Cultivation, characterization, and properties of

Environmental Science and Pollution Research https://doi.org/10.1007/s11356-018-2368-5

RESEARCH ARTICLE

Cultivation, characterization, and properties of Chlorella vulgaris microalgae with different lipid contents and effect on fast pyrolysis oil composition Ioannis-Dimosthenis Adamakis 1 & Polykarpos A. Lazaridis 2 & Evangelia Terzopoulou 1 & Stylianos Torofias 2 & Maria Valari 1 & Photeini Kalaitzi 1 & Vasilis Rousonikolos 1 & Dimitris Gkoutzikostas 1 & Anastasios Zouboulis 2 & Georgios Zalidis 1 & Konstantinos S. Triantafyllidis 2 Received: 13 November 2017 / Accepted: 22 May 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract A systematic study of the effect of nitrogen levels in the cultivation medium of Chlorella vulgaris microalgae grown in photobioreactor (PBR) on biomass productivity, biochemical and elemental composition, fatty acid profile, heating value (HHV), and composition of the algae-derived fast pyrolysis (bio-oil) is presented in this work. A relatively high biomass productivity and cell concentration (1.5 g of dry biomass per liter of cultivation medium and 120 × 106 cells/ml, respectively) were achieved after 30 h of cultivation under N-rich medium. On the other hand, the highest lipid content (ca. 36 wt.% on dry biomass) was obtained under N-depletion cultivation conditions. The medium and low N levels favored also the increased concentration of the saturated and mono-unsaturated C16:0 and C18:1(n-9) fatty acids (FA) in the lipid/oil fraction, thus providing a raw lipid feedstock that can be more efficiently converted to high-quality biodiesel or green diesel (via hydrotreatment). In terms of overall lipid productivity, taking in consideration both the biomass concentration in the medium and the content of lipids on dry biomass, the most effective system was the N-rich one. The thermal (non-catalytic) pyrolysis of Chlorella vulgaris microalgae produced a highly complex bio-oil composition, including fatty acids, phenolics, ethers, ketones, etc., as well as aromatics, alkanes, and nitrogen compounds (pyrroles and amides), originating from the lipid, protein, and carbohydrate fractions of the microalgae. However, the catalytic fast pyrolysis using a highly acidic ZSM-5 zeolite, afforded a bio-oil enriched in mono-aromatics (BTX), reducing at the same time significantly oxygenated compounds such as phenolics, acids, ethers, and ketones. These effects were even more pronounced in the catalytic fast pyrolysis of Chlorella vulgaris residual biomass (after extraction of lipids), thus showing for the first time the potential of transforming this low value by-product towards high added value platform chemicals. Keywords Microalgae . Chlorella vulgaris . Nitrogen-depleted cultivation . Lipids and residual biomass . Fast pyrolysis and catalytic fast pyrolysis . Aromatic hydrocarbons

Responsible editor: Gerald Thouand

Introduction

* Anastasios Zouboulis [email protected]

The intensive use of fossil fuels has contributed to climate change, environmental pollution, and related health issues. The partial replacement of conventional petroleum fuels with biofuels has been adopted over the last years as one of the measures to mitigate these problems. Biodiesel (fatty acid methyl esters, FAME) and HVO (hydrogenated vegetable oil, often called as Bgreen diesel^), both being substitutes of petroleum-derived transportation diesel, are currently produced from rapeseed oil (mainly), palm oil, used cooking oil, animal fats, and soybean oil (Flach et al. 2017). Microalgae have been recently considered as a promising,

* Georgios Zalidis [email protected] * Konstantinos S. Triantafyllidis [email protected] 1

Laboratory of Applied Soil Science, School of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece

2

Laboratory of Chemical and Environmental Technology, Department of Chemistry, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece

Environ Sci Pollut Res

alternative oil source because they can grow rapidly and convert solar energy to chemical energy by CO2 fixation (Chen et al. 2011; Mata et al. 2010; Morales-Sánchez et al. 2017). They do not compete with food or feed crops and can be produced in various, often polluted, water systems offering at the same time relatively high oil/lipid yield, thus making them an ideal source for biodiesel or HVO production (Chisti 2007; Lu et al. 2015; Luque 2010; Yang et al. 2016; Zhao et al. 2013). Still, a sustainable microalgae biorefinery concept would require the efficient utilization of the whole microalgae biomass via selective fractionation and conversion/use of its main components, i.e., lipids/oil for biodiesel/HVO, proteins, and other high-value compounds, i.e., carotenoids for food and health applications, and remaining carbohydrate-rich biomass for the production of additional valuable chemicals/fuels, i.e., H2, ethanol, methane, bio-oil, etc. (Dibenedetto et al. 2016; Ho et al. 2011; Mata et al. 2010; Pragya and Pandey 2016; Singh et al. 2011). In order to enhance microalgae growth and lipid accumulation, several strategies have been applied. Some examples are the optimization of the medium composition (e.g., the type of carbon source, vitamins, salts, and nutrients), the improvement of its physical parameters (e.g., pH, temperature, and light intensity), or the modification of microalgae metabolism (e.g., phototrophic, heterotrophic, mixotrophic, and photoheterotrophic growth) (Dibenedetto et al. 2016; Ho et al. 2010; Jiménez et al. 2003; Langley et al. 2012; Negi et al. 2016; Yeh and Chang 2012; You and Barnett 2004). The type of cultivation method, the energy sources (light or organic), and the carbon sources (inorganic or organic) used are known to significantly influence the growth and lipid content of several microalgae species (Chen et al. 2010; Přibyl et al. 2012; Talukder et al. 2012; Wang et al. 2007). With regard to lipid enhancement, various strategies have been applied, including combined nutrient and cultivation condition stress, microalgae–bacteria interactions, use of phytohormones EDTA and chemical additives, and improved light conditions using LED, dyes and paints, as well as gene expression and genetic and metabolic engineering studies (Singh et al. 2016). Nitrogen starvation has been recognized as an effective method for increasing lipid accumulation, although this may depend on the type of microalgae species. However, as Ndepleted growth media usually restrict total biomass productivity, various combined approaches have been proposed, such as a two-stage process, in which microalgae cells are first grown under nitrogen-rich conditions to stimulate biomass growth and are then transferred to nitrogen-depleted medium for increasing the production of lipids (Minhas et al. 2016; Singh et al. 2016). A continuous nitrogen-limitation (CNL) strategy was also proposed to enhance lipid accumulation in oleaginous Chlorella vulgaris, succeeding 1.35-fold higher lipid accumulation and similar biomass productivity with those obtained by the traditional batch nitrogen-starvation

strategy, thus indicating the potential of continuous largescale process (Liu et al. 2016). In addition to the effect on total lipid/biomass productivity, environmental stress conditions, including nitrogen starvation, influence also the production of other high-value components, such as triacylglycerols, carotenoids, and long-chain polyunsaturated fatty acids (LCPUFA) (Minhas et al. 2016; Shekh et al. 2016). In addition to microalgae fractionation and utilization of lipids, proteins, carotenoids, and other high-value components, the fast pyrolysis and catalytic fast pyrolysis of microalgal biomass have been also reported as a potential valorization process, leading to the production of crude biooil. The composition of microalgal bio-oil is different from that of a lignocellulosic biomass-derived bio-oil, and contains mainly aliphatics (alkanes), fatty acids, phenolics, aromatics, N-containing compounds (i.e., pyrroles, nitriles, and amides), alcohols, sugars, furans, and acetic acid (Babich et al. 2011; Chagas et al. 2016; Francavilla et al. 2015; ThangalazhyGopakumar et al. 2012). In some cases, as with heterotrophic Chlorella protothecoides, the bio-oil produced by fast pyrolysis had much better properties (i.e., lower oxygen content, higher heating value, lower density, and lower viscosity) compared to the bio-oil from the corresponding autotrophic cells as well as from wood (lignocellulosic biomass), being closer to the properties of fossil oil (Miao and Wu 2004). A much higher energy recovery efficiency (44%) was also determined for the fast pyrolysis of a fast growing low-lipid Chlorella algae compared to that calculated for biodiesel production (13%) (Babich et al. 2011). One disadvantage of algal biomass as a fast pyrolysis feedstock is its high nitrogen content, which can be transferred to the bio-oil as various nitrogen-containing compounds. Most of this nitrogen exists in proteins of fastgrowing, autotrophic microalgae (Becker 2007). Based on the developments of catalytic fast pyrolysis of lignocellulosic biomass mainly with acidic zeolites and various aluminosilicates that lead to the in situ deoxygenation of bio-oil and the formation of mono-aromatics, similar efforts have been recently published using microalgae feedstocks. Zeolites such as ZSM-5, H-Y, H-Beta, etc. (Chagas et al. 2016; Du et al. 2013; Thangalazhy-Gopakumar et al. 2012; Wang and Brown 2013), as well as basic catalytic materials, such as Na2CO3 (Babich et al. 2011), have been tested, leading to reduction of fatty acids and N-containing compounds and increase of mono- and di-aromatics (BTX, naphthalenes, etc.), alkanes, and phenolics as well as of ammonia and hydrogen cyanide in the gas products, depending mainly on the catalyst type and amount, pyrolysis temperature, and type of feedstock microalgae. In order to further increase the value and sustainability of the microalgae as a source of fuels and chemicals, the remaining biomass after extraction of lipids and proteins needs to be also valorized in an efficient way (Chisti 2007; Luque 2010). Its use as animal feed (Becker 2007) or for biogas production


via compost and digestion processes (Park and Li 2012) (Mussgnug et al. 2010) has been proposed. Fast pyrolysis of these biomass remnants has been shown to provide a bio-oil with various oxygenated compounds, i.e., aliphatics and fatty acids (originating from the remaining lipids), aromatics, pyrroles, phenolics, nitriles and amides (deriving from the remaining proteins) and sugars, furans, acetic acid, and aldehydes (deriving from the carbohydrate fraction) (Francavilla et al. 2015; Wang et al. 2013). Such an oxygenated bio-oil with a highly complex composition cannot be easily utilized and needs further upgrading. Thus, in analogy to the parent microalgae, the catalytic fast pyrolysis of microalgae remnants would constitute a promising option for further increasing the sustainability of microalgae biorefinery and has not been studied yet. In the present work, the cultivation of Chlorella vulgaris has been systematically studied in a FMT-150 photobioreactor (PBR), emphasizing the effect of nitrogen levels (low, moderate, high) of modified Bold’s media on biomass productivity, biochemical and elemental composition, fatty acid profile, and heating value (HHV) of the microalgae. Furthermore, the potential of valorizing the microalgae biomass residues, after extraction of lipids, via fast pyrolysis and especially catalytic fast pyrolysis, has been illustrated.

Materials and methods Algal strains Cultures of Chlorella vulgaris (initial cultures were commercially available, Algal Research and Supply Co.) were maintained in 25-mL clear plastic tissue culture flasks at 17 °C in a light∶dark cycle of 12∶12 h with a photon flux density of 48–55 μE m−2 s−1 in a Bold’s medium as previously described (Illman et al. 2000). In a typical cultivation procedure, 25 mL of aerated cultures (106 cells/ml) of the above Chlorella vulgaris strain was diluted in 1.2 L Bold’s medium and grown in aerated 2-L Schott bottles at 17 °C with a photon flux density of 60–70 μE m−2 s−1. The aeration was used both as a source of carbon as well as for agitating the algal cells in the culture. These cultures were allowed to grow for 7–10 days.

Operation of photobioreactor and production of Chlorella vulgaris samples under different nitrogen levels A conventional photobioreactor (FMT-150 PBR; Photon Systems Instruments, Drasov, Czech Republic) was used in this study, comprising of a 1-L glass-made vessel illuminated with an external light source (LED light source) mounted on both sides of the PBR. The light intensity used on the PBR was 150 μE m−2 s−1. The PBR (1 L working volume) was

inoculated with 400 mg L−1 of the precultured Chlorella vulgaris and was operated for up to 30 h, at 20 °C and pH 7.5 with an agitation rate of 300 rpm. The microalgae cell concentration and the pH of the medium were in situ determined in the PBR by means of UV analysis (560 nm, as described by the manufacturer) and mounted pH meter, respectively. High-nitrogen, typical Bold’s medium (Nichols and Bold 1965), 15 mM NO3−, cultures were developed and the respective Chlorella vulgaris samples produced were named as High Nitrogen – Low Lipids, HN-LL. Chlorella vulgaris cultures were also developed in two Bold’s media with lower nitrogen concentration, at 5 mM NO3− (the respective Chlorella vulgaris samples produced were named as Medium Nitrogen – Medium Lipids, MN-ML), and at 0.5 mM NO3− (N-depleted cultivation, respective Chlorella vulgaris samples were named as Low Nitrogen – High Lipids, LN-HL). The above two lower NO3− concentrations provided sufficient nitrogen for the cultures to grow within the time range of the experiments (i.e., up to 30 h). Microalgae growth under nitrate stress is linked to rise in pH (Liu et al. 2016); therefore, elevated aeration was applied in order to control pH changes through carbonic acid-bicarbonate buffering, providing at the same time carbon to avoid its limitation in the culture medium, as mentioned above. At the end of the cultivation period, the microalgae biomass was harvested by continuous centrifugation (9000 rpm for 10 min). All the experiments were conducted in three replicates, and data are presented as mean values with the corresponding standard error. Statistical analysis was conducted with the XLSTAT 2018 (New York, NY, USA) software.

Proximate and ultimate analysis, biochemical composition, and thermal decomposition properties of microalgae biomass The Chlorella vulgaris cell paste obtained after centrifuging of the cultures was washed twice with deionized water and oven dried at 80 °C for 48 h. The moisture content of the algal biomass equilibrated at ambient conditions was determined by drying tests of 100 mg biomass at 105 °C overnight. Ash determination was performed by a method similar to ASTM E1755-01 via gravimetric analysis by heating the biomass samples in a muffle furnace in air at 575 °C for 48 h. The total organic matter, which includes volatile and fixed carbon, was determined by subtracting the percentage of ash and moisture from 100. Higher heating values (HHV) were measured using an oxygen bomb calorimeter (Parr Instrument Company, USA). The ultimate (elemental) analysis of dried algal biomass samples was performed by a carbon/hydrogen/nitrogen elemental analyzer (LECO 628, USA). Oxygen content was calculated by difference.


The determination of the biochemical composition was based on Soxhlet extraction of lipids (D’Oca et al. 2011) and nitrogen content measurement by the Kjeldahl method. More specifically, 1.5 g of the dried microalgae biomass was placed in a Soxhlet extraction thimble and the solvent (90 mL of a mixture of chloroform and methanol (2:1, v/v)) was placed in the round-bottom flask. The continuous extraction process was performed for 24 h in evaporation/condensation/percolation cycles. The crude lipid was recovered by solvent evaporation in a rotary evaporator. Sonication of the recovered lipid/ oil in CHCl3 led to the separation of a solid fraction insoluble in CHCl3 that was isolated by filtration. Crude protein was determined by the Kjeldahl method and the conversion factor from nitrogen to protein was 5.8 (Gnaiger and Bitterlich 1984). The residual biomass, which is mainly composed of carbohydrates, was calculated by difference, by subtracting the percentages of protein, lipid, and ash from 100 (Waghmare et al. 2016). Thermogravimetric analysis (TGA, NETZSCH STA 449 F5 Jupiter) of the dried microalgae biomass samples was carried out using N2 as carrier gas (purity > 99.99 vol%) at a flow rate of 50 mL/min. The samples were heated from room temperature to 850 °C at heating rate of 10 °C/min.

Fatty acid composition profile The composition of the lipid/oil fraction and the selectivity to the various fatty acids were determined by GC-MS analysis of the corresponding fatty acid methyl esters (FAMEs). More specifically, the lipid/oil fraction recovered was treated with MeOH/H2SO4 (95:5 v/v) solution under reflux at 80 °C for 4 h, followed by addition of water and cooling to room temperature. The FAMEs produced were extracted with n-hexane and were analyzed by gas chromatography–mass spectroscopy (GC-MS, Agilent 7890A/5975C; electron energy 70 eV; emission 300 V; helium flow rate: 0.7 cm3 min−1; column: HP-5MS, 30 m × 0.25 mm, ID × 0.25 μm). The NIST05 mass spectral library was used for the identification of the compounds.

mass spectrometer (GCMS-QP2010, Shimadzu). Py/GC-MS systems are widely used for studying the fast pyrolysis (in the absence or presence of a catalyst) of various types of lignocellulosic biomass and more specifically for fundamental investigations of the effect of feed type, pyrolysis temperature, and catalyst properties on the composition of the produced bio-oil vapors (Chagas et al. 2016; Du et al. 2013; Gamliel et al. 2016). The interface temperature between the micropyrolyzer and GC was set to 300 °C and pyrolysis tests were conducted at 500 or 600 °C for 12 s. For the thermal fast pyrolysis experiments, approximately ~ 0.5 mg of dried algae or residue was loaded into stainless-steel cups which were automatically lowered into the preheated furnace. For the catalytic fast pyrolysis experiments, a commercially available NH4+-ZSM-5 zeolite catalyst (CBV8014 with SiO2/Al2O3 ratio of 80, Zeolyst, USA) was used. The catalyst was calcined at 550 °C for 3 h in a muffle furnace to produce the acidic H+form prior to its use. A mixture of 2 mg calcined catalyst and 0.5 mg dried algae or residue (mass ratio 4:1) was loaded in the cups for the respective catalytic fast pyrolysis experiments. Helium (99.999%) was employed as the carrier gas at a flow rate of 100 mL min−1 in the micropyrolyzer, with injector split ratio of 1:100 and 1 mL min−1 in the GC column. A capillary column was used (Ultra Alloy-5, Frontier Laboratories, Japan) with stationary phase consisting of 5% diphenyl and 95% dimethylpolysiloxane (30 m × 0.25 mm and 0.25 μm film thickness). The GC oven was programmed for 1-min hold at 50 °C followed by heating (10 °C min−1) up to 300 °C (held at this temperature for 4 min). The injector and detector temperatures were kept at 300 °C. The mass spectra were recorded in the range of 45 to 500 m z −1 with the scan speed of 5000 amu s−1. Identification of mass spectra peaks was achieved by the use of the scientific library NIST11s. The derived compounds were classified into the following nine groups: mono-aromatics (AR), aliphatics (ALI), phenols (PH), acids (AC), alcohols (AL), oxygenates (OXYG, including furans, aldehydes, ketones, and ethers), polycyclic aromatic hydrocarbons (PAHs), nitrogen compounds (NIT), and unidentified compounds (UN).

Pyrolysis experiments using the Py/GC-MS system

Results and discussion Both the parent dried Chlorella vulgaris microalgae samples cultivated at different N-levels as well as their residues after lipid extraction were studied in fast pyrolysis experiments. The residues were produced via extraction of dried microalgae (100 mg) by 10 mL of 1:2 (v/v) chloroform and methanol at 200 rpm for 30 min, followed by a second extraction with the same volume of chloroform. The residues of the above extraction procedure were used in the pyrolysis experiment. Fast pyrolysis experiments were performed on a MultiShot Micro-Pyrolyzer (EGA/PY-3030D, Frontier Laboratories, Japan) connected to a gas chromatographer–

Yield and composition of Chlorella vulgaris microalgae cultivated under different N-levels Chlorella vulgaris was grown under high, medium, and low nitrogen-containing modified Bold’s media and its biomass productivity (concentration of dry algal biomass in the cultivation broth) and cell density were monitored up to 30 h, as already described in the section BMaterials and methods^ (Fig. 1). In the low nitrogen LN-HL cultures, growth ceased at a relatively short cultivation time, with biomass


(a)

LN-HL

Cell density (106 x cell/ml)

140

MN-ML

HN-LL

120 100 80 60 40 20 0 0

(b)

10 15 Time (h)

LN-HL

1.6

Biomass concentration (g dry biomass/L)

5

20

MN-ML

25

30

HN-LL

1.4 1.2 1

0.8

0.6 0.4 0.2 0 0

5

10

15

20

25

30

Fig. 1 Growth characteristics (cell density and biomass yield) of Chlorella vulgaris microalgae under low (LN-HL), medium (MN-ML), and high (HN-LL) nitrate concentrations. The values are the average of three replicates; standard error is also indicated by the error bars

concentration and cell density reaching a plateau after 5 and 10 h of cultivation, respectively, in accordance with previously reported studies (Phukan et al. 2011). The biomass concentration and cell density of the HN-LL cultures were continuously increasing with time, reaching the highest values among the three Chlorella vulgaris samples, i.e., 1.5 g of dry biomass per liter of cultivation medium and 120 × 106 cells/mL−1, respectively. The observed slight decrease of cell density, still with increased biomass production, at the longest time tested (i.e., 30 h) could be attributed to changes in the relative rates of cell division (which is faster at the initial growth phase and leads to higher number of small cells) and cell mass/size increase (which is more pronounced at the stationary phase), as was also previously discussed (Chioccioli et al. 2014). The medium nitrogen system (sample MN-ML) exhibited an intermediate performance with the LN-HL and HN-LL samples with respect to the above two parameters. High cell density and biomass productivity cannot be obtained under nitrogen limitation since the content of chlorophyll under these conditions is negatively affected (Geider et al. 1993; Herrig and Falkowski 1989; Zhila et al. 2005), due to the decreased biosynthesis of proteins, such as RuBisCo (Beardall et al. 2005; Ikaran et al. 2015).

The proximate, ultimate, and biochemical analyses of the three samples of Chlorella vulgaris algae are shown in Table 1. In general, the main components of microalgae are proteins, lipids, and carbohydrates. Protein contents of the LN-HL, MN-ML, and HN-LL samples, grown in increasing nitrogen levels of the cultivation medium, were 24.09, 27.11, and 29.29 wt.%, respectively. The total lipid content followed the reverse trend and the sample HN-LL grown under high nitrogen conditions contained relatively low amount of lipids (16.6 wt.%). By decreasing the nitrogen levels, the lipid content increased substantially to 21.6 wt.% in the MN-ML sample and 36.6 wt.% in the LN-HL sample. The lipid content of sample LN-HL is among the higher values reported for the cultivation of Chlorella vulgaris under similar conditions. Furthermore, the increased content of lipids under nitrogen depletion has been mainly reported for longer cultivation periods of 1–10 days, compared to those applied in our study (i.e., 30 h max) (Granata 2017). The overall productivity of lipids was 0.18, 0.20, and 0.25 g of lipids per liter of cultivation medium for the LN-HL, MN-ML, and HN-LL samples, respectively, being estimated on the basis of the biomass concentration in the medium (g dry biomass/L, Fig. 1) and the content of lipids in the algal biomass (wt.% on dry biomass, Table 1). Based on these data, the N-rich medium may be the preferred option for a potential large-scale Chlorella vulgaris production. The ash contents of all three Chlorella vulgaris samples LN-HL, MN-ML, and HN-LL samples, grown in different nitrogen levels, were similar, i.e., 19.2, 18.6, and 18.4 wt.%, respectively. Ash in the microalgae biomass is due to the accumulation of inorganic salts in the cells from the culture media during the growth phase. The heating values (HHL) of the three microalgae samples range from 18.6 to 20.5 MJ/kg and it is higher with the high lipid content samples. With regard to the ultimate (elemental) analysis of the microalgae samples, a small but constant increase of C (43.4 to 47.6 wt.%) and H (6.4 to 6.7 wt.%) can be observed with decreasing nitrogen levels and consequently higher lipid content of the samples (Table 1). At the same time, N exhibits the reverse trend in accordance with the protein content of the samples. These results are in agreement with those of other related works in the literature and show that the various strains of microalgae biomass has carbon and hydrogen contents similar to those of lignocellulosic biomass; however, the nitrogen content of microalgal biomass is much higher (Francavilla et al. 2015; Wang and Brown 2013). In this study, the nitrogen content of the three Chlorella vulgaris samples produced was in the range of 5.8–6.7 wt.%. Most of the nitrogen in the algal biomass exists in the form of proteins. Other nitrogenous constituents of microalgae may include chlorophyll, nucleic acids, glucoseamides, and cell wall materials, all with relatively low levels of nitrogen compared to the levels in the proteins (Becker 2007).

Environ Sci Pollut Res Table 1 Proximate, ultimate, and biochemical analyses of Chlorella vulgaris samples

Chlorella vulgaris samples

LN-HL

Proximate analysis (wt.%; relative standard error ≤ 2%) Moisture

MN-ML

HN-LL

9.5

6.46

6.9

Ash

19.22

18.63

18.44

Organic matter (volatiles and fixed carbon) Higher heating value, dry basis (MJ kg−1)

71.28 20.49

74.91 19.70

74.66 18.63

Ultimate (elemental) analysis, dry basis (wt.%; relative standard error ≤ 2%) C H

47.61 6.72

46.66 6.58

43.39 6.41

N O

5.80 39.87

6.12 40.64

6.67 43.53

Biochemical composition, dry basis (wt.%; relative standard error ≤ 3%) Protein Lipids

24.09 36.60

27.11 21.67

29.29 16.60

Residue

23.72

32.12

33.40

LN-HL, low nitrogen – high lipids; MN-ML, medium nitrogen – medium lipids; HN-LL, high nitrogen – low lipids

Fatty acid distribution in the lipid/oil fraction The fatty acid (FA) profile of the lipid fraction of the three Chlorella vulgaris samples produced under different nitrogen levels was determined by GC-MS analysis of the respective fatty acid methyl esters (FAME), as described in BMaterials and methods.^ As can be seen from the data of Table 2, the major FAs present in all three Chlorella vulgaris samples were the C16 and C18 acids with none (aliphatic acids), one, or multiple double-bonds. The FA profiles of low and medium nitrate grown cells (i.e., samples LN-HL and MN-ML) were

Table 2 Fatty acid concentration (% GC-MS peak area) of the lipid/oil fraction of the Chlorella vulgaris samples grown under different nitrogen levels. The values are the average of three replicates with a relative standard error of ≤ 5% Chlorella vulgaris samples

HN-LL

MN-ML

LN-HL

Fatty acids 16:0 16:1 (n-9) 16:2 (n-6) 16:3 (n-3) 18:0 18:1 (n-9) 18:2 (n-6) 18:3 (n-3) Othersa

20.32 2.14 10.72 7.35 1.67 15.33 11.07 30.11 1.24

20.70 1.12 3.74 4.20 1.85 38.62 10.54 18.26 1.51

19.70 1.12 2.97 3.54 1.44 42.77 9.54 16.24 1.46

LN-HL, low nitrogen – high lipids; MN-ML, medium nitrogen – medium lipids; HN-LL, high nitrogen – low lipids a

Others: comprised fatty acids such as 14:0, 16:1(n-7), and 18:1(n-7) present at low concentrations

similar, particularly with regard to C16:0 and C18:1(n-9), and to polyunsaturated fatty acids (PUFAs), i.e., C18:2(n-6) and C18:3(n-3). Furthermore, the saturated and mono-unsaturated C16:0 and C18:1(n-9), which are preferred as feedstocks towards biofuels such as biodiesel and green diesel (produced via hydrotreatment of lipids), are more abundant in the medium and high lipid content samples (62–65% of total FAMEs) compared to the low lipid sample. The FAs developed in microalgae cells represent essentially a form of energy storage and carrier for further use and utilization (Rodolfi et al. 2009; Roessler 1990). The high nitrogen grown cells showed the reverse FA profile, with a lower percentage of saturated and mono-unsaturated FAs (39% of total FAMEs) and a higher percentage of PUFAs (60% of total FAMEs). This high PUFA content can negatively impact the cetane number and oxidation stability of fuels (Ramos et al. 2009) but can be utilized as food additives and in other related applications (Minhas et al. 2016). Similar effects of nitrate levels on FA profile have been observed by other researchers for Chlorella vulgaris (Miura et al. 1993) as well as for other microalgae species (Hu and Gao 2006; Li et al. 2008; Zhila et al. 2005). The thermal decomposition profile of the LN-HL, MNML, and HN-LL Chlorella vulgaris samples was investigated by TGA experiments and the respective TGA curves as well as their derivative (derivative thermogravimetric, DTG) curves are shown in Fig. 2. A stepwise weight loss can be observed, starting with 4–5% loss up to ca. 150 °C due to evaporation of humidity, followed by a steep decrease of weight (~ 60% loss) initiating at about 185–235 °C and ending at about 500 °C which corresponds to the decomposition of the organic matter of algae (volatiles). A third less pronounced step is observed between 600 and 850 °C which is probably attributed to the slow pyrolysis/gasification of carbonaceous


Fast pyrolysis of initial microalgae and residues (after lipid extraction)

Fig. 2 Thermal decomposition profiles (TGA, solid curves; and DTG, dashed curves) of the three Chlorella vulgaris microalgae samples (LNHL, MN-ML, and HN-LL) with different biochemical compositions; heating rate 10 °C/min, N2 atmosphere

material. No significant difference in the thermal decomposition profile could be observed among the three microalgae samples despite their different content of mainly lipids and residual biomass (Table 1), thus indicating that these two organic fractions, i.e., lipids and residual biomass, exhibit similar thermal stability and decomposition characteristics. Still, however, small differences between the samples can be identified on the basis of the relatively limited variation in the shape and maximum temperature of the DTG curves. Fig. 3 Composition of fast pyrolysis vapors (bio-oil) at 600 °C for Chlorella vulgaris microalgal biomass cultivated under different nitrogen levels (HN-LL, MN-ML, LN-HL) (a) and for their residues after lipid extraction (HN-LL-Res, MNML-Res, LN-HL-Res) (b). The values are the average of three replicate experiments; standard error is also indicated by the error bars

The thermal fast pyrolysis (Py-GC/MS) results at 600 °C for the three Chlorella vulgaris microalgal biomass samples with varying lipid, protein, and residue contents (LN-HL, HN-LL, MNML) and of the corresponding residues after lipid extraction are shown in Fig. 3, as percent peak area of the GC-MS spectra. The identified compounds of bio-oil vapors have been grouped in mono-aromatics (AR), aliphatics (ALI), phenols (PH), acids (AC), alcohols (AL), oxygenates (OXYG, including furans, aldehydes, ketones, and ethers), polycyclic aromatic hydrocarbons (PAHs), nitrogen compounds (NIT), and unidentified compounds (UN). It can be seen in Fig. 3a that the increase of lipid content from HN-LL to MN-ML to LN-HL (i.e., by reducing the available nitrogen in the cultivation medium) leads to decrease of mono-aromatics (12.3 to 8.6%) and increase of aliphatics (7.9 to 20.6%) and acids (25.4 to 33.1%), especially with the high lipid LN-HL sample. Nitrogen-containing compounds seem to be higher in the low lipid/high protein sample HN-LL (24%) compared to the medium and high lipid alga samples (10–15%), as expected on the basis of the biochemical analysis results (Table 1). Other oxygenates (furans, ketones, aldehydes, and ethers) are also in considerable relative concentration (ca. 12–27%) but do not exhibit a constant trend with the lipid/protein content of initial alga biomass, while alcohols and phenols are below ca. 7%.


The most representative (most abundant) compounds in the bio-oil vapors of the three Chlorella vulgaris microalgal biomass samples are shown in Fig. 4, i.e., toluene, ethyl benzene (AR), heptadecane, tetradecane (ALI), phenol, 2-methoxy-4vinylphenol (PH), acetic acid, n-hexadecanoic acid, cisvaccenic acid (AC), small alcohols such as 4-penten-1-ol, 3penten-1-ol (AL), oxygenated compounds, such as phytol-acetate, oleyl alcohol methyl ether, and nitrogen compounds, such as hexadecanamide (NIT). The obtained composition of the Chlorella vulgaris bio-oil samples shows the presence of the same groups of compounds reported in previous studies but of course at different relative concentrations, being dependent on the type of microalgal biomass and pyrolysis reactor and conditions (Babich et al. 2011; Chagas et al. 2016; Francavilla et al. 2015; Thangalazhy-Gopakumar et al. 2012). The fatty acids, i.e., n-hexadecanoic acid, cisvaccenic acid, and 9,12-octadecadienoic acid (Z,Z), found in the fast pyrolysis oil of the three Chlorella vulgaris microalgae samples of our study originate from the lipid fraction of algal biomass. Furthermore, the acetic acid could be derived from the decomposition of the fatty acids and not from the carbohydrate fraction, as relevant data on cellulose fast pyrolysis do not support the production of acetic acid (Stefanidis et al. 2014). The observed aromatics and phenolics may originate from the aromatic amino acids such as tyrosine and phenylalanine present in the microalgal proteins, pyrroles may derive from proline in the protein fraction, fatty amides can be

formed via reaction between the produced NH3 and fatty acids and aliphatics (alkanes) via removal of CO2 from fatty acids (Chagas et al. 2016; Wang et al. 2013). The respective composition data for the fast pyrolysis oil of the microalgae residues (or often called as residual biomass) after extraction of lipids (i.e., HN-LL-Res, MN-ML-Res, LNHL-Res) can be seen in Fig. 3b. If compared to the product distribution of the parent microalgae bio-oil (Fig. 3a), it can be seen that the acids and nitrogen compounds are less but still present while the concentrations of aliphatics, alcohols, and various oxygenates (i.e., furans, ketones, ether, and aldehydes) have been increased. These results are in accordance with the extraction of the lipid fraction, and part of the proteins, from the algal biomass, but also indicate their incomplete removal and their presence in the residues, as has also been reported in most of the related previous works on the thermal fast pyrolysis of algal residues (Francavilla et al. 2015; Wang et al. 2013). The most representative (most abundant) compounds are presented in Fig. 4 and comprise toluene, ethyl benzene, and benzene (AR), tetradecane, octadecane, and 1-hexene (ALI), 2-methoxy-4-vinylphenol (PH), acetic acid, nhexadecanoic acid (AC), small alcohols such as 4-penten-1ol, octacosanol (AL), oxygenated compounds, such as phytolacetate, 2-methylfuran (OXYG), and nitrogen compounds, such as ammonium acetate, 2-methyl-butanenitrile, etc. (NIT). The above data, as well as previously published results with Chlorella vulgaris or other microalgae strains, clearly

HN-LL, MN-ML & LN-HL

HN-LL-Res, MN-ML-Res & LN-HL-Res

Fig. 4 Representative (most abundant) compounds in fast pyrolysis oil (bio-oil) at 600 °C for Chlorella vulgaris microalgal biomass cultivated under different nitrogen levels (HN-LL, MN-ML, LN-HL) and for their residues after lipid extraction (HN-LL-Res, MN-ML-Res, LN-HL-Res)

Environ Sci Pollut Res Table 3 Most abundant compounds (GC-MS peak area, %) in the bio-oil produced from thermal and catalytic fast pyrolysis of Chlorella vulgaris sample MN-ML (moderate lipid content) and its residue (after lipid extraction) Categories/compounds

GC-MS peak area (%); relative standard error ≤ 8% MN-ML

MN-ML-Res

MN-ML+ZSM-5

MN-ML-Res+ZSM-5

Aromatics (AR) Toluene

8.63

6.41

13.78

Ethylbenzene Benzene

1.43

2.57 7.16

2.84 5.75

Octa-2,4,6-triene p-Xylene

0.11 9.64

o-Xylene

0.73

Styrene Benzene, propyl-

3.02 0.58

0.53

Benzene, 1-ethyl-2-methylBenzene, 1,2,3-trimethyl-

4.55 3.69

5.39 5.54

Benzene, cyclopropylBenzene, 2-ethyl-1,3-dimethylBenzene, 1,2,3,4-tetramethyl-

0.11 0.55 0.52

Benzene, (1-methylene-2-propenyl)Indane Indene Benzene, 1,2-diethylBenzene 1,3 dimethyl

1.32 2.13 3.3

15.91

1H-Indene, 2,3-dihydro-5-methyl1H-Indene, 2,3-dihydro-4-methyl1H-Indene, 2,3-dihydro-4,7-dimethyl1H-Indene, 1-methylAzulene 1H-Indene, 1,3-dimethylIndole Aliphatics (ALI) cis-4-Nonene

1.09 2.74 1.97

0.77

0.74

1.00 2.83 0.67 2.19

1.14 0.6 0.61 0.68

trans--4-Nonene 2-Butene, (E)1-Nonene Cyclopentene, 4-methyl1,3,5,7-Cyclooctatetraene Cyclopropane, 1-methyl-1-isopropenylD-Limonene 1,3,5,7-Cyclooctatetraene 3-Hexadecene, (Z)-

0.23

1-Tridecene 1-Pentadecene Pentadecane Heptadecane 1-Pentadecene 1-Tetradecene 2-Hexadecene, 3,7,11,15-tetramethyl-, [R-[R*,R*-(E)]]3,7,11-Trimethyl-2,4-dodecadiene 3-Vinyl-1-cyclobutene

0.34 0.36 0.28 1.07 0.79 0.21 2.04 0.47

1.73 0.54 0.69 1.57 1.11 0.98 1.52 0.27

1.36 2.87

Environ Sci Pollut Res Table 3 (continued) Categories/compounds


MN-ML-Res

Cyclobutane, 1,1-dimethyl-3-methylene1,3-Cyclopentadiene, 5,5-dimethyl2.16

Heneicosane

7.8 1.42

Eicosane Phenols (PH)

1.23 3.96

Oleyl alcohol, methyl ether

5.62

Furan, 2-methylButanal, 3-methyl-

2.2 1.27

Butanal, 2-methylPhytol, acetate

2.43 14.35

Tridecanal Glutaraldehyde Benzofuran Benzofuran, 4,7-dimethylAlcohols (AL) 4-Penten-1-ol 1,3-Cyclobutanediol, 2,2,4,4-tetramethylCyclohexanol, 4-methyl1-Heptacosanol 4-Penten-1-ol 1-Butanol, 3-methylE-6-Octadecen-1-ol acetate 3,7,11,15-Tetramethyl-2-hexadecen-1-ol Phytol

15.95

1.03

19.71

0.12

0.42 1.16 0.89 0.25 4.45 2.2 0.51 0.21 4.45 14.81 0.21 8.95 0.52

Polycyclic aromatic hydrocarbons (PAHs) Naphthalene, 1-methylNaphthalene, 1,3-dimethylNaphthalene, 1,2-dihydroNaphthalene Naphthalene, 1,2-dihydro-3-methylNaphthalene, 2-methylNaphthalene, 2,6-dimethylNitrogen-containing compounds(NIT) 5-Nonylamine Butanenitrile, 2-methylPyridine Indole 1-Dimethylaminohexane Tetradecanenitrile Pentanenitrile Butanenitrile, 2-methylHexadecanenitrile Methylamine, N,N-dimethyl-

MN-ML-Res+ZSM-5

3.43 0.36

1,3,5-Cycloheptatriene Squalene

2-Methoxy-4-vinylphenol Oxygenates (OXYG)

MN-ML+ZSM-5

1.72 0.84 0.92 2.06 1.9 4.38 2.37 2.1 0.33 0.25 1.06 2.73 0.9 5.26 0.79 1.15 6.5

Environ Sci Pollut Res Table 3 (continued) Categories/compounds


Ethylamine Acids (AC) Acetic acid Hexadecanoic acid, methyl ester 9-Hexadecenoic acid n-Hexadecanoic acid Octadecanoic acid, 2-propenyl ester Gamolenic Acid 9,12-Octadecadienoic acid (Z,Z)-

MN-ML-Res

MN-ML+ZSM-5

MN-ML-Res+ZSM-5

3.58 5.86 0.3

24.48

5.36

1.31 12.69 0.36

5.71

0.34 1.4

9,12,15-Octadecatrienoic acid, (Z,Z,Z)-

show that the bio-oil produced by thermal fast pyrolysis of the parent microalgae or even the lipid-extracted residue, is a complex crude oil that cannot be utilized directly as fuel (due to the high content of oxygenated and nitrogencontaining compounds) while it is equally difficult to selectively isolate/extract groups of high-value chemicals, i.e., phenols, aliphatics, etc. The catalytic fast pyrolysis, as a method of in situ deoxygenation and in some cases aromatization of the bio-oil, has been already suggested and investigated with raw microalgae biomass, as described in BIntroduction.^ In the following section, we further describe the effect of catalyst use in the fast pyrolysis oil derived from the microalgae residual biomass, after extraction of the lipid fraction.

Catalytic fast pyrolysis of initial microalgae and residual biomass The catalytic fast pyrolysis of lignocellulosic biomass for the in situ upgrading of bio-oil has been extensively described using mainly acidic zeolites and various aluminosilicates (Mihalcik et al. 2011; Stephanidis et al. 2011; Wang et al. 2014). Similar studies have been recently published using various microalgae as feedstock (Babich et al. 2011; Chagas et al. 2016; Du et al. 2013; Wang and Brown 2013), leading to reduction of fatty acids and N-containing compounds and increase of mono- and di-aromatics (BTX, naphthalenes, etc.), alkanes, and phenolics in the produced bio-oils. In the present work, we selected the Chlorella vulgaris sample grown in medium nitrogen level, leading to medium lipid content (sample MN-ML), as well as its residue after lipid extraction, as feedstock for the catalytic fast pyrolysis experiments at 500 °C, in comparison to the thermal (non-catalytic) experiments at the same temperature. The most abundant compounds in the bio-oil vapors are presented in Table 3. The major compounds in the thermal fast pyrolysis oil of the parent MN-ML sample were toluene and ethylbenzene (mono-

6.13

aromatics, AR), 2-hexadecene, 3,7,11,15-tetramethyl-, 1,3,5,7-cyclooctatetraene (aliphatics, ALI), 2-methoxy-4vinylphenol (phenolics, PH), phytol-acetate, 2-methylfuran, and oleyl alcohol methyl ether (oxygenates, OXYG), 4penten-1-ol (alcohols, AL), indole (nitrogen compounds, NIT), acetic acid, and n-hexadecanoic acid (acids, AC). In the case of the residue (MN-ML-Res)-derived thermal biooil, the most representative compounds were squalene and heneicosane (aliphatics, ALI), 2-methoxy-4-vinylphenol (phenolics, PH), phytol-acetate (oxygenates, OXYG), 3,7,11,15-tetramethyl-2-hexadecen-1-ol (alcohols, AL), methylamine, N,N-dimethyl- (nitrogen compounds, NIT), acetic acid, and n-hexadecanoic acid (acids, AC). The catalyst used in the catalytic fast pyrolysis experiments was a typical relatively strongly acidic ZSM-5 zeolite, with the following characteristics: Si/Al = 40, specific surface area 437 m2/g, total acid sites 0.27 mmoles NH3/g (measured by TPD-NH3), and Brönsted/Lewis acid sites mol ratio of 6.6 (determined by FTIR-pyridine measurements). The unique features of ZSM-5, such as medium size (~ 0.55 nm) tubular pores with larger size intersections (~ 9 nm) and strong Brønsted acidity, make it ideal for the deep deoxygenation of the intermediate thermal pyrolysis oxygenated products (vapors) followed by cracking towards smaller C2-C3 alkenes which can then be converted to mono-aromatics on the acid sites of ZSM-5. As a result, ZSM-5 is the catalyst of choice when a highly aromatic bio-oil is targeted at least in fast pyrolysis of lignocellulosic biomass (Mihalcik et al. 2011). The composition of catalytic bio-oil derived from the initial MN-ML microalgae is presented in Fig. 5, in the form of groups of compounds (i.e., aromatics, aliphatics, phenolics, etc.), while the most representative individual compounds are shown in Table 3 and Fig. 6. A significant increase of mono-aromatics from 10% in the thermal bio-oil to 49% in the catalytic bio-oil has occurred, with p-xylene, benzene, and toluene being the most abundant compounds. Aliphatics (i.e.,


Fig. 5 Composition of fast pyrolysis vapors (bio-oil) at 500 °C of Chlorella vulgaris microalgal biomass cultivated under moderate nitrogen levels (MN-ML) of its residue after lipid extraction (MN-ML-

Res) and by the use of zeolite ZSM-5 (Si/Al = 40) as catalyst (MN-ML+ ZSM-5 and MN-ML-Res+ZSM-5). The values are the average of three replicates experiments; standard error is also indicated by the error bars

1,3,5-cycloheptatriene and 1,1-dimethyl-3-methylenecyclobutane) and alcohols (i.e., 3-methyl-1-butanol) were also increased to a lesser extent while phenolics, acids, and oxygenates (i.e., ketones, ethers, furans) were almost completely eliminated. Nitrogen compounds were also reduced from 10 to 7% (i.e., hexadecanenitrile and pentanenitrile). A low concentration of polycyclic aromatic hydrocarbons (PAHs) such as 1-methyl-naphthalene was also detected. The above product distribution in the catalytic bio-oil and the differences from the thermal (non-catalytic) bio-oil are in accordance with previous related studies on catalytic pyrolysis of various types of

microalgae biomass and verify the expected performance of ZSM-5 as observed in lignocellulosic biomass pyrolysis. As discussed above, there is only a limited number of studies in the literature that deal with the thermal (non-catalytic) fast pyrolysis of microalgae residues, i.e., the biomass left after extraction of lipids (Francavilla et al. 2015; Wang et al. 2013) but none focusing on their catalytic fast pyrolysis. The composition data for the bio-oil produced from the catalytic fast pyrolysis of Chlorella vulgaris residue by the use of ZSM-5 are shown in Figs. 5 and 6, and Table 3. The effects of ZSM-5 observed in the bio-oil of the initial microalgae were also

MN-ML & MN-ML-Res

MN-ML & MN-ML-Res using ZSM-5

Fig. 6 Representative (most abundant) compounds in fast pyrolysis oil (bio-oil) at 500 °C of Chlorella vulgaris microalgal biomass cultivated under moderate nitrogen levels (MN-ML) of its residue after lipid

extraction (MN-ML-Res) and with the use of zeolite ZSM-5 (Si/Al = 40) as catalyst (MN-ML+ZSM-5 and MN-ML-Res+ZSM-5)


detected here and were even more pronounced. The monoaromatics concentration reached the high value of 67%, being negligible in the thermal bio-oil of the residue. Most of the oxygenated compounds, i.e., phenolics, alcohols, ketones, etc., were eliminated while the nitrogen-containing compounds were also significantly reduced. As a consequence of the high mono-aromatics formation and their condensation on ZSM-5, PAHs (mainly naphthalenes) were also slightly increased in the catalytic bio-oil. The most representative (most abundant) compounds in the catalytic bio-oil vapors of the Chlorella vulgaris residue comprise 1,3-dimethylbenzene (15.9%), toluene (13.8%), benzene (5.6%), 1-ethyl-2-methylbenzene (5.4%), and 1,2,3-trimethylbenzene (5.5%) as mono-aromatics (AR), 2-butene, and 1,3,5,7-cyclooctatetraene (ALI), glutaraldehyde and benzofuran (OXYG), acetic acid (AC), 1dimethylaminohexane (NIT), 2-methylnaphthalene, and 2,6dimethylnaphthalene (PAHs). From the above data, it becomes evident that ZSM-5 favors deoxygenation and denitrogenation, cracking, and aromatization, as well as condensation reactions, in the catalytic fast pyrolysis of microalgae residues. By optimizing further the process parameters, such as increasing the catalyst to biomass ratio and/or increasing the pyrolysis temperature to ca. 550 °C, a deeper deoxygenation of the bio-oil with concomitant enhanced production of aromatics may be achieved. Thus, the complexity of the thermal (non-catalytic) bio-oil composition is avoided and the produced catalytic biooil can serve as a source of high added value bio-aromatics to be utilized in the chemical and polymer industry.

Conclusions The results of this study showed that Chlorella vulgaris cell density and biomass productivity augmented with increased nitrogen levels of the cultivation medium. The higher nitrogen levels lead to higher protein and lower lipid content, while the highest lipid content (36 wt.% on dry biomass) was achieved in N-depleted conditions at a relatively short cultivation time (i.e., 30 h). Still, the highest overall lipid productivity, taking in consideration both the biomass concentration in the cultivation medium and the content of lipids on dry biomass, was achieved by the N-rich system. It was also shown that heating value (HHV) was favored by the higher lipid content. With regard to the elemental composition of the microalgae, an increasing content of C and H was observed in the Chlorella vulgaris samples with increasing lipid content while the reverse trend was found for N which was favored by the higher protein content, as expected. With regard to the fatty acids selectivity in the lipid fraction, it was shown that the medium and low nitrogen media favor the formation of saturated and mono-unsaturated FAs (C16:0 and C18:1(n-9)), while the high nitrogen system induced higher concentration of PUFAs.

The increase of lipid content in the Chlorella vulgaris microalgae by reducing the nitrogen levels in the cultivation medium induces a decrease of mono-aromatics and increase of aliphatics and acids in the composition of bio-oil produced via the thermal (non-catalytic) fast pyrolysis of the parent microalgae. On the other hand, the nitrogen-containing compounds are higher in the bio-oil derived from the high protein microalgae sample that was grown under high nitrogen levels. Despite the clearly observed effects of the biochemical composition of microalgae on the bio-oil composition, it is clear that such types of bio-oils comprising of a wide variety of oxygenated compounds, i.e., fatty acids, phenolics, ethers, ketones, etc., as well as aromatics, aliphatics, and nitrogen compounds (pyrroles and amides), cannot be easily utilized directly as fuel or source of specific high-value chemicals and its downstream upgrading might be costly and ineffective. Based on the more mature process of catalytic fast pyrolysis of lignocellulosic biomass, the use of a strongly acidic ZSM-5 zeolite favored the deoxygenation and aromatization reactions of the intermediate oxygenated compounds present in the microalgae fast pyrolysis vapors towards valuable BTX aromatics, eliminating at the same time phenolics, acids, and various other oxygenates, such as ethers and ketones. These effects of ZSM-5 were even more pronounced in the case of catalytic fast pyrolysis of the microalgae residue leading to the production of a highly aromatic bio-oil with minimum concentration of oxygenates and nitrogen compounds. The potential of upgrading the microalgae residue (after lipid extraction) via catalytic fast pyrolysis may increase significantly the value of the Bwhole^ microalgae biomass valorization chain in the concept of (bio)refining. Funding information This research was funded by ENERGEIA project: a Strategic Co-Funded Project of the European Territorial Cooperation Programme Greece-Bulgaria 2007–2013.

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