Enhanced thermal destruction of toxic microalgal biomass by using CO2

Science of the Total Environment 566–567 (2016) 575–583

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Enhanced thermal destruction of toxic microalgal biomass by using CO2 Jong-Min Jung a, Jechan Lee a, Jieun Kim a, Ki-Hyun Kim b, Hyung-Wook Kim c, Young Jae Jeon d,⁎, Eilhann E. Kwon a,⁎ a

Department of Environment and Energy, Sejong University, Seoul 05006, Republic of Korea Department of Civil and Environmental Engineering, Hanyang University, Seoul 04763, Republic of Korea Department of Biological Science and Technology, Sejong University, Seoul 05006, Republic of Korea d Department of Microbiology, Pukyong National University, Busan 48513, Republic of Korea b c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Identification of dominant microalgal strains as M. aeruginosa was carried out. • Enhanced thermal destruction induced by CO2 • Direct gas phase reaction between CO2 and volatile organic carbons (VOCs) • Significant tar reduction in the presence of CO2

a r t i c l e

i n f o

Article history: Received 26 January 2016 Received in revised form 16 May 2016 Accepted 22 May 2016 Available online xxxx Editor: Simon Pollard Keywords: Microalgae Microcystis aeruginosa (M. aeruginosa) Waste disposal Thermo-chemical treatment CO2 Energy recovery

a b s t r a c t This work confirmed that dominant microalgal strain in the eutrophic site (the Han River in Korea) was Microcystis aeruginosa (M. aeruginosa) secreting toxins. Collected and dried microalgal biomass had an offensive odor due to microalgal lipid, of which the content reached up to 2 ± 0.2 wt.% of microalgal biomass (dry basis). This study has validated that the offensive odor is attributed to the C3–6 range of volatile fatty acids (VFAs), which was experimentally identified by the non-catalytic transformation of triglycerides (TGs) and free fatty acids (FFAs) in microalgal biomass into fatty acid methyl esters (FAMEs). In particular, this study mechanistically investigated the influence of CO2 in the thermal destruction (i.e., pyrolysis) of hazardous microalgal biomass in order to achieve dual purposes (i.e., thermal disposal of hazardous microalgal biomass and energy recovery). The influence of CO2 in pyrolysis of microalgal biomass was identified as 1) the enhanced thermal cracking behaviors of volatile organic compounds (VOCs) from the thermal degradation of microalgal biomass and 2) the direct gas phase reaction between CO2 and VOCs. These identified influences of CO2 in pyrolysis of microalgal biomass significantly enhanced the generation of CO: the enhanced generation of CO in the presence of CO2 was 590% at 660 °C, 1260% at 690 °C, and 3200% at 720 °C. In addition, two identified influences of CO2 (i.e., enhanced thermal cracking and direct gas phase reaction) occurred simultaneously and independently. The identified gas phase reaction in the presence of CO2 was only initiated at temperatures higher than 500 °C, which was different from the Boudouard reaction. Lastly, the experimental work justified that exploiting CO2 as a reaction medium and/or chemical feedstock will provide new technical approaches for controlling syngas ratio and in-situ air pollutant control without using catalysts. © 2016 Elsevier B.V. All rights reserved.

⁎ Corresponding authors. E-mail addresses: [email protected] (Y.J. Jeon), [email protected] (E.E. Kwon).

http://dx.doi.org/10.1016/j.scitotenv.2016.05.161 0048-9697/© 2016 Elsevier B.V. All rights reserved.

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1. Introduction A massive South Korean project to dam and dredge four major rivers was completed in 2012 as part of the “Green New Deal” policy in Korea. More than 929 km of streams in Korea were restored as part of the project, with a follow-operation planned to restore more than 10,000 km of local streams (Normile, 2010). More than 35 riparian wetlands were also reconstructed (Normile, 2010). This massive project has not only enhanced the national water supply security, but also has been evaluated positively for contributing to national economic aspects. However, the unexpected and/or unprecedented algal blooms in four major rivers have been occurring consecutively since 2009. Among microalgal strains, cyanobacteria such as Microcystis aeruginosa (M. aeruginosa) (Harding et al., 2014; Kemka et al., 2006; Lone et al., 2015) were identified as dominant strains in the Korean fresh water system and a substantial amount of microcystin was sporadically detected at the level of 0.5–1.2 μg L−1. Microcystin-LR (MC-LR) that is secreted from M. aeruginosa is known to be hepatotoxic and cause neural toxicity (Bhattacharya et al., 1996; Ekvall et al., 2014a; Ekvall et al., 2014b; Lone et al., 2015; Pitois et al., 2014; Shen et al., 2003; Yoshida et al., 1997). Chronic oral administration of MC-LR to rats was reported to induce the impairment of spatial learning and memory, inflammatory response by astrocyte activation, and liver dysfunction (Li et al., 2014a; Li et al., 2014b; Yoshida et al., 1997). Furthermore, numerous studies reported that inevitable environmental exposure to cyanobacterial MC-LR could result in Alzheimer's disease-related neuroinflammation, tauopathy, oxidative stress, apoptosis and microglial activation (Li et al., 2014b; Sun et al., 2011). Underlying mechanisms are still unclear but a great deal of data suggested that the involvement of impaired lipid metabolizing enzymes, protein phosphatase, increased reactive oxygen species (ROS), imbalanced pro-/anti-inflammatory factors (Bieczynski et al., 2014; Bury et al., 1998; Jaumot and Hancock, 2001; Katsuyama and Morgan, 1993; Li et al., 2012; Vesterkvist and Meriluoto, 2003). Moreover, some studies reported that microcystin exposure could lead to growth inhibition, immune dysfunction and imbalanced neuroendocrine system via the altered expression of related-genes (Lankoff et al., 2004; Liu et al., 2014; Rymuszka, 2013; Yea et al., 2001). Therefore, in case of microalgal blooms, microalgae from four major rivers in Korea was collected and disposed for the purpose of hygiene. However, the appropriate disposal techniques for the collected microalgal biomass have not been fully established since the recently identified algal blooms were unprecedented. Among various disposal options (i.e., anaerobic digestion, landfilling, and so on) (Lin et al., 2007; Passos et al., 2014; Passos et al., 2015) for collected microalgal biomass, the thermo-chemical process (i.e., incineration, pyrolysis, and gasification) (Conesa and Domene, 2015; Liu and Ma, 2008; Yakaboylu et al., 2015) would be the most feasible option due to significant volume reduction and thermal destruction of toxic materials such as MC-LR. Furthermore, energy recovery via the thermo-chemical treatment of microalgal biomass is possible. Despite these positive advantages stated above, the thermo-chemical process has been known to be an energy intensive process and energy production via the thermo-chemical process has been known as being suitable only for mass production (Conesa and Domene, 2015; Liu and Ma, 2008). Thus, exploiting microalgal biomass as an initial feedstock for the thermo-chemical process could not be an appropriate option due to its seasonal occurrence and insufficient amount as an initial feedstock for the thermo-chemical process. Its compatibility for treating microalgal biomass with the existing infrastructure of thermo-chemical process must be considered. Moreover, establishing environmentally benign process would be preferable. However, as compared to the conventional fuels such as coal, previous works done by other authors associated the thermo-chemical process of microalgal biomass was quite limited (Ceylan and Kazan, 2015; Conesa and Domene, 2015; Kim et al., 2015; Lopez-Gonzalez et al., 2014; Sharara et al., 2014; Wang et al., 2015a; Wang et al., 2015b).

Thus, this study was oriented to investigate the thermo-chemical process of microalgal biomass to achieve the dual purposes associated with hazardous waste disposal and energy recovery. In order to avoid the complexities arising from various reactions in the gasification process, this work fundamentally investigated the thermal degradation of microalgal biomass (i.e., pyrolysis) since pyrolysis has been known to be the critical intermediate step for the gasification process. Thus, our work was done to mechanistically explore the influence of CO2 as reaction medium and/or feedstock in pyrolysis of microalgal biomass to maximize the thermal efficiency and to achieve the environmentally benign process. The mechanistic understandings of CO2 in pyrolysis of microalgal biomass will be a milestone for the utilization of CO2 in the gasification. In this regard, this work intentionally limited the scope within the influence of CO2 in the pyrolysis process. 2. Material & methods 2.1. Sample collection and DNA extraction The samples were collected from the Han River in Korea with seasonal blooms on various spot in the middle of summer (July 2015). Microalgal biomass centrifuged at 20,000 rpm for 20 min. The collected samples were kept on −20 °C until the further analysis. The method for DNA extraction used have been described in Saker et al. (2005), 10 mg of wet sample was used. The samples were combined with 500 mL of XS buffer (1% potassium–methylxanthogenate; 800 mM ammonium acetate; 20 mM EDTA; 1% SDS; 100 mM Tris–HCl, pH 7.4) and incubated at 65 °C for 2 h (vortexed after 1 h). The solutions were then placed on ice for 10 min, and centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was harvested, and the DNA was precipitated by adding 1 volume of isopropanol and 1/10 volume of 4 M KOAc for 15 min at 4 °C followed by a centrifugation step at 12,000 rpm for 20 min. The collected DNA was washed with 70% ethanol, centrifuged at 12,000 rpm for 15 min, dried, and resuspended in 50 μL of milli-Q water. 2.2. Polymerase chain reaction (PCR) and sequencing analysis 16S rDNA amplification was performed in 50 μL reactions using primers previously reported (Saker et al., 2005), 27F (5′ 5′-AGA GTTTGATCCTGGCTCAG-3′) and 809R (5′-GCTTCGGCACGGCTCGG GTCGATA-3′) with an initial denaturation step at 92 °C for 5 min followed by 35 cycles of 94 °C for 10 s, 60 °C for 20 s and 72 °C for 5 min. Hepatotoxin (Hep)-PCR reactions were performed using primers previously reported for the detection of mycE encoding aminotransferase involved in microcystin synthesis (Saker et al., 2005), HepF (5′-TTTGG GGTTAACTTTTTTGGCCATAGTC-′3) and HepR (5′-AATTCTTGAGGC TGTAAATCGGGTTT-′3). The initial denaturation step at 92 °C for 2 min was followed by 35 cycles of denaturation at 92 °C for 20 s, annealing at 52 °C for 30 s, extension at 72 °C for 1 min and a final extension step at 72 °C for 5 min. For the identification of eukaryotic microorganisms in the sample, Internal Transcribed Spacer (ITS) region using the ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTT ATTGATATGC-3′) primers were used, which have been reported elsewhere (Preetha et al., 2012). All PCR reactions were performed using 0.2 units of Tag polymerase (Blend Taq, Toyobo, Japan) in a 50 μL reaction volume containing 2.5 mM MgCl2, 1 × Taq polymerase buffer (Toyobo), 0.2 mM of dNTPs and 0.5 pM of forward and reverse primers. For all PCR reactions, 1 ng of chromosomal DNA was used. The amplified PCR products from the fresh water bloom of 16S rDNA, ITS region and the region encoding for microcystin synthase were respectively created for clone library using RBC T/A cloning kit according to the manufacturer's manual. To perform this, the HIT competent cells of Escherichia coli DH5α purchased from RBC was used and the E. coli transformations were carried out according to manufacturer's instructions. M13F and M13R primers were used to screen the clone library and sequence the individual clone library. To identify the

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microorganism responsible for fresh water bloom, all PCR products were analyzed on 1% agarose gels with 1 × TAE buffer and visualized with ethidium bromide (1 μg mL−1) for 10 min. Sequence data were analyzed using the BioEdit program. The identities of the sequenced amplicons were determined using a BLAST search on GenBank. All sequences were checked manually with regard to quality of the data. 2.3. Sample preparation for the thermo-chemical treatment Collected microalgal biomass centrifuged at 8000 rpm for 10 min and microalgal biomass was dried with a freeze dryer (TFD series, Seoul, Korea) at − 80 °C and less than ~ 1 Torr for 48 h. The dried microalgal biomass was stored at 20 °C in the desiccator filled with silica gel. 2.4. Non-catalytic derivatization of triglycerides into FAMEs Organic solvents (e.g., n-hexane) were purchased from Sigma Aldrich (St. Louis, USA) and were used for solvent extraction with the Soxhlet device equipped with a reflux condenser. The extraction temperatures were varied on the boiling point (Tb) of the organic solvent, but temperature was adjusted at Tb + 5 °C. Microalgal biomass was placed in the Soxhlet unit and then solvent extraction was conducted for 10 h. Then, the organic solvent was recovered via using the rotary evaporator (Cole palmer, USA). A bulkhead union (6.35 mm (0.25 in) - part no.: 2507-400-61 (Swagelok (USA)) was used for a tubular reactor (TR) in our experimental work. The volumetric ratio of triglycerides (TGs) to MeOH was 1:2. First, one side of the bulkhead was sealed with the Swagelok fitting, and then 0.18 g of porous materials (silica, 60 Å) was inserted. Then, 200 μL mixture of the extracted solution from TGs and MeOH was carefully inserted into the bulkhead, and then 0.18 g of porous silica was inserted again. The other side of the bulkhead was sealed with Swagelok fitting, and the bulkhead was placed in the muffle furnace. After that, the sealed bulkhead was chilled with 4 °C of water. In order to ensure reproducibility, the experimental work based on each test matrix was conducted in triplicate. The converted FAMEs were collected with organic solvent (i.e., dichloromethane). 2.5. Thermo-gravimetric analysis (TGA)

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was used for the analytic measurement. The temperature programming of GC was set to run isothermally (60 °C for 1 min, 20 °C min−1), to ramp (up to 240 °C), and to run isothermally (240 °C for 50 min) for a total elapsed time of 60 min. Multiple calibrations of FAME were conducted with FAME STD (37 FAME MIX, CRM47885, Sigma Aldrich, St. Louis, USA). The effluent of TGA unit was sent to a GC/MS (Agilent 9890/4973) or micro-GC (Agilent 3000A) for identification and quantification of the chemical species from the TGA unit. The lag time of the sample from the TGA unit to the injection block, located on the GC/MS, was calculated to be less than 1 s, based on a 3 mL volume transfer line. The sampling system was maintained at greater than 300 °C to mitigate condensation and/or adsorption of chemical species onto the system surface (Kwon and Castaldi, 2008). The GC was equipped with a capillary column (0.25 mm × 30 m HP-5MS), which was directly interfaced to a quadrupole mass spectrometer. Identification of the species was accomplished by matching the gas chromatographic retention times to those of the pure components and the mass spectral fragmentation patterns to species found in standard MS libraries.

3. Results & discussion 3.1. Microorganism composition analysis and toxigenicity test by PCR To identify the potential microorganism populations in the collected microalgal samples, the PCRs were conducted using the cyanobacteriaspecific 16S rDNA, Hep, and ITS primers respectively on the total DNA extracted from the microalgal samples. As positive controls for the 16S rDNA and Hep PCRs, to determine whether the potential harmful microalgae were present in the collected microalgal samples, the genomic DNA of M. aeruginosa PCC 7806, the known microcystin producer (Stefanelli et al., 2014) was used. The results are illustrated in Fig. 1. Approximately 800 bp- and 700 bp-fragments respectively were amplified from the microalgal samples as shown in the lanes of 2 and 4 in Fig. 1. The positive controls also generated the same sizes of PCR fragments found in the microalgal samples shown in the lanes 5 and 6. These results indicate the potential microcystin producing cyanobacteria were present in the microalgae samples.

Experiments were carried out in N2 and CO2 using a Mettler Toledo TGA/DSC star system (Mettler, Switzerland). A series of TGA tests were carried out at a heating rate of 10 °C min−1 over a temperature range of ambient temperature to 900 °C. The flow rate of the purge (reactive) and protective gas was 60 mL min−1 and ~10 ± 0.05 mg of sample was loaded into the TGA unit. 2.6. Tubular reactor setting for pyrolysis of microalgal biomass A tubular reactor made of a quartz tube (Chemglass CGQ-0900T-13, USA) and its dimension was 1 in (25.4 mm) of outer diameter and 24 in (0.61 m) of length. 1 in stainless Ultra Torr Vacuum Fitting (Swagelok SS-4-UT-6-400) was assembled to build the TR. The sample of 1.5 ± 0.01 g was loaded inside of the TR. The required experimental temperatures were provided using a programmable tube furnace (Wisetherm, Korea). The purge and reactive gases were controlled with mass flow controller (Brooks, 6850E series, USA) and its flowrate was set as 500 mL min−1. The condensable hydrocarbons (i.e., tar) were collected with a condenser, and the temperature of the condenser was maintained at 4 °C. 2.7. GC measurements The injection of samples into the GC unit was made after the dilution (i.e., 1:15). GC/TOF-MS (Agilent 7850B and Bench TOF) equipped with DB-Wax column (Agilent J&W GC column, 60 m × 0.25 mm × 0.25 μm)

Fig. 1. 1% agarose gel showing PCR products amplified from the total DNA of fresh water sample from the Han River; 1. Kb Plus DNA size maker (SMOBIO); 2. 782-bp PCR fragments amplified by the cyanobacterial specific 16S rDNA 27F and 809R primers using the total DNA of fresh water sample from the Han River; 3. 472-bp PCR fragments amplified by the Hep primer set using the total DNA of fresh water sample from the Han River; 4. 698-bp PCR fragments amplified by the ITS1 and ITS4 primers using the total DNA of fresh water sample from the Han River; 5. 782-bp PCR fragments amplified by the cyanobacterial specific 16S rDNA 27F and 609R primers using the total DNA of toxic M. aeruginosa PCC 7806; 6. 472-bp PCR fragments amplified by the Hep primer set using the total DNA of the toxic M. aeruginosa PCC 7806.

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To identify the potential microcystin producer in the microalgae samples, these PCR products were used to create a clone library. About 280 colonies were isolated from the cyanospecific 16S rDNA library and these colonies were severed as templates for the PCRs using M13 primers to screen the cyanobacterial populations. The PCRs products from the colonies were used for DNA sequencing analysis. The sequencing analysis results indicated the potential toxic species of Microcystis sp. were predominantly populated at approximately 100% in the microalgae samples. Most DNA sequences derived from the cyanobacterial clone library showed the 99–100 similarities with uncultured Microcystis sp., M. aeruginosa NIES-2549 and M. panniformis FACHB-1757 in the GenBank. Also, the Hep PCRs clone library was used characterized from about 20 colonies generated and these were characterized via DNA sequencing analysis. The results indicates that most of sequences from Hep PCR clone library showed 99–100% sequence similarities with Microcystis sp. mcyE genes in the GenBanks. The 44 DNA sequences from ITS clone library generated from the PCR shown in the lane 3, Fig. 1, predominantly showed the 80–87% sequence similarities of Chlamydomonas sp. in the Genbank. Other authors reported that Microcystis sp. is one of the most common bloom-forming cyanobacteria in freshwater ecosystems, and is widely distributed on the five continents. Members of this genus are known to be able to synthesize numerous secondary metabolites, including microcystins, which are hepatotoxins frequently involved in animal and human poisoning (Briand et al., 2003). Our results from the microorganism composition analysis and toxigenicity test indicated that the potential toxic Microcystis sp. was predominantly present in the microalgae sample from the Han River in Korea. Over the years, various field studies have been conducted for understanding the diverse interactions among physicochemical and biological variables leading to the proliferation of cyanobacterial blooms in Korean freshwater bodies. Our results were also agreed upon by other authors' reports that Microcystis sp. was a major causative reagent for the fresh water blooms in the Han River in Korea, although the appearance of other members of genus such Anabaena and Oscillatoria with the potential for anatoxin-a had been reported with percent dominance of 30 and 10%, respectively, apart from Microcystis in other regions of water systems in Korea (Srivastava et al., 2015). However, our results indicates that the microalgal samples collected from the Han river only showed the potential of microcystin production in the river system. 3.2. Microalgal lipid analysis characterization

and basic

thermal degradation

Collected and dried microalgal biomass had a very offensive odor, which would be attributed to microalgal lipid: this offensive odor would be highly contingent on constituents of fatty acids (FAs) in microalgal lipid (Nguyen et al., 2009). In order to explore this, the total lipid content was measured after solvent extraction and the constituents of FAs were determined by using the non-catalytic

transformation of extracted microalgal lipid (i.e., triglycerides (TGs) and free fatty acids (FFAs)) into fatty acid methyl esters (FAMEs) (Jung et al., 2015; Kim et al., 2016). In order to ensure the reproducibility, the experimental work based on each test matric was conducted as triplicates, which indicated that the total lipid content in microalgal biomass was 2 ± 0.2 wt.% based on dry basis of microalgal biomass. The representative chromatogram showing the constituents of FAs was depicted in Fig. 2 and their identifications of major FA components were summarized in Table 1. Fig. 2 and Table 1 indicates the high content of C14–16 range of FAs in microalgal lipid, which is different from edible vegetable oil (i.e., C16–18 range of FAs) (Kwon et al., 2012b; Kwon et al., 2013b; Kwon et al., 2013c). This high content of C14–16 range of FAs in microalgal lipid is well consistent with the discussion in Section 3.1 since the dominant microalgal strain is identified as M. aeruginosa (i.e., cyanobacteria): the major composition of FAs in bacterial lipid has been known as C14–16 range of FAs (Bury et al., 1998; Kwon et al., 2012a). Furthermore, the short-chained FAs such as 2-butenoic acid (C4-FA) and hexanoic acid (C6-FA) were identified as the major FAs. Propionic acid (C3-FA) and valeric acid (C5-FA) were also detected as the minor FAs. These shortchained FAs are referred as volatile fatty acids (VFAs) and are well known as their offensive odors (Lu et al., 2008; Nguyen et al., 2009). Considering the toxicity (i.e., MC-LR) and offensive odor, landfilling of microalgal biomass would not be the best option for the final disposal of hazardous microalgal biomass. For instance, recognition of offensive odorants and resulting nuisance is considered one of the most serious issues triggering public complaints in urban environment and community odors remain one of the top three complaints to air quality regulators and government bodies in different countries (Lu et al., 2008; Nguyen et al., 2009; Shusterman, 1992). Furthermore, the possible environmental and ecological disturbance by MC-LR would be possible, which partially justified that the thermo-chemical treatment of microalgal biomass could be a reasonable disposal option for hazardous microalgal biomass. Thus, a series of thermo-gravimetric analysis (TGA) tests at the heating rate of 10 °C min− 1 from 25 to 900 °C with the microalgal sample were conducted to characterize the thermal degradation of microalgal biomass. The representative thermograms in N2 and CO2 are shown in Fig. 3: the experimental atmospheric conditions of N2 and CO2 are used for reference and control in this work, respectively. The representative thermograms in N2 and CO2 are nearly identical, which indicates that the influence of CO2 does not affect any physical aspects such as onset and end temperature of the thermal decomposition of microalgal biomass: the thermal degradation rate in N2 and CO2, which is represented as a slope, is identical. These observations were very consistent with the previous publications done by the authors (Cho et al., 2015; Kwon et al., 2015; Kwon et al., 2013a). This observation indirectly implies that the influence of CO2 on the sample surface (i.e., direct reaction between CO2 and microalgal biomass) should be excluded since the any reactions between CO2 and the sample surface as

Fig. 2. Representative chromatogram of lipid in the collected and dried microalgal biomass.

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Table 1 Identification of the major fatty acids (FAs) in microalgal lipid. tR, [min]

Identified chemical species

Chemical formula

Cas no.

3.19 3.63 4.69 10.27 10.45 11.54 12.24 12.45 14.28 14.78 14.96 16.45 16.86 17.81 19.49 20.02 20.13 20.59 21.04 21.35 22.81 23.93 30.41 34.46

2-Butenoic acid methyl ester Hexanoic acid methyl ester Octanoic acid methyl ester 4-oxo-Pentanoic acid methyl ester Butanedioic acid dimethyl ester Pentanedioic acid dimethyl ester Benzeneacetic acid methyl ester Dodecanoic acid methyl ester Tetradecanoic acid methyl ester Cyclopentanetridecanoic acid methyl ester Pentadecanoic acid methyl ester Hexadecanoic acid methyl ester 9-Hexadecenoic acid methyl ester Heptadecanoic acid methyl ester Octadecanoic acid methyl ester 9-Octadecenoic acid methyl ester 16-Octadecenoic acid methyl ester 11,14-Octadecadienoic acid methyl ester 9,12-Octadecadienoic acid methyl ester 12,15-Octadecadienoic acid methyl ester 9,12,15-Octadecatrienoic acid methyl ester Eicosanoic acid methyl ester Docosanoic acid methyl ester 10-oxo-Octadecanoic acid methyl ester

C5H8O2 C7H14O2 C9H18O2 C6H10O3 C6H10O4 C7H12O4 C9H10O2 C13H26O2 C15H30O2 C19H36O2 C16H32O2 C17H34O2 C17H32O2 C18H36O2 C19H38O2 C19H36O2 C19H36O2 C19H34O2 C19H34O2 C19H34O2 C19H32O2 C21H42O2 C23H46O2 C19H36O3

623-43-8 106-70-7 111-11-5 624-45-3 106-65-0 1119-40-0 101-41-7 111-82-0 124-10-7 24828-61-3 7132-64-1 112-39-0 1120-25-8 1731-92-6 112-61-8 1937-62-8 56554-49-5 999442-59-7 112-63-0 57156-97-5 301-00-8 1120-28-1 929-77-1 870-10-0

well as energy inputs from the furnace in the TGA equipment unit are reflected as the different thermal degradation rate (i.e., different slope). Moreover, the expected Boudouard reaction (i.e., C(s) + CO2 ➔ 2CO) did not occur. For example, the Boudouard reaction is thermodynamically favorable at temperatures higher than 720 °C (Chmielniak et al., 2014; Kaini and Mondal, 2014). However, the mass decay is indeed occurred at temperatures higher than 720 °C. This

mass decay is not attributed to the Boudouard reaction since the mass decay in N2 and CO2 is identical. This significantly suggests that the reaction kinetics of Boudouard reaction is very low (i.e., thermodynamically favorable, but kinetically not favorable), which is consistent with the previous work (Cho et al., 2015; Kwon et al., 2015). In addition to the physical aspects, in order to investigate the possible influence of CO2, the effluent of the TGA unit was monitored since the TGA experiment reflected the only physical aspects (i.e., mass change over the temperature changes). Interestingly, the concentration of major pyrolytic gases (i.e., H2, CH4, and CO) in N2 was different from those in CO2 (data not shown in this work) at temperatures higher than 500 °C, but these differences were not substantial due to our experimental limitations (i.e., dilution and small amount of sample). Thus, based on all discussions above, these observations suggest that the influence of CO2 is limited to the certain gaseous phase reactions between CO2 and volatile organic compounds (VOCs) evolved from the thermal degradation of microalgal biomass. In other words, the influence of CO2 is limited to the chemical aspects by means of the homogeneous reaction (i.e., gas phase reaction between VOCs and CO2), which indicates that additional energy requirement is not existed in a series of TGA experimental work.

3.3. Influence of CO2 in pyrolysis of microalgal biomass

Fig. 3. Representative thermograms of microalgal biomass in N2 and CO2.

In order to explore the chemical aspects induced by CO2 during the thermal degradation (i.e., pyrolysis) of microalgal biomass, the scaleup experiment was conducted with the tubular reactor (TR). The sample loading of microalgal biomass was 1.5 ± 0.01 g, and the major pyrolytic gases (i.e., H2, CH4, and CO) were quantified at various temperatures. Their concentration profiles are established in Fig. 4, and the concentration profiles of the major pyrolytic gases in N2 and CO2 in this study are the reference and control, respectively. The concentration profiles of major pyrolytic gases of microalgal biomass using the TR in N2 reflected the typical pyrolytic behaviors (Kwon and Castaldi, 2008); the generation of H2 is proportional to the experimental temperatures due to the thermal cracking via dehydrogenation, thus, the experimental temperature indicating the highest concentration of CH4 is significantly lower than that of H2. This observation suggests that the dominant thermal degradation mechanism at temperatures higher than 500 °C is hydrogenation. Thus, the

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Fig. 4. Concentration profiles of major pyrolytic gases evolved from the thermal degradation of microalgal biomass in N2 and CO2.

concentration profiles of CO in N2 in the entire experimental temperatures were significantly lower than those of H2 in N2 since dehydrogenation expedited the generation of chemical species containing a low ratio of H to C and carbon residue (Kwon and Castaldi, 2008): the chemical species containing a low ratio of H to C would be aromatics, that is, the main constituents of condensable hydrocarbons (i.e., tar). Unlike the typical evolution of pyrolytic gases in N2, the evolution of major pyrolytic gases in CO2 are significantly different from that in N2. As evidenced in Fig. 4, the evolution of major pyrolytic gases at temperatures lower than 500 °C was very similar in N2 and CO2, but the enhanced generation of CO in the presence of CO2 is identified at temperatures higher than 500 °C. This implies that the influence of CO2 is only effective at temperatures higher than 500 °C, which is consistent with the previous discussion in Section 3. 2. One interesting observation in Fig. 4 is that the concentration of H2 in the presence of CO2 at temperatures higher than 500 °C is substantially lower than those of H2 in N2. Unlike the pyrolytic gas evolution of H2 and CO, the concentration profiles of CH4 seem to be not sensitive to the experimental temperatures and atmospheres. However, the lower concentrations of H2 at temperatures higher than 500 °C in the CO2 environment would be attributed to the dilution factor attributed to the enhanced generation of CO since the GC analysis provides only relative mole fraction. In order to justify this, the pyrolytic experiment of microalgal biomass under equivalent volumetric mixture of N2 and CO2 was conducted, but the total flow rate of this mixture was the same (i.e., 500 mL min− 1) as other sets of the experiment in Fig. 4: the

concentration of N2 was used as an internal standard to justify the dilution factor attributed to the enhanced generation of CO in CO2. Thus, this experimentally justifies the dilution factor and suggests that the generation of H2 and CH4 at temperatures higher than 500 °C in CO2 is significantly enhanced. Thus, it is desirable to establish the syngas production with the ratio of CO and H2 and with the ratio of CH4 to H2 to differentiate the dilution factor attributed to the enhanced generation of CO in the CO2 environment. These genuine dissimilarities in the pyrolysis of microalgal biomass in CO2 can be attributed to the specified reactions between VOCs evolved from the thermal degradation and CO2 since the influence of CO2 is limited to the gas phase reaction, which was justified in Section 3.1. In addition, the effect of water gas shift (WGS) reaction should be excluded since water was not provided during the experiment and there is no possibility to provide water from microalgal biomass due to the batch-type experiment. Thus, for further investigate the influence of CO2 in pyrolysis of microalgal biomass, the pyrolytic oil in N2 and CO2 generated at 650 °C was collected and compared since the authors hypothesized that the specified gas phase reaction between VOCs and CO2 would affect the composition of pyrolytic oils. In other words, it is postulated that the enhanced generation of syngas originates from VOCs via providing additional C, H, and O. The representative chromatogram of pyrolytic oil in N2 and CO2 is compared in Fig. 5 and the representative chromatograms qualitatively show the differences of condensable hydrocarbons (i.e., tar) derived in N2 and CO2. In addition to comparison of the representative

Fig. 5. Representative chromatogram of pyrolytic oil generated at 650 °C in N2 and CO2.

Table 2 Summary of identified chemical species in pyrolytic oil generated in N2 and CO2. Chemical species

Peak area in N2

Peak area in CO2

Area change (%)

TR

Chemical species

Peak area in N2

Peak area in CO2

Area change (%)

9.37 9.73 10.35 10.48 10.90 10.95 11.16 11.40 12.31 12.45 12.65 12.85 13.18 13.68 14.54 14.67 14.71 15.11 15.30 15.42 15.65 15.73 16.20 16.20 16.64 16.85 17.35 17.38 17.53 17.69 18.23 18.59 18.96 19.38 20.22 20.57 20.84 21.57 21.71 22.17 22.59 22.97 23.54 23.97

2-Propenenitrile Isobutyronitrile 1-Decene Toluene Vinylfuran Schizanthine x 2,3-Dithiabutane 2-Cyanobutane Methyllaurate 1-Undecene m-Xylene 3-Butenenitrile Dimethylcyclotetrazenoborane Pyridine 2-Methylpyridine o-Ethyltoluene 1,2,4-Trimethylbenzene 4-Methylpentanenitrile 5-Dodecene Styrene 2-Propenylbenzene Methylpyrazine 1,3,5-Trimethylbenzene 1-Ethyl-3-methylbenzene 3-Methylpyridine 4-Methylpyridine 2-Phenylpropene p-Methylstyrene 3,4-Dimethylpyridine 1,2,3-Trimethylbenzene 2-Cyclopenten-1-one 2-Methyl-2-cyclopenten-1-one Dimethyl trisulfide 2-propenylbenzene Acetic acid 2-Furancarboxaldehyde 1-Tetradecene Methylphenylacetylene Pyrrole Pentadecane 3-Methylpyrrole 2-Methylpyrrole 2,5-Dimethylpyrrole 2,4-Diamine-6-chloro-1,3,5-triazine

230,457,000 323,033,000 212,525,000 2,083,610,000 24,703,300 – 54,453,700 299,762,000 377,621,000 206,084,000 272,627,000 45,683,200 77,649,800 451,426,000 178,795,000 52,895,800 – 340,326,000 38,010,300 472,134,000 26,910,900 146,810,000 70,223,800 – 56,653,700 15,381,800 – 37,776,200 22,514,600 16,300,900 79,022,400 35,014,300 42,122,300 32,823,100 316,554,000 17,200,700 57,858,800 33,782,700 458,309,000 28,852,500 218,456,000 124,202,000 65,435,000 15,822,100

183,042,000 271,065,000 140,655,000 1,876,830,000 – 19,039,600 39,870,800 – 334,210,000 120,563,000 189,002,000 – 58,575,800 307,790,000 96,808,400 – 42,524,300 205,010,000 – 894,710,000 – 72,967,000 – 32,676,200 21,409,700 – 35,928,200 – – – 24,170,400 – – – 49,779,100 – – – 287,005,000 – 85,597,000 78,894,700 42,772,600 –

−20.57 −16.09 −33.82 −9.92 – – −26.78 – −11.50 −41.50 −30.67 – −24.56 −31.82 −45.86 – – −39.76 – 89.50 – −50.30 – – −62.21 – – – – – −69.41 – – – −84.27 – – – −37.38 – −60.82 −36.48 −34.63 –

24.18 24.25 24.36 24.69 24.93 25.27 25.96 26.79 27.98 28.38 29.89 30.34 30.45 31.13 31.31 31.54 32.00 32.68 32.73 33.00 34.04 34.18 34.51 34.73 34.87 35.75 36.11 36.64 36.92 37.81 38.57 38.82 39.28 39.29 40.05 40.21 40.45 40.87 41.07 41.82 43.60 46.89 47.09

Benzonitrile 5-Methoxy-2-methylpyridine Butyric acid 2-Furanmethanol 1-Ethylpyrrole Valeric acid Heptadecane Acetamide Octadecane Corylon Isovaleramide Neophytadiene Benzeneacetonitrile 2-Acetylpyrrole 2,4-Phytadiene Phenol 2,4-Phytadiene Benzenepropanenitrile Dodecanamide 4-Methylphenol 4-Chloro-2.3-dimethyl-1.3-hexadiene Hexahydrofarnesyl acetone 2,6,6-Trimethyl-3-methylenecyclohexene 4-Ethyl-phenol 2,4-Dimethylimidazole Methyl palmitate 3,4-Dimethyl-1H-pyrrole-2.5-dione Methylethylmaleimide 2-methyl-3-Pyridinol 2,6-Piperidinedione 2,6-Piperidinedione Methyl-2.5-dimethylpyrrole 4(1H)-Pyridone 4-Pyridinol Indole 1-methylpiperidine 2,5-Pyrrolidinedione 2-Methyl-3-buten-2-ol 4-Methyl-1H-Indole 6-Undecylamine Isophytol Hexadecanamide Benzeneacetamide

15,937,500 30,876,300 423,367,000 36,310,200 11,063,200 424,736,000 750,141,000 270,083,000 117,559,000 48,588,900 33,054,700 138,789,000 41,998,300 12,516,600 17,675,400 183,511,000 14,408,100 18,934,100 40,991,100 162,178,000 17,307,800 28,988,100 14,919,700 42,382,800 30,475,000 27,855,800 16,107,000 17,213,600 14,536,700 11,158,700 44,384,300 55,631,400 129,389,000 – 93,197,400 13,180,900 33,105,500 23,515,300 54,945,800 38,516,400 20,943,900 282,811,000 23,888,400

– –

– – −86.96 – – −81.63 −84.62 – – – – – – – – −91.97 – – – – – – – – –

55,192,600 – – 78,039,600 115,357,000 – – – – – – – – 14,728,100 – – – – – – – – – – – – – – – – – 13,932,100 – – – – – – – 115,498,000 –

– – – – – – – – – – – – – – – −59.16 –

J.-M. Jung et al. / Science of the Total Environment 566–567 (2016) 575–583

TR

581

582

J.-M. Jung et al. / Science of the Total Environment 566–567 (2016) 575–583

thermogram in N2 and CO2, the identified chemical species in Fig. 5 was summarized via comparing the peak area of each identified chemical species in Table 2. One interesting observation in Fig. 5 and Table 2 is that the influence of CO2 on the chemical species in pyrolytic oil is identified at different magnitudes. As compared the concentration of each chemical species in Fig. 5, the difference in peak area of chemical species having a relatively short retention time is much less than that of chemical species having a relatively long retention time. In other words, the influence of CO2 is more sensitive to heavy molecular chemical species (i.e., chemical species having high boiling point). This observation could be attributed to the enhanced thermal cracking behaviors induced by CO2 via blocking the gas phase addition reaction of VOCs evolved from the thermal degradation of microalgal biomass. For example, the gas phase addition reactions are generally observed in the thermal degradation of carbonaceous materials, which provides the favorable conditions to form the substituted aromatics via simultaneous hydrogenation (Kwon et al., 2012c). As evidenced in Table 2, the identified chemical species in pyrolytic oil derived from the CO2 environment showed less variety of substituted aromatics compared to those derived from the N2 environment. Thus, this observation led to significant tar reduction in CO2 at the magnitude of ~70%, which was also clearly evidenced in Fig. 5 and Table 2. In other words, CO2 not only expedites the thermal cracking of VOCs evolved from the thermal degradation of microalgal biomass, but also reacts with VOCs. This may induce the different color of pyrolytic oils and may decrease the concentration of identified chemical species in the presence of CO2. Other reactions also can be attributed the enhanced generation of CO, but it is hard to explained at this stage of work. The experimental results in Fig. 4 and the significant reduction of tar in Fig. 5 and Table 2 partially justify the enhanced generation of major pyrolytic gases (H2, CH4, and CO) since the enhanced generation of major pyrolytic gases via the expedited thermal cracking justified the additional sources of C and H. However, the identified chemical species containing the O atom in Table 2 is very few, and the Boudouard reaction is not thermodynamically favorable at temperatures lower than 720 °C. Thus, the additional source of the O atom should be existed to explain the enhanced generation of CO in the CO2 environment.

Considering our experimental setup, the chemical species possibly providing the addition source of the O atom is CO2. In order to illustrate the additional supply of C and O from CO2, and the reaction between CO2 and VOCs, the syngas ratio of CO to H2 is plotted at temperature from 420 to 720 °C. As discussed above, it is desirable to establish the syngas production with the ratio of CO and H2 and with the ratio of CH4 to H2 to differentiate the dilution factor attributed to the enhanced generation of CO in the CO2 environment. As indicated in Fig. 6, the enhanced generation of CO in CO2 at temperatures higher than 500 °C is substantial. Unlike the enhanced generation of CO, the concentration of CH4 was not enhanced. This significantly suggests that the direct reaction between VOCs and CO2 is occurred simultaneously and independently with the thermal cracking behavior (i.e., dehydrogenation). Thus, the influence of CO2 is not only the enhanced thermal cracking of VOCs, but also the direct reaction between VOCs and CO 2 to form CO. At this stage, the underlying reaction mechanisms have not been fully established, but these findings are very significant in three respects. First, CO2 can increase the thermal efficiency of the thermo-chemical process (pyrolysis and gasification) since pyrolysis is the intermediate step in the gasification process. The enhanced syngas generation in the CO2 environment will lead to the enhanced generation of syngas in the gasification process via reducing condensable HCs (i.e., tar). Second, the identified influence of CO2 is applicable to in-situ air pollution control in various industries via the identified enhanced thermal cracking behaviors. Third, the ratio of CO to H2 could be adjustable by means of using the different amount of CO2. 4. Conclusions This work confirmed that the dominant microalgal strain in the eutrophic site was M. aeruginosa secreting toxins. Collected microalgal biomass from the eutrophic site had an offensive odor due to the lipid content, which reached 2 ± 0.2 wt.% of microalgal biomass (dry basis). This work also confirmed that the offensive odor was attributed to C3–6 range of volatile fatty acids (VFAs) via the non-catalytic transformation of triglycerides (TGs) and free fatty acids (FFAs) into fatty acid methyl esters (FAMEs). In particular, this work mechanistically

Fig. 6. Molar ratio of CH4 to H2 and CO to H2 at various temperatures.

J.-M. Jung et al. / Science of the Total Environment 566–567 (2016) 575–583

investigated the influence of CO2 addition in pyrolysis of hazardous microalgal biomass to achieve a dual purpose (i.e., thermal disposal of microalgal biomass and energy recovery). The influence of CO2 addition in pyrolysis of microalgal biomass was the enhanced thermal cracking behaviors of volatile organic compounds (VOCs) evolved from the thermal degradation and the direct gas phase reaction between CO2 and VOCs that enhance the generation of CO. This identified gas phase reaction in the presence of CO2 was only triggered at temperatures higher than 500 °C, which was different from the Boudouard reaction. Thus, this observation significantly suggests that using CO2 as reaction medium in pyrolysis enhance the thermal efficiency in pyrolysis without the extra heating source.

Acknowledgements This work was supported by a National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIP) (no. NRF-2014RA1A004893 and NRF-2015H1D3A1066513). This work was also supported by the Pukyong National University Research Fund in 2013 (CD20131333).

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