Simultaneous Extraction, Fractionation, and Enrichment of Microalgal

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Simultaneous Extraction, Fractionation and Enrichment of Microalgal Triacylglyerides by Exploiting the Tunability of Neat Supercritical Carbon Dioxide Thomas Alan Kwan, Qingshi Tu, and Julie Zimmerman ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b02214 • Publication Date (Web): 12 Oct 2016 Downloaded from http://pubs.acs.org on October 20, 2016

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ACS Sustainable Chemistry & Engineering

Simultaneous Extraction, Fractionation and Enrichment of Microalgal Triacylglyerides by Exploiting the Tunability of Neat Supercritical Carbon Dioxide

1 2 3 4

Thomas A. Kwan a, Qingshi Tu a and Julie B. Zimmerman a,b

a

5 6 7 8 9 10

Department of Chemical and Environmental Engineering, Yale University, 9 Hillhouse Avenue, New Haven, CT, USA.

11

Keywords: solubility separation, biorefinery, energy analysis

12 13

Abstract: Continuous flow supercritical carbon dioxide (scCO2) was used to simultaneously

14

extract, fractionate, and enrich triacylglycerides (TAGs) of different carbon chain lengths and

15

degrees of unsaturation from a 6-component mixture of analytical standards and from Chlorella

16

sp., a common microalgae used as biodiesel feedstock. Evidence is presented that solubility for a

17

given TAG can be enhanced or diminished in the presence of other, dissimilar TAGs due to

18

solute to solute interactions and scCO2 densities.

19

Chlorella sp. extracts suggests that TAG mixtures and biomass matrices influence extraction

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behavior with the same TAG behaving differently depending on the starting material. This was

21

validated by fitting Chrastil’s solubility parameters to indicate which TAGs were most

22

susceptible to solute to solute interactions and neat CO2 density.

23

comparing the energy requirements for the downstream purification of TAGs trimyristin (C14:0),

24

tripalmitin (C16:0), and trierucin (C22:1) from mixtures extracted with the scCO2 method

25

developed here. The model outputs suggest trade-offs for over- or under-enriching certain TAGs

26

using neat scCO2. Both the experimental and modeling results demonstrate the potential of

b

School of Forestry and Environmental Studies, Yale University, 195 Prospect St, New Haven, CT, USA. E-mail: [email protected], Tel: +1-203-432-9703

Comparison of analytical standard and

1 ACS Paragon Plus Environment

A model was developed


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scCO2 to be used as an initial fractionation and enrichment tool whereby the associated energy

28

and economic costs of downstream processing, such as distillation, can be reduced.

29 30 31

1. Introduction Increasing awareness of the adverse environmental, economic, social, and political impacts

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resulting from our dependence on fossil fuels is driving the need to develop greener products and

33

processes in support of a bioeconomy.1-3 Development of these clean technologies presents

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significant opportunities for environmental and societal benefit as well as economic growth

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through advancing a renewable chemicals industry.4 However, supplanting petroleum-derived

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fuels and chemicals is challenging as modern refineries efficiently produce a multitude of

37

valuable products by utilizing every fraction of crude oil.5 Recent research has aimed to develop

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analogous biorefinery technologies to fully transform biomass into biofuels and useful chemicals

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while realizing returns on energy investments.6 A number of challenges remain in the

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development of biorefineries; chief among them is the efficient and cost-effective separation and

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purification of the targeted compounds.7

42 43

Lipids are one group of compounds that has been targeted for biorefining as they can be used for

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the production of biofuels, renewable chemicals, and dietary supplements.8-9 Due in part to their

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variety of triacylglyceride (TAG) lipid content, including saturated and unsaturated fatty acids

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with medium-chain (C10-C14), long-chain (C16-C18), and very-long-chain (C≥20) lengths,9-10

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microalgae have gained attention as a sustainable feedstock for a multitude of uses. Of particular

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interest is Chlorella sp., a microalgae, that produces reasonable quantities of polyunsaturated

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fatty acids (PUFAs) (e.g., octadecadienoic (C18:2); octadecatrienoic (C18:3)), monounsaturated

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fatty acids (MUFAs) (e.g., hexadecenoic (C16:1); octadecenoic (C18:1)), and saturated fatty 2 ACS Paragon Plus Environment

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acids (e.g., tetradecanoic (C14:0); eicosanoic (C20:0)).11 Depending on chain length and degree

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of unsaturation, lipid preservation or transformation may be desirable. For example, the PUFA,

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octadecatrienoic acid (C18:3) is used directly as a nutritional supplement in infant formulas12 and

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MUFAs, octadecenoic (C18:1) and hexadecenoic (C16:1), can be transformed to alkyl esters via

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transesterification to produce an optimal biodiesel because of their ignition and cold-flow

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properties.13 Saturated medium-chain and long-chain fatty acids are utilized in pharmaceutical,

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chemical, and food applications while upgrading stearic acid (C18:0) and palmitic acid (C16:0)

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to iso-alkanes for transportation fuels is actively being investigated.14-15

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Enhancing the efficient and cost-effective extraction, fractionation, and purification of TAGs

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will facilitate their exploitation, a significant step towards a viable biorefinery and ultimately a

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biobased economy.16 However, cost-effective and sustainable methods are still needed as

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traditional organic solvents are non-selective for TAG, necessitate further downstream separation

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processes, are toxic, and are costly to recycle.17 Despite high capital cost and potentially high

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energy requirements, supercritical carbon dioxide (scCO2) has been heralded as a scalable,

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tunable, and green technology for the extraction of microalgae lipids.18 Unlike organic solvents,

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scCO2 is non-toxic, produces solvent-free lipids, has a high selectivity for TAG, and is suitable

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to produce extracts for human consumption.19 The efficacy of scCO2 as a solvent primarily

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depends on its density, which can be controlled by varying pressure and/or temperature.20 This

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tunability has been leveraged to optimize total TAG yield, but not necessarily fractionation and

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enrichment, for various microalgae and extraction conditions.7

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A number of models have been developed to explain the extraction kinetics of a biomass/scCO2

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system describing an initial, rapid extraction phase limited by solute solubility followed by a

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slower extraction phase limited by internal mass transfer.21 Controlling extraction kinetics is

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essential for scaling biomass/scCO2 technologies, optimizing fractionation, and evaluating

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industrial scale economics.19, 22 Consequently, understanding TAG solubility behavior in scCO2,

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particularly from microalgal biomass, is key as it regulates kinetics during the first phase of

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extraction (Figure 1; inset). Previous studies using binary and tertiary mixtures of analytical

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standard TAGs have shown the solubility of a given TAG is affected by solute-solute

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interactions with other TAGs of various chain lengths and degrees of saturation.23-24 This effect

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can diminish or enhance relative solubility such that a TAG of lesser solubility in scCO2 at a

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given density can demonstrate enhanced solubility in the presence of another, more soluble

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TAG. Conversely, more soluble TAGs can exhibit diminished solubility in the presence of a less

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soluble TAG.24 These results have implications toward separation efficiency and fractionation

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using scCO2. Extraction systems can potentially be designed and operated to favor or disfavor

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solubilizing certain TAGs based on chain length and degree of saturation to minimize or negate

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downstream separation processes. However, this requires greater understanding of the behavior

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of complex TAG mixtures extracted from actual biomass as well as biomass matrix effects.

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This study evaluated the tunability of neat scCO2 to extract and fractionate TAGs from Chlorella

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sp. in a continuous flow system with a high resolution of temporal information. These results are

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then compared to the behavior of mixtures of TAG analytical standards. Combinations of

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pressures and temperatures were used to achieve different scCO2 densities. Results were

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assessed by solubility separation factors and solvato complex behavior (the necessary molecular


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association between solvent and solute to transition the TAG into scCO2) as well as the heats of

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solvation and vaporization. The potential of scCO2 to be used as an initial fractionation and

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enrichment tool is demonstrated whereby the associated energy and economic costs of

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downstream processing, such as distillation, can be reduced.

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101 102 103 104

2. Experimental materials and methods

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solvents with HPLC or better grade were purchased from Sigma-Aldrich. TAG standards were

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obtained from Nu-Chek Prep, Inc.; bone dry (≥99.8%) liquid carbon dioxide and ultra-high

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purity (99.999%) nitrogen gas was purchased from Airgas, Inc. Microalgae Chlorella sp. was

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grown in the University of Arizona’s Aquaculture Raceway Integrated Design (ARID),25

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lyophilized, ground with a pestle and mortar, and sieved to a particle size between 250 and 355

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microns.

111 112

2.2 Extraction apparatus

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through a stainless steel tube, heated with a CAL ETC1311 controller and insulated heating tape,

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to a 25 mL SFT-100 extractor vessel from Supercritical Fluid Technologies, Inc. In addition to

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the two SFT-100 temperature probes, three additional probes monitored temperature at the inlet

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of the SFT-100, the extraction cylinder, and outlet line after the restrictor valve.

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temperatures were held constant, never exceeding 3⁰C deviation from the extraction vessel inlet,

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extraction vessel, and outlet line. Constant temperature from the point of biomass extraction to

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collection was setup to prevent reflux. It should be noted the temperature set point for the fluid

120

delivery and restrictor/outlet lines were higher than the targeted value to heat the incoming flow

2.1 Standards, reagents, and biomass Fatty acid methyl ester (FAME) analytical standards, boron trifluoride (BF3), and organic

Two ISCO 100DX pumps setup for continuous constant pressure delivered carbon dioxide


System


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from room temperature and prevent freezing of the outlet due to the Joule-Thompson effect. All

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samples were collected in 5mL glass vial sample traps placed in an ice bath, excavated with N2

123

gas and sealed with Teflon lined caps and stored at -20 ⁰C until derivatization (Chlorella sp.

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extracts) or analysis (analytical standard extracts).

125 126

2.3 Supercritical carbon dioxide

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temperature and pressure extraction conditions using the National Institute of Standards and

128

Technology (NIST) Reference Fluid Thermodynamic and Transport Properties Database

129

(REFPROP).27 Analytical standards or microalgae biomass were loaded in the extraction vessel

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and the system was heated to the specified temperature. Pressurized CO2 was then introduced

131

and equilibrated for 15 minutes after which the restrictor and outlet valves were opened and

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sample collection of the continuous flow began. CO2 flowrate was monitored from the ISCO

133

pump controller and a flowmeter connected to the sample trap.

134 135

2.4 Experimental conditions

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component mixture of trimyristin, tripalmitin, tripalmitolein, tristearin, triolein, and trierucin.

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300 mg of each TAG was loaded in the extraction vessel and each extraction was triplicated. For

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biomass extractions, 5 grams of Chlorella sp. were loaded and the extractions triplicated for each

139

system condition evaluated. Extractions of analytical standards were all set to 40 ⁰C with

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pressures of 8.31, 9.3, 13.4, and 17.2 MPa for system condition referred to as very low, medium,

141

high, and very high density respectively. Extractions of Chlorella sp. were run at 37 ⁰C and

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8.3MPa, 60 ⁰C and 13.8 MPa, and 40 ⁰C and 13.4 MPa for system conditions referred to with

143

low, medium, and high density respectively. scCO2 density was thus 384 (very low), 550-556

Span and Wagner’s carbon dioxide equation of state26 was used to calculate densities based on

Extractions of TAG standards used a binary mixture of tripalmitin and triolein and a six


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(medium), 750 (high), and 812 (very high) mg/ml. The flowrate for all extractions was 2.8 ± 0.3

145

mg/min.

146 147

Samples were collected in ~5 minute intervals for the first 30 minutes, then ~10 minute intervals

148

for the next 30 minutes. After the initial hour of collection, samples were collected in ~15

149

minute intervals. The initial, rapid sample collection was necessary to characterize the solubility

150

during the first phase of Chlorella sp. extractions.

151 152

2.5 Analysis

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6890 gas chromatography (GC) unit equipped with an Agilent Select Biodiesel (15 m, 0.32 mm,

154

0.10 µm) column and a flame ionization detector (FID) set to 400⁰C. The inlet temperature was

155

set to 250⁰C with an 80:1 split, helium carrier gas at 4ml/min, the initial oven temperature was

156

set to 40⁰C and held for 5 minutes, ramped up to 180⁰C at 15⁰C per minute, then to 280⁰C at 5⁰C

157

per minute, then to 380⁰C at 15⁰C per minute and held for 10 minutes. Lipid extracts of

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Chlorella sp. were reconstituted in chloroform and transesterified to FAME for analysis using

159

14% boron trifluoride (BF3) in methanol for 1 hour at 95 ⁰C.28-29 The FAME was collected in

160

hexane and analyzed via the GC-FID equipped with an SP-2560 Capillary Column (100 m ×

161

0.25 mm, df 0.20 µm). The inlet temperature was set to 250⁰C with an 80:1 split, helium carrier

162

gas at 4ml/min, the initial oven temperature was set to 140⁰C and held for 5 minutes, ramped to

163

180⁰C at 8⁰C per minute, then to 210⁰C at 8⁰C per minute, then to 250⁰C at 20⁰C per minute and

164

held for 10 minutes. FID temperature was 250⁰C. TAG extracts and FAME were identified and

165

quantified by comparing retention times and responses to known analytical standards.

TAG standard extracts were reconstituted in a 1:1 chloroform/hexane and analyzed with an HP

166



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The obtained FAME profile of Chlorella sp. was used as a proxy for the TAG content; intact

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TAG tend to have the same or similar fatty acid groups, particularly ones that predominate the

169

lipid profile such as the profiles presented in this study.30 Chlorella sp. extracts were calculated

170

assuming a TAG composed of identical fatty acid chains. In this way, the transesterification

171

served only to identify chain length and degree of saturation of the solutes and enabled a

172

comparison to the extractions of TAG standards where each fatty acid chain was known to be

173

identical.

174 175 176

2.6 Solubility data

177

extraction results described by (Eq. 1):

Continuous flow solubility for each compound was calculated using the linear first phase

∗ =

178

(Eq. 1)

179

where yi* is the apparent solubility of solute i in scCO2 at a given density. Additionally, a

180

solubility separation factor, αij, was calculated as a ratio of apparent solubility of solutes i and j,

181

yi* and yj*, respectively (Eq. 2). ∗

182

= ∗

183

First phase extraction results were also used to fit Chrastil’s equation for compound solubility.

184

Chrastil’s equation (Eq. 5) is valid for TAG in scCO2 with solubilities less than 100-200 g/L

185

where good fits with experimental data may indicate no solute to solute interaction and poor fits

186

suggesting that solute-solute interactions affect solubility. The formation of a solvato complex in

187

equilibrium with no solute to solute interaction is defined by (Eq. 3.):

188

∆ !"#$%&"' ()

+ + , + -./ + 0-1/ = -.12 /


(Eq. 2)

(Eq. 3)

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where Y is the solute, Z is the dense gas, k is the number of molecules of gas to form a solvato

190

complex, R is the gas constant, T is temperature, qs is a constant, and YZk is the solute

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concentration in the dense gas. Chrastil approximated solute concentration in the supercritical

192

fluid by the Clapeyron-Clausius equation: -./ =

193 194

∆ $%3"45%&"' ()

+ +

(Eq. 4)

where qv is a constant. By combining Eq. 3 and Eq. 4, Chrastil’s equation31 is:

∗ = 6 2 exp ( + ;) )

195

(Eq. 5)

196

where d is the density of the gas and k is a density dependent association number between solute

197

and solvent. a accounts for the enthalpy of solute vaporization and enthalpy of a single solute to

198

form a solvato complex in a neat, dense gas and is equivalent to: ==

199

∆ !"#$%&"' >∆ $%3"45%&"' (

(Eq. 6)

200

An association parameter, k, describes the number of molecules required to form a solvato

201

complex with a single solute. The b parameter characterizes the effect of molecular weights and

202

includes constants from the Clapeyron-Clausius equation and the equilibrium heat of solvation as

203

described by: ; = ln(AB + 0AC ) + + + + − 0ln (AC )

204 205

(Eq. 7)

where MY and MZ are the solute and solvent molecular weights.

206 207

Additionally, Stahl32-33 showed that at constant temperature, Equation 6 is linear for single

208

substances:

209

ln(∗ ) = 0 E ln 6 + ; E


(Eq. 8)


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210

Experimental data from analytical standard TAG was used to fit parameter k’ using the slope of

211

the solubility isotherm, ln(y*) vs. ln(d), and the b’ parameter is reported as the intercept of the y*

212

axis.

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Experimental data from Chlorella sp. was used to initially fit k using the slope of the solubility

215

isotherm, ln(y*) vs. ln(d) while a was fit using the slope of ln(y*) plotted against 1/T according

216

to Chrastil’s equation.31,

217

(GRG) algorithm was used to estimate b with the freedom to adjust a and k. Convergence was

218

achieved for all b parameters such that a and k did not deviate from the initial experimental data

219

values.

33

With initial values for k and a, the Generalized Reduced Gradient

220 221 222

2.7 Downstream energy consumption

223

was modeled in Aspen Plus V9. The process flow diagram is in supporting information S.1.

224

Process energy consumption of four initial TAG mixtures were based on the first phase

225

extraction results from this study using three different scCO2 densities and a traditional Bligh and

226

Dyer solvent extraction.34 The objective modeled was to separate trimyristin (C14:0), tripalmitin

227

(C16:0) and Trierucin (C22:1) into high purity (>99%) streams from the rest of the TAGs. The

228

process flow diagram (PFD) and initial composition of the TAG mixture for each scenario is

229

available in supporting information.

Energy consumption for the downstream separation of TAG mixtures by molecular distillation

230 231

The Peng-Robinson equation of state35 was used for TAG property estimation; due to the missing

232

parameters in the Aspen component database, the concentration of tripalmitolein (C16:1) was

233

combined with trimyristin (C14:0) based on the Equivalent Carbon Number (ECN)36 and the 10 ACS Paragon Plus Environment

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234

concentration of triarachidin (C:20:0) and tribehenin (C:22:0) were combined with trierucin

235

(C22:1). Three distillation columns were assumed to have 20 trays and a pressure of 1.3e-6 MPa.

236

Reflux ratios were varied to minimize the energy consumption while maintaining the targeted

237

purity for the trimyristin and tripalmitin streams.

238

239 240 241 242 243

3. Results and Discussion

244

analytical standards at a mass ratio of 1:1:1:1:1:1 were loaded into the vessel and extracted at

245

three different CO2 densities, 550, 750, and 812 mg/ml CO2. These densities were chosen to

246

represent the tunability of scCO2 and are referred to as medium, high, and very high density

247

respectively. Previous studies have shown that the solubility of analytical standards will be

248

unchanged for initial mass loading differences of up to 20%.37 The apparent solubility of each

249

TAGs using 2.8 mg/min of CO2 is shown in Figure 2 and supports that the solubility of various

250

TAGs changes with CO2 density. This is determined by the slope of the linear portion of the

251

initial extraction phase (Figure 1).

3.1 Impact of CO2 density on solubility of analytical standard mixtures To more readily approximate the complex mixtures that result from biomass, a mix of six


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Extract (mg)


252 253 254 255 256 257 258

Figure 1: First phase extraction behavior of a mixture of six analytical standard triacylglycerides at high CO2 density (750 mg/ml) with a flow rate of 2.8 mg/min over 107 minutes as an example. Linear first phase behavior was measured for each triacylglyceride extracted from a mix of analytical standards and from the microalgae Chlorella sp. in this study. Insert: A representation of the first phase linear relationship between the amount of TAG extracted and the amount of solvent delivered, which is characterized by being solubility limited, whereas the second phase is mass transfer limited.

259 260

While trierucin (C20:1), the longest chain TAG evaluated in the analytical standards mixture,

261

demonstrates the highest solubility at both densities, the solubility of the other TAGs changes in

262

relation to each other at the various densities (Figure 2; analytical standards). It is interesting to

263

note, that the shortest TAG chain, trimyristin (C14:0), consistently demonstrates an apparent

264

solubility that is in the middle of the group. Further, tripalmitolein (C16:1), a mid-chain length

265

TAG in this mixture, has its apparent solubility reduced from 0.11 at medium CO2 density to the

266

lowest measured solubilities of 0.05 and 0.11 for high and very high densities of CO2, 12 ACS Paragon Plus Environment

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267

respectively. (The tabulated y* data is available in supporting information S.2.) This indicates a

268

potential strategy to fractionate complex TAG mixtures by changing the density of CO2 during a

269

dynamic extraction. In this way, the density of the CO2 can be tuned to increase the separation of

270

certain MUFAs (i.e., enriching for C20:1 and deselecting for C16:1 at very high CO2 densities)

271

before altering the density of the CO2 (i.e., lowering the density to 550 mg/ml CO2) to then

272

selectively remove other TAGs (i.e., C16:1) from the remaining mixture (i.e., depleted of C20:1).

273

Solubilities at very high density were close to or exceeded the recommended valid range for

274

Chrastil’s equation of 100-200 g/L (y* values greater than 0.12-0.25 at this density). For this

275

reason, the very high density condition was not evaluated for Chlorella sp. extraction

276

experiments.

0.3

Analytical Standards

Chlorella sp. Saturated TAG C14:0 C16:0 C18:0 C20:0

0.2

C22:0 C24:0 MUFA TAG C16:1 C18:1

0.1

C20:1 C22:1 PUFA TAG C18:2 C18:3

0.0 Medium

High

Low

Medium

High

Supercritical Carbon Dioxide Density 277 278 279

Figure 2: Measured solubility, y*, and scCO2 densities for multi-component analytical standard TAG mixtures and TAGs extracted from the microalgae Chlorella sp. (Note: extracts from Chlorella sp. were



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280 281

modeled to be a triacylglyceride with identical fatty acids and reported as the modeled chain length and saturation.)

282 283 284 285 286

3.2 Solubility separation

287

traditionally assumed to exhibit similar properties as a solute38. The separation factors for

288

tripalmitin and triolein from the binary, six analytical standards mixture, and Chlorella sp.

289

extractions are presented in Figure 3. For all conditions, scCO2 density had an effect on the

290

separation factor. At lower density conditions, the extract was enriched for C18:1 while the at

291

higher densities the extract was enriched for C16:0 in all of the analytical standard mixtures.

292

Interestingly, at a CO2 density of 750 mg/ml, the separation factor, αC16:0 / C18:0, was similar at 1.3

293

and 1.4 for the binary and multi component mixture respectively, but was 0.65 (enriching for

294

C18:1) when extracting from Chlorella sp.. The enrichment trend was different for the

295

microalgae extracts such that at lower densities C16:0 was favored and at higher densities C18:1

296

was favored. Solubility separation factors for each TAG pair at all of the conditions evaluated in

297

this study are available in the supporting information S.3.

Tripalmitin (C16:0) and triolein (C18:1) have an equivalent carbon number (ECN) and are

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299 300 301

Figure 3: Relative amounts of tripalmitin (C16:0) and triolein (C18:1) extracted depend on the state of CO2 as well as the matrix from which it is being extracted from. Densities of scCO2 evaluated were 384, 463, 550, 750, and 812 mg/ml labeled as very low, low, medium, high, and very high respectively.

302 303

Total yield during the first phase extraction for all system conditions increased with increasing

304

density. However, the lipid composition of the extract differed for each density condition. The

305

analytical standards mixture saw an increase in trimystirin (C14:0), tripalmitin (C16:0), tristearin

306

(C18:0), and trierucin (C20:1) from medium to high density while tripalmitolein (C16:1)

307

decreased and triolein (C18:1) was unchanged. Similar compounds extracted from Chlorella sp.

308

with medium and high CO2 densities showed that C16:1 and C18:1 increased, C18:0 decreased,

309

and C16:0 was unchanged.

310 311

The dissimilar solubility trends of analytical standards and Chlorella sp. extracts at different

312

density conditions suggest that the mixture (and matrix) from which TAG are extracted has an

313

impact on solubility behavior. Because the number of unique TAGs in the microalgae was

314

greater than that of the analytical standards, more solute/solute interactions may be modifying

315

their solubility. Additionally, at a high scCO2 density the apparent solubility of C16:1 and C18:1

316

(MUFAs) are higher from the microalgae extraction compared to the analytical standards. While

317

the TAG extracts from the analytical standard mixture showed no clear MUFA behavior trend,

318

both MUFA and PUFA compounds all increased in yield and solubility with increasing density

319

when extracted from Chlorella sp.

320 321

Interestingly, at medium and high scCO2 densities, the apparent solubility for similar compounds

322

extracted from microalgae compared to the analytical standards mixtures are dissimilar. The

323

apparent solubility of C16:0 extracted from the analytical standards mixture increased with



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324

increasing density while a slight depression was observed for extractions from Chlorella sp..

325

Note that in the analytical standards mixture, C16:0 was approximately 17% of the total mass but

326

accounted for almost 50% of the microalgae lipids. Because of the differences in initial mass

327

loadings, as well as the absence or presence of a complex biomass matrix, the different behaviors

328

and trends of the C16:0 TAG suggest that the matrix has an effect on the apparent solubility of

329

this TAG in scCO2 regardless of density.

330 331 332

3.3 Solvato complex and solute to solute interactions

333

solute to solute interactions. The parameters described in Eqs. 5 and 8 are listed in Table 1,

334

ordered from poorest fit to best fit, and were derived from the results presented in Figure 2

335

(complete data set available in supporting information S.2). Results from Chrastil’s original

336

investigation31 and Güclu-Üstundag and Temelli’s review33 for select individual TAGs where

337

solute to solute interactions do not exist are also presented for comparison.

Chrastil’s solubility parameters are derived from equilibrium thermodynamics and assume no

338 339 340 341 342 343 344

Table 1: Solubility parameters and their respective coefficient of determination from this study (indicated by *) and from other studies for select individual TAG to compare values derived from a mixture versus single component extractions where no solute-solute interactions exist. Results are ordered from poorest to best fit. †the authors cite a low R2 value as a result of combining data sets from different

studies

C16:1 (Tripalmitolein)* C18:1 (Triolein)* C20:1 (Trierucin)* C16:0 (Tripalmitin)* C18:0 (Tristearin)* C14:0 (Trimyristin)* C14:0 (Trimyristin)33 C16:0 (Tripalmitin)33

Class MUFA MUFA MUFA Saturated Saturated Saturated Saturated Saturated

Analytical standards k' b' 0.4 1.7 2.9 -14.7 4.2 -22.4 6.3 -36.5 6.0 -35.0 3.7 -20.1 9.27 -61.0 5.50 -32.1


R2 0.036 0.635 0.749 0.776 0.960 0.984 0.991 0.601†

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C20:0* C18:0* C14:0* C22:0* C18:2* C18:1* C24:0* C16:0* C18:3* C20:1* C16:1* C16:031 C18:131

Class Saturated Saturated Saturated Saturated PUFA MUFA Saturated Saturated PUFA MUFA MUFA Saturated MUFA

b -10.6 0.7 -3.0 4.3 -2.9 -2.5 -8.8 4.6 -0.7 -0.9 0.7 -12.15 2.42

Chlorella sp. k a 1.5 808.4 0.7 -694.5 -1.7 4801.5 2.1 -5157.9 6.3 -10717.2 6.5 -11207.3 17.3 -32297.2 0.8 -1634.0 9.4 -18029.9 5.0 -9607.7 7.8 -15273.6 2.98 -2387.8 5.22 -11386.5

R2 (k) 0.042 0.208 0.287 0.478 0.807 0.824 0.971 0.985 0.994 0.997 0.999 -

R2 (a) 0.003 0.045 0.539 0.729 0.571 0.592 0.826 0.981 0.891 0.903 0.923 -

345 346

The coefficient of determination (R2), which indicates limited solute to solute interaction as it

347

approaches 1, ranged from 0.036 to 0.984 (Eq. 8) for extracts from the analytical standards

348

mixture. The k and a parameter values ranged from 0.042 to 0.999 and 0.003 to 0.923 (Eq. 6),

349

respectively, for Chlorella sp. extracts. MUFA in the analytical standards mixture generally had

350

poor fits to Eq. 8 while saturated TAGs had better fits. In contrast, MUFA and PUFA TAG

351

extracted from the biomass tended to have better fits for k and saturated TAG tended not to fit

352

Eq. 6 with the exception of C24:0 and C16:0 with k fits of 0.9712 and 0.9852 and a fits of 0.8262

353

and 0.9812 respectively. The a parameter suggests which TAGs are likely to be affected by

354

temperature and solutes with poor fits, such as C20:0 and C18:0, are more difficult to predict in

355

terms of first phase solubility behavior. This variability may be due to the affect mixtures have

356

on the heat of solvation and vaporization needed to transition into the scCO2 solvent. R2 values

357

for k and a parameters trended together as expected given that TAGs susceptible to solubility

358

effects stemming from mixture impacts would also be susceptible to enthalpy effects.

359

Intermolecular interactions between dissimilar TAG can change their solubility behavior in



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360

scCO237 and may be explained mechanistically by the energy needed to form a solvato complex,

361

or the condensation of the solvent around a solute molecule.39 Evidence presented here shows

362

that dissimilar TAG can modify the formation of the solvato complex and consequently alter the

363

apparent solubility behavior in scCO2. R2 values for k and a parameters trended together as

364

expected given that TAGs susceptible to solubility effects stemming from mixture impacts would

365

also be susceptible to enthalpy effects.

366 367

These results suggest that MUFA and PUFA TAG extracted from microalgae may be less

368

affected by potential solute to solute interactions in the solvato complex and mixture impacts due

369

to the enthalpy of vaporization. However, saturated TAG tend to be more affected by solute to

370

solute interactions which may be acting as entrainers, potentially due to hydrogen bonding.40

371

Additionally, CO2 can form a weak, cooperative, hydrogen bond with carbonyl groups as shown

372

by a longer C-O bond of CO2 involved with a C-H•••O hydrogen bond.41 The absence of a

373

double bond in a saturated TAG provides more opportunities for hydrogen bonding. Saturated

374

TAG tend to be straighter and longer than unsaturated TAG, providing more surface area for

375

hydrogen bonding when compared to a fatty acid chain with the same carbon chain length. TAG

376

interaction with CO2 and TAG entrainer effects would corroborate the observed different

377

apparent solubilites of the same TAG extracted from Chlorella sp. compared to analytical

378

standards mixtures for similar operating conditions and scCO2 density.

379 380

MUFAs C16:1 and C20:1 extracted from Chlorella sp. yielded relatively high a (0.923 and

381

0.903, respectively) and k (0.999 and 0.997, respectively) fits indicating their extraction behavior

382

is effected by their physical properties and less susceptible to solute to solute interactions.


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383

Because the solvation power of scCO2 is attributed to density, and consequently its polarity, logP

384

values for MUFAs C16:1, C18:1, and C20:1 may provide additional insight to their apparent

385

solubility (Figure 4).

386 387

Figure 4: Apparent solubility of MUFA extracts from Chlorella sp. and relationship with their polarity.

388 389

The trend appears to show an optimal value of logP, between 19 and 21, which is responsive to

390

CO2 density. Though the apparent solubility of C20:1 was relatively low regardless of CO2

391

density, it fit Chrastil’s model well, implying that extracting other TAG at lower densities first,

392

then switching conditions to enrich for the long chain MUFA may offer another handle for TAG

393

extraction enrichment. Additionally, the logP and y* relationships indicate a very high density

394

will be required for optimal extraction of compounds with relatively high logP values. Lastly,

395

C16:1 demonstrated similar solubility at low and medium density conditions, but at high density

396

its solubility is increased seven-fold. This indicates that C16:1 can withstand a range of low to

397

medium densities if operators seek to enrich the extract for other TAGs at lower density

398

conditions.

399



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400 401

3.4 Downstream energy impacts and trade-offs

402

potential reductions of energy and economic costs associated with downstream processing, such

403

as distillation, were modeled. Both organic and scCO2 extraction systems can use wet or dry

404

biomass but the organic system will have ~18% less overall extraction efficiency.42

405

upstream energy required to dry microalgae was not considered in the analysis but has been

406

shown to be ~2.6 MJ/kg of water dried in both cases.43-44 However, even “wet” biomass is still

407

dewatered and can be considered a biomass slurry of 10-20% water.45 Because of this, dry

408

biomass was used to maximize the lipid yield from the organic solvent system and the energy

409

required for drying was assumed to be the same for both technologies. This results in the most

410

conservative estimate of energy comparisons for the two systems.

411

compositions were used in the analysis including those resulting from the low, medium, and high

412

scCO2 density extractions of Chlorella sp. as determined in this study as well as one resulting

413

from conventional organic solvent extraction using dried algae (Table 2).

414

Table 2: Initial composition of the TAG mixture prior to the modeled distillation purification.

Given the possible use of scCO2 as an initial fractionation and enrichment tool, an analysis of the

The

Four initial TAG

C14:0

C16:0

C16:1

C18:0

C18:1

C18:2

C18:3

C20:0

C22:0

C22:1

C24:0

Solvent extraction

7.09%

57.26%

3.14%

17.78%

4.76%

1.52%

2.69%

0.00%

0.00%

0.03%

5.73%

scCO2 low density

2.26%

67.97%

1.98%

13.67%

5.32%

3.68%

1.04%

0.96%

2.60%

0.50%

0.00%

scCO2 medium density

1.98%

26.55%

3.20%

10.55%

24.49%

16.66%

2.89%

12.39%

0.51%

0.50%

0.27%

scCO2 high density

0.19%

16.07%

14.37%

3.46%

24.61%

15.26%

16.68%

0.53%

0.99%

0.92%

6.93%

415 416

In all cases, the first distillation column purified C14:0 to at least 99.4%, the second column

417

purified C16:0 to at least 98.8%, and the third column purified C22:1 to at least 99.2%. Results

418

from the energy analysis showed that altering the TAG composition in the extraction step by

419

scCO2 can provide energy savings for downstream processing. Table 3 summarizes the reflux

420

ratio of each column as well as the energy consumption to purify the selected TAG reported as 20 ACS Paragon Plus Environment

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421

total and normalized to 1kg of purified TAG. Tripalmitin (C16:0) and trierucin (C22:1) were

422

relatively more concentrated after the low density scCO2 extraction; accordingly, the energy

423

consumption for the distillation process was lower compared to the other starting TAG

424

composition scenarios. On the other hand, when the relative tripalmitin concentration in the

425

initial TAG mixture was significantly reduced, as is the case for the initial composition of the

426

medium and high density scCO2 extracts, the energy consumption for separation was increased.

427 428 429

Table 3: Summary of normalized results for each TAG analyzed and starting with the four relative mixtures of TAG from the three different densities in this study and an organic extraction. Reflux ratio

Organic solvent scCO2 low density scCO2 medium density scCO2 high density

MJ/kg TAG purified

MJ/hr

Column 1

Column 2

Column 3

Column 1

Column 2

Column 3

Energy consumption

0.7

2.5

0.5

5.422

6.536

0.988

1.29

0.7

2.0

0.5

5.083

6.103

0.847

1.20

0.5

7.0

0.5

5.140

7.177

1.918

1.42

0.5

20

0.5

5.507

12.656

2.213

2.04

Energy consumption normalized to mass of TAG separated

Organic solvent

scCO2 low density

scCO2 medium density

scCO2 high density

kg/hr

MJ/kg

kg/hr

MJ/kg

kg/hr

MJ/kg

kg/hr

MJ/kg

C14:0

1.070

5.065

0.418

12.175

0.512

10.036

1.545

3.565

C16:0

5.976

1.094

6.694

0.912

2.619

2.740

1.682

7.523

C22:1

0.003

339.179

0.399

2.121

1.321

1.452

0.257

8.610

430 431 432

Energy consumption per mass of purified TAG did not always follow a linear relationship; that

433

is, higher initial enrichment did not necessarily mean a lower energy requirement to complete the

434

purification downstream. Non-linear trends may be attributed to the different lipid profiles being

435

distilled. For example, C22:1 accounts for 0.50% of the extract in both the low and medium

436

scCO2 density lipid profile but the relative amount of C18:1, C18:2, and C20:0 is higher and

437

C16:0 is lower in the medium density extract. Subsequently, the energy per mass of C22:1 21 ACS Paragon Plus Environment


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Page 22 of 26

438

purified is 2.121 and 1.452 MJ/kg for the low and medium density conditions respectively. The

439

trends are presented in Figure 5 on a log-log plot. C16:0 showed a linear trend meaning higher

440

extraction enrichments would reduce the energy needed for downstream purification. However,

441

for C14:0 and C22:1, a non-linear trend indicates an optimal amount of enrichment can be

442

targeted to reduce downstream energy consumption for purification. Because the analysis used

443

relative amounts of TAG mixtures from this study, extractions from other biomass and/or with

444

non-TAG fractions may make separation more or less energy intensive. These results, based on

445

extraction profiles from Chlorella sp., however do provide insight to the complex nature of

446

enriching for certain TAG in an extraction. Additional, more robust investigations to the

447

extraction and purification energy impacts and trade-offs in a biorefinery setting will be essential

448

to guide development of methods and technologies supporting a sustainable bioeconomy.

449

450 451 452 453

Figure 5: Downstream energy required to purify select TAG in relation to the relative amount of the TAG in the starting mixture extracted from microalgae. The trends indicate energy trade-offs may exist for over or under enriching various TAGs with scCO2 as a pre-treatment for distillation.

454 455 456 22 ACS Paragon Plus Environment

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457 458

Acknowledgements: This work was partially supported by the National Science Foundation

459

under Grant #152432 and Graduate Research Fellowship, Grant #DGE-1122492. Any opinions,

460

findings, and conclusions or recommendations expressed in this material are those of the

461

author(s) and do not necessarily reflect the views of the Nation Science Foundation.

462 463

Supporting Information: Process flow diagram of downstream distillation process and the

464

unabridged solubility separation factors.

465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492

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11. Wang, L.; Li, Y.; Chen, P.; Min, M.; Chen, Y.; Zhu, J.; Ruan, R. R., Anaerobic digested dairy manure as a nutrient supplement for cultivation of oil-rich green microalgae Chlorella sp. Bioresour. Technol. 2010, 101 (8), 2623-2628. 12. Spolaore, P.; Joannis-Cassan, C.; Duran, E.; Isambert, A., Commercial applications of microalgae. J Biosci Bioeng 2006, 101 (2), 87-96. 13. Stansell, G. R.; Gray, V. M.; Sym, S. D., Microalgal fatty acid composition: implications for biodiesel quality. J. Appl. Phycol. 2011, 24 (4), 791-801. 14. Biermann, U.; Bornscheuer, U.; Meier, M. A.; Metzger, J. O.; Schafer, H. J., Oils and fats as renewable raw materials in chemistry. Angew. Chem. Int. Ed. Engl. 2011, 50 (17), 3854-3871. 15. Shi, Y.; Cao, Y.; Duan, Y.; Chen, H.; Chen, Y.; Yang, M.; Wu, Y., Upgrading of palmitic acid to iso-alkanes over bi-functional Mo/ZSM-22 catalysts. Green Chem. 2016, (18), 46334648. 16. Brennan, L.; Owende, P., Biofuels from microalgae—A review of technologies for production, processing, and extractions of biofuels and co-products. Renewable Sustainable Energy Rev. 2010, 14 (2), 557-577. 17. Cooney, M.; Young, G.; Nagle, N., Extraction of Bio‐oils from Microalgae. Sep. Purif. Rev. 2009, 38 (4), 291-325. 18. Soh, L.; Zimmerman, J., Biodiesel production: the potential of algal lipids extracted with supercritical carbon dioxide. Green Chem. 2011, 13 (6), 1422-1429. 19. Halim, R.; Danquah, M. K.; Webley, P. A., Extraction of oil from microalgae for biodiesel production: A review. Biotechnol. Adv. 2012, 30 (3), 709-732. 20. Eckert, C. A.; Bush, D.; Brown, J. S.; Liotta, C. L., Tuning Solvents for Sustainable Technology. Ind. Eng. Chem. Res. 2000, 39 (12), 4615-4621. 21. Huang, Z.; Shi, X. H.; Jiang, W. J., Theoretical models for supercritical fluid extraction. J. Chromatogr. A 2012, 1250, 2-26. 22. del Valle, J. M., Extraction of natural compounds using supercritical CO2: Going from the laboratory to the industrial application. J. Supercrit. Fluid 2015, 96, 180-199. 23. Bharath, R.; Yamane, S.; Inomata, H.; Adschiri, T.; Arai, K., Phase equilibria of supercritical CO2 - fatty oil component binary systems. Fluid Phase Equilib. 1993, 83, 183-192. 24. Güçlü-Üstündağ, Ö.; Temelli, F., Solubility behavior of ternary systems of lipids in supercritical carbon dioxide. J. Supercrit. Fluid 2006, 38 (3), 275-288. 25. Waller, P.; Ryan, R.; Kacira, M.; Li, P., The algae raceway integrated design for optimal temperature management. Biomass Bioenergy 2012, 46, 702-709. 26. Span, R.; Wagner, W., A New Equation of State for Carbon Dioxide Covering the Fluid Region from the Triple-Point Temperature to 1100 K at Pressures up to 800 MPa. J. Phys. Chem. Ref. Data 1996, 25 (6), 1509. 27. Lemmon, E.; Huber, M.; McLinden, M., NIST Standard Reference Database 23: Reference Fluid Thermodynamic and Transport Properties-REFPROP, Version 9.1. Gaithersburg, MD: National Institute of Standards and Technology, Standard Reference Data Program. 2013. 28. Morrison, W. R.; Smith, L. M., Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride–methanol. J. Lipid Res. 1964, 5 (4), 600-608. 29. Ackman, R., Remarks on official methods employing boron trifluoride in the preparation of methyl esters of the fatty acids of fish oils. J. Am. Oil Chem. Soc. 1998, 75 (4), 541-545.


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Title: Simultaneous Extraction, Fractionation and Enrichment of Microalgal Triacylglyerides by Exploiting the Tunability of Neat Supercritical Carbon Dioxide Authors: Thomas A. Kwan, Qingshi Tu, and Julie B. Zimmerman Brief synopsis: Supercritical carbon dioxide is shown to have potential as an initial fractionation and enrichment approach reducing the energy consumption and costs in an integrated biorefinery model.


Simultaneous Extraction, Fractionation, and Enrichment of Microalgal

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