. There are other pathways from other sources such as chlorophyll and carotenoids (Barreiro et al., 2015) which are not included in the six identified pathways of this study. The six suggested pathways are illusturated in the Fig 2. The identified compounds for each pathway are separated in Table 6. First pathway considers carbohydrate as the source for producing compounds such as furans and

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cyclopenten derivatives. Carbohydrate or polysaccharide in macro-algae is usually founded in four types: alginate, mannitol, laminarin and fucoidan (Ross et al., 2009). After the reaction starts, these polymers hydrolyze to monosaccharide (such as glucose and fructose) (Watanabe et al., 2006). These compounds dehydrate and decompose to furans or furfurals like compounds which are unstable in alkali condition. They degrade by losing water and produce phenolic compounds (Barreiro et al., 2015; Toor et al., 2011). The difference between the biocrude produced from Cladophora and Gracilaria is the slight alkali condition in G.gracilis. Due to this fact, all of furfural and furan like compounds could be converted to phenolic compounds. Moreover, due to low amount of lipid and protein in G.gracilis which can limit other reaction plans for HTL process the yield of products for G.gracilis in the first pathway is more than other pathways. The second reaction pathway is related to the reaction between carbohydrate and protein under HTL condition. Protein is the main biochemical compound of macro-algae. After temperature increases in HTL reactor, protein dehydrates to peptides and amino acids and then based on Maillard reaction goes on to react with the monosaccharaides (Toor et al., 2014). The Maillard reaction could produce several type of N-contained heterocycle compounds which are consisted of significant volumes of biocrude oil in both macro-algaes. The produced compounds based on second reaction pathway are listed in Table 6. Due to higher amount of protein in the Cladophora, more N-contained heterocycles compounds have been identified in its biocrude. All the products in this category are Pyrazine derivatives. In the other suggested pathway, peptides could convert to amines by decarboxylation reaction (Toor et al., 2014). In both biocrude samples, 4-methoxy benzenamine has been formed by this pathway under macro-algae HTL process. 17

The amount of lipid in feedstock plays the major role in liquefaction of biomass. However, it is very low in macro-algae species. At the beginning of HTL process, lipid under high temperature reacts with water and produces fatty acids (Barreiro et al., 2015). Then, These compounds react with saccharides or peptides and other compounds could be composed. On their way to produce nitriles in fuel compounds, first fatty acids synthesize with amines produced by peptides which could make amides. The created amides then produce nitrile through dehydration reaction (Toor et al., 2011). Various compounds created in this process are shown in Table 6. The next pathway in producing compounds from lipids could be the decarboxylation of fatty acids which results in a chemical reaction that could make alkanes and alkynes (Changi et al., 2012). The amount of aliphatic hydrocarbons is considerably more in green Cladophora micro-algae than in G.gracilis which has a direct link to the amount of biochemical lipid compounds present in this species. G.gracilis with less lipid and therefore less fatty acids, has less aliphatic compounds compared with Cladophora glomerata. The last path investigated here is the production of methyl esters of fatty acids. In this process that begins with the chemical reaction of Aldol saccharides, some amount of carbohydrates could react with fatty acids produced from the hydrolysis of lipids and create methyl esters of fatty acids (Rushdi and Simoneit, 2006). 3.4. Recycling of aqueous phase In order to obtain maximum yield in biocrude production, the HTL experiments replete by consecutive recycle of aqueous solution in 15 minutes retention time and 350 ºC. As seen in Figure 3, the biocrude yield increases from 16.9 and 15.71 wt% in the first run to a

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maximum of 25 and 24.6 wt% after 2 runs in biocrude obtained from C.glomerata and G.gracilis, respectively. The biochar yield in both samples was almost constant, although it decreased in aqueous and gaseous phases. Compounds such as organic carbon and nitrogen increase in the process waters and are detectcted with TOC and TN analysis. The presence of organic molecules increases the biocrude yields in the recycling process of aqueous phase which makes the aqueous phase more suitable for energy recovery. A previous study on micro-algae show that reclycling the aqueous phase has a significant effect on the biocrude yield. The biocrude yield of Chlorella vulgaris rose from 38 to 55 after one recycle (Hu et al., 2017). Studies on other biomasses such as Blackcurrant ( Ribes nigrum L.) Pomace (Deniel et al., 2016) and dried distillers grains with solubles (Biller et al., 2016) also show a significant increase in biocrude yield due to recycling of aqueous phase. 4. Conclusion HTL conversion of two low lipid macro-algae has produced biocrude with high HHV values and low yield. High concentration of nitrogen contents identified in biocrude samples and the necessity of decreasing them, caused the need for further bio-refinery process. The formation pathway compounds in biocrude were suggested based on degradation of raw material and variation of biochemicals (lipid, protein and carbohydrate) in macro-algae. Moreover, a high rise in biocrude yields was observed when recycling the aqueous solution. Maximum biocrude yield was 34 and 29% after two time recycling of aqueous solution for C.glomerata and G.gracilis, respectively. Appendix A. Supplementary data

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22

List of Table

Brown Macro-algae

Macro-algae species

HTL condition

Bio-oil yield ( wt.% )

HHV (MJ/Kg)

References

Latissima saccharina Laminaria digitata Laminaria saccharina

350 ºC, 15 min

19.3 17.6 13

36.5 32 33.9

(Anastasakis and Ross, 2011)

Laminaria saccharina

Fast HTL

79

35.97

(Bach et al., 2014)

Laminaria hyperborea Alaria Esculenta

350 ºC, 15 min

8.1 13

33 33.8

(Anastasakis and Ross, 2015)

Sargassum patens

340 ºC, 15 min

32.3

27.1

(Li et al., 2012)

Saccharina spp.

continuous flow reactors

22

-

(Elliott et al., 2013)

21 22 28

33.4 35.2 33.9

(Barreiro et al., 2015)

19.7 18.7 9.7 13.5

33.2 33.8 32.5 33.3

19.7

33.5

26.2

33.7

23.0

30

focus vesiculosus Laminaria saccharina Alaria esculenta

Green macro-lagae

Derbesia tenuissima Ulva ohnoi Chaetomorpha linum Cladophora coelothrix Cladophora vagabunda(fw) Oedogonnium sp. Enteromorpha prolifera

350 ºC, 15 min

350 ºC, 5 min

300 ºC, 30 min

Table 1. Literature review of reported researches on macro-algae HTL process.

23

(Neveux et al., 2014)

(Zhou et al., 2010)

Table 2. Characterization analysis of macro-algae feedstocks.

*

Biomass characterization

G. gracilis

C. glomerata

Moisture (wt %) Ash (wt %)

5.88 36

4.4 26.1

VM

53.1

44.8

FC

10.9

29.1

Lipid

1.7 ± 0.2

2.4 ± 0.15

Protein

13.7 ± 0.45

26.3 ± 0.36

Carbohydrate

28.6 ± 0.35

34.7 ± 0.4

C (wt %)

36.75

31.33

H (wt %)

5.86

4.99

N (wt %)

2.88

4.90

S (wt %)

1.99

1.99

O* (wt %)

17.51

30.67

HHV (MJ/Kg)

11.7

13.7

Calculated by difference: O=100-C-H-N-S-ash;

24

Table 3. Metal analysis of macro-algae using ICP-OES. Mineral metal content G. gracilis

C. glomerata

Al

4.9

23.1

Na

6.2

11.5

K

53.1

94.1

Mg

14.6

13.5

Fe

3.8

26.5

Ca

71.4

56.4

Cr

0.001

0.247

(g/Kg of the dry weight)

25

Table 4. Product yield for Gracilaria gracilis and Cladophora glomerata under HTL process. Species source

G. gracilis

C. glomerata

Biocrude yield (wt %)

15.7 ± 0.9

16.9 ± 1.0

Bio-char yield (wt %)

15.1 ± 2.2

15.0 ± 1.8

Aqueous + gas

69.2 ± 1.3

68.1 ± 0.8

26

Table 5. Ultimate analysis and energy recovery of the product, total organic carbon and total nitrogen concentrations of aqueous phase following HTL of the macro-algae.

product

TOC (mg/ L)

TN (mg/ L)

C.glomerata

2800

6400

G.gracilis

1750

2100

Macroalgae

HHV (MJ/ Kg)

C (wt %)

H (wt %)

N (wt %)

S (wt %)

O (wt %)

ER (%)

C.glomerata

70.38

8.42

4.02

2.02

12.34

33.06

48.3

G.gracilis

71.59

10.19

7.14

1.02

13.06

36.01

40.8

C.glomerata

35.39

5.18

5.53

2.27

51.63

13.1

14.3

G.gracilis

32.69

4.48

1.45

0.98

60.39

10.1

13.0

Biocrude

Biochar

Aqueous

27

Table 6. Tentative identities and area % of major peaks in total ion chromatograms for biooils produced from C.glomerata, G.gracilis. Source and

RT

C.glomerata

G.gracilis

Area (%)

Area (%)

Compound name pathway

(min)

7.73

-

2-cyclopenten-1-one

-

4.56

11.30

2- cyclopenton-1 one, 2- methyl-

-

2.81

13.17

2-Cyclopenten-1-one, 3-ethyl-2-hydroxy-

-

1.96

11.98

2, 3-dimethyl -2-cyclopenten-1-one

-

4.82

21.00

[1,1' -biphenyl]-4-ol

-

2.67

7.73%

19.62%

2.3

1.61

9.88

2-furancarboxaldehyde

9.87

Carbohydrate

Sum=

Millard reaction

8.12

Pyrazine

6.69

pyrazine, methyl

7.6

4.28

10.94

pyrzaine, 2,6-dimethyl

4.05

-

11.98

Acetylpyrazine

6.8

-

20.75%

5.89%

(Carbohydrate and protein)

Sum=

Protein

13.62

benzenamine, 4-methoxy

2.90

0.7

Lipid and

19.51

hydantoin, 1-butyl-

5.33

-

protein

14.68

Benzeneacetonitrile

-

2.14

5.33%

2.14%

Sum=

Lipid

28

15.09

Tetradecane

0.91

0.49

15.82

Pentadecane

0.54

-

16.61

Hexadecane

0.88

-

17.50

Heptadecane

0.79

-

18.56

Octadecane

0.51

-

19.88

Nonadecane

8.85

-

21.59

Eicosane

1.35

-

23.79

Heneicosane

5.25

-

19.61

1 -heptadecene

1.41

-

19.77

1- hexadcene

-

3.08

20.49%

3.57%

Sum=

24.15

11-octadecenoic, methyl ester

1.48

1.68

Lipid and

20.39

Hexadecanoic acid, methyl ester

1.05

1.19

Carbohydrate

21.52

dibutyl phthalate

-

6.95

16.89

Diisobutyl phthalate

-

9.68

2.53%

19.5%

Sum=

29

Figure Captions Fig. 1. The percentage of carbon and nitrogen accumulation in macro-algae HTL product under 350 ºC after 15 minutes. Fig. 2. The schematic of Six main suggested pathways identified for recognized compounds in biocrude. Fig. 3. Gaseous and aqueous, biocrude and biochar yields along HTL process without aqueous phase recycling (R0), after recycling aqueous phase (R2) and after recycling aqueous phase twice (R3) at 350 °C for 15 minutes.

30

Fig 1. The percentage of carbon and nitrogen accumulation in macro-algae HTL product under 350 ºC after 15 minutes.

31

Fig. 2. The schematic of Six main suggested pathways identified for recognized compounds in biocrude.

32

Fig. 3. Gaseous and aqueous, biocrude and biochar yields along HTL process without aqueous phase recycling (R0), after recycling aqueous phase (R2) and after recycling aqueous phase twice (R3) at 350 °C for 15 minutes.

33

Highlights •

Two Caspian Sea macroalgae were converted to biocrude by HTL conversion.

•

Red macroalgae has high biocrude HHV, 36 Mj/Kg.

•

The formation pathways of biocrude compounds were studied.

•

HTL aqueous residue recycle significantly increases biocrude yields.

34