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Convergence of direct-transesterification and anaerobic digestion for improved bioenergy potentials of microalgae
Accepted Manuscript Convergence of direct-transesterification and anaerobic digestion for improved bioenergy potentials of microalgae Jae-Cheol Lee, Hyun-Woo Kim PII:
S0959-6526(18)30047-7
DOI:
10.1016/j.jclepro.2018.01.036
Reference:
JCLP 11716
To appear in:
Journal of Cleaner Production
Received Date: 1 August 2017 Revised Date:
4 January 2018
Accepted Date: 7 January 2018
Please cite this article as: Lee J-C, Kim H-W, Convergence of direct-transesterification and anaerobic digestion for improved bioenergy potentials of microalgae, Journal of Cleaner Production (2018), doi: 10.1016/j.jclepro.2018.01.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Convergence of direct-transesterification and anaerobic digestion for improved
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bioenergy potentials of microalgae
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Jae-Cheol Lee and Hyun-Woo Kim*
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Department of Environmental Engineering, Soil Environment Research Center, Chonbuk
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National University, 567, Baekje-daero, Deokjin-gu, Jeonju, Jeollabuk-do 54896, Republic of
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Korea
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* Corresponding author (HW Kim)
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Tel. +82-63-270-2444; Fax: +82-63-270-2449; E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract
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Direct-transesterification is an effective and economical method to extract fatty acid methyl
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ester (FAME) from microalgal biomass by omitting lipid separation step, but it still generates
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glycerol residue requiring further treatment. To minimize the waste, this study investigates
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the synergistic coupling of direct-transesterification and anaerobic digestion (AD). Four types
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of catalysts (HCl, H2SO4, NaOH, and K2CO3) were tested for better direct-transesterification
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followed by the AD of lyophilized Botryococcus braunii. Results indicate that base catalysts
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can extract more FAME than acid ones. The ranges of FAME yield were recorded as 6.0~9.2 %
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of the inoculum biomass, equivalent to >50% of FAME contained in inoculum biomass.
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Fourier transform infrared spectroscopy (FT-IR) spectra reveals that the FAME production
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from microalgae can be inhibited by existing proteinaceous soluble microbial products (SMP)
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or extracellular polymeric substances (EPS). The AD demonstrates a good methane potential
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of residual by-product, glycerol. The biochemical methane potential (BMP) test show that 90%
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of organic matter was converted to methane, and prove that the direct-transesterification
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contributes to following anaerobic digestion without inhibition. This result indicates that
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additional >22% of total energy recovery is possible by the AD. Overall results imply that
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this convergence of direct-transesterification and AD offers an economic and efficient way to
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control the bioenergy recovery from microalgal biomass.
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Keywords
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Microalgae, FAME, Direct transesterification, anaerobic digestion, bioenergy
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FAME
Fatty acid methyl ester, biodiesel
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C. vulgaris
Chlorella vulgaris
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C. sorokiniana Chlorella sorokiniana
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B. braunii
Botryococcus braunii
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Chlorella sp.
Chlorella species
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LED
light emitting diode
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GC
Gas chromatograph
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FID
Flame ionization detector
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SV
Saponification value
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IV
Iodine value
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CN
Cetane number
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LCSF
Long-chain saturated factor
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CFPP
Cold filter plugging point
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CP
Cloud point
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PP
Pour point
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ν
Kinetic viscosity at 40°C
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ρ
Density at 20°C
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HHV
Higher heating value
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ASTM
American Society for Testing and Materials
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AD
Anaerobic digestion
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BMP
Biochemical methane potential
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COD
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FT-IR
Fourier transform infrared spectroscopy
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SMP
Soluble microbial products
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EPS
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Chemical oxygen demand
Extracellular polymeric substance
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ACCEPTED MANUSCRIPT 1. Introduction
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The rapid increase of energy consumptions has become major consequences for the world
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energy crisis. As the development of environmentally-friendly, social-adequate, and cost-
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effective technologies, bioenergy from microalgae has been getting a significant attention.
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Tsolcha et al. (2017) indicated that the microalgal biotechnologies will be useful for
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producing bioenergy as a form of biodiesel (fatty acid methyl ester, FAME) because they
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require lower land, water, and energy usages than crop-based biodiesel.
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Conventional configurations for cultivating microalgae were open pond, raceway, microalgal turf scrubber, and photobioreactor. Several highly promising approaches have
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been introduced to combine the cultivation of microalgae with wastewater treatment targeting
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municipal, industrial, agricultural, and piggery wastewater (Salama et al., 2017). The
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microalgal treatment of livestock wastewater has gained much attention since a large amount
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of inorganic nitrogen and phosphorous could be assimilated into biomass via
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photoautotrophic metabolisms together with atmospheric CO2, which are ones of major
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pollutants (Kim and Kim, 2017).
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From the harvested microalgal biomass, carbohydrates, proteins, and lipids can be
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extracted for their beneficial uses. Carbohydrates can be convertible to alcohols, several
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individual proteins are extractable as value-added bioproducts, and lipids to biodiesel via
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transesterification is well-established (Katiyar et al., 2017). In one hand biodiesel production
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has gotten more attention since biofuel supply will be essential to prepare against future
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energy crisis (Mahmudul et al., 2017). However, Halim et al. (2011) reported the low
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extraction efficiency of lipid from the biomass as a problem to overcome.
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Previous studies have suggested various ways to extract lipid for biodiesel production but their cost-effectiveness is still questionable. Lepage and Roy (1986) and 4
ACCEPTED MANUSCRIPT Harrington and D’Arcy-Evans (1985) originally responded to the need for enhanced biodiesel
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production by using a direct (or in-situ) transesterification, which can omit cell disruption,
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solvent extraction, and solvent recovery. This direct-transesterification could reduce energy
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consumption and processing costs significantly. However, the method still has a room for
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improvement of efficiency in the usage of catalysts and solvents thus further optimization
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seems still necessary.
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Glycerol waste is an inevitable by-product of transesterification and contains various components such as alcohols, free fatty acids, un-reacted triglycerides, and methyl esters.
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Although the glycerol waste can be used as raw material for alimentary, cosmetic or drug
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industry, additional purification or refinery steps are essential for utilization (Rodriguez-
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Abalde et al., 2017). Anaerobic digestion (AD) is an attractive option to utilize the glycerol
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waste for energy valorization since no purification process is required. In addition, anaerobic
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microorganism has tolerance to various impurities, and most of the organic compounds in the
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glycerol waste is effectively convertible to biogas. The AD of harvested microalgal biomass
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has been widely applied, but glycerol waste from microalgal biodiesel production has yet to
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be fully investigated. Yang et al. (2012) pointed out that valorization and energy production
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of glycerol waste from biodiesel production is an important key of industrialization on a large
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scale.
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In this study, four types of catalysts (HCl, H2SO4, NaOH, and K2CO3) were tested to
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compare the productivity, adequacy, and FAME quality of microalgal biomass obtained from
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the converged process of direct-transesterification followed by anaerobic digestion. Fuel
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characteristics of the produced biodiesel were evaluated based on the composition and yield
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of each FAME. In addition, we further investigated how the decrease of FAME yield is
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associated with the contents of soluble microbial products (SMP) or extracellular polymeric 5
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light on the additional bioenergy recovery from the inevitably generated glycerol waste by
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demonstrating overall improvement on the energy potential based on the biochemical
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methane potential (BMP) test.
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2. Materials & Methods
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2.1. Inoculum
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Three species of microalgae were studied: Chlorella vulgaris (C. vulgaris), Chlorella
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sorokiniana (C. sorokiniana), and Botryococcus braunii (B. braunii). Since these microalgae
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had been used for the treatment of wastewater containing nitrogen and phosphorus (Xu et al.,
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2014), the removal efficiencies of organics and nutrients were characterized according to
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photosynthetic growth. Gupta et al. (2016) reported that C. sorokiniana and C. vulgaris have
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strong tolerance to organic loading and physiological stresses while removing nitrogen and
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phosphorus. B. braunii shows a high ratio of hydrocarbon that is a rich renewable source of
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biorefinery (An et al., 2003).
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All of the microalgae were isolated from Korea research institute of bioscience and
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biotechnology (KRIBB, Korea) and they were separately cultivated using a common liquid
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BG-11 medium which is widely used for cultivating phototrophic microorganisms (Qi et al.,
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2016). Inoculum cultures were grown under continuous illumination with light emitting diode
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(LED) modules (220 µmol E/m2/s) at maintained in constant temperature 25±5°C. Each stock
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cultures were harvested regularly after 1- to 2-week interval.
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2.2. Experimental procedure of the direct-transesterification
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The overall experimental procedure was shown in Fig. 1. Harvested wet biomass was 6
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Freeze-dried powder of biomass was used as a source for direct-transesterification. Four
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catalysts were applied in the direct-transesterification process. As a reaction catalyst, two
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types of acid catalysts (H2SO4 and HCl) and base catalysts (NaOH and K2CO3) were used.
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Acid catalysts can reduce the free fatty acids of lipids and have not significant effects from
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water content in biomass. Base homogeneous catalysis containing hydroxyl group can
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promote the reaction between lipid and glycerol (Santacesaria et al., 2012). Sodium
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hydroxide (NaOH) is very well accepted and widely used because of its low cost and high
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production yield (Demirbas, 2003). Recently, potassium carbonate (K2CO3) was newly
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suggested instead of hydroxyl base to reduce saponification and to increase FAME yield
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(Sung and Han, 2016).
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To demonstrate direct-transesterification in wastewater-treated biomass, we obtained other three types of microalgal biomass from continuously operated photobioreactors:
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cultivated in livestock wastewater (with phototrophic and mixotrophic mode of growth) and
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slaughterhouse wastewater. The characteristics of wastewater before treatment was shown in
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Table 1. After 10 days of operation, all the biomass was harvested and centrifuged at 9,000
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rpm for 10 min.
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2.3. Analytical procedure
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Extracted FAME was analyzed using gas chromatograph (GC) and a flame ionization
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detector (FID) (GC-2010, Shimadzu, Japan) equipped with column SPTM-2330 (Sigma
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Aldrich, USA) (McWilliams and Angelici, 2016). Characteristics of the column were as
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follow: its type was capillary highly polar, material was fused silica, length × inner diameter
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was 30 m × 0.25 mm, and film thickness was 0.20 µm. FID detector temperature was set at 7
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to increase at a rate of 4 °C /min up to 220 °C. The 1.0 µL of each sample was automatically
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injected using an auto-sampler with the split ratio of 1:10. Carrier gas was He (99.999%) and
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flame ignition gases were air (99.999%) and H2 (99.999%).
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2.4. Properties of FAMEs
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Quantification of FAME was conducted by comparing peak retention times with certified
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reference material ampule for FAME mix (CRM 18918, Supelco, USA) that contains C8 –
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C24. In addition, the quantitative composition was also calculated based on standard
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calibration curve between area and a percentage by mass. All samples were analyzed in
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triplicate, and the average results were reported with standard deviation.
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Based on composition and saturation of FAME, various fuel properties were
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calculated using empirical equations reported by other researches: saponification value (SV),
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iodine value (IV), cetane number (CN), long-chain saturated factor (LCSF), cold filter
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plugging point (CFPP), cloud point (CP), pour point (PP), kinematic viscosity (at 40℃, ν),
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density (at 20℃, ρ), and higher heating value (HHV) (Knothe, 2002; Krisnangkura, 1986;
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Ramirez-Verduzco et al., 2012; Ramos et al., 2009; Sarin et al., 2009). The detailed empirical
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equations were listed in Table S1. The yield of FAME was calculated according to Equation
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(1), and the recovery efficiency of FAME was calculated according to Equation (2):
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FAME yield (%) =
Total mass of FAME (g) Total mass of dried microalgal biomass (g)
FAME recovery % =
×100
! " !#$%&% ! "
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8
(1) × 100
(2)
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The BMP test was conducted to evaluate the potential of biogas production using glycerol
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waste from the direct-transesterification process. Anaerobic granular inoculum, taken from
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the brewery wastewater plant in Wanju-gun, Korea, was used to perform the BMP assays.
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The inoculum and substrate were added in serum bottles of 160 mL total volume. Working
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volume was 100mL that consist of the mineral solution suggested by Owen et al. (1979) and
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substrate based on 2,000 mg O2/L of chemical oxygen demand (COD). Blank bottles were
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filled up using inoculum and deionized water. All BMP tests were done in triplicates and
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were incubated in thermostat shaking incubator (Hanbaek science, Korea, HB-201SLI) at
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37°C and 120 rpm.
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3. Results & Discussion
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3.1. Yield of FAME from inoculum
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To compare FAME yield following the experimental design, four kinds of catalysts were used
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in direct-transesterification process followed by AD. The FAME yield in this study means
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that total mass of produced FAME over the total mass of dried microalgal biomass (Eq. 1).
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As shown in Fig. 2, acid catalysts need elevated temperature conditions (80°C) while base catalysts are active even at room temperature (25°C). In a transesterification process,
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temperature is an essential factor and plays a constructive role in lipid extraction, degradation,
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and solubility increase (Hidalgo et al., 2013). It was confirmed that base catalysts use less
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intensive energy for the reaction though more than 6% of FAME can be extractable from all
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the species tested.
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Table 2 compares the obtained FAME yield with other studies. While the main
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research trend was in conducting transesterification in elevated temperature, critical condition, 9
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sonication to increase yield, which increases the production cost, this study focused on the
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producing maximum FAME yield with the lowest cost by using simple catalyst, cheaper
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alcohol, shortened time, reduced procedure, and no additional control. Base catalyst results of
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this study achieved 25~76% FAME yield compared to the literature which used expensive
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and energy intensive (70~220oC) condition.
The FT-IR spectra provided various stretching bonds in each FAME samples. Fig. 3
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shows the FT-IR spectra of biodiesel from B. braunii strain in the spectral region of 4,000-
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400 cm-1. Main characteristic peaks in each spectrum were compared in 2,923, 2,854, 1,742,
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1,436, 1,170 and 721 cm-1, which were identified in a previous study (Silverstein et al., 2014).
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The peaks at 2,923 cm-1 show the unsaturated =C-H bond stretching. The peaks at
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2,800~2,900 and 1,742 cm-1 indicate the symmetrical and asymmetrical stretching vibrations
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of a methylene group (–CH2) and the stretching vibration of carbonyl C=O bond from ester
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group from FAME (Kumar et al., 2017). The band at 1,170 cm-1 can be assigned to the
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symmetric stretching vibration of C-O bonds from the ester.
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These stretching bond of FT-IR spectra supports the extraction mechanism of FAME
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from microalgal biomass. Reaction at room temperature could be slower than that of acid
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catalysis at 80℃ but the FT-IR spectra clearly indicates that the transesterification of methyl
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ester (1,742 cm-1) must be much faster in the case of alkaline catalysts (Fig. 3A) than that of
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acid ones (Fig. 3B). It was observed that the bond of methyl ester (1,742 cm-1) could be
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broken (Fig. 3B) at the temperature at 80℃, which led to FAME yield decrease.
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3.2 FAME production from wastewater treated microalgae biomass
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Fig. 4 shows the effect of B. braunii’s metabolic variation on FAME recoveries via the direct10
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of FAME recovery were obtained at both phototrophic and mixotrophic conditions. The
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results of acid catalysts presented very low recovery rates (~20%) though phototrophic
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growth (10~20%) was better than mixotrophic one (<5%). However, no difference was
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observed between acid and base catalysts when treating slaughterhouse wastewater under
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phototrophic condition.
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The spectra of FT-IR give supporting explanations about the difference as shown in Fig. 5. The strong peaks at 1,020 cm-1 were attributed to hemicellulose and carbohydrate in
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each biomass (Badhan et al., 2017). The peak ranges from 1,421 to 1,483 cm-1 show
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characteristic bands about amide group (or proteins) (Sadiq et al., 2011). Both carbohydrate
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and proteins are the major components of microalgal cell walls. Polysaccharides, proteins,
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and/or amino sugar-like compounds were also detected possibly due to SMP and EPS from
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biofoulants (Ke et al., 2013). Those peaks indicate that the potential inhibition of
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transesterification could be occurred while treating wastewater since the growth of biomass
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inevitably accompanies the production of SMP or EPS when compared to inoculum biomass.
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The decrease of FAME recovery in acid catalysis seemed to be caused by the inhibition of
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proteinaceous substances from B. braunii. During the acid transesterification, the donation of
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the proton must have occurred to SMP- or EPS-protein instead of the carboxyl group.
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3.3. Characterization and properties of FAME
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Compositions of produced FAME for three different microalgal species were shown in Fig.
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S1 (inoculum) and Fig. S2 (biomass treating wastewater). The major FAMEs were palmitic
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acid (C16:0), oleic acid (C18:1), and linoleic acid (C18:2), that are generally detected in the
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biodiesel products. For C. vulgaris, the average percentage of C16:0, C18:1, and C18:2 were 11
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shown 18.6±5.8%, 1.6±0.1%, and 31.6±2.3% (w/w), while those of C16:0, C18:1, and C18:2
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for C. sorokiniana and B. braunii were 41.6±4.3%, 11.4±0.6%, and 12.7±2.3% (w/w), and
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37.9±6.9%, 8.2±1.0%, and 10.6±0.9% (w/w), respectively.
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Based on the composition of FAME, fuel properties were calculated using empirical equations as shown in Table 3. Nine different fuel properties were compared with ASTM
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standards (ASTM D6751), EU standards (EN 14214), petroleum diesel, and results reported
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by other researchers using microalgal biomass (Guldhe et al., 2017; Pham et al., 2017; Yu et
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al., 2017). Conventional biodiesel fuel according to ASTM standard requires iodine value,
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cetane number, viscosity, and density. The EU standards suggest the minimum value of the
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higher heating value of 35 MJ/kg. Results indicate that the produced biodiesel in this study
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was sufficiently satisfactory to most of the standards.
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3.4 Methane potential from glycerol waste
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After the direct-transesterification, residual glycerol waste was applied to AD for additional
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energy recovery. Produced methane was compared to theoretical methane potential. The
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average concentration of the glycerol was 900 mg/L as COD. Because COD based theoretical
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methane production rate is 0.35 L CH4/g COD at standard temperature and pressure condition
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(Rittmann and McCarty, 2012), the theoretical methane potential could be estimated as 70
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mL.
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Figure 6 illustrates the cumulative methane production curves. Results indicate that
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methane production was increased rapidly after 4 days of lag period and reach stationary
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phase after 8 days. Produced methane at final phase was about 61 ml (acid catalyst) and 57
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ml (base catalyst), respectively. The comparison results indicated that methane production
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presents no significant difference according to the type of catalysts. The average 12
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concentration of remained COD after methane production was 120 ml (acid catalyst) and 135
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ml (base catalyst) mg O2/L, respectively. These results confirm that above 90% of residual
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glycerol waste can be biologically convertible to methane without inhibition. The potential of energy production was computed based on experimental data about
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biodiesel and biogas production. Energy potential from biodiesel was obtained using data
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from the maximum heating value (29.1 MJ/kg) and the mass of FAME at optimal condition
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(30 mg) and calculated as 0.9 kJ. Energy output from AD was calculated by using the
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equations suggested by Passos and Ferrer (2014). This equation calculates total output energy
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(kJ) using data from volume methane production (m3), the lower heating energy value of
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methane (=37.7 MJ/m3 of methane gas), reactor volume (m3), and energy conversion
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efficiency (90%). As a result, calculated energy potential from methane gas was 0.2 kJ that is
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equivalent to 22% of energy from biodiesel.
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3.5 Economical implication and future perspectives
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Various researchers have reported the use of wastewaters such as municipal, piggery,
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industrial, and AD effluent as the growth medium for microalgae (Salama et al., 2017) due to
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cheaper price and sustainability. However, this study shows that, with the >50% recovery
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among available FAME, suggested direct-transesterification is economically comparable to
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other transesterification methods. In addition, the transesterification for the biodiesel
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production from the harvested biomass can be inhibited by the SMP or EPS produced while
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growing the microalgae, which possibly lead to cost increase and efficiency decline.
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Harvesting and drying biomass are major factors to determine the total cost of
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biofuel production. The results of this study clearly demonstrate that about 22% additional
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energy recovery via anaerobic digestion makes this synergistic coupling of direct13
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transesterification and AD more competitive.
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future research should focus on optimization of direct-transesterification, additional energy or
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material recovery from residual glycerol waste, and the direct use of wet biomass or
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unsaponifiable lipids.
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From a scientific engineering standpoint,
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4. Conclusion
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The direct-transesterification process was coupled with the AD process to recover more
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energy from the microalgal biomass of B. braunii strain. For direct-transesterification, the
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range of overall FAME yield was recorded between 6 and 10% of total biomass due to the
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inhibition by microalgal SMP or EPS. The results indicate that base catalysts are preferable
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for efficient and economical direct-transesterification, which recovers above 50% of FAMEs
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available from inoculum biomass. Converging the direct-transesterification and the AD
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produces additional energy (~22%) via AD process. The conversion efficiency of residual
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glycerol waste to biogas was above 80% from theoretical methane production. The results of
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this study help establishing a better means to treat wastewater with microalgae while
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recovering renewable biofuels.
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Acknowledgement
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This research was supported by Basic Science Research Program through the National
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Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-
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2014R1A1A2057997 and NRF-2017R1A2B4012762).
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We also thank Dabin Kim for conducting BMP test.
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References
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An, J.Y., Sim, S.J., Lee, J.S., Kim, B.W., 2003. Hydrocarbon production from secondarily
326
treated piggery wastewater by the green alga Botryococcus braunii. J Appl Phycol 15,
327
185-191. Badhan, A., Wang, Y., McAllister, T.A., 2017. Analysis of Complex Carbohydrate
329
Composition in Plant Cell Wall Using Fourier Transformed Mid-Infrared Spectroscopy
330
(FT-IR), in: Abbott, D.W., Lammerts van Bueren, A. (Eds.), Protein-Carbohydrate
331
Interactions: Methods and Protocols. Springer, New York, pp. 209-214.
RI PT
328
Demirbas, A., 2003. Biodiesel fuels from vegetable oils via catalytic and non-catalytic
333
supercritical alcohol transesterifications and other methods: a survey. Energ Convers
334
Manage 44, 2093-2109.
SC
332
Guldhe, A., Singh, P., Ansari, F.A., Singh, B., Bux, F., 2017. Biodiesel synthesis from
336
microalgal lipids using tungstated zirconia as a heterogeneous acid catalyst and its
337
comparison with homogeneous acid and enzyme catalysts. Fuel 187, 180-188.
M AN U
335
Gupta, S.K., Ansari, F.A., Shriwastav, A., Sahoo, N.K., Rawat, I., Bux, F., 2016. Dual role of
339
Chlorella sorokiniana and Scenedesmus obliquus for comprehensive wastewater treatment
340
and biomass production for bio-fuels. J Clean Prod 115, 255-264.
341 342
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338
Halim, R., Gladman, B., Danquah, M.K., Webley, P.A., 2011. Oil extraction from microalgae for biodiesel production. Bioresour Technol 102, 178-185. Harrington, K.J., D’Arcy-Evans, C., 1985. A comparison of conventional and in situ methods
344
of transesterification of seed oil from a series of sunflower cultivars. Journal of the
345
American Oil Chemists Society 62, 1009-1013.
347
Hidalgo, P., Toro, C., Ciudad, G., Navia, R., 2013. Advances in direct transesterification of microalgal biomass for biodiesel production. Rev Environ Sci Bio 12, 179-199.
AC C
346
EP
343
348
Jo, Y.J., Lee, O.K., Lee, E.Y., 2014. Dimethyl carbonate-mediated lipid extraction and lipase-
349
catalyzed in situ transesterification for simultaneous preparation of fatty acid methyl esters
350
and glycerol carbonate from Chlorella sp. KR-1 biomass. Bioresource Technol 158, 105-
351
110.
352
Katiyar, R., Gurjar, B.R., Biswas, S., Pruthi, V., Kumar, N., Kumar, P., 2017. Microalgae: An
353
emerging source of energy based bio-products and a solution for environmental issues.
354
Renew Sust Energ Rev 72, 1083-1093. 15
ACCEPTED MANUSCRIPT
358 359 360 361 362 363 364 365 366 367 368
Kim, J.Y., Kim, H.W., 2017. Photoautotrophic Microalgae Screening for Tertiary Treatment of Livestock Wastewater and Bioresource Recovery. Water-Sui 9, 192. Kim, T.-H., Oh, Y.-K., Lee, J.W., Chang, Y.K., 2017. Levulinate production from algal cell hydrolysis using in situ transesterification. Algal Research 26, 431-435.
RI PT
357
in municipal secondary effluent reclamation. Environ Sci Pollut Res Int 20, 771-777.
Knothe, G., 2002. Structure indices in FA chemistry. How relevant is the iodine value? Journal of the American Oil Chemists' Society 79, 847-854.
Krisnangkura, K., 1986. A Simple Method for Estimation of Cetane Index of Vegetable Oil Methyl-Esters. Journal of the American Oil Chemists Society 63, 552-553.
SC
356
Ke, X., Hongqiang, R., Lili, D., Jinju, G., Tingting, Z., 2013. A review of membrane fouling
Kumar, S., Kumar, A., Mandal, A., 2017. Characterizations of surfactant synthesized from Jatropha oil and its application in enhanced oil recovery. Aiche J 63, 2731-2741.
M AN U
355
Lepage, G., Roy, C.C., 1986. Direct transesterification of all classes of lipids in a one-step reaction. Journal of Lipid Research 27, 114-120.
369
Mahmudul, H.M., Hagos, F.Y., Mamat, R., Adam, A.A., Ishak, W.F.W., Alenezi, R., 2017.
370
Production, characterization and performance of biodiesel as an alternative fuel in diesel
371
engines - A review. Renew Sust Energ Rev 72, 497-509.
McWilliams, K.M., Angelici, R.J., 2016. Batchwise extraction of methyl linolenate (18:3,
373
ALA) from fatty acid methyl esters derived from soybean and canola oils using silver
374
nitrate/silica gel. Eur J Lipid Sci Tech 118, 252-261.
TE D
372
Owen, W.F., Stuckey, D.C., Healy, J.B., Young, L.Y., Mccarty, P.L., 1979. Bioassay for
376
Monitoring Biochemical Methane Potential and Anaerobic Toxicity. Water Res 13, 485-
377
492.
EP
375
Pan, Y., Alam, M.A., Wang, Z., Huang, D., Hu, K., Chen, H., Yuan, Z., 2017. One-step
379
production of biodiesel from wet and unbroken microalgae biomass using deep eutectic
380
solvent. Bioresour Technol 238, 157-163.
381 382
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378
Passos, F., Ferrer, I., 2014. Microalgae conversion to biogas: thermal pretreatment contribution on net energy production. Environ Sci Technol 48, 7171-7178.
383
Pham, H.M., Kwak, H.S., Hong, M.E., Lee, J., Chang, W.S., Sim, S.J., 2017. Development of
384
an X-Shape airlift photobioreactor for increasing algal biomass and biodiesel production.
385
Bioresour Technol 239, 211-218.
386
Qi, F., Pei, H.Y., Hu, W.R., Mu, R.M., Zhang, S., 2016. Characterization of a microalgal 16
ACCEPTED MANUSCRIPT 387
mutant for CO2 biofixation and biofuel production. Energ Convers Manage 122, 344-349.
388
Ramirez-Verduzco, L.F., Rodriguez-Rodriguez, J.E., Jaramillo-Jacob, A.D., 2012. Predicting
389
cetane number, kinematic viscosity, density and higher heating value of biodiesel from its
390
fatty acid methyl ester composition. Fuel 91, 102-111. Ramos, M.J., Fernandez, C.M., Casas, A., Rodriguez, L., Perez, A., 2009. Influence of fatty
392
acid composition of raw materials on biodiesel properties. Bioresour Technol 100, 261-
393
268.
394 395
RI PT
391
Rittmann, B.E., McCarty, P.L., 2012. Environmental biotechnology: principles and applications. McGraw-Hill Education, New York.
Rodriguez-Abalde, A., Flotats, X., Fernandez, B., 2017. Optimization of the anaerobic co-
397
digestion of pasteurized slaughterhouse waste, pig slurry and glycerine. Waste Manag 61,
398
521-528.
M AN U
SC
396
399
Sadiq, I.M., Pakrashi, S., Chandrasekaran, N., Mukherjee, A., 2011. Studies on toxicity of
400
aluminum oxide (Al2O3) nanoparticles to microalgae species: Scenedesmus sp and
401
Chlorella sp. J Nanopart Res 13, 3287-3299.
Salama, E.S., Kurade, M.B., Abou-Shanab, R.A.I., El-Dalatony, M.M., Yang, I.S., Min, B.,
403
Jeon, B.H., 2017. Recent progress in microalgal biomass production coupled with
404
wastewater treatment for biofuel generation. Renew Sust Energ Rev 79, 1189-1211.
405 406
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402
Santacesaria, E., Vicente, G.M., Di Serio, M., Tesser, R., 2012. Main technologies in biodiesel production: State of the art and future challenges. Catal Today 195, 2-13. Sarin, A., Arora, R., Singh, N.P., Sarin, R., Malhotra, R.K., Kundu, K., 2009. Effect of blends
408
of Palm-Jatropha-Pongamia biodiesels on cloud point and pour point. Energy 34, 2016-
409
2021.
411 412
Silverstein, R.M., Webster, F.X., Kiemle, D.J., Bryce, D.L., 2014. Spectrometric
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identification of organic compounds. John wiley & sons, New York. Sivaramakrishnan, R., Incharoensakdi, A., 2017. Direct transesterification of Botryococcus sp.
413
catalysed by immobilized lipase: Ultrasound treatment can reduce reaction time with high
414
yield of methyl ester. Fuel 191, 363-370.
415 416
Sung, M., Han, J.I., 2016. Alkaline in situ transesterification of Aurantiochytrium sp. KRS 101 using potassium carbonate. Bioresour Technol 205, 250-253.
417
Tran, H.-L., Hong, S.-J., Lee, C.-G., 2009. Evaluation of extraction methods for recovery of
418
fatty acids from Botryococcus braunii LB 572 and Synechocystis sp. PCC 6803. 17
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Biotechnology and Bioprocess Engineering 14, 187-192.
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Tsolcha, O.N., Tekerlekopoulou, A.G., Akratos, C.S., Aggelis, G., Genitsaris, S., Moustaka-
421
Gouni, M., Vayenas, D.V., 2017. Biotreatment of raisin and winery wastewaters and
422
simultaneous biodiesel production using a Leptolyngbya-based microbial consortium. J
423
Clean Prod 148, 185-193. Xu, M., Bernards, M., Hu, Z., 2014. Algae-facilitated chemical phosphorus removal during
425
high-density Chlorella emersonii cultivation in a membrane bioreactor. Bioresour Technol
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153, 383-387.
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Yang, F., Hanna, M.A., Sun, R., 2012. Value-added uses for crude glycerol--a byproduct of biodiesel production. Biotechnol Biofuels 5, 13.
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Yu, Z., Song, M.M., Pei, H.Y., Han, F., Jiang, L.Q., Hou, Q.J., 2017. The growth
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characteristics and biodiesel production of ten algae strains cultivated in anaerobically
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digested effluent from kitchen waste. Algal Res 24, 265-275.
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Table
436 437
Table 1. Characteristics of wastewater before treatment
438
Total nitrogen (mg N/L)
Total phosphorus (mg P/L)
877.4 ± 1.9
251.1 ± 0.1
16.1 ± 0.7
817.8 ± 26.0
112.2 ± 2.9
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Livestock wastewater (LWW) Slaughterhouse wastewater (SWW)
CODCr (mg O2/L)
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5.1 ± 0.6
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Table 2. Comparison of biodiesel production yield using the microalgal biomass Conditions
Yield
C. vulgaris
25℃/2h/Acid and base catalysts
9.2% a
C. sorokiniana
25℃/2h/ Acid and base catalysts
6.2% a
B. braunii
25℃/2h/ Acid and base catalysts
6% a
Chlorella sp.
70℃/24h/Lipase catalyst
36.7% a
Chlorella sp.
220℃/30min/DES (deep eutectic solvent)
13.9%a
Pan et al. (2017)
12.1%a
Tran et al. (2009)
Kim et al. (2017)
100℃/1h/ Solvent B. braunii (mixture of acetyl chloride and methanol)
442 443
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100℃/1h/ Acid catalyst (methanol, H2SO4)
81.2%b (Lipid content: 28%)
B. braunii
40℃/6h/ Lipase catalyst (with sonication)
88% b (Lipid content: 23%)
This study This study This study Jo et al. (2014)
Sivaramakrishnan and Incharoensakdi (2017)
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Chlorella sp.
Relative to dry biomass percentage (%), b Relative to lipid (%).
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Species
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Table 3. Fuel properties of biodiesel from microalgal biomass (SV: Saponification value, IV: Iodine value, CN: Cetane number, LCSF: Long-chain
445
saturated factor, CFPP: Cold filter plugging point, CP: Cloud point, N: Kinetic viscosity, P: Density, HHV: Higher heating value) SV
IV
Unit
mg KOH/g
g I2/100g
This study
119.5~191.5
24.2~41.1
66.0~84.3
9.8~18.4
ASTM D6751
-
120 max
47 min
-
EN 14214
-
120 max
51 max
-
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Petroleum diesel
-
-
-
-
93.7
52.7
208.2
185.2
30.9
-
19.1
62.6
-
32.0
-
32.8
(Monoraphidium sp.) 446
- Not specified
LCSF
CFPP ℃
61.1
14.4~41.4
CP
N
P
HHV
℃
40℃, mm2/s
g/cm3
MJ/kg
3.9~20.5
2.1~3.4
0.50~0.80
22.4~35.7
-
1.9~6.0
0.80~0.90
-
-
-
3.5~5.0
0.86~0.9
35 min
-
-
-
1.9~6.0
0.82~0.86
44 max
-
-
-
-
-
38.4
7.0
5.44
-
-
-
-
-
-
18.9
5.2
0.87
38.7
-
-
16.6
5.1
0.87
39.0
-
-
16.4
5.1
0.87
39.0
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Biodiesel (S. obliquus) Biodiesel (Chlamydomonas reinhardtii) Biodiesel (Chlorella sp.) Biodiesel (Scenedesmus sp.) Biodiesel
CN
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447
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ACCEPTED MANUSCRIPT Figure captions Fig. 1. Schematic flow chart of the experimental procedure. Fig. 2. Effects of catalysts on yield of fatty acids methyl esters (FAME) in three species of microalgae:
C. sorokiniana at 80 , and (F) B. braunii at 80
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Fig. 3. FT-IR spectra of FAME from B. brauniii strain in various conditions: (A) FAME from B. braunii at 25 , (B) FAME from B. braunii at 80
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Fig. 4. Inoculum basis FAME recoveries obtained from wastewater treated microalgal biomass via direct-transesterification process: (A) Livestock wastewater treated B. braunii (phototrophic), (B)
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Fig. 5. FT-IR spectra of B. braunii biomass: (A) Inoculum, (B) Livestock wastewater treated biomass, and (C) Slaughterhouse treated biomass
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Highlights Direct-transesterification and anaerobic digestion were converged for enhancement
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FAME recovery from microalgae grown by LWW reaches 50% of inoculum biomass
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Transesterification inhibition by EPS- and SMP-proteins were newly detected
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Coupling anaerobic digestion synergistically convert glycerol waste into bioenergy
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S0959-6526(18)30047-7
DOI:
10.1016/j.jclepro.2018.01.036
Reference:
JCLP 11716
To appear in:
Journal of Cleaner Production
Received Date: 1 August 2017 Revised Date:
4 January 2018
Accepted Date: 7 January 2018
Please cite this article as: Lee J-C, Kim H-W, Convergence of direct-transesterification and anaerobic digestion for improved bioenergy potentials of microalgae, Journal of Cleaner Production (2018), doi: 10.1016/j.jclepro.2018.01.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Convergence of direct-transesterification and anaerobic digestion for improved
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bioenergy potentials of microalgae
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Jae-Cheol Lee and Hyun-Woo Kim*
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Department of Environmental Engineering, Soil Environment Research Center, Chonbuk
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National University, 567, Baekje-daero, Deokjin-gu, Jeonju, Jeollabuk-do 54896, Republic of
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Korea
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* Corresponding author (HW Kim)
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Tel. +82-63-270-2444; Fax: +82-63-270-2449; E-mail: [email protected]
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1
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Direct-transesterification is an effective and economical method to extract fatty acid methyl
14
ester (FAME) from microalgal biomass by omitting lipid separation step, but it still generates
15
glycerol residue requiring further treatment. To minimize the waste, this study investigates
16
the synergistic coupling of direct-transesterification and anaerobic digestion (AD). Four types
17
of catalysts (HCl, H2SO4, NaOH, and K2CO3) were tested for better direct-transesterification
18
followed by the AD of lyophilized Botryococcus braunii. Results indicate that base catalysts
19
can extract more FAME than acid ones. The ranges of FAME yield were recorded as 6.0~9.2 %
20
of the inoculum biomass, equivalent to >50% of FAME contained in inoculum biomass.
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Fourier transform infrared spectroscopy (FT-IR) spectra reveals that the FAME production
22
from microalgae can be inhibited by existing proteinaceous soluble microbial products (SMP)
23
or extracellular polymeric substances (EPS). The AD demonstrates a good methane potential
24
of residual by-product, glycerol. The biochemical methane potential (BMP) test show that 90%
25
of organic matter was converted to methane, and prove that the direct-transesterification
26
contributes to following anaerobic digestion without inhibition. This result indicates that
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additional >22% of total energy recovery is possible by the AD. Overall results imply that
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this convergence of direct-transesterification and AD offers an economic and efficient way to
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control the bioenergy recovery from microalgal biomass.
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Keywords
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Microalgae, FAME, Direct transesterification, anaerobic digestion, bioenergy
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FAME
Fatty acid methyl ester, biodiesel
36
C. vulgaris
Chlorella vulgaris
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C. sorokiniana Chlorella sorokiniana
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B. braunii
Botryococcus braunii
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Chlorella sp.
Chlorella species
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LED
light emitting diode
41
GC
Gas chromatograph
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FID
Flame ionization detector
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SV
Saponification value
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IV
Iodine value
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CN
Cetane number
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LCSF
Long-chain saturated factor
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CFPP
Cold filter plugging point
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CP
Cloud point
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PP
Pour point
50
ν
Kinetic viscosity at 40°C
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ρ
Density at 20°C
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HHV
Higher heating value
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ASTM
American Society for Testing and Materials
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AD
Anaerobic digestion
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BMP
Biochemical methane potential
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COD
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FT-IR
Fourier transform infrared spectroscopy
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SMP
Soluble microbial products
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EPS
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Chemical oxygen demand
Extracellular polymeric substance
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The rapid increase of energy consumptions has become major consequences for the world
63
energy crisis. As the development of environmentally-friendly, social-adequate, and cost-
64
effective technologies, bioenergy from microalgae has been getting a significant attention.
65
Tsolcha et al. (2017) indicated that the microalgal biotechnologies will be useful for
66
producing bioenergy as a form of biodiesel (fatty acid methyl ester, FAME) because they
67
require lower land, water, and energy usages than crop-based biodiesel.
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Conventional configurations for cultivating microalgae were open pond, raceway, microalgal turf scrubber, and photobioreactor. Several highly promising approaches have
70
been introduced to combine the cultivation of microalgae with wastewater treatment targeting
71
municipal, industrial, agricultural, and piggery wastewater (Salama et al., 2017). The
72
microalgal treatment of livestock wastewater has gained much attention since a large amount
73
of inorganic nitrogen and phosphorous could be assimilated into biomass via
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photoautotrophic metabolisms together with atmospheric CO2, which are ones of major
75
pollutants (Kim and Kim, 2017).
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From the harvested microalgal biomass, carbohydrates, proteins, and lipids can be
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extracted for their beneficial uses. Carbohydrates can be convertible to alcohols, several
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individual proteins are extractable as value-added bioproducts, and lipids to biodiesel via
79
transesterification is well-established (Katiyar et al., 2017). In one hand biodiesel production
80
has gotten more attention since biofuel supply will be essential to prepare against future
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energy crisis (Mahmudul et al., 2017). However, Halim et al. (2011) reported the low
82
extraction efficiency of lipid from the biomass as a problem to overcome.
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Previous studies have suggested various ways to extract lipid for biodiesel production but their cost-effectiveness is still questionable. Lepage and Roy (1986) and 4
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86
production by using a direct (or in-situ) transesterification, which can omit cell disruption,
87
solvent extraction, and solvent recovery. This direct-transesterification could reduce energy
88
consumption and processing costs significantly. However, the method still has a room for
89
improvement of efficiency in the usage of catalysts and solvents thus further optimization
90
seems still necessary.
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Glycerol waste is an inevitable by-product of transesterification and contains various components such as alcohols, free fatty acids, un-reacted triglycerides, and methyl esters.
93
Although the glycerol waste can be used as raw material for alimentary, cosmetic or drug
94
industry, additional purification or refinery steps are essential for utilization (Rodriguez-
95
Abalde et al., 2017). Anaerobic digestion (AD) is an attractive option to utilize the glycerol
96
waste for energy valorization since no purification process is required. In addition, anaerobic
97
microorganism has tolerance to various impurities, and most of the organic compounds in the
98
glycerol waste is effectively convertible to biogas. The AD of harvested microalgal biomass
99
has been widely applied, but glycerol waste from microalgal biodiesel production has yet to
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be fully investigated. Yang et al. (2012) pointed out that valorization and energy production
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of glycerol waste from biodiesel production is an important key of industrialization on a large
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scale.
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In this study, four types of catalysts (HCl, H2SO4, NaOH, and K2CO3) were tested to
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compare the productivity, adequacy, and FAME quality of microalgal biomass obtained from
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the converged process of direct-transesterification followed by anaerobic digestion. Fuel
106
characteristics of the produced biodiesel were evaluated based on the composition and yield
107
of each FAME. In addition, we further investigated how the decrease of FAME yield is
108
associated with the contents of soluble microbial products (SMP) or extracellular polymeric 5
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110
light on the additional bioenergy recovery from the inevitably generated glycerol waste by
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demonstrating overall improvement on the energy potential based on the biochemical
112
methane potential (BMP) test.
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2. Materials & Methods
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2.1. Inoculum
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Three species of microalgae were studied: Chlorella vulgaris (C. vulgaris), Chlorella
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sorokiniana (C. sorokiniana), and Botryococcus braunii (B. braunii). Since these microalgae
118
had been used for the treatment of wastewater containing nitrogen and phosphorus (Xu et al.,
119
2014), the removal efficiencies of organics and nutrients were characterized according to
120
photosynthetic growth. Gupta et al. (2016) reported that C. sorokiniana and C. vulgaris have
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strong tolerance to organic loading and physiological stresses while removing nitrogen and
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phosphorus. B. braunii shows a high ratio of hydrocarbon that is a rich renewable source of
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biorefinery (An et al., 2003).
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All of the microalgae were isolated from Korea research institute of bioscience and
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biotechnology (KRIBB, Korea) and they were separately cultivated using a common liquid
126
BG-11 medium which is widely used for cultivating phototrophic microorganisms (Qi et al.,
127
2016). Inoculum cultures were grown under continuous illumination with light emitting diode
128
(LED) modules (220 µmol E/m2/s) at maintained in constant temperature 25±5°C. Each stock
129
cultures were harvested regularly after 1- to 2-week interval.
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2.2. Experimental procedure of the direct-transesterification
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The overall experimental procedure was shown in Fig. 1. Harvested wet biomass was 6
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134
Freeze-dried powder of biomass was used as a source for direct-transesterification. Four
135
catalysts were applied in the direct-transesterification process. As a reaction catalyst, two
136
types of acid catalysts (H2SO4 and HCl) and base catalysts (NaOH and K2CO3) were used.
137
Acid catalysts can reduce the free fatty acids of lipids and have not significant effects from
138
water content in biomass. Base homogeneous catalysis containing hydroxyl group can
139
promote the reaction between lipid and glycerol (Santacesaria et al., 2012). Sodium
140
hydroxide (NaOH) is very well accepted and widely used because of its low cost and high
141
production yield (Demirbas, 2003). Recently, potassium carbonate (K2CO3) was newly
142
suggested instead of hydroxyl base to reduce saponification and to increase FAME yield
143
(Sung and Han, 2016).
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To demonstrate direct-transesterification in wastewater-treated biomass, we obtained other three types of microalgal biomass from continuously operated photobioreactors:
146
cultivated in livestock wastewater (with phototrophic and mixotrophic mode of growth) and
147
slaughterhouse wastewater. The characteristics of wastewater before treatment was shown in
148
Table 1. After 10 days of operation, all the biomass was harvested and centrifuged at 9,000
149
rpm for 10 min.
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2.3. Analytical procedure
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Extracted FAME was analyzed using gas chromatograph (GC) and a flame ionization
153
detector (FID) (GC-2010, Shimadzu, Japan) equipped with column SPTM-2330 (Sigma
154
Aldrich, USA) (McWilliams and Angelici, 2016). Characteristics of the column were as
155
follow: its type was capillary highly polar, material was fused silica, length × inner diameter
156
was 30 m × 0.25 mm, and film thickness was 0.20 µm. FID detector temperature was set at 7
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158
to increase at a rate of 4 °C /min up to 220 °C. The 1.0 µL of each sample was automatically
159
injected using an auto-sampler with the split ratio of 1:10. Carrier gas was He (99.999%) and
160
flame ignition gases were air (99.999%) and H2 (99.999%).
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2.4. Properties of FAMEs
163
Quantification of FAME was conducted by comparing peak retention times with certified
164
reference material ampule for FAME mix (CRM 18918, Supelco, USA) that contains C8 –
165
C24. In addition, the quantitative composition was also calculated based on standard
166
calibration curve between area and a percentage by mass. All samples were analyzed in
167
triplicate, and the average results were reported with standard deviation.
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Based on composition and saturation of FAME, various fuel properties were
169
calculated using empirical equations reported by other researches: saponification value (SV),
170
iodine value (IV), cetane number (CN), long-chain saturated factor (LCSF), cold filter
171
plugging point (CFPP), cloud point (CP), pour point (PP), kinematic viscosity (at 40℃, ν),
172
density (at 20℃, ρ), and higher heating value (HHV) (Knothe, 2002; Krisnangkura, 1986;
173
Ramirez-Verduzco et al., 2012; Ramos et al., 2009; Sarin et al., 2009). The detailed empirical
174
equations were listed in Table S1. The yield of FAME was calculated according to Equation
175
(1), and the recovery efficiency of FAME was calculated according to Equation (2):
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FAME yield (%) =
Total mass of FAME (g) Total mass of dried microalgal biomass (g)
FAME recovery % =
×100
! " !#$%&% ! "
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8
(1) × 100
(2)
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The BMP test was conducted to evaluate the potential of biogas production using glycerol
182
waste from the direct-transesterification process. Anaerobic granular inoculum, taken from
183
the brewery wastewater plant in Wanju-gun, Korea, was used to perform the BMP assays.
184
The inoculum and substrate were added in serum bottles of 160 mL total volume. Working
185
volume was 100mL that consist of the mineral solution suggested by Owen et al. (1979) and
186
substrate based on 2,000 mg O2/L of chemical oxygen demand (COD). Blank bottles were
187
filled up using inoculum and deionized water. All BMP tests were done in triplicates and
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were incubated in thermostat shaking incubator (Hanbaek science, Korea, HB-201SLI) at
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37°C and 120 rpm.
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3. Results & Discussion
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3.1. Yield of FAME from inoculum
193
To compare FAME yield following the experimental design, four kinds of catalysts were used
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in direct-transesterification process followed by AD. The FAME yield in this study means
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that total mass of produced FAME over the total mass of dried microalgal biomass (Eq. 1).
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As shown in Fig. 2, acid catalysts need elevated temperature conditions (80°C) while base catalysts are active even at room temperature (25°C). In a transesterification process,
198
temperature is an essential factor and plays a constructive role in lipid extraction, degradation,
199
and solubility increase (Hidalgo et al., 2013). It was confirmed that base catalysts use less
200
intensive energy for the reaction though more than 6% of FAME can be extractable from all
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the species tested.
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Table 2 compares the obtained FAME yield with other studies. While the main
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research trend was in conducting transesterification in elevated temperature, critical condition, 9
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sonication to increase yield, which increases the production cost, this study focused on the
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producing maximum FAME yield with the lowest cost by using simple catalyst, cheaper
207
alcohol, shortened time, reduced procedure, and no additional control. Base catalyst results of
208
this study achieved 25~76% FAME yield compared to the literature which used expensive
209
and energy intensive (70~220oC) condition.
The FT-IR spectra provided various stretching bonds in each FAME samples. Fig. 3
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shows the FT-IR spectra of biodiesel from B. braunii strain in the spectral region of 4,000-
212
400 cm-1. Main characteristic peaks in each spectrum were compared in 2,923, 2,854, 1,742,
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1,436, 1,170 and 721 cm-1, which were identified in a previous study (Silverstein et al., 2014).
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The peaks at 2,923 cm-1 show the unsaturated =C-H bond stretching. The peaks at
215
2,800~2,900 and 1,742 cm-1 indicate the symmetrical and asymmetrical stretching vibrations
216
of a methylene group (–CH2) and the stretching vibration of carbonyl C=O bond from ester
217
group from FAME (Kumar et al., 2017). The band at 1,170 cm-1 can be assigned to the
218
symmetric stretching vibration of C-O bonds from the ester.
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from microalgal biomass. Reaction at room temperature could be slower than that of acid
221
catalysis at 80℃ but the FT-IR spectra clearly indicates that the transesterification of methyl
222
ester (1,742 cm-1) must be much faster in the case of alkaline catalysts (Fig. 3A) than that of
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acid ones (Fig. 3B). It was observed that the bond of methyl ester (1,742 cm-1) could be
224
broken (Fig. 3B) at the temperature at 80℃, which led to FAME yield decrease.
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3.2 FAME production from wastewater treated microalgae biomass
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Fig. 4 shows the effect of B. braunii’s metabolic variation on FAME recoveries via the direct10
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229
of FAME recovery were obtained at both phototrophic and mixotrophic conditions. The
230
results of acid catalysts presented very low recovery rates (~20%) though phototrophic
231
growth (10~20%) was better than mixotrophic one (<5%). However, no difference was
232
observed between acid and base catalysts when treating slaughterhouse wastewater under
233
phototrophic condition.
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The spectra of FT-IR give supporting explanations about the difference as shown in Fig. 5. The strong peaks at 1,020 cm-1 were attributed to hemicellulose and carbohydrate in
236
each biomass (Badhan et al., 2017). The peak ranges from 1,421 to 1,483 cm-1 show
237
characteristic bands about amide group (or proteins) (Sadiq et al., 2011). Both carbohydrate
238
and proteins are the major components of microalgal cell walls. Polysaccharides, proteins,
239
and/or amino sugar-like compounds were also detected possibly due to SMP and EPS from
240
biofoulants (Ke et al., 2013). Those peaks indicate that the potential inhibition of
241
transesterification could be occurred while treating wastewater since the growth of biomass
242
inevitably accompanies the production of SMP or EPS when compared to inoculum biomass.
243
The decrease of FAME recovery in acid catalysis seemed to be caused by the inhibition of
244
proteinaceous substances from B. braunii. During the acid transesterification, the donation of
245
the proton must have occurred to SMP- or EPS-protein instead of the carboxyl group.
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3.3. Characterization and properties of FAME
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Compositions of produced FAME for three different microalgal species were shown in Fig.
249
S1 (inoculum) and Fig. S2 (biomass treating wastewater). The major FAMEs were palmitic
250
acid (C16:0), oleic acid (C18:1), and linoleic acid (C18:2), that are generally detected in the
251
biodiesel products. For C. vulgaris, the average percentage of C16:0, C18:1, and C18:2 were 11
ACCEPTED MANUSCRIPT 252
shown 18.6±5.8%, 1.6±0.1%, and 31.6±2.3% (w/w), while those of C16:0, C18:1, and C18:2
253
for C. sorokiniana and B. braunii were 41.6±4.3%, 11.4±0.6%, and 12.7±2.3% (w/w), and
254
37.9±6.9%, 8.2±1.0%, and 10.6±0.9% (w/w), respectively.
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Based on the composition of FAME, fuel properties were calculated using empirical equations as shown in Table 3. Nine different fuel properties were compared with ASTM
257
standards (ASTM D6751), EU standards (EN 14214), petroleum diesel, and results reported
258
by other researchers using microalgal biomass (Guldhe et al., 2017; Pham et al., 2017; Yu et
259
al., 2017). Conventional biodiesel fuel according to ASTM standard requires iodine value,
260
cetane number, viscosity, and density. The EU standards suggest the minimum value of the
261
higher heating value of 35 MJ/kg. Results indicate that the produced biodiesel in this study
262
was sufficiently satisfactory to most of the standards.
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3.4 Methane potential from glycerol waste
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After the direct-transesterification, residual glycerol waste was applied to AD for additional
266
energy recovery. Produced methane was compared to theoretical methane potential. The
267
average concentration of the glycerol was 900 mg/L as COD. Because COD based theoretical
268
methane production rate is 0.35 L CH4/g COD at standard temperature and pressure condition
269
(Rittmann and McCarty, 2012), the theoretical methane potential could be estimated as 70
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mL.
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Figure 6 illustrates the cumulative methane production curves. Results indicate that
272
methane production was increased rapidly after 4 days of lag period and reach stationary
273
phase after 8 days. Produced methane at final phase was about 61 ml (acid catalyst) and 57
274
ml (base catalyst), respectively. The comparison results indicated that methane production
275
presents no significant difference according to the type of catalysts. The average 12
ACCEPTED MANUSCRIPT 276
concentration of remained COD after methane production was 120 ml (acid catalyst) and 135
277
ml (base catalyst) mg O2/L, respectively. These results confirm that above 90% of residual
278
glycerol waste can be biologically convertible to methane without inhibition. The potential of energy production was computed based on experimental data about
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biodiesel and biogas production. Energy potential from biodiesel was obtained using data
281
from the maximum heating value (29.1 MJ/kg) and the mass of FAME at optimal condition
282
(30 mg) and calculated as 0.9 kJ. Energy output from AD was calculated by using the
283
equations suggested by Passos and Ferrer (2014). This equation calculates total output energy
284
(kJ) using data from volume methane production (m3), the lower heating energy value of
285
methane (=37.7 MJ/m3 of methane gas), reactor volume (m3), and energy conversion
286
efficiency (90%). As a result, calculated energy potential from methane gas was 0.2 kJ that is
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equivalent to 22% of energy from biodiesel.
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3.5 Economical implication and future perspectives
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Various researchers have reported the use of wastewaters such as municipal, piggery,
291
industrial, and AD effluent as the growth medium for microalgae (Salama et al., 2017) due to
292
cheaper price and sustainability. However, this study shows that, with the >50% recovery
293
among available FAME, suggested direct-transesterification is economically comparable to
294
other transesterification methods. In addition, the transesterification for the biodiesel
295
production from the harvested biomass can be inhibited by the SMP or EPS produced while
296
growing the microalgae, which possibly lead to cost increase and efficiency decline.
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Harvesting and drying biomass are major factors to determine the total cost of
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biofuel production. The results of this study clearly demonstrate that about 22% additional
299
energy recovery via anaerobic digestion makes this synergistic coupling of direct13
ACCEPTED MANUSCRIPT 300
transesterification and AD more competitive.
301
future research should focus on optimization of direct-transesterification, additional energy or
302
material recovery from residual glycerol waste, and the direct use of wet biomass or
303
unsaponifiable lipids.
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From a scientific engineering standpoint,
304
4. Conclusion
306
The direct-transesterification process was coupled with the AD process to recover more
307
energy from the microalgal biomass of B. braunii strain. For direct-transesterification, the
308
range of overall FAME yield was recorded between 6 and 10% of total biomass due to the
309
inhibition by microalgal SMP or EPS. The results indicate that base catalysts are preferable
310
for efficient and economical direct-transesterification, which recovers above 50% of FAMEs
311
available from inoculum biomass. Converging the direct-transesterification and the AD
312
produces additional energy (~22%) via AD process. The conversion efficiency of residual
313
glycerol waste to biogas was above 80% from theoretical methane production. The results of
314
this study help establishing a better means to treat wastewater with microalgae while
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recovering renewable biofuels.
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Acknowledgement
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This research was supported by Basic Science Research Program through the National
319
Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-
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2014R1A1A2057997 and NRF-2017R1A2B4012762).
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We also thank Dabin Kim for conducting BMP test.
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References
325
An, J.Y., Sim, S.J., Lee, J.S., Kim, B.W., 2003. Hydrocarbon production from secondarily
326
treated piggery wastewater by the green alga Botryococcus braunii. J Appl Phycol 15,
327
185-191. Badhan, A., Wang, Y., McAllister, T.A., 2017. Analysis of Complex Carbohydrate
329
Composition in Plant Cell Wall Using Fourier Transformed Mid-Infrared Spectroscopy
330
(FT-IR), in: Abbott, D.W., Lammerts van Bueren, A. (Eds.), Protein-Carbohydrate
331
Interactions: Methods and Protocols. Springer, New York, pp. 209-214.
RI PT
328
Demirbas, A., 2003. Biodiesel fuels from vegetable oils via catalytic and non-catalytic
333
supercritical alcohol transesterifications and other methods: a survey. Energ Convers
334
Manage 44, 2093-2109.
SC
332
Guldhe, A., Singh, P., Ansari, F.A., Singh, B., Bux, F., 2017. Biodiesel synthesis from
336
microalgal lipids using tungstated zirconia as a heterogeneous acid catalyst and its
337
comparison with homogeneous acid and enzyme catalysts. Fuel 187, 180-188.
M AN U
335
Gupta, S.K., Ansari, F.A., Shriwastav, A., Sahoo, N.K., Rawat, I., Bux, F., 2016. Dual role of
339
Chlorella sorokiniana and Scenedesmus obliquus for comprehensive wastewater treatment
340
and biomass production for bio-fuels. J Clean Prod 115, 255-264.
341 342
TE D
338
Halim, R., Gladman, B., Danquah, M.K., Webley, P.A., 2011. Oil extraction from microalgae for biodiesel production. Bioresour Technol 102, 178-185. Harrington, K.J., D’Arcy-Evans, C., 1985. A comparison of conventional and in situ methods
344
of transesterification of seed oil from a series of sunflower cultivars. Journal of the
345
American Oil Chemists Society 62, 1009-1013.
347
Hidalgo, P., Toro, C., Ciudad, G., Navia, R., 2013. Advances in direct transesterification of microalgal biomass for biodiesel production. Rev Environ Sci Bio 12, 179-199.
AC C
346
EP
343
348
Jo, Y.J., Lee, O.K., Lee, E.Y., 2014. Dimethyl carbonate-mediated lipid extraction and lipase-
349
catalyzed in situ transesterification for simultaneous preparation of fatty acid methyl esters
350
and glycerol carbonate from Chlorella sp. KR-1 biomass. Bioresource Technol 158, 105-
351
110.
352
Katiyar, R., Gurjar, B.R., Biswas, S., Pruthi, V., Kumar, N., Kumar, P., 2017. Microalgae: An
353
emerging source of energy based bio-products and a solution for environmental issues.
354
Renew Sust Energ Rev 72, 1083-1093. 15
ACCEPTED MANUSCRIPT
358 359 360 361 362 363 364 365 366 367 368
Kim, J.Y., Kim, H.W., 2017. Photoautotrophic Microalgae Screening for Tertiary Treatment of Livestock Wastewater and Bioresource Recovery. Water-Sui 9, 192. Kim, T.-H., Oh, Y.-K., Lee, J.W., Chang, Y.K., 2017. Levulinate production from algal cell hydrolysis using in situ transesterification. Algal Research 26, 431-435.
RI PT
357
in municipal secondary effluent reclamation. Environ Sci Pollut Res Int 20, 771-777.
Knothe, G., 2002. Structure indices in FA chemistry. How relevant is the iodine value? Journal of the American Oil Chemists' Society 79, 847-854.
Krisnangkura, K., 1986. A Simple Method for Estimation of Cetane Index of Vegetable Oil Methyl-Esters. Journal of the American Oil Chemists Society 63, 552-553.
SC
356
Ke, X., Hongqiang, R., Lili, D., Jinju, G., Tingting, Z., 2013. A review of membrane fouling
Kumar, S., Kumar, A., Mandal, A., 2017. Characterizations of surfactant synthesized from Jatropha oil and its application in enhanced oil recovery. Aiche J 63, 2731-2741.
M AN U
355
Lepage, G., Roy, C.C., 1986. Direct transesterification of all classes of lipids in a one-step reaction. Journal of Lipid Research 27, 114-120.
369
Mahmudul, H.M., Hagos, F.Y., Mamat, R., Adam, A.A., Ishak, W.F.W., Alenezi, R., 2017.
370
Production, characterization and performance of biodiesel as an alternative fuel in diesel
371
engines - A review. Renew Sust Energ Rev 72, 497-509.
McWilliams, K.M., Angelici, R.J., 2016. Batchwise extraction of methyl linolenate (18:3,
373
ALA) from fatty acid methyl esters derived from soybean and canola oils using silver
374
nitrate/silica gel. Eur J Lipid Sci Tech 118, 252-261.
TE D
372
Owen, W.F., Stuckey, D.C., Healy, J.B., Young, L.Y., Mccarty, P.L., 1979. Bioassay for
376
Monitoring Biochemical Methane Potential and Anaerobic Toxicity. Water Res 13, 485-
377
492.
EP
375
Pan, Y., Alam, M.A., Wang, Z., Huang, D., Hu, K., Chen, H., Yuan, Z., 2017. One-step
379
production of biodiesel from wet and unbroken microalgae biomass using deep eutectic
380
solvent. Bioresour Technol 238, 157-163.
381 382
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378
Passos, F., Ferrer, I., 2014. Microalgae conversion to biogas: thermal pretreatment contribution on net energy production. Environ Sci Technol 48, 7171-7178.
383
Pham, H.M., Kwak, H.S., Hong, M.E., Lee, J., Chang, W.S., Sim, S.J., 2017. Development of
384
an X-Shape airlift photobioreactor for increasing algal biomass and biodiesel production.
385
Bioresour Technol 239, 211-218.
386
Qi, F., Pei, H.Y., Hu, W.R., Mu, R.M., Zhang, S., 2016. Characterization of a microalgal 16
ACCEPTED MANUSCRIPT 387
mutant for CO2 biofixation and biofuel production. Energ Convers Manage 122, 344-349.
388
Ramirez-Verduzco, L.F., Rodriguez-Rodriguez, J.E., Jaramillo-Jacob, A.D., 2012. Predicting
389
cetane number, kinematic viscosity, density and higher heating value of biodiesel from its
390
fatty acid methyl ester composition. Fuel 91, 102-111. Ramos, M.J., Fernandez, C.M., Casas, A., Rodriguez, L., Perez, A., 2009. Influence of fatty
392
acid composition of raw materials on biodiesel properties. Bioresour Technol 100, 261-
393
268.
394 395
RI PT
391
Rittmann, B.E., McCarty, P.L., 2012. Environmental biotechnology: principles and applications. McGraw-Hill Education, New York.
Rodriguez-Abalde, A., Flotats, X., Fernandez, B., 2017. Optimization of the anaerobic co-
397
digestion of pasteurized slaughterhouse waste, pig slurry and glycerine. Waste Manag 61,
398
521-528.
M AN U
SC
396
399
Sadiq, I.M., Pakrashi, S., Chandrasekaran, N., Mukherjee, A., 2011. Studies on toxicity of
400
aluminum oxide (Al2O3) nanoparticles to microalgae species: Scenedesmus sp and
401
Chlorella sp. J Nanopart Res 13, 3287-3299.
Salama, E.S., Kurade, M.B., Abou-Shanab, R.A.I., El-Dalatony, M.M., Yang, I.S., Min, B.,
403
Jeon, B.H., 2017. Recent progress in microalgal biomass production coupled with
404
wastewater treatment for biofuel generation. Renew Sust Energ Rev 79, 1189-1211.
405 406
TE D
402
Santacesaria, E., Vicente, G.M., Di Serio, M., Tesser, R., 2012. Main technologies in biodiesel production: State of the art and future challenges. Catal Today 195, 2-13. Sarin, A., Arora, R., Singh, N.P., Sarin, R., Malhotra, R.K., Kundu, K., 2009. Effect of blends
408
of Palm-Jatropha-Pongamia biodiesels on cloud point and pour point. Energy 34, 2016-
409
2021.
411 412
Silverstein, R.M., Webster, F.X., Kiemle, D.J., Bryce, D.L., 2014. Spectrometric
AC C
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EP
407
identification of organic compounds. John wiley & sons, New York. Sivaramakrishnan, R., Incharoensakdi, A., 2017. Direct transesterification of Botryococcus sp.
413
catalysed by immobilized lipase: Ultrasound treatment can reduce reaction time with high
414
yield of methyl ester. Fuel 191, 363-370.
415 416
Sung, M., Han, J.I., 2016. Alkaline in situ transesterification of Aurantiochytrium sp. KRS 101 using potassium carbonate. Bioresour Technol 205, 250-253.
417
Tran, H.-L., Hong, S.-J., Lee, C.-G., 2009. Evaluation of extraction methods for recovery of
418
fatty acids from Botryococcus braunii LB 572 and Synechocystis sp. PCC 6803. 17
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Biotechnology and Bioprocess Engineering 14, 187-192.
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Tsolcha, O.N., Tekerlekopoulou, A.G., Akratos, C.S., Aggelis, G., Genitsaris, S., Moustaka-
421
Gouni, M., Vayenas, D.V., 2017. Biotreatment of raisin and winery wastewaters and
422
simultaneous biodiesel production using a Leptolyngbya-based microbial consortium. J
423
Clean Prod 148, 185-193. Xu, M., Bernards, M., Hu, Z., 2014. Algae-facilitated chemical phosphorus removal during
425
high-density Chlorella emersonii cultivation in a membrane bioreactor. Bioresour Technol
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153, 383-387.
428
Yang, F., Hanna, M.A., Sun, R., 2012. Value-added uses for crude glycerol--a byproduct of biodiesel production. Biotechnol Biofuels 5, 13.
SC
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Yu, Z., Song, M.M., Pei, H.Y., Han, F., Jiang, L.Q., Hou, Q.J., 2017. The growth
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characteristics and biodiesel production of ten algae strains cultivated in anaerobically
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digested effluent from kitchen waste. Algal Res 24, 265-275.
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Table
436 437
Table 1. Characteristics of wastewater before treatment
438
Total nitrogen (mg N/L)
Total phosphorus (mg P/L)
877.4 ± 1.9
251.1 ± 0.1
16.1 ± 0.7
817.8 ± 26.0
112.2 ± 2.9
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Livestock wastewater (LWW) Slaughterhouse wastewater (SWW)
CODCr (mg O2/L)
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5.1 ± 0.6
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Table 2. Comparison of biodiesel production yield using the microalgal biomass Conditions
Yield
C. vulgaris
25℃/2h/Acid and base catalysts
9.2% a
C. sorokiniana
25℃/2h/ Acid and base catalysts
6.2% a
B. braunii
25℃/2h/ Acid and base catalysts
6% a
Chlorella sp.
70℃/24h/Lipase catalyst
36.7% a
Chlorella sp.
220℃/30min/DES (deep eutectic solvent)
13.9%a
Pan et al. (2017)
12.1%a
Tran et al. (2009)
Kim et al. (2017)
100℃/1h/ Solvent B. braunii (mixture of acetyl chloride and methanol)
442 443
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100℃/1h/ Acid catalyst (methanol, H2SO4)
81.2%b (Lipid content: 28%)
B. braunii
40℃/6h/ Lipase catalyst (with sonication)
88% b (Lipid content: 23%)
This study This study This study Jo et al. (2014)
Sivaramakrishnan and Incharoensakdi (2017)
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Relative to dry biomass percentage (%), b Relative to lipid (%).
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Reference
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Species
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Table 3. Fuel properties of biodiesel from microalgal biomass (SV: Saponification value, IV: Iodine value, CN: Cetane number, LCSF: Long-chain
445
saturated factor, CFPP: Cold filter plugging point, CP: Cloud point, N: Kinetic viscosity, P: Density, HHV: Higher heating value) SV
IV
Unit
mg KOH/g
g I2/100g
This study
119.5~191.5
24.2~41.1
66.0~84.3
9.8~18.4
ASTM D6751
-
120 max
47 min
-
EN 14214
-
120 max
51 max
-
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Petroleum diesel
-
-
-
-
93.7
52.7
208.2
185.2
30.9
-
19.1
62.6
-
32.0
-
32.8
(Monoraphidium sp.) 446
- Not specified
LCSF
CFPP ℃
61.1
14.4~41.4
CP
N
P
HHV
℃
40℃, mm2/s
g/cm3
MJ/kg
3.9~20.5
2.1~3.4
0.50~0.80
22.4~35.7
-
1.9~6.0
0.80~0.90
-
-
-
3.5~5.0
0.86~0.9
35 min
-
-
-
1.9~6.0
0.82~0.86
44 max
-
-
-
-
-
38.4
7.0
5.44
-
-
-
-
-
-
18.9
5.2
0.87
38.7
-
-
16.6
5.1
0.87
39.0
-
-
16.4
5.1
0.87
39.0
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CN
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447
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C. sorokiniana at 80 , and (F) B. braunii at 80
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Fig. 3. FT-IR spectra of FAME from B. brauniii strain in various conditions: (A) FAME from B. braunii at 25 , (B) FAME from B. braunii at 80
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Fig. 4. Inoculum basis FAME recoveries obtained from wastewater treated microalgal biomass via direct-transesterification process: (A) Livestock wastewater treated B. braunii (phototrophic), (B)
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Fig. 5. FT-IR spectra of B. braunii biomass: (A) Inoculum, (B) Livestock wastewater treated biomass, and (C) Slaughterhouse treated biomass
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Highlights Direct-transesterification and anaerobic digestion were converged for enhancement
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FAME recovery from microalgae grown by LWW reaches 50% of inoculum biomass
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Transesterification inhibition by EPS- and SMP-proteins were newly detected
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Coupling anaerobic digestion synergistically convert glycerol waste into bioenergy
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