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Promoting Synechocystis sp. PCC 6803 Harvesting by Cationic Surfactants: Alkyl-chain Length and Dose Control the Release of Extracellular Polymeric Substances and Biomass Aggregation Yun Zhou, YenJung Sean Lai, Everett Eustance, and Bruce E. Rittmann ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b04776 • Publication Date (Web): 18 Dec 2018 Downloaded from http://pubs.acs.org on December 19, 2018
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Promoting Synechocystis sp. PCC 6803 Harvesting by Cationic Surfactants:
Alkyl-chain Length and Dose Control the Release of
Extracellular Polymeric Substances and Biomass Aggregation
Yun Zhou a, b, YenJung Sean Lai a, *, Everett Eustance a, Bruce E. Rittmann a
a
Biodesign Swette Center for Environmental Biotechnology, Arizona State University, 1001 S McAllister Avenue, Tempe, AZ 85287-5701, United States
b
State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China
Email address for all the co-authors:
[email protected] (Y. Zhou);
[email protected] (Y.J.S. Lai);
[email protected] (E. Eustance);
[email protected] (B.E. Rittmann) * Corresponding author.
Address: Biodesign Swette Center for Environmental
Biotechnology, Arizona State University, Tempe, AZ 85287-5701, United States. E-mail addresses:
[email protected] (Y.J.S. Lai).
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ABSTRACT:
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Development of efficient biomass-harvesting technology for
microalgae would achieve cost and energy savings in large-scale microalgae biomass cultivation.
Cationic surfactants could improve biomass harvesting, but determining
the optimal type and dose of surfactant requires mechanistic understanding.
In this
study, we evaluated how the alkyl-chain length and dose of three cationic surfactants -- hexadecyltrimethylammonium bromide (CTAB), myristyltrimethylammonium bromide (MTAB), and dodecyltrimethylammonium bromide (DTAB) -- affected biomass harvesting of Synechocystis.
Flow cytometry (FC) with the nucleic-acid
(NA) stain SYTOX Green (SG) was used to differentiate the release of extracellular polymeric substances (EPS) from cell lysis.
All of the cationic surfactants could
dramatically improve the biomass harvesting efficiency, and harvesting kinetics were represented well with a first-order kinetic model.
The efficiency of biomass
harvesting correlated positively with the alkyl-chain length: DTAB.
i.e., CTAB > MTAB >
A longer alkyl-chain increased EPS release, which made it easier to achieve
a less-negative zeta potential, but without cell lysis.
For CTAB, the largest cationic
surfactant tested, a dose of 4.5 mM and treatment for 60 min, achieved the maximum harvesting efficiency of ~91%.
This work lays the foundation for optimizing
surfactant species and dose for biomass harvesting.
KEYWORDS: Synechocystis; Biomass harvesting; Cationic surfactants; Flow cytometry; Cyanobacteria
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INTRODUCTION Microalgal biomass has the potential to be a renewable energy source, and it also can contain numerous high-value bioproducts, such as proteins, chlorophyll, carotenoids, and antioxidants.1,
2
microalgae lies in biomass harvesting.3
However, a major challenge for utilizing An efficient biomass-harvesting technology
would achieve cost and energy savings for large-scale microalgae cultivation.4 Synechocystis sp. PCC 6803 (hereafter called Synechocystis) is an important source of bioproducts that span energy feedstock, cosmetics, and nutraceuticals.5,
6
Synechocystis is a spherical, unicellular cyanobacterium with an average cell size of ~2 m.7
Like almost all bacteria, Synechocystis produces extracellular polymeric
substances (EPS) that it uses for aggregation and protection against environment toxicants.8
EPS, which can comprise as much as 8.4% of the dry weight of
Synechocystis,9 contain carboxyl (X-COOH) and phosphoryl (X-PO4H) groups that are negatively charged at slightly acidic to alkaline conditions.10-12
Due to its small
cell size and negative charge from EPS, Synechocystis does not readily self-flocculate,13 and the biomass-harvesting process incurs high cost.
Thus,
removing EPS to reduce the cells’ negative charge should improve biomass harvesting. Among a number of approaches for enhancing the aggregation of biomass,8, adding cationic surfactants has particular promise in this context.13, 15-17
14
When the
surfactant is adsorbed to cells, its quaternary-ammonium cation makes the cell’s charge less negative, which enhances the onset of aggregation.18
The long alkyl
chain is an inter-particle bridge that links the cells together, which further enhances 3 ACS Paragon Plus Environment
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aggregation.
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Moreover, the long linear hydrocarbon chains form micelles that
accelerate EPS release from biomass through the principle of inter-miscibility.18, 19 Charge neutralization and EPS release shifts the cells’ zeta potential toward zero, leading to improvements in biomass aggregation and harvesting efficiency. Although surfactant-induced release of EPS is beneficial, too much addition and micelle formation can bring about cell lysis and the release of intracellular polymeric substances (IPS),16, 17 which is unfavorable for biomass harvesting.13 A means to distinguish EPS release from cell lysis is flow cytometry (FC) combined with the SYTOX Green (SG) dye.20, 21
Flow cytometry (FC) is a powerful
tool to determine physical and chemical characteristics of single particles, including intact cells and cellular debris after lysis.22
FC can be used to characterize cell
features, such as cell size and granularity and cell membrane integrity.21, 23
SG binds
strongly with nucleic acid (NA),24 but cannot penetrate an intact cell membranes due to its large molecular size;25 therefore, the emitted fluorescence is due only to NA in EPS for intact biomass.
However, SG can bind with intracellular NA when the cell’s
membrane is made permeable or the cell is lysed.
The large increase in SG-bound
intracellular NA is readily detected by higher fluorescence intensity.20
Thus, FC
with SG can sensitively and accurately differentiate cell disruption and EPS release. While the general trends outlined in this introduction are established,4,
13, 26,
detailed knowledge of how alkyl chain-length and surfactant dose affect biomass harvesting is absent.
We comprehensively studied the mechanisms of surfactants
alkyl chain-length and dose on harvesting of Synechocystis. cationic
surfactants
hexadecyltrimethylammonium 4 ACS Paragon Plus Environment
The widely used
bromide
(CTAB),
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myristyltrimethylammonium bromide (MTAB), and dodecyltrimethylammonium bromide
(DTAB)
contain
a
long
alkyl
chain
(C12
to
C16)
with
a
quaternary-ammonium cation;27 We evaluated the biomass-harvesting efficiency based upon the different lengths of their hydrophobic tails, but with the same hydrophilic head.4,
13, 26
To gain mechanistic understanding, we related improved
harvest efficiency with surfactant properties, zeta potential, and EPS release, which was determined by FC with SG.
The study provides guidelines for optimizing
surfactant species and dose for efficient biomass harvesting.
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MATERIALS AND METHODS Chemicals and Synechocystis sp. PCC 6803 Culturing. of analytical grade.
All chemicals were
Hexadecyltrimethylammonium bromide (CTAB, C19H42NBr,
364.4 g/mol), myristyltrimethylammonium bromide (MTAB, C17H38NBr, 336.4 g/mol), and dodecyltrimethylammonium bromide (DTAB, C15H34NBr, 308.4 g/mol) (alkyl-chain lengths of C16, C14, and C12, respectively) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Wild-type Synechocystis sp. PCC 6803 (i.e., Synechocystis) was grown in a 16-L flat-panel photobioreactor utilizing standard BG-11 medium28 and bubbled with air filtered through a 1.0-µm air filter (Pall, Port Washington, NY, USA) at a flow rate of about 0.1 L/min.
The culturing conditions were:
temperature of 30oC, maintained
by Nestlab RTE 7 chillers; incident light provided from T5 fluorescent plant grow lamps (Envirogro Hydrofarm, USA) at 120 µE/m2.s from each side of the reactor; and pH of 8.0 maintained using a pH-Stat sparging pure CO2 when the pH was higher than 8.01.29
The optical density of culture at 730 nm (OD730) and biomass dry weight
were about 3.6 and 1.15 g/L after incubated for two weeks, respectively, which are prepared well for biomass harvesting testing. Biomass Harvesting and Kinetic Analysis.
We prepared stock solutions of
each surfactant with concentrations of 25, 10, 5, 1, and 0.5 mM using tap water.
We
mixed the biomass with each of the five surfactant concentrations using a surfactant-and-biomass volume ratio of 9:1 in 10-mL graduated centrifuge tubes (45200-10, Kimble/Knote, USA).
This yielded a biomass concentration of 0.75 g/L
and surfactant concentrations of 22.5, 9, 4.5, 0.9, or 0.45 mM. 6 ACS Paragon Plus Environment
We mixed the
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biomass with tap water using the same dilution ratio (tap water/biomass = 9:1, v/v) as the control.
During the harvesting period (up to 180 min), we withdrew duplicate
100-μL biomass samples from the middle of the glass tube and transferred each to a 96-well plate (655101, Greiner bio-one, USA).
We measured each sample’s
turbidity at 750 nm using a microplate reader (Spectra Max190, Molecular Devices, Sunnyvale, CA, USA) and calculated the harvesting efficiency using: Harvesting efficiency (%) =
OD750(t0) - OD750(t) OD750(t0)
× 100
(1)
where OD750(t0) is the OD of the sample at time zero, and OD750(t) is the OD of the sample after t min. In order to quantify the effect of surfactant type and dose on the biomass harvesting, we analyzed the batch results using first-order kinetics: Ct = C0e - k1t
(2)
where C0 and Ct are the biomass concentrations (OD units) at time zero and t, respectively.
k1 is the first-order biomass-harvesting coefficient (1/min), and higher
k1 means better biomass harvesting.
The biomass harvesting efficiency at time is:
Harvesting efficiency (%) = (1 - e -kt) × 100% SYTOX Green Staining and Flow Cytometry. and flow-cytometry approach13, surfactant treatment.
20, 21
(3)
We adapted a SG-staining
to identify cell size and EPS release after
We applied the fluorescent dye SG according to the
manufacturer’s guidelines (Invitrogen, Carlsbad, CA, USA).
After treatment with
each surfactant for 5 hours, we withdrew a 2-mL sample, mixed it with 1 µL SG, and then allowed the reaction to proceed for 15 min in the dark on a rocker mixer
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(Lab-Line, TX, USA).
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We used Synechocystis biomass without treatment or SG
stain to zero the fluorescent intensity (FI).
After staining, we performed FC using a
FACSAria flow cytometer (BD Biosciences, CA, USA) having an air-cooled 20-mW argon ion laser with an excitation wavelength of 488 nm.
We used a fluorescein
isothionyanate (FITC) filter with a wavelength band of 510-550 nm to detect the SG emission.
We diluted the samples stained with SG to a concentration suitable for the
instrument’s counting speed of 300 to 400 events/s, and we counted 10,000 events for each sample.
We performed the data analysis and graphical outputs with FlowJo
7.6.1 software (Treestar, Inc., San Carlos, CA, USA). Analytical Methods.
Optical Density (OD) of the incubation culture was
measured at 730 nm using a UV-vis BioSpec-mini spectrometer (Shimadzu Corp., Japan).
The dry weight of biomass was determined using total suspended solids,
assayed by Method 2450D in Standard Methods.30
The zeta potential was measured
using a Zetasizer (Nano-ZS, Malvern, Britain) after the biomass was diluted to a manufacture recommended concentration, between 0.1 g/L and 1 g/L.4 Statistical Analysis.
For surfactant-treatment experiments, we quantified the
zeta potential and turbidity at 750 nm (OD750) in triplicate.
Results are expressed as
the mean and standard deviation of the three measured samples (mean ± SD).
When
presenting the results of Synechocystis light scattering and the spectra from FC, we show one result for each sample.
Statistical analysis with SPSS software for
Windows (SPSS, Chicago, Illinois, USA) was used to identify the strength of the relationship between zeta potential and biomass harvesting efficiency for Synechocystis.
The Pearson’s correlation coefficient, R2, was used to estimate the 8 ACS Paragon Plus Environment
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linear correlation between two parameters.
Correlations were considered statistically
significance at a 95% confidence interval (P < 0.05).
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RESULTS AND DISCUSSION Effects of Surfactant Type and Doses on Biomass Harvesting.
Fig. 1 shows
the time course of Synechocystis harvesting efficiency with CTAB, MTAB, and DTAB at the various surfactant doses. using first-order kinetics.
It also presents modeling representations
Synechocystis had only ~8% self-flocculation efficiency
(without added surfactant) after 180 min.
Self-flocculation of Synechocystis in a
previous study was negligible,13 and the small increase in self-flocculation here was due to using tap water, which contained higher concentrations of divalent cations (Ca2+ and Mg2+) that improved charge neutralization and flocculation.13, 31, 32 CTAB, MTAB, and DTAB enhanced coagulation and flocculation in ways that depended on alkyl-chain length and dose:
Longer chain length was more effective
with a smaller dose, but also could break up large aggregates and lead to cell lysis with a high dose.
For example, the surfactant with the longest chain length, CTAB,
could achieve 80% harvest efficiency within 60 min at a dose of 0.9 mM, but a dose ≥ 4.5 mM had a negative effect on the biomass harvesting.
In addition, doses ≥ 4.5
mM showed gradually declining harvesting efficiencies beyond 90 min due to disaggregation of large aggregates.
MTAB ≤ 0.9 mM had no effect on biomass
harvesting, but could achieve 90% harvesting efficiency within 60 min at a dose ≥ 4.5 mM.
DTAB had almost no effect on biomass harvesting for ≤ 9.0 mM, but 22.5 mM
could achieve 90% harvesting efficiency within 40 min. Table 1 shows the best-fit kinetic parameters (k1) for fitting the experimental data by the first-order model.
Visually good fits in Figure 1 and having R2 > 0.92 for all
of experiments support that first-order kinetics provided a good representation of the 10 ACS Paragon Plus Environment
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experimental trends.
The kinetic parameters reinforce that it took a lower surfactant
dose to achieve better flocculation for the longer-chain surfactants and an excessively high dose causes deterioration in flocculation effectiveness.
For CATB, k1 increased
to 0.030 min-1 as the surfactant dose rose to 4.5 mM, but gradually decreased to 0.018 min-1 as the CATB dose increased to 22.5 mM.
For MTAB, k1 was lower than 0.001
min-1 for MTAB ≤ 0.9 mM, but reached the highest measured value, 0.049 min-1 for a dose of 4.5 mM.
Small declines in k1 occurred for larger MTAB doses.
For
DTAB, k1 was lower than 0.001 min-1 for doses less than ≤ 4.5 mM, and a high k1 required the maximum does of 22.5 mM. Zeta Potential at 60 min and Pearson Correlations between Biomass Harvesting Efficiency and Zeta Potential.
Fig. 2 shows the zeta potentials of
Synechocystis after treatment for 60 min with CTAB, MTAB, and DTAB at various surfactant doses, along with the relationship between zeta potential and biomass harvesting efficiency.
Zeta potential dramatically increased with increasing
surfactant dose in each case, and the surfactant with the longer alkyl-chain achieved a higher zeta potential.
For example, the zeta potential for untreated Synechocystis
was -37.2 mV, but it dramatically increased to 26.6, 22.8, and -7.76 mV after adding 4.5 mM CTAB, MTAB, and DTAB, respectively. The relationship between the zeta potential and harvesting efficiency was strong and nearly linear with MTAB (R2 = 0.917, P < 0.01) and DTAB (R2 = 0.914, P < 0.01) treatments (Fig. 2(d)), but CTAB did not exhibit a strong relationship (R2 = 0.154, P = 0.38) (Fig. 2(c)).
Zeta potential higher than 12 mV led to the highest
biomass harvesting efficiency (~ 90%) for MTAB and DTAB, but, for CTAB, zeta 11 ACS Paragon Plus Environment
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potential greater than about 25 mV led to a poorer harvesting efficiency.
This
reversal may have been related to a steric effect that camouflaged charge neutralization33 and the disaggregation of large aggregates due to EPS release or even cell lysis.13 Flow Cytometry Analysis of Surfactant-treated Synechocystis.
Fig. 3(a)
shows selected FC results for light scattering of Synechocystis before and after treatment for 1 day with 10 mM of CTAB, MTAB, or DTAB.
In FC, the slope (SL)
of the linear relationship between the side scatter (SSC, vertical axis) and forward scatter (FSC, horizontal axis) in the region with the highest density of points corresponds to particle size (i.e., larger slope corresponds to smaller particle size).13, 21, 22
The SL was 0.96 for Synechocystis after two weeks cultivation without
surfactant treatment.
All surfactant treatments decreased cell size by releasing EPS
or even causing cell lysis.13,
16, 17
After adding 10 mM of surfactant, the SL
dramatically increased from 0.96 (control) to 1.28 for CTAB, but the increases were only to 1.14 and 1.02 for MTAB and DTAB, respectively (Fig. 3(b)).
SL increased
with a strong linear relationship (R2 = 0.99, P < 0.01) with CTAB dose up to 1.0 mM due to the release of EPS.13, 21
When the CTAB dose was higher than 5.0 mM, SL
dramatically increased, indicating a sharp decrease in particle size caused by cell lysis.13, 34
SL dramatically increased with a strong linear relationship (R2 = 0.99, P
10 FIU (M2 region), which was from SG binding with NA in the fraction of increased membrane permeability or dead cells naturally present.13, 36 After CTAB treatment, the fluorescence peak gradually shifted to the right as the surfactant dose rose to 0.9 mM, signifying complete EPS removal, but minimal cell lysis.13
The fluorescence peak steadily increased in intensity, indicating that CTAB
led to cell-membrane permeability37, 38 that allowed SG to pass through the membrane and bind with intracellular nucleic acid (NA).
At CTAB doses higher than 0.9 mM,
the peak gradually shifted to the left, a sign of increasing cell lysis and the resulting loss of IPS and NA inside the cells.25, 36 For MTAB treatment, the shifts in the fluorescence peak mirrored those for CTAB, but doses ≥ 4.5 mM were needed to cause cell lysis.
In the case of DTAB,
the fluorescence peak gradually shifted to the right as the surfactant dose increased from 0 to 22.5 mM.
Overall, the results indicate that the longest alkyl-chain
surfactant (CTAB) could achieve complete release of EPS, but with minimal cell lysis at the lowest surfactant concentration. Fig. 5 shows the distribution of SG-emission intensity between the M1 and M2 regions after adding various doses of the surfactants.
For CATB, the proportion of
M1 (low FI region) dramatically decreased to 3.8% as the CTAB dose increased to 0.9 mM; the steep decline of M1 signifies nearly complete removal of EPS.13 13 ACS Paragon Plus Environment
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However, M2 (high FI region) gradually decreased for a higher dose of CTAB, which could have resulted from cell lysis and the release of intracellular NA.25,
36
For
MTAB and DTAB, the proportion of M1 also decreased, but for much higher doses, 4.5 mM and 9.0 for MTAB and DTAB, respectively.
Results also confirmed that
long alkyl-chain surfactant could achieve the complete release of EPS at low surfactant dose. Synthesizing the Results.
Fig. 6 synthesizes the results in terms of the
mechanisms acting and how they relate to alkyl-chain length of the cationic surfactants.
Because negatively charged functional groups in EPS prevent biomass
aggregation, adsorption of cationic surfactants can dramatically improve biomass harvesting through charge neutralization and making the zeta potential less negative. A second phenomenon occurs when the surfactant’s alkyl chains form micelles that release EPS from the cells, which also contributes to charge neutralization and improved biomass harvesting.
A surfactant with a longer alkyl chain has better
ability to increase EPS release.
Thus, the longer alkyl-chain surfactants could
achieve complete EPS release and high biomass-harvesting efficiency at a low surfactant dose.
However, longer chain length surfactant also could breakup large
aggregates and leads to cell lysis with a high dose, which had a negative effect on the biomass harvesting. Implications of This Work.
Microalgae biomass could be a promising
alternative clean and renewable energy, but biomass harvesting is costly.13,
39
By
using FC with SG and zeta potential analysis, our study revealed that improved efficiency of biomass harvesting was correlated positively with the alkyl-chain length: 14 ACS Paragon Plus Environment
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CTAB > MTAB > DTAB.
A major factor was that the longer-alkyl chain surfactant
could dramatically improve EPS release, which helped remove negative charge on the biomass surface.
From an economic standpoint, using a lower dose of longer-alkyl
chain surfactant should be beneficial for achieving cost-effective biomass harvesting. CTAB, MTAB, and DTAB are biodegradable,40, 41 and Lai et al42 demonstrated that an oxygen-based membrane biofilm reactor (O2-MBfR) could achieve continuous biodegradation of CTAB.
Zhou et al16 also reported that 91.2% of cocoamidopropyl
betaine (CAPB) was removed after treatment for 24 h in an aerobic digestion system. Thus, biodegradation of quaternary ammonium compounds (QACs) should be achievable, thus preventing their discharge to aquatic environments.
CONCLUSIONS Development of efficient biomass harvesting for microalgae can lead to major cost and energy savings in large-scale applications.
Our study emphasizes how the
alkyl-chain length and dose of cationic surfactants affect biomass harvesting of Synechocystis.
The efficiency of biomass harvesting was correlated positively with
the alkyl-chain length:
i.e., CTAB > MTAB > DTAB.
A longer alkyl-chain
increased EPS release, which made it easier to achieve a less negative zeta potential, but without cell lysis.
For CTAB, the largest cationic surfactant tested, a dose of 4.5
mM and treatment for 60 min achieved the maximum harvesting efficiency of ~ 91%.
ACKNOWLEDGEMENTS This work was supported by LightWorks, Arizona State University, and 15 ACS Paragon Plus Environment
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Shanghai Tongji Gao Tingyao Environmental Science & Technology Development Foundation.
We thank Dr. Willem Vermaas and his laboratory in the School of Life
Sciences at Arizona State University for providing Synechocystis sp. PCC6803 wild type; and Dr. Dong Fu at the Center of Infectious Diseases and Vaccinology, Biodesign Institute at Arizona State University, for her expertise in flow cytometry for sample quantification.
SUPPORTING INFORMATION There is no SI file for publication.
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TABLES Table 1
Parameter values of the first-order kinetics for biomass harvesting a k1 (min-1)
R2
Surfactant dose (mM)
CTAB
MTAB
DTAB
0 (Control)
5.7 10-4
5.7 10-4
5.7 10-4
1.8
90
9.2
0.45
10-3
10-4
CTAB
MTAB
DTAB
0.933
0.933
0.933
10-4
0.927
0.925
0.979
9.3 10-4
0.943
0.933
0.926
6.2
0.987
0.958
0.961
0.90
0.016
5.6 10-4
4.5
0.030
0.049
9.0
0.020
0.035
0.019
0.973
0.934
0.976
22.5
0.018
0.040
0.048
0.966
0.946
0.941
10-4
a
k1 (min-1), biomass harvesting coefficient in the first-order kinetics; CTAB, hexadecyltrimethylammonium bromide; MTAB, myristyltrimethylammonium bromide; DTAB, dodecyltrimethylammonium bromide. Boldface indicated the fastest harvesting kinetics for each surfactant.
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FIGURES
100
CTAB
75 50 25 0 100 75
MTAB
Harvesting efficiency (%)
22.5 mM 4.5 mM 0.45 mM
50 25
9.0 mM 0.9 mM Control
0 100 75
DTAB
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Experimental data Modeling outputs
50 25 0 0
30
60 90 120 150 180 Harvesting time (min)
Figure 1. Time course of Synechocystis harvesting efficiency and modeling reprentations after treatment with CTAB, MTAB, and DTAB over the noted range of surfactant doses. Half-solid symbols are the experimental outputs, and dashed lines are the modeling fits using first-order kinetics.
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Zeta potential (mV)
a 40 20 0 CTAB MTAB DTAB
-20 Control
-40 0
b Harvesting efficiency (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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80 60
5
10 15 Surfactant dose (mM)
20
Model outputs R2 = 0.917, P < 0.01 R2 = 0.914, P < 0.01 R2 = 0.154, P = 0.38
40 20 0 -30
-15 0 15 Zeta potential (mV)
30
Figure 2. (a) Zeta potentials of Synechocystis after treatment for 60 min with CTAB, MTAB, and DTAB at various surfactant doses, and (b) the relationship between the zeta potential and biomass harvesting efficiency treated by CTAB, MTAB, and DTAB.
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b 1.6 1.4
SL
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
CTAB y=0.010x+0.584 R2=0.99, P