Research Article pubs.acs.org/journal/ascecg
Synergistic Integration of C12−C16 Cationic Surfactants for Flocculation and Lipid Extraction from Chlorella Biomass YenJung Sean Lai,*,† Yun Zhou,†,‡ Rebecca Martarella,† Zhaocheng Wang,†,§ and Bruce E. Rittmann† †
Swette Center for Environmental Biotechnology, The Biodesign Institute at Arizona State University, 1001 S McAllister Avenue, Tempe, Arizona 85287-5701, United States ‡ State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China § Department of Water Engineering and Science, College of Civil Engineering, Hunan University, 2 Lushan South Road, Changsha 410082, China S Supporting Information *
ABSTRACT: Microalgae lipids could be a good alternative feedstock for liquid fuel, but complex processing steps with significant energy demands present roadblocks. Combining biomass harvesting and lipid recovery into one simple step can lower complexity and energy costs. This study evaluated the use of C12−C16 cationic surfactants in this synergistic manner. First, we determined that a dose as low as 0.45 mM of C16-alkyl-chain cationic surfactant (hexadecyltrimethylammonium, CTAB) led to >85% biomass-harvesting rate for Chlorella biomass, and good harvest was correlated to a slightly positive zeta potential. Second, the cationic surfactants disrupted cell structures (detected by transmission electron microscopy) and led to lipid recovery (measured as fatty acid methyl esters, FAME) as high as 90% using nontoxic ethyl acetate (EA) as the solvent and without altering the FAME distribution; for context, EA was able to extract less than 1% of FAME from control (not surfactant-treated) Chlorella. Disruption and high FAME yield were associated with surfactants’ critical micelle concentration (CMC): a lower CMC required a lower concentration of surfactant to disrupt the cells. The synergistic benefits of cationic surfactants can be attained by maintaining a slightly positive zeta potential for effective flocculation and adding the minimum concentration of surfactant needed for cell disruption. KEYWORDS: Cationic surfactant, Microalga biomass, Harvesting, Lipid extraction, Synergistic integration
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INTRODUCTION
the particles can aggregate when the zeta potential is close to zero. When cationic surfactants are used as microalgae flocculants, their hydrophobicity and micelle properties affect the zeta potential and flocculation efficiency. Longer alkyl chains lead to greater hydrophobicity and a lower critical micelle concentration (CMC).11−14 In addition to flocculation, cationic surfactants have the potential to disrupt algal cells, which should improve lipid recovery.4,5,15 Surfactants have hydrophobic ends that can bind to or insert into the hydrophobic cell membranes. Once membrane components and surfactants develop micelles, they can be separated from microalgae membranes.4 An effective dose for cell disruption has been related to a surfactant’s CMC, but it also depends on properties of the microalga’s membrane,15−17 which vary with species and culturing
While microalgae-derived biofuel is a promising alternative to petroleum-based fuel, its feasibility depends on overcoming bottlenecks in cultivation, harvest, lipid extraction, and fuel conversion.1−3 Integrating harvesting and lipid extraction into one simple step could cut financial and energy costs in a major way.4,5 Efficient harvest requires that the algal biomass be separated quickly from the cultivation medium. Biomass flocculation, a simple and cost-effective means to enhance separation, begins with particle-charge neutralization, which then leads to aggregation and bridging.6−8 Because the algal biomass carries a negative surface charge, cations such as calcium or quaternary ammonium salts are good for neutralizing the surface charge6−10 and have been used to enhance the performance of separation by settling and dissolved air flotation.9,10 The zeta potential is a good indicator for particle stabilization:6 e.g., particles with >25 mV zeta potential (positive or negative) maintain stable suspensions due to strong electric repulsion, but © XXXX American Chemical Society
Received: August 31, 2016 Revised: October 29, 2016 Published: October 31, 2016 A
DOI: 10.1021/acssuschemeng.6b02095 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering Table 1. Characteristics of the Cationic Surfactants
a
surfactant
formula
alkyl-chain length
CMC, 20−25 °C
formula
dodecyltrimethylammonium bromide myristyltrimethylammonium bromide hexadecyltrimethylammonium bromide
C15H34NBr C17H38NBr C19H42NBr
C12 C14 C16
14.4 mM22 4−5 mMa 0.9 mMa
DTAB MTAB CTAB
The values obtained from the manufacturer, Sigma-Aldrich.
Table 2. Characteristics of the Nutrient-Depleted Chlorella Biomass
conditions. For example, nonhydrolyzable polymers, such as the trilaminar structures (TLS) of algaenan, may make certain algae more resistant to disruption by a detergent.18,19 Thus, effective cell disruption depends on using a surfactant compatible with the properties of harvested microalgae’s membranes. Hexadecyltrimethylammonium bromide (CATB), a cationic surfactant having an alkyl-chain length of 16 (i.e., C16), is one of the common flocculants utilized for harvesting algal biomass.4,5,9,10 Alternatives are myristyltrimethylammonium bromide (C14, MTAB) and dodecyltrimethylammonium bromide (C12, DTAB), which have shorter alkyl chains and are less hydrophobic, but have fewer steric effects compared to CTAB.11−14 These differences may affect microalgae flocculation and disruption. A related factor is the solvent applied for lipid extraction. In particular, polar and nonpolar solvents extract different lipid fractions.20,21 A low-toxicity solvent like ethyl acetate (EA) offers benefits for environmental and worker safety, but it is less polar than alcohol,21 which may make EA less effective for disrupting hydrogen bonds and electrostatic forces that stabilize cell membranes.20 Thus, cell disruption with cationic surfactants could have a strong benefit for lipid extraction by EA. We evaluated the trade offs between biomass harvesting and lipid extraction among three surfactants, DTAB, MTAB, and CTAB, for nutrient-depleted Chlorella biomass. First, we examined how surfactant dose affects flocculation and zeta potential. Second, we used transmission electron microscopy (TEM) to examine the degree of cell disruption caused by surfactants. Third, we assessed how surfactant dose affected lipid recovery during wet-biomass extraction using EA. Finally, we developed guidelines for determining the surfactant dose to achieve an optimal outcome in terms of microalgae flocculation for harvest and disruption for lipid extraction.
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total FAME to TSS (%)
VSS (g/L)
TSS (g/L)
C (%)
H (%)
N (%)
5.8 ± 0.3
7.5 ± 0.0
7.7 ± 0.0
49.8
7.7
6.0
of 7.5 g/L VSS. We prepared stock solutions of each surfactant with concentrations of 25, 10, 5, 1, and 0.5 mM. We mixed the biomass with each of the five surfactant concentrations with a ratio of 9:1 (volume surfactant/volume biomass) in 10 mL graduated centrifuge tubes (45200-10, Kimble/Knote). This yielded biomass concentrations of 0.75 g/L for Chlorella and surfactant concentration of 22.5, 9, 4.5, 0.9, and 0.45 mM. Controls were biomass mixed with tap water using the same dilution ratio (tap water/biomass = 9:1, v/v). We gently mixed the samples up and down by hand until the samples were well-mixed and withdrew duplicate 100 μL biomass samples from the middle height of the tube at time zero.26,27 During the settling period (up to 180 min), duplicate samples were withdrawn from the middle level and transferred to a 96-well plate (655101, Greiner bio-one), where the turbidity was measured at 750 nm using a microplate reader (Spectra Max190, Molecular Devices, Sunnyvale, CA). The harvesting efficiency, or biomass-recovery ratio, was computed with eq 1 adapted from the previous work.26,27
harvesting efficiency (%) =
OD750 (t0) − OD750 (t ) × 100 OD750 (t0)
(1)
OD750(t0) was the OD of the aliquot withdrawn at time zero (t0), and OD750(t) was the OD of the biomass withdrawn at time = t. The experiments on flocculation kinetics had a duration of at least 120 min and were terminated when the percent recovery reached a stable value. The zeta potential was measured using a Zetasizer (nano-ZS, Malvern) after the biomass was diluted to a manufacturerrecommended concentration at least 0.1 g/L and up to 1 g/L. Photographic images were taken to document the flocculation kinetics for Chlorella biomass; examples are in S Figures 1−3 in the Supporting Information. Characterization of Cell Disruption by Transmission Electron Microscopy (TEM). We adapted the TEM approach of Lai et al. (2016)15 and Sheng et al. (2011)28 to identify morphology changes for Chlorella after surfactant addition. The microalgal samples (control or surfactant-treated) were incubated with 2% glutaraldehyde in 50 mM sodium phosphate at pH 7.2 for 1 h, washed with buffer, and fixed with 1% osmium tetroxide for 1 h. Fixed cells were fully dehydrated with an ascending series of acetone solutions. Spurr’s epoxy resin29 was used to infiltrate and embed the samples, which were polymerized at 60 °C for 36 h. Finally, samples were cut into 60 nm sections using a Leica Ultracut-R microtome (Wetzlar) and poststained in uranyl acetate and lead citrate. Images were generated using a Philips CM12 TEM operated at 80 kV with a Gatan model 791 CCD camera. Effect of Surfactant Doses on Wet-Biomass Extraction. Interactions between the surfactant and solvent during lipid recovery were evaluated by adapting the method of wet-biomass extraction of Lai et al. (2016).15 The surfactant powder was weighted to give 25, 10, 5, 1, or 0.5 mM for a 30 mL microalgal slurry, which was placed into a 50 mL centrifuge tube (VWR). The slurries were mixed within an incubator (New Brunswick, Scientific, Enfield, CT) at 210 rpm and at room temperature (23−24 °C). Samples at time = 0 were gently mixed by vortexing until all surfactant powder was fully dissolved. At
EXPERIMENTAL SECTION
Chemicals. Dodecyltrimethylammonium bromide (DTAB), myristyltrimethylammonium bromide (MTAB), and hexadecyltrimethylammonium bromide (CTAB) were obtained from Sigma-Aldrich (St. Louis, MO), and their characteristics are summarized in Table 1. Microalgal Biomass/Elemental Analysis. Chlorella biomass was harvested in a nutrient-depleted condition from a pilot-scale photobioreactor at the Arizona Center for Algae Technology and Innovation (AzCATI), located at the Polytechnic Campus of Arizona State University (Mesa, AZ). Nutrient-depleted is defined as a growth phase in which nitrate is fully consumed; nutrient depletion is used to generate algal biomass that has a high lipid content, although the lipid content of nutrient-depleted biomass varies with the species and duration of depletion.23,24 Biomass concentrations were assayed as total suspended solids (TSS) and volatile suspended solids (VSS) according to Standard Methods.25 The elemental composition of the biomass was quantified using a CHN elemental analyzer (CE-440, Exeter Analytical Inc.). Table 2 summarizes the biomass characteristics of Chlorella. Flocculation Assay and Zeta Potential. We evaluated flocculation kinetics using Chlorella biomass having a concentration B
DOI: 10.1021/acssuschemeng.6b02095 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering specified incubation times, a 1 mL sample (surfactant plus microalga slurry) was withdrawn by the pipet and mixed with 3 mL of the ethyl acetate (EA) solvent. The mixtures of solvent and biomass were vortexed for 1 min, and then 1 mL of clear separated solvent was taken out after centrifugation (Eppendorf 5810R, NY, USA) at 4000 rpm for 5 min. The lipids in the solvent were assayed for fatty acid methyl esters (FAME) after being air-dried. FAME were quantified for the solvent extract and also directly for the control and surfactant-treated samples. Direct transesterification (DT) was carried out with a 1 mL slurry sample of freeze-dried biomass (FreeZone Benchtop instrument (Labconco, MO, USA)). Samples were amended with 2 mL of 3 N methanolic HCl (SigmaAldrich, MO, USA) and incubated at 85 °C in the oven for 2.5 h.15,30 The DT assay allowed us to define the maximum FAME, and FAME obtained after solvent extraction could be compared against the total FAME obtained from DT. Following Sheng et al. (2011),30 FAME components were quantified using a gas chromatograph (Shimadzu GC 2010, Japan) equipped with a Supelco SP-2380 capillary column (30 m × 0.25 mm × 0.20 μm) and flame ionization detector (FID) (Sheng et al., 2011) against a 37-Component FAME Mix standard (Supelco, PA, USA).
degree of self-flocculation, the harvesting efficiency was much greater with addition of each cationic surfactant. Harvesting efficiency was affected by alkyl-chain length and surfactant dose. About 80% harvesting efficiency could be achieved for all concentrations of CTAB within 60 min, and most of the benefits of surfactant were realized by ∼40 min. Hence, harvest efficiency >80% could be achieved with a minimum dose of 0.45 mM CTAB within 40 min. About 80% harvesting efficiency also could be achieved with MTAB and DTAB, but both required higher doses, at least 4.5 and 9 mM for MTAB and DTAB, respectively. Harvesting Efficiency and Zeta Potential at 40 min. Figure 2 summarizes the impact of surfactant concentration for
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RESULTS AND DISCUSSION Screening Surfactant Doses for Harvesting Efficiency. Figure 1 summarizes the biomass-harvesting efficiency for Chlorella using C12 (DTAB), C14 (MTAB), and C16 (CTAB) cationic surfactants. Although the microalgae exhibited a small
Figure 2. (a) Harvesting efficiency of Chlorella with increasing concentration of the three cationic surfactants and (b) the corresponding zeta potentials.
Chlorella biomass after 40 min. Figure 2a presents the harvesting efficiency, and Figure 2b presents the corresponding zeta potentials. Harvesting efficiency was enhanced up to 10fold over biomass without surfactant amendment. The length of the alkyl chain dominated the effects on the harvesting efficiency (Figure 2a). In particular, CTAB was much more effective than MTAB and DTAB: 1 mM CTAB could change zeta potential to 3.6 mV from −24.0 mV (for the control biomass), and this was associated with nearly 80% harvesting efficiency. For comparison, ∼80% harvesting efficiency required 22.5 mM for DTAB (shifting the zeta potential to 3.5 mV) and 4.5 mM for MTAB (shifting the zeta potential to 4.6 mV). For DTAB and MTAB, the trend of harvesting efficiency correlated well with zeta potential (Figure 2b). Slightly positive zeta potential, ≤∼5 mV, led to high harvesting efficiency. In the case of CTAB, the highest dose led to a reduction in harvesting efficiency and an increase in zeta potential to ≥7 mV. The negative impact of a high CTAB dose may have been related to a steric effect in which the longer alkyl chain camouflaged charge neutralization, an effect observed before with CTAB.31 Another factor could be excess adsorption and charge
Figure 1. Biomass-harvesting recovery efficiency for the indicated range of concentrations (mM) with three cationic surfactants for Chlorella biomass: (a) CTAB, (b) MTAB, and (c) DTAB. C
DOI: 10.1021/acssuschemeng.6b02095 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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ACS Sustainable Chemistry & Engineering
Figure 3. TEM images of Chlorella incubated with 25 mM surfactant, at room temperature, and for 1 day incubations: (a) control and (b) CTAB-, (c) MTAB-, and (d) DTAB-treated biomass.
reversal,11 since the zeta potential was higher than 5 mM, and it was ∼10 mV for the highest dose. TEM Ultrastructure. Figure 3 shows TEM images of Chlorella biomass without and with the addition of cationic surfactants after 1 day incubations. Before addition of surfactant, microalgae had typical nutrient-depleted morphology (Figure 3a) that included lipid inclusions (gray color).18 Chlorella did not have a thick and clear cell membrane, like Scenedesmus’,15,18 and this probably was linked to a lack of algaenan in Chlorella.18,32 Surfactant treatment dramatically altered the cell morphology of Chlorella, whose internal cell structures lost their shapes or even disappeared completely (Figure 3b−d). Chlorella was disrupted quickly by surfactant treatment: Green-yellowish supernatant began appearing during the harvesting test ( MTAB ≥ DTAB. Wet-Biomass Extraction. Figure 4 shows that adding cationic surfactants improved FAME recovery by several orders of magnitude compared with control biomass without treatment. EA-extractable FAME was nearly 90% of total FAME by DT. FAME recovery correlated positively with longer alkylchain length: i.e., CTAB > MTAB > DTAB. To the best of our knowledge, this is the first report of EA extraction yielding substantial FAME recovery.
Figure 4. FAME recoveries obtained using ethyl acetate (100%) with 5 h incubation using a 25 mM surfactant concentration.
FAME Profiles. Figure 5 compares extractable FAME profiles between the control and surfactant-treated biomass. The FAME profiles were nearly identical between DT of control biomass and EA-extracted biomass. Furthermore, the three surfactants did not alter the profiles obtained by direct transesterification (in S Figure 5). Effect of CMC on FAME Recovery and Their Integration with Harvesting Process. Figure 6 relates harvesting efficiency and FAME recovery for the different surfactants and doses. CTAB at as little as 0.45 mM gave nearly D
DOI: 10.1021/acssuschemeng.6b02095 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering
CTAB (5 mM) to effect good flocculation and lipid extraction with EA. It may not always be possible to find a single surfactant dose that provides excellent flocculation and lipid extraction. For instance, a dose high enough for good lipid extraction might result in charge reversal for flocculation (e.g., 22.5 mM of CTAB, Figure 2). In this situation, we could achieve high harvesting efficiencies with a lower dose (e.g., 0.45 mM CTAB) and then gain effective cell disruption by adding a larger dose before extraction. In addition, surfactant demand could be substantially reduced by applying the large dose for lipid extraction only to the harvested and concentrated biomass. With either strategy, the goal is to attain a zeta potential ≤∼5 mV for good flocculation and the minimum surfactant dose needed for cell disruption. For good sustainability, the discharge of a surfactant-bearing waste stream after harvesting and lipid extraction is not acceptable due to the surfactants’ adverse impact on aquatic biota. Furthermore, recycling medium containing surfactant for cultivation could negatively affect microalga growth. Fortunately, most cationic surfactants are biodegradable in aerobic conditions,33 which means that medium reuse or discharge could be feasible following aerobic biotreatment. Future studies should focus on whether recycled medium containing surfactant affects microalga growth and biodegradation of residual surfactant.
Figure 5. FAME profiles obtained from wet-biomass extraction using ethyl acetate (EA), along with profiles of total FAME obtained from direct transesterification (DT). Surfactant incubation was for 5 h with a 25 mM surfactant concentration.
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CONCLUSIONS Addition of cationic surfactants dramatically improved biomass flocculation and lipid extraction for nutrient-depleted Chlorella biomass. Flocculation and harvesting were improved dramatically by a dose of as little as 0.45 mM CTAB, which gave a zeta potential ≤∼5 mV. EA extraction of surfactant-treated Chlorella biomass achieved nearly 90% lipid recovery, several orders of magnitude higher than with no surfactant treatment, and surfactant treatment did not alter the FAME profile of EAextracted biomass. The improvement in lipid recovery corresponded to extensive cell disruption, identified by TEM images, which required a CTAB dose larger than its CMC. For the nutrient-depleted Chlorella biomass, we could achieve high harvesting and lipid extraction into one simple step by adding 5 mM CTAB. The keys were to maintain a slightly positive zeta potential (
