Cationic Cellulose Nanocrystals for Flocculation of Microalgae: Effect

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Cationic Cellulose Nanocrystals for Flocculation of Microalgae: Effect of Degree of Substitution and Crystallinity Jonas Blockx,†,∥,‡ An Verfaillie,†,∥,‡ Samuel Eyley,∥ Olivier Deschaume,§ Carmen Bartic,§ Koenraad Muylaert,*,† and Wim Thielemans*,∥ †

Laboratory for Aquatic Biology, KU Leuven, Campus Kulak Kortrijk, Etienne Sabbelaan 53, box 7659, B-8500 Kortrijk, Belgium Renewable Materials and Nanotechnology Research Group, Department of Chemical Engineering, KU Leuven, Campus Kulak Kortrijk, Etienne Sabbelaan 53, box 7659, B-8500 Kortrijk, Belgium § Department of Physics and Astronomy, Soft-Matter Physics and Biophysics Section, KU Leuven, Celestijnenlaan 200 D, box 2416, B-3000 Heverlee, Belgium ∥

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S Supporting Information *

ABSTRACT: Flocculation could offer a low-cost and straightforward solution to harvest microalgae in an economic and energy-efficient way. Cationically modified cellulose nanocrystals (CNCs) have been proposed as an alternative to biopolymer-based flocculants such as chitosan. The aim of this study was to fine-tune the cationic modification of CNCs for use as flocculants for harvesting the freshwater microalgae Chlorella vulgaris. CNCs were functionalized with two cationic groups, pyridinium (PYR) or methylimidazolium (MIM), and the degree of substitution (DS) was varied by controlling reaction conditions (stoichiometry, reaction time, and temperature). The DS ranged from 0.08 to 0.34 for PYR modifications and from 0.10 to 0.29 for MIM modifications. All cationic CNCs achieved flocculation efficiencies of >95% at optimal dosage. The required dose to induce flocculation decreased with increasing DS and did not differ between the PYR and MIM modified CNCs. The relation between DS and the flocculant dose points to the importance of electrostatic interactions in the flocculation mechanism. To check the advantages of a rigid flocculant compared to a flexible flocculant, we compared flocculation between rigid cationic CNCs and the flexible molecular polymer chitosan. While CNCs required a slightly higher dose by weight compared to chitosan, CNCs required a lower dose than chitosan when expressed per number of cationic charges added. Moreover, overdosing resulted in dispersion restabilization when chitosan was used but not when cationic CNCs were used, pointing to a different mechanism of flocculation between rigid cationic CNCs and the molecular polymer chitosan. Opposed to flexible flocculants, CNCs cannot reorient all their charges toward the microalgal cell. This results in patches of positive charges onto the surface. Because of the rigidity of CNCs, we propose an electrostatic patch mechanism as the mechanism of flocculation for CNC-based flocculants. KEYWORDS: flocculation mechanism, Chlorella vulgaris, cellulose nanocrystals, electrostatic interactions, rigidity

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industrial processes.6 Although these flocculants are effective for microalgae, their use in downstream processing of microalgae is not recommended.7 It results in contamination of the microalgal biomass with metals or potentially toxic acrylamide residues that may interfere with further downstream processing of the biomass or with the use of biomass fractions in food or feed applications.7 Natural flocculants have received close attention in recent years.8 Flocculants that are derived from natural biopolymers are preferred over synthetic flocculants to avoid contamination of the biomass. An example of a biopolymerbased flocculant widely used in microalgae harvesting is chitosan, which is derived from chitin.9−13 Other examples of

INTRODUCTION Microalgae are a new biomass resource that can complement conventional biomass feedstocks for the production of food, feed, fuel, and building blocks for the chemical industry.1 Largescale deployment of microalgae production, however, is hindered by the high cost and energy demand of downstream processing of microalgal biomass. Despite the fact that microalgae are more productive compared to conventional agricultural crops, harvesting the biomass is a challenge, mainly because biomass concentration in liquid cultures is low (0.5−5 g L−1) and microalgal cells are small (3−30 μm).2,3 The cost and energy demand for harvesting might be significantly reduced if individual cells can be aggregated into larger particles by means of flocculation.4,5 Metal salt coagulants and synthetic polyacrylamide-based polymers are today the most commonly used flocculants in © XXXX American Chemical Society

Received: February 18, 2019 Accepted: May 13, 2019

A

DOI: 10.1021/acsanm.9b00315 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

Article

ACS Applied Nano Materials biopolymers used for flocculation are derived from starch (cationic starch),14,15 poly-ϒ-glutamic acid,16 or tannins.17 Banerjee et al.18,19 also explored the use of modified guar gum or cassia gum as flocculant for microalgae harvesting. Cellulose, as the most abundant natural polymer on earth, is an attractive biopolymer. It is the main component of plant cell walls, including the cell wall of most algae.20 Cellulose is a linear homopolysaccharide consisting of β-D-anhydroglucopyranose units (AGUs), linked by β(1 → 4) ether bonds (glycosidic links). In plant cell walls, individual cellulose polymers are linked by hydrogen bonds to form fibrils that consist of amorphous and crystalline regions.20,21 Cellulose nanocrystals (CNCs) can be obtained by selective acid hydrolysis of the amorphous regions of these fibrils.20 Cationic groups can be linked to the free hydroxyl groups on the surface of the CNCs to yield a cationic material that induces flocculation. As an example, Kan et al.22 coated polyvinylpyridine on CNCs, resulting in a pH-responsive flocculant. Liu et al.23 synthesized polyacrylamide-based CNCs to successfully flocculate kaolin dispersions, while Akhlaghi et al.24 modified CNCs with amine groups, providing a flocculant for the negatively charged surfactant sodium dodecyl sulfate. Jin et al.25 grafted ethylenediamine onto CNCs which could remove anionic dyes in solution. These CNC-based flocculants could find applications in wastewater treatment or could also have potential in microalgal flocculation. Recently, Vandamme et al.21 introduced a novel flocculant based on CNCs for flocculation applied in microalgae harvesting The CNCs are modified with a quaternary amine group. These CNCs have a high aspect ratio (3−5 nm × 50−500 nm) and a similar length compared to molecular polymer flocculants such as chitosan.26,27 In contrast to molecular polymer flocculants, however, CNCs have a high rigidity due to their crystallinity. Before this work, it was not clear how this rigidity influences the flocculation properties of cationic CNCs when compared to cationic polymer flocculants. Research on biopolymer flocculants indicates that a higher amount of cationic charges (higher degree of substitution or DS) often leads to a lower flocculant dose needed, if the interactions between algal cells and the flocculant are predominantly due to electrostatic interactions.11,21,28−30 It is not known whether this also applies to CNC-based flocculants. We therefore set out in this work to evaluate the efficiency of cationic CNCs with a range of DS to confirm and further explore an increase in flocculation efficiency. Two types of grafts, pyridinium- and methylimidazolium-based, were tested to assess potential differences between different grafts. In addition, we compared the flocculation properties of cationic CNCs and cationic biopolymer flocculant chitosan to assess the advantages of a rigid flocculant when compared to a flexible polymer flocculant. In future work, we can use the acquired knowledge to vary the cationic functionality on CNCs to create effective benign flocculants by investigating the toxicity of the different flocculants and choosing the most benign.

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molecular weight: 190−375 kDa), and sodium hydroxide (≥98%, reagent grade, in pellets) were purchased from Sigma-Aldrich BVBA. pToluenesulfonyl chloride (98%) was purchased from Alfa Aesar. The degree of deacetylation (DD) of the chitosan was found in an earlier study to be between 90% and 98%, and the Mn and Mw were determined to be 151.3 and 345.2 kDa, respectively, resulting in a polydispersity index (PDI) of 2.28.31 All products, including chitosan as reference for flocculation performance, were used as received. Synthesis of Cellulose Nanocrystals (CNCs). CNCs were prepared by hydrolysis of cellulose from cotton wool. Aqueous hydrochloric acid (500 mL, 4 M) was heated to 80 °C, and cotton wool (25 g) was slowly added. The reaction was subsequently stirred mechanically for 4 h. Dilution with 2 L of deionized water stopped the reaction. Several washing steps with deionized water followed to separate the acidic solution from the cellulose material, using centrifugation (8500 rpm or 12 000g, 4 °C, 10 min) until the pH of the supernatant was above 4. The product was dialyzed against running deionized water for 72 h in a regenerated cellulose dialysis tube (MWCO 12−14 kDa). Afterward, Amberlite MB-6113 mixed bed ionexchange resin (50 g) was added to the deionized water outside the dialysis tubing to further purify for 24 h. The CNCs were sonicated with a tip sonicator (5 min pulsing 2 s ON, 1 s OFF at a frequency of 20.000 kHz on a Branson Sonifier 250 operating at 25% power), frozen in liquid nitrogen, and freeze-dried (−56 °C for 48 h). The dried nanocrystals were Soxhlet extracted with ethanol for 48 h in a cellulose extraction thimble and dried in the vacuum oven (40 °C) to remove adsorbed organic impurities.32 All starting material was collected, which served as stock CNCs for all following modifications. Synthesis of Benzylpyridinium Grafted CNCs ([Br][BnPy]-gCNCs). The one-pot reaction for the modification of CNCs with a pyridinium salt was described earlier by Jasmani et al.33 The modification was performed five times, under different reaction conditions as described in Table 1 to achieve different degrees of

Table 1. Reaction Conditions for the Pyridinium and Methylimidazolium Grafted CNCsa CNCs (g) 1

5

2

5

3

5

4

5

5

5

4-(1bromomethyl)benzoic acid (g, mmol) 5.9 g (27.4 mmol) 5.9 g (27.4 mmol) 5.9 g (27.4 mmol) 5.9 g (27.4 mmol) 2.6 g (13.6 mmol)

ptoluenesufonylchloride (g, mmol) 5.2 g (27.3 mmol) 5.2 g (27.3 mmol) 5.2 g (27.3 mmol) 5.2 g (27.3 mmol) 3.0 g (13.9 mmol)

temperature (°C)

reaction time (h)

70

16

25

16

80

48

80

16

70

16

The reaction was carried out five times in 150 mL of pyridine or 1methylimidazole as reagent and solvent under different reaction conditions. a

substitution in line with work by Lombardo et al.34 Compared to Lombardo et al.’s report, one additional reaction at 25 °C was performed in this work. Figure 1 illustrates an overview of the reaction. CNCs, 4-(1-bromomethy)benzoic acid, and p-toluenesulfonyl chloride were suspended in dry pyridine (150 mL) under an argon atmosphere. The suspension was heated at the given temperature for the reported length of time before filtering the solid product through a cellulose Soxhlet thimble. Purification with dichloromethane (24 h) and ethanol (48 h) via Soxhlet extraction and drying in vacuo (24 h at 40 °C) resulted in a white powder. These benzylpyridinium grafted CNCs ([Br][BnPy]-g-CNCs) are further referred to as pyridinium (PYR) modified CNCs throughout the manuscript. Synthesis of N-Benzylmethylimidazolium Grafted CNCs ([Br][BnIm]-g-CNCs). The procedure for the synthesis of Nbenzylmethylimidazolium grafted CNCs is a modified procedure

MATERIALS AND METHODS

Materials. Cotton wool (German Pharmacopeia grade) and KBr (>99%, IR spectroscopy grade) were obtained from Chem-lab Analytical. Pyridine (99.5%, extra dry) and 4-(bromomethyl)benzoic acid (97%) were purchased from Acros Organics NV. Dichloromethane (GPR Rectapur), HCl (37%), and ethanol (99.8%, absolute) were purchased from VWR International. Methylimidazole (≥99%, purified), chitosan (from shrimp, practical grade, ≥75% deacetylated; B

DOI: 10.1021/acsanm.9b00315 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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ACS Applied Nano Materials

Figure 1. One-pot reaction to graft pyridinium or methylimidazolium cationic charges onto the cellulose nanocrystals (CNCs). reported by Jasmani et al.35 Similar to the benzylpyridinium grafted CNCs, the reaction was carried out five times under different reaction conditions to obtain different degrees of substitution as reported in Table 1. The conditions were the same as for the pyridinium reaction, except that pyridine was replaced with methyl imidazole. Figure 1 illustrates an overview of the reaction. CNCs, 4-(1-bromomethyl)benzoic acid, and p-toluenesulfonyl chloride were suspended in dry 1methylimidazole (150 mL) under an argon atmosphere. The suspension was heated to the given temperature for the reported amount of time before filtering the solid product through a cellulose Soxhlet thimble. Purification with dichloromethane (24 h) and ethanol (48 h) via Soxhlet extraction and drying in vacuo resulted in a white powder. These benzylmethylimidazolium grafted CNCs ([Br][BnIm]g-CNCs) are further referred to as methylimidazolium (MIM) modified CNCs throughout the manuscript. Characterization of the CNCs. All of the cellulose nanocrystals used in this paper were fully characterized by methods developed in our laboratory combining different techniques that cross-correlate.36 The infrared spectra were measured on a Bruker ALPHA FT-IR spectrophotometer to assess the success of the modifications. The samples were prepared by grinding dried CNCs with potassium bromide (1:133, w/w) and pressing the mixture into transparent pellets. The spectra were acquired in transmission mode as a sum of 16 scans over a frequency range from 4000 to 400 cm−1. The percentages of the elements C, H, N, and S were determined with a Thermo Scientific FLASH 2000 elemental analyzer (EA) using ±1 mg of dry sample. 2,5-Bis(5-tert-butyl-2-benzo-oxazol-2-yl) thiophene (BBOT) (Elemental Microanalysis, UK) was used as a standard for calibration, and vanadium(V) oxide was added to all samples to aid in sulfur determination. Reported values are the average of triplicate measurements. Thermogravimetrical analyses (TGA) were carried out with a Netzsch TG 209 F3 Tarsus. The samples (±5 mg) were heated in platinum crucibles at a ramp rate of 10 °C/min from 10 to 600 °C under an argon atmosphere. Water content of the different samples was determined as the mass loss at 120 °C. In order to evaluate the purity of the samples and the presence of counterions, X-ray photoelectron spectroscopy (XPS) was performed on a Kratos Axis Supra photoelectron spectrometer using a monochromated Al Kα (hν = 1486.7 eV, 5 mA) X-ray source, hybrid (magnetic/electrostatic) optics, and a hemispherical analyzer with a pass energy of 160 eV for survey spectra and 20 eV for high resolution spectra. Spectra were acquired under charge neutralization conditions using an electron flood gun within the field of the magnetic lens. Spectra were charge corrected to aliphatic carbon at 285.0 eV. Spectra were processed in CasaXPS with Tougaard 2-parameter backgrounds used for integration and LA(α, m) lineshapes corresponding with a Voigtian function with Lorentzian exponent α and Gaussian width m used for fitting high resolution spectra. Empirical relative sensitivity factors supplied by Kratos Analytical (Manchester, UK) were used for quantification.

To determine the crystallinity, X-ray diffraction measurements were performed on a Xenocs Xeuss 2.0 laboratory beamline with a Cu Kα Xray source and DECTRIS Eiger 1 M detector. Samples were mounted in between two kapton films and data acquired in transmission geometry. Nine images (300 s per frame) from each sample were stitched together to cover the angular range from 8 to 45° 2θ and integrated to produce 1D diffractograms using Foxtrot software (Xenocs/Synchrotron du Soleil). After dark current correction, intensities were corrected to the absolute intensity scale taking into account transmission of the direct beam (measured at the detector) and sample thickness. A background measurement consisting of two clean kapton foils was subtracted from sample measurements prior to further analysis. Rietveld refinement was performed using TOPAS academic v6 to fit the cellulose Iβ crystal structure, previously published by Nishiyama et al., to the data.37 Refinement was performed in multiple steps. Initially, cellulose Iβ and a flat background were fitted to the data. All unit cell parameters of cellulose Iβ were allowed to vary throughout the refinement, along with a scale factor, two March-Dollase preferred orientation parameters (010 and 001 directions), and a Lorentzian size-broadening parameter. The background scale factor was then fixed, and amorphous content was defined using an hkl phase in P4 space group with a = 2 Å, c = 50 Å, and a peak width limited to a maximum equivalent crystallite size of 1 nm. Finally, the background scale factor was manually reduced until the calculated profile matched the data in the low angular range (8−10° 2θ). Crystallinity was calculated from the integrated crystalline and amorphous intensities according to the method of Thygesen et al.38 The presence of positive charges on the CNC surface was verified with ζ-potential measurements. The ζ-potential of all modifications was measured in a diluted suspension in ultrapure water (0.01 wt % CNCs) carried out with a Brookhaven NanoBrook Omni instrument in phase analysis light scattering mode. Prior to measurements, the suspension was sonicated (Agar Scientific bath sonicator, 60 W, 40 kHz, for 5 min) to disperse the CNCs. Equilibration was allowed prior to collection of the ζ-potential. The ζ-potential was estimated over an average of three measurements of 30 cycles each. Determination of Degree of Substitution (DS). The degree of substitution was calculated using the procedure reported by Eyley and Thielemans from the EA results after correction of the sample for water content (obtained from TGA data).20,35 The XPS data were used to determine the ratio between chlorine and bromine anions as counterions present in the material. Briefly, the empirical formula of the cationic CNCs was calculated by addition of the formula of the grafted species (pyridinium (PYR) or methylimidazolium (MIM)) to the formula of the AGU and calculation of the expected elemental composition (in mass percent), followed by minimization of the difference between this expected composition and the determined composition for all detected elements. Cultivation of Microalgae. The green freshwater microalgae Chlorella vulgaris 211-11b (SAG) was used as the model species for flocculation experiments. The species was cultivated in Wright’s cryptophyte (WC) medium prepared in deionized water.39 Bubble C

DOI: 10.1021/acsanm.9b00315 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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ACS Applied Nano Materials column photobioreactors (30 L) were used for the cultivation, mixed by purging 0.2 μm filtered air (5 L/min) through the culture. The pH was controlled at 8.5 by addition of 2−3% CO2 using a pH-stat system. Experiments were carried out with exponential phase batch cultures (days 6−8) with a biomass concentration of about 0.28 g/L. Flocculation Experiments. The performance of the cationic CNCs and chitosan as flocculants was tested in standardized jar test experiments that were carried out in duplicate. The pH of the cultures was standardized at pH 6 in all experiments. The flocculation efficiency (ηa) was evaluated on the basis of changes in the optical density (OD) of the algal suspension before and after flocculation. OD750 nm was measured with a UV−vis spectrometer (Genesys 10s, Thermo Fisher Scientific). Beakers filled with 50 mL of microalgae culture (ODi 0.750 nm = ±0.7, 0.28 g/L microalgae), adjusted at a starting pH of 6, were mixed intensively using magnetic stirring (550 rpm). This pH was chosen to efficiently compare the flocculation of CNCs and chitosan. Chitosan is only positively charged at a lower pH.32 Stock solutions (5 g/L) of the flocculants were prepared. The CNC-based flocculants were suspended in MQ water and sonicated (Agar Scientific bath sonicator, 60 W, 40 kHz, for 10 min). Chitosan was dissolved in 0.04 M HCl and stirred for 30 min. From the stock solutions, different volumes were added to the beakers with microalgae, resulting in a dose ranging from 0 to 200 mg/ L. The small change in pH due to addition of the chitosan solution was neglected considering the change in pH during the experiment caused by the photosynthetic metabolism of the microalgae. For the modified CNCs, doses of 0, 10, 20, 40, 50, 60, 75, 100, 150, and 200 mg/L were added in duplicate. For chitosan, doses of 0, 1, 3, 7, 10, 15, 20, 40, 70, and 150 mg/L were added in duplicate. After addition of the flocculant, the suspensions were mixed gently (200 rpm) for 20 min. The suspensions were decanted in 50 mL tubes and allowed to settle for 30 min. The difference between initial and final OD measurement was used as a measure of the flocculation efficiency. The percentage of microalgae removed from the suspension was calculated as formulated in eq 1. ηa (%) = 100 ×

(ODi − ODf ) ODi

predicted values. Also, the correlation between the data and predicted values was checked, and the residual standard error (rse) was calculated. The inflection point of the sigmoidal curve corresponds to the ratio a/b and equals the dose needed to achieve 50% flocculation efficiency. This point indicates the minimum flocculation dosage needed to induce flocculation.40 A general linear model was used to test the effect of DS as well as the type of charge on minimum dosage needed to induce flocculation. The model was calculated in R (R version 3.4.1). The initial model included the inflection point as the response variable and the DS and type of charge as independent variables and tested for direct effects as well as their interactions. The most nonsignificant and more complex (as the interaction) variables were left out stepwise, until only variables that significantly contributed to the model (p-value 800 nm) dust particles. The hydrodynamic radius was determined as the average of 5 measurements of 120 s each in triplicate. Measurements were carried out on a Brookhaven NanoBrook Omni instrument.

(1)

Calculation of the Equivalent Charges for Chitosan and CNCs Required (in mol) for the Flocculation of 1 L of Microalgae. The amount of cationic charges required to achieve >90% flocculation was calculated for chitosan and the PYR- and MIM CNC-based flocculants with the highest flocculation efficiency. A degree of deacetylation (DD) of 90% was considered for chitosan based on an earlier study using the same material.31 The molecular weight of the modified saccharide monomers was assumed to be 164.3 g/mol for chitosan. For the CNC-based flocculants, the DS was calculated considering the entire crystal (and not only the surface) and estimated to be 0.20 (1) for PYR modified CNCs and 0.29 for MIM modified CNCs. The calculated molecular weights of the modified AGUs were 216.1 g/mol for PYR and 245.6 g/mol for MIM. The total charge per weight of the flocculant Npos was calculated according to eq 3. Npos =

M n × L0 MM (modified AGU)

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RESULTS AND DISCUSSION Characterization of the Cationic CNCs. CNCs were obtained by acid hydrolysis from pure cotton wool. The CNCs were subsequently functionalized with cationic pyridinium or methylimidazolium groups using a one-pot reaction. The cellulose modifications were verified using different analytical techniques as described above. All analytical results can be found in Tables S2−S15 and Figures S1−S39. FTIR analysis confirmed the modification of the CNCs with pyridinium (PYR) or methylimidazolium (MIM) grafts. The appearance of bands around 1718−1720 cm−1 and around 1279 cm−1 when compared with the unmodified CNCs indicates the formation of a carbonyl ester bond through the attachment of the benzoic acid group. ζ-Potential measurements of the cationic CNCs confirmed that the material carried positive charges on the surface: all modified samples resulted in positive values while the unmodified cellulose had a negative ζ-potential (see Figure 2). The results of elemental analysis are in accordance with the

(2)

Statistical Analysis. For each cellulose-based flocculant, the relation between the added dose of flocculant and the flocculation percentage (FP) was described by a sigmoidal model (eq 2), similar to Lama et al.:40 c FP = (3) 1 + e(dose × b) − a with c as the upper limit of the sigmoidal curve, b as the slope of the curve at the inflection point, and a as the position of the inflection point. On the basis of the average data points of both duplicates, the parameters a, b, and c were estimated from the data using a nonlinear least-squares regression in R (R version 3.4.1). For all models, following starting values were attributed to each parameter: a = −1, b = −0.08, and c = 74. To check the accuracy of the models, a chi square test was performed to check if the data points did not differ significantly from the D

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Table 2. Crystallinity Index (χc) and Calculated Mass Fractions for the Unmodified CNCs and All Cationic CNCsa DS

crystallinity index (χc)

calculated amorphous mass fraction

total

Unmodified 1.0

0

1.0

0.43 0.28 0.27 0.21 0.16

0.98 1.02 0.98 1.00 1.02

0.36 0.38 0.36 0.33 0.19

0.97 0.99 1.02 1.00 0.95

PYR 0.34 0.20 (1) 0.20 (2) 0.13 0.08

0.55 0.74 0.69 0.79 0.86

0.29 0.28 0.26 0.23 0.10

0.61 0.61 0.66 0.67 0.76

MIM

Figure 2. Degree of substituted positive charges (DS) versus ζpotential of all MIM (black squares) and PYR (red dots) cationic CNCs as well as of the unmodified CNCs (blue triangle). Unmodified CNCs show a negative ζ-potential, while all modifications have a positive ζpotential. The plotted values are the average and standard deviation over three measurements of the ζ-potential.

a

The sum of both values is in agreement with the crystallinity index of the unmodified CNCs within the accepted error of the method of ±5%.

both samples within the estimated error of the used method (±5%, Table 2). These results are in line with an earlier work.21 For the PYR modified CNCs, two modifications with a calculated DS of 0.20 were obtained. Within the error of DS determination, these two samples could not be distinguished, although the reaction conditions were different (Table 1). For future reference, the two samples will be indicated with DS = 0.20 (1) and DS = 0.20 (2). The highest DS that was achieved in this study was 0.34 for the PYR modified CNCs. Lombardo et al.45 reported a maximal DS of 0.230 ± 0.008 for PYR modified cellulose. The slightly higher amount of positive charges obtained in this study can be explained by the difference in the hydrolysis process. With sulfuric acid, some of the hydroxyl groups might be substituted by sulfate groups, resulting in part of the hydroxyl groups that are unavailable for substitution (as in Lombardo et al.45) This is not the case when using hydrochloric acid to hydrolyze the cellulose.46 For MIM modified cellulose, the highest DS equaled 0.29. Evaluation of Cationic CNCs as Flocculant. In jar test experiments, the flocculation efficiency of all CNC modifications was evaluated using the microalgae Chlorella vulgaris as a model. All cationic CNCs induced flocculation of Chlorella suspension and achieved a flocculation efficiency exceeding 95% at the optimal dosage. The unmodified CNCs without a positive charge did not induce flocculation (Figure 3). The CNC-based flocculants achieved a flocculation efficiency of >80% at a dosage of 50 mg/L. One exception is the MIM modified CNCs (DS = 0.10) which reaches a 50% efficiency at a dosage of 50 mg/L. This applied range (10−50 mg/L) is in agreement with other studies on natural flocculants applied in microalgae harvesting.47 A general trend could be observed that increasing DS resulted in a decrease in required flocculant dose. The dose−response curves for all flocculants were modeled using nonlinear least-squares regression, assuming a sigmoidal function (eq 2). A chi square test confirmed no significant differences between the data points and the predicted values (pvalue = 0.24 ± 0.01) (see Table S1). The high correlation (>0.99) between measured data and predicted values illustrates that this model is useful to predict the flocculation performance for all flocculants.

modifications: compared to the unmodified CNCs, all modifications show a higher C and N content and slightly lower H content. On the basis of the reaction components, chloride, bromide, and p-toluenesulfonate anions are potential counterions present on the cationic CNC surface. Zooming in on the XPS peak of bromine 3d 5/2, ranging from 67.29 to 67.93 eV, reveals the presence of only bromide anions, without any neutral bromine atoms present, in line with earlier work.21 The absence of neutral bromine indicates full conversion of the bromomethyl benzoic acid to benzylpyridinium and benzyl methylimidazolium for both respective reactions. The chlorine 2p 3/2 peak shows two chemical environments for all of the cationic CNCs. An environment at 196.62−197.65 eV corresponds with the chloride anion (j = 3/2), while another environment at 198.40−200.28 eV could be explained by partial chlorination of cellulose by p-toluene sulfonyl chloride.21 All XPS data of the cationic cellulose indicated an S 2p peak around the 168 eV environment, except for the PYR modified CNCs with a DS = 0.08. The presence of the element sulfur could be attributed to the p-toluenesulfonate present as counterion for the cationic cellulose. Taking the relative amounts of the counterions from XPS data made it possible to account for their contributions in elemental analysis for more accurate DS calculations. The DS was corrected for water content determined by thermogravimetric analysis (water content was determined from the mass loss at 120 °C). A clear positive relation was observed between the DS estimated using this method and the ζ-potential (see Figure 2) as well as the N content based on the elemental analysis. Unmodified nanocellulose had a crystallinity index (χc) of 1.0 (Table 2 and Figure S4). Since the graft onto CNCs is amorphous, the crystallinity index of all cationic CNCs decreased when compared to the unmodified CNCs. This decrease in the crystallinity index should roughly agree with the added mass fraction of the grafts.36 This was confirmed for all CNC modifications with PYR and MIM used in this paper (Figures S5−S14). The calculated mass fraction of the PYR and MIM grafts corresponds to the apparent crystallinity loss for E

DOI: 10.1021/acsanm.9b00315 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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The relationship between the dose to induce flocculation (inflection point, ratio a/b) and the DS as well as type of charge and their interaction was tested statistically using general linear modeling. The type of cationic charge had no significant influence in the model (p = 0.170); i.e., cationic charges due to PYR or MIM were equally effective. There was also no interaction between DS and type of cationic charge on the dose needed (p = 0.620). This resulted in the simple linear model only including DS as influencing parameter on the dose needed to induce flocculation (p = 0.006, adjusted R2 = 0.58, rse = 8.44). These results clearly confirm that a lower dose is needed to induce flocculation when the amount of cationic charges on the CNC surface is higher. The data points as well as the fitted model can be found in Figure 5. These findings show that an increase in positive charges promotes the electrostatic interactions of the flocculant with the negatively charged microalgae, leading to a lower effective dose needed. Studies that evaluated the effect of DS in (semi)flexible polymer flocculants (cationic starch or quaternary chitosan) also showed a lower dose needed to induce flocculation for a higher DS compared to a lower DS.21,28,30,48 These results were explained by a higher charge density on the polyelectrolyte, leading to a stronger electrostatic interaction. By including a range of DS instead of only two values, this trend is confirmed in our study for crystalline cellulose-based flocculants. The influence of DS on flocculation suggests that an electrostatic interaction mechanism plays the lead role in flocculation with CNCs. Comparison between CNC-Based Flocculants and the Molecular Polymer Flocculant Chitosan. Cationic CNCbased flocculants are similar to biopolymer-based flocculants in that the polymer backbone is based on a natural material. They differ from biopolymer-based flocculants by the polymer backbone that has a crystalline structure and therefore a high rigidity. To evaluate the advantages or disadvantages of nanocrystalline versus molecular biopolymer-based flocculants, a comparison was made between cationic CNCs and chitosan, which is the most frequently investigated biopolymer flocculant for harvesting microalgae.5,13 The cationic CNC flocculants prepared in this study consist of β(1−4)-linked D-glucose units with a cationic group replacing a certain amount of surface hydroxyl groups, most likely the primary hydroxyl group (on C6) and the secondary hydroxyl group on the C2 (Figure 6a). The secondary hydroxyl group on the C3 is much less available due to the hydrogen bonding system on the CNC surface. and its modification would result in the loss of crystallinity.20 Chitosan is composed of β(1−4)linked D-glucosamine (deacetylated) and β(1−4)-linked Nacetyl-D-glucosamine (acetylated) groups (Figure 6b). While chitosan is a polymer chain with freedom of rotation and coiling of the chain, the cellulose chains in the cationic CNCs are fixed in a crystalline structure. On the basis of size exclusion chromatography measurements performed in an earlier study,31 the contour length of the chitosan was calculated to be 479 nm on average (with eq 4). The length of chitosan in solution, on the other hand, is reduced due to coiling of the polymer. Indeed, the hydrodynamic radius of chitosan in solution as determined with DLS was only 221.1 ± 11.0 nm. In contrast, the length of CNCs is fixed due to their high rigidity, and their length in solution will not vary with solution conditions (e.g., ionic strength). The length of the modified CNCs determined by AFM ranged between 71 nm (PYR, DS = 0.20 (1)) and 101 nm (PYR, DS = 0.13). Because CNCs are only

Figure 3. Dose−response curves of all cationic cellulose and unmodified cellulose. (a) Dose−response curves for PYR modified cellulose flocculants with different degrees of substitution (DSs) at pH 6 and (b) dose response curves for MIM modified cellulose flocculants with different degrees of substitution (DSs) at pH 6. The plotted values are the averages and standard deviation of the measurements in duplicate. All modifications show flocculation, and the unmodified CNCs do not.

The parameters a, b, and c of the sigmoidal model of the dose−response curve were compared for all flocculants. Parameter c corresponds to the maximum flocculation efficiency and is shown in Figure 4. Both MIM and PYR modified CNCs had similar maximum flocculation efficiencies (95 ± 2.5%). The inflection point of the sigmoidal curve, or the CNC dosage where 50% of the flocculation efficiency is reached, corresponds to the ratio a/b and was used as the minimum flocculation dosage needed to induce flocculation.40 When the ratio of a/b is low, a lower dose is needed to induce flocculation. As the ratio illustrates, this parameter was used to evaluate the effect of DS as well as the effect of type of charge. The lowest degrees of substitution (DS ≤ 0.13) for both PYR and MIM modified cellulose resulted in a higher dose needed to induce flocculation, compared to the substitutions with a DS > 0.13 (see Figure 5). For PYR DS = 0.34, a dose of only 10.2 mg/ L was needed, while a dose of 43.5 mg/L was needed for PYR DS = 0.13, on the basis of the inflection point. For MIM modified cellulose, the lowest dose needed to reach 50% flocculation efficiency was 18.8 mg/L (DS = 0.29) and the highest dose was 48.4 mg/L (DS = 0.10). F

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Figure 4. Degree of substituted positive charges (DS) vs maximum flocculation efficiency (%) for PYR (red dots) and MIM (black squares) modified cellulose. The maximum flocculation efficiency is derived as parameter c, which equals the limit of the modeled sigmoidal curves. The estimated parameter value and standard deviation of this value are plotted. All maximum flocculation efficiencies lie between 90% and 99%.

with DS = 0.34) (0.92 × 10−3 charges/g) when compared to the amount of charges per weight for chitosan (5.48 × 10−3 charges/ g). Moreover, the cationic CNCs have a pH independent charge, while chitosan gets deprotonated with increasing pH, resulting in loss of charges. This limits the use of chitosan for flocculation to a narrow pH range; often, pH adjustments of the growth medium are needed before harvesting. Cationic CNCs, on the other hand, could be applicable at any cultivation pH. Because the number of charges per mass unit of flocculant is different between chitosan and CNCs, the flocculation efficiency as a function of flocculant dose was compared on the basis of the mass of flocculant added as well as on the number of charges added (Figure 7). When expressed on the basis of mass dose of flocculant, the optimal chitosan dose to induce flocculation was slightly lower than for the CNC-based flocculants. For a dose of 10 mg/L, a flocculation efficiency of 96.43 ± 0.14% was reached for chitosan, while at the same dose for the CNC-based flocculant, this was significantly lower, only 47.75 ± 1.79% (unpaired t test, p = 0.022). When the same comparison is made for the number of charges added, however, the minimum number of charges needed to induce flocculation is lower for the CNC-based flocculants compared to chitosan. For a charge of 1.83 × 10−5 mol charges/L, a flocculation efficiency of 61.07 ± 3.76% was reached for chitosan, which was lower compared to the CNC-based flocculant (unpaired t test, p = 0.072). For the same number of charges, the CNC-based flocculant reached a flocculation efficiency of 94.04 ± 0.15%. When chitosan was used for flocculation, the flocculation efficiency declined when the optimal dose was exceeded (Figure 7a). This phenomenon is known as dispersion restabilization and occurs when overdosing of cationic polymer leads to a charge reversal on the cell surface. This was confirmed by ζpotential measurements, which indicated that restabilization of the suspension occurred when the charge of the supernatant changed from negative to positive (Figure S51). Dispersion restabilization is commonly reported when chitosan is used to flocculate microalgae.12,48,49 It also occurs when other biopolymer flocculants are used such as cationic starch28 or

Figure 5. Degree of substituted positive charges (DS) versus the minimal dose needed to induce flocculation at pH 6. This dose equals the inflection point (a/b) of the modeled sigmoidal curves. Black full squares represent MIM modified cellulose; red full dots represent PYR modified cellulose. The full line indicates the linear fitted model.

Figure 6. Chemical structure of (a) cationic CNCs and (b) chitosan.

modified on the surface, the total amount of charges per weight is substantially lower for the cationic CNCs (PYR modification G

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Figure 7. (a) Dose response curve based on the added amount of flocculant for chitosan, PYR modified CNCs (DS = 0.20 (1)), and MIM modified CNCs (DS = 0.29). (b) Dose response curve based on the added amount of charges of each flocculant, for chitosan, PYR modified CNCs (DS = 0.20 (1)), and MIM modified CNCs (DS = 0.29). The average and standard deviation of the measurements in duplicate are plotted.

molecular cationic cellulose.50 When cationic CNCs were used for flocculation, on the contrary, no restabilization occurred, even when the dosage exceeded 200 mg/L. However, at this dose, the ζ-potential of the supernatant became positive (Figure S51). The absence of restabilization at high doses, as well as the difference on a per weight basis and amount of charges, points to a different flocculation mechanism for CNCs compared to polymers like chitosan. There are in general four different flocculation mechanisms mentioned in the literature.3 Three mechanisms are based on electrostatic interactions. In charge neutralization, positively charged flocculants adsorb on the negative cell surface, eliminating the negative charge and destabilizing the microalgal suspension. In the electrostatic patch mechanism, adsorption of the cationic flocculant creates positively charged patches on an otherwise negatively charged surface, resulting in a surface with negatively and positively charged patches. The positively and negatively charged patches on different cells interact, resulting in floc formation. In the bridging mechanism, the flocculant interacts with the surface of two different particles. Finally, in sweeping flocculation, the particles are caught by precipitation of the flocculant, which forms a network dragging particles larger than the network pores with it, and this does not require electrostatic interactions between the microalgae and the flocculant network.31 For polymer flocculation, most studies indicate charge neutralization as the electrostatic interaction mechanism of cationic flocculants, often in combination with bridging.28,45,51−53 Vandamme et al.21 also suggested charge neutralization when using CNCs as flocculant. However, the high rigidity of the CNCs prevents coiling and bending of the polymeric chain. The grafted charges on the CNC surface cannot easily be reoriented spatially as they can for flexible polymers. We hypothesize that polymers thus occupy a larger surface area of the cells, compared to CNCs for the same

efficiency. Cationic CNCs carry charges on opposite sides of the cellulose crystal.20 As a result, when cationic charges on one side of the crystal interact with the microalgal cell surface, cationic charges on the other side of the crystal point away from the cell surface. This is illustrated in Figure 8a. This creates a positive

Figure 8. Schematic representation of the possible flocculation mechanism of cationic modified CNCs (a) compared with chitosan (b). CNCs always have positive charges pointing outward, resulting in positive patches on the cell’s surface, leading to a patch mechanism. Chitosan wraps around the cells and reorients its positive charges toward the cells, resulting in a combination of charge neutralization and a bridging mechanism.

patch that can interact with other microalgal cells. This is different from flexible polymers, which can wrap around a surface, resulting in shielding of all or most of their charges. As Figure 8b illustrates, the chitosan polymer bridges multiple cells, and it reorients its positive charges toward the cell’s surface. Microalgae attached CNCs have remaining positive charges pointing outward, leaving patches of positive charges onto the H

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ACS Applied Nano Materials negatively charged algal cell wall, creating a patchy surface. This would explain the lower charge dose required for CNCs, compared to chitosan: Aided by polymer bridging between algal cells, chitosan neutralizes the algal cell wall, while this is not required for the CNC-based flocculants. Crystalline or rigid flocculants therefore show a great potential for flocculation. This study gained insight into the flocculation mechanism of CNC-based flocculants and the fine-tuning of the modifications. The latter will allow the improvement of the application of CNC-based flocculants in microalgae harvesting. For example, the recovery of the flocculant after harvesting can be addressed using pH-reversible charged CNCs as suggested in the literature.54,55 Previously, the toxicity of the CNC-based flocculants was less of an issue, as the applications focused on wastewater treatment.23−25 To apply these flocculants in microalgae processing, further studies on the possible toxicity of these flocculants as well as nontoxic alternative modifications are suggested.

Carmen Bartic: 0000-0001-9577-2844 Koenraad Muylaert: 0000-0001-9645-4063 Wim Thielemans: 0000-0003-4451-1964 Author Contributions ‡

J.B. and A.V. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors want to acknowledge financial support for this work from Research Foundation Flanders (grant G.0608.16N) and from the EU Interreg France-Wallonie-Vlaanderen program through the ALPO project. W.T. further acknowledges the Provincie West-Vlaanderen for his Chair in Advance Materials, Research Foundation Flanders for his Odysseus fellowship (grant G.0C60.13N), and the European Union’s European Fund for Regional Development, Flanders Innovation & Entrepreneurship, and the Province of West-Flanders for financial support in the Accelerate3 project (Interreg Vlaanderen-Nederland program).

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CONCLUSION The degree of substitution of positive charges on the rigid CNC surfaces plays a prominent role in their flocculation efficiency. A higher degree of substitution leads to a lower dose needed to flocculate microalgae. This suggests that electrostatic interaction plays a role in the flocculation mechanism. No differences between the PYR or MIM type of charges on CNCs was found. Compared to flexible polymers, such as chitosan, CNCs did not show any restabilization: the attachment of other microalgae occurs before the cell surface is completely saturated by the rigid flocculants. The absence of restabilization and the lower charge dose needed to induce flocculation could suggest another flocculation mechanism for CNCs, compared to conventional cationic polymers. CNCs leave patches of positive charges onto the negatively charged algae cell wall by their unattached charges pointing outward. We hypothesize that the patch mechanism could be occurring as the electrostatic interaction. Since flocculation with cationic CNCs is more efficient (charge based) and more widely usable than chitosan (no restabilization, no pH dependence), it can be argued that CNC-based flocculants should be the better option than other biopolymers such as chitosan.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.9b00315.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jonas Blockx: 0000-0002-7991-7676 Samuel Eyley: 0000-0002-1929-8455 Olivier Deschaume: 0000-0001-6222-0947 I

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